A phylogenetic approach to explore the Aspergillus fumigatus conidial surface-associated proteome and its role in pathogenesis

A phylogenetic approach to explore the Aspergillus fumigatus conidial surface-associated proteome and its role in pathogenesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A phylogenetic approach to explore the Aspergillus fumigatus conidial surface-associated proteome and its role in pathogenesis Gustavo Goldman, Clara Valero, Camila Pinzan, Patrícia de Castro, and 22 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3306535/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Aug, 2024 Read the published version in Nature Microbiology → Version 1 posted You are reading this latest preprint version Abstract Aspergillus fumigatus , an important pulmonary fungal pathogen causing several diseases collectively called aspergillosis, relies on asexual spores (conidia) for initiating host infection. Here, we used a phylogenomic approach to compare proteins in the conidial surface of A. fumigatus , two closely related non-pathogenic species, Aspergillus fischeri and Aspergillus oerlinghausenensis , and the cryptic pathogen Aspergillus lentulus . After identifying 62 proteins uniquely expressed on the A. fumigatus conidial surface, we assessed null mutants for 42 genes encoding conidial proteins. Deletion of 33 of these genes altered susceptibility to macrophage killing, penetration and damage to epithelial cells, and cytokine production. Notably, a gene that encodes glycosylasparaginase, which modulates levels of the host pro-inflammatory cytokine IL-1β, is important for infection in an immunocompetent murine model of fungal disease. These results suggest that A. fumigatus conidial surface proteins and effectors are important for evasion and modulation of the immune response at the onset of fungal infection. Biological sciences/Microbiology/Fungi/Fungal pathogenesis Biological sciences/Microbiology/Pathogens Aspergillus fumigatus conidial surface-associated proteome surfome conidia asexual spore pathogenicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Pulmonary fungal diseases caused by the environmental mold Aspergillus fumigatus lead to significant morbidity and mortality. In patients with weakened lung defenses arising from immunosuppression, a chronic respiratory condition, or a prior respiratory infection, asexual spores or conidia of A. fumigatus can evade the lung defenses, germinate, and cause disease 1 . However, our understanding of what makes A. fumigatus a successful pathogen compared to other species remains incomplete. Current evidence suggests that several different Aspergillus species, including A. fumigatus , likely independently evolved the ability to cause human disease 2 (Fig. 1 A), raising the hypothesis that species-specific genes contribute to disease. The early stages of disease—marked by the interaction between the inhaled conidia and the host—may prove insightful for unraveling A. fumigatus pathogenicity. Among studies of other microbial pathogens, there has been an increasing focus on conidial surface protein characterization since they mediate the first encounter with the host immune system. The conidial cell wall comprises a β-1,3-glucan and chitin body, which is covered by rodlet and melanin layers, where proteins are anchored 3,4 . These and other surface proteins play pivotal roles in morphogenesis, resistance to environmental stressors, substrate adherence, and virulence 5 . The hydrophobin RodA is a significant component of the rodlet layer and is essential for cell wall physical resistance and permeability, preventing immune recognition 6,7 . Another conidial surface protein, CcpA, is essential to maintain the correct surface structure and prevent immune recognition 8 . Recently, HscA has been demonstrated to anchor human p11 on phagosomal membranes, rewiring the vesicular trafficking to the non-degradative pathway, allowing the escape of conidia 9 . Other studies aimed to identify potential vaccine candidates, allergens, and biomarkers for diagnosis or characterization of A. fumigatus cell wall dynamics under different stresses or biological processes 10,11,12 . Despite the importance of the conidia for disease, a comprehensive examination of conidial surface proteins and their contribution to pathogenicity in A. fumigatus and closely related species is lacking. Here, we analyzed the conidial surface proteome of A. fumigatus and three closely related species: (i) Aspergillus lentulus , which is a cryptic pathogen and close relative of A. fumigatus 13 ; (ii) Aspergillus oerlinghausenensis , the closest known relative of A. fumigatus , which is azole-resistant but non-pathogenic 14,15 ; and (iii) Aspergillus fischeri , a close relative of A. fumigatus that is less virulent in several animal models and rarely causes disease in humans 16 (Fig. 1 a). Results A. fumigatus has increased virulence in a chemotherapeutic murine model of Invasive Pulmonary Aspergillosis (IPA) and elicits higher cytokine production by bone marrow-derived macrophages (BMDMs). To understand whether the four species have in vivo and in vitro differences in pathogenicity, we comparatively analyzed the capacity of A. fumigatus , A. fischeri (Afi), A. oerlinghausensis (Aoe), and A. lentulus (Ale) to infect macrophages and susceptible hosts. A. fumigatus conidia are less engulfed and killed by Bone Marrow derived macrophages (BMDMs) than Afi, Aoe, and Ale (Figs. 1 b and 1 c). A. fumigatus conidia are recognized by macrophages and are intracellularly degraded by the endocytic pathway through the fusion of conidia-containing phagosomes and lysosomes, forming an acidic phagolysosome (PL) 17 . Melanin can facilitate evading this mechanism, as the A. fumigatus Δ pksP mutant (which lacks a key enzyme involved in melanin biosynthesis) induces higher levels of PL acidification than the corresponding wild-type strain ATCC46645 (Fig. 1 d). However, there are no differences in PL acidification in A. fumigatus , Afi, Aoer, and Ale, indicating that these four species' mechanisms of inhibiting PL acidification are conserved (Fig. 1 d). In contrast, A. fumigatus conidia induced higher levels of four cytokines—TNF-α, IL-6, IL-1β, and 1L-18—in BMDMs than the other three species (Figs. 1 e to 1 h). Interestingly, A. fumigatus and Ale uniquely induced production of inflammasome cytokines IL-1β and IL-18, while Afi and Aoer conidia led to the production of lower levels of TNF-α than A. fumigatus and Afi conidia (Figs. 1 e to 1 h). Virulence of each species was assessed in a clinically relevant chemotherapeutic BALB/c murine model of IPA. The A. fumigatus strain was significantly more virulent than Afi, Aoe, and Ale strains ( p -value > 0.005, Fig. 1 i) and killed all mice after 5 days post-infection (d.p.i.). However, while there was no statistical difference among the other species, an increased virulence caused by Ale strain was observed when compared to the Aoe and Afi strains (40% vs. 50–60% of survival at 15 d.p.i., Fig. 1 i). Taken together, these results strongly indicate that A. fumigatus is more virulent in a chemotherapeutic murine model of IPA, is more efficiently recognized by BMDMs, and can elicit higher levels of cytokine production than the other three species. The conidial surfome of A. fumigatus contains 62 unique proteins. Variation in host recognition observed between A. fumigatus and the other three species raises the hypothesis that conidial surface proteins may underlie differences in host recognition variation. To test this hypothesis, we conducted a trypsin-shaving proteomic analysis and identified A. fumigatus unique proteins on the conidial surface. To do so, samples of resting (0h) and swollen (4h, 37°C) conidia from the four Aspergillus species included in the study ( A. fumigatus , Afi, Aoe, and Ale; Fig. 2 a) were collected and processed for LC-MS/MS analysis as described in 5 (Fig. 2 b). A total of 354 proteins were identified in A. fumigatus conidial surface [193 in resting and 161 in swollen conidia, and 122 (52.6%) shared between the two conditions]. Similarly, in Afi, 594 proteins were identified as part of the conidial surfome [309 in resting and 285 in swollen conidia, and 204 (52.3%) shared between the two conditions]. For Ale, we identified 1,027 conidial surface proteins [285 in resting and 742 in swollen conidia, but only 240 (30.1%) shared between the two conditions]. Finally, 763 proteins were identified in Aoe conidial surface [393 in resting and 370 in swollen conidia, and 269 (54.5%) shared between the two conditions] ( Supplementary Figures S1 ; Supplementary Table S1 ). Comparative analyses of the surface-associated proteome or "surfome" revealed 62 conidial surface proteins were A. fumigatus -specific, i.e., only detected in A. fumigatus conidia. Fifty-six of the genes encoding these proteins are shared by the majority of 206 A. fumigatus isolates, whereas six (AFUB_028320, AFUB_039180, AFUB_045200, AFUB_050510, AFUB_069520, and AFUB_094680) were more variable, belonging to the accessory pangenome (pangenome information from 18 ). In the other three Aspergillus species, 53, 118, and 385 conidial proteins were unique to Afi, Aoer, and Ale, respectively. Numerous conidial proteins were also broadly shared; 188 proteins were shared by at least two species, 146 were shared by at least three species, and 75 were shared by all four species (Fig. 2 c). Functional categorization of the A. fumigatus -specific conidial surfome was carried out manually according to the information available in FungiDB database ( https://fungidb.org/fungidb/app/ ). Most genes encode enzymes involved in many biological processes such as cell wall modification, metabolism, cell signaling, and secondary metabolite biosynthesis. Some of these enzymes have previously been identified as allergens and proteases (Fig. 2 c; Table 1 ). Other surface proteins belonged to less enriched functional categories, such as structural proteins and transporters. However, more than a third of the proteins identified as part of the A. fumigatus -specific conidial surfome had unknown functions (Fig. 2 c). To further explore the putative function of identified proteins, we predicted the presence of secretion and GPI-anchor peptides using SignalP − 5.0 ( https://services.healthtech.dtu.dk/service.php?SignalP-5.0 ) and PredGPI ( http://gpcr2.biocomp.unibo.it/gpipe/index.htm ). Thirty-one out of the 62 proteins are predicted to have a signal peptide cleavage site, while only two have high probability (> 99%) of harboring a GPI-anchoring signal (Table 1 ). One protein (encoded by Afu3g13755) did not have a homologue in the A. fumigatus A1163 background strain and was not examined further. We examined the expression of these 62 genes in a recently published RNAseq dataset that describes gene modulation during conidial germination 19 (Fig. 2 d). About 27% of the surfome genes (16 genes) do not change their gene expression during the first 16 h of germination while 33% (21 genes) have increased expression during the 2 to 16 h germination window (Fig. 2 d). About 24% of the surfome genes (15 genes) are expressed late, while 16% (10 genes) are expressed early (Fig. 2 d). Further characterization of A. fumigatus -specific conidial surface proteins was carried out using gene-deletion strains and phenotypic assays. To do so, we accessed strains from the COFUN homozygous deletion library for 42 genes identified in our previous proteomic analysis following the high-throughput methodology described in 20 . After three attempts, we were unable to construct deletion mutants for the reminaing 20 of the 62 A. fumigatus -specific genes that were not in the library. It remains to be determined if we were not able to delete the 20 remaining genes because they encode essential proteins. Deletions were PCR validated (primers listed in Supplementary Table S2 ). An exhaustive phenotypic screen was carried out for each deletion mutant to identify the involvement of the A. fumigatus surfome in diverse bioprocesses related to virulence (e.g., growth at the temperature of the human body). Growth rates in solid MM at 37°C revealed a defect in growth for ΔAFUB_058080 mutant strain, while ΔAFUB_047280 displayed increased or faster growth compared to the parental A1160 strain. None of the mutants exhibited increased sensitivity to heat (44°C) (Fig. 2 e and Table 1 ). However, ΔAFUB_058080 was found to be more sensitive than the A1160 wild-type strain to the cell wall stressor Congo Red (CR). Similarly, ΔAFUB_040910 and ΔAFUB_100920 showed decreased and increased susceptibility to oxidative stress, respectively (Fig. 2 e and Table 1 ). Germination rates for all the mutants and wild-type strain were also determined; most null mutant strains (33/42) behaved similarly to A1160 reference strain. The remaining deletion strains could be differentiated into two groups: i) slow-germinating (below 30% of A1160 germination rate; ΔAFUB_035440, ΔAFUB_057080, ΔAFUB_058080, ΔAFUB_100460) and ii) fast-germinating (above 30% of A1160 germination rate; ΔAFUB_010730, ΔAFUB_039800, ΔAFUB_040910; ΔAFUB_043810, ΔAFUB_095010) (Fig. 2 f and Table 1 ). The crystal violet (CV) assay was used as an indirect measurement of the adhesion properties of conidia at both resting and swollen stages for all the strains. Interestingly, significant differences between the wild-type and deletion strains were only observed in two instances: ΔAFUB_000410 displayed reduced adhesion properties in comparison with A1160, while ΔAFUB_041890 showed a higher capacity to adhere to surfaces than the wild-type strain (Fig. 2 g and Table 1 ). Abnormalities in conidial cell wall organization were investigated by staining different components of the cell wall. Calcofluor White (CFW) binds and blocks chitin biosynthesis, and FITC-conjugated wheat germ agglutinin (WGA), a lectin that recognizes exposed chitin and chitooligomers were used for staining chitin. Chitooligomer exposure is better quantified using WGA; total cell wall chitooligomer content is better assessed by CFW. CFW revealed a group of 8 strains (ΔAFUB_030040, ΔAFUB_035440, ΔAFUB_039180, ΔAFUB_039800, ΔAFUB_040910, ΔAFUB_041890, ΔAFUB_043810 and ΔAFUB_045200) that exhibited lower chitin exposure than the wild-type strain, and WGA a set of 11 mutants (ΔAFUB_030040, ΔAFUB_031630, ΔAFUB_035440, ΔAFUB_039180, ΔAFUB_065340, ΔAFUB_068800, ΔAFUB_069520, ΔAFUB_076190, ΔAFUB_078110 and ΔAFUB_079300) that showed higher chitin exposure (Fig. 2 h and Table 1 ). Intriguingly, three of these mutants showed opposing chitin exposure phenotypes (ΔAFUB_030040, ΔAFUB_035440 and ΔAFUB_039180). Using a dectin-based immunofluorescent assay, we did not find any consistent phenotypes for β-D-glucan exposure (Fig. 2 h and Table 1 ). Finally, only one mutant (ΔAFUB_050510) showed altered hydrophobicity properties when compared with the A1160 wild-type strain ( Supplementary Figure S2 ). Taken together, these results suggest that A. fumigatus conidial surface-associated proteins are also involved in the correct assembly of the cell wall and have an important role in combatting stress and adhering to surfaces. A subset of A. fumigatus surfome proteins is involved in host-pathogen interactions. To understand the role of the A. fumigatus surfome in the establishment of infection, we individually assessed the role of each of the 42 A. fumigatus-specific surfome proteins in survival and tissue invasion using in vitro models of infection. A. fumigatus killing was evaluated by the number of CFUs after challenging murine macrophages (Raw 264.7) with conidia from each of the null mutants at 6 h post-infection (hpi), while A. fumigatus invasion was determined by differential staining after infecting human lung epithelial cells (A549) with germinating conidia from all deletion strains at 3 hpi. For statistical analyses, we only considered those strains showing a 30% increase or decrease in conidial survival and invasion compared to the parental strain (A1160). Overall, we identified 18 A. fumigatus genes encoding surface proteins that contribute to survival to macrophage killing (9 conidial surface null strains were more susceptible to macrophage killing and another 9 were more resistant to being cleared) (Fig. 3 a, Table 1 ). On the other hand, 27 A. fumigatus genes encoding for surface proteins were important for epithelial cell invasion (3 mutants were more efficiently taken up by the epithelial cells whilst 24 exhibited defects in cell invasion) (Fig. 3 b, Table 1 ). Although there is no correlation between macrophage survival and epithelial cell invasion (Fig. 3 c), interestingly, 14 A. fumigatus surfome proteins were identified to be involved in both killing and invasion (ΔAFUB_000410, ΔAFUB_004010, ΔAFUB_012100, ΔAFUB_014430, ΔAFUB_028320, ΔAFUB_040910, ΔAFUB_045200, ΔAFUB_047280, ΔAFUB_056750, ΔAFUB_059140, ΔAFUB_059210, ΔAFUB_069520, ΔAFUB_078110 and ΔAFUB_094680) (Figs. 3 a and 3 b, Table 1 ), half of them with unknown function, suggesting that a subset of A. fumigatus conidial surfome proteins identified in our proteomic analysis contributes to triggering immune cell response. The capacity of the null surface protein strains to induce epithelial cell damage was also evaluated with 3 strains producing less damage than the A1160 strain and only one triggering higher cell toxicity (Fig. 3 d, Table 1 ). Our data in Fig. 1 e to h indicate A. fumigatus induces a stronger immune response in BMDMs compared to three other closely related species. To investigate whether increased induction of immune response in A. fumigatus is driven by specific conidial surface protein(s), we investigated if the absence of these 42 proteins could play a role in cytokine response by determining the IL1-β and TNF-α production in BMDMs (Figs. 3 e and 3 f and Table 1 ). We observed a significantly increased 1L-1β production in 11 mutants (AFUB_000410, AFUB_028320, AFUB_031630, AFUB_035440, AFUB_036380, AFUB_039800, AFUB_059140, AFUB_059210, AFUB_068800, AFUB_078110, and AFUB_094680) and decreased production in 3 mutants (AFUB_020170, AFUB_045200, and AFUB_050560) (Fig. 3 f and Table 1 ). TNF-α production was significantly increased in AFUB_012100 and decreased in AFUB_010730, AFUB_039800, and AFUB_059210 (Fig. 3 f and Table 1 ). We also evaluated the BMDMs survival in the presence of these mutants by looking at their lactate dehydrogenase (LDH) activity during infection with the mutants compared to the wild-type strain; only the AFU_065340 (a putative zinc-containing alcohol dehydrogenase) mutant had about 80% inhibited LDH. Using information about A. fumigatus regulatory networks publicly available at the National Center for Biotechnology Information (NCBI) and the clustering online tool FungiExpressZ ( https://cparsania.shinyapps.io/FungiExpresZ/ ), we identified two clusters enriched for RNAseq datasets of A. fumigatus exposed to animal cells or invasive aspergillosis conditions (Fig. 4 a, highlighted in red circles ). These regulatory datasets were obtained after challenging human dendritic and lung epithelial cells with A. fumigatus strains (NCBI Bioprojects PRJEB1583 and PRJNA399754). These two clusters contain 22 genes, 11 of which are also among the 62 A. fumigatus -specific genes, suggesting that the protein products of these 11 surfome genes could be important for host-pathogen interactions and the establishment of the infection (Figs. 4 b and 4 c, Table 1 ). Other upregulated clusters are mainly related to response to temperature changes, oxidative and osmotic stresses, or nutritional availability, thus illustrating many other functions of A. fumigatus conidial surface proteins ( Supplementary Figure S3 ; Supplementary Table S3 ). Furthermore, 7 of the 62 A. fumigatus -specific genes were within the 1,700 genes whose evolutionary rate differed between pathogenic and non-pathogenic species from Aspergillus section Fumigati 21 , suggesting that they may be associated with the repeated evolution of pathogenicity in Aspergillus spp. (Fig. 4 d). These results suggest that some of the proteins specifically identified in the A. fumigatus surfome, but which are absent from three other closely related species, are important for mediating host interactions and eliciting cytokines, and are modulated at transcriptional level in the presence of animal cells. Characterization of the glycosylasparaginase null mutant. As an initial step to investigate in more detail the surfome mutants, we prioritize the mutants that elicit increased IL-1β production (Fig. 3 g and Table 1 ) and decided to start by the characterization of ΔAFUB_059140 mutant (here named Δ aspA ). The aspA null mutant showed increased killing by BMDMs compared to the wild-type strain but decreased invasion of A549 alveolar epithelial cells (Figs. 3 a to 3 c and Table 1 ). We constructed a second independent Δ aspA mutant and both independent aspA null mutants have the same phenotypes, strongly suggesting that the observed phenotypes are only due to the aspA single deletion and not to secondary mutations present in these strains. The aspA gene encodes a putative glycosylasparaginase (Fig. 4 e) that catalyzes the hydrolysis of N4-(beta-N-acetyl-D-glucosaminyl)-L-asparagine yielding as products N-acetyl-beta-glucosaminylamine plus L-aspartate cleaving the GlcNAc-Asn bond that links oligosaccharides to asparagine in N-linked glycoproteins, playing a major role in the degradation of glycoproteins ( http://pfam-legacy.xfam.org/ ; PF01112). AspA is distributed among several fungal classes (687 sequences) but enriched in Eurotiomycetes and Sordariomycetes where most of the plant and animal pathogens are present (Fig. 4 f). The Δ aspA mutants are more phagocytosed by BMDMs than the wild-type (Fig. 5 a) and have increased percentage of adherent conidia on the BMDMs surface, and decreased cell viability as measured by CFW fluorescence and metabolic activity by MTT, respectively (Figs. 5 a to 5 c). The increased expression of 1L-1β in the Δ aspA mutants is dependent on their viability since UV-killed conidia have no IL-1β and TNF-α activities comparable to the wild-type strain (Fig. 5 d). Although BMDMs have comparable Reactive Oxygen Species (ROS) production upon 4 and 24 h exposure to either the wild-type or Δ aspA mutant, BMDMs have significant increased production of ROS when 8 h in the presence of Δ aspA mutant, suggesting that this mutant is either not able to cope with ROS detoxification at this time-point or induces increased ROS production (Fig. 5 e). The lack of aspA does not change A. fumigatus ability to evade phagolysosome acidification in contrast to the A. fumigatus Δ pksP mutant (Fig. 5 f). Transwell migration assays show that most of the increased production of IL-1β by aspA null mutants need contact with BMDMs (Fig. 5 g). In contrast, TNF-α production is equally dependent on contact for either the wild-type and Δ aspA mutants (Fig. 5 h). Taken together, these results suggest that AspA activity is important for BMDMs phagocytosis and viability of A. fumigatus conidia, and that AspA can modulate the IL-1β production by direct contact of the conidia with BMDMs. AspA is important for establishment of virulence in an immunocompetent murine model of aspergillosis. A. fumigatus Δ aspA have the same virulence than the wild-type in a chemotherapeutic murine model but they are less virulent than the wild-type strain in an immunocompetent murine model as measured by fungal burden (Figs. 6 a and 6 b). We measured the production of cytokines TNF-α, IL-1β, IL-18, IL-12, INF-ϒ, IL-6, and the chemokine CXCL-1 in the lungs homogenates of the immunocompetent model infected by the wild-type and Δ aspA mutants (Figs. 6 c to 6 i ) . The lack of AspA caused increased production of proinflammatory cytokines and CXCL-1 that acts as a chemoattractant for several immune cells, and it has already been observed as induced upon A. fumigatus lung infection 22 . Our results indicate that AspA is important for attenuating the inflammatory responses and decreasing the neutrophil migration during the early steps of A. fumigatus infection in the lungs. Characterization of the heterologously expressed glycosylasparaginase. We heterologously expressed AspA in Pichia pastoris and verified if the enzyme was active by checking its putative asparaginase activity 23 ( Supplementary Figure S4 ). Incubation of BMDMs with increasing AspA concentrations had no significant impact on LDH activity (Fig. 7 a) AspA 0.2 or 2 µg decreased BMDMs viability by about 1 and 10%, respectively, while DMSO decreased by 80% (Fig. 7 b). AspA can modulate IL-1β and TNF-α production by using different AspA concentrations in a linear dose-response relationship (Figs. 7 c and 7 d). The cytokine induction by AspA is not related to its glycosylation since AspA exposure to Peptide:N-glycosidase F (PNGase F), which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins and glycopeptides 24 , did not abolish this induction ( Supplementary Figure S5 ). We were unable to identify asparaginase activity in AspA, and we decided to inactivate the enzyme by boiling it for 5 minutes. Boiling of the AspA decreased IL-1β production by about 50% (Fig. 7 e). Most surprisingly, AspA was also able to induce TNF-α production and AspA inactivation decreased its accumulation by 30% (Fig. 7 f). Next, we investigated if previous (by 8 h) or concomitant exposure of BMDMs to 2µg of AspA and conidia could change the conidial viability (Fig. 7 g). Both treatments increased conidial viability by about 20%, strongly indicating that AspA contributes to conidial viability (Fig. 7 g). Taken together these results suggest AspA modulates BMDM cytokines and it is important to increase conidial viability. Proteomic profiling of BMDMs exposed to AspA. To begin investigating which macrophage metabolic pathways are modulated by AspA, we incubated BMDMs in the presence of AspA (0.2 µg) for 24 h, extracted the proteins, and identified them by mass spectrometry ( Supplementary Table S4 ). Principal Component Analysis (PCA) and quantitative analysis of these samples showed that BMDM proteins and BMDM proteins exposed to AspA displayed very different distributions (Fig. 8 a). Differentially expressed proteins are defined as those with a minimum of two-fold change in protein abundance (log2FC ≥ 1.0 and ≤ -1.0; FDR of 0.05) when compared to the BMDMs under the equivalent conditions. We observed 101 proteins upregulated and 259 downregulated in the BMDMs exposed to AspA (Fig. 8 b and Supplementary Table S4 ). We have used the Reference Database of Immune cells ( http://refdic.rcai.riken.jp/document.cgi ) for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of distinct biological functions of the shared and specific proteomic differences. The BMDM proteins downregulated in the presence of AspA are involved in (i) cell redox homeostasis, such as glutathione, metabolic process, oxidation reduction, and response to oxidative stress; (ii) carbohydrate metabolism, such as pentose-phosphate shunt, tricarboxylic acid cycle, and glycolysis/gluconeogenesis; (iii) protein synthesis; (iv) pyruvate metabolism and (v) ubiquitin-dependent protein catabolism (Figs. 8 c and 8 d). In contrast, protein transport, vesicle-mediated transport, mRNA processing, and metabolic processes are upregulated (Fig. 8 c). We have also visually detected the downregulation of 18 proteins specifically related to the modulation of the innate immune and lysosomal functions (Table 2 and Supplementary Table S4 ), including: (i) Csf1r, macrophage colony-stimulating factor 1 receptor, a tyrosine-protein kinase that acts as a cell surface receptor and plays a role in the regulation of survival, proliferation and differentiation of mononuclear phagocytes, such as macrophages and monocytes ( www.uniprot.org/uniprotkb/P09581/entry ); (ii) Ncf1, neutrophil cytosol factor 1, required for the activation of the latent NADPH oxidase ( www.uniprot.org/uniprotkb/Q09014/entry ); (iii) Cotl1, coactosin-like protein that influences leukotrienes synthesis ( www.uniprot.org/uniprotkb/Q9CQI6/entry ); (iv) Mtdh, protein LYRIC that activates the nuclear factor kappa-B (NF-kappa B) transcription factor ( www.uniprot.org/uniprotkb/Q80WJ7/entry ), and (v) and a few lysosomal proteases cathepsins; and upregulated, (i) Ptprc, a receptor-type tyrosine-protein phosphatase C that is required for T-cell activation through the antigen receptor ( www.uniprot.org/uniprotkb/P06800/entry ) and (ii) Ctss, cathepsin S, a key protease responsible for the removal of the invariant chain from MHC class II molecules and MHC class II antigen presentation ( www.uniprot.org/uniprotkb/O70370/entry ). Examination of the general mouse functional protein association network, retrieved from STRING ( https://string-db.org ) showed that 15 of these proteins have functional associations, suggesting that AspA impacts specific protein interaction networks (Fig. 8 e). Taken together, our results suggest that AspA modulates several BMDM metabolic pathways specifically affecting the abundance of proteins important for immune function. DISCUSSION A. fumigatus is a prominent human fungal pathogen that infects and frequently kills hundreds of thousands yearly. Most other closely related species, on the other hand, are not pathogenic 2,15,25 . The dozen or so clinically relevant Aspergillus species are scattered among hundreds of non-pathogenic species across the Aspergillus phylogeny, implying that the capacity to cause illness or pathogenicity has evolved multiple times in this lineage 2 . The observed pathogenicity spectrum cannot be solely explained by changes in species ecologies or ascertainment bias, suggesting that the repeated evolution of Aspergillus pathogenicity has a genetic foundation, at least in part 2 . A. fumigatus conidia are the first fungal structure that encounters the host. In Immunocompetent hosts, conidia are cleared and do not affect host health. However, there are several mechanisms of conidial and germling recognition and evasion based mostly on melanin and polysaccharides, such as β-1,3 glucan, chitin, and galactosaminogalactan present on the conidial surface. Although the importance of protein effectors for pathogenesis and virulence have been extensively described in plant fungal pathogens, there are very few reports about conidial surface proteins or protein effectors in human fungal pathogens. In A. fumigatus , these include hydrophobin 6 , the conidial surface protein cpcA 8 that prevents immune recognition of β-1,3-glucans, and the HscA protein that anchors human p11 on phagosomal membranes, rewiring the vesicular trafficking to the non-degradative pathway, allowing escape of conidia 9 . More recently, a Cryptococcus neoformans secreted effector that triggers allergic inflammation 26 was also identified. Here, we used a phylogenetic approach to investigate proteins specifically expressed in A. fumigatus conidial surface by comparing A. fumigatus proteins with two closely non-pathogenic related species, Afi and Aoe, and one more distantly-related pathogenic species, Ale. These surface proteins could be potential surface proteins or protein effectors that modulate different aspects of virulence and pathogenicity or alternatively new genetic determinants important for maintaining the correct conidial structure allowing attachment to host surfaces or preventing immune recognition. Using this approach, we identified 62 A. fumigatus -specific conidial surface proteins and were able to delete 42 of them. Some of these proteins affect the correct assembly of the conidial surface components since they have increased or decreased chitin exposure or different hydrophobic properties. However, several of them affect the interaction with the host cells since they modulate survival in the presence of macrophages, invasion and/or damage to epithelial cells, and cytokine production. This collection of genes and mutants provides an opportunity for the characterization of genetic determinants important for the A. fumigatus -host interaction. We focused on one of these genetic determinants, a gene encoding a putative glycosylasparaginase, named AspA. To our knowledge this is the first characterized fungal glycosylasparaginase. Homologues of this gene are not present in Saccharomyces cerevisiae and when mutated in humans (AGA gene encodes the glycosylasparaginase) cause the most frequent type of recessively inherited lysosomal storage disease named Aspartylglucosaminuria (AGU) 27 . AGU is a disorder related to degradation of glycoproteins. Carbohydrate moieties are typically found in glycoproteins and are linked to the protein moiety by an N-glycosidic bond formed by the amino acid L-asparagine and the monosaccharide N-acetylglucosamine. The lysosomal resident enzyme glycosylasparaginase, which cleaves the N-glycosidic link between the L-asparagine and N-acetylglucosamine moieties of GlcNAc-Asn, is lacking in AGU. This enzyme deficiency causes a buildup of undegraded aspartylglucosamine and several other glycoasparagines, i.e., glycoconjugates having a L-asparagine moiety attached to the carbohydrate chain, in the affected person's tissues and body fluids 28,29 . AGU patients have developmental delays, hyperactivity, early growth spurt, inguinal and abdominal hernias, clumsiness, characteristic facial features, recurrent respiratory and ear infections, and chronic arthritis 30 . As far as we know there is no previous information about the involvement of glycosylasparaginases in bacterial and fungal pathogenesis. However, there are several reports about possible roles played by asparaginases in bacterial virulence 31–35 , but not in fungal virulence. Several bacterial virulence factors have evolved to use enzymatic deamidation, i.e., the irreversible conversion of the amino acids glutamine and asparagine to glutamic acid and aspartic acid, respectively 32 . Helicobacter pylori cytotoxic activity was significantly decreased by an asparaginase-deficient strain 31 while Mycobacterium tuberculosis exploits asparaginase to assimilate nitrogen to produce ammonium and resist acid stress during infection 33 ; Salmonellae asparaginases mediate virulence and inhibit T cell responses 34,35 . A. fumigatus has two putative glycosylasparaginase (AFUB_059140, which is described here, and AFUB_021340, none of them with a putative signal peptide) and two asparaginase encoding-genes (AFUB_003170 and AFUB_051450, with and without a putative signal peptide, respectively). It remains to be investigated if these genes are also important for A. fumigatus virulence. The Δ aspA mutants are more phagocytosed and killed than the wild-type, elicit more ROS production and need BMDMs contact to have increased IL-1β production, but have identical percentages of acidification of the phagolysosomes. These results suggest that AspA has a role in earlier or later steps after the formation of phagolysosomes. AspA is important for the establishment of the infection in an immunocompetent mouse model but not in the chemotherapeutic mouse model, and the lack of AspA also plays an important role in modulating proinflammatory cytokines in vivo . The lack of AspA causes increased production of proinflammatory cytokines and CXCL-1 that acts as a chemoattractant for several immune cells, especially neutrophils, and it has already been observed as induced upon A. fumigatus lung infection 22 . Our results indicate that AspA is important for modulating the inflammatory response and neutrophils migration upon A. fumigatus infection in the lungs. We also investigated AspA through its heterologous expression and the influence of AspA on cytokine production and macrophage metabolism. Although the human glycosylasparaginase has both deglycosylation and asparaginase activities 36 , we were not able to detect asparaginase activity in A. fumigatus AspA. However, inactivated boiled AspA decreases BMDM cytokine induction, suggesting that it is still active, and this activity is not due to possible glycosylated residues present in AspA since PNGase treatment has not abolished the cytokine induction. AspA has no putative signal peptide, was identified only at the surface of the dormant conidia and does not seem to be secreted. When BMDMs were exposed to AspA, there was a dramatic change in its proteomic profiling, with decreased cell redox and carbohydrate, and pyruvate metabolism, and protein synthesis suggesting that AspA is affecting the BMDMs aerobic metabolism and ROS production, and decreasing the expression of proteins important for ROS production, such as Ncf1, Neutrophil cytosol factor 1, a cytosolic subunit of neutrophil NADPH oxidase, a multicomponent enzyme that is activated to produce superoxide anion. It has been previously shown that patients with hypersensitivity pneumonitis (HP), a rare initial presentation in chronic granulomatous disease (CGD) have mutations in NCF1 and NCF2 genes and are more susceptible to invasive pulmonary A. fumigatus infection 37 . Actually, the Δ aspA shows higher induction of ROS in BMDMs. We also observed that BMDMs exposure to AspA decreased the production of several lysosomal proteases, cathepsins, important for protein degradation. Upon conidial phagocytosis, phagosomes are fused with lysosomes resulting in functional phagolysosomes that are integral part of a degradative pathway that kills and destroys conidia. Is AspA important for escaping phagolysosomal killing? An attractive model for AspA mechanism of action could be that upon conidial phagocytosis, AspA is already interacting with proteins at the macrophage surface deglycosylating asparagine residues and modifying protein activity. Since glycosylasparaginases are described as lysosomal resident proteins, they must be resistant to acid pH (AspA has an isoelectric point of 5.4). Upon conidial phagocytosis and during the formation of phagolysosomes, AspA remains active in the phagolysosome, eventually deglycosylating asparagine residues affecting conidial survival. This apparently has no effect on the percentage of acidification of the phagolysosome since there are no differences between the mutant and wild-type strains. However, it is possible after partial conidial destruction, AspA remains active in the host lysosome affecting the glycosylation metabolism in the cell, and consequently allowing some conidia to survive. A. fumigatus conidia can survive for some time in the phagosomes of immune cells and epithelial cells 38–41 . AspA could affect host’s membrane trafficking system or redirect the conidia-containing phagosomes toward exocytosis and release of conidia. This has already been reported for a novel A. fumigatus surface effector, the heat-shock protein HscA that interacts with the human p11 protein, a decisive regulatory node for directing endosomes to different pathways 9 . Different aspects of our proposed model remain to be investigated. METHODS Fungal strains. All strains included in this work are listed in Table 1 . Knockout mutant strains belong to the Manchester Infection Fungal Group (MFIG) collection and were constructed as described in 20 . Surface proteome analysis. Freshly harvested conidia of A. fumigatus A1163, A. oerlinghausenensis CBS 139183 T , A. fischeri NRRL 181, and A. lentulus CNM-CM6069 were cultivated on potato dextrose agar at 37°C for 0 h (resting conidia) or 4 h (swollen conidia). 1 × 10 9 conidia of each species in three biological replicates were washed twice with 25 mM ammonium bicarbonate (AB) and centrifuged at 1,800 × g for 10 min. Conidia were resuspended in 800 µl of 25 mM AB and incubated with 5 µg trypsin (MS approved, Serva) for 5 min at 37°C. Cell suspensions were immediately passed through 0.2 µm syringe filters (cellulose acetate, Sartorius) to separate conidia from the digestion buffer followed by washing the filters with 200 µl of 25 mM AB. Subsequently, 10 µl of 90% (v/v) formic acid was added to the cell-free solution to stop the proteolytic digestion. Samples were evaporated to dryness in a vacuum concentrator (Eppendorf), resuspended in 30 µl of 2% (v/v) acetonitrile (ACN) and 0.05%(v/v) trifluoroacetic acid (TFA), centrifuged for 15 min at 14,000 × g through 10 kDa molecular weight cut-off filters (modified PES, VWR), and transferred into HPLC vials. LC − MS/MS analysis was performed on an Ultimate 3000 RSLC nano instrument coupled to a QExactive HF mass spectrometer (Thermo Fisher Scientific) as described previously 12 . Tryptic peptides were trapped for 4 min on an Acclaim Pep Map 100 column (2 cm × 75 µm, 3 µm) at a flow rate of 5 µl/min. Peptides were further separated on an Acclaim Pep Map column (50 cm × 75 µm, 2 µm) using a binary gradient of eluents A (0.1% (v/v) formic acid in H 2 O) and B (0.1% (v/v) formic acid in 90:10 (v/v) ACN/H2O): 0–4 min at 4% B, 5 min at 8% B, 20 min at 12% B, 30 min at 18% B, 40 min at 25% B, 50 min at 35% B,57 min at 50% B, 62 − 65 min at 96% B, 65.1 − 90 min at 4% B. Positively charged ions were generated by a Nanospray FlexIon Source (Thermo Fisher Scientific) using a stainless steel emitter with 2.2 kV spray voltage. Ions were measured in the Full MS / data-dependent MS2 (Top15) mode. Precursor ions were scanned at m/z 300–1500, a mass resolution of 60,000 full width at half maximum (FWHM), an automatic gain control (AGC) target of 1 × 10 6 , and a maximum injection time (maxIT) of 100 ms. Precursor ions were isolated with a width of m/z 2.0. Fragment ions generated in the higher-energy collisional dissociation (HCD) cell at 30% normalized collision energy using N 2 gas were scanned at 15,000 FWHM, an AGC target of 2×10 5 , and a maxIT of 80 ms. Dynamic exclusion was set to 25 s. The MS/MS data were searched against the FungiDB databases of A. fumigatus Af293 and A. fischeri NRRL_181, the NCBI database of A. lentulus and the JGI database of A. oerlinghausenensis (all downloaded on 2020/10/27) using Proteome Discoverer 2.4 and the algorithms of Mascot 2.4.1, Sequest HT, and MS Amanda 2.0 and MS Fragger 3.0. Two missed cleavages were allowed for the tryptic digestion. The precursor mass tolerance was set to 10 ppm and the fragment mass tolerance was set to 0.02 Da. Modifications were defined as dynamic Met oxidation, protein N-term acetylation and/or loss of methionine. A strict false discovery rate (FDR) 30 (Mascot), > 4 (Sequest HT), > 300 (MS Amanda) or > 8 (MS Fragger) were required for positive protein hits. The Percolator node of PD2.4 and a reverse decoy database was used for qvalue validation of spectral matches. Only rank 1 proteins and peptides of the top scored proteins were counted ( Supplementary Table S1 ). The infected macrophage proteome was performed through lysis in 8M urea in 25 mM AB containing protease inhibitor cocktail using a probe tip sornicator: 40% amplitude for 3 cycles for 10 s and intervals of 10 s. After sonication, samples were centrifuged, the supernatant was transferred to a new tube and quantified using Bradford reagent. Proteins were reduced with 10mM DTT for 30 min at 30 degrees, alkylated with 40mM iodoacetamide for 40 min at room temperature in the dark and overnight digested with trypsin in the ratio 1:50 (enzyme to protein ratio). The tryptic peptides were desalted with Oasis HLB Cartridges (Waters) according to the manufacturer instructions, dried down by speed-vac, and then resuspended in 0.1% formic. LC-MS/MS analysis was performed in an EASY-nLC system (Thermo Scientific) coupled to LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific). Peptides were separated on C18 PicoFrit column (C18 PepMap, 75 µm id × 10 cm, 3.5 µm particle size, 100 Å pore size; New Objective) using a gradient of A and B buffers (buffer A: 0.1% formic acid; Buffer B: 95% CAN, 0.1% formic acid) at a flow rate of 300nL/min: from 2–30% B over 80 min and from 30–90% B over 5 min. The LTQ-Orbitrap Velos was operated in positive ion mode with data-dependent acquisition. The full scan was obtained in the Orbitrap with an automatic gain control (AGC) target value of 10e6 ions and a maximum fill time of 500 ms. Each precursor ion scan was acquired at a resolution of 60,000 FWHM in the 400–1500 m/z mass range. Peptide ions were fragmented by CID MS/MS using a normalized collision energy of 35. The 20 most abundant peptides were selected for MS/MS and dynamically excluded during 30 sec. All raw data were accessed in the Xcalibur software (Thermo Scientific). For protein identification, raw data were processed using MaxQuant software version 1.5.3.8. The MS/MS spectra were searched against a protein database composed of Aspergillus fumigatus and Human sequences with the addition of common contaminants with a tolerance level of 4.5 ppm for MS and 0.5 Da for MS/MS. Trypsin was selected as a specific enzyme with a maximum of two missed cleavages. Carbamidomethylation of cysteine (57.021 Da) was set as a fixed modification, and oxidation of methionine (15.994 Da), deamidation NQ (+ 0.984 Da) and protein N-terminal acetylation (42.010 Da) were set as variable modifications. PSMs, peptides and proteins were accepted at FDR less than 1%. Multivariate statistical analyses were performed on the protein groups using the label-free quantification (LFQ) results. The analyses were conducted using the LFQ-Analyst web platform 42 with default parameters, including a p-value cutoff of 0.05 and a Log2 fold change cutoff of 1. BMDMs protein extraction. The BMDMs protein extraction was performed in 8M urea containing protease inhibitor cocktail using a probe tip sornicator: 40% amplitude for 3 cycles for 10 s and intervals of 10 s. After sonication, samples were centrifuged, the supernatant was transferred to a new tube and quantified using Bradford reagent. Proteins were reduced with 10mM DTT, alkylated with 40mM iodoacetamide and overnight digested with trypsin in the ratio 1:50 (enzyme:protein). The tryptic peptides were desalted with Oasis HLB Cartridges, dried down by speed-vac, and then resuspended in 0.1% formic to the LC-MS/MS analysis that was performed in an EASY-nLC system (Thermo Scientific) coupled to LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific). Peptides were separated on C18 PicoFrit column (C18 PepMap, 75 µm id × 10 cm, 3.5 µm particle size, 100 Å pore size; New Objective) using a gradient of A and B buffers (buffer A: 0.1% formic acid; Buffer B: 95% ACN, 0.1% formic acid) at a flow rate of 300nL/min: from 2–30% B over 80 min and from 30–90% B over 5 min. The LTQ-Orbitrap Velos was operated in positive ion mode with data-dependent acquisition. The full scan was obtained in the Orbitrap with an automatic gain control (AGC) target value of 10e6 ions and a maximum fill time of 500 ms. Each precursor ion scan was acquired at a resolution of 60,000 FWHM in the 400–1500 m/z mass range. Peptide ions were fragmented by CID MS/MS using a normalized collision energy of 35. The 20 most abundant peptides were selected for MS/MS and dynamically excluded during 30 sec. All raw data were accessed in the Xcalibur software (Thermo Scientific). For protein identification, raw data were processed using MaxQuant software version 1.5.3.8. The MS/MS spectra were searched against a protein database composed of human sequences with the addition of common contaminants with a tolerance level of 4.5 ppm for MS and 0.5 Da for MS/MS. Trypsin was selected as a specific enzyme with a maximum of two missed cleavages. Carbamidomethylation of cysteine (57.021 Da) was set as a fixed modification, and oxidation of methionine (15.994 Da), deamidation NQ (+ 0.984 Da) and protein N-terminal acetylation (42.010 Da) were set as variable modifications. Proteins and peptides were accepted at FDR less than 1%. Glycosylasparaginase heterologous expression. The synthetic asparaginase gene (AFUB_059140) cloned in the expression vector pPICZαA (Invitrogen) was synthesized by the company Genescript. The vector has optimized codons for the expression of Komagataella phaffii and the α factor secretion signal in the N-terminal portion and Zeocin resistance gene. The plasmid obtained was propagated in competent Escherichia coli (DH10β) cells (Thermo Fisher Scientific, Waltham, USA), resistant to zeocin. The purified plasmid was linearized by Anza 24 MssI restriction enzyme (Invitrogen), purified again, and transformed into competent K. phaffii (KM71H) by electroporation (1.5 kV, 25 mF and 200 Ω) in a 0.2 µm cuvette using an electroporator (MicroPulser Electroporator, Bio-Rad). The recombinant strain was selected, cultivated and induced to express the rAsparaginase protein following the EasySelect Pichia Expression Kit manual (Invitrogen, Waltham, USA). After 120 h of induction with 1% (v/v) methanol, the material was centrifuged (5,000 × g , 45 min at 4°C) and the supernatant was filtered through a 0.22 µm filter. The filtered material was dialyzed against 50 mM Bicine buffer (pH 8.5) and subjected to anion exchange purification. The Mono Q 5/50 GL column (GE Healthcare) was equilibrated with 50 mM Bicine buffer (pH 8.5) and the enzyme eluted with 180 mM NaCl. Protein integrity and sample purity were confirmed by running a 12% SDS-PAGE 43 . Purified protein fractions were dialyzed against 1X PBS (pH 7.4) using the Vivaspin 10 kDa centrifuge system (Sartorius). Protein quantification was performed in a spectrophotometer at 280 nm and the assays were performed with the enzyme at a concentration of 5.88 µM. AspA phylogenetic analysis. The visualize the distribution of the glycosylasparaginase gene presence among fungi, the AspA amino acid sequence (AFUB_059140) was used for BLAST search 44 against the NCBI RefSeq database 45 . The 687 sequences found were aligned using MAFFT v7.508 46 for the inference of a maximum likelihood phylogenetic tree using IQ-TREE v1.7 47 with the substitution model JTT + I + G4, determined to be the best by the program. The calculated tree was visualized and edited using iTOL v6 48 . Macrophage Culture. BALB/c bone marrow-derived macrophages (BMDMs) were obtained as previously described 49 . Briefly, bone marrow cells were cultured for 7–9 days in RPMI 20/30, which consists of RPMI-1640 medium (Gibco, Thermo Fisher Scientific Inc.), supplemented with 20% (vol/ vol) FBS and 30% (vol/vol) L-Cell Conditioned Media (LCCM) as a source of macrophage colony-stimulating factor (M-CSF) on non-treated Petri dishes (Optilux - Costar, Corning Inc. Corning, NY). Twenty-four h before experiments, BMDM monolayers were detached using cold phosphate-buffered saline (PBS) (Hyclone, GE Healthcare Inc. South Logan, UT) and cultured, as specified, in RPMI-1640 (Gibco, Thermo Fisher Scientific Inc.) supplemented with 10% (vol/vol) FBS, 10 U/mL penicillin, and 10 mg/mL streptomycin, (2 mM) L-glutamine, (25 mM) HEPES, pH 7.2 (Gibco, Thermo Fisher Scientific Inc.) at 37°C in 5% (vol/vol) CO 2 for the indicated periods. Macrophage infection, cytokine and LDH determination. BMDMs were cultured as described before and were seeded at a density of 10 6 cells/ml in 24-well plates (Greiner Bio-One, Kremsmünster, Austria). The cells were challenged with the conidia of different strains at a multiplicity of infection of 1:10 and incubated a 37°C with 5% (vol/vol) CO 2 for 24h. BMDMs were also stimulated with different concentrations (mM) of AspA protein (desnaturated or not) by boilling per 10 min 100 degrees Celsius. LPS (standard LPS, E. coli 0111: B4; Sigma-Aldrich, 500 ng/mL) plus Nigericin (tlrl-nig, InvivoGen 5 µM/mL) and medium alone were used respectively as the positive and negative controls. Cell culture supernatants were collected and stored at − 80°C until they were assayed for TNF-α, IL-1 and LDH release using Mouse DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA and CyQUANT™ lactate dehydrogenase (LDH) Cytotoxicity Assay (Invitrogen), according to the manufacturer’s instructions. For cytokine determination, plates were analysed by using a microplate reader (Synergy™ HTX Multi-Mode, BioTek) measuring absorbance at 450 nm. Cytokine concentrations were interpolated from a standard curve and statistical significance was determined using an ANOVA (GraphPad Prism 8.0, La Jolla, CA). The level of LDH was determined by measuring absorbance at 490 and 680nm using a microplate reader (Synergy™ HTX Multi-Mode, BioTek). All assays were performed in triplicate in three independent experiments. Aspergillus growth condition and Fluorescein isothiocyanate (FIT C) label. Aspergillus strains were cultivated on minimal medium (AMM) agar plates at 37°C for 3 days. Conidia were harvested in sterile water with 0.05% (vol/vol) Tween20.The resulting suspension was filtered through two layers of gauze (Miracloth, Calbiochem). FITC-labelling of conidia was performed with 0.1 mg/ml FITC (Sigma) in 0.1 M Na 2 CO 3 at 37°C for 30 min. Labelled conidia were washed three times with PBS, 0.1% (vol/vol) Tween20. The conidia concentration was determined using a hemocytometer. Phagocytosis and adhesion assays. BMDMs were cultivated in Dulbecco's Modified Eagle Medium ( DMEM) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum, 2mM glutamine and penicillin-streptomycin. For infection experiments, macrophages were seeded on glass cover slips in 24 well plates at a density of 5×10 5 cells per well and allowed to grow adherently overnight. Following washing with prewarmed medium, FITC-labelled conidia were added at a multiplicity of infection of 10. The infection experiment was synchronized for 30 min at 4°C. Unbound conidia were removed by washing with pre-warmed medium and phagocytosis was initiated by shifting the coincubation to 37°C in a humidified CO 2 incubator. After 1 h the phagocytosis was stopped by washing with ice-cold PBS. Labelling of extracellular conidia was performed by incubation with PBS, 0.25 mg/ml calcofluor white (Sigma) for 30 min at 4°C to avoid further. The cells were washed twice with PBS and fixed with 3.7% (vol/vol) formaldehyde/PBS for 15 min followed by two washes with PBS. Microscopic photographs were taken on a Zeiss microscope. For statistical reproducibility two biological replicates and in each case two technical replicates were made and analyzed for each strain. The phagocytic index was enumerated by counting 100 macrophages per cover slip from duplicate wells. Phagocytic index was calculated by the average number of conidia that had been phagocytosed for each macrophage. Macrophage killing assay. BMDMs were seeded at a density of 10 6 cells/ml in 24-well plates (Corning Costar) and were challenged with conidia at a multiplicity of infection of 1:10 and incubated a 37°C with 5% (vol/vol) CO 2 for 24h. After incubation media was removed the cells were washed with ice-cold PBS and finally 2ml of sterile water was added to the wells. A P1000 tip was then used to scrape away the cell monolayer and the cell suspension was collected. This suspension was then diluted 1:1000 and 100 µl was plated on Sabouraud agar before the plates were incubated a 37°C overnight and the colonies were counted. 50 µl of the inoculum adjusted to 10 3 /ml was also plated on SAB agar to correct CFU counts. The CFU/ml for each sample was calculated and compared to the A1160 wild-type strain. Acidification of phagolysosomes. Acidification of phagolysosomes was essentially determined as previously described 17 . In brief, RAW 264.7 murine macrophages were incubated in Dulbecco's Modified Eagle Medium (DMEM, Gibco) with 27.5 mg/mL gentamicin sulfate (Gibco), 10% (vol/vol) fetal bovine serum (FBS, GE Healthcare Life Sciences), and 1% (wt/vol) ultraglutamine (Gibco) at 37°C and 5% (vol/vol) CO 2 in humidified incubator. Cells were pre-stained with 50 nM LysoTracker Red DND-99 (Invitrogen) for 1 h before infection. Conidia were stained with 0.1 mg/mL Calcofluor White (CFW, Sigma Aldrich) for 10–15 min before infection. 1 x 10 5 cells per well were added to 8 well ibidi slides (ibidi GmbH) and infected with an MOI of 3 with CFW stained conidia for 2 h at 37°C and 5% (vol/vol) CO 2 in a humidified incubator. Uptake of conidia was synchronized by centrifugation at 100 x g for 5 min. After infection, cells were washed with PBS and fixed using 3.7% formaldehyde in PBS for 10 min, followed by a washing step with PBS. Slides were directly imaged using a Zeiss LSM 780 microscope and analyzed using Zeiss ZEN software. At least, 100 conidia were counted for each biological replicate. Clear red signals surrounding conidia within phagosomes were evaluated as acidified conidia. Three independent experiments were performed. BMDMs antifungal activity. BMDMs were isolated as described before and 5×105 cells were seeded in 96 round bottom wells in complete DMEM (DMEM (Gibco), 10% (vol/vol) FBS (Gibco), 1% (wt/vol) penicillin/streptomycin (Sigma), 1% HEPES (Gibco). After adhesion the BMDMs were infected with 5×10 6 conidia (MOI 1:10). Upon 24 h of co- incubation the cells were lysed with sterile water and conidia were stained for 5 min with 50 µL of a filter sterilized 1mg/mL Calcofluor white solution (Sigma-Aldrich). Conidia were washed with sterile water and the staining was quantified using a fluorometer at 360 nm excitation and 440 nm emission. XTT assay was performed as described 50,51 . Briefly, 5 × 10 5 BMDMs were plated in 96-well and then infected with 5 × 10 6 spores in a final volume of 100 µl DMEM media. Control wells contained spores but no BMDMs or only BMDMs. Following 2 hr incubation, the macrophages were subjected to hypotonic lysis by three gentle washes with distilled water followed by a 30 min incubation with distilled water at 37°C. Supernatants then were removed, with great care taken not to remove the spores. DMEM media without Phenol Red containing 400 µg/ml of XTT and 50 µg/ml of Coenzyme Q, were added, and the wells were incubated for 2 hr at 37°C. The OD 450 and OD 650 were then measured, and data were expressed as the percent of antifungal activity according to the published formula 50 . Cytokine and chemokine quantification. For lung homogenates, the lungs of all experimental groups were homogenized in PBS supplemented with Complete Mini protease inhibitor tablets (Roche), clarified by centrifugation, and stored at − 80°C. A panel of cytokines and chemokines were quantified by ELISA (R&D Systems) according to the manufacturer’s instructions. Detection of reactive oxygen species (ROS). In 96-well tissue culture (TC)-treated dark clear bottom plates (Sigma-Aldrich), 5×105 BMDMs per 100 µL were infected with 5×106 conidia followed by 4, 8, and 24 h of incubation at 37 0 C. The cells were treated with the ROS detection dye CellROX ® Reagent (ThermoFisher) at a final concentration of 5 µM for 30 min at 37 ° C. ROS detection was performed using a fluorescent plate reader (Thermo Fisher), with an excitation of 485 nm and an emission of 520 nm. BMDMs stimulated with 50 nM phorbol- 12-myristate-13-acetate (PMA) (Sigma-Aldrich) for 20 min at 37 ° C were used as positive control. Transwell Assay. BMDMs were seeded at a density of 10 6 cells/ml in 24-well plates (Corning Costar) and the assay was performed as the addition each well containing a transwell insert (0.2-µm filter; Corning) containing medium alone, Δ aspA-1 , Δ aspA-2 or wild-type conidia at a multiplicity of infection of 1:10 and incubated a 37°C with 5% (vol/vol) CO 2 for 24h. After incubation cell culture supernatants were collected and stored at − 80°C until they were assayed for TNF-α, IL-1 production. In the transwell system the small and soluble compounds are able to migrate between the upper transwell and lower chamber, whereas spores are prevented from moving between the chambers. Epithelial cell invasion assay. The capacity of the various strains to invade the A549 cells was determined using previously described methods with some modification 52,53 . The A. fumigatus wild-type and mutant strains were grown on Sabouraud dextrose agar (Difco) at 37°C for 7 d prior to use. Conidia were harvested with phosphate-buffered saline (PBS) containing 0.1% (vol/vol) Tween 80 (Sigma-Aldrich) and enumerated with a hemacytometer. The A549 pulmonary epithelial cell line (American Type Culture Collection) was cultured in F12 medium (American Type Culture Collection) containing 10% (vol/vol) fetal bovine serum (Gemini Bio-Products), and 2 mM L-glutamine with penicillin and streptomycin (Irvine Scientific) in 5% CO 2 at 37°C. Before the assay, 2 x 10 5 A549 cells were cultured in 24-well tissue culture plates containing fibronectin coated circular glass coverslips in each well for overnight. A. fumigatus conidia were pre-germinated in Sabouraud dextrose broth (Difco) at 37°C for 5.5 h, counted and suspended in F12k medium. Next, 10 5 germlings of each strain in 1 ml F12 K medium were added to A549 cells that had been grown to confluency on the glass coverslips. After incubation for 3 h, the cells were rinsed with 1 ml HBSS in a standardized manner and then fixed with 4% (vol/vol) paraformaldehyde. The noninternalized portions of the organisms were stained with a polyclonal rabbit anti- A. fumigatus primary antibody (Meridian Life Science, Inc.) followed by an AlexaFluor 568-labeled secondary antibody (Life Technologies). After the coverslips were mounted inverted on microscope slides, they were viewed by epifluorescence. The number of cell-associated organisms was determined by counting the number of red fluoresced organisms per high-powered field (HPF). The number of endocytosed organisms was determined by subtracting the number of non-internalized organisms (staining of entire germlings) from the number of cell-associated organisms. At least 100 organisms per coverslip were scored and each strain was tested in triplicate. Epithelial cell damage assay. To evaluate the capacity of various strains to damage A549 epithelial cells, we used our standard 51 Cr release assay 53–55 . A. fumigatus conidia and A549 were prepared as described before. The A549 cells were grown to confluency in a 24-well tissue culture plate and then loaded with 51 Cr overnight. The following day, the cells were rinsed twice with HBSS to remove the unincorporated 51 Cr and 5 x 10 5 conidia in 1 ml of F12K medium of each strain were added to triplicate wells. After 16 h of incubation, 500 µl of medium above the cells were collected and transferred to glass test tube A. Next, the remaining medium in the wells was collected and placed in glass test tube B. After lysing the A549 cells with 6 N NaOH, the lysate was collected and the wells were rinsed twice with RadiacWash (Biodex Medical Systems). The lysate and rinses were added to test tube B. The amount of 51 Cr in the medium and the cell lysate was measured using a gamma counter. The spontaneous release of 51 Cr was determined using uninfected A549 cells that were processed in parallel. The specific release of 51 Cr was calculated using our previous described formula. Each experiment was performed in triplicate. Mutants that caused less damage to the A549 cells than the control strain were tested at least one more time to verify the results. Animal survival curves and fungal burden. Inbred female mice (BALB/c or C57BL/6 strains; body weight, 20–22 g) were housed in vented cages containing five animals. Mice were immunosuppressed with cyclophosphamide (150 mg/kg of body weight), which was administered intraperitoneally on days − 4, -1 and 2 prior to and post infection (infection day is “day 0”). Hydrocortisonacetate (200 mg/kg body weight) was injected subcutaneously on day − 3. Mice (10 mice per group) were anesthetized by halothane inhalation and infected by intranasal instillation of 20 µL of 1.0 x 10 5 conidia of A. fumigatus wild-type or mutant strains, A. lentulus , A. oerlinghausenensis , or A. fischeri (the viability of the administered inoculum was determined by incubating a serial dilution of the conidia on MM medium, at 37°C). As a negative control, a group of 10 mice received PBS only. Animals were sacrificed 15 days post-infection. To investigate fungal burden in murine lungs, mice are immunosuppressed as described previously or not, and mice were intranasally inoculated with 1 x 10 6 conidia/20 µl of suspension for the chemotherapeutic murine model and with 5 x 10 8 conidia/20 µl for the immunocompetent murine model. Animals were sacrificed 48 h post-infection, and the lungs were harvested and immediately frozen in liquid nitrogen. DNA was extracted via the phenol/chloroform method and 400 mg of total DNA from each sample were used for quantitative PCRs using primers to amplify the 18S rRNA region of A. fumigatus and an intronic region of mouse GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Six-point standard curves were calculated using serial dilutions of gDNA from A. fumigatus strain and the uninfected mouse lung. Fungal and mouse DNA quantities were obtained from the threshold cycle (CT) values from the appropriate standard curves. The qPCR analysis was performed using the SYBR green PCR master mix kit (Applied Biosystems) in the ABI 7500 Fast real-time PCR system (Applied Biosystems, Foster City, CA, USA). The principles that guide our studies are based on the Declaration of Animal Rights ratified by UNESCO on January 27, 1978 in its 8th and 14th articles. All protocols adopted in this study were approved by the local ethics committee for animal experiments from the University of São Paulo, Campus of Ribeirão Preto (Permit Number: 08.1.1277.53.6; Studies on the interaction of A. fumigatus with animals). Groups of five animals were housed in individually ventilated cages and were cared for in strict accordance with the principles outlined by the Brazilian College of Animal Experimentation (COBEA) and Guiding Principles for Research Involving Animals and Human Beings, American Physiological Society. All efforts were made to minimize suffering. Animals were clinically monitored at least twice daily and humanely sacrificed if moribund (defined by lethargy, dyspnea, hypothermia and weight loss). All stressed animals were sacrificed by cervical dislocation. Phenotypic assay. Plates containing solid MM were inoculated with 10 4 spores per strain and left to grow for 72 h at 37 or 44°C. When required MM was supplemented with Congo Red (10 µg/ml) or H 2 O 2 (1.5 mM). All radial growths were expressed as ratios, dividing colony radial diameter of growth in the stress condition by colony radial diameter in the control (MM at 37°C) condition. Germination assay. The germination process was followed for the A1160 strain over a 16 h time period under a Nikon TI microscope equipped with a 37°C incubator and a 40× objective, with 1 picture captured every 30 min by using NIS-Elements 4.0 (Nikon) software. Cell Counter plug-in of ImageJ platform ( http://rsb.info.nih.gov/ij/index.html ) was employed to differentially count resting or swollen conidia versus germinated conidia. A time point was selected (7h) in which A1160 achieved 50–60% of germination and the knockout mutant strains were assayed for the same time point included A1160 as a control. 10 4 conidia were inoculated into 200 µl of liquid MM in 96-well plates and each strain was tested in technical duplicates. Staining for cell surface components. Cell wall surface polysaccharide staining was performed as described previously 56 . Briefly, 10 4 spores for each deletion mutant and A1160 strains were inoculated in 200µl of MM liquid medium and incubated for 4 h at 37°C (swollen conidia) and 4°C (resting conidia) before the culture medium was removed and conidia were UV irradiated (600,000mJ). For dectin staining, 200 µl of a blocking solution (2% [wt/vol] goat serum, 1% [wt/vol] bovine serum albumin [BSA], 0.1% [vol/vol] Triton X-100, 0.05% [vol/vol] Tween 20, 0.05% [vol/vol] sodium azide, and 0.01 M PBS) was added to each working well. Samples were incubated for 30 min at room temperature (RT) and 0.2 µg/ml of Fc-h-dectin-hFc (Invivogen) was added to the UV-irradiated conidia and incubated for 1 h at RT, followed by the addition of 1:1,000 DyLight 594-conjugated goat anti-human IgG1 (Abcam) for 1 h at RT. Conidia were then washed with phosphate-buffered saline (PBS), and fluorescence was read at 587-nm excitation and 615-nm emission. For chitin staining, 200µl of a PBS solution with 10mg/ml of calcofluor white (CFW) was added to the UV-irradiated conidia, which were incubated for 5 min at RT and washed with PBS before fluorescence was read at 380-nm excitation and 450-nm emission. For N-acetyl-D-glucosamine (GlcNAc) staining, 200 µl of PBS supplemented with 0.1 mg/ml of wheat germ agglutinin (WGA) lectin (lectin-FITC L4895; Sigma) was added to the UV-irradiated germlings for 1 h at RT. Germlings were washed with PBS, and fluorescence was read at 492 nm excitation and 517 nm emission. All experiments were performed using at least 4 repetitions, and fluorescence was read in a microtiter plate reader (Synergy HTX Multimode Reader; Agilent Biotek or EnSpire Multimode Plate Reader; Perkin Elmer). Fungal adhesion assay. In order to determine the adhesion capacity of conidia from deleted mutant and A1160 strains, 10 4 conidia were inoculated into 200 ul of MM liquid medium in a 96-well polystyrene microtiter plate. Following an initial adherence phase of 4 h during static incubation at 37°C (swollen conidia) or 4°C (resting conidia), unbound conidia were washed with sterile PBS. Fresh MM liquid medium was added to the adhered conidia, and static submerged cultures were grown for up to 24 h at 37°C. Subsequently, the plate was washed exhaustively with PBS prior to incubation with 200 ul 0.5% (wt/vol) crystal violet solution for 5 min at room temperature. The stained mycelia were then exhaustively washed with sterile water and air dried. Finally, the crystal violet was eluted from the wells using 100% ethanol, and the absorbance was measured at 590 nm in a Synergy HTX Multimode Reader (Agilent Biotek). Assessment of conidial hydrophobic properties. The distribution of the conidia on a water-oil interface was performed by the addition of 1x10 8 conidia of the wild-type and each deleted strain in a solution containing water and tributyrin (1:1 [vol/vol]). The mixture was vortexed during 1 min and set to allow the conidia to disperse for 1 h. The distribution of the conidia for each mutant were then visually compared to the A1160 wild-type strain. Statistical analysis. Statistical significance for each experiment was established by one-way or two-way ANOVA using the built-in tools of Prism 6. Statistical tests are indicated in the figure legends and were chosen based on the nature of the experiment and the standard tests employed in the field. Underlying assumptions for these tests, including sample independence, variance equality, and normality were assumed to be met. One-way ANOVA was followed by Tukey’s or Dunnett’s multiple comparison test when appropriate. Asterisks denote p value as follows: ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Declarations Data Availability The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository 57 with the dataset identifiers PXD031199 (Username: [email protected] ; Password: HjEpfE78) and PXD044190 (Username: [email protected] ; Password: WNvL2PfA). Acknowledgements We thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grants numbers 2021/04977-5 (G.H.G.), 2018/18257-1 (GP), 2018/15549-1 (GP), 2020/04923-0 (GP) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) grant numbers 301058/2019-9 and 404735/2018-5 (G.H.G.), both from Brazil, and the National Institutes of Health/National Institute of Allergy and Infectious Diseases grant R01 AI153356 from the USA (to A.R. and G.H.G.). This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) Collaborative Research Centre/Transregio FungiNet 124 ‘Pathogentic fungi and their human host: Networks of Interaction’ (project number 210879364; projects A1 and Z2). Work was also supported by a grant from the Wellcome Trust ‘The Aspergillus fumigatus gene and non-coding RNA knockout library “COFUN”’ (Project number 208396/Z/17/Z to MB). SG was cofounded by NIHR Manchester Biomedical Research Centre, the Manchester Academy of Health Sciences, the Fungal Infection Trust, the Dowager Countess Eleanor Peel trust and the Medical Research Council (MC_PC_21021). KE was supported by an NC3Rs studentship [NC/T001798/1]. References Earle, K. et al. Pathogenicity and virulence of Aspergillus fumigatus . Virulence 14 , 2172264 (2023). Rokas, A. et al. Evolving moldy murderers: Aspergillus section Fumigati as a model for studying the repeated evolution of fungal pathogenicity. PLoS Pathog. 16 , e1008315 (2020). Bayry, J. et al. Surface structure characterization of Aspergillus fumigatus conidia mutated in the melanin synthesis pathway and their human cellular immune response. Infect. Immun. 82 , 3141–3153 (2014) Heinekamp, T. et al. Interference of Aspergillus fumigatus with the immune response. Semin. Immunopathol. 37 , 141-152 (2015). Blango, M.G. et al. Dynamic surface proteomes of allergenic fungal conidia. J. Proteome Res. 19 , 2092-2104 (2020). Aimanianda, V. et al. Surface hydrophobin prevents immune recognition of airborne fungal spores. Nature 460 , 1117-1121 (2009). Valsecchi, I. et al. Role of Hydrophobins in Aspergillus fumigatus . J. Fungi (Basel) 4 : 2 (2017). Voltersen, V. et al. Proteome Analysis Reveals the Conidial Surface Protein CcpA Essential for Virulence of the Pathogenic Fungus Aspergillus fumi gatus . mBio 9 , e01557-18 (2018). Jia, L.J. et al. Aspergillus fumigatus hijacks human p11 to redirect fungal-containing phagosomes to non-degradative pathway. Cell Host Microbe 31 , 373-388 (2023). Asif, A.R. et al. Proteome of conidial surface associated proteins of Aspergillus fumigatus reflecting potential vaccine candidates and allergens. J. Proteome Res. 5 , 954-962 (2006). Jia, L.J. et al. Biotinylated Surfome Profiling Identifies Potential Biomarkers for Diagnosis and Therapy of Aspergillus fumigatus Infection. mSphere 5 , e00535-20 (2020). Blango, M.G. et al. Dynamic Surface Proteomes of Allergenic Fungal Conidia. J. Proteome Res. 19 , 2092-2104 (2020). Yu, S.Y. et al. Clinical and Microbiological Characterization of Invasive Pulmonary Aspergillosis Caused by Aspergillus lentulus in China. Front. Microbiol. 11 , 1672 (2020). Houbraken J., Weig M., Groβ U. Aspergillus oerlinghausenensis , a new mould species closely related to A. fumigatus . FEMS Microbiology Letters 363 ,fnv236 (2016). Steenwyk, J.L. et al. Variation Among Biosynthetic Gene Clusters, Secondary Metabolite Profiles, and Cards of Virulence Across Aspergillus Species. Genetics 216 , 481-497 (2020). Mead, M.E. et al. Characterizing the Pathogenic, Genomic, and Chemical Traits of Aspergillus fischeri , a Close Relative of the Major Human Fungal Pathogen Aspergillus fumigatus . mSphere 4 , e00018-19 (2019). Thywißen, A. et al. Conidial Dihydroxynaphthalene Melanin of the Human Pathogenic Fungus Aspergillus fumigatus Interferes with the Host Endocytosis Pathway. Front. Microbiol. 2 , 96 (2011). Horta, M.A.C. et al. Examination of Genome-Wide Ortholog Variation in Clinical and Environmental Isolates of the Fungal Pathogen Aspergillus fumigatus . mBio 13 , e0151922 (2022). Baltussen, T.J.H. et al. The C 2 H 2 transcription factor SltA is required for germination and hyphal development in Aspergillus fumigatus . mSphere 1 , e0007623 (2023). Zhao, C. et al. High-throughput gene replacement in Aspergillus fumigatus. Curr. Protoc. Microbiol. 54 , e88 (2019). Mead, M.E. et al. An evolutionary genomic approach reveals both conserved and species-specific genetic elements related to human disease in closely related Aspergillus fungi. Genetics 218 , iyab066 (2021). Bouyssi, A. et al. Characterization of Lung Inflammatory Response to Aspergillus fumigatus Spores. J. Fungi (Basel) 9 , 682 (2023). Shifrin, S., Parrott, C. L., Luborsky, S. W. Substrate binding and intersubunit interactions in L asparaginase. Journal of Biological Chemistry , 249 , 1335–1340 (1974). Tarentino AL, Plummer TH Jr. Enzymatic deglycosylation of asparagine-linked glycans: purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum . Methods Enzymol. 230 , 44-57 (1994). Steenwyk, J.L. et al. A Robust Phylogenomic Time Tree for Biotechnologically and Medically Important Fungi in the Genera Aspergillus and Penicillium . mBio 10 , e00925-19 (2019). Dang, E.V. et al. Secreted fungal virulence effector triggers allergic inflammation via TLR4. Nature 608, 161-167 (2022). Arvio, M, Mononen, I. Aspartylglycosaminuria: a review. Orphanet J. Rare Dis. 11 , 162 (2016). Mononen, I. et al. Aspartylglycosaminuria: protein chemistry and molecular biology of the most common lysosomal storage disorder of glycoprotein degradation. FASEB J. 7 , 1247–1256 (1993). Mononen, I. Aronson, N.N., editors. Lysosomal storage disease: Aspartylglycosaminuria. Heidelberg: Spirenger-Verlag; 1997. Goodspeed, K., Feng, C., Laine, M., Lund, T.C. Aspartylglucosaminuria: Clinical Presentation and Potential Therapies. J. Child Neurol. 36 , 403-414 (2021). Shibayama, K. et al. Biochemical and pathophysiological characterization of Helicobacter pylori asparaginase. Microbiol. Immunol. 55 , 408-417 (2011). Washington, E.J., Banfield, M.J., Dangl, J.L. What a difference a Dalton makes: bacterial virulence factors modulate eukaryotic host cell signaling systems via deamidation. Microbiol. Mol. Biol. Rev. 77 , 527-39 (2013). Gouzy, A. et al. Mycobacterium tuberculosis exploits asparagine to assimilate nitrogen and resist acid stress during infection. PLoS Pathog. 10 , e1003928 (2014). McLaughlin, P.A. et al. Contribution of Asparagine Catabolism to Salmonella Virulence. Infect. Immun. 85 , e00740-16 (2017). Torres, A. et al. Asparagine deprivation mediated by Salmonella asparaginase causes suppression of activation-induced T cell metabolic reprogramming. J. Leukoc. Biol. 99 , 387-98 (2016). Kelo, E., Noronkoski, T., Mononen, I. Depletion of L-asparagine supply and apoptosis of leukemia cells induced by human glycosylasparaginase. Leukemia 23 , 1167-71 (2009). Liu, H. et al. Mimicking hypersensitivity pneumonitis as an uncommon initial presentation of chronic granulomatous disease in children. Orphanet J. Rare Dis . 12 , 169 (2017). Amin, S. et al. Melanin dependent survival of Apergillus fumigatus conidia in lung epithelial cells. Int. J. Med. Microbiol. 304 , 626–636 (2014). Seidel, C. et al. Phagolysosomal survival enables non-lytic hyphal escape and ramification through lung epithelium during Aspergillus fumigatus infection. Front. Microbiol. 11 , 1955 (2020). Thywißen, A. et al. Conidial dihydroxynaphthalene melanin of the human pathogenic fungus Aspergillus fumigatus interferes with the host endocytosis pathway. Front. Microbiol. 2 , 96. (2011). Jahn, B. et al. PKSP-dependent reduction of phagolysosome fusion and intracelular kill of Aspergillus fumigatus conidia by human monocyte-derived macrophages. Cell. Microbiol. 4 , 793–803 (2002). Shah, A.D. et al. LFQ-Analyst: An Easy-To-Use Interactive Web Platform To Analyze and Visualize Label-Free Proteomics Data Preprocessed with MaxQuant. J. Proteome Res. 19, 204-211 (2020). Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 , 680-685 (1970). Johnson, M. et al. NCBI BLAST: a better web interface. Nucleic Acids Res. 36 , W5-9 (2008). Sayers, E.W. et al. Database resources of the national center for biotechnology information. Nucleic Acids Res . 50 , D20-D26 (2022). Katoh, K., Rozewicki, J., Yamada, K.D. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform. 20 , 1160-1166 (2019). Nguyen, L.T., Schmidt, H.A., von Haeseler, A., Minh, B.Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32 , 268-74 (2015). Letunic, I., Bork, P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 49 , W293-W296 (2021). Marim, F.M., Silveira, T.N., Lima, D.S. Jr., Zamboni, D.S. A Method for Generation Bone Marrow-Derived Macrophages from Cryopreserved Mouse Bone Marrow Cells. PloS One 5 , e15263 (2010). Ramirez-Ortiz, Z.G. et al. A nonredundant role for plasmacytoid dendritic cells in host defense against the human fungal pathogen Aspergillus fumigatus . Cell Host Microbe 9 , 415–424 (2011). Meshulam, T., Levitz, S.M., Christin, L., Diamond, R.D. A simplified new assay for assessment of fungal cell damage with the tetrazolium dye, (2,3)-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide (XTT). J. Infect. Dis. 172 , 1153–1156 (1995). Liu, H. et al. Determining Aspergillus fumigatus transcription factor expression and function during invasion of the mammalian lung. PLoS Pathog. 17 , e1009235 (2021). Liu, H. et al. Aspergillus fumigatus CalA binds to integrin α5β1 and mediates host cell invasion. Nat. Microbiol. 2 ,16211 (2016). Ejzykowicz, D.E. et al. The Aspergillus fumigatus transcription factor Ace2 governs pigment production, conidiation and virulence. Mol. Microbiol. 72 , 155-169 (2009). Bertuzzi, M. et al. The pH-responsive PacC transcription factor of Aspergillus fumigatus governs epithelial entry and tissue invasion during pulmonary aspergillosis. PLoS Pathog. 10 , e1004413 (2014). Winkelstroter, L.K. et al. High osmolarity glycerol response PtcB phosphatase is important for Aspergillus fumigatus virulence. Mol. Microbiol . 96 , 42–54 (2015). Perez-Riverol, Y. et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 47, D442-D450 (2019). Brown, A. et al. Extensive non-coding sequence divergence between the major human pathogen Aspergillus fumigatus and its relatives. Front. Fungal Biol. 3 , 802494 (2022) Tables Tables 1 and 2 are available in the Supplementary Files section. Additional Declarations There is NO Competing Interest. J.L.S. is a scientific advisor for WittGen Biotechnologies. J.L.S. is an advisor for ForensisGroup Incorporated. A.R. is a scientific consultant for LifeMine Therapeutics, Inc. G.H.G. is a scientific consultant for Innovation Pharmaceuticals Inc. The other authors declare no other competing interests. Supplementary Files SupplementaryTableS1.xlsx Supplementary Table S1 SupplementaryTableS2.xlsx Supplementary Table S2 SupplementaryTableS3.xlsx Supplementary Table S3 SupplementaryTableS4.xlsx Supplementary Table S4 SupplementaryFigureS1.pdf SupplementaryFigureS2.pdf SupplementaryFigureS3.pdf SupplementaryFigureS4.pdf SupplementaryFigureS5.pdf Tables.docx Cite Share Download PDF Status: Published Journal Publication published 27 Aug, 2024 Read the published version in Nature Microbiology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Delbaje","email":"","orcid":"https://orcid.org/0000-0002-0313-9046","institution":"University of São Paulo","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Endrews","middleName":"","lastName":"Delbaje","suffix":""},{"id":233651219,"identity":"75210545-79a1-452d-ba49-2b4087e2d7f3","order_by":20,"name":"Michael Bromley","email":"","orcid":"https://orcid.org/0000-0002-7611-0201","institution":"University of Manchester","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Bromley","suffix":""},{"id":233651220,"identity":"82700c82-39db-4660-868a-a068be35138a","order_by":21,"name":"Claudia Angeli","email":"","orcid":"https://orcid.org/0000-0002-3906-1973","institution":"ICB, Universidade de Sao Paulo","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Angeli","suffix":""},{"id":233651221,"identity":"bbd4603f-349a-4440-bbe4-5eaff738a05a","order_by":22,"name":"Giuseppe Palmisano","email":"","orcid":"","institution":"ICB-USP","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Giuseppe","middleName":"","lastName":"Palmisano","suffix":""},{"id":233651222,"identity":"cfc39c77-c689-4cb7-a99d-ef8e4c6aa144","order_by":23,"name":"Ashraf Ibrahim","email":"","orcid":"https://orcid.org/0000-0003-3787-8530","institution":"The Lundquist Institute at Harbor-University of California Los Angeles Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ashraf","middleName":"","lastName":"Ibrahim","suffix":""},{"id":233651223,"identity":"172409b2-7ad5-4d77-8234-02735faf19d8","order_by":24,"name":"Sara Gago","email":"","orcid":"https://orcid.org/0000-0002-7027-4598","institution":"University of Manchester","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sara","middleName":"","lastName":"Gago","suffix":""},{"id":233651224,"identity":"e780c171-fcdf-4d0d-8929-c49cf82323e3","order_by":25,"name":"Thaila does Reis","email":"","orcid":"","institution":"University of Sao Paulo","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Thaila","middleName":"does","lastName":"Reis","suffix":""}],"badges":[],"createdAt":"2023-08-29 11:26:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3306535/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3306535/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41564-024-01782-y","type":"published","date":"2024-08-27T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":43307327,"identity":"356817ab-a3f5-4978-b35d-3a604b3f8f20","added_by":"auto","created_at":"2023-09-18 16:16:13","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":434870,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eA. fumigatus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ehas increased virulence and cytokine production by BMDMs than the other three species. a. \u003c/strong\u003ePhylogeny of \u003cem\u003eAspergillus\u003c/em\u003e\u0026nbsp;section\u0026nbsp;\u003cem\u003eFumigati\u003c/em\u003e\u0026nbsp;representative species constructed from concatenation analysis of a 5,215-gene data matrix. Branch lengths correspond to nucleotide substitutions / site.\u0026nbsp;Adapted from \u003csup\u003e58\u003c/sup\u003e. \u003cstrong\u003eb and c.\u003c/strong\u003e Phagocytic index and viability of conidia after exposure to BMDMs. \u003cstrong\u003ed. \u003c/strong\u003ePercentage of acidifed phagolysosomes (PLs). \u003cstrong\u003ee to h. \u003c/strong\u003eCytokine production elicited by Afu, Afi, Aoer, and Ale conidia by BMDMs. \u003cstrong\u003ei.\u003c/strong\u003e Survival curves for \u003cem\u003eA. fumigatus \u003c/em\u003e(Afu), \u003cem\u003eA. fischeri\u003c/em\u003e (Afi), \u003cem\u003eA. oerlinghausenensis\u003c/em\u003e (Aoer), and \u003cem\u003eA. lentulus\u003c/em\u003e (Ale) in a chemotherapeutic murine model of Invasive Pulmonary Aspergillosis (IPA). The results are the average of three repetitions and are expressed as average ± standard deviation. Statistical analysis was performed using a one-way ANOVA (Dunnett’s test) for multiple comparisons. ns not significant, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. Survival curves (n=10 mice/strain) infected with the indicated \u003cem\u003eA. fumigatus\u003c/em\u003e strain. Phosphate buffered saline (PBS) was administered in a negative control group. The indicated \u003cem\u003ep-\u003c/em\u003evalues are based on the log-rank, Mantel-Cox, and Gehan-Breslow-Wilcoxon tests comparing the Afi, Aoer, and Ale strains with Afu strain.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/02afe7a8408134f7624b77b3.jpg"},{"id":43307329,"identity":"1e796675-cadc-48f5-9e1c-1183118932cb","added_by":"auto","created_at":"2023-09-18 16:16:13","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":766994,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative trypsin-shaving proteomics to study the surface associated proteome “surfome” of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. fumigatus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003ea. \u003c/strong\u003eRepresentative images of Afu, Afi, Aoer, and Ale\u003cem\u003e \u003c/em\u003econidia at time 0 and 4h post-incubation at 37°C (early germination) in liquid potato dextrose (PD) media and radial growth (10\u003csup\u003e4\u003c/sup\u003e conidia were displayed at the center of MM plates and incubated for 5 days at 37°C). \u003cstrong\u003eb. \u003c/strong\u003eTrypsin shaving proteomic analysis workflow. Proteins obtained from trypsin-treated resting and swollen conidia were analyzed by LC–MS/MS. Created with BioRender.com. \u003cstrong\u003ec. \u003c/strong\u003e\u0026nbsp;Sixty-two\u003cstrong\u003e \u003c/strong\u003eunique proteins constitute the \u003cem\u003eA. fumigatus \u003c/em\u003econidial surfome.\u003cstrong\u003e \u003c/strong\u003eVenn diagram ilustrating the intersection of proteins identified by trypsin-shaving of resting and swollen conidia of \u003cem\u003eAspergillus\u003c/em\u003e spp. strains. The conidial surfome of \u003cem\u003eA. fumigatus \u003c/em\u003eis composed of 62 unique proteins (in red).\u0026nbsp; Functional categorization of the 62 proteins that comprise \u003cem\u003eA. fumigatus \u003c/em\u003esurfome according to their description in FungiDB. \u003cstrong\u003ed. \u003c/strong\u003eHeat map of the mRNA accumulation during conidial germination of 62 genes encoding the \u003cem\u003eA. fumigatus\u003c/em\u003e surfome proteins. The RNAseq database according to Baltussen \u003cem\u003eet al.\u003c/em\u003e (2023). \u003cstrong\u003ee. \u003c/strong\u003eGrowth phenotypes of the wild-type A1160 strain (in red) and deleted mutants grown for 72 h at 37˚C or 44˚C in solid MM media and MM supplemented with Congo Red (CR; 10µg/ml) or hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e; 1.5mM). Red circle represents A1163 strain. \u003cstrong\u003ef. \u003c/strong\u003eGermination rates of the wild-type A1160 strain (in blue) and deleted mutants. Only those hits below or above the 30%-fold change threshold and stastistically different from the parental strain A1160 are displayed in the figure. Blue circle represents A1163 strain. \u003cstrong\u003eg. \u003c/strong\u003eAdhesion, masured by CV assay, of resting (○) and swollen (△) conidia for all all mutant strains and A1160 (in blue). Only hits that shared the same significant phenotype (lower and higher detection in green and red, respectively) in both stages are highlighted in the figure. Red circle and triangle represent A1163 strain. \u003cstrong\u003eh\u003c/strong\u003e. Detection of chitin (CFW), N-acetylglucosamine (GlcNAc) (WGA) and β-(1,3)-glucan (Dectin) contents on the conidial surface of all mutant strains and A1160 (in blue) in resting (○) and swollen (△) conidia. Blue circle and triangle represent A1163 strain. Only hits that shared the same significant phenotype (lower and higher detection in green and red, respectively) in both stages are highlighted in the figure.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/f2bf65eb891993df2a5de61d.jpg"},{"id":43307330,"identity":"874be94e-0ffb-475b-94f6-2b8fe455f034","added_by":"auto","created_at":"2023-09-18 16:16:13","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":478978,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eA. fumigatus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esurface-associated proteins are involved in host-pathogen interactions. a. \u003c/strong\u003eConidia survival of deleted mutants in murine Raw 264.7 macrophages at 6 hpi expressed as fold change against A1160 wild-type strain. \u003cstrong\u003eb. \u003c/strong\u003eConidia internalization in A549 epithelial cells at 3 hpi expressed as fold change against A1160 (wild-type) strain. \u003cstrong\u003ec\u003c/strong\u003e. Simple linear regression models of \u003cstrong\u003ea. \u003c/strong\u003eand \u003cstrong\u003eb. \u003c/strong\u003edatasets (no correlation). Thresholds of 30 %-fold change above (red) and below (green) the wild-type strain (A1160) condition were established for statistical significance testing. \u003cstrong\u003ed.\u003c/strong\u003e Damage to A549 epithelial cells. \u003cstrong\u003ee and f.\u003c/strong\u003e \u003cem\u003eA. fumigatus \u003c/em\u003ewild-type and mutant conidia eliciting cytokines IL-1β and TNF-α production by BMDMs. The results are the average of three independent biological repetitions and are expressed as average ± standard deviation. Statistical analysis was performed using one-tailed, paired t-tests for comparisons to the wild-type strain (A1160) (*p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/bf4beee85d9d77ff236e603f.jpg"},{"id":43307335,"identity":"343f3bc6-4428-4b39-830b-4b71a0981a04","added_by":"auto","created_at":"2023-09-18 16:16:13","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":523907,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSome of the surfome genes are positively modulated at transcriptional level in the presence of the host cells and a few encoded proteins apparently display faster evolution in pathogenic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAspergillus spp\u003c/strong\u003e\u003c/em\u003e. \u003cstrong\u003ea.\u003c/strong\u003e Heat map clustering of 242 \u003cem\u003eA. fumigatus \u003c/em\u003eRNAseq datasets available at FungiExpresZ (\u003ca href=\"https://cparsania.shinyapps.io/FungiExpresZ/\"\u003ehttps://cparsania.shinyapps.io/FungiExpresZ/\u003c/a\u003e). \u003cstrong\u003eb and c.\u003c/strong\u003e Clustering of the two subclusters highlighted in the red circles enriched only for RNAseq datasets of \u003cem\u003eA. fumigatus \u003c/em\u003eexposed to animal cells or invasive aspergillosis conditions. Heatmap and clustering were generated using the online tool FungiExpressZ (https://cparsania.shinyapps.io/FungiExpresZ/) with the publicly available regulatory information of \u003cem\u003eA. fumigatus.\u003c/em\u003e \u003cstrong\u003ed. \u003c/strong\u003eViolin and box plots showing the ω values (representing the rate of sequence evolution for each gene in every strain) for genes that exhibited different ω values (p-value \u0026lt;0.01) in the three groups of strains (\u003cem\u003eA. fumigatus\u003c/em\u003e, other pathogens, and non-pathogens). Seven out of the 62 unique genes that encode the \u003cem\u003eA. fumigatus \u003c/em\u003esurfome exhibit different evolutionary rates in the conserved pathogenicity evolution model. Note that genes with omega values greater than 0.6 are not shown for clarity. The full image is available in \u003csup\u003e21\u003c/sup\u003e. \u003cstrong\u003ee\u003c/strong\u003e. Protein organization of the AspA according to \u003ca href=\"http://smart.embl-heidelberg.de/\"\u003ehttp://smart.embl-heidelberg.de/\u003c/a\u003e. \u003cstrong\u003ef.\u003c/strong\u003e Phylogenetic distribution of AspA in fungal classes.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/fd82f868404f3d1ed7788166.jpg"},{"id":43307333,"identity":"d0d8cc2a-d8a6-4e38-944b-2485cb129f4c","added_by":"auto","created_at":"2023-09-18 16:16:13","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":504129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eA. fumigatus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003easpA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant has reduced viability in the presence of BMDMs and can modulate IL-1β production. a. \u003c/strong\u003eThe Δ\u003cem\u003easpA\u003c/em\u003emutants have increased engulfment and adherence, and reduced viability by the BMDMs when compared to the wild-type strain.\u003cstrong\u003e b.\u003c/strong\u003e and \u003cstrong\u003ec. \u003c/strong\u003eFungal viability upon 24 h exposure to BMDMs measured by MTT and Calcofluor While (CFW) accumulation, respectively. \u003cstrong\u003ed. \u003c/strong\u003eUV-killed conidia show that Δ\u003cem\u003easpA\u003c/em\u003e conidia need to be alive to modulate IL-1β production. \u003cstrong\u003ee. \u003c/strong\u003eReactive Oxygen Species (ROS) accumulation upon exposure of the wild-type and Δ\u003cem\u003easpA\u003c/em\u003e mutants to BMDMs for 24 h. PMA (phorbol-12-myristate-13-acetate) was used as a positive control for ROS induction. \u003cstrong\u003ef.\u003c/strong\u003e \u0026nbsp;Percentage of acidifed phagolysosomes (PLs). \u003cstrong\u003eg. and h. \u003c/strong\u003eTranswell migration assays. Wild-type and Δ\u003cem\u003easpA \u003c/em\u003emutants were exposed to BMDMs and IL-1β and TNF-α production was measured. (+) means when the transwell insert was added to the well plate and (-) means the well plate whitout transwell insert. The results are the average of three independent biological repetitions and are expressed as average ± standard deviation. Statistical analysis was performed using a one-way ANOVA (Dunnett’s test) for multiple comparisons. ns not significant, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/f39372b0f411f1d31531951d.jpg"},{"id":43307342,"identity":"8671fc47-3582-44af-a4a4-4a901ecaf29e","added_by":"auto","created_at":"2023-09-18 16:16:13","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":463101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eA. fumigatus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eΔ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003easpA \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emutants have decreased fungal burden in an immunocompetent murine model. a. \u003c/strong\u003eFungal burden of the wild-type and Δ\u003cem\u003easpA\u003c/em\u003e mutants in a murine chemotherapeutic mouse infection model and \u003cstrong\u003eb. \u003c/strong\u003eimmunocompetent model. \u003cstrong\u003ec. to h.\u003c/strong\u003e Cytokine production (TNF-α, IL-1β, IL-18, IL-12, INF-ϒ, and IL-6) in the immunocompetent mouse model infected by the wild type and Δ\u003cem\u003easpA\u003c/em\u003e mutants. \u003cstrong\u003ei.\u003c/strong\u003e Chemokine production (CXCL-1) in the immunocompetent mouse model infected by the wild type and Δ\u003cem\u003easpA \u003c/em\u003emutants. The results graphic shows the value of three to five technical replicates ±SD deviation. Statistical analysis was performed using a one-way ANOVA (Turkey’s test) for multiple comparisons. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/9bf53f0a92072be1f728daa2.jpg"},{"id":43307341,"identity":"d65833fd-37c7-4b86-814a-014061408168","added_by":"auto","created_at":"2023-09-18 16:16:13","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":400445,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeterologously expressed AspA can modulate IL-1β and TNF-α levels and suffers pinocytosis by BMDMs.\u003c/strong\u003e \u003cstrong\u003ea. \u003c/strong\u003eViability of BMDMs exposed to 24 h at 37\u003csup\u003eo\u003c/sup\u003eC to different concentrations of AspA and measured by lactate dehydrogenase (LDH) activity or \u003cstrong\u003eb.\u003c/strong\u003e exposed to DMSO, AspA:GFP 0.2 or 2 µg, and measured by MTT assay. \u003cstrong\u003ec. \u003c/strong\u003eIncreasing concentrations of AspA can modulate IL-1β and TNF-α production by BMDMs. \u003cstrong\u003ee and f. \u003c/strong\u003eDenatured AspA has decreased IL-1β and TNF-α in BMDMs.\u003cstrong\u003e g. \u003c/strong\u003eConidial viability of BMDMs exposed or not to AspA 2µg for 8h and subsequent addition of conidia or AspA 2µg together with conidia evaluated after 32 and 24 h. The results are the average of three repetitions and are expressed as individual data points or average ± standard deviation. Statistical analysis was performed using a one-way ANOVA (Dunnett’s test) for multiple comparisons. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. \u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/fcf6adf3f9fcb6b2cb7c821a.jpg"},{"id":43308488,"identity":"96c2d3a6-0470-4dd5-beb8-89ef5635592c","added_by":"auto","created_at":"2023-09-18 16:24:13","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":421832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteomic profiling of BMDMs exposed to AspA\u003c/strong\u003e. \u003cstrong\u003ea.\u003c/strong\u003e PCA distribution of three repetitions of BMDM and BMDM proteins exposed to AspA. \u003cstrong\u003eb.\u003c/strong\u003e Heat map of protein abundance of BMDM and BMDM exposed to AspA. \u003cstrong\u003ec. and d. \u003c/strong\u003eCategorization of BMDM and BMDM proteins exposed to AspA. \u003cstrong\u003ee. \u003c/strong\u003eMouse functional protein association network based on selected 15 proteins that are modulated in the BMDMs in the presence of AspA. Each node represents a protein that is differentially expressed in the BMDMs upon exposure to AspA. Each edge represents a functional protein association retrieved from the STRING (\u003ca href=\"https://string-db.org/\"\u003ehttps://string-db.org\u003c/a\u003e) server (medium confidence threshold of 0.4 for the interaction score), and node sizes represent the degree of each node (number of edges connected to the node). Proteins were annotated based on \u003ca href=\"https://www.genecards.org/\"\u003ehttps://www.genecards.org\u003c/a\u003e and are: SIGLEC1, Msr1, Csf1r, TfrC, PtprC, Hcls1, Lsp1, Lamp2, Ctss, Ncf4, Colt1, CtsA, CtsB, CtsZ, and Ncf1.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/0d313f9dd10e62a274881674.jpg"},{"id":63439352,"identity":"7ec727a0-7e85-4ce4-9c8f-e4b9813f1489","added_by":"auto","created_at":"2024-08-28 07:06:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5571105,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/0660faa3-503d-4b9d-afe2-dc56c2ed360f.pdf"},{"id":43307331,"identity":"8218a6b0-691f-4a68-b40c-a8452602b0dd","added_by":"auto","created_at":"2023-09-18 16:16:13","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12393584,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table S1\u003c/p\u003e","description":"","filename":"SupplementaryTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/19697a9c9c6b31ec6da948cd.xlsx"},{"id":43307328,"identity":"e510dc5e-b435-45b7-8345-78cc1aeb7ce8","added_by":"auto","created_at":"2023-09-18 16:16:13","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":31366,"visible":true,"origin":"","legend":"Supplementary Table S2","description":"","filename":"SupplementaryTableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/175ca7fe020787501bf7364e.xlsx"},{"id":43308486,"identity":"82e38421-b5cf-496b-be8d-96e62d971745","added_by":"auto","created_at":"2023-09-18 16:24:13","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1136840,"visible":true,"origin":"","legend":"Supplementary Table S3","description":"","filename":"SupplementaryTableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/3dc390629d95b39dde70998d.xlsx"},{"id":43307343,"identity":"5912f384-b0c7-435a-b13e-0aa8196eefb4","added_by":"auto","created_at":"2023-09-18 16:16:13","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1129115,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table S4\u003c/p\u003e","description":"","filename":"SupplementaryTableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/91e1be1e648d11f883c3c07c.xlsx"},{"id":43307340,"identity":"9b24eb94-3431-4f80-83e8-352faf493d7f","added_by":"auto","created_at":"2023-09-18 16:16:13","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":317502,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/de0c827221b16c65fcf37092.pdf"},{"id":43308487,"identity":"1811ec50-6ff3-4656-968e-995e89308f21","added_by":"auto","created_at":"2023-09-18 16:24:13","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":673498,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/673026a81f90628556f394e9.pdf"},{"id":43307346,"identity":"1e14c81f-ef33-4208-acc4-afa43d53701c","added_by":"auto","created_at":"2023-09-18 16:16:14","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":524470,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/a1cb07d181c6d48095e8b483.pdf"},{"id":43307334,"identity":"5da8f05b-8ae5-43f3-abc4-ed8b258bf672","added_by":"auto","created_at":"2023-09-18 16:16:13","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":20992,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/3fe06f91891c46c8804df8a7.pdf"},{"id":43307345,"identity":"0f2c1e41-a6a5-4b40-9d2f-27fbbf1a2130","added_by":"auto","created_at":"2023-09-18 16:16:14","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":100050,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/64aaec6c5d1eecfec4414dfa.pdf"},{"id":43307337,"identity":"6eeebdae-6ac8-44bf-96cd-71a980056a8e","added_by":"auto","created_at":"2023-09-18 16:16:13","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":43475,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-3306535/v1/6681a9e4dc63f51f776261b2.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.\nJ.L.S. is a scientific advisor for WittGen Biotechnologies. J.L.S. is an advisor for ForensisGroup Incorporated. A.R. is a scientific consultant for LifeMine Therapeutics, Inc. G.H.G. is a scientific consultant for Innovation Pharmaceuticals Inc. The other authors declare no other competing interests. ","formattedTitle":"A phylogenetic approach to explore the \u003ci\u003eAspergillus fumigatus\u003c/i\u003e conidial surface-associated proteome and its role in pathogenesis","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePulmonary fungal diseases caused by the environmental mold \u003cem\u003eAspergillus fumigatus\u003c/em\u003e lead to significant morbidity and mortality. In patients with weakened lung defenses arising from immunosuppression, a chronic respiratory condition, or a prior respiratory infection, asexual spores or conidia of \u003cem\u003eA. fumigatus\u003c/em\u003e can evade the lung defenses, germinate, and cause disease \u003csup\u003e1\u003c/sup\u003e. However, our understanding of what makes \u003cem\u003eA. fumigatus\u003c/em\u003e a successful pathogen compared to other species remains incomplete. Current evidence suggests that several different \u003cem\u003eAspergillus\u003c/em\u003e species, including \u003cem\u003eA. fumigatus\u003c/em\u003e, likely independently evolved the ability to cause human disease \u003csup\u003e2\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), raising the hypothesis that species-specific genes contribute to disease.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe early stages of disease\u0026mdash;marked by the interaction between the inhaled conidia and the host\u0026mdash;may prove insightful for unraveling \u003cem\u003eA. fumigatus\u003c/em\u003e pathogenicity. Among studies of other microbial pathogens, there has been an increasing focus on conidial surface protein characterization since they mediate the first encounter with the host immune system. The conidial cell wall comprises a β-1,3-glucan and chitin body, which is covered by rodlet and melanin layers, where proteins are anchored \u003csup\u003e3,4\u003c/sup\u003e. These and other surface proteins play pivotal roles in morphogenesis, resistance to environmental stressors, substrate adherence, and virulence \u003csup\u003e5\u003c/sup\u003e. The hydrophobin RodA is a significant component of the rodlet layer and is essential for cell wall physical resistance and permeability, preventing immune recognition \u003csup\u003e6,7\u003c/sup\u003e. Another conidial surface protein, CcpA, is essential to maintain the correct surface structure and prevent immune recognition \u003csup\u003e8\u003c/sup\u003e. Recently, HscA has been demonstrated to anchor human p11 on phagosomal membranes, rewiring the vesicular trafficking to the non-degradative pathway, allowing the escape of conidia \u003csup\u003e9\u003c/sup\u003e. Other studies aimed to identify potential vaccine candidates, allergens, and biomarkers for diagnosis or characterization of \u003cem\u003eA. fumigatus\u003c/em\u003e cell wall dynamics under different stresses or biological processes\u003csup\u003e10,11,12\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite the importance of the conidia for disease, a comprehensive examination of conidial surface proteins and their contribution to pathogenicity in \u003cem\u003eA. fumigatus\u003c/em\u003e and closely related species is lacking. Here, we analyzed the conidial surface proteome of \u003cem\u003eA. fumigatus\u003c/em\u003e and three closely related species: (i) \u003cem\u003eAspergillus lentulus\u003c/em\u003e, which is a cryptic pathogen and close relative of \u003cem\u003eA. fumigatus\u003c/em\u003e\u003csup\u003e13\u003c/sup\u003e; (ii) \u003cem\u003eAspergillus oerlinghausenensis\u003c/em\u003e, the closest known relative of \u003cem\u003eA. fumigatus\u003c/em\u003e, which is azole-resistant but non-pathogenic \u003csup\u003e14,15\u003c/sup\u003e; and (iii) \u003cem\u003eAspergillus fischeri\u003c/em\u003e, a close relative of \u003cem\u003eA. fumigatus\u003c/em\u003e that is less virulent in several animal models and rarely causes disease in humans \u003csup\u003e16\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eA. fumigatus\u003c/strong\u003e \u003cstrong\u003ehas increased virulence in a chemotherapeutic murine model of Invasive Pulmonary Aspergillosis (IPA) and elicits higher cytokine production by bone marrow-derived macrophages (BMDMs).\u003c/strong\u003e To understand whether the four species have \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e differences in pathogenicity, we comparatively analyzed the capacity of \u003cem\u003eA. fumigatus\u003c/em\u003e, \u003cem\u003eA. fischeri\u003c/em\u003e (Afi), \u003cem\u003eA. oerlinghausensis\u003c/em\u003e (Aoe), and \u003cem\u003eA. lentulus\u003c/em\u003e (Ale) to infect macrophages and susceptible hosts. \u003cem\u003eA. fumigatus\u003c/em\u003e conidia are less engulfed and killed by Bone Marrow derived macrophages (BMDMs) than Afi, Aoe, and Ale (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). \u003cem\u003eA. fumigatus\u003c/em\u003e conidia are recognized by macrophages and are intracellularly degraded by the endocytic pathway through the fusion of conidia-containing phagosomes and lysosomes, forming an acidic phagolysosome (PL) \u003csup\u003e17\u003c/sup\u003e. Melanin can facilitate evading this mechanism, as the \u003cem\u003eA. fumigatus\u003c/em\u003e \u0026Delta;\u003cem\u003epksP\u003c/em\u003e mutant (which lacks a key enzyme involved in melanin biosynthesis) induces higher levels of PL acidification than the corresponding wild-type strain ATCC46645 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed). However, there are no differences in PL acidification in \u003cem\u003eA. fumigatus\u003c/em\u003e, Afi, Aoer, and Ale, indicating that these four species' mechanisms of inhibiting PL acidification are conserved (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed). In contrast, \u003cem\u003eA. fumigatus\u003c/em\u003e conidia induced higher levels of four cytokines\u0026mdash;TNF-\u0026alpha;, IL-6, IL-1\u0026beta;, and 1L-18\u0026mdash;in BMDMs than the other three species (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee to \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eh). Interestingly, \u003cem\u003eA. fumigatus\u003c/em\u003e and Ale uniquely induced production of inflammasome cytokines IL-1\u0026beta; and IL-18, while Afi and Aoer conidia led to the production of lower levels of TNF-\u0026alpha; than \u003cem\u003eA. fumigatus\u003c/em\u003e and Afi conidia (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee to \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eh).\u003c/p\u003e\n\u003cp\u003eVirulence of each species was assessed in a clinically relevant chemotherapeutic BALB/c murine model of IPA. The \u003cem\u003eA. fumigatus\u003c/em\u003e strain was significantly more virulent than Afi, Aoe, and Ale strains (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026gt;\u0026thinsp;0.005, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ei) and killed all mice after 5 days post-infection (d.p.i.). However, while there was no statistical difference among the other species, an increased virulence caused by Ale strain was observed when compared to the Aoe and Afi strains (40% vs. 50\u0026ndash;60% of survival at 15 d.p.i., Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ei).\u003c/p\u003e\n\u003cp\u003eTaken together, these results strongly indicate that \u003cem\u003eA. fumigatus\u003c/em\u003e is more virulent in a chemotherapeutic murine model of IPA, is more efficiently recognized by BMDMs, and can elicit higher levels of cytokine production than the other three species.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe conidial surfome of\u003c/strong\u003e \u003cstrong\u003eA. fumigatus\u003c/strong\u003e \u003cstrong\u003econtains 62 unique proteins.\u003c/strong\u003e Variation in host recognition observed between \u003cem\u003eA. fumigatus\u003c/em\u003e and the other three species raises the hypothesis that conidial surface proteins may underlie differences in host recognition variation. To test this hypothesis, we conducted a trypsin-shaving proteomic analysis and identified \u003cem\u003eA. fumigatus\u003c/em\u003e unique proteins on the conidial surface. To do so, samples of resting (0h) and swollen (4h, 37\u0026deg;C) conidia from the four \u003cem\u003eAspergillus\u003c/em\u003e species included in the study (\u003cem\u003eA. fumigatus\u003c/em\u003e, Afi, Aoe, and Ale; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea) were collected and processed for LC-MS/MS analysis as described in \u003csup\u003e5\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). A total of 354 proteins were identified in \u003cem\u003eA. fumigatus\u003c/em\u003e conidial surface [193 in resting and 161 in swollen conidia, and 122 (52.6%) shared between the two conditions]. Similarly, in Afi, 594 proteins were identified as part of the conidial surfome [309 in resting and 285 in swollen conidia, and 204 (52.3%) shared between the two conditions]. For Ale, we identified 1,027 conidial surface proteins [285 in resting and 742 in swollen conidia, but only 240 (30.1%) shared between the two conditions]. Finally, 763 proteins were identified in Aoe conidial surface [393 in resting and 370 in swollen conidia, and 269 (54.5%) shared between the two conditions] (\u003cstrong\u003eSupplementary Figures \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e; Supplementary Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eComparative analyses of the surface-associated proteome or \"surfome\" revealed 62 conidial surface proteins were \u003cem\u003eA. fumigatus\u003c/em\u003e-specific, i.e., only detected in \u003cem\u003eA. fumigatus\u003c/em\u003e conidia. Fifty-six of the genes encoding these proteins are shared by the majority of 206 \u003cem\u003eA. fumigatus\u003c/em\u003e isolates, whereas six (AFUB_028320, AFUB_039180, AFUB_045200, AFUB_050510, AFUB_069520, and AFUB_094680) were more variable, belonging to the accessory pangenome (pangenome information from \u003csup\u003e18\u003c/sup\u003e). In the other three \u003cem\u003eAspergillus\u003c/em\u003e species, 53, 118, and 385 conidial proteins were unique to Afi, Aoer, and Ale, respectively. Numerous conidial proteins were also broadly shared; 188 proteins were shared by at least two species, 146 were shared by at least three species, and 75 were shared by all four species (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e\n\u003cp\u003eFunctional categorization of the \u003cem\u003eA. fumigatus\u003c/em\u003e-specific conidial surfome was carried out manually according to the information available in FungiDB database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://fungidb.org/fungidb/app/\u003c/span\u003e\u003c/span\u003e). Most genes encode enzymes involved in many biological processes such as cell wall modification, metabolism, cell signaling, and secondary metabolite biosynthesis. Some of these enzymes have previously been identified as allergens and proteases (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec; Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Other surface proteins belonged to less enriched functional categories, such as structural proteins and transporters. However, more than a third of the proteins identified as part of the \u003cem\u003eA. fumigatus\u003c/em\u003e-specific conidial surfome had unknown functions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec). To further explore the putative function of identified proteins, we predicted the presence of secretion and GPI-anchor peptides using SignalP \u0026minus;\u0026thinsp;5.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/service.php?SignalP-5.0\u003c/span\u003e\u003c/span\u003e) and PredGPI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gpcr2.biocomp.unibo.it/gpipe/index.htm\u003c/span\u003e\u003c/span\u003e). Thirty-one out of the 62 proteins are predicted to have a signal peptide cleavage site, while only two have high probability (\u0026gt;\u0026thinsp;99%) of harboring a GPI-anchoring signal (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). One protein (encoded by Afu3g13755) did not have a homologue in the \u003cem\u003eA. fumigatus\u003c/em\u003e A1163 background strain and was not examined further. We examined the expression of these 62 genes in a recently published RNAseq dataset that describes gene modulation during conidial germination \u003csup\u003e19\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). About 27% of the surfome genes (16 genes) do not change their gene expression during the first 16 h of germination while 33% (21 genes) have increased expression during the 2 to 16 h germination window (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). About 24% of the surfome genes (15 genes) are expressed late, while 16% (10 genes) are expressed early (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e\n\u003cp\u003eFurther characterization of \u003cem\u003eA. fumigatus\u003c/em\u003e-specific conidial surface proteins was carried out using gene-deletion strains and phenotypic assays. To do so, we accessed strains from the COFUN homozygous deletion library for 42 genes identified in our previous proteomic analysis following the high-throughput methodology described in \u003csup\u003e20\u003c/sup\u003e. After three attempts, we were unable to construct deletion mutants for the reminaing 20 of the 62 \u003cem\u003eA. fumigatus\u003c/em\u003e-specific genes that were not in the library. It remains to be determined if we were not able to delete the 20 remaining genes because they encode essential proteins. Deletions were PCR validated (primers listed in \u003cstrong\u003eSupplementary Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/strong\u003e). An exhaustive phenotypic screen was carried out for each deletion mutant to identify the involvement of the \u003cem\u003eA. fumigatus\u003c/em\u003e surfome in diverse bioprocesses related to virulence (e.g., growth at the temperature of the human body). Growth rates in solid MM at 37\u0026deg;C revealed a defect in growth for \u0026Delta;AFUB_058080 mutant strain, while \u0026Delta;AFUB_047280 displayed increased or faster growth compared to the parental A1160 strain. None of the mutants exhibited increased sensitivity to heat (44\u0026deg;C) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee \u003cstrong\u003eand\u003c/strong\u003e Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). However, \u0026Delta;AFUB_058080 was found to be more sensitive than the A1160 wild-type strain to the cell wall stressor Congo Red (CR). Similarly, \u0026Delta;AFUB_040910 and \u0026Delta;AFUB_100920 showed decreased and increased susceptibility to oxidative stress, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee \u003cstrong\u003eand\u003c/strong\u003e Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Germination rates for all the mutants and wild-type strain were also determined; most null mutant strains (33/42) behaved similarly to A1160 reference strain. The remaining deletion strains could be differentiated into two groups: i) slow-germinating (below 30% of A1160 germination rate; \u0026Delta;AFUB_035440, \u0026Delta;AFUB_057080, \u0026Delta;AFUB_058080, \u0026Delta;AFUB_100460) and ii) fast-germinating (above 30% of A1160 germination rate; \u0026Delta;AFUB_010730, \u0026Delta;AFUB_039800, \u0026Delta;AFUB_040910; \u0026Delta;AFUB_043810, \u0026Delta;AFUB_095010) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef \u003cstrong\u003eand\u003c/strong\u003e Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe crystal violet (CV) assay was used as an indirect measurement of the adhesion properties of conidia at both resting and swollen stages for all the strains. Interestingly, significant differences between the wild-type and deletion strains were only observed in two instances: \u0026Delta;AFUB_000410 displayed reduced adhesion properties in comparison with A1160, while \u0026Delta;AFUB_041890 showed a higher capacity to adhere to surfaces than the wild-type strain (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eg \u003cstrong\u003eand\u003c/strong\u003e Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eAbnormalities in conidial cell wall organization were investigated by staining different components of the cell wall. Calcofluor White (CFW) binds and blocks chitin biosynthesis, and FITC-conjugated wheat germ agglutinin (WGA), a lectin that recognizes exposed chitin and chitooligomers were used for staining chitin. Chitooligomer exposure is better quantified using WGA; total cell wall chitooligomer content is better assessed by CFW. CFW revealed a group of 8 strains (\u0026Delta;AFUB_030040, \u0026Delta;AFUB_035440, \u0026Delta;AFUB_039180, \u0026Delta;AFUB_039800, \u0026Delta;AFUB_040910, \u0026Delta;AFUB_041890, \u0026Delta;AFUB_043810 and \u0026Delta;AFUB_045200) that exhibited lower chitin exposure than the wild-type strain, and WGA a set of 11 mutants (\u0026Delta;AFUB_030040, \u0026Delta;AFUB_031630, \u0026Delta;AFUB_035440, \u0026Delta;AFUB_039180, \u0026Delta;AFUB_065340, \u0026Delta;AFUB_068800, \u0026Delta;AFUB_069520, \u0026Delta;AFUB_076190, \u0026Delta;AFUB_078110 and \u0026Delta;AFUB_079300) that showed higher chitin exposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eh \u003cstrong\u003eand\u003c/strong\u003e Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Intriguingly, three of these mutants showed opposing chitin exposure phenotypes (\u0026Delta;AFUB_030040, \u0026Delta;AFUB_035440 and \u0026Delta;AFUB_039180). Using a dectin-based immunofluorescent assay, we did not find any consistent phenotypes for \u0026beta;-D-glucan exposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eh \u003cstrong\u003eand\u003c/strong\u003e Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Finally, only one mutant (\u0026Delta;AFUB_050510) showed altered hydrophobicity properties when compared with the A1160 wild-type strain (\u003cstrong\u003eSupplementary Figure \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTaken together, these results suggest that \u003cem\u003eA. fumigatus\u003c/em\u003e conidial surface-associated proteins are also involved in the correct assembly of the cell wall and have an important role in combatting stress and adhering to surfaces.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA subset of\u003c/strong\u003e \u003cstrong\u003eA. fumigatus\u003c/strong\u003e \u003cstrong\u003esurfome proteins is involved in host-pathogen interactions.\u003c/strong\u003e To understand the role of the \u003cem\u003eA. fumigatus\u003c/em\u003e surfome in the establishment of infection, we individually assessed the role of each of the 42 \u003cem\u003eA. fumigatus-specific\u003c/em\u003e surfome proteins in survival and tissue invasion using \u003cem\u003ein vitro\u003c/em\u003e models of infection. \u003cem\u003eA. fumigatus\u003c/em\u003e killing was evaluated by the number of CFUs after challenging murine macrophages (Raw 264.7) with conidia from each of the null mutants at 6 h post-infection (hpi), while \u003cem\u003eA. fumigatus\u003c/em\u003e invasion was determined by differential staining after infecting human lung epithelial cells (A549) with germinating conidia from all deletion strains at 3 hpi. For statistical analyses, we only considered those strains showing a 30% increase or decrease in conidial survival and invasion compared to the parental strain (A1160). Overall, we identified 18 \u003cem\u003eA. fumigatus\u003c/em\u003e genes encoding surface proteins that contribute to survival to macrophage killing (9 conidial surface null strains were more susceptible to macrophage killing and another 9 were more resistant to being cleared) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). On the other hand, 27 \u003cem\u003eA. fumigatus\u003c/em\u003e genes encoding for surface proteins were important for epithelial cell invasion (3 mutants were more efficiently taken up by the epithelial cells whilst 24 exhibited defects in cell invasion) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb, Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Although there is no correlation between macrophage survival and epithelial cell invasion (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec), interestingly, 14 \u003cem\u003eA. fumigatus\u003c/em\u003e surfome proteins were identified to be involved in both killing and invasion (\u0026Delta;AFUB_000410, \u0026Delta;AFUB_004010, \u0026Delta;AFUB_012100, \u0026Delta;AFUB_014430, \u0026Delta;AFUB_028320, \u0026Delta;AFUB_040910, \u0026Delta;AFUB_045200, \u0026Delta;AFUB_047280, \u0026Delta;AFUB_056750, \u0026Delta;AFUB_059140, \u0026Delta;AFUB_059210, \u0026Delta;AFUB_069520, \u0026Delta;AFUB_078110 and \u0026Delta;AFUB_094680) (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb, Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), half of them with unknown function, suggesting that a subset of \u003cem\u003eA. fumigatus\u003c/em\u003e conidial surfome proteins identified in our proteomic analysis contributes to triggering immune cell response. The capacity of the null surface protein strains to induce epithelial cell damage was also evaluated with 3 strains producing less damage than the A1160 strain and only one triggering higher cell toxicity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed, Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eOur data in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee \u003cstrong\u003eto h\u003c/strong\u003e indicate \u003cem\u003eA. fumigatus\u003c/em\u003e induces a stronger immune response in BMDMs compared to three other closely related species. To investigate whether increased induction of immune response in \u003cem\u003eA. fumigatus\u003c/em\u003e is driven by specific conidial surface protein(s), we investigated if the absence of these 42 proteins could play a role in cytokine response by determining the IL1-\u0026beta; and TNF-\u0026alpha; production in BMDMs (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef \u003cstrong\u003eand\u003c/strong\u003e Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). We observed a significantly increased 1L-1\u0026beta; production in 11 mutants (AFUB_000410, AFUB_028320, AFUB_031630, AFUB_035440, AFUB_036380, AFUB_039800, AFUB_059140, AFUB_059210, AFUB_068800, AFUB_078110, and AFUB_094680) and decreased production in 3 mutants (AFUB_020170, AFUB_045200, and AFUB_050560) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef \u003cstrong\u003eand\u003c/strong\u003e Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). TNF-\u0026alpha; production was significantly increased in AFUB_012100 and decreased in AFUB_010730, AFUB_039800, and AFUB_059210 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef \u003cstrong\u003eand\u003c/strong\u003e Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). We also evaluated the BMDMs survival in the presence of these mutants by looking at their lactate dehydrogenase (LDH) activity during infection with the mutants compared to the wild-type strain; only the AFU_065340 (a putative zinc-containing alcohol dehydrogenase) mutant had about 80% inhibited LDH.\u003c/p\u003e\n\u003cp\u003eUsing information about \u003cem\u003eA. fumigatus\u003c/em\u003e regulatory networks publicly available at the National Center for Biotechnology Information (NCBI) and the clustering online tool FungiExpressZ (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cparsania.shinyapps.io/FungiExpresZ/\u003c/span\u003e\u003c/span\u003e), we identified two clusters enriched for RNAseq datasets of \u003cem\u003eA. fumigatus\u003c/em\u003e exposed to animal cells or invasive aspergillosis conditions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea, \u003cstrong\u003ehighlighted in red circles\u003c/strong\u003e). These regulatory datasets were obtained after challenging human dendritic and lung epithelial cells with \u003cem\u003eA. fumigatus\u003c/em\u003e strains (NCBI Bioprojects PRJEB1583 and PRJNA399754). These two clusters contain 22 genes, 11 of which are also among the 62 \u003cem\u003eA. fumigatus\u003c/em\u003e-specific genes, suggesting that the protein products of these 11 surfome genes could be important for host-pathogen interactions and the establishment of the infection (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec, Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Other upregulated clusters are mainly related to response to temperature changes, oxidative and osmotic stresses, or nutritional availability, thus illustrating many other functions of \u003cem\u003eA. fumigatus\u003c/em\u003e conidial surface proteins (\u003cstrong\u003eSupplementary Figure \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e; Supplementary Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/strong\u003e). Furthermore, 7 of the 62 \u003cem\u003eA. fumigatus\u003c/em\u003e-specific genes were within the 1,700 genes whose evolutionary rate differed between pathogenic and non-pathogenic species from \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eFumigati\u003c/em\u003e\u003csup\u003e21\u003c/sup\u003e, suggesting that they may be associated with the repeated evolution of pathogenicity in \u003cem\u003eAspergillus\u003c/em\u003e spp. (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e\n\u003cp\u003eThese results suggest that some of the proteins specifically identified in the \u003cem\u003eA. fumigatus\u003c/em\u003e surfome, but which are absent from three other closely related species, are important for mediating host interactions and eliciting cytokines, and are modulated at transcriptional level in the presence of animal cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of the glycosylasparaginase null mutant.\u003c/strong\u003e As an initial step to investigate in more detail the surfome mutants, we prioritize the mutants that elicit increased IL-1\u0026beta; production (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eg \u003cstrong\u003eand\u003c/strong\u003e Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) and decided to start by the characterization of \u0026Delta;AFUB_059140 mutant (here named \u0026Delta;\u003cem\u003easpA\u003c/em\u003e). The \u003cem\u003easpA\u003c/em\u003e null mutant showed increased killing by BMDMs compared to the wild-type strain but decreased invasion of A549 alveolar epithelial cells (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea to \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec \u003cstrong\u003eand\u003c/strong\u003e Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). We constructed a second independent \u0026Delta;\u003cem\u003easpA\u003c/em\u003e mutant and both independent \u003cem\u003easpA\u003c/em\u003e null mutants have the same phenotypes, strongly suggesting that the observed phenotypes are only due to the \u003cem\u003easpA\u003c/em\u003e single deletion and not to secondary mutations present in these strains. The \u003cem\u003easpA\u003c/em\u003e gene encodes a putative glycosylasparaginase (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee) that catalyzes the hydrolysis of N4-(beta-N-acetyl-D-glucosaminyl)-L-asparagine yielding as products N-acetyl-beta-glucosaminylamine plus L-aspartate cleaving the GlcNAc-Asn bond that links oligosaccharides to asparagine in N-linked glycoproteins, playing a major role in the degradation of glycoproteins (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam-legacy.xfam.org/\u003c/span\u003e\u003c/span\u003e; PF01112). AspA is distributed among several fungal classes (687 sequences) but enriched in Eurotiomycetes and Sordariomycetes where most of the plant and animal pathogens are present (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ef). The \u0026Delta;\u003cem\u003easpA\u003c/em\u003e mutants are more phagocytosed by BMDMs than the wild-type (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea) and have increased percentage of adherent conidia on the BMDMs surface, and decreased cell viability as measured by CFW fluorescence and metabolic activity by MTT, respectively (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea to \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec). The increased expression of 1L-1\u0026beta; in the \u0026Delta;\u003cem\u003easpA\u003c/em\u003e mutants is dependent on their viability since UV-killed conidia have no IL-1\u0026beta; and TNF-\u0026alpha; activities comparable to the wild-type strain (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed). Although BMDMs have comparable Reactive Oxygen Species (ROS) production upon 4 and 24 h exposure to either the wild-type or \u0026Delta;\u003cem\u003easpA\u003c/em\u003e mutant, BMDMs have significant increased production of ROS when 8 h in the presence of \u0026Delta;\u003cem\u003easpA\u003c/em\u003e mutant, suggesting that this mutant is either not able to cope with ROS detoxification at this time-point or induces increased ROS production (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee). The lack of \u003cem\u003easpA\u003c/em\u003e does not change \u003cem\u003eA. fumigatus\u003c/em\u003e ability to evade phagolysosome acidification in contrast to the \u003cem\u003eA. fumigatus\u003c/em\u003e \u0026Delta;\u003cem\u003epksP\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ef). Transwell migration assays show that most of the increased production of IL-1\u0026beta; by \u003cem\u003easpA\u003c/em\u003e null mutants need contact with BMDMs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eg). In contrast, TNF-\u0026alpha; production is equally dependent on contact for either the wild-type and \u0026Delta;\u003cem\u003easpA\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eh). Taken together, these results suggest that AspA activity is important for BMDMs phagocytosis and viability of \u003cem\u003eA. fumigatus\u003c/em\u003e conidia, and that AspA can modulate the IL-1\u0026beta; production by direct contact of the conidia with BMDMs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAspA is important for establishment of virulence in an immunocompetent murine model of aspergillosis.\u003c/strong\u003e \u003cem\u003eA. fumigatus\u003c/em\u003e \u0026Delta;\u003cem\u003easpA\u003c/em\u003e have the same virulence than the wild-type in a chemotherapeutic murine model but they are less virulent than the wild-type strain in an immunocompetent murine model as measured by fungal burden (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). We measured the production of cytokines TNF-\u0026alpha;, IL-1\u0026beta;, IL-18, IL-12, INF-\u0026upsih;, IL-6, and the chemokine CXCL-1 in the lungs homogenates of the immunocompetent model infected by the wild-type and \u0026Delta;\u003cem\u003easpA\u003c/em\u003e mutants (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec to \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ei\u003cstrong\u003e)\u003c/strong\u003e. The lack of AspA caused increased production of proinflammatory cytokines and CXCL-1 that acts as a chemoattractant for several immune cells, and it has already been observed as induced upon \u003cem\u003eA. fumigatus\u003c/em\u003e lung infection \u003csup\u003e22\u003c/sup\u003e. Our results indicate that AspA is important for attenuating the inflammatory responses and decreasing the neutrophil migration during the early steps of \u003cem\u003eA. fumigatus\u003c/em\u003e infection in the lungs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of the heterologously expressed glycosylasparaginase.\u003c/strong\u003e We heterologously expressed AspA in \u003cem\u003ePichia pastoris\u003c/em\u003e and verified if the enzyme was active by checking its putative asparaginase activity \u003csup\u003e23\u003c/sup\u003e (\u003cstrong\u003eSupplementary Figure \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/strong\u003e). Incubation of BMDMs with increasing AspA concentrations had no significant impact on LDH activity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea) AspA 0.2 or 2 \u0026micro;g decreased BMDMs viability by about 1 and 10%, respectively, while DMSO decreased by 80% (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eb). AspA can modulate IL-1\u0026beta; and TNF-\u0026alpha; production by using different AspA concentrations in a linear dose-response relationship (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ec and \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ed). The cytokine induction by AspA is not related to its glycosylation since AspA exposure to Peptide:N-glycosidase F (PNGase F), which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins and glycopeptides \u003csup\u003e24\u003c/sup\u003e, did not abolish this induction (\u003cstrong\u003eSupplementary Figure \u003cspan class=\"InternalRef\"\u003eS5\u003c/span\u003e\u003c/strong\u003e). We were unable to identify asparaginase activity in AspA, and we decided to inactivate the enzyme by boiling it for 5 minutes. Boiling of the AspA decreased IL-1\u0026beta; production by about 50% (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ee). Most surprisingly, AspA was also able to induce TNF-\u0026alpha; production and AspA inactivation decreased its accumulation by 30% (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ef). Next, we investigated if previous (by 8 h) or concomitant exposure of BMDMs to 2\u0026micro;g of AspA and conidia could change the conidial viability (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eg). Both treatments increased conidial viability by about 20%, strongly indicating that AspA contributes to conidial viability (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eg).\u003c/p\u003e\n\u003cp\u003eTaken together these results suggest AspA modulates BMDM cytokines and it is important to increase conidial viability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProteomic profiling of BMDMs exposed to AspA.\u003c/strong\u003e To begin investigating which macrophage metabolic pathways are modulated by AspA, we incubated BMDMs in the presence of AspA (0.2 \u0026micro;g) for 24 h, extracted the proteins, and identified them by mass spectrometry (\u003cstrong\u003eSupplementary Table \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/strong\u003e). Principal Component Analysis (PCA) and quantitative analysis of these samples showed that BMDM proteins and BMDM proteins exposed to AspA displayed very different distributions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ea). Differentially expressed proteins are defined as those with a minimum of two-fold change in protein abundance (log2FC\u0026thinsp;\u0026ge;\u0026thinsp;1.0 and \u0026le; -1.0; FDR of 0.05) when compared to the BMDMs under the equivalent conditions. We observed 101 proteins upregulated and 259 downregulated in the BMDMs exposed to AspA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eb \u003cstrong\u003eand Supplementary Table \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eWe have used the Reference Database of Immune cells (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://refdic.rcai.riken.jp/document.cgi\u003c/span\u003e\u003c/span\u003e) for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of distinct biological functions of the shared and specific proteomic differences. The BMDM proteins downregulated in the presence of AspA are involved in (i) cell redox homeostasis, such as glutathione, metabolic process, oxidation reduction, and response to oxidative stress; (ii) carbohydrate metabolism, such as pentose-phosphate shunt, tricarboxylic acid cycle, and glycolysis/gluconeogenesis; (iii) protein synthesis; (iv) pyruvate metabolism and (v) ubiquitin-dependent protein catabolism (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ec and \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ed). In contrast, protein transport, vesicle-mediated transport, mRNA processing, and metabolic processes are upregulated (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ec). We have also visually detected the downregulation of 18 proteins specifically related to the modulation of the innate immune and lysosomal functions (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cstrong\u003eand Supplementary Table \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/strong\u003e), including: (i) Csf1r, macrophage colony-stimulating factor 1 receptor, a tyrosine-protein kinase that acts as a cell surface receptor and plays a role in the regulation of survival, proliferation and differentiation of mononuclear phagocytes, such as macrophages and monocytes (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://fungidb.org/fungidb/app/\" target=\"_blank\"\u003ewww.uniprot.org/uniprotkb/P09581/entry\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e); (ii) Ncf1, neutrophil cytosol factor 1, required for the activation of the latent NADPH oxidase (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://fungidb.org/fungidb/app/\" target=\"_blank\"\u003ewww.uniprot.org/uniprotkb/Q09014/entry\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e); (iii) Cotl1, coactosin-like protein that influences leukotrienes synthesis (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://fungidb.org/fungidb/app/\" target=\"_blank\"\u003ewww.uniprot.org/uniprotkb/Q9CQI6/entry\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e); (iv) Mtdh, protein LYRIC that activates the nuclear factor kappa-B (NF-kappa B) transcription factor (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://fungidb.org/fungidb/app/\" target=\"_blank\"\u003ewww.uniprot.org/uniprotkb/Q80WJ7/entry\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e), and (v) and a few lysosomal proteases cathepsins; and upregulated, (i) Ptprc, a receptor-type tyrosine-protein phosphatase C that is required for T-cell activation through the antigen receptor (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://fungidb.org/fungidb/app/\" target=\"_blank\"\u003ewww.uniprot.org/uniprotkb/P06800/entry\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e) and (ii) Ctss, cathepsin S, a key protease responsible for the removal of the invariant chain from MHC class II molecules and MHC class II antigen presentation (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://fungidb.org/fungidb/app/\" target=\"_blank\"\u003ewww.uniprot.org/uniprotkb/O70370/entry\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e). Examination of the general mouse functional protein association network, retrieved from STRING (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org\u003c/span\u003e\u003c/span\u003e) showed that 15 of these proteins have functional associations, suggesting that AspA impacts specific protein interaction networks (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ee).\u003c/p\u003e\n\u003cp\u003eTaken together, our results suggest that AspA modulates several BMDM metabolic pathways specifically affecting the abundance of proteins important for immune function.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e \u003cem\u003eA. fumigatus\u003c/em\u003e is a prominent human fungal pathogen that infects and frequently kills hundreds of thousands yearly. Most other closely related species, on the other hand, are not pathogenic \u003csup\u003e2,15,25\u003c/sup\u003e. The dozen or so clinically relevant \u003cem\u003eAspergillus\u003c/em\u003e species are scattered among hundreds of non-pathogenic species across the \u003cem\u003eAspergillus\u003c/em\u003e phylogeny, implying that the capacity to cause illness or pathogenicity has evolved multiple times in this lineage \u003csup\u003e2\u003c/sup\u003e. The observed pathogenicity spectrum cannot be solely explained by changes in species ecologies or ascertainment bias, suggesting that the repeated evolution of \u003cem\u003eAspergillus\u003c/em\u003e pathogenicity has a genetic foundation, at least in part \u003csup\u003e2\u003c/sup\u003e. \u003cem\u003eA. fumigatus\u003c/em\u003e conidia are the first fungal structure that encounters the host. In Immunocompetent hosts, conidia are cleared and do not affect host health. However, there are several mechanisms of conidial and germling recognition and evasion based mostly on melanin and polysaccharides, such as β-1,3 glucan, chitin, and galactosaminogalactan present on the conidial surface. Although the importance of protein effectors for pathogenesis and virulence have been extensively described in plant fungal pathogens, there are very few reports about conidial surface proteins or protein effectors in human fungal pathogens. In \u003cem\u003eA. fumigatus\u003c/em\u003e, these include hydrophobin \u003csup\u003e6\u003c/sup\u003e, the conidial surface protein \u003cem\u003ecpcA\u003c/em\u003e\u003csup\u003e8\u003c/sup\u003e that prevents immune recognition of β-1,3-glucans, and the HscA protein that anchors human p11 on phagosomal membranes, rewiring the vesicular trafficking to the non-degradative pathway, allowing escape of conidia \u003csup\u003e9\u003c/sup\u003e. More recently, a \u003cem\u003eCryptococcus neoformans\u003c/em\u003e secreted effector that triggers allergic inflammation \u003csup\u003e26\u003c/sup\u003e was also identified.\u003c/p\u003e \u003cp\u003eHere, we used a phylogenetic approach to investigate proteins specifically expressed in \u003cem\u003eA. fumigatus\u003c/em\u003e conidial surface by comparing \u003cem\u003eA. fumigatus\u003c/em\u003e proteins with two closely non-pathogenic related species, Afi and Aoe, and one more distantly-related pathogenic species, Ale. These surface proteins could be potential surface proteins or protein effectors that modulate different aspects of virulence and pathogenicity or alternatively new genetic determinants important for maintaining the correct conidial structure allowing attachment to host surfaces or preventing immune recognition. Using this approach, we identified 62 \u003cem\u003eA. fumigatus\u003c/em\u003e-specific conidial surface proteins and were able to delete 42 of them. Some of these proteins affect the correct assembly of the conidial surface components since they have increased or decreased chitin exposure or different hydrophobic properties. However, several of them affect the interaction with the host cells since they modulate survival in the presence of macrophages, invasion and/or damage to epithelial cells, and cytokine production. This collection of genes and mutants provides an opportunity for the characterization of genetic determinants important for the \u003cem\u003eA. fumigatus\u003c/em\u003e-host interaction.\u003c/p\u003e \u003cp\u003eWe focused on one of these genetic determinants, a gene encoding a putative glycosylasparaginase, named AspA. To our knowledge this is the first characterized fungal glycosylasparaginase. Homologues of this gene are not present in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and when mutated in humans (AGA gene encodes the glycosylasparaginase) cause the most frequent type of recessively inherited lysosomal storage disease named Aspartylglucosaminuria (AGU) \u003csup\u003e27\u003c/sup\u003e. AGU is a disorder related to degradation of glycoproteins. Carbohydrate moieties are typically found in glycoproteins and are linked to the protein moiety by an N-glycosidic bond formed by the amino acid L-asparagine and the monosaccharide N-acetylglucosamine. The lysosomal resident enzyme glycosylasparaginase, which cleaves the N-glycosidic link between the L-asparagine and N-acetylglucosamine moieties of GlcNAc-Asn, is lacking in AGU. This enzyme deficiency causes a buildup of undegraded aspartylglucosamine and several other glycoasparagines, i.e., glycoconjugates having a L-asparagine moiety attached to the carbohydrate chain, in the affected person's tissues and body fluids \u003csup\u003e28,29\u003c/sup\u003e. AGU patients have developmental delays, hyperactivity, early growth spurt, inguinal and abdominal hernias, clumsiness, characteristic facial features, recurrent respiratory and ear infections, and chronic arthritis \u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs far as we know there is no previous information about the involvement of glycosylasparaginases in bacterial and fungal pathogenesis. However, there are several reports about possible roles played by asparaginases in bacterial virulence \u003csup\u003e31\u0026ndash;35\u003c/sup\u003e, but not in fungal virulence. Several bacterial virulence factors have evolved to use enzymatic deamidation, i.e., the irreversible conversion of the amino acids glutamine and asparagine to glutamic acid and aspartic acid, respectively \u003csup\u003e32\u003c/sup\u003e. \u003cem\u003eHelicobacter pylori\u003c/em\u003e cytotoxic activity was significantly decreased by an asparaginase-deficient strain \u003csup\u003e31\u003c/sup\u003e while \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e exploits asparaginase to assimilate nitrogen to produce ammonium and resist acid stress during infection \u003csup\u003e33\u003c/sup\u003e; \u003cem\u003eSalmonellae\u003c/em\u003e asparaginases mediate virulence and inhibit T cell responses \u003csup\u003e34,35\u003c/sup\u003e. \u003cem\u003eA. fumigatus\u003c/em\u003e has two putative glycosylasparaginase (AFUB_059140, which is described here, and AFUB_021340, none of them with a putative signal peptide) and two asparaginase encoding-genes (AFUB_003170 and AFUB_051450, with and without a putative signal peptide, respectively). It remains to be investigated if these genes are also important for \u003cem\u003eA. fumigatus\u003c/em\u003e virulence.\u003c/p\u003e \u003cp\u003eThe Δ\u003cem\u003easpA\u003c/em\u003e mutants are more phagocytosed and killed than the wild-type, elicit more ROS production and need BMDMs contact to have increased IL-1β production, but have identical percentages of acidification of the phagolysosomes. These results suggest that AspA has a role in earlier or later steps after the formation of phagolysosomes. AspA is important for the establishment of the infection in an immunocompetent mouse model but not in the chemotherapeutic mouse model, and the lack of AspA also plays an important role in modulating proinflammatory cytokines \u003cem\u003ein vivo\u003c/em\u003e. The lack of AspA causes increased production of proinflammatory cytokines and CXCL-1 that acts as a chemoattractant for several immune cells, especially neutrophils, and it has already been observed as induced upon \u003cem\u003eA. fumigatus\u003c/em\u003e lung infection \u003csup\u003e22\u003c/sup\u003e. Our results indicate that AspA is important for modulating the inflammatory response and neutrophils migration upon \u003cem\u003eA. fumigatus\u003c/em\u003e infection in the lungs.\u003c/p\u003e \u003cp\u003eWe also investigated AspA through its heterologous expression and the influence of AspA on cytokine production and macrophage metabolism. Although the human glycosylasparaginase has both deglycosylation and asparaginase activities \u003csup\u003e36\u003c/sup\u003e, we were not able to detect asparaginase activity in \u003cem\u003eA. fumigatus\u003c/em\u003e AspA. However, inactivated boiled AspA decreases BMDM cytokine induction, suggesting that it is still active, and this activity is not due to possible glycosylated residues present in AspA since PNGase treatment has not abolished the cytokine induction. AspA has no putative signal peptide, was identified only at the surface of the dormant conidia and does not seem to be secreted. When BMDMs were exposed to AspA, there was a dramatic change in its proteomic profiling, with decreased cell redox and carbohydrate, and pyruvate metabolism, and protein synthesis suggesting that AspA is affecting the BMDMs aerobic metabolism and ROS production, and decreasing the expression of proteins important for ROS production, such as Ncf1, Neutrophil cytosol factor 1, a cytosolic subunit of neutrophil NADPH oxidase, a multicomponent enzyme that is activated to produce superoxide anion. It has been previously shown that patients with hypersensitivity pneumonitis (HP), a rare initial presentation in chronic granulomatous disease (CGD) have mutations in NCF1 and NCF2 genes and are more susceptible to invasive pulmonary \u003cem\u003eA. fumigatus\u003c/em\u003e infection \u003csup\u003e37\u003c/sup\u003e. Actually, the Δ\u003cem\u003easpA\u003c/em\u003e shows higher induction of ROS in BMDMs. We also observed that BMDMs exposure to AspA decreased the production of several lysosomal proteases, cathepsins, important for protein degradation.\u003c/p\u003e \u003cp\u003eUpon conidial phagocytosis, phagosomes are fused with lysosomes resulting in functional phagolysosomes that are integral part of a degradative pathway that kills and destroys conidia. Is AspA important for escaping phagolysosomal killing? An attractive model for AspA mechanism of action could be that upon conidial phagocytosis, AspA is already interacting with proteins at the macrophage surface deglycosylating asparagine residues and modifying protein activity. Since glycosylasparaginases are described as lysosomal resident proteins, they must be resistant to acid pH (AspA has an isoelectric point of 5.4). Upon conidial phagocytosis and during the formation of phagolysosomes, AspA remains active in the phagolysosome, eventually deglycosylating asparagine residues affecting conidial survival. This apparently has no effect on the percentage of acidification of the phagolysosome since there are no differences between the mutant and wild-type strains. However, it is possible after partial conidial destruction, AspA remains active in the host lysosome affecting the glycosylation metabolism in the cell, and consequently allowing some conidia to survive. \u003cem\u003eA. fumigatus\u003c/em\u003e conidia can survive for some time in the phagosomes of immune cells and epithelial cells \u003csup\u003e38\u0026ndash;41\u003c/sup\u003e. AspA could affect host\u0026rsquo;s membrane trafficking system or redirect the conidia-containing phagosomes toward exocytosis and release of conidia. This has already been reported for a novel \u003cem\u003eA. fumigatus\u003c/em\u003e surface effector, the heat-shock protein HscA that interacts with the human p11 protein, a decisive regulatory node for directing endosomes to different pathways \u003csup\u003e9\u003c/sup\u003e. Different aspects of our proposed model remain to be investigated.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e \u003cb\u003eFungal strains.\u003c/b\u003e All strains included in this work are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Knockout mutant strains belong to the Manchester Infection Fungal Group (MFIG) collection and were constructed as described in \u003csup\u003e20\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSurface proteome analysis.\u003c/b\u003e Freshly harvested conidia of \u003cem\u003eA. fumigatus\u003c/em\u003e A1163, \u003cem\u003eA. oerlinghausenensis\u003c/em\u003e CBS 139183\u003csup\u003eT\u003c/sup\u003e, \u003cem\u003eA. fischeri\u003c/em\u003e NRRL 181, \u003cem\u003eand A. lentulus\u003c/em\u003e CNM-CM6069 were cultivated on potato dextrose agar at 37\u0026deg;C for 0 h (resting conidia) or 4 h (swollen conidia). 1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e conidia of each species in three biological replicates were washed twice with 25 mM ammonium bicarbonate (AB) and centrifuged at 1,800 \u0026times; g for 10 min. Conidia were resuspended in 800 \u0026micro;l of 25 mM AB and incubated with 5 \u0026micro;g trypsin (MS approved, Serva) for 5 min at 37\u0026deg;C. Cell suspensions were immediately passed through 0.2 \u0026micro;m syringe filters (cellulose acetate, Sartorius) to separate conidia from the digestion buffer followed by washing the filters with 200 \u0026micro;l of 25 mM AB. Subsequently, 10 \u0026micro;l of 90% (v/v) formic acid was added to the cell-free solution to stop the proteolytic digestion. Samples were evaporated to dryness in a vacuum concentrator (Eppendorf), resuspended in 30 \u0026micro;l of 2% (v/v) acetonitrile (ACN) and 0.05%(v/v) trifluoroacetic acid (TFA), centrifuged for 15 min at 14,000 \u0026times; g through 10 kDa molecular weight cut-off filters (modified PES, VWR), and transferred into HPLC vials.\u003c/p\u003e \u003cp\u003eLC\u0026thinsp;\u0026minus;\u0026thinsp;MS/MS analysis was performed on an Ultimate 3000 RSLC nano instrument coupled to a QExactive HF mass spectrometer (Thermo Fisher Scientific) as described previously \u003csup\u003e12\u003c/sup\u003e. Tryptic peptides were trapped for 4 min on an Acclaim Pep Map 100 column (2 cm \u0026times; 75 \u0026micro;m, 3 \u0026micro;m) at a flow rate of 5 \u0026micro;l/min. Peptides were further separated on an Acclaim Pep Map column (50 cm \u0026times; 75 \u0026micro;m, 2 \u0026micro;m) using a binary gradient of eluents A (0.1% (v/v) formic acid in H\u003csub\u003e2\u003c/sub\u003eO) and B (0.1% (v/v) formic acid in 90:10 (v/v) ACN/H2O): 0\u0026ndash;4 min at 4% B, 5 min at 8% B, 20 min at 12% B, 30 min at 18% B, 40 min at 25% B, 50 min at 35% B,57 min at 50% B, 62\u0026thinsp;\u0026minus;\u0026thinsp;65 min at 96% B, 65.1\u0026thinsp;\u0026minus;\u0026thinsp;90 min at 4% B. Positively charged ions were generated by a Nanospray FlexIon Source (Thermo Fisher Scientific) using a stainless steel emitter with 2.2 kV spray voltage. Ions were measured in the Full MS / data-dependent MS2 (Top15) mode. Precursor ions were scanned at \u003cem\u003em/z\u003c/em\u003e 300\u0026ndash;1500, a mass resolution of 60,000 full width at half maximum (FWHM), an automatic gain control (AGC) target of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e, and a maximum injection time (maxIT) of 100 ms. Precursor ions were isolated with a width of \u003cem\u003em/z\u003c/em\u003e 2.0. Fragment ions generated in the higher-energy collisional dissociation (HCD) cell at 30% normalized collision energy using N\u003csub\u003e2\u003c/sub\u003e gas were scanned at 15,000 FWHM, an AGC target of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e, and a maxIT of 80 ms. Dynamic exclusion was set to 25 s.\u003c/p\u003e \u003cp\u003eThe MS/MS data were searched against the FungiDB databases of \u003cem\u003eA. fumigatus\u003c/em\u003e Af293 and \u003cem\u003eA. fischeri\u003c/em\u003e NRRL_181, the NCBI database of \u003cem\u003eA. lentulus\u003c/em\u003e and the JGI database of \u003cem\u003eA. oerlinghausenensis\u003c/em\u003e (all downloaded on 2020/10/27) using Proteome Discoverer 2.4 and the algorithms of Mascot 2.4.1, Sequest HT, and MS Amanda 2.0 and MS Fragger 3.0. Two missed cleavages were allowed for the tryptic digestion. The precursor mass tolerance was set to 10 ppm and the fragment mass tolerance was set to 0.02 Da. Modifications were defined as dynamic Met oxidation, protein N-term acetylation and/or loss of methionine. A strict false discovery rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;1% (peptide and protein level) and a search engine score of \u0026gt;\u0026thinsp;30 (Mascot), \u0026gt; 4 (Sequest HT), \u0026gt;\u0026thinsp;300 (MS Amanda) or \u0026gt;\u0026thinsp;8 (MS Fragger) were required for positive protein hits. The Percolator node of PD2.4 and a reverse decoy database was used for qvalue validation of spectral matches. Only rank 1 proteins and peptides of the top scored proteins were counted (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe infected macrophage proteome was performed through lysis in 8M urea in 25 mM AB containing protease inhibitor cocktail using a probe tip sornicator: 40% amplitude for 3 cycles for 10 s and intervals of 10 s. After sonication, samples were centrifuged, the supernatant was transferred to a new tube and quantified using Bradford reagent. Proteins were reduced with 10mM DTT for 30 min at 30 degrees, alkylated with 40mM iodoacetamide for 40 min at room temperature in the dark and overnight digested with trypsin in the ratio 1:50 (enzyme to protein ratio). The tryptic peptides were desalted with Oasis HLB Cartridges (Waters) according to the manufacturer instructions, dried down by speed-vac, and then resuspended in 0.1% formic. LC-MS/MS analysis was performed in an EASY-nLC system (Thermo Scientific) coupled to LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific). Peptides were separated on C18 PicoFrit column (C18 PepMap, 75 \u0026micro;m id \u0026times; 10 cm, 3.5 \u0026micro;m particle size, 100 \u0026Aring; pore size; New Objective) using a gradient of A and B buffers (buffer A: 0.1% formic acid; Buffer B: 95% CAN, 0.1% formic acid) at a flow rate of 300nL/min: from 2\u0026ndash;30% B over 80 min and from 30\u0026ndash;90% B over 5 min. The LTQ-Orbitrap Velos was operated in positive ion mode with data-dependent acquisition. The full scan was obtained in the Orbitrap with an automatic gain control (AGC) target value of 10e6 ions and a maximum fill time of 500 ms. Each precursor ion scan was acquired at a resolution of 60,000 FWHM in the 400\u0026ndash;1500 m/z mass range. Peptide ions were fragmented by CID MS/MS using a normalized collision energy of 35. The 20 most abundant peptides were selected for MS/MS and dynamically excluded during 30 sec. All raw data were accessed in the Xcalibur software (Thermo Scientific). For protein identification, raw data were processed using MaxQuant software version 1.5.3.8. The MS/MS spectra were searched against a protein database composed of Aspergillus fumigatus and Human sequences with the addition of common contaminants with a tolerance level of 4.5 ppm for MS and 0.5 Da for MS/MS. Trypsin was selected as a specific enzyme with a maximum of two missed cleavages. Carbamidomethylation of cysteine (57.021 Da) was set as a fixed modification, and oxidation of methionine (15.994 Da), deamidation NQ (+\u0026thinsp;0.984 Da) and protein N-terminal acetylation (42.010 Da) were set as variable modifications. PSMs, peptides and proteins were accepted at FDR less than 1%.\u003c/p\u003e \u003cp\u003eMultivariate statistical analyses were performed on the protein groups using the label-free quantification (LFQ) results. The analyses were conducted using the LFQ-Analyst web platform \u003csup\u003e42\u003c/sup\u003e with default parameters, including a p-value cutoff of 0.05 and a Log2 fold change cutoff of 1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBMDMs protein extraction.\u003c/b\u003e The BMDMs protein extraction was performed in 8M urea containing protease inhibitor cocktail using a probe tip sornicator: 40% amplitude for 3 cycles for 10 s and intervals of 10 s. After sonication, samples were centrifuged, the supernatant was transferred to a new tube and quantified using Bradford reagent. Proteins were reduced with 10mM DTT, alkylated with 40mM iodoacetamide and overnight digested with trypsin in the ratio 1:50 (enzyme:protein). The tryptic peptides were desalted with Oasis HLB Cartridges, dried down by speed-vac, and then resuspended in 0.1% formic to the LC-MS/MS analysis that was performed in an EASY-nLC system (Thermo Scientific) coupled to LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific). Peptides were separated on C18 PicoFrit column (C18 PepMap, 75 \u0026micro;m id \u0026times; 10 cm, 3.5 \u0026micro;m particle size, 100 \u0026Aring; pore size; New Objective) using a gradient of A and B buffers (buffer A: 0.1% formic acid; Buffer B: 95% ACN, 0.1% formic acid) at a flow rate of 300nL/min: from 2\u0026ndash;30% B over 80 min and from 30\u0026ndash;90% B over 5 min. The LTQ-Orbitrap Velos was operated in positive ion mode with data-dependent acquisition. The full scan was obtained in the Orbitrap with an automatic gain control (AGC) target value of 10e6 ions and a maximum fill time of 500 ms. Each precursor ion scan was acquired at a resolution of 60,000 FWHM in the 400\u0026ndash;1500 m/z mass range. Peptide ions were fragmented by CID MS/MS using a normalized collision energy of 35. The 20 most abundant peptides were selected for MS/MS and dynamically excluded during 30 sec. All raw data were accessed in the Xcalibur software (Thermo Scientific).\u003c/p\u003e \u003cp\u003eFor protein identification, raw data were processed using MaxQuant software version 1.5.3.8. The MS/MS spectra were searched against a protein database composed of human sequences with the addition of common contaminants with a tolerance level of 4.5 ppm for MS and 0.5 Da for MS/MS. Trypsin was selected as a specific enzyme with a maximum of two missed cleavages. Carbamidomethylation of cysteine (57.021 Da) was set as a fixed modification, and oxidation of methionine (15.994 Da), deamidation NQ (+\u0026thinsp;0.984 Da) and protein N-terminal acetylation (42.010 Da) were set as variable modifications. Proteins and peptides were accepted at FDR less than 1%.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGlycosylasparaginase heterologous expression.\u003c/b\u003e The synthetic asparaginase gene (AFUB_059140) cloned in the expression vector pPICZαA (Invitrogen) was synthesized by the company Genescript. The vector has optimized codons for the expression of \u003cem\u003eKomagataella phaffii\u003c/em\u003e and the α factor secretion signal in the N-terminal portion and Zeocin resistance gene. The plasmid obtained was propagated in competent \u003cem\u003eEscherichia coli\u003c/em\u003e (DH10β) cells (Thermo Fisher Scientific, Waltham, USA), resistant to zeocin. The purified plasmid was linearized by Anza 24 MssI restriction enzyme (Invitrogen), purified again, and transformed into competent \u003cem\u003eK. phaffii\u003c/em\u003e (KM71H) by electroporation (1.5 kV, 25 mF and 200 Ω) in a 0.2 \u0026micro;m cuvette using an electroporator (MicroPulser Electroporator, Bio-Rad). The recombinant strain was selected, cultivated and induced to express the rAsparaginase protein following the EasySelect \u003cem\u003ePichia Expression\u003c/em\u003e Kit manual (Invitrogen, Waltham, USA). After 120 h of induction with 1% (v/v) methanol, the material was centrifuged (5,000 \u0026times; \u003cem\u003eg\u003c/em\u003e, 45 min at 4\u0026deg;C) and the supernatant was filtered through a 0.22 \u0026micro;m filter.\u003c/p\u003e \u003cp\u003eThe filtered material was dialyzed against 50 mM Bicine buffer (pH 8.5) and subjected to anion exchange purification. The Mono Q 5/50 GL column (GE Healthcare) was equilibrated with 50 mM Bicine buffer (pH 8.5) and the enzyme eluted with 180 mM NaCl. Protein integrity and sample purity were confirmed by running a 12% SDS-PAGE \u003csup\u003e43\u003c/sup\u003e. Purified protein fractions were dialyzed against 1X PBS (pH 7.4) using the Vivaspin 10 kDa centrifuge system (Sartorius). Protein quantification was performed in a spectrophotometer at 280 nm and the assays were performed with the enzyme at a concentration of 5.88 \u0026micro;M.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAspA phylogenetic analysis.\u003c/b\u003e The visualize the distribution of the glycosylasparaginase gene presence among fungi, the AspA amino acid sequence (AFUB_059140) was used for BLAST search \u003csup\u003e44\u003c/sup\u003e against the NCBI RefSeq database \u003csup\u003e45\u003c/sup\u003e. The 687 sequences found were aligned using MAFFT v7.508 \u003csup\u003e46\u003c/sup\u003e for the inference of a maximum likelihood phylogenetic tree using IQ-TREE v1.7 \u003csup\u003e47\u003c/sup\u003e with the substitution model JTT\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G4, determined to be the best by the program. The calculated tree was visualized and edited using iTOL v6 \u003csup\u003e48\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMacrophage Culture.\u003c/b\u003e BALB/c bone marrow-derived macrophages (BMDMs) were obtained as previously described \u003csup\u003e49\u003c/sup\u003e. Briefly, bone marrow cells were cultured for 7\u0026ndash;9 days in RPMI 20/30, which consists of RPMI-1640 medium (Gibco, Thermo Fisher Scientific Inc.), supplemented with 20% (vol/ vol) FBS and 30% (vol/vol) L-Cell Conditioned Media (LCCM) as a source of macrophage colony-stimulating factor (M-CSF) on non-treated Petri dishes (Optilux - Costar, Corning Inc. Corning, NY). Twenty-four h before experiments, BMDM monolayers were detached using cold phosphate-buffered saline (PBS) (Hyclone, GE Healthcare Inc. South Logan, UT) and cultured, as specified, in RPMI-1640 (Gibco, Thermo Fisher Scientific Inc.) supplemented with 10% (vol/vol) FBS, 10 U/mL penicillin, and 10 mg/mL streptomycin, (2 mM) L-glutamine, (25 mM) HEPES, pH 7.2 (Gibco, Thermo Fisher Scientific Inc.) at 37\u0026deg;C in 5% (vol/vol) CO\u003csub\u003e2\u003c/sub\u003e for the indicated periods.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMacrophage infection, cytokine and LDH determination.\u003c/b\u003e BMDMs were cultured as described before and were seeded at a density of 10\u003csup\u003e6\u003c/sup\u003e cells/ml in 24-well plates (Greiner Bio-One, Kremsm\u0026uuml;nster, Austria). The cells were challenged with the conidia of different strains at a multiplicity of infection of 1:10 and incubated a 37\u0026deg;C with 5% (vol/vol) CO\u003csub\u003e2\u003c/sub\u003e for 24h. BMDMs were also stimulated with different concentrations (mM) of AspA protein (desnaturated or not) by boilling per 10 min 100 degrees Celsius. LPS (standard LPS, \u003cem\u003eE. coli\u003c/em\u003e 0111: B4; Sigma-Aldrich, 500 ng/mL) plus Nigericin (tlrl-nig, InvivoGen 5 \u0026micro;M/mL) and medium alone were used respectively as the positive and negative controls. Cell culture supernatants were collected and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until they were assayed for TNF-α, IL-1 and LDH release using Mouse DuoSet ELISA kits (R\u0026amp;D Systems, Minneapolis, MN, USA and CyQUANT\u0026trade; lactate dehydrogenase (LDH) Cytotoxicity Assay (Invitrogen), according to the manufacturer\u0026rsquo;s instructions. For cytokine determination, plates were analysed by using a microplate reader (Synergy\u0026trade; HTX Multi-Mode, BioTek) measuring absorbance at 450 nm. Cytokine concentrations were interpolated from a standard curve and statistical significance was determined using an ANOVA (GraphPad Prism 8.0, La Jolla, CA). The level of LDH was determined by measuring absorbance at 490 and 680nm using a microplate reader (Synergy\u0026trade; HTX Multi-Mode, BioTek). All assays were performed in triplicate in three independent experiments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAspergillus\u003c/b\u003e \u003cb\u003egrowth condition and Fluorescein isothiocyanate (FIT\u003c/b\u003e \u003cb\u003eC)\u003c/b\u003e \u003cb\u003elabel.\u003c/b\u003e \u003cem\u003eAspergillus\u003c/em\u003e strains were cultivated on minimal medium (AMM) agar plates at 37\u0026deg;C for 3 days. Conidia were harvested in sterile water with 0.05% (vol/vol) Tween20.The resulting suspension was filtered through two layers of gauze (Miracloth, Calbiochem). FITC-labelling of conidia was performed with 0.1 mg/ml FITC (Sigma) in 0.1 M Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e at 37\u0026deg;C for 30 min. Labelled conidia were washed three times with PBS, 0.1% (vol/vol) Tween20. The conidia concentration was determined using a hemocytometer.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhagocytosis and adhesion assays.\u003c/b\u003e BMDMs were cultivated in Dulbecco's Modified Eagle Medium \u003cem\u003e(\u003c/em\u003eDMEM) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum, 2mM glutamine and penicillin-streptomycin. For infection experiments, macrophages were seeded on glass cover slips in 24 well plates at a density of 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well and allowed to grow adherently overnight. Following washing with prewarmed medium, FITC-labelled conidia were added at a multiplicity of infection of 10. The infection experiment was synchronized for 30 min at 4\u0026deg;C. Unbound conidia were removed by washing with pre-warmed medium and phagocytosis was initiated by shifting the coincubation to 37\u0026deg;C in a humidified CO\u003csub\u003e2\u003c/sub\u003e incubator. After 1 h the phagocytosis was stopped by washing with ice-cold PBS. Labelling of extracellular conidia was performed by incubation with PBS, 0.25 mg/ml calcofluor white (Sigma) for 30 min at 4\u0026deg;C to avoid further. The cells were washed twice with PBS and fixed with 3.7% (vol/vol) formaldehyde/PBS for 15 min followed by two washes with PBS. Microscopic photographs were taken on a Zeiss microscope. For statistical reproducibility two biological replicates and in each case two technical replicates were made and analyzed for each strain. The phagocytic index was enumerated by counting 100 macrophages per cover slip from duplicate wells. Phagocytic index was calculated by the average number of conidia that had been phagocytosed for each macrophage.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMacrophage killing assay.\u003c/b\u003e BMDMs were seeded at a density of 10\u003csup\u003e6\u003c/sup\u003e cells/ml in 24-well plates (Corning Costar) and were challenged with conidia at a multiplicity of infection of 1:10 and incubated a 37\u0026deg;C with 5% (vol/vol) CO\u003csub\u003e2\u003c/sub\u003e for 24h. After incubation media was removed the cells were washed with ice-cold PBS and finally 2ml of sterile water was added to the wells. A P1000 tip was then used to scrape away the cell monolayer and the cell suspension was collected. This suspension was then diluted 1:1000 and 100 \u0026micro;l was plated on Sabouraud agar before the plates were incubated a 37\u0026deg;C overnight and the colonies were counted. 50 \u0026micro;l of the inoculum adjusted to 10\u003csup\u003e3\u003c/sup\u003e/ml was also plated on SAB agar to correct CFU counts. The CFU/ml for each sample was calculated and compared to the A1160 wild-type strain.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAcidification of phagolysosomes.\u003c/b\u003e Acidification of phagolysosomes was essentially determined as previously described \u003csup\u003e17\u003c/sup\u003e. In brief, RAW 264.7 murine macrophages were incubated in Dulbecco's Modified Eagle Medium (DMEM, Gibco) with 27.5 mg/mL gentamicin sulfate (Gibco), 10% (vol/vol) fetal bovine serum (FBS, GE Healthcare Life Sciences), and 1% (wt/vol) ultraglutamine (Gibco) at 37\u0026deg;C and 5% (vol/vol) CO\u003csub\u003e2\u003c/sub\u003e in humidified incubator. Cells were pre-stained with 50 nM LysoTracker Red DND-99 (Invitrogen) for 1 h before infection. Conidia were stained with 0.1 mg/mL Calcofluor White (CFW, Sigma Aldrich) for 10\u0026ndash;15 min before infection. 1 x 10\u003csup\u003e5\u003c/sup\u003e cells per well were added to 8 well ibidi slides (ibidi GmbH) and infected with an MOI of 3 with CFW stained conidia for 2 h at 37\u0026deg;C and 5% (vol/vol) CO\u003csub\u003e2\u003c/sub\u003e in a humidified incubator. Uptake of conidia was synchronized by centrifugation at 100 x g for 5 min. After infection, cells were washed with PBS and fixed using 3.7% formaldehyde in PBS for 10 min, followed by a washing step with PBS. Slides were directly imaged using a Zeiss LSM 780 microscope and analyzed using Zeiss ZEN software. At least, 100 conidia were counted for each biological replicate. Clear red signals surrounding conidia within phagosomes were evaluated as acidified conidia. Three independent experiments were performed.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBMDMs antifungal activity.\u003c/b\u003e BMDMs were isolated as described before and 5\u0026times;105 cells were seeded in 96 round bottom wells in complete DMEM (DMEM (Gibco), 10% (vol/vol) FBS (Gibco), 1% (wt/vol) penicillin/streptomycin (Sigma), 1% HEPES (Gibco). After adhesion the BMDMs were infected with 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e conidia (MOI 1:10). Upon 24 h of co- incubation the cells were lysed with sterile water and conidia were stained for 5 min with 50 \u0026micro;L of a filter sterilized 1mg/mL Calcofluor white solution (Sigma-Aldrich). Conidia were washed with sterile water and the staining was quantified using a fluorometer at 360 nm excitation and 440 nm emission.\u003c/p\u003e \u003cp\u003eXTT assay was performed as described \u003csup\u003e50,51\u003c/sup\u003e. Briefly, 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e BMDMs were plated in 96-well and then infected with 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e spores in a final volume of 100 \u0026micro;l DMEM media. Control wells contained spores but no BMDMs or only BMDMs. Following 2 hr incubation, the macrophages were subjected to hypotonic lysis by three gentle washes with distilled water followed by a 30 min incubation with distilled water at 37\u0026deg;C. Supernatants then were removed, with great care taken not to remove the spores. DMEM media without Phenol Red containing 400 \u0026micro;g/ml of XTT and 50 \u0026micro;g/ml of Coenzyme Q, were added, and the wells were incubated for 2 hr at 37\u0026deg;C. The OD\u003csub\u003e450\u003c/sub\u003e and OD\u003csub\u003e650\u003c/sub\u003e were then measured, and data were expressed as the percent of antifungal activity according to the published formula \u003csup\u003e50\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCytokine and chemokine quantification.\u003c/b\u003e For lung homogenates, the lungs of all experimental groups were homogenized in PBS supplemented with Complete Mini protease inhibitor tablets (Roche), clarified by centrifugation, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. A panel of cytokines and chemokines were quantified by ELISA (R\u0026amp;D Systems) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of reactive oxygen species (ROS).\u003c/b\u003e In 96-well tissue culture (TC)-treated dark clear bottom plates (Sigma-Aldrich), 5\u0026times;105 BMDMs per 100 \u0026micro;L were infected with 5\u0026times;106 conidia followed by 4, 8, and 24 h of incubation at 37 \u003csup\u003e0\u003c/sup\u003eC. The cells were treated with the ROS detection dye CellROX\u003csup\u003e\u0026reg;\u003c/sup\u003e Reagent (ThermoFisher) at a final concentration of 5 \u0026micro;M for 30 min at 37\u003csup\u003e\u0026deg;\u003c/sup\u003eC. ROS detection was performed using a fluorescent plate reader (Thermo Fisher), with an excitation of 485 nm and an emission of 520 nm. BMDMs stimulated with 50 nM phorbol- 12-myristate-13-acetate (PMA) (Sigma-Aldrich) for 20 min at 37\u003csup\u003e\u0026deg;\u003c/sup\u003eC were used as positive control.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranswell Assay.\u003c/b\u003e BMDMs were seeded at a density of 10\u003csup\u003e6\u003c/sup\u003e cells/ml in 24-well plates (Corning Costar) and the assay was performed as the addition each well containing a transwell insert (0.2-\u0026micro;m filter; Corning) containing medium alone, Δ\u003cem\u003easpA-1\u003c/em\u003e, Δ\u003cem\u003easpA-2\u003c/em\u003e or wild-type conidia at a multiplicity of infection of 1:10 and incubated a 37\u0026deg;C with 5% (vol/vol) CO\u003csub\u003e2\u003c/sub\u003e for 24h. After incubation cell culture supernatants were collected and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until they were assayed for TNF-α, IL-1 production. In the transwell system the small and soluble compounds are able to migrate between the upper transwell and lower chamber, whereas spores are prevented from moving between the chambers.\u003c/p\u003e \u003cp\u003e\u003cb\u003eEpithelial cell invasion assay.\u003c/b\u003e The capacity of the various strains to invade the A549 cells was determined using previously described methods with some modification \u003csup\u003e52,53\u003c/sup\u003e. The \u003cem\u003eA. fumigatus\u003c/em\u003e wild-type and mutant strains were grown on Sabouraud dextrose agar (Difco) at 37\u0026deg;C for 7 d prior to use. Conidia were harvested with phosphate-buffered saline (PBS) containing 0.1% (vol/vol) Tween 80 (Sigma-Aldrich) and enumerated with a hemacytometer. The A549 pulmonary epithelial cell line (American Type Culture Collection) was cultured in F12 medium (American Type Culture Collection) containing 10% (vol/vol) fetal bovine serum (Gemini Bio-Products), and 2 mM L-glutamine with penicillin and streptomycin (Irvine Scientific) in 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. Before the assay, 2 x 10\u003csup\u003e5\u003c/sup\u003e A549 cells were cultured in 24-well tissue culture plates containing fibronectin coated circular glass coverslips in each well for overnight. \u003cem\u003eA. fumigatus\u003c/em\u003e conidia were pre-germinated in Sabouraud dextrose broth (Difco) at 37\u0026deg;C for 5.5 h, counted and suspended in F12k medium. Next, 10\u003csup\u003e5\u003c/sup\u003e germlings of each strain in 1 ml F12 K medium were added to A549 cells that had been grown to confluency on the glass coverslips. After incubation for 3 h, the cells were rinsed with 1 ml HBSS in a standardized manner and then fixed with 4% (vol/vol) paraformaldehyde. The noninternalized portions of the organisms were stained with a polyclonal rabbit anti-\u003cem\u003eA. fumigatus\u003c/em\u003e primary antibody (Meridian Life Science, Inc.) followed by an AlexaFluor 568-labeled secondary antibody (Life Technologies). After the coverslips were mounted inverted on microscope slides, they were viewed by epifluorescence. The number of cell-associated organisms was determined by counting the number of red fluoresced organisms per high-powered field (HPF). The number of endocytosed organisms was determined by subtracting the number of non-internalized organisms (staining of entire germlings) from the number of cell-associated organisms. At least 100 organisms per coverslip were scored and each strain was tested in triplicate.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEpithelial cell damage assay.\u003c/b\u003e To evaluate the capacity of various strains to damage A549 epithelial cells, we used our standard \u003csup\u003e51\u003c/sup\u003eCr release assay \u003csup\u003e53\u0026ndash;55\u003c/sup\u003e. \u003cem\u003eA. fumigatus\u003c/em\u003e conidia and A549 were prepared as described before. The A549 cells were grown to confluency in a 24-well tissue culture plate and then loaded with \u003csup\u003e51\u003c/sup\u003eCr overnight. The following day, the cells were rinsed twice with HBSS to remove the unincorporated \u003csup\u003e51\u003c/sup\u003eCr and 5 x 10\u003csup\u003e5\u003c/sup\u003e conidia in 1 ml of F12K medium of each strain were added to triplicate wells. After 16 h of incubation, 500 \u0026micro;l of medium above the cells were collected and transferred to glass test tube A. Next, the remaining medium in the wells was collected and placed in glass test tube B. After lysing the A549 cells with 6 N NaOH, the lysate was collected and the wells were rinsed twice with RadiacWash (Biodex Medical Systems). The lysate and rinses were added to test tube B. The amount of \u003csup\u003e51\u003c/sup\u003eCr in the medium and the cell lysate was measured using a gamma counter. The spontaneous release of \u003csup\u003e51\u003c/sup\u003eCr was determined using uninfected A549 cells that were processed in parallel. The specific release of \u003csup\u003e51\u003c/sup\u003eCr was calculated using our previous described formula. Each experiment was performed in triplicate. Mutants that caused less damage to the A549 cells than the control strain were tested at least one more time to verify the results.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnimal survival curves and fungal burden.\u003c/b\u003e Inbred female mice (BALB/c or C57BL/6 strains; body weight, 20\u0026ndash;22 g) were housed in vented cages containing five animals. Mice were immunosuppressed with cyclophosphamide (150 mg/kg of body weight), which was administered intraperitoneally on days \u0026minus;\u0026thinsp;4, -1 and 2 prior to and post infection (infection day is \u0026ldquo;day 0\u0026rdquo;). Hydrocortisonacetate (200 mg/kg body weight) was injected subcutaneously on day \u0026minus;\u0026thinsp;3. Mice (10 mice per group) were anesthetized by halothane inhalation and infected by intranasal instillation of 20 \u0026micro;L of 1.0 x 10\u003csup\u003e5\u003c/sup\u003e conidia of \u003cem\u003eA. fumigatus\u003c/em\u003e wild-type or mutant strains, \u003cem\u003eA. lentulus\u003c/em\u003e, \u003cem\u003eA. oerlinghausenensis\u003c/em\u003e, or \u003cem\u003eA. fischeri\u003c/em\u003e (the viability of the administered inoculum was determined by incubating a serial dilution of the conidia on MM medium, at 37\u0026deg;C). As a negative control, a group of 10 mice received PBS only. Animals were sacrificed 15 days post-infection. To investigate fungal burden in murine lungs, mice are immunosuppressed as described previously or not, and mice were intranasally inoculated with 1 x 10\u003csup\u003e6\u003c/sup\u003e conidia/20 \u0026micro;l of suspension for the chemotherapeutic murine model and with 5 x 10\u003csup\u003e8\u003c/sup\u003e conidia/20 \u0026micro;l for the immunocompetent murine model. Animals were sacrificed 48 h post-infection, and the lungs were harvested and immediately frozen in liquid nitrogen. DNA was extracted via the phenol/chloroform method and 400 mg of total DNA from each sample were used for quantitative PCRs using primers to amplify the 18S rRNA region of \u003cem\u003eA. fumigatus\u003c/em\u003e and an intronic region of mouse GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Six-point standard curves were calculated using serial dilutions of gDNA from \u003cem\u003eA. fumigatus\u003c/em\u003e strain and the uninfected mouse lung. Fungal and mouse DNA quantities were obtained from the threshold cycle (CT) values from the appropriate standard curves. The qPCR analysis was performed using the SYBR green PCR master mix kit (Applied Biosystems) in the ABI 7500 Fast real-time PCR system (Applied Biosystems, Foster City, CA, USA).\u003c/p\u003e \u003cp\u003e The principles that guide our studies are based on the Declaration of Animal Rights ratified by UNESCO on January 27, 1978 in its 8th and 14th articles. All protocols adopted in this study were approved by the local ethics committee for animal experiments from the University of S\u0026atilde;o Paulo, Campus of Ribeir\u0026atilde;o Preto (Permit Number: 08.1.1277.53.6; Studies on the interaction of \u003cem\u003eA. fumigatus\u003c/em\u003e with animals). Groups of five animals were housed in individually ventilated cages and were cared for in strict accordance with the principles outlined by the Brazilian College of Animal Experimentation (COBEA) and Guiding Principles for Research Involving Animals and Human Beings, American Physiological Society. All efforts were made to minimize suffering. Animals were clinically monitored at least twice daily and humanely sacrificed if moribund (defined by lethargy, dyspnea, hypothermia and weight loss). All stressed animals were sacrificed by cervical dislocation.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhenotypic assay.\u003c/b\u003e Plates containing solid MM were inoculated with 10\u003csup\u003e4\u003c/sup\u003e spores per strain and left to grow for 72 h at 37 or 44\u0026deg;C. When required MM was supplemented with Congo Red (10 \u0026micro;g/ml) or H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (1.5 mM). All radial growths were expressed as ratios, dividing colony radial diameter of growth in the stress condition by colony radial diameter in the control (MM at 37\u0026deg;C) condition.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGermination assay.\u003c/b\u003e The germination process was followed for the A1160 strain over a 16 h time period under a Nikon TI microscope equipped with a 37\u0026deg;C incubator and a 40\u0026times; objective, with 1 picture captured every 30 min by using NIS-Elements 4.0 (Nikon) software. Cell Counter plug-in of ImageJ platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rsb.info.nih.gov/ij/index.html\u003c/span\u003e\u003cspan address=\"http://rsb.info.nih.gov/ij/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was employed to differentially count resting or swollen conidia versus germinated conidia. A time point was selected (7h) in which A1160 achieved 50\u0026ndash;60% of germination and the knockout mutant strains were assayed for the same time point included A1160 as a control. 10\u003csup\u003e4\u003c/sup\u003e conidia were inoculated into 200 \u0026micro;l of liquid MM in 96-well plates and each strain was tested in technical duplicates.\u003c/p\u003e \u003cp\u003e\u003cb\u003eStaining for cell surface components.\u003c/b\u003e Cell wall surface polysaccharide staining was performed as described previously \u003csup\u003e56\u003c/sup\u003e. Briefly, 10\u003csup\u003e4\u003c/sup\u003e spores for each deletion mutant and A1160 strains were inoculated in 200\u0026micro;l of MM liquid medium and incubated for 4 h at 37\u0026deg;C (swollen conidia) and 4\u0026deg;C (resting conidia) before the culture medium was removed and conidia were UV irradiated (600,000mJ). For dectin staining, 200 \u0026micro;l of a blocking solution (2% [wt/vol] goat serum, 1% [wt/vol] bovine serum albumin [BSA], 0.1% [vol/vol] Triton X-100, 0.05% [vol/vol] Tween 20, 0.05% [vol/vol] sodium azide, and 0.01 M PBS) was added to each working well. Samples were incubated for 30 min at room temperature (RT) and 0.2 \u0026micro;g/ml of Fc-h-dectin-hFc (Invivogen) was added to the UV-irradiated conidia and incubated for 1 h at RT, followed by the addition of 1:1,000 DyLight 594-conjugated goat anti-human IgG1 (Abcam) for 1 h at RT. Conidia were then washed with phosphate-buffered saline (PBS), and fluorescence was read at 587-nm excitation and 615-nm emission. For chitin staining, 200\u0026micro;l of a PBS solution with 10mg/ml of calcofluor white (CFW) was added to the UV-irradiated conidia, which were incubated for 5 min at RT and washed with PBS before fluorescence was read at 380-nm excitation and 450-nm emission. For N-acetyl-D-glucosamine (GlcNAc) staining, 200 \u0026micro;l of PBS supplemented with 0.1 mg/ml of wheat germ agglutinin (WGA) lectin (lectin-FITC L4895; Sigma) was added to the UV-irradiated germlings for 1 h at RT. Germlings were washed with PBS, and fluorescence was read at 492 nm excitation and 517 nm emission. All experiments were performed using at least 4 repetitions, and fluorescence was read in a microtiter plate reader (Synergy HTX Multimode Reader; Agilent Biotek or EnSpire Multimode Plate Reader; Perkin Elmer).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFungal adhesion assay.\u003c/b\u003e In order to determine the adhesion capacity of conidia from deleted mutant and A1160 strains, 10\u003csup\u003e4\u003c/sup\u003e conidia were inoculated into 200 ul of MM liquid medium in a 96-well polystyrene microtiter plate. Following an initial adherence phase of 4 h during static incubation at 37\u0026deg;C (swollen conidia) or 4\u0026deg;C (resting conidia), unbound conidia were washed with sterile PBS. Fresh MM liquid medium was added to the adhered conidia, and static submerged cultures were grown for up to 24 h at 37\u0026deg;C. Subsequently, the plate was washed exhaustively with PBS prior to incubation with 200 ul 0.5% (wt/vol) crystal violet solution for 5 min at room temperature. The stained mycelia were then exhaustively washed with sterile water and air dried. Finally, the crystal violet was eluted from the wells using 100% ethanol, and the absorbance was measured at 590 nm in a Synergy HTX Multimode Reader (Agilent Biotek).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAssessment of conidial hydrophobic properties.\u003c/b\u003e The distribution of the conidia on a water-oil interface was performed by the addition of 1x10\u003csup\u003e8\u003c/sup\u003e conidia of the wild-type and each deleted strain in a solution containing water and tributyrin (1:1 [vol/vol]). The mixture was vortexed during 1 min and set to allow the conidia to disperse for 1 h. The distribution of the conidia for each mutant were then visually compared to the A1160 wild-type strain.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e Statistical significance for each experiment was established by one-way or two-way ANOVA using the built-in tools of Prism 6. Statistical tests are indicated in the figure legends and were chosen based on the nature of the experiment and the standard tests employed in the field. Underlying assumptions for these tests, including sample independence, variance equality, and normality were assumed to be met. One-way ANOVA was followed by Tukey\u0026rsquo;s or Dunnett\u0026rsquo;s multiple comparison test when appropriate. Asterisks denote \u003cem\u003ep\u003c/em\u003e value as follows: ns\u0026thinsp;=\u0026thinsp;not significant, *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository \u003csup\u003e57\u003c/sup\u003e with the dataset identifiers PXD031199 (Username: [email protected]; Password: HjEpfE78) and PXD044190 (Username: [email protected]\u003cu\u003e; \u003c/u\u003ePassword: WNvL2PfA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de S\u0026atilde;o Paulo (FAPESP) grants numbers 2021/04977-5 (G.H.G.), 2018/18257-1 (GP), 2018/15549-1 (GP), 2020/04923-0 (GP) and the Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq) grant numbers 301058/2019-9 and 404735/2018-5 (G.H.G.), both from Brazil, and the National Institutes of Health/National Institute of Allergy and Infectious Diseases grant R01 AI153356 from the USA (to A.R. and G.H.G.). This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) Collaborative Research Centre/Transregio FungiNet 124 \u0026lsquo;Pathogentic fungi and their human host: Networks of Interaction\u0026rsquo; (project number 210879364; projects A1 and Z2). Work was also supported by a grant from the Wellcome Trust \u0026lsquo;The Aspergillus fumigatus gene and non-coding RNA knockout library \u0026ldquo;COFUN\u0026rdquo;\u0026rsquo; (Project number 208396/Z/17/Z to MB). SG was cofounded by NIHR Manchester Biomedical Research Centre, the Manchester Academy of Health Sciences, the Fungal Infection Trust, the Dowager Countess Eleanor Peel trust and the Medical Research Council (MC_PC_21021). KE was supported by an NC3Rs studentship [NC/T001798/1]. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEarle, K. et al. Pathogenicity and virulence of \u003cem\u003eAspergillus fumigatus\u003c/em\u003e. \u003cem\u003eVirulence\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 2172264 (2023).\u003c/li\u003e\n\u003cli\u003eRokas, A. et al. Evolving moldy murderers: \u003cem\u003eAspergillus\u003c/em\u003e section Fumigati as a model for studying the repeated evolution of fungal pathogenicity. \u003cem\u003ePLoS Pathog.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, e1008315 (2020).\u003c/li\u003e\n\u003cli\u003eBayry, J. et al. Surface structure characterization of Aspergillus fumigatus conidia mutated in the melanin synthesis pathway and their human cellular immune response. \u003cem\u003eInfect. Immun.\u003c/em\u003e \u003cstrong\u003e82\u003c/strong\u003e, 3141\u0026ndash;3153 (2014)\u003c/li\u003e\n\u003cli\u003eHeinekamp, T. et al. Interference of \u003cem\u003eAspergillus fumigatus\u003c/em\u003e with the immune response. \u003cem\u003eSemin. Immunopathol.\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 141-152 (2015). \u003c/li\u003e\n\u003cli\u003eBlango, M.G. et al. Dynamic surface proteomes of allergenic fungal conidia. \u003cem\u003eJ. Proteome Res.\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 2092-2104 (2020).\u003c/li\u003e\n\u003cli\u003eAimanianda, V. \u003cem\u003eet al.\u003c/em\u003e Surface hydrophobin prevents immune recognition of airborne fungal spores. \u003cem\u003eNature \u003c/em\u003e\u003cstrong\u003e460\u003c/strong\u003e, 1117-1121 (2009). \u003c/li\u003e\n\u003cli\u003eValsecchi, I. et al. Role of Hydrophobins in \u003cem\u003eAspergillus fumigatus\u003c/em\u003e. J. Fungi (Basel) \u003cstrong\u003e4\u003c/strong\u003e: 2 (2017). \u003c/li\u003e\n\u003cli\u003eVoltersen, V. \u003cem\u003eet al.\u003c/em\u003e Proteome Analysis Reveals the Conidial Surface Protein CcpA Essential for Virulence of the Pathogenic Fungus \u003cem\u003eAspergillus fumi\u003c/em\u003e\u003cem\u003egatus\u003c/em\u003e. \u003cem\u003emBio \u003c/em\u003e\u003cstrong\u003e9\u003c/strong\u003e, e01557-18 (2018).\u003c/li\u003e\n\u003cli\u003eJia, L.J. et al. \u003cem\u003eAspergillus fumigatus\u003c/em\u003e hijacks human p11 to redirect fungal-containing phagosomes to non-degradative pathway. \u003cem\u003eCell Host Microbe\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 373-388 (2023).\u003c/li\u003e\n\u003cli\u003eAsif, A.R. et al. Proteome of conidial surface associated proteins of \u003cem\u003eAspergillus fumigatus\u003c/em\u003e reflecting potential vaccine candidates and allergens. \u003cem\u003eJ. Proteome Res.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 954-962 (2006).\u003c/li\u003e\n\u003cli\u003eJia, L.J. et al. Biotinylated Surfome Profiling Identifies Potential Biomarkers for Diagnosis and Therapy of \u003cem\u003eAspergillus fumigatus\u003c/em\u003e Infection. \u003cem\u003emSphere\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, e00535-20 (2020).\u003c/li\u003e\n\u003cli\u003eBlango, M.G. \u003cem\u003eet al.\u003c/em\u003e Dynamic Surface Proteomes of Allergenic Fungal Conidia. \u003cem\u003eJ. Proteome Res.\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 2092-2104 (2020).\u003c/li\u003e\n\u003cli\u003eYu, S.Y. et al. Clinical and Microbiological Characterization of Invasive Pulmonary Aspergillosis Caused by \u003cem\u003eAspergillus lentulus\u003c/em\u003e in China. \u003cem\u003eFront. Microbiol.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1672 (2020). \u003c/li\u003e\n\u003cli\u003eHoubraken J., Weig M., Gro\u0026beta; U. \u003cem\u003eAspergillus oerlinghausenensis\u003c/em\u003e, a new mould species closely related to \u003cem\u003eA. fumigatus\u003c/em\u003e. \u003cem\u003eFEMS Microbiology Letters\u003c/em\u003e \u003cstrong\u003e363\u003c/strong\u003e,fnv236 (2016). \u003c/li\u003e\n\u003cli\u003eSteenwyk, J.L. et al. Variation Among Biosynthetic Gene Clusters, Secondary Metabolite Profiles, and Cards of Virulence Across \u003cem\u003eAspergillus\u003c/em\u003e Species. \u003cem\u003eGenetics\u003c/em\u003e \u003cstrong\u003e216\u003c/strong\u003e, 481-497 (2020). \u003c/li\u003e\n\u003cli\u003eMead, M.E. et al. Characterizing the Pathogenic, Genomic, and Chemical Traits of \u003cem\u003eAspergillus fischeri\u003c/em\u003e, a Close Relative of the Major Human Fungal Pathogen \u003cem\u003eAspergillus fumigatus\u003c/em\u003e. \u003cem\u003emSphere \u003c/em\u003e\u003cstrong\u003e4\u003c/strong\u003e, e00018-19 (2019). \u003c/li\u003e\n\u003cli\u003eThywi\u0026szlig;en, A. et al. Conidial Dihydroxynaphthalene Melanin of the Human Pathogenic Fungus \u003cem\u003eAspergillus fumigatus\u003c/em\u003e Interferes with the Host Endocytosis Pathway. \u003cem\u003eFront. Microbiol.\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 96 (2011).\u003c/li\u003e\n\u003cli\u003eHorta, M.A.C. et al. Examination of Genome-Wide Ortholog Variation in Clinical and Environmental Isolates of the Fungal Pathogen \u003cem\u003eAspergillus fumigatus\u003c/em\u003e. \u003cem\u003emBio\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, e0151922 (2022). \u003c/li\u003e\n\u003cli\u003eBaltussen, T.J.H. et al. The C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e transcription factor SltA is required for germination and hyphal development in \u003cem\u003eAspergillus fumigatus\u003c/em\u003e. \u003cem\u003emSphere\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, e0007623 (2023). \u003c/li\u003e\n\u003cli\u003eZhao, C. et al. High-throughput gene replacement in Aspergillus fumigatus. \u003cem\u003eCurr. Protoc. Microbiol.\u003c/em\u003e \u003cstrong\u003e54\u003c/strong\u003e, e88 (2019). \u003c/li\u003e\n\u003cli\u003eMead, M.E. et al. An evolutionary genomic approach reveals both conserved and species-specific genetic elements related to human disease in closely related \u003cem\u003eAspergillus\u003c/em\u003e fungi. \u003cem\u003eGenetics\u003c/em\u003e \u003cstrong\u003e218\u003c/strong\u003e, iyab066 (2021).\u003c/li\u003e\n\u003cli\u003eBouyssi, A. et al. Characterization of Lung Inflammatory Response to \u003cem\u003eAspergillus fumigatus\u003c/em\u003e Spores. \u003cem\u003eJ. Fungi\u003c/em\u003e (Basel) \u003cstrong\u003e9\u003c/strong\u003e, 682 (2023). \u003c/li\u003e\n\u003cli\u003eShifrin, S., Parrott, C. L., Luborsky, S. W. Substrate binding and intersubunit interactions in L asparaginase. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e, \u003cstrong\u003e249\u003c/strong\u003e, 1335\u0026ndash;1340 (1974).\u003c/li\u003e\n\u003cli\u003eTarentino AL, Plummer TH Jr. Enzymatic deglycosylation of asparagine-linked glycans: purification, properties, and specificity of oligosaccharide-cleaving enzymes from \u003cem\u003eFlavobacterium meningosepticum\u003c/em\u003e. \u003cem\u003eMethods Enzymol.\u003c/em\u003e \u003cstrong\u003e230\u003c/strong\u003e, 44-57 (1994). \u003c/li\u003e\n\u003cli\u003eSteenwyk, J.L. et al. A Robust Phylogenomic Time Tree for Biotechnologically and Medically Important Fungi in the Genera \u003cem\u003eAspergillus\u003c/em\u003e and \u003cem\u003ePenicillium\u003c/em\u003e. \u003cem\u003emBio\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, e00925-19 (2019). \u003c/li\u003e\n\u003cli\u003eDang, E.V. et al. Secreted fungal virulence effector triggers allergic inflammation via TLR4. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e608,\u003c/strong\u003e161-167 (2022). \u003c/li\u003e\n\u003cli\u003eArvio, M, Mononen, I. Aspartylglycosaminuria: a review. \u003cem\u003eOrphanet J. Rare Dis.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 162 (2016). \u003c/li\u003e\n\u003cli\u003eMononen, I. et al. Aspartylglycosaminuria: protein chemistry and molecular biology of the most common lysosomal storage disorder of glycoprotein degradation. \u003cem\u003eFASEB J.\u003c/em\u003e\u003cstrong\u003e 7\u003c/strong\u003e, 1247\u0026ndash;1256 (1993).\u003c/li\u003e\n\u003cli\u003eMononen, I. Aronson, N.N., editors. Lysosomal storage disease: Aspartylglycosaminuria. Heidelberg: Spirenger-Verlag; 1997.\u003c/li\u003e\n\u003cli\u003eGoodspeed, K., Feng, C., Laine, M., Lund, T.C. Aspartylglucosaminuria: Clinical Presentation and Potential Therapies. \u003cem\u003eJ. Child Neurol.\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 403-414 (2021). \u003c/li\u003e\n\u003cli\u003eShibayama, K. et al. Biochemical and pathophysiological characterization of \u003cem\u003eHelicobacter pylori\u003c/em\u003e asparaginase. \u003cem\u003eMicrobiol. Immunol.\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 408-417 (2011).\u003c/li\u003e\n\u003cli\u003eWashington, E.J., Banfield, M.J., Dangl, J.L. What a difference a Dalton makes: bacterial virulence factors modulate eukaryotic host cell signaling systems via deamidation. \u003cem\u003eMicrobiol. Mol. Biol. Rev.\u003c/em\u003e \u003cstrong\u003e77\u003c/strong\u003e, 527-39 (2013).\u003c/li\u003e\n\u003cli\u003eGouzy, A. et al. \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e exploits asparagine to assimilate nitrogen and resist acid stress during infection. \u003cem\u003ePLoS Pathog.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, e1003928 (2014). \u003c/li\u003e\n\u003cli\u003eMcLaughlin, P.A. et al. Contribution of Asparagine Catabolism to \u003cem\u003eSalmonella\u003c/em\u003e Virulence. \u003cem\u003eInfect. Immun.\u003c/em\u003e \u003cstrong\u003e85\u003c/strong\u003e, e00740-16 (2017). \u003c/li\u003e\n\u003cli\u003eTorres, A. et al. Asparagine deprivation mediated by \u003cem\u003eSalmonella\u003c/em\u003e asparaginase causes suppression of activation-induced T cell metabolic reprogramming. \u003cem\u003eJ. Leukoc. Biol.\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e, 387-98 (2016). \u003c/li\u003e\n\u003cli\u003eKelo, E., Noronkoski, T., Mononen, I. Depletion of L-asparagine supply and apoptosis of leukemia cells induced by human glycosylasparaginase. \u003cem\u003eLeukemia\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 1167-71 (2009).\u003c/li\u003e\n\u003cli\u003eLiu, H. et al. Mimicking hypersensitivity pneumonitis as an uncommon initial presentation of chronic granulomatous disease in children. \u003cem\u003eOrphanet J. Rare Dis\u003c/em\u003e. \u003cstrong\u003e12\u003c/strong\u003e, 169 (2017).\u003c/li\u003e\n\u003cli\u003eAmin, S. et al. Melanin dependent survival of \u003cem\u003eApergillus fumigatus \u003c/em\u003econidia in lung epithelial cells. \u003cem\u003eInt. J. Med. Microbiol.\u003c/em\u003e \u003cstrong\u003e304\u003c/strong\u003e, 626\u0026ndash;636 (2014).\u003c/li\u003e\n\u003cli\u003eSeidel, C. et al. Phagolysosomal survival enables non-lytic hyphal escape and ramification through lung epithelium during \u003cem\u003eAspergillus fumigatus\u003c/em\u003e infection.\u003cem\u003e Front. Microbiol.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1955 (2020).\u003c/li\u003e\n\u003cli\u003eThywi\u0026szlig;en, A. et al. Conidial dihydroxynaphthalene melanin of the human pathogenic fungus \u003cem\u003eAspergillus fumigatus\u003c/em\u003e interferes with the host endocytosis pathway. \u003cem\u003eFront. Microbiol.\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 96. (2011).\u003c/li\u003e\n\u003cli\u003eJahn, B. et al. PKSP-dependent reduction of phagolysosome fusion and intracelular kill of \u003cem\u003eAspergillus fumigatus\u003c/em\u003e conidia by human monocyte-derived macrophages. \u003cem\u003eCell. Microbiol.\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 793\u0026ndash;803 (2002).\u003c/li\u003e\n\u003cli\u003eShah, A.D. et al. LFQ-Analyst: An Easy-To-Use Interactive Web Platform To Analyze and Visualize Label-Free Proteomics Data Preprocessed with MaxQuant. \u003cem\u003eJ. Proteome Res.\u003c/em\u003e 19, 204-211 (2020).\u003c/li\u003e\n\u003cli\u003eLaemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e227\u003c/strong\u003e, 680-685 (1970).\u003c/li\u003e\n\u003cli\u003eJohnson, M. et al. NCBI BLAST: a better web interface. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, W5-9 (2008).\u003c/li\u003e\n\u003cli\u003eSayers, E.W. \u003cem\u003eet al.\u003c/em\u003e Database resources of the national center for biotechnology information. \u003cem\u003eNucleic Acids Res\u003c/em\u003e. \u003cstrong\u003e50\u003c/strong\u003e, D20-D26 (2022).\u003c/li\u003e\n\u003cli\u003eKatoh, K., Rozewicki, J., Yamada, K.D. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. \u003cem\u003eBrief Bioinform.\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 1160-1166 (2019).\u003c/li\u003e\n\u003cli\u003eNguyen, L.T., Schmidt, H.A., von Haeseler, A., Minh, B.Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. \u003cem\u003eMol. Biol. Evol.\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 268-74 (2015).\u003c/li\u003e\n\u003cli\u003eLetunic, I., Bork, P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, W293-W296 (2021).\u003c/li\u003e\n\u003cli\u003eMarim, F.M., Silveira, T.N., Lima, D.S. Jr., Zamboni, D.S. A Method for Generation Bone Marrow-Derived Macrophages from Cryopreserved Mouse Bone Marrow Cells. \u003cem\u003ePloS One\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, e15263 (2010). \u003c/li\u003e\n\u003cli\u003eRamirez-Ortiz, Z.G. et al. A nonredundant role for plasmacytoid dendritic cells in host defense against the human fungal pathogen \u003cem\u003eAspergillus fumigatus\u003c/em\u003e. \u003cem\u003eCell Host Microbe\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 415\u0026ndash;424 (2011). \u003c/li\u003e\n\u003cli\u003eMeshulam, T., Levitz, S.M., Christin, L., Diamond, R.D. A simplified new assay for assessment of fungal cell damage with the tetrazolium dye, (2,3)-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide (XTT). \u003cem\u003eJ. Infect. Dis.\u003c/em\u003e \u003cstrong\u003e172\u003c/strong\u003e, 1153\u0026ndash;1156 (1995).\u003c/li\u003e\n\u003cli\u003eLiu, H. et al. Determining \u003cem\u003eAspergillus fumigatus\u003c/em\u003e transcription factor expression and function during invasion of the mammalian lung.\u003cem\u003e PLoS Pathog.\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, e1009235 (2021).\u003c/li\u003e\n\u003cli\u003eLiu, H. et al. \u003cem\u003eAspergillus fumigatus\u003c/em\u003e CalA binds to integrin \u0026alpha;5\u0026beta;1 and mediates host cell invasion. \u003cem\u003eNat. Microbiol.\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e,16211 (2016).\u003c/li\u003e\n\u003cli\u003eEjzykowicz, D.E. et al. The \u003cem\u003eAspergillus fumigatus\u003c/em\u003e transcription factor Ace2 governs pigment production, conidiation and virulence. \u003cem\u003eMol. Microbiol.\u003c/em\u003e \u003cstrong\u003e72\u003c/strong\u003e, 155-169 (2009).\u003c/li\u003e\n\u003cli\u003eBertuzzi, M. et al. The pH-responsive PacC transcription factor of \u003cem\u003eAspergillus fumigatus\u003c/em\u003e governs epithelial entry and tissue invasion during pulmonary aspergillosis. \u003cem\u003ePLoS Pathog.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, e1004413 (2014).\u003c/li\u003e\n\u003cli\u003eWinkelstroter, L.K. et al. High osmolarity glycerol response PtcB phosphatase is important for \u003cem\u003eAspergillus fumigatus\u003c/em\u003e virulence. \u003cem\u003eMol. Microbiol\u003c/em\u003e. \u003cstrong\u003e96\u003c/strong\u003e, 42\u0026ndash;54 (2015).\u003c/li\u003e\n\u003cli\u003ePerez-Riverol, Y. et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e 47, D442-D450 (2019).\u003c/li\u003e\n\u003cli\u003eBrown, A. et al. Extensive non-coding sequence divergence between the major human pathogen \u003cem\u003eAspergillus fumigatus\u003c/em\u003e and its relatives. \u003cem\u003eFront. Fungal Biol.\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 802494 (2022)\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e "}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Aspergillus fumigatus, conidial surface-associated proteome, surfome, conidia, asexual spore, pathogenicity","lastPublishedDoi":"10.21203/rs.3.rs-3306535/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3306535/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eAspergillus fumigatus\u003c/em\u003e, an important pulmonary fungal pathogen causing several diseases collectively called aspergillosis, relies on asexual spores (conidia) for initiating host infection. Here, we used a phylogenomic approach to compare proteins in the conidial surface of \u003cem\u003eA. fumigatus\u003c/em\u003e, two closely related non-pathogenic species, \u003cem\u003eAspergillus fischeri\u003c/em\u003e and \u003cem\u003eAspergillus oerlinghausenensis\u003c/em\u003e, and the cryptic pathogen \u003cem\u003eAspergillus lentulus\u003c/em\u003e. After identifying 62 proteins uniquely expressed on the \u003cem\u003eA. fumigatus\u003c/em\u003e conidial surface, we assessed null mutants for 42 genes encoding conidial proteins. Deletion of 33 of these genes altered susceptibility to macrophage killing, penetration and damage to epithelial cells, and cytokine production. Notably, a gene that encodes glycosylasparaginase, which modulates levels of the host pro-inflammatory cytokine IL-1β, is important for infection in an immunocompetent murine model of fungal disease. These results suggest that \u003cem\u003eA. fumigatus\u003c/em\u003e conidial surface proteins and effectors are important for evasion and modulation of the immune response at the onset of fungal infection.\u003c/p\u003e","manuscriptTitle":"A phylogenetic approach to explore the Aspergillus fumigatus conidial surface-associated proteome and its role in pathogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-18 16:16:08","doi":"10.21203/rs.3.rs-3306535/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-microbiology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nmicrobiol","sideBox":"Learn more about [Nature Microbiology](http://www.nature.com/nmicrobiol/)","snPcode":"","submissionUrl":"","title":"Nature Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"685372eb-1e57-4f63-83e8-5ee45c55ebb7","owner":[],"postedDate":"September 18th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":24729016,"name":"Biological sciences/Microbiology/Fungi/Fungal pathogenesis"},{"id":24729017,"name":"Biological sciences/Microbiology/Pathogens"}],"tags":[],"updatedAt":"2024-08-28T07:06:16+00:00","versionOfRecord":{"articleIdentity":"rs-3306535","link":"https://doi.org/10.1038/s41564-024-01782-y","journal":{"identity":"nature-microbiology","isVorOnly":false,"title":"Nature Microbiology"},"publishedOn":"2024-08-27 04:00:00","publishedOnDateReadable":"August 27th, 2024"},"versionCreatedAt":"2023-09-18 16:16:08","video":"","vorDoi":"10.1038/s41564-024-01782-y","vorDoiUrl":"https://doi.org/10.1038/s41564-024-01782-y","workflowStages":[]},"version":"v1","identity":"rs-3306535","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3306535","identity":"rs-3306535","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}