MUC16-dependent Renal Vascular Adhesion of Candida Promotes Tissue Invasion and Predicts Clinical Outcome in Candidemia

MUC16-dependent Renal Vascular Adhesion of Candida Promotes Tissue Invasion and Predicts Clinical Outcome in Candidemia | 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 MUC16-dependent Renal Vascular Adhesion of Candida Promotes Tissue Invasion and Predicts Clinical Outcome in Candidemia Peter Williamson, Jin Qiu, Shellee Grim, Guowu Hu, Yoon-Dong Park, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8810991/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Bloodstream infection with Candida species is associated with high mortality, yet the molecular basis of vascular adhesion and invasion remains unclear. We identify the endothelial surface mucin MUC16 as a key host factor enabling fungal vascular adherence. The MUC16 SEA domain directly engages the Candida adhesin Als3p and endothelial N-cadherin to form a ternary complex that promotes endothelial attachment and vascular invasion. Genetic deletion of Muc16 in mice markedly reduced fungal vascular binding, renal fungal burden, and Ly6Ghi CD11b+ neutrophil recruitment following intravenous infection, and diminished oral colonization in an oral model. Inflammatory induction of MUC16 occurred via MINCLE-, TLR9-, and Dectin-1–dependent signaling, converging on CARD9. In clinical cohorts, a MUC16 T10155I variant and elevated circulating MUC16 were associated with increased 30-day mortality. These findings establish MUC16 as a mechanistic link between endothelial sensing and Candida vascular invasion, with prognostic relevance in candidemia. Health sciences/Medical research Biological sciences/Microbiology/Fungi Virulence Candida MUC16 CA125 Endothelium Cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Candida spp. are the fourth most common cause of nosocomial bloodstream infection in the US, with crude mortality of 25-40% and causing an estimated 400,000 life threatening systemic infections per year 1 with an additive adverse impact during the COVID-19 era 2 . However, despite its significance, key elements of pathogenesis remain poorly understood, especially in disseminated bloodstream infections in which mortality remains high despite therapy. Risk factors for mortality include malignancy, presence of chronic indwelling central catheters and prolonged antibiotic therapy associated with bacterial infections 3 . Recently, genetic risk factors have been identified that include the single nucleotide polymorphisms (SNPs) in PLA2G4B with reactive oxygen species (ROS) and IL-6 production in response to Candida infections 4 and relationships of CXCR1 and CX3CR1 with the acquisition of infections 5,6 . These studies suggest that better understanding of pathogenic molecular mechanisms may identify patients at highest risk of death from candidemia that would1) allow intensification of therapy in the clinical setting or 2) enable better stratification of patients in clinical treatment trials. Transient Candida in the bloodstream may occur after colonization of and release from central venous catheters, or translocation through intestinal barriers after pathogenic epithelial cell damage 7 . Once in the bloodstream, dissemination throughout the host requires adherence followed by invasion through the vascular endothelium which occurs through endocytosis and is induced most efficiently by hyphae 8,9 . Successful endothelial endocytosis is dependent on fungal adhesins such as Als3p with a requirement for N-cadherin for fungal endocytosis 10 . Invasion through the endothelial layer into tissue, principally the kidney in mice 11 , results in significant tissue damage and is mediated by a plethora of factors-- proteases, lipases and haemolysins 12 as well as toxins including the hyphae-specific inflammasome-activating candidalysin 13,14 , important for neutrophil recruitment 15 . Tissue invasion by neutrophils is thought to cause paradoxical collateral tissue damage during fungal killing, constituting a predominant mechanism of deep tissue injury 16 . However, despite the role of N-cadherin in the endocytosis of Candida hyphae that follows initial endothelial binding, the identity of the mammalian-endothelial cell adhesion molecule remains unknown. In light of surface mucin’s role modulating infections by Staphylococcus 17 , which also expresses a bacterial adhesin with structural similarity to C. albicans Als3 18 , the present study sought a possible role for surface renal mucins in bloodstream infections with Candida albicans as the kidney has been identified as a key organ involved in initial pathogenesis 11 . Of the mucins expressed in endothelial cells (ECs) our studies found that MUC16 was both expressed on the surface of ECs by immunohistochemistry and flow cytometry of mouse kidney primary CD31+ ECs and required for endothelial binding of Candida hyphae. Additional mechanistic studies identified the formation of a Als3-SEA-N-cadherin ternary complex, facilitating sequential binding and endocytosis of the fungal pathogen. The presence of MUC16 in WT mice resulted in increased tissue burden and mortality compared to Muc16 -/- littermates in a disseminated candidiasis model, and increased weight loss and tissue burden in an oral candidiasis model. Further clinical studies identified a key T10155I heterozygous polymorphism to be associated with 30-day mortality in two independent cohorts of patients with candidemia and established this polymorphism as having a role in Candida -endothelial cell adherence in model studies. Interestingly, modeling of candidemia risk factors--exposure of ECs to bacterial and fungal products, resulted in increased expression of MUC16, mediated prominently by the receptors MINCLE, TLR9 and DECTIN-1 in a Card9 -dependent fashion. Hypothesizing that increased MUC16 expression in patients via such mechanisms could result in poor outcomes, we tested and found in a cohort of 134 patients that increased serum concentrations of MUC16, measured by a commercial CA125 antibody at the time of diagnosis, demonstrated a strong association with 30-day mortality. In summary, these data point to a role for MUC16 in Candida binding to ECs and may provide a readily available biomarker for outcome in bloodstream infections. Results MUC16 is required for adherence of Candida albicans to purified primary renal ECs Because of previously described roles of surface mucins, particularly MUC16 in modulating interactions with pathogens 17 as well as interactions with surface cadherins 19 , we studied the role of mucins in Candida adherence using primary mouse kidney ECs 20 . As shown in Fig. S1a-c, purified primary mouse kidney ECs did not express CD45 and displayed typical properties of ECs 21-26 27 . Primary purified kidney ECs were then assessed for expression of renal-specific mucins identified in whole-genome expression studies 28 in the BioGBS database 29 which identified expression of Muc1 , Muc14 and Muc16 that was suppressible by siRNA (Fig. S1e). Surface Muc16 protein was identified by flow cytometry which was not present in ECs purified from Muc16 -/- mice (Fig. S1d). Muc13 displayed little qRT-PCR signal (> 45 cycles). Confluent EC monolayers of each suppressed group were then incubated with hyphal elements of Candida albicans expressing fluorescent d-tomato followed by extensive washing 30 10 , which demonstrated reduced fungal adherence after Muc16 suppression but not Muc1 , Muc13 or Muc14 vs non-targeted controls (Fig. 1a; Fig. S1f). In addition, primary EC monolayers from Muc16 -/- KO mice demonstrated reductions in adherence at 30, 60 or 90 minutes compared to WT litter-mate controls (Fig. 1b). As expected, incubation of WT ECs with the calcium chelator ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) which inhibits subsequent N-cadherin endocytosis did not inhibit adherence in WT mouse kidney ECs (Fig. 1c). Assays combining adherence and endocytosis were then conducted using a previously described dual assay utilizing a fluorescent-d-Tomato C. albicans strain followed by addition of a green-fluorescent Alexa 488-labeled non-permeable antibody against intact Candida , the latter of which labels surface adherent but non-endocytosed cells 10 . As shown in Fig. 1d, d-Tomato-labeled C. albicans hyphae exhibited both adherence to WT ECs, identified by green fluorescence (left panel, green arrows), as well as endocytosis demonstrated by red fluorescent hyphae not labeled by green fluorescence (left panel, red arrows). Endocytosis was inhibited by EGTA (+EGTA). In contrast, ECs from Muc16 -/- KO mice demonstrated little adherence or subsequent endocytosis of hyphae-induced fungal cells in the presence (+) or absence (-) of EGTA (Fig. 1d). Endocytosis intensity ratios calculated as described 10 were significantly reduced by EGTA for WT ECs, as expected, but the small amount of residual unlabeled fungal cells found in the Muc16 -/- ECs were not further reduced by EGTA (Fig. 1e). To molecularly probe interactions between fungal Als3, MUC16 and N-cadherin, a proximity ligation assay was utilized after incubation of primary murine ECs with recombinant Als3, the latter described previously 31 . As shown in Fig. 1f and g, strong signal was recorded evidenced by multiple visible puncta, linking Als3 to MUC16 in WT mouse ECs compared to equivalent assays using Muc16 -/- KO mouse ECs. Detectable signal was also exhibited between Als3 and N-cadherin in WT mice consistent with earlier studies 32 which was abolished in the Muc16 -/- KO ECs. Signal was also registered between MUC16 and N-cadherin, which was absent in the Muc16 -/- KO EC’s. In summary, these data demonstrate a role for mammalian MUC16 in Candida adherence to mouse primary kidney ECs and is required for subsequent endovascular invasion by a complex linking fungal Als3 and mammalian MUC16 and N-cadherin. Molecular mediators of MUC16-dependent binding of C. albicans to mouse primary ECs. Additional studies sought to confirm a role for the C. albicans hypha-specific surface adhesin Als3 32-34 in MUC16-specific adhesion to mouse kidney ECs. As shown in Fig. 2a, the normally non-adherent Saccharomyces cerevisiae fungal cells expressing ALS3 ( +ALS3 ) were found to exhibit significant primary mouse kidney EC adherence compared to equivalent strains expressing empty vector (EV) alone. However, the same S. cerevisiae ALS3 -expressing strains showed little augmentation of binding to Muc16 -/- KO ECs, suggesting a role of Als3 binding to MUC16-expressing ECs in mice. Some augmentation in ALS3 -dependent binding to the Muc16 -/- KO ECs, suggesting additional minor constituents involved in ALS3 -dependent adherence; however, MUC16 appears to be the dominant molecule involved in adherence. Further molecular interaction studies sought to identify structural elements within the MUC16 molecule involved in adherence. As shown in Fig. 2b, MUC16 is a large surface-bound mucin with a significant number of N-terminal glycosylation and multiple conserved SEA domains (Fig. S2a), the latter so called because of a structural similarity to the sea urchin sperm protein, enterokinase and agrin 35,36 . Initial experiments utilized a combination of neuraminidase + b1,4 galactosidase to reduce N and O-labeled glycosylation of the surface mucin to determine if adherence might also be reduced. Surprisingly, this treatment increased adherence (Fig. 2c), which suggested an alternative domain that may have been sterically shielded by the extensively glycosylated N-terminus. Since autocatalytic processing of MUC16 is known to expose pauci-glycosylated SEA domains, we expressed a fusion protein of 8 murine SEA domains (a.a. 8682-8805; Fig. S2b), previously found to mimic the antigenic site of the anti-human CA125 antibody by NMR spectroscopy, 37 with the affinity tag maltose-binding protein (MBP). As shown in Fig. S2c, the rMBP-SEA8 purified as a 145kD protein with some step-ladder proteolysis typical of the properties of this self-cleaving protein 38 , compared to the 42-kDa MBP. As shown in Fig. 2d (green arrows), rMBP-SEA8, but not rMBP alone was found to exhibit strong binding to hyphae of C. albicans but, interestingly, not the yeast forms of the fungus that are nonpathogenic during invasive disease and do not express Als3p. Flow cytometry confirmed the increased overall binding of the rMBP-SEA8 compared to rMBP alone (Fig. 2e,f). Addition of rMBP-SEA8 was also found to block endothelial adherence of C. albicans hyphae to levels equivalent to that of Muc16 /- ECs whereas rMBP alone showed no significant reductions in adherence (Fig. 2g). These results were duplicated using a single SEA domain protein fusion to MBP at either the N or the C-terminus (Fig S2d-g). rMBP-SEA1 binding to Als3p was further confirmed by co-immunoprecipitation experiments which showed little binding to rMBP alone (Fig. S2h, i). Interestingly, rMBP-SEA1 also retained binding to Als3-GK which has a mutation within the complement binding pocket described previously, suggesting an alternative novel binding site 31 . In summary, these data suggest a role of fungal Als3 in MUC16-dependent fungal adherence to primary mouse kidney ECs as well as a role for the MUC16 SEA domains in C. albicans -EC binding. MUC16 is expressed in CD31+ renal ECs and co-localizes with renal vasculature and invading Candida hyphal elements. While MUC16 is a well-known epithelial-related mucin, less is known about its role as a renal endothelial mucin. As shown in Fig. 3a, kidney tissue demonstrated robust expression of MUC16 as well as N-cadherin within glomeruli and surrounding structures of mouse kidneys by immunostaining. In addition, immunofluorescence (IFA) demonstrated colocalization of MUC16 with CD31+ staining ECs in vessel walls, without significant MUC16 staining in Muc16 -/- mutant mice (Fig. 3b). Furthermore, 2 hours after intravenous inoculation of C. albicans hyphae expressing fluorescent d-Tomato (Fig. 3c), fungal cells (red arrows) localized within CD31+ renal vascular structures in WT mice (green arrows), expressing MUC16 (center and left panel, purple arrow). Endothelial adherence was persistent at 4 days (Fig. 3d) with occasionally observed penetration of the endothelial layer by fungal hyphal structures (* and red arrows). In contrast, fungal organisms were not observed localized to vascular endothelium in the Muc16 -/- KO animals. In summary, these data suggest that MUC16 is expressed in renal ECs of intact mouse kidneys and intravenously-inoculated fungal cells co-localize to the MUC16-expressing EC layer, consistent with the MUC16-dependent adherence observed in purified mouse kidney ECs. Deletion of MUC16 in mice conveys protection in an intravenous dissemination model of Candida albicans infections The significance of MUC16 in systemic Candida pathogenesis was further studied using an intravenous dissemination candidemia model that results in mortality mediated primarily by fungal renal vascular invasion followed by pathogenic renal neutrophil invasion 11 . As shown in Fig. 4a, Muc16 -/- 129 x C57BL/6N strain F1 progeny exhibited significant protection after intravenous challenge with 1 x 10 5 CFU of C. albicans compared to littermate WT F1 controls. To confirm the phenotypic difference, Muc16 -/- mice were then backcrossed to C57B6 mice 5x using a high-speed congenic strategy and first-wave male germ cells with 100% B6-homozygous loci (84 markers) 39 and were again found to exhibit significant protection compared to WT littermate control mice at even a higher inoculum of 2.5 x 10 5 CFU (Fig. 4b). Fungal burdens were shown to be reduced in the Muc16 -/- mouse kidneys by CFU (Fig. 4c) with WT excised kidneys demonstrating increased edema and hypopigmentation compared to Muc16 -/- mice (Fig. 4d) and histology (Fig. 4e) showing increased Grocott’s methenamine silver (GMS)-staining fungal elements in the WT mice compared to the Muc16 -/- mice. In addition, fungal burdens in Muc16 -/- mice showed reductions in brain, liver or lung compared to WT, though less consistent in brain (Fig. S4). Cellular studies also demonstrated reduced accumulation of ly6C int CD11b + Ly6G hi neutrophils (Fig. 4f), Ly6C hi CD11b + inflammatory monocytes (Fig. 4g) and MHCII + F4/F80 + CD11c - macrophages (Fig. 4h) in Muc16 -/- mice commensurate with the reduced fungal burden found in this organ and improved survivals. These data demonstrates a role for MUC16 in potentiating fungal renal vascular invasion, associated with increased neutrophil accumulation and mortality. Deletion of MUC16 in mice conveys protection in an oral model of Candida albicans infections Since MUC16 is well-known as an epithelial mucin 35 , the Muc16 -/- strain was also compared to littermate WT controls using two adapted oral models of C. albicans including one recently used to demonstrate a role for innate lymphoid cells in oral candidiasis 40 . Without immunosuppression, WT mice inoculated orally with a solution of C. albicans displays a transient reduction in weight (Fig. 5a). In addition, since corticosteroids are a known risk factor for severe mucocutaneous candidiasis 41 , an additional oral model of candidiasis in mice treated with cortisone acetate was utilized 42 , which demonstrated significant protection from infection as measured by percent of body weight loss and reduced tongue colonization measured by fungal CFU (Fig. 5b, c) or histology of GMS-stained tongues at 4 days (Fig. 4d). Commensurate with less fungal burden, Muc16 -/- mice demonstrated less cellular recruitment of neutrophils (Fig. 5e), monocytes (Fig. 5f) and macrophages (Fig. 5g) within the tongue tissue [summary statistics day 1 (Fig. 5h) and day 4 (Fig. 5i)] Expression of MUC16 is induced via TLR and C-type lectin receptor ligands and is CARD9-dependent. Since MUC16 plays a role in initiating and determining severity of candidal infection in mice, we hypothesized that MUC16 expression could be induced by factors associated with increased mortality of candidemia in patients, including pathogen products released during either transient seeding from central venous catheters or bacterial infections associated with antibiotic use 43 . Indeed, hematologic malignancies are well known to induce MUC16 measured by the CA125 levels in blood which are known risk factors for candidemia 44 . In the present studies intravenous exposure of mice with C. albicans increased kidney levels of MUC16 after 4 days (Fig. 6a) and incubation of fungal cells with purified mouse EC monolayers for 3 hours also induced MUC16 (Fig. 6b). Similarly, incubation of heat-killed E. coli resulted in a dose-dependent induction of Muc16 expression in primary mouse kidney ECs at 3 h (Fig. 6c). Since pathogen-associated molecular patterns (PAMPs) are prominent signaling ligands of pathogens, we assessed for induction of Muc16 by selected PAMPs within purified mouse kidney ECs for 1 hour and 3 hours and found a striking induction of this mucin after stimulation of a number of TLR and C-type lectin receptors (Fig. 6d), including Candida -associated ligands Mincle, TLR9 and Dectin1 (Fig. 6e). Induction was dependent on Card 9 (Fig. 6f), suggesting the role of this pathway in PAMP-dependent signaling of MUC16. In addition, adherence to primary mouse ECs was found to be reduced in Card9 -/- mice, almost to a degree found in the Muc16 -/- mice (Fig. 6g). In conclusion, MUC16 was found to be an inducible endothelial mucin, mediated by both TLR and C-type lectin receptors and is Card9 dependent. This could suggest mechanisms whereby inflammatory signals prime MUC16 to provide increased receptors to bind transient fungal cells released during illness, perhaps facilitating endothelial adhesion prior to endothelial endocytosis and tissue invasion. MUC16 -rs11670318 single nucleotide polymorphism and serum concentrations of MUC16/CA125 are associated with mortality in humans. To identify possible human MUC16 SNPs that could contribute to severity of human disease, we analyzed human DNA samples from a prospective genetic discovery study of non-neutropenic patients with bloodstream infections of Candida . Over a 3-year period in 2005, 37 consecutive eligible patients were recruited at the University of Illinois at Chicago after being identified by the microbiological laboratory and informed consent obtained (see entry criteria in Table S6). Demographic data demonstrated similarity between patients that died versus survivors, although there was a difference in APACHE II score (Table S7). Characteristics of the pathogen obtained in cultures are described in Table S7. The predominant organism was Candida albicans , although there were also isolates of C. glabrata and other strains. Using an additive genetic model adjusted for age and gender, two SNPs (rs2547065, rs7508222) among 80 tested SNPs (Table S8) were associated with mortality with a nominal p < 0.05. These two SNPs remained significant after further adjustment for the APACHE II score. Validation of three of the top 4 SNPs from the discovery cohort were analyzed in a second previously-described cohort of candidemia patients 4 , selecting for patients matching that of the discovery cohort. The top candidate SNP associated with protection from mortality was identified as rs11670318-A with an adjusted p value of 0.07. In humans, the MUC16-rs11670318 SNP results in an adenine-to-guanidine substitution at position 19:9056982 that changes a threonine at position 10155 to an isoleucine, found to be a site of O-GalNAc modification by a whole genome study of the glycoproteome 45 . To determine whether the rs11670318 polymorphism might have the capacity to affect MUC16 Candida adherence, a human EC line, HMEC-1 was subjected to CRISPR-cas9-directed substitution of the rs11670318 SNP utilizing the guide RNAs described in Methods. Three clones were selected by limiting dilution and isolated, verified to have similar observed growth and morphology at confluence and assessed by PCR followed by sequencing to establish the correct substitution. As shown in Fig. 6A, adherence assays demonstrated modest but significant reductions in fungal binding, that could contribute to less burden of renal disease after transient intravenous fungal exposure. While serum was not collected in the discovery cohort I, it was available in Cohort II which allowed examination of serum concentrations of MUC16 protein in 134 patients by a standard CA125 assay of MUC16 (Quantikine ELISA Human CA125/MUC, R&D Systems, Minneapolis, MN). CA125 is a widely-used assay of the MUC16 soluble SEA domain that facilitates staging and follow-up of malignancies, principally ovarian cancer, whose prognosis and outcomes are strongly associated with levels of MUC16 46 . As shown in Fig. 6i concentrations of MUC16 in serum were highly associated with 30-day survival in non-neutropenic patients with candidemia (p = 0.01) with an ROC of 0.7 (Fig. 6j), suggesting that higher levels of MUC16 expression, perhaps mediated by risk conditions such as transient bacterial or fungal products, may have resulted in poor clinical outcome similar to the mouse models. Further study in additional cohorts may be required to delineate more fully the role of CA125 monitoring in patients with candidemia, but, to our knowledge, these data provide the first readily available commercial assay to stratify outcome in human candidemia. Discussion ECs are among the first host cells encountered by Candida during hematogenous dissemination, and adhesion is the critical initiating step for invasion and tissue damage. Previous studies demonstrated that endothelial endocytosis of Candida is calcium dependent and requires N-cadherin engagement, with Als3 functioning as a key fungal invasion 10 34 . However, the molecular mechanisms governing the initial attachment of Candida to endothelial surfaces have remained undefined. Our data identify MUC16 as a central mediator of this process, demonstrated through adherence studies, proximity ligation assays, siRNA knockdown, genetic deletion, and functional inhibition. Our findings support a model (Fig. 6k) in which infection-associated inflammatory cues induce endothelial MUC16 expression, thereby creating a permissive adhesive platform for fungal Als3-mediated binding, endocytosis, and tissue invasion. This work establishes MUC16 as a “pathogen-inducible endothelial adhesin” that links host risk factors, innate immune signaling, and fungal virulence mechanisms to disease severity. MUC16 is a large molecule which contains a distal glycoprotein as well as 16 proximal SEA (sea urchin sperm protein, enterokinase and agrin)-domains internal to the glycoprotein which are self-proteolytically cleaved at specific intervals to yield soluble and residual cell surface pauci-glycosylated fragments containing one or more residual SEA domains 47 . The SEA domain is highly conserved evolutionarily and is known to assist or regulate binding a number of carbohydrate moieties 48 , E and L-Selectins 49 , as well as mammalian proteins including mesothelian, facilitating cell-cell adhesion and cancer metastasis 50,51 . In the present study, recombinant SEA domains bound preferentially to Candida hyphae, the morphological form responsible for endothelial adherence and invasion. These findings support a model in which exposed SEA domains provide a compatible binding interface for fungal adhesins. Als3 is the best-characterized member of the ALS family of Candida adhesins and plays a central role in endothelial adherence and invasion 34 . Structural and functional studies of Als3 have demonstrated its capacity to engage multiple host ligands through distinct interfaces, enabling multivalent adhesion 32 . Interestingly, we observed SEA domain binding to the Als3 gatekeeper (GK) variant S179Y 31 , suggesting that binding of this ligan may be independent of the Als3 peptide binding cavity. Clinically, Als3 is highly relevant, as vaccination with the Als3-derived NDV-3A reduces recurrent vulvovaginal candidiasis and induces protective antibody responses 52 . Our identification of MUC16 as an endothelial binding partner of Als3 extends these findings and provides a mechanistic basis for targeting host–fungal adhesion as a therapeutic strategy. Mechanistically, our data support a ternary adhesion complex involving fungal Als3, endothelial MUC16 SEA domains, and N-cadherin. N-cadherin mediates calcium-dependent homophilic adhesion and undergoes lateral clustering and polymerization at the EC surface, processes that are dynamically regulated during junction remodeling and endocytic uptake 53-55 . We propose that MUC16 facilitates stable initial attachment of Candida , positioning Als3 for productive engagement of N-cadherin and subsequent endocytosis. This multi-receptor model provides a ‘missing link,’ facilitating Als3–N-cadherin interactions required for invasion by supporting early adhesion events and recruitment of cadherins involved in endocytosis and tissue invasion 32 . The kidney is a principal target organ in disseminated candidiasis, and endothelial interactions are critical during early infection. Using purified murine kidney ECs and intact mouse models, we demonstrate that MUC16 is expressed on renal vascular endothelium, co-localizes with CD31, and facilitates rapid fungal adherence and endocytosis. Genetic deletion of Muc16 resulted in significantly reduced renal fungal burdens, diminished neutrophil accumulation, and improved survival following intravenous Candida challenge. These findings provide mechanistic details supporting kidney-specific endothelial invasion as a driver of immunopathology and organ failure 11 . In contrast, MUC16 plays a more limited role in oral candidiasis models, consistent with a plethora of additional epithelial-specific receptors and virulence mechanisms at mucosal surfaces, including EphA2-mediated β-glucan sensing and candidalysin-induced epithelial damage 13,15 . These observations highlight the compartment-specific nature of host–fungal interactions and underscore the unique importance of endothelial adhesion mechanisms in disseminated disease. However, a paucity of Candida -binding receptors in ECs is consistent with the primary property of Candida as a commensal organism that would likely gain little evolutionarily by facilitating vascular interactions that could kill the host. The present findings thus help to understand a long-known but poorly understood property of Candida pathogenesis. A key finding of this study is that endothelial MUC16 expression is inducible by microbial and inflammatory signals associated with candidemia risk. Bacterial lysates, LPS, and multiple fungal PAMPs triggered robust MUC16 upregulation in ECs. Notably, induction by ligands for Dectin-1, Mincle, and TLR9 was CARD9 dependent. While CARD9 signaling downstream of C-type lectin receptors is well established, TLR9 has not previously been linked to CARD9-mediated pathways 56 suggesting a novel signaling axis regulating endothelial adhesion molecule expression. Blocking this pathway in CARD9-deficient ECs abrogated Candida adherence, supporting a functional role for PRR/CARD9 MUC16 induction. These findings extend emerging concepts that ECs act as active innate immune sensors rather than passive barriers during fungal infection 57,58 including recent evidence that serum bridging molecules can drive endocytosis through specific endothelial receptors 59 . These results also shed light on an important Candida -specific disease related to CARD9 . For example, in a cohort of patients with CARD9 polymorphisms no significant association was identified between CARD9 variation and the prevalence of candidemia, even though the Dectin-1/CARD9 pathway was nonredundant in mucosal immunity to Candida albicans 60 Such a result could be due to limited CARD9 -dependent expression of endothelial receptors such as MUC16. Given the clinical relevance of endothelial adhesion in dissemination, we investigated whether MUC16 variation influences outcomes in human candidemia in the most common risk group today, non-neutropenic patients with risk factors of intensive care residence or hematologic malignancies. In two independent cohorts, we identified three non-synonymous MUC16 polymorphisms associated with mortality in a discovery cohort and a fourth variant (T10155I) that showed consistent trends across cohorts. Functional validation using CRISPR–Cas9–engineered HMEC-1 cells demonstrated that the T10155I mutation significantly reduced Candida adherence, supporting a mechanistic link between genotype and endothelial function. While the observed p values did not uniformly meet conventional thresholds, the consistency across cohorts and functional corroboration argue for biological relevance, consistent with modern statistical perspectives that emphasize effect sizes and reproducibility over arbitrary cutoffs 61 . In addition to genetic risk, we identified a strong association between elevated serum MUC16/CA125 concentrations and mortality in candidemia. This finding is particularly intriguing given that known risk factors for invasive candidiasis, including hematologic malignancies, inflammation, and prior infections, are themselves associated with increased MUC16 expression. We propose that these conditions may “prime” the endothelium by increasing MUC16 availability, thereby facilitating fungal adhesion and invasion once candidemia occurs (Fig. 6k). While cohort size limits definitive conclusions, further studies could suggest a role for CA125 in biomarker-stratified clinical designs where selection of higher risk patients may facilitate the use of smaller patient sample sizes and/or improved power as recently suggested for biomarker-driven clinical studies 62 . In summary, this study identifies MUC16 as a pathogen-inducible endothelial adhesin that integrates host inflammatory signals, innate immune pathways, and fungal virulence mechanisms to drive disseminated candidiasis. By defining a novel Als3–MUC16–N-cadherin adhesion axis and linking endothelial biology to clinical outcomes, our findings open new avenues for host-directed therapies, anti-adhesion strategies, and biomarker-guided clinical trial design in a disease that continues to carry unacceptably high mortality. Materials and Methods Mice, yeast and bacterial strains Wild-type C57BL/6 mice were obtained from Taconic or Jackson Laboratory. Muc16 -/- mutant mice in a 129 background were a gracious gift from the Department of Genetics, University of Texas M. D. Anderson Cancer Center, Houston, Texas and were backcrossed to C57B6 mice 5x using a high-speed congenic strategy and first-wave male germ cells with 100% B6-homozygous loci (84 markers) 39 C57/Bl/6N card9 -/- mice were the generous gift of M. Lionakis. Candida albicans strain SC5314 was obtained from American Type Culture Collection (Manassas, VA), C. albicans -dTomato was a kind gift of R. Wheeler, 30 , and E. coli strain 25922 was a generous gift from the NIH clinical center microbiology laboratory (Bethesda, MD). Isolation of mouse primary kidney ECs Mouse tissue ECs were isolated as described 20 with some modification. Briefly, mouse kidneys were aseptically removed washed, dissected, finely minced and washed, digested with Liberase at 37°C with agitation for 25-35 min. The suspensions were filtered twice centrifuged at 300 g for 10 minutes and the pellets washed three times in phosphate buffer saline (PBS) containing 0.5% bovine serum albumin (BSA) and 2 mM ethylenediamine-tetraacetic acid (EDTA). EC purifications were performed using CD146 MicroBeads positive selection kit according to the manufacturer’s instructions. Isolated cells were plated onto T25 flasks precoated with 1% gelatin and cultured at 37 0 C in a 5% CO2 in DMEM/F12 containing EC growth supplements. To verify the cell preparation, cells were tested for uptake of Dil-Ac-LDL according to the method of Voyta 27 , and cells were also stained with anti-CD45-PE-Cy7A, CD146 PE-A and Muc16 APC-A (Table S4) and subjected to flow cytometry. In addition, mouse kidney ECs demonstrated induction of E-selectin and VCAM-1 26,63 as well as the endothelial marker, CD31/PECAM-1 22 . Gene silencing of renal endothelial-associated mucins in mouse ECs Gene silencing was performed using four siRNA plasmids targeting the mouse Muc16, Muc1, Muc14 and Muc13 genes and Accell Non-targeting Control Pool (D-001910-10-05, Dhar Macon) according to Materials and Methods. ECs were transfected with endotoxin-free siRNA plasmids containing the proprietary nucleotide sequences. Accell™siRNA delivery). Transiently transfected cells were assayed 72 h post-transfection.knockdown was quantified using real-time RT-PCR. Knockdown efficiencies ⩾70% were regarded as significant. Adherence Assays of Candida to primary mouse kidney ECs The method of Phan et al was utilized 10 . Briefly, 10 5 mouse purified ECs were cultured in 6-well tissue culture plate until cells reached confluence. To determine Candida adherence, endothelial monolayers were washed twice with 2 ml of PBS, and 1 ml of the singlet Candida suspension (10 2 cfu/ml of HBSS) was added to replicate monolayers. The inoculum size of each suspension was confirmed by culturing aliquots in YPD agar plates. Following incubation at 37°C, the nonadherent organisms were aspirated and the EC monolayers were rinsed with 10 ml of HBSS in a standard manner. Two milliliters of warm 2% glucose, 2% bactopeptone, 1% yeast extract agar (YPD) agar was poured over the surface and allowed to solidify. After incubation at 37°C for 24 h, colonies (representing adherent organisms) were counted, and the results were expressed as a percentage of the inoculum. Measurement of endocytoses of C. albicans by ECs. The number of organisms endocytosed by ECs was determined using the method of a previously described differential fluorescence assay with minor modifications 10 . Briefly, 10 3 ECs were grown to confluency on 8 well chamber slides. The cells were rinsed once and infected with 10 2 cells of dTomato- C. albicans in RPMI 1640 medium plus or minus 1.9 mM ethylene glycol tetraacetic acid (EGTA) for 45 min at 37 °C in 5% CO2. Next, the cells were rinsed twice with 0.5 ml of PBS in a standardized manner and then fixed with 3% paraformaldehyde. The adherent but non-endocytosed organisms were labeled with polyclonal rabbit anti- C. albicans antibodies (Table S2) that had been conjugated with green fluorescing AF488 (Molecular Probes). ECs were then permeabilized with 0.5% Triton X-100 (Merck-Sigma) in PBS, after which both the endocytosed and non-endocytosed organisms were visualized by microscopy using Imaris Software. The number of cell-associated organisms (endocytosed plus adherent organisms) was determined by counting the number of organisms that were labeled with Alexa 488. The number of endocytosed organisms was determined by subtracting the number of organisms labeled with red fluorescent dTomato from the number of cell-associated organisms. Proximity Ligation Assay. The method of Soderberg et al 64 was adapted using the commercial Duolink reagents (Millepore Sigma/Merck KGaA, Darmstadt Germany). Briefly, purified mouse ECs were grown to 70-80% confluence on 12-well culture slides, rinsed once and 5x10 4 hyphae of C. albicans SC5314 were incubated with EC monolayers in DMEM/F12 medium alone. Cells were rinsed twice with PBS and then fixed for 15 min at room temperature. For primary antibody incubation, the cells were treated with primary antibodies respectively for 2-3 hours in 3 groups: 1) rabbit anti-MUC16 antibody and mouse anti-Als3 antibody described previously 65 (a generous gift from L Hoyer). 2) Rabbit anti N-cadherin Polyclonal Antibody (company PA5-19486) and mouse anti-Als3 antibody. 3) rabbit anti-MUC16 antibody and mouse anti-N-cadherin monoclonal antibody. Subsequently, probe incubation, ligation, and amplification were performed according to the manufacturer's operating procedures. Puncta were captured by fluorescent (FITC) microscopy using Imaris Software. Comparison of human and mouse MUC16 and expression and fungal binding of recombinant huSEA domains: Recombinant SEA domains were expressed as a synthetic gene comprising 8 human SEA domains (aa 8682-8805; Fig. S2B) (GenScript, Piscataway, NJ), as a fusion protein with maltose-binding protein (MBP) by using the pIH902 expression system (New England Biolabs, Beverly MA) purified on amylose-Sepharose according to the manufacture’s directions. Alternatively, a single SEA domain was expressed with either an N-terminal or C-terminal MBP. Intact sequence was verified by Sanger sequencing prior to use. SEA domain binding to C. albicans was performed as follows: d-Tomato-expressing C. albicans described above was induced for hyphal formation followed by incubation of 10 3 CFU with 100 mg/ml of rSEA-MBP or MBP alone for 2 h at 37C followed by extensive washing and incubation with anti-MBP antibody (Table S2) and visualized by epifluorescence or by flow cytometry. Isolated Kidney Perfusion and Tissue Clearing Mouse kidney perfusions were performed as described 66 with modifications . Briefly, wild-type mice were anesthetized. Mouse kidneys were perfused through the abdominal/renal artery with 10 ml of ammonium-chloride-potassium lysing buffer containing 10 unit of heparin followed by 1ml of 10 6 CFU/ml of C. albicans -dTomato in PBS. After 2 h kidneys were perfused with PBS to remove unbound fungi. Excised mouse kidneys were post-fixed overnight and processed utilizing the method of Clear T2 tissue-clearing as described 67 . Briefly, kidneys were transferred to 25% formamide/10% polyethylene glycol (PEG) solution, 50% formamide/20% PEG solution, then clear in new 50% formamide/20% PEG solution. After sectioning (300 μm) on a vibrating microtome, the obtained kidney slices were blocked in blocking solution for 1 hour at room temperature. Goat anti-mouse CD31 and rabbit anti-mouse MUC16 were incubated overnight at 4°C. After washes AF488 conjugated anti-goat and AF647 conjugated anti-rabbit secondary antibodies were applied, incubated in 1%BSA/1%Tween/PBS overnight at 4°C. 4’6-diamindino-2-phenylindole (DAPI) was used for nuclear staining followed by washes in phosphate-buffered saline before epifluorescent imaging on an inverted SP8 microscope. Mouse model of systemic candidiasis and oropharyngeal candidiasis (OPC) The model of Huang et al was utilized 40 . Fungal burden was determined by excision of tissue and determination of colony forming units. Histopathological and immunohistochemistry (IHC) for N-cadherin and Muc16 expression Studies The method of Lionakis et al was adapted 11 . Briefly, control uninfected mice and infected mice were euthanized on days 1, 4, and 7 after infection and the kidney was removed, fixed with 10% formalin. The samples underwent sectioning with H&E or PAS staining. For IHC of N-cadherin and MUC16 expression, tissue sections were placed on poly- L -lysine-coated glass slides, deparaffinized in xylene and rehydrated. Following antigen retrieval in citrate buffer endogenous peroxidase was blocked and the slides were then incubated with 5% bovine serum albumin followed by washing with buffer containing sheep anti-N-Cadherin antibody or Rabbit anti-CA-125(MUC16) antibody. BSA (1%) without primary antibody was used as negative control. The slides were then washed twice and detection of immunoreaction was achieved using the streptavidin-HRP (horse radish peroxidase) containing the streptavidin-peroxidase complex after a 40-min incubation at RT. Color was developed with DAB 3 DAB (3,3'-diaminobenzidine tetrahydrochloride) substrate kit (Thermofisher, 34002) and slides were subsequently counterstained with hematoxylin, dehydrated, and mounted. Two different sections of each model were tested per experiment. Flow cytometry Control uninfected mice and infected mice were euthanized on days 1, 4, and 7 after infection. Then, the animals were perfused with normal saline and brain, spleen, liver and kidneys were harvested. Single-cell suspensions were obtained using methods previously described by Lionakis 11 . Further details including antibodies utilized can be found in Supplemental Materials. FACS was performed on an BD Fortessa (BD Biosciences) and data were analyzed using FlowJo software. Induction of Muc16 in intact mice and in purified mouse kidney ECs. For Candida induction of Muc16 in intact mice, C57/Bl/6 mice were inoculated intravenously with 2.5 x 10 5 C. albicans , kidneys excised as described above and RNA assayed by qRT-PCR using primers described above. For Candida induction of Muc16 in ECs, 2 x10 5 CFU of C. albicans was incubated with 10 6 ECs for 8 h in cell culture media and RNA assayed for Muc16 expression by qRT-PCR. For E. coli induction of Muc16 in ECs, E. coli strain 25922 (10 6 CFU) were heat treated (65 C for 1h) and indicated equivalent amount of organism lysate dilution (0.01-100 organisms equivalent) incubated with 10 6 of ECs and RNA assessed for Muc16 expression as above. For PAMP ligand induction, Pam3CSK4, Poly (I:C), lipopolysaccharide (LPS), fibroblast-stimulating lipopeptide (FSL), CpG oligodeoxynucleotide trehalose dibehenate FurFurman, Laminarin, Poly IC-LMW bacterial flagellin or single stranded RNA40 (see Table S5) were incubated with EC from either C57/Bl/6N or littermate C57/Bl/6N card9 -/- mice according to the manufacturer’s instructions for either 1 h or 3 h and RNA isolated and assayed for MUC16 expression by qRT-PCR. CRISPR Cas9 mediated T10155I KI of MUC16 in HMEC1 cells. To generate cells, ribonucleoproteins containing Cas9 protein and synthetically chemically modified sgRNA produced by the manufacturer (Synthego, Redwood City, CA) was electroporated into HMEC1 cells. Guide RNA cut location was chr19:8,946,320. Editing efficiency was assessed upon recovery and genomic DNA was extracted from a portion of the cells, PCR amplified and subjected to Sanger sequencing. The pool was subjected to repetitive dilutional cloning and clones screened for the appropriate KI primers and Sanger sequencing and equivalent growth rates and confluent monolayer productions to that of the wild-type cells. Single cell clonogenic assays were performed as described 68 . Ethics Statement The human prospective observational cohort studies were approved by the respective institutional review board at each institution (Cohort 1, The University of Illinois at Chicago and Cohort 2, Duke University Health System). All subjects or their legally authorized representatives provided written informed consent, or had a waiver as approved by the IRB. Study Design and Participants The UIC candidemia discovery study (Cohort I) was a prospective, observational study involving non-neutropenic patients admitted to UIC over a three year period and developed Candida bloodstream infections reported by the UIC clinical laboratory. Inclusion criteria are described in Table S6. (Demographic data in Table S7). Further details are described in Supplemental Materials. All infected patients were followed for up to 12 weeks to assess for clinical outcome. Case-association study of mortality in Cohort I candidemia patients. DNA collected at entry was genotyped. Eighty non-synonymous alleles of the MUC16 gene (Table S8) were identified from the GnomAD database 69 , and assessed using DNA from 37 patients using Taqman (ThermoFisher) and Sequenom (San Diego, CA) platforms according to the manufacturer’s instructions. Within-case association study of mortality in Cohort II candidemia patients The genotyping, quality control, and imputation of the genetic data was previously described 4 Briefly, DNA was genotyped using Illumina HumanCoreExome-12 v1.0 and HumanCoreExome-24v1.0 BeadChip Snp chips. Genotypes were imputed using the human reference consortium reference panel 70 using the Michigan imputation server 71 . Quality control filters resulted in a dataset of 156 candidemia patients. Of these 156 patients, 148 had available mortality data. We tested for association with mortality at 14, 30 and 90 days within the patient cohort using 31 non-survivors and 117 survivors, 49 non-survivors and 99 survivors, and 65 non-survivors and 83 survivors respectively. The association between the genetic variants and mortality were tested by Fisher’s exact test using PLINK v1.9 (www.cog-genomics.org/plink/1.9/) 72 . The corresponding receiver operating characteristic (ROC) and precision–recall (PR) curves were generated using the cumulative prtC abundance as the predictor and actual disease status (based on the clinical metadata) as the ground truth. Declarations Acknowledgement: This research was supported [in part]* by the Intramural Research Program of the National Institutes of Health (NIH). The contributions of the NIH author(s) are considered Works of the United States Government. The findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. References McCarty, T.P., White, C.M. & Pappas, P.G. Candidemia and Invasive Candidiasis. Infect Dis Clin North Am 35 , 389-413 (2021). Seagle, E.E. , et al. The landscape of candidemia during the COVID-19 pandemic. Clin Infect Dis (2021). Keighley, C. , et al. Candidaemia and a risk predictive model for overall mortality: a prospective multicentre study. BMC Infect Dis 19 , 445 (2019). Jaeger, M. , et al. A Genome-Wide Functional Genomics Approach Identifies Susceptibility Pathways to Fungal Bloodstream Infection in Humans. J Infect Dis 220 , 862-872 (2019). Lionakis, M.S. , et al. CX3CR1-dependent renal macrophage survival promotes Candida control and host survival. J Clin Invest 123 , 5035-5051 (2013). Swamydas, M. , et al. CXCR1-mediated neutrophil degranulation and fungal killing promote Candida clearance and host survival. Sci Transl Med 8 , 322ra310 (2016). Allert, S. , et al. Candida albicans-Induced Epithelial Damage Mediates Translocation through Intestinal Barriers. MBio 9 (2018). Sheppard, D.C. & Filler, S.G. Host cell invasion by medically important fungi. Cold Spring Harb Perspect Med 5 , a019687 (2014). Chen, J. , et al. Crk1, a novel Cdc2-related protein kinase, is required for hyphal development and virulence in Candida albicans. Mol Cell Biol 20 , 8696-8708 (2000). Phan, Q.T., Fratti, R.A., Prasadarao, N.V., Edwards, J.E., Jr. & Filler, S.G. N-cadherin mediates endocytosis of Candida albicans by endothelial cells. J Biol Chem 280 , 10455-10461 (2005). Lionakis, M.S., Lim, J.K., Lee, C.C. & Murphy, P.M. Organ-specific innate immune responses in a mouse model of invasive candidiasis. J Innate Immun 3 , 180-199 (2011). Monika, S. Virulence factors in Candida species. Curr Protein Pept Sci (2019). Moyes, D.L. , et al. Candidalysin is a fungal peptide toxin critical for mucosal infection. Nature 532 , 64-68 (2016). Kasper, L. , et al. The fungal peptide toxin Candidalysin activates the NLRP3 inflammasome and causes cytolysis in mononuclear phagocytes. Nat Commun 9 , 4260 (2018). Swidergall, M. , et al. Candidalysin Is Required for Neutrophil Recruitment and Virulence During Systemic Candida albicans Infection. J Infect Dis 220 , 1477-1488 (2019). Desai, J.V. & Lionakis, M.S. The role of neutrophils in host defense against invasive fungal infections. Curr Clin Microbiol Rep 5 , 181-189 (2018). Blalock, T.D. , et al. Functions of MUC16 in corneal epithelial cells. Invest Ophthalmol Vis Sci 48 , 4509-4518 (2007). Yeaman, M.R. , et al. Applying Convergent Immunity to Innovative Vaccines Targeting Staphylococcus aureus. Front Immunol 5 , 463 (2014). Giannakouros, P., Comamala, M., Matte, I., Rancourt, C. & Piche, A. MUC16 mucin (CA125) regulates the formation of multicellular aggregates by altering beta-catenin signaling. Am J Cancer Res 5 , 219-230 (2015). Demeule, M., Labelle, M., Regina, A., Berthelet, F. & Beliveau, R. Isolation of endothelial cells from brain, lung, and kidney: expression of the multidrug resistance P-glycoprotein isoforms. Biochem Biophys Res Commun 281 , 827-834 (2001). Radek, M., Babunkova, E., Spacek, M., Kvasnicka, T. & Kvasnicka, J. Determination of Circulating Endothelial Cells and Endothelial Progenitor Cells Using Multicolor Flow Cytometry in Patients with Thrombophilia. Acta Haematol 142 , 113-119 (2019). Woodfin, A., Voisin, M.B. & Nourshargh, S. PECAM-1: a multi-functional molecule in inflammation and vascular biology. Arterioscler Thromb Vasc Biol 27 , 2514-2523 (2007). Gholizadeh, S. , et al. E-selectin targeted immunoliposomes for rapamycin delivery to activated endothelial cells. Int J Pharm 548 , 759-770 (2018). Liu, C. , et al. Human endomucin is an endothelial marker. Biochem Biophys Res Commun 288 , 129-136 (2001). Eder, P. , et al. The influence of anti-TNF therapy on CD31 and VEGF expression in colonic mucosa of Crohn's disease patients in relation to mucosal healing. Folia Histochem Cytobiol 54 , 75-80 (2016). Phan, Q.T., Belanger, P.H. & Filler, S.G. Role of hyphal formation in interactions of Candida albicans with endothelial cells. Infect Immun 68 , 3485-3490 (2000). Voyta, J.C., Via, D.P., Butterfield, C.E. & Zetter, B.R. Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J Cell Biol 99 , 2034-2040 (1984). Su, A.I. , et al. Large-scale analysis of the human and mouse transcriptomes. Proc Natl Acad Sci U S A 99 , 4465-4470 (2002). Wu, C., Jin, X., Tsueng, G., Afrasiabi, C. & Su, A.I. BioGPS: building your own mash-up of gene annotations and expression profiles. Nucleic Acids Res 44 , D313-316 (2016). Gratacap, R.L., Rawls, J.F. & Wheeler, R.T. Mucosal candidiasis elicits NF-kappaB activation, proinflammatory gene expression and localized neutrophilia in zebrafish. Dis Model Mech 6 , 1260-1270 (2013). Lin, J. , et al. The peptide-binding cavity is essential for Als3-mediated adhesion of Candida albicans to human cells. J Biol Chem 289 , 18401-18412 (2014). Phan, Q.T. , et al. Als3 is a Candida albicans invasin that binds to cadherins and induces endocytosis by host cells. PLoS Biol 5 , e64 (2007). Liu, Y. & Filler, S.G. Candida albicans Als3, a multifunctional adhesin and invasin. Eukaryot Cell 10 , 168-173 (2011). Zhao, X. , et al. ALS3 and ALS8 represent a single locus that encodes a Candida albicans adhesin; functional comparisons between Als3p and Als1p. Microbiology (Reading) 150 , 2415-2428 (2004). van Putten, J.P.M. & Strijbis, K. Transmembrane Mucins: Signaling Receptors at the Intersection of Inflammation and Cancer. J Innate Immun 9 , 281-299 (2017). Bork, P. & Patthy, L. The SEA module: a new extracellular domain associated with O-glycosylation. Protein Sci 4 , 1421-1425 (1995). Maeda, T. , et al. Solution structure of the SEA domain from the murine homologue of ovarian cancer antigen CA125 (MUC16). J Biol Chem 279 , 13174-13182 (2004). Levitin, F. , et al. The MUC1 SEA module is a self-cleaving domain. J Biol Chem 280 , 33374-33386 (2005). Ogonuki, N. , et al. A high-speed congenic strategy using first-wave male germ cells. PLoS One 4 , e4943 (2009). Huang, Y. , et al. IL-25-responsive, lineage-negative KLRG1(hi) cells are multipotential 'inflammatory' type 2 innate lymphoid cells. Nat Immunol 16 , 161-169 (2015). Underwood, J.A., Williams, J.W. & Keate, R.F. Clinical findings and risk factors for Candida esophagitis in outpatients. Dis Esophagus 16 , 66-69 (2003). Solis, N.V. & Filler, S.G. Mouse model of oropharyngeal candidiasis. Nat Protoc 7 , 637-642 (2012). Hajjeh, R.A. , et al. Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program. J Clin Microbiol 42 , 1519-1527 (2004). Fehm, T., Beck, E., Valerius, T., Gramatzki, M. & Jager, W. CA 125 elevations in patients with malignant lymphomas. Tumour Biol 19 , 283-289 (1998). Steentoft, C. , et al. Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology. EMBO J 32 , 1478-1488 (2013). Zhang, M., Cheng, S., Jin, Y., Zhao, Y. & Wang, Y. Roles of CA125 in diagnosis, prediction, and oncogenesis of ovarian cancer. Biochim Biophys Acta Rev Cancer 1875 , 188503 (2021). Das, S. & Batra, S.K. Understanding the Unique Attributes of MUC16 (CA125): Potential Implications in Targeted Therapy. Cancer Res 75 , 4669-4674 (2015). Mbanefo, E.C. , et al. Characterization of a gene family encoding SEA (sea-urchin sperm protein, enterokinase and agrin)-domain proteins with lectin-like and heme-binding properties from Schistosoma japonicum. PLoS Negl Trop Dis 8 , e2644 (2014). Chen, S.H., Dallas, M.R., Balzer, E.M. & Konstantopoulos, K. Mucin 16 is a functional selectin ligand on pancreatic cancer cells. FASEB J 26 , 1349-1359 (2012). Muniyan, S. , et al. MUC16 contributes to the metastasis of pancreatic ductal adenocarcinoma through focal adhesion mediated signaling mechanism. Genes Cancer 7 , 110-124 (2016). Rump, A. , et al. Binding of ovarian cancer antigen CA125/MUC16 to mesothelin mediates cell adhesion. J Biol Chem 279 , 9190-9198 (2004). Edwards, J.E., Jr. , et al. A Fungal Immunotherapeutic Vaccine (NDV-3A) for Treatment of Recurrent Vulvovaginal Candidiasis-A Phase 2 Randomized, Double-Blind, Placebo-Controlled Trial. Clin Infect Dis 66 , 1928-1936 (2018). Takeichi, M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251 , 1451-1455 (1991). Strale, P.O. , et al. The formation of ordered nanoclusters controls cadherin anchoring to actin and cell-cell contact fluidity. J Cell Biol 210 , 333-346 (2015). Dejana, E., Orsenigo, F. & Lampugnani, M.G. The role of adherens junctions and VE-cadherin in the control of vascular permeability. J Cell Sci 121 , 2115-2122 (2008). Zhong, X., Chen, B., Yang, L. & Yang, Z. Molecular and physiological roles of the adaptor protein CARD9 in immunity. Cell Death Dis 9 , 52 (2018). Swidergall, M. , et al. EphA2 Is a Neutrophil Receptor for Candida albicans that Stimulates Antifungal Activity during Oropharyngeal Infection. Cell Rep 28 , 423-433 e425 (2019). Orozco, A.S., Zhou, X. & Filler, S.G. Mechanisms of the proinflammatory response of endothelial cells to Candida albicans infection. Infect Immun 68 , 1134-1141 (2000). Phan, Q.T. , et al. Serum bridging molecules drive candidal invasion of human but not mouse endothelial cells. PLoS Pathog 18 , e1010681 (2022). Rosentul, D.C. , et al. Genetic variation in the dectin-1/CARD9 recognition pathway and susceptibility to candidemia. J Infect Dis 204 , 1138-1145 (2011). Amrhein, V., Greenland, S. & McShane, B. Scientists rise up against statistical significance. Nature 567 , 305-307 (2019). Polley, M.C., Korn, E.L. & Freidlin, B. Phase III Precision Medicine Clinical Trial Designs That Integrate Treatment and Biomarker Evaluation. JCO Precis Oncol 3 (2019). Filler, S.G., Pfunder, A.S., Spellberg, B.J., Spellberg, J.P. & Edwards, J.E., Jr. Candida albicans stimulates cytokine production and leukocyte adhesion molecule expression by endothelial cells. Infect Immun 64 , 2609-2617 (1996). Soderberg, O. , et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3 , 995-1000 (2006). Coleman, D.A. , et al. Monoclonal antibodies specific for Candida albicans Als3 that immunolabel fungal cells in vitro and in vivo and block adhesion to host surfaces. J Microbiol Methods 78 , 71-78 (2009). Czogalla, J., Schweda, F. & Loffing, J. The Mouse Isolated Perfused Kidney Technique. J Vis Exp (2016). Kuwajima, T. , et al. ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue. Development 140 , 1364-1368 (2013). Alvarez, D.F. , et al. Lung microvascular endothelium is enriched with progenitor cells that exhibit vasculogenic capacity. Am J Physiol Lung Cell Mol Physiol 294 , L419-430 (2008). Karczewski, K.J. , et al. The mutational constraint spectrum quantified from variation in 141,456 humans. Nature 581 , 434-443 (2020). McCarthy, S. , et al. A reference panel of 64,976 haplotypes for genotype imputation. Nat Genet 48 , 1279-1283 (2016). Das, S. , et al. Next-generation genotype imputation service and methods. Nat Genet 48 , 1284-1287 (2016). Purcell, S. , et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 81 , 559-575 (2007). Additional Declarations There is NO Competing Interest. 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Nijmegen","correspondingAuthor":false,"prefix":"","firstName":"Vinod","middleName":"","lastName":"Kumar","suffix":""},{"id":600158767,"identity":"ceeca102-264a-42c7-9913-6df0d9fc9949","order_by":11,"name":"Frank van de Veerdonk","email":"","orcid":"https://orcid.org/0000-0002-1121-4894","institution":"Radboud University Nijmegen Medical Centre","correspondingAuthor":false,"prefix":"","firstName":"Frank","middleName":"van","lastName":"de Veerdonk","suffix":""},{"id":600158768,"identity":"cb0eeaf1-39cb-4617-8264-610fca4bb6f8","order_by":12,"name":"Mihai Netea","email":"","orcid":"https://orcid.org/0000-0003-2421-6052","institution":"Radboud University Nijmegen Medical Centre","correspondingAuthor":false,"prefix":"","firstName":"Mihai","middleName":"","lastName":"Netea","suffix":""},{"id":600158769,"identity":"3be83ccf-3542-4697-b6ca-25b3f6eb8fbe","order_by":13,"name":"Michail Lionakis","email":"","orcid":"https://orcid.org/0000-0003-4994-9500","institution":"National Institute of Allergy and Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Michail","middleName":"","lastName":"Lionakis","suffix":""},{"id":600158770,"identity":"0328f319-2580-46d9-9b49-fbed22c19b9c","order_by":14,"name":"Adebo Adebowale","email":"","orcid":"","institution":"National Institutes of Health","correspondingAuthor":false,"prefix":"","firstName":"Adebo","middleName":"","lastName":"Adebowale","suffix":""},{"id":600158771,"identity":"686a3469-528c-4952-a044-c6ea512a8af2","order_by":15,"name":"Scott Filler","email":"","orcid":"","institution":"Lundquist Institute for Biomedical Innovation at Harbor-UCLA","correspondingAuthor":false,"prefix":"","firstName":"Scott","middleName":"","lastName":"Filler","suffix":""},{"id":600158772,"identity":"963040cb-f475-4d0f-abc9-0deab61ed392","order_by":16,"name":"Nina Clark","email":"","orcid":"","institution":"University of Illinois at Chicago","correspondingAuthor":false,"prefix":"","firstName":"Nina","middleName":"","lastName":"Clark","suffix":""},{"id":600158773,"identity":"18c89a02-ba5e-4c22-a1ea-de5b0aa5f540","order_by":17,"name":"Ernesto Cota","email":"","orcid":"","institution":"Imperial College, London","correspondingAuthor":false,"prefix":"","firstName":"Ernesto","middleName":"","lastName":"Cota","suffix":""}],"badges":[],"createdAt":"2026-02-06 22:05:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8810991/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8810991/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103886358,"identity":"bc62ce68-7b6b-403e-9da2-5a720ba63d5d","added_by":"auto","created_at":"2026-03-04 07:03:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6637408,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMUC16 is required for adhesion of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCandida\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e hyphae to mouse primary renal ECs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea) Primary mouse kidney endothelial were grown to confluence (10\u003csup\u003e4\u003c/sup\u003e cells/well) and subjected to the indicated siRNA and incubated with 10\u003csup\u003e2\u003c/sup\u003e \u003cem\u003eC. albicans\u003c/em\u003e induced for hyphae formation for 60 min, washed extensively and adherence determined by colony forming units (CFU) as described in Methods. b) Adherence assays conducted as in a) for the indicated times. c) Adherence performed as in a) in the presence (+) or absence (-) of EGTA. d) Merged fluorescent micrographs of \u003cem\u003eCandida albicans\u003c/em\u003e cells expressing d-Tomato (red arrows) and incubated on confluent endothelial monolayers from indicated mouse strains for 45 min in media alone (-) EGTA or in the presence of 1.9 mM EGTA (+) according to Methods. \u0026nbsp;Endothelial nuclei were labeled with 4’6- diamidino-2-phenylindole (DAPI) and non-endocytosed fungal cells were identified with anti-\u003cem\u003eCandida\u003c/em\u003e AF488-labeled antibody (green arrows) and observed by fluorescence microscopy.\u0026nbsp; Hyphae unlabeled by antibody represents endocytosed \u003cem\u003eC. albicans\u003c/em\u003e hyphae (red arrows). \u0026nbsp;e) Results from d), expressed as a percentage of the number of organisms per high-powered field that were endocytosed by or cell-associated with primary ECs from indicated mouse strains incubated with or without EGTA. Data pooled from 20 random low-powered fields and results expressed as average of 3 independent experiments. Student’s t-test (a, b, c) and two-way Anova (e), N = 3 independent experiments, * p \u0026lt; 0.05, ** p \u0026lt; 0.001, NS non-significant. f) Proximity ligation assay: Primary ECs monolayers from WT (top panels) or \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice (bottom panels) were incubated with 5 x 10\u003csup\u003e4\u003c/sup\u003e \u003cem\u003eC. albicans\u003c/em\u003e hyphae for 2 hours, rinsed and fixed as per Methods. Indicated primary antibody was incubated for 2-3 hrs with the indicated antibodies with subsequent incubation, ligating, amplification and microscopy (red arrows point to positive signal consisting of small puncta) according to Methods. g) summary statistics, (anti-Als3p, A), (anti-MUC16, M) or (anti-N-cadherin, C): Error bars indicate s.d.; Student’s t-test, N= 6 (a, b, c) or two-way Anova (e, g), N = 6 (a, left 2 panels of b, c) 8 (right panel of b) or 3 (e) independent experiments, 6-15 independent experiments for each condition counting between 114 to 220 cells each (g). ** p \u0026lt;0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001. Bar = 50 (d), 20 (f) mm\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8810991/v1/776ef66178d4d4fea2f6df2b.png"},{"id":103886356,"identity":"a49684b8-686a-441c-b30f-5ee96478a1fa","added_by":"auto","created_at":"2026-03-04 07:03:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2321568,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCandida albicans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e binding to mouse kidney ECs is dependent on fungal Als3 and endothelial MUC16-SEA domains.\u003c/strong\u003e a) Adherence to indicated primary mouse kidney ECs of \u003cem\u003eS. cerevisiae\u003c/em\u003e expressing either \u003cem\u003eALS3\u003c/em\u003e or empty vector control (EV).\u0026nbsp; b) Schematic of MUC16 showing glycosylated internal repeat domain, removable by neuraminidase and b1,4 galactosidase, cytoplasmic tail domain, signal peptide and autoproteolytically cleaved SEA domains. \u0026nbsp;Adapted from van Putten and Strijbis \u003csup\u003e35\u003c/sup\u003e. c) Fungal cells induced for hyphae formation (10\u003csup\u003e2\u003c/sup\u003e) were inoculated on a monolayer of 10\u003csup\u003e4\u003c/sup\u003e primary ECs either untreated (UT) or treated with 1 mg neuraminidase + b1,4 galactosidase treatment (N/G) for 1 hr. Adherence assays conducted as in Fig. 1a. \u0026nbsp;d) \u003cem\u003eC. albicans\u003c/em\u003e expressing d-Tomato induced for hyphae formation was incubated with 100 mg/ml rSEA-MBP or MBP alone, washed and labeled with FITC-labeled anti-MBP antibody (green arrows) and observed by fluorescent microscopy. Red arrow demonstrates hyphae element without FITC staining. e) Equivalent conditions as in d) but indicated proteins were assessed by flow cytometry after staining with FITC-labeled anti-MBP antibody. f. Summary data from Fig. 2e (MBP alone, M) or (rSEA8-MBP, S-M). g) Adherence assays of \u003cem\u003eC. albicans\u003c/em\u003e hyphae to primary mouse kidney WT or \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e ECs (ECs) at 60 min after inoculation conducted as in Fig. 1a in the presence of (Tx) 100 mg/ml of rSEA-MBP (+S-M), 100 mg/ml MBP alone (+M) or untreated (UT). Error bars indicate s.d.; Student’s t-test (a, c, f) and two-way Anova (g), N = 3 independent experiments, * p\u0026lt; 0.05, ** p \u0026lt;0.01.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8810991/v1/8a24009499754ee133362e23.png"},{"id":104401506,"identity":"e2f01d47-cf82-4177-9920-f3713fa5ae5c","added_by":"auto","created_at":"2026-03-11 12:12:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":13209427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMUC16 is expressed in glomeruli and in perfused kidney ECs lining vascular structures and co-localizes to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCandida\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e binding\u003c/strong\u003e a) WT mouse kidney was stained with anti N-cadherin, \u0026nbsp;anti-MUC16 (black arrows) or IgG controls and developed by immunoperoxidase staining according to Methods. Bar = 50 mm. b) Immunofluorescence of excised perfused kidneys of indicated strains were stained with AF488-labled anti-CD31 (green arrows) or AF647-labled anti-MUC16 (purple arrows) and observed by epifluorescence microscopy. Indicated mouse strains were inoculated intravenously with \u0026nbsp;2.5 x 10\u003csup\u003e5\u003c/sup\u003e CFU of \u003cem\u003eC. albicans\u003c/em\u003e expressing d-Tomato. After 2 h (c), or 4 days (d) mice were sacrificed and excised kidney tissue stained with antibodies against CD31 (with additional anti-MUC16 in right panel WT). Bar = 20 (b, c, d) except middle panel of (c) 10 mm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8810991/v1/1408741af3719d94f924c147.png"},{"id":104401257,"identity":"e48fffa9-b905-4290-b093-88c9ea3eaf63","added_by":"auto","created_at":"2026-03-11 12:12:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6243615,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMuc16\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-/- \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003emice demonstrate resistance to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCandida albicans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e intravenous infection.\u003c/strong\u003e a) F1 cross of C57/b6 x129 \u003cem\u003eMuc16\u003c/em\u003e \u003csup\u003e-/- \u003c/sup\u003emice (\u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e-F1) \u0026nbsp;vs wild-type (WT-F1) F1 control littermates were inoculated with 1 x 10\u003csup\u003e5\u003c/sup\u003e \u003cem\u003eC. albicans\u003c/em\u003e by tail vein and their progress followed until moribund b) \u003cem\u003eMuc16\u003c/em\u003e \u003csup\u003e-/- \u003c/sup\u003emice backcrossed 5x vs wild-type litter mate controls were inoculated with 2.5 x 10\u003csup\u003e5\u003c/sup\u003e \u003cem\u003eC. albicans\u003c/em\u003e by tail vein and observed until moribund. c) Fungal burdens of kidney of indicated strains and equivalent inocula as in B vs. time after infection. d) Representative gross and e) Histology of kidneys at indicated times post-infection. (black arrows indicates fungal structures on low power (lower panels) and representative higher power (upper panels); Grocott’s methenamine silver stain). Bar = 3 mm. Representative flow cytometry (left panels) and summary statistics (right panels) of f) Ly6G\u003csup\u003ehi\u003c/sup\u003e CD11b+ neutrophils, g) Ly6C\u003csup\u003ehi\u003c/sup\u003e CD11b+ inflammatory monocytes and h) CD11C-, MHC+, F4/80 macrophages after infection as in b) at the indicated times. Error bars indicate s.d.; Log rank (a, b), Student’s t-test (c, f, g, h), N = 9 mice (c, f, g, h), * p \u0026lt; 0.05, ** p \u0026lt; 0.01, **** p \u0026lt; 0.0001. Bar = 0.3 cm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8810991/v1/bfcc8d180208b2f0c880d026.png"},{"id":103886357,"identity":"d92699f1-25b1-4e45-b7a4-4284b86aff73","added_by":"auto","created_at":"2026-03-04 07:03:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2779127,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMuc16\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-/- \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003emice demonstrate resistance to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCandida albicans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in an oral colonization model.\u003c/strong\u003e a) Body weight at indicated times of \u003cem\u003eMuc16\u003c/em\u003e \u003csup\u003e-/- \u003c/sup\u003emice or littermate controls (WT) infected sublingually with 10\u003csup\u003e5\u003c/sup\u003e CFU/ml of \u003cem\u003eC. albicans\u003c/em\u003e. b) Percent body weight loss of indicated mouse strains after sublingual inoculations of 10\u003csup\u003e5\u003c/sup\u003e CFU/ml of \u003cem\u003eC. albicans\u003c/em\u003e. Mice were treated with cortisone acetate on day -3, -1, +1 and +3 relative to inoculation. Fungal burden (c) and histology (d) of the tongues of mice on day 4 post infection of mice treated as in Fig. 4b. (Grocott’s methanamine silver stain, black arrows identify hyphal elements). \u0026nbsp;Representative flow cytometry of e) Ly6G+ CD11b+ neutrophils, f) Ly6C\u003csup\u003ehi \u003c/sup\u003eCD11b+ inflammatory monocytes and h) CD11C-, MHC+, g) F4/80 macrophages. Summary statistics of indicated cells at h) day 1 and i) day 4. \u0026nbsp;Error bars indicate s.d.; Student’s t-test, (a, b) 6 mice per group, (a, b, e, f, g), * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8810991/v1/ad36c5619e5cd17f3d470a4c.png"},{"id":103886362,"identity":"e343364b-ace9-4f1d-92f2-4a53f1ccbd85","added_by":"auto","created_at":"2026-03-04 07:03:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1869639,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMuc16\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e is induced by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCandida albicans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and heat-killed \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in primary mouse kidney ECs as well as TLR and C-type lectin receptor ligands and expression and fungal binding is \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCard9\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-dependent.\u003c/strong\u003e a) Mice were inoculated intravenously with 2.5x10\u003csup\u003e5\u003c/sup\u003e \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eMuc16\u003c/em\u003e expression in excised kidneys was assayed by qRT-PCR. b) Primary mouse kidney ECs (10\u003csup\u003e6 \u003c/sup\u003ecells/well) from indicated strains were incubated with (6.6x10\u003csup\u003e5\u003c/sup\u003e CFU \u003cem\u003eC. albicans\u003c/em\u003e) for 2 h and \u003cem\u003eMuc16\u003c/em\u003e assessed by qRT-PCR.\u0026nbsp; c) 2 x 10\u003csup\u003e6\u003c/sup\u003e mouse kidney ECs incubated with indicated number of organism equivalents of heat-killed \u003cem\u003eE. coli\u003c/em\u003e strain 25922 for 3 h and \u003cem\u003eMuc16\u003c/em\u003e assessed by qRT-PCR. d) Mouse ECs either untreated (UT) or treated with indicated ligands according to Methods for indicated times and \u003cem\u003eMuc16 \u003c/em\u003eexpression assessed by qRT-PCR. Expression profile of \u003cem\u003eMuc16\u003c/em\u003e by cells of wild-type strains of \u003cem\u003eCard9\u003c/em\u003e-related ligands e) as well as indicated KO strains (f). g) Adherence of \u003cem\u003eC. albicans\u003c/em\u003e cells to indicated primary mouse kidney ECs by the method of Fig. 1a.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eh) Adherence assays of indicated cells as in Fig. 1a). Error bars indicate s.d.; Student’s t-test, (a, b) 2-way Anova (c); 9 (a, b), 4 (e, f, g) mice or 9 (c), 3 (d) or 18 (h) independent experiments per group (e) * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001. i) MUC16 plasma concentrations and receiver operating characteristic curve (j) assessed from a cohort of 134 non-neutropenic candidemia patients by mortality assessed at 30 days post diagnosis. Student’s t-test (i) ** p \u0026lt; 0.01. k) Working hypothesis scheme: C-terminal MUC16 bound to renal vascular ECs (1.) is induced by products associated with \u003cem\u003eCandida\u003c/em\u003e risk factors including hematological cancers, as well as bacterial and \u003cem\u003eCandida\u003c/em\u003e products released during past infections or seeding from central lines, the latter infectious products through PRRs signaled through CARD9 (2.). Transient \u003cem\u003eCandida\u003c/em\u003e organisms undergo hyphae formation, expressing and exposing the adhesin Als3 (3.). Increased MUC16 undergoes autocatalysis, exposing pauci-glycosylated SEA domains, facilitating fungal binding (5.) and released SEA domains are detectable as CA125 antigen (6.). \u003cem\u003eCandida\u003c/em\u003e-bound SEA domains complexes then bind to surface molecules including calcium activated N-cadherin polymers (7.), resulting in formation of an Als3-SEA-N-cadherine ternary complex, facilitating \u003cem\u003eCandida \u003c/em\u003einvasion (9) followed by recruitment of damaging neutrophils, and potential mortality.\u0026nbsp;\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8810991/v1/864af98f9d662f19d839dbb8.png"},{"id":104410756,"identity":"7506774d-ccdf-4d57-b8f8-238f37b676eb","added_by":"auto","created_at":"2026-03-11 12:53:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":43510384,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8810991/v1/79e0d294-3126-4a5c-a18a-4fe0b2f53daf.pdf"},{"id":103886355,"identity":"8231669c-988a-4db3-9cd1-115a344b58aa","added_by":"auto","created_at":"2026-03-04 07:03:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":125617,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementalMaterialsFinalish.docx","url":"https://assets-eu.researchsquare.com/files/rs-8810991/v1/b8244048c4445300fe8b4476.docx"},{"id":103886359,"identity":"65e9ee8c-d7f2-4384-82f3-279763598e84","added_by":"auto","created_at":"2026-03-04 07:03:27","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10867341,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresallfinalishcopy.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8810991/v1/2ae2d7bfb03bb34efb338a75.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"MUC16-dependent Renal Vascular Adhesion of Candida Promotes Tissue Invasion and Predicts Clinical Outcome in Candidemia","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eCandida\u0026nbsp;\u003c/em\u003espp. are the fourth most common cause of nosocomial bloodstream infection in the US, with crude mortality of 25-40% and causing an estimated 400,000 life threatening systemic infections per year \u003csup\u003e1\u003c/sup\u003e with an additive adverse impact during the COVID-19 era \u003csup\u003e2\u003c/sup\u003e. \u0026nbsp; However, despite its significance, key elements of pathogenesis remain poorly understood, especially in disseminated bloodstream infections in which mortality remains high despite therapy. Risk factors for mortality include malignancy, presence of chronic indwelling central catheters and prolonged antibiotic therapy associated with bacterial infections \u003csup\u003e3\u003c/sup\u003e. Recently, genetic risk factors have been identified that include the single nucleotide polymorphisms (SNPs) in \u003cem\u003ePLA2G4B\u003c/em\u003e with reactive oxygen species (ROS) and IL-6 production in response to \u003cem\u003eCandida\u003c/em\u003e infections \u003csup\u003e4\u003c/sup\u003e and relationships of CXCR1 and CX3CR1 with the acquisition of infections \u003csup\u003e5,6\u003c/sup\u003e. \u0026nbsp;These studies suggest that better understanding of pathogenic molecular mechanisms may identify patients at highest risk of death from candidemia that would1) allow intensification of therapy in the clinical setting or 2) enable better stratification of patients in clinical treatment trials.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTransient \u003cem\u003eCandida\u003c/em\u003e in the bloodstream may occur after colonization of and release from central venous catheters, or translocation through intestinal barriers after pathogenic epithelial cell damage \u003csup\u003e7\u003c/sup\u003e. Once in the bloodstream, dissemination throughout the host requires adherence followed by invasion through the vascular endothelium which occurs through endocytosis and is induced most efficiently by hyphae \u003csup\u003e8,9\u003c/sup\u003e.\u0026nbsp;Successful endothelial endocytosis is dependent on fungal adhesins such as Als3p with a requirement for N-cadherin for fungal endocytosis\u0026nbsp;\u003csup\u003e10\u003c/sup\u003e. Invasion through the endothelial layer into tissue, principally the kidney in mice\u0026nbsp;\u003csup\u003e11\u003c/sup\u003e, results in significant tissue damage and is mediated by a plethora of factors-- proteases, lipases and haemolysins\u0026nbsp;\u003csup\u003e12\u003c/sup\u003e as well as toxins including the hyphae-specific inflammasome-activating candidalysin\u0026nbsp;\u003csup\u003e13,14\u003c/sup\u003e, important for neutrophil recruitment\u0026nbsp;\u003csup\u003e15\u003c/sup\u003e. Tissue invasion by neutrophils is thought to cause paradoxical collateral tissue damage during fungal killing, constituting a predominant mechanism of deep tissue injury\u0026nbsp;\u003csup\u003e16\u003c/sup\u003e. However, despite the role of N-cadherin in the endocytosis of \u003cem\u003eCandida\u003c/em\u003e hyphae that follows initial endothelial binding, the identity of the mammalian-endothelial cell adhesion molecule remains unknown.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn light of surface mucin\u0026rsquo;s role modulating infections by \u003cem\u003eStaphylococcus\u003c/em\u003e \u003csup\u003e17\u003c/sup\u003e, which also expresses a bacterial adhesin with structural similarity to \u003cem\u003eC. albicans\u003c/em\u003e Als3 \u003csup\u003e18\u003c/sup\u003e, the present study sought a possible role for surface renal mucins in bloodstream infections with \u003cem\u003eCandida albicans\u0026nbsp;\u003c/em\u003eas the kidney has been identified as a key organ involved in initial pathogenesis \u003csup\u003e11\u003c/sup\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003e Of the mucins expressed in endothelial cells (ECs) our studies found that MUC16 was both expressed on the surface of ECs by immunohistochemistry and flow cytometry of mouse kidney primary CD31+ ECs and required for endothelial binding of \u003cem\u003eCandida\u003c/em\u003e hyphae. Additional mechanistic studies identified the formation of a Als3-SEA-N-cadherin ternary complex, facilitating sequential binding and endocytosis of the fungal pathogen. The presence of MUC16 in WT mice resulted in increased tissue burden and mortality compared to \u003cem\u003eMuc16\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003elittermates in a disseminated candidiasis model, and increased weight loss and tissue burden in an oral candidiasis model. Further clinical studies identified a key T10155I heterozygous polymorphism to be associated with 30-day mortality in two independent cohorts of patients with candidemia and established this polymorphism as having a role in \u003cem\u003eCandida\u003c/em\u003e-endothelial cell adherence in model studies. Interestingly, modeling of candidemia risk factors--exposure of ECs to bacterial and fungal products, resulted in increased expression of MUC16, mediated prominently by the receptors MINCLE, TLR9 and DECTIN-1 in a \u003cem\u003eCard9\u003c/em\u003e-dependent fashion. Hypothesizing that increased MUC16 expression in patients via such mechanisms could result in poor outcomes, we tested and found in a cohort of 134 patients that increased serum concentrations of MUC16, measured by a commercial CA125 antibody at the time of diagnosis, demonstrated a strong association with 30-day mortality. In summary, these data point to a role for MUC16 in \u003cem\u003eCandida\u003c/em\u003e binding to ECs and may provide a readily available biomarker for outcome in bloodstream infections.\u0026nbsp;\u003c/p\u003e\n"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMUC16 is required for adherence of Candida albicans to purified primary renal ECs\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBecause of previously described roles of surface mucins, particularly MUC16 in modulating interactions with pathogens \u003csup\u003e17\u003c/sup\u003e as well as interactions with surface cadherins \u003csup\u003e19\u003c/sup\u003e, we studied the role of mucins in \u003cem\u003eCandida \u003c/em\u003eadherence using primary mouse kidney ECs \u003csup\u003e20\u003c/sup\u003e. As shown in Fig. S1a-c, purified primary mouse kidney ECs did not express CD45 and displayed typical properties of ECs \u003csup\u003e21-26\u003c/sup\u003e \u003csup\u003e27\u003c/sup\u003e. Primary purified kidney ECs were then assessed for expression of renal-specific mucins identified in whole-genome expression studies \u003csup\u003e28\u003c/sup\u003e in the BioGBS database \u003csup\u003e29\u003c/sup\u003e which identified expression of \u003cem\u003eMuc1\u003c/em\u003e, \u003cem\u003eMuc14\u003c/em\u003e and \u003cem\u003eMuc16 \u003c/em\u003ethat was suppressible by siRNA (Fig. S1e). Surface Muc16 protein was identified by flow cytometry which was not present in ECs purified from \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice (Fig. S1d). Muc13 displayed little qRT-PCR signal (\u0026gt; 45 cycles). Confluent EC monolayers of each suppressed group were then incubated with hyphal elements of \u003cem\u003eCandida albicans\u003c/em\u003e expressing fluorescent d-tomato followed by extensive washing \u003csup\u003e30\u003c/sup\u003e \u003csup\u003e10\u003c/sup\u003e, which demonstrated reduced fungal adherence after \u003cem\u003eMuc16\u003c/em\u003e suppression but not \u003cem\u003eMuc1\u003c/em\u003e, \u003cem\u003eMuc13\u003c/em\u003e or \u003cem\u003eMuc14 \u003c/em\u003evs non-targeted controls (Fig. 1a; Fig. S1f). In addition, primary EC monolayers from \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e KO mice demonstrated reductions in adherence at 30, 60 or 90 minutes compared to WT litter-mate controls (Fig. 1b). As expected, incubation of WT ECs with the calcium chelator ethylene glycol-bis(\u0026beta;-aminoethyl ether)-N,N,N\u0026prime;,N\u0026prime;-tetraacetic acid (EGTA) which inhibits subsequent N-cadherin endocytosis did not inhibit adherence in WT mouse kidney ECs (Fig. 1c). \u003c/p\u003e\n\u003cp\u003eAssays combining adherence and endocytosis were then conducted using a previously described dual assay utilizing a fluorescent-d-Tomato \u003cem\u003eC. albicans\u003c/em\u003e strain followed by addition of a green-fluorescent Alexa 488-labeled non-permeable antibody against intact \u003cem\u003eCandida\u003c/em\u003e, the latter of which labels surface adherent but non-endocytosed cells \u003csup\u003e10\u003c/sup\u003e. As shown in Fig. 1d, d-Tomato-labeled \u003cem\u003eC. albicans\u003c/em\u003e hyphae exhibited both adherence to WT ECs, identified by green fluorescence (left panel, green arrows), as well as endocytosis demonstrated by red fluorescent hyphae not labeled by green fluorescence (left panel, red arrows). Endocytosis was inhibited by EGTA (+EGTA). In contrast, ECs from \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e KO mice demonstrated little adherence or subsequent endocytosis of hyphae-induced fungal cells in the presence (+) or absence (-) of EGTA (Fig. 1d). Endocytosis intensity ratios calculated as described \u003csup\u003e10\u003c/sup\u003e were significantly reduced by EGTA for WT ECs, as expected, but the small amount of residual unlabeled fungal cells found in the \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e ECs were not further reduced by EGTA (Fig. 1e). \u003c/p\u003e\n\u003cp\u003eTo molecularly probe interactions between fungal Als3, MUC16 and N-cadherin, a proximity ligation assay was utilized after incubation of primary murine ECs with recombinant Als3, the latter described previously \u003csup\u003e31\u003c/sup\u003e. As shown in Fig. 1f and g, strong signal was recorded evidenced by multiple visible puncta, linking Als3 to MUC16 in WT mouse ECs compared to equivalent assays using \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e KO mouse ECs. Detectable signal was also exhibited between Als3 and N-cadherin in WT mice consistent with earlier studies \u003csup\u003e32\u003c/sup\u003e which was abolished in the \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e KO ECs. Signal was also registered between MUC16 and N-cadherin, which was absent in the \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e KO EC\u0026rsquo;s. In summary, these data demonstrate a role for mammalian \u003cem\u003eMUC16\u003c/em\u003e in \u003cem\u003eCandida\u003c/em\u003e adherence to mouse primary kidney ECs and is required for subsequent endovascular invasion by a complex linking fungal Als3 and mammalian MUC16 and N-cadherin. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eMolecular mediators of MUC16-dependent binding of \u003cem\u003eC. albicans\u003c/em\u003e to mouse primary ECs. \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional studies sought to confirm a role for the \u003cem\u003eC. albicans \u003c/em\u003ehypha-specific surface adhesin Als3 \u003csup\u003e32-34\u003c/sup\u003e in MUC16-specific adhesion to mouse kidney ECs. As shown in Fig. 2a, the normally non-adherent \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e fungal cells expressing \u003cem\u003eALS3\u003c/em\u003e (\u003cem\u003e+ALS3\u003c/em\u003e) were found to exhibit significant primary mouse kidney EC adherence compared to equivalent strains expressing empty vector (EV) alone. However, the same \u003cem\u003eS. cerevisiae\u003c/em\u003e \u003cem\u003eALS3\u003c/em\u003e-expressing strains showed little augmentation of binding to \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e KO ECs, suggesting a role of Als3 binding to MUC16-expressing ECs in mice. Some augmentation in \u003cem\u003eALS3\u003c/em\u003e-dependent binding to the \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e KO ECs, suggesting additional minor constituents involved in \u003cem\u003eALS3\u003c/em\u003e-dependent adherence; however, MUC16 appears to be the dominant molecule involved in adherence. \u003c/p\u003e\n\u003cp\u003eFurther molecular interaction studies sought to identify structural elements within the MUC16 molecule involved in adherence. As shown in Fig. 2b, MUC16 is a large surface-bound mucin with a significant number of N-terminal glycosylation and multiple conserved SEA domains (Fig. S2a), the latter so called because of a structural similarity to the sea urchin sperm protein, enterokinase and agrin \u003csup\u003e35,36\u003c/sup\u003e. Initial experiments utilized a combination of neuraminidase + b1,4 galactosidase to reduce N and O-labeled glycosylation of the surface mucin to determine if adherence might also be reduced. Surprisingly, this treatment increased adherence (Fig. 2c), which suggested an alternative domain that may have been sterically shielded by the extensively glycosylated N-terminus. Since autocatalytic processing of MUC16 is known to expose pauci-glycosylated SEA domains, we expressed a fusion protein of 8 murine SEA domains (a.a. 8682-8805; Fig. S2b), previously found to mimic the antigenic site of the anti-human CA125 antibody by NMR spectroscopy, \u003csup\u003e37\u003c/sup\u003e with the affinity tag maltose-binding protein (MBP). As shown in Fig. S2c, the rMBP-SEA8 purified as a 145kD protein with some step-ladder proteolysis typical of the properties of this self-cleaving protein \u003csup\u003e38\u003c/sup\u003e, compared to the 42-kDa MBP. As shown in Fig. 2d (green arrows), rMBP-SEA8, but not rMBP alone was found to exhibit strong binding to hyphae of \u003cem\u003eC. albicans\u003c/em\u003e but, interestingly, not the yeast forms of the fungus that are nonpathogenic during invasive disease and do not express Als3p. Flow cytometry confirmed the increased overall binding of the rMBP-SEA8 compared to rMBP alone (Fig. 2e,f). Addition of rMBP-SEA8 was also found to block endothelial adherence of \u003cem\u003eC. albicans \u003c/em\u003ehyphae to levels equivalent to that of \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e/- \u003c/sup\u003eECs whereas rMBP alone showed no significant reductions in adherence (Fig. 2g). These results were duplicated using a single SEA domain protein fusion to MBP at either the N or the C-terminus (Fig S2d-g). rMBP-SEA1 binding to Als3p was further confirmed by co-immunoprecipitation experiments which showed little binding to rMBP alone (Fig. S2h, i). Interestingly, rMBP-SEA1 also retained binding to Als3-GK which has a mutation within the complement binding pocket described previously, suggesting an alternative novel binding site \u003csup\u003e31\u003c/sup\u003e. In summary, these data suggest a role of fungal Als3 in MUC16-dependent fungal adherence to primary mouse kidney ECs as well as a role for the MUC16 SEA domains in \u003cem\u003eC. albicans\u003c/em\u003e-EC binding. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMUC16 is expressed in CD31+ renal ECs and co-localizes with renal vasculature and invading Candida hyphal elements. \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhile MUC16 is a well-known epithelial-related mucin, less is known about its role as a renal endothelial mucin. As shown in Fig. 3a, kidney tissue demonstrated robust expression of MUC16 as well as N-cadherin within glomeruli and surrounding structures of mouse kidneys by immunostaining. In addition, immunofluorescence (IFA) demonstrated colocalization of MUC16 with CD31+ staining ECs in vessel walls, without significant MUC16 staining in \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mutant mice (Fig. 3b). Furthermore, 2 hours after intravenous inoculation of \u003cem\u003eC. albicans\u003c/em\u003e hyphae expressing fluorescent d-Tomato (Fig. 3c), fungal cells (red arrows) localized within CD31+ renal vascular structures in WT mice (green arrows), expressing MUC16 (center and left panel, purple arrow). Endothelial adherence was persistent at 4 days (Fig. 3d) with occasionally observed penetration of the endothelial layer by fungal hyphal structures (* and red arrows). In contrast, fungal organisms were not observed localized to vascular endothelium in the \u003cem\u003eMuc16\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e \u003c/sup\u003eKO animals. In summary, these data suggest that MUC16 is expressed in renal ECs of intact mouse kidneys and intravenously-inoculated fungal cells co-localize to the MUC16-expressing EC layer, consistent with the MUC16-dependent adherence observed in purified mouse kidney ECs. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDeletion of MUC16 in mice conveys protection in an intravenous dissemination model of Candida albicans infections\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe significance of MUC16 in systemic \u003cem\u003eCandida\u003c/em\u003e pathogenesis was further studied using an intravenous dissemination candidemia model that results in mortality mediated primarily by fungal renal vascular invasion followed by pathogenic renal neutrophil invasion \u003csup\u003e11\u003c/sup\u003e. As shown in Fig. 4a, \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e 129 x C57BL/6N strain F1 progeny exhibited significant protection after intravenous challenge with 1 x 10\u003csup\u003e5\u003c/sup\u003e CFU of \u003cem\u003eC. albicans\u003c/em\u003e compared to littermate WT F1 controls. To confirm the phenotypic difference, \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice were then backcrossed to C57B6 mice 5x using a high-speed congenic strategy and first-wave male germ cells with 100% B6-homozygous loci (84 markers) \u003csup\u003e39\u003c/sup\u003e and were again found to exhibit significant protection compared to WT littermate control mice at even a higher inoculum of 2.5 x 10\u003csup\u003e5\u003c/sup\u003e CFU (Fig. 4b). Fungal burdens were shown to be reduced in the \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mouse kidneys by CFU (Fig. 4c) with WT excised kidneys demonstrating increased edema and hypopigmentation compared to \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice (Fig. 4d) and histology (Fig. 4e) showing increased Grocott\u0026rsquo;s methenamine silver (GMS)-staining fungal elements in the WT mice compared to the \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice. In addition, fungal burdens in \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice showed reductions in brain, liver or lung compared to WT, though less consistent in brain (Fig. S4). Cellular studies also demonstrated reduced accumulation of ly6C\u003csup\u003eint\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003ehi\u003c/sup\u003e neutrophils (Fig. 4f), Ly6C\u003csup\u003ehi\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e inflammatory monocytes (Fig. 4g) and MHCII\u003csup\u003e+\u003c/sup\u003eF4/F80\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e-\u003c/sup\u003e macrophages (Fig. 4h) in \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice commensurate with the reduced fungal burden found in this organ and improved survivals. These data demonstrates a role for MUC16 in potentiating fungal renal vascular invasion, associated with increased neutrophil accumulation and mortality. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDeletion of MUC16 in mice conveys protection in an oral model of Candida albicans infections\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince MUC16 is well-known as an epithelial mucin \u003csup\u003e35\u003c/sup\u003e, the \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e strain was also compared to littermate WT controls using two adapted oral models of \u003cem\u003eC. albicans\u003c/em\u003e including one recently used to demonstrate a role for innate lymphoid cells in oral candidiasis \u003csup\u003e40\u003c/sup\u003e. Without immunosuppression, WT mice inoculated orally with a solution of \u003cem\u003eC. albicans\u003c/em\u003e displays a transient reduction in weight (Fig. 5a). In addition, since corticosteroids are a known risk factor for severe mucocutaneous candidiasis \u003csup\u003e41\u003c/sup\u003e, an additional oral model of candidiasis in mice treated with cortisone acetate was utilized \u003csup\u003e42\u003c/sup\u003e, which demonstrated significant protection from infection as measured by percent of body weight loss and reduced tongue colonization measured by fungal CFU (Fig. 5b, c) or histology of GMS-stained tongues at 4 days (Fig. 4d). Commensurate with less fungal burden, \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice demonstrated less cellular recruitment of neutrophils (Fig. 5e), monocytes (Fig. 5f) and macrophages (Fig. 5g) within the tongue tissue [summary statistics day 1 (Fig. 5h) and day 4 (Fig. 5i)]\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eExpression of MUC16 is induced via TLR and C-type lectin receptor ligands and is CARD9-dependent. \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince MUC16 plays a role in initiating and determining severity of candidal infection in mice, we hypothesized that MUC16 expression could be induced by factors associated with increased mortality of candidemia in patients, including pathogen products released during either transient seeding from central venous catheters or bacterial infections associated with antibiotic use \u003csup\u003e43\u003c/sup\u003e. Indeed, hematologic malignancies are well known to induce MUC16 measured by the CA125 levels in blood which are known risk factors for candidemia \u003csup\u003e44\u003c/sup\u003e. In the present studies intravenous exposure of mice with \u003cem\u003eC. albicans\u003c/em\u003e increased kidney levels of MUC16 after 4 days (Fig. 6a) and incubation of fungal cells with purified mouse EC monolayers for 3 hours also induced MUC16 (Fig. 6b). Similarly, incubation of heat-killed \u003cem\u003eE. coli\u003c/em\u003e resulted in a dose-dependent induction of \u003cem\u003eMuc16\u003c/em\u003e expression in primary mouse kidney ECs at 3 h (Fig. 6c). Since pathogen-associated molecular patterns (PAMPs) are prominent signaling ligands of pathogens, we assessed for induction of \u003cem\u003eMuc16\u003c/em\u003e by selected PAMPs within purified mouse kidney ECs for 1 hour and 3 hours and found a striking induction of this mucin after stimulation of a number of TLR and C-type lectin receptors (Fig. 6d), including \u003cem\u003eCandida\u003c/em\u003e-associated ligands Mincle, TLR9 and Dectin1 (Fig. 6e). Induction was dependent on \u003cem\u003eCard 9\u003c/em\u003e (Fig. 6f), suggesting the role of this pathway in PAMP-dependent signaling of MUC16. In addition, adherence to primary mouse ECs was found to be reduced in \u003cem\u003eCard9\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice, almost to a degree found in the \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice (Fig. 6g). In conclusion, MUC16 was found to be an inducible endothelial mucin, mediated by both TLR and C-type lectin receptors and is \u003cem\u003eCard9\u003c/em\u003e dependent. This could suggest mechanisms whereby inflammatory signals prime MUC16 to provide increased receptors to bind transient fungal cells released during illness, perhaps facilitating endothelial adhesion prior to endothelial endocytosis and tissue invasion. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMUC16\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-rs11670318 single nucleotide polymorphism and serum concentrations of MUC16/CA125 are associated with mortality in humans.\u003c/strong\u003e To identify possible human MUC16 SNPs that could contribute to severity of human disease, we analyzed human DNA samples from a prospective genetic discovery study of non-neutropenic patients with bloodstream infections of \u003cem\u003eCandida\u003c/em\u003e. Over a 3-year period in 2005, 37 consecutive eligible patients were recruited at the University of Illinois at Chicago after being identified by the microbiological laboratory and informed consent obtained (see entry criteria in Table S6). Demographic data demonstrated similarity between patients that died versus survivors, although there was a difference in APACHE II score (Table S7). Characteristics of the pathogen obtained in cultures are described in Table S7. The predominant organism was \u003cem\u003eCandida albicans\u003c/em\u003e, although there were also isolates of \u003cem\u003eC. glabrata \u003c/em\u003eand other strains. Using an additive genetic model adjusted for age and gender, two SNPs (rs2547065, rs7508222) among 80 tested SNPs (Table S8) were associated with mortality with a nominal p \u0026lt; 0.05. These two SNPs remained significant after further adjustment for the APACHE II score. Validation of three of the top 4 SNPs from the discovery cohort were analyzed in a second previously-described cohort of candidemia patients \u003csup\u003e4\u003c/sup\u003e, selecting for patients matching that of the discovery cohort. The top candidate SNP associated with protection from mortality was identified as rs11670318-A with an adjusted p value of 0.07. In humans, the MUC16-rs11670318 SNP results in an adenine-to-guanidine substitution at position 19:9056982 that changes a threonine at position 10155 to an isoleucine, found to be a site of O-GalNAc modification by a whole genome study of the glycoproteome \u003csup\u003e45\u003c/sup\u003e. \u003c/p\u003e\n\u003cp\u003eTo determine whether the rs11670318 polymorphism might have the capacity to affect MUC16 \u003cem\u003eCandida\u003c/em\u003e adherence, a human EC line, HMEC-1 was subjected to CRISPR-cas9-directed substitution of the rs11670318 SNP utilizing the guide RNAs described in Methods. Three clones were selected by limiting dilution and isolated, verified to have similar observed growth and morphology at confluence and assessed by PCR followed by sequencing to establish the correct substitution. As shown in Fig. 6A, adherence assays demonstrated modest but significant reductions in fungal binding, that could contribute to less burden of renal disease after transient intravenous fungal exposure. \u003c/p\u003e\n\u003cp\u003eWhile serum was not collected in the discovery cohort I, it was available in Cohort II which allowed examination of serum concentrations of MUC16 protein in 134 patients by a standard CA125 assay of MUC16 (Quantikine ELISA Human CA125/MUC, R\u0026amp;D Systems, Minneapolis, MN). CA125 is a widely-used assay of the MUC16 soluble SEA domain that facilitates staging and follow-up of malignancies, principally ovarian cancer, whose prognosis and outcomes are strongly associated with levels of MUC16 \u003csup\u003e46\u003c/sup\u003e. As shown in Fig. 6i concentrations of MUC16 in serum were highly associated with 30-day survival in non-neutropenic patients with candidemia (p = 0.01) with an ROC of 0.7 (Fig. 6j), suggesting that higher levels of MUC16 expression, perhaps mediated by risk conditions such as transient bacterial or fungal products, may have resulted in poor clinical outcome similar to the mouse models. Further study in additional cohorts may be required to delineate more fully the role of CA125 monitoring in patients with candidemia, but, to our knowledge, these data provide the first readily available commercial assay to stratify outcome in human candidemia. \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eECs are among the first host cells encountered by \u003cem\u003eCandida\u003c/em\u003e during hematogenous dissemination, and adhesion is the critical initiating step for invasion and tissue damage. Previous studies demonstrated that endothelial endocytosis of \u003cem\u003eCandida\u003c/em\u003e is calcium dependent and requires N-cadherin engagement, with Als3 functioning as a key fungal invasion \u003csup\u003e10\u003c/sup\u003e \u003csup\u003e34\u003c/sup\u003e. \u0026nbsp;However, the molecular mechanisms governing the initial attachment of \u003cem\u003eCandida\u003c/em\u003e to endothelial surfaces have remained undefined. Our data identify MUC16 as a central mediator of this process, demonstrated through adherence studies, proximity ligation assays, siRNA knockdown, genetic deletion, and functional inhibition. Our findings support a model (Fig. 6k) in which infection-associated inflammatory cues induce endothelial MUC16 expression, thereby creating a permissive adhesive platform for fungal Als3-mediated binding, endocytosis, and tissue invasion. This work establishes MUC16 as a \u0026ldquo;pathogen-inducible endothelial adhesin\u0026rdquo; that links host risk factors, innate immune signaling, and fungal virulence mechanisms to disease severity.\u003c/p\u003e\n\u003cp\u003eMUC16 is a large molecule which contains a distal glycoprotein as well as 16 proximal SEA (sea urchin sperm protein, enterokinase and agrin)-domains internal to the glycoprotein which are self-proteolytically cleaved at specific intervals to yield soluble and residual cell surface pauci-glycosylated fragments containing one or more residual SEA domains \u003csup\u003e47\u003c/sup\u003e. The SEA domain is highly conserved evolutionarily and is known to assist or regulate binding a number of carbohydrate moieties \u003csup\u003e48\u003c/sup\u003e, E and L-Selectins \u003csup\u003e49\u003c/sup\u003e, as well as mammalian proteins including mesothelian, facilitating cell-cell adhesion and cancer metastasis \u003csup\u003e50,51\u003c/sup\u003e. In the present study, recombinant SEA domains bound preferentially to \u003cem\u003eCandida\u0026nbsp;\u003c/em\u003ehyphae, the morphological form responsible for endothelial adherence and invasion. These findings support a model in which exposed SEA domains provide a compatible binding interface for fungal adhesins.\u003c/p\u003e\n\u003cp\u003eAls3 is the best-characterized member of the ALS family of \u003cem\u003eCandida\u003c/em\u003e adhesins and plays a central role in endothelial adherence and invasion \u003csup\u003e34\u003c/sup\u003e. Structural and functional studies of Als3 have demonstrated its capacity to engage multiple host ligands through distinct interfaces, enabling multivalent adhesion \u003csup\u003e32\u003c/sup\u003e. Interestingly, we observed SEA domain binding to the Als3 gatekeeper (GK) variant S179Y \u003csup\u003e31\u003c/sup\u003e, suggesting that binding of this ligan may be independent of the Als3 peptide binding cavity. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eClinically, Als3 is highly relevant, as vaccination with the Als3-derived NDV-3A reduces recurrent vulvovaginal candidiasis and induces protective antibody responses \u003csup\u003e52\u003c/sup\u003e. Our identification of MUC16 as an endothelial binding partner of Als3 extends these findings and provides a mechanistic basis for targeting host\u0026ndash;fungal adhesion as a therapeutic strategy. Mechanistically, our data support a ternary adhesion complex involving fungal Als3, endothelial MUC16 SEA domains, and N-cadherin. N-cadherin mediates calcium-dependent homophilic adhesion and undergoes lateral clustering and polymerization at the EC surface, processes that are dynamically regulated during junction remodeling and endocytic uptake \u003csup\u003e53-55\u003c/sup\u003e. We propose that MUC16 facilitates stable initial attachment of \u003cem\u003eCandida\u003c/em\u003e, positioning Als3 for productive engagement of N-cadherin and subsequent endocytosis. This multi-receptor model provides a \u0026lsquo;missing link,\u0026rsquo; facilitating Als3\u0026ndash;N-cadherin interactions required for invasion by supporting early adhesion events and recruitment of cadherins involved in endocytosis and tissue invasion \u003csup\u003e32\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe kidney is a principal target organ in disseminated candidiasis, and endothelial interactions are critical during early infection. Using purified murine kidney ECs and intact mouse models, we demonstrate that MUC16 is expressed on renal vascular endothelium, co-localizes with CD31, and facilitates rapid fungal adherence and endocytosis. Genetic deletion of Muc16 resulted in significantly reduced renal fungal burdens, diminished neutrophil accumulation, and improved survival following intravenous\u003cem\u003e\u0026nbsp;Candida\u003c/em\u003e challenge. These findings provide mechanistic details supporting kidney-specific endothelial invasion as a driver of immunopathology and organ failure \u003csup\u003e11\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn contrast, MUC16 plays a more limited role in oral candidiasis models, consistent with a plethora of additional epithelial-specific receptors and virulence mechanisms at mucosal surfaces, including EphA2-mediated \u0026beta;-glucan sensing and candidalysin-induced epithelial damage \u003csup\u003e13,15\u003c/sup\u003e. These observations highlight the compartment-specific nature of host\u0026ndash;fungal interactions and underscore the unique importance of endothelial adhesion mechanisms in disseminated disease. However, a paucity of \u003cem\u003eCandida\u003c/em\u003e-binding receptors in ECs is consistent with the primary property of \u003cem\u003eCandida\u003c/em\u003e as a commensal organism that would likely gain little evolutionarily by facilitating vascular interactions that could kill the host. The present findings thus help to understand a long-known but poorly understood property of \u003cem\u003eCandida\u003c/em\u003e pathogenesis. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA key finding of this study is that endothelial MUC16 expression is inducible by microbial and inflammatory signals associated with candidemia risk. Bacterial lysates, LPS, and multiple fungal PAMPs triggered robust MUC16 upregulation in ECs. Notably, induction by ligands for Dectin-1, Mincle, and TLR9 was CARD9 dependent. While CARD9 signaling downstream of C-type lectin receptors is well established, TLR9 has not previously been linked to CARD9-mediated pathways \u003csup\u003e56\u003c/sup\u003e suggesting a novel signaling axis regulating endothelial adhesion molecule expression. Blocking this pathway in CARD9-deficient ECs abrogated \u003cem\u003eCandida\u0026nbsp;\u003c/em\u003eadherence, supporting a functional role for PRR/CARD9 MUC16 induction. These findings extend emerging concepts that ECs act as active innate immune sensors rather than passive barriers during fungal infection \u003csup\u003e57,58\u003c/sup\u003e including recent evidence that serum bridging molecules can drive endocytosis through specific endothelial receptors \u003csup\u003e59\u003c/sup\u003e. These results also shed light on an important \u003cem\u003eCandida\u003c/em\u003e-specific disease related to \u003cem\u003eCARD9\u003c/em\u003e. For example, in a cohort of patients with \u003cem\u003eCARD9\u003c/em\u003e polymorphisms\u0026nbsp;no significant association was identified between \u003cem\u003eCARD9\u003c/em\u003e variation and the prevalence of candidemia, even though the Dectin-1/CARD9 pathway was nonredundant in mucosal immunity to \u003cem\u003eCandida albicans\u003c/em\u003e \u003csup\u003e60\u003c/sup\u003e Such a result could be due to limited \u003cem\u003eCARD9\u003c/em\u003e-dependent expression of endothelial receptors such as MUC16.\u003c/p\u003e\n\u003cp\u003eGiven the clinical relevance of endothelial adhesion in dissemination, we investigated whether MUC16 variation influences outcomes in human candidemia in the most common risk group today, non-neutropenic patients with risk factors of intensive care residence or hematologic malignancies. In two independent cohorts, we identified three non-synonymous \u003cem\u003eMUC16\u003c/em\u003e polymorphisms associated with mortality in a discovery cohort and a fourth variant (T10155I) that showed consistent trends across cohorts. Functional validation using CRISPR\u0026ndash;Cas9\u0026ndash;engineered HMEC-1 cells demonstrated that the T10155I mutation significantly reduced \u003cem\u003eCandida\u003c/em\u003e adherence, supporting a mechanistic link between genotype and endothelial function. While the observed p values did not uniformly meet conventional thresholds, the consistency across cohorts and functional corroboration argue for biological relevance, consistent with modern statistical perspectives that emphasize effect sizes and reproducibility over arbitrary cutoffs \u003csup\u003e61\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn addition to genetic risk, we identified a strong association between elevated serum MUC16/CA125 concentrations and mortality in candidemia. This finding is particularly intriguing given that known risk factors for invasive candidiasis, including hematologic malignancies, inflammation, and prior infections, are themselves associated with increased MUC16 expression. We propose that these conditions may \u0026ldquo;prime\u0026rdquo; the endothelium by increasing MUC16 availability, thereby facilitating fungal adhesion and invasion once candidemia occurs (Fig. 6k). While cohort size limits definitive conclusions, further studies could suggest a role for CA125 in biomarker-stratified clinical designs where selection of higher risk patients may facilitate the use of smaller patient sample sizes and/or improved power as recently suggested for biomarker-driven clinical studies \u003csup\u003e62\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn summary, this study identifies MUC16 as a pathogen-inducible endothelial adhesin that integrates host inflammatory signals, innate immune pathways, and fungal virulence mechanisms to drive disseminated candidiasis. By defining a novel Als3\u0026ndash;MUC16\u0026ndash;N-cadherin adhesion axis and linking endothelial biology to clinical outcomes, our findings open new avenues for host-directed therapies, anti-adhesion strategies, and biomarker-guided clinical trial design in a disease that continues to carry unacceptably high mortality.\u003c/p\u003e\n"},{"header":"Materials and Methods ","content":"\u003cp\u003e\u003cstrong\u003eMice, yeast and bacterial strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWild-type C57BL/6 mice were obtained from Taconic or Jackson Laboratory. \u003cem\u003eMuc16\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mutant mice in a 129 background were a gracious gift from the Department of Genetics, University of Texas M. D. Anderson Cancer Center, Houston, Texas and were backcrossed to C57B6 mice 5x using a high-speed congenic strategy and first-wave male germ cells with 100% B6-homozygous loci (84 markers) \u003csup\u003e39\u003c/sup\u003e C57/Bl/6N \u003cem\u003ecard9\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice were the generous gift of M. Lionakis. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCandida albicans\u003c/em\u003e strain SC5314 was obtained from American Type Culture Collection (Manassas, VA), \u003cem\u003eC. albicans\u003c/em\u003e-dTomato was a kind gift of R. Wheeler, \u003csup\u003e30\u003c/sup\u003e, and \u003cem\u003eE. coli\u003c/em\u003e strain 25922 was a generous gift from the NIH clinical center microbiology laboratory (Bethesda, MD).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eIsolation of mouse primary kidney ECs \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse tissue ECs were isolated as described \u003csup\u003e20\u003c/sup\u003e with some modification. Briefly, mouse kidneys were aseptically removed washed, dissected, finely minced and washed, digested with Liberase at 37\u0026deg;C with agitation for 25-35 min. The suspensions were filtered twice centrifuged at 300\u003cem\u003eg \u003c/em\u003efor 10 minutes and the pellets washed three times in phosphate buffer saline (PBS) containing 0.5% bovine serum albumin (BSA) and 2 mM ethylenediamine-tetraacetic acid (EDTA). EC purifications were performed using CD146 MicroBeads positive selection kit according to the manufacturer\u0026rsquo;s instructions. Isolated cells were plated onto T25 flasks precoated with 1% gelatin and cultured at 37\u003csup\u003e0\u003c/sup\u003e C in a 5% CO2 in DMEM/F12 containing EC growth supplements. To verify the cell preparation, cells were tested for uptake of Dil-Ac-LDL according to the method of Voyta \u003csup\u003e27\u003c/sup\u003e, and cells were also stained with anti-CD45-PE-Cy7A, CD146 PE-A and Muc16 APC-A (Table S4) and subjected to flow cytometry. In addition, mouse kidney ECs demonstrated induction of E-selectin and VCAM-1 \u003csup\u003e26,63\u003c/sup\u003e as well as the endothelial marker, CD31/PECAM-1 \u003csup\u003e22\u003c/sup\u003e. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eGene silencing of renal endothelial-associated mucins in mouse ECs \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene silencing was performed using four siRNA plasmids targeting the mouse \u003cem\u003eMuc16, Muc1, Muc14 and Muc13\u003c/em\u003e genes and Accell Non-targeting Control Pool (D-001910-10-05, Dhar Macon) according to Materials and Methods. ECs were transfected with endotoxin-free siRNA plasmids containing the proprietary nucleotide sequences. Accell\u0026trade;siRNA delivery). Transiently transfected cells were assayed 72 h post-transfection.knockdown was quantified using real-time RT-PCR. Knockdown efficiencies ⩾70% were regarded as significant.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAdherence Assays of \u003cem\u003eCandida\u003c/em\u003e to primary mouse kidney ECs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe method of Phan et al was utilized \u003csup\u003e10\u003c/sup\u003e. Briefly, 10\u003csup\u003e5\u003c/sup\u003e mouse purified ECs were cultured in 6-well tissue culture plate until cells reached confluence. To determine \u003cem\u003eCandida\u003c/em\u003e adherence, endothelial monolayers were washed twice with 2 ml of PBS, and 1 ml of the singlet \u003cem\u003eCandida\u003c/em\u003e suspension (10\u003csup\u003e2\u003c/sup\u003e cfu/ml of HBSS) was added to replicate monolayers. The inoculum size of each suspension was confirmed by culturing aliquots in YPD agar plates. Following incubation at 37\u0026deg;C, the nonadherent organisms were aspirated and the EC monolayers were rinsed with 10 ml of HBSS in a standard manner. Two milliliters of warm 2% glucose, 2% bactopeptone, 1% yeast extract agar (YPD) agar was poured over the surface and allowed to solidify. After incubation at 37\u0026deg;C for 24 h, colonies (representing adherent organisms) were counted, and the results were expressed as a percentage of the inoculum.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eMeasurement of endocytoses of \u003cem\u003eC. albicans\u003c/em\u003e by ECs. \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe number of organisms endocytosed by ECs was determined using the method of a previously described differential fluorescence assay with minor modifications \u003csup\u003e10\u003c/sup\u003e. Briefly, 10\u003csup\u003e3\u003c/sup\u003e ECs were grown to confluency on 8 well chamber slides. The cells were rinsed once and infected with 10\u003csup\u003e2\u003c/sup\u003e cells of dTomato-\u003cem\u003eC. albicans\u003c/em\u003e in RPMI 1640 medium plus or minus 1.9 mM ethylene glycol tetraacetic acid (EGTA) for 45 min at 37 \u0026deg;C in 5% CO2. Next, the cells were rinsed twice with 0.5 ml of PBS in a standardized manner and then fixed with 3% paraformaldehyde. The adherent but non-endocytosed organisms were labeled with polyclonal rabbit anti-\u003cem\u003eC. albicans\u003c/em\u003e antibodies (Table S2) that had been conjugated with green fluorescing AF488 (Molecular Probes). ECs were then permeabilized with 0.5% Triton X-100 (Merck-Sigma) in PBS, after which both the endocytosed and non-endocytosed organisms were visualized by microscopy using Imaris Software. The number of cell-associated organisms (endocytosed plus adherent organisms) was determined by counting the number of organisms that were labeled with Alexa 488. The number of endocytosed organisms was determined by subtracting the number of organisms labeled with red fluorescent dTomato from the number of cell-associated organisms. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eProximity Ligation Assay. \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe method of Soderberg et al \u003csup\u003e64\u003c/sup\u003e was adapted using the commercial Duolink reagents (Millepore Sigma/Merck KGaA, Darmstadt Germany). Briefly, purified mouse ECs were grown to 70-80% confluence on 12-well culture slides, rinsed once and 5x10\u003csup\u003e4\u003c/sup\u003e hyphae of \u003cem\u003eC. albicans\u003c/em\u003e SC5314 were incubated with EC monolayers in DMEM/F12 medium alone. Cells were rinsed twice with PBS and then fixed for 15 min at room temperature. For primary antibody incubation, the cells were treated with primary antibodies respectively for 2-3 hours in 3 groups: 1) rabbit anti-MUC16 antibody and mouse anti-Als3 antibody described previously \u003csup\u003e65\u003c/sup\u003e (a generous gift from L Hoyer). 2) Rabbit anti N-cadherin Polyclonal Antibody (company PA5-19486) and mouse anti-Als3 antibody. 3) rabbit anti-MUC16 antibody and mouse anti-N-cadherin monoclonal antibody. Subsequently, probe incubation, ligation, and amplification were performed according to the manufacturer\u0026apos;s operating procedures. Puncta were captured by fluorescent (FITC) microscopy using Imaris Software. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eComparison of human and mouse \u003cem\u003eMUC16\u003c/em\u003e and expression and fungal binding of recombinant huSEA domains:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecombinant SEA domains were expressed as a synthetic gene comprising 8 human SEA domains (aa 8682-8805; Fig. S2B) (GenScript, Piscataway, NJ), as a fusion protein with maltose-binding protein (MBP) by using the pIH902 expression system (New England Biolabs, Beverly MA) purified on amylose-Sepharose according to the manufacture\u0026rsquo;s directions. Alternatively, a single SEA domain was expressed with either an N-terminal or C-terminal MBP. Intact sequence was verified by Sanger sequencing prior to use. \u003c/p\u003e\n\u003cp\u003eSEA domain binding to \u003cem\u003eC. albicans\u003c/em\u003e was performed as follows: d-Tomato-expressing C. albicans described above was induced for hyphal formation followed by incubation of 10\u003csup\u003e3\u003c/sup\u003e CFU with 100 mg/ml of rSEA-MBP or MBP alone for 2 h at 37C followed by extensive washing and incubation with anti-MBP antibody (Table S2) and visualized by epifluorescence or by flow cytometry. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolated Kidney Perfusion and Tissue Clearing \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse kidney perfusions were performed as described \u003csup\u003e66\u003c/sup\u003e with modifications\u003cstrong\u003e. \u003c/strong\u003eBriefly, wild-type mice were anesthetized. Mouse kidneys were perfused through the abdominal/renal artery with 10 ml of ammonium-chloride-potassium lysing buffer containing 10 unit of heparin followed by 1ml of 10\u003csup\u003e6\u003c/sup\u003e CFU/ml of \u003cem\u003eC. albicans\u003c/em\u003e-dTomato in PBS. After 2 h kidneys were perfused with PBS to remove unbound fungi. Excised mouse kidneys were post-fixed overnight and processed utilizing the method of \u003cem\u003eClear\u003csup\u003eT2\u003c/sup\u003e\u003c/em\u003e tissue-clearing as described \u003csup\u003e67\u003c/sup\u003e. Briefly, kidneys were transferred to 25% formamide/10% polyethylene glycol (PEG) solution, 50% formamide/20% PEG solution, then clear in new 50% formamide/20% PEG solution. After sectioning (300 \u0026mu;m) on a vibrating microtome, the obtained kidney slices were blocked in blocking solution for 1 hour at room temperature. Goat anti-mouse CD31 and rabbit anti-mouse MUC16 were incubated overnight at 4\u0026deg;C. After washes AF488 conjugated anti-goat and AF647 conjugated anti-rabbit secondary antibodies were applied, incubated in 1%BSA/1%Tween/PBS overnight at 4\u0026deg;C. 4\u0026rsquo;6-diamindino-2-phenylindole (DAPI) was used for nuclear staining followed by washes in phosphate-buffered saline before epifluorescent imaging on an inverted SP8 microscope.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eMouse model of systemic candidiasis and oropharyngeal candidiasis (OPC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe model of Huang et al was utilized \u003csup\u003e40\u003c/sup\u003e. Fungal burden was determined by excision of tissue and determination of colony forming units. \u003c/p\u003e\n\n\n\n\u003cp\u003e\u003cstrong\u003eHistopathological and immunohistochemistry (IHC) for N-cadherin and Muc16 expression Studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe method of Lionakis et al was adapted \u003csup\u003e11\u003c/sup\u003e. Briefly, control uninfected mice and infected mice were euthanized on days 1, 4, and 7 after infection and the kidney was removed, fixed with 10% formalin. The samples underwent sectioning with H\u0026amp;E or PAS staining. For IHC of N-cadherin and MUC16 expression, tissue sections were placed on poly- L -lysine-coated glass slides, deparaffinized in xylene and rehydrated. Following antigen retrieval in citrate buffer endogenous peroxidase was blocked and the slides were then incubated with 5% bovine serum albumin followed by washing with buffer containing sheep anti-N-Cadherin antibody or Rabbit anti-CA-125(MUC16) antibody. BSA (1%) without primary antibody was used as negative control. The slides were then washed twice and detection of immunoreaction was achieved using the streptavidin-HRP (horse radish peroxidase) containing the streptavidin-peroxidase complex after a 40-min incubation at RT. Color was developed with DAB 3 DAB (3,3\u0026apos;-diaminobenzidine tetrahydrochloride) substrate kit (Thermofisher, 34002) and slides were subsequently counterstained with hematoxylin, dehydrated, and mounted. Two different sections of each model were tested per experiment.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eControl uninfected mice and infected mice were euthanized on days 1, 4, and 7 after infection. Then, the animals were perfused with normal saline and brain, spleen, liver and kidneys were harvested. Single-cell suspensions were obtained using methods previously described by Lionakis \u003csup\u003e11\u003c/sup\u003e. Further details including antibodies utilized can be found in Supplemental Materials. FACS was performed on an BD Fortessa (BD Biosciences) and data were analyzed using FlowJo software.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eInduction of \u003cem\u003eMuc16\u003c/em\u003e in intact mice and in purified mouse kidney ECs. \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eCandida\u003c/em\u003e induction of \u003cem\u003eMuc16\u003c/em\u003e in intact mice, C57/Bl/6 mice were inoculated intravenously with 2.5 x 10\u003csup\u003e5\u003c/sup\u003e \u003cem\u003eC. albicans\u003c/em\u003e, kidneys excised as described above and RNA assayed by qRT-PCR using primers described above. For \u003cem\u003eCandida\u003c/em\u003e induction of \u003cem\u003eMuc16\u003c/em\u003e in ECs, 2 x10\u003csup\u003e5\u003c/sup\u003e CFU of \u003cem\u003eC. albicans\u003c/em\u003e was incubated with 10\u003csup\u003e6\u003c/sup\u003e ECs for 8 h in cell culture media and RNA assayed for \u003cem\u003eMuc16\u003c/em\u003e expression by qRT-PCR. For \u003cem\u003eE. coli\u003c/em\u003e induction of \u003cem\u003eMuc16\u003c/em\u003e in ECs, \u003cem\u003eE. coli\u003c/em\u003e strain 25922 (10\u003csup\u003e6\u003c/sup\u003e CFU) were heat treated (65 C for 1h) and indicated equivalent amount of organism lysate dilution (0.01-100 organisms equivalent) incubated with 10\u003csup\u003e6\u003c/sup\u003e of ECs and RNA assessed for \u003cem\u003eMuc16\u003c/em\u003e expression as above. For PAMP ligand induction, Pam3CSK4, Poly (I:C), lipopolysaccharide (LPS), fibroblast-stimulating lipopeptide (FSL), CpG oligodeoxynucleotide trehalose dibehenate FurFurman, Laminarin, Poly IC-LMW bacterial flagellin or single stranded RNA40 (see Table S5) were incubated with EC from either C57/Bl/6N or littermate C57/Bl/6N \u003cem\u003ecard9\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice according to the manufacturer\u0026rsquo;s instructions for either 1 h or 3 h and RNA isolated and assayed for \u003cem\u003eMUC16\u003c/em\u003e expression by qRT-PCR. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCRISPR Cas9 mediated T10155I KI of \u003cem\u003eMUC16\u003c/em\u003e in HMEC1 cells. \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo generate cells, ribonucleoproteins containing Cas9 protein and synthetically chemically modified sgRNA produced by the manufacturer (Synthego, Redwood City, CA) was electroporated into HMEC1 cells. Guide RNA cut location was chr19:8,946,320. Editing efficiency was assessed upon recovery and genomic DNA was extracted from a portion of the cells, PCR amplified and subjected to Sanger sequencing. The pool was subjected to repetitive dilutional cloning and clones screened for the appropriate KI primers and Sanger sequencing and equivalent growth rates and confluent monolayer productions to that of the wild-type cells. \u003c/p\u003e\n\u003cp\u003eSingle cell clonogenic assays were performed as described \u003csup\u003e68\u003c/sup\u003e. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human prospective observational cohort studies were approved by the respective institutional review board at each institution (Cohort 1, The University of Illinois at Chicago and Cohort 2, Duke University Health System). All subjects or their legally authorized representatives provided written informed consent, or had a waiver as approved by the IRB.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eStudy Design and Participants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe UIC candidemia discovery study (Cohort I) was a prospective, observational study involving non-neutropenic patients admitted to UIC over a three year period and developed \u003cem\u003eCandida\u003c/em\u003e bloodstream infections reported by the UIC clinical laboratory. Inclusion criteria are described in Table S6. (Demographic data in Table S7). Further details are described in Supplemental Materials. All infected patients were followed for up to 12 weeks to assess for clinical outcome. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCase-association study of mortality in Cohort I candidemia patients.\u003c/strong\u003e DNA collected at entry was genotyped. Eighty non-synonymous alleles of the \u003cem\u003eMUC16\u003c/em\u003e gene (Table S8) were identified from the GnomAD database \u003csup\u003e69\u003c/sup\u003e, and assessed using DNA from 37 patients using Taqman (ThermoFisher) and Sequenom (San Diego, CA) platforms according to the manufacturer\u0026rsquo;s instructions. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eWithin-case association study of mortality in Cohort II candidemia patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genotyping, quality control, and imputation of the genetic data was previously described \u003csup\u003e4\u003c/sup\u003e Briefly, DNA was genotyped using Illumina HumanCoreExome-12 v1.0 and HumanCoreExome-24v1.0 BeadChip Snp chips. Genotypes were imputed using the human reference consortium reference panel \u003csup\u003e70\u003c/sup\u003e using the Michigan imputation server \u003csup\u003e71\u003c/sup\u003e. Quality control filters resulted in a dataset of 156 candidemia patients. Of these 156 patients, 148 had available mortality data. We tested for association with mortality at 14, 30 and 90 days within the patient cohort using 31 non-survivors and 117 survivors, 49 non-survivors and 99 survivors, and 65 non-survivors and 83 survivors respectively. The association between the genetic variants and mortality were tested by Fisher\u0026rsquo;s exact test using PLINK v1.9 (www.cog-genomics.org/plink/1.9/) \u003csup\u003e72\u003c/sup\u003e. The corresponding receiver operating characteristic (ROC) and precision\u0026ndash;recall (PR) curves were generated using the cumulative prtC abundance as the predictor and actual disease status (based on the clinical metadata) as the ground truth.\u003c/p\u003e\n\n\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u003c/strong\u003e This research was supported [in part]* by the Intramural Research Program of the National Institutes of Health (NIH). The contributions of the NIH author(s) are considered Works of the United States Government. The findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMcCarty, T.P., White, C.M. \u0026amp; Pappas, P.G. Candidemia and Invasive Candidiasis. \u003cem\u003eInfect Dis Clin North Am\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 389-413 (2021).\u003c/li\u003e\n\u003cli\u003eSeagle, E.E.\u003cem\u003e, et al.\u003c/em\u003e The landscape of candidemia during the COVID-19 pandemic. \u003cem\u003eClin Infect Dis\u003c/em\u003e (2021).\u003c/li\u003e\n\u003cli\u003eKeighley, C.\u003cem\u003e, et al.\u003c/em\u003e Candidaemia and a risk predictive model for overall mortality: a prospective multicentre study. \u003cem\u003eBMC Infect Dis\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 445 (2019).\u003c/li\u003e\n\u003cli\u003eJaeger, M.\u003cem\u003e, et al.\u003c/em\u003e A Genome-Wide Functional Genomics Approach Identifies Susceptibility Pathways to Fungal Bloodstream Infection in Humans. \u003cem\u003eJ Infect Dis\u003c/em\u003e \u003cstrong\u003e220\u003c/strong\u003e, 862-872 (2019).\u003c/li\u003e\n\u003cli\u003eLionakis, M.S.\u003cem\u003e, et al.\u003c/em\u003e CX3CR1-dependent renal macrophage survival promotes Candida control and host survival. \u003cem\u003eJ Clin Invest\u003c/em\u003e \u003cstrong\u003e123\u003c/strong\u003e, 5035-5051 (2013).\u003c/li\u003e\n\u003cli\u003eSwamydas, M.\u003cem\u003e, et al.\u003c/em\u003e CXCR1-mediated neutrophil degranulation and fungal killing promote Candida clearance and host survival. \u003cem\u003eSci Transl Med\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 322ra310 (2016).\u003c/li\u003e\n\u003cli\u003eAllert, S.\u003cem\u003e, et al.\u003c/em\u003e Candida albicans-Induced Epithelial Damage Mediates Translocation through Intestinal Barriers. \u003cem\u003eMBio\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e(2018).\u003c/li\u003e\n\u003cli\u003eSheppard, D.C. \u0026amp; Filler, S.G. Host cell invasion by medically important fungi. \u003cem\u003eCold Spring Harb Perspect Med\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, a019687 (2014).\u003c/li\u003e\n\u003cli\u003eChen, J.\u003cem\u003e, et al.\u003c/em\u003e Crk1, a novel Cdc2-related protein kinase, is required for hyphal development and virulence in Candida albicans. \u003cem\u003eMol Cell Biol\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 8696-8708 (2000).\u003c/li\u003e\n\u003cli\u003ePhan, Q.T., Fratti, R.A., Prasadarao, N.V., Edwards, J.E., Jr. \u0026amp; Filler, S.G. N-cadherin mediates endocytosis of Candida albicans by endothelial cells. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e280\u003c/strong\u003e, 10455-10461 (2005).\u003c/li\u003e\n\u003cli\u003eLionakis, M.S., Lim, J.K., Lee, C.C. \u0026amp; Murphy, P.M. Organ-specific innate immune responses in a mouse model of invasive candidiasis. \u003cem\u003eJ Innate Immun\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 180-199 (2011).\u003c/li\u003e\n\u003cli\u003eMonika, S. Virulence factors in Candida species. \u003cem\u003eCurr Protein Pept Sci\u003c/em\u003e (2019).\u003c/li\u003e\n\u003cli\u003eMoyes, D.L.\u003cem\u003e, et al.\u003c/em\u003e Candidalysin is a fungal peptide toxin critical for mucosal infection. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e532\u003c/strong\u003e, 64-68 (2016).\u003c/li\u003e\n\u003cli\u003eKasper, L.\u003cem\u003e, et al.\u003c/em\u003e The fungal peptide toxin Candidalysin activates the NLRP3 inflammasome and causes cytolysis in mononuclear phagocytes. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 4260 (2018).\u003c/li\u003e\n\u003cli\u003eSwidergall, M.\u003cem\u003e, et al.\u003c/em\u003e Candidalysin Is Required for Neutrophil Recruitment and Virulence During Systemic Candida albicans Infection. \u003cem\u003eJ Infect Dis\u003c/em\u003e \u003cstrong\u003e220\u003c/strong\u003e, 1477-1488 (2019).\u003c/li\u003e\n\u003cli\u003eDesai, J.V. \u0026amp; Lionakis, M.S. The role of neutrophils in host defense against invasive fungal infections. \u003cem\u003eCurr Clin Microbiol Rep\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 181-189 (2018).\u003c/li\u003e\n\u003cli\u003eBlalock, T.D.\u003cem\u003e, et al.\u003c/em\u003e Functions of MUC16 in corneal epithelial cells. \u003cem\u003eInvest Ophthalmol Vis Sci\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 4509-4518 (2007).\u003c/li\u003e\n\u003cli\u003eYeaman, M.R.\u003cem\u003e, et al.\u003c/em\u003e Applying Convergent Immunity to Innovative Vaccines Targeting Staphylococcus aureus. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 463 (2014).\u003c/li\u003e\n\u003cli\u003eGiannakouros, P., Comamala, M., Matte, I., Rancourt, C. \u0026amp; Piche, A. MUC16 mucin (CA125) regulates the formation of multicellular aggregates by altering beta-catenin signaling. \u003cem\u003eAm J Cancer Res\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 219-230 (2015).\u003c/li\u003e\n\u003cli\u003eDemeule, M., Labelle, M., Regina, A., Berthelet, F. \u0026amp; Beliveau, R. Isolation of endothelial cells from brain, lung, and kidney: expression of the multidrug resistance P-glycoprotein isoforms. \u003cem\u003eBiochem Biophys Res Commun\u003c/em\u003e \u003cstrong\u003e281\u003c/strong\u003e, 827-834 (2001).\u003c/li\u003e\n\u003cli\u003eRadek, M., Babunkova, E., Spacek, M., Kvasnicka, T. \u0026amp; Kvasnicka, J. Determination of Circulating Endothelial Cells and Endothelial Progenitor Cells Using Multicolor Flow Cytometry in Patients with Thrombophilia. \u003cem\u003eActa Haematol\u003c/em\u003e \u003cstrong\u003e142\u003c/strong\u003e, 113-119 (2019).\u003c/li\u003e\n\u003cli\u003eWoodfin, A., Voisin, M.B. \u0026amp; Nourshargh, S. PECAM-1: a multi-functional molecule in inflammation and vascular biology. \u003cem\u003eArterioscler Thromb Vasc Biol\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 2514-2523 (2007).\u003c/li\u003e\n\u003cli\u003eGholizadeh, S.\u003cem\u003e, et al.\u003c/em\u003e E-selectin targeted immunoliposomes for rapamycin delivery to activated endothelial cells. \u003cem\u003eInt J Pharm\u003c/em\u003e \u003cstrong\u003e548\u003c/strong\u003e, 759-770 (2018).\u003c/li\u003e\n\u003cli\u003eLiu, C.\u003cem\u003e, et al.\u003c/em\u003e Human endomucin is an endothelial marker. \u003cem\u003eBiochem Biophys Res Commun\u003c/em\u003e \u003cstrong\u003e288\u003c/strong\u003e, 129-136 (2001).\u003c/li\u003e\n\u003cli\u003eEder, P.\u003cem\u003e, et al.\u003c/em\u003e The influence of anti-TNF therapy on CD31 and VEGF expression in colonic mucosa of Crohn\u0026apos;s disease patients in relation to mucosal healing. \u003cem\u003eFolia Histochem Cytobiol\u003c/em\u003e \u003cstrong\u003e54\u003c/strong\u003e, 75-80 (2016).\u003c/li\u003e\n\u003cli\u003ePhan, Q.T., Belanger, P.H. \u0026amp; Filler, S.G. Role of hyphal formation in interactions of Candida albicans with endothelial cells. \u003cem\u003eInfect Immun\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 3485-3490 (2000).\u003c/li\u003e\n\u003cli\u003eVoyta, J.C., Via, D.P., Butterfield, C.E. \u0026amp; Zetter, B.R. Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. \u003cem\u003eJ Cell Biol\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e, 2034-2040 (1984).\u003c/li\u003e\n\u003cli\u003eSu, A.I.\u003cem\u003e, et al.\u003c/em\u003e Large-scale analysis of the human and mouse transcriptomes. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e, 4465-4470 (2002).\u003c/li\u003e\n\u003cli\u003eWu, C., Jin, X., Tsueng, G., Afrasiabi, C. \u0026amp; Su, A.I. BioGPS: building your own mash-up of gene annotations and expression profiles. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, D313-316 (2016).\u003c/li\u003e\n\u003cli\u003eGratacap, R.L., Rawls, J.F. \u0026amp; Wheeler, R.T. Mucosal candidiasis elicits NF-kappaB activation, proinflammatory gene expression and localized neutrophilia in zebrafish. \u003cem\u003eDis Model Mech\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 1260-1270 (2013).\u003c/li\u003e\n\u003cli\u003eLin, J.\u003cem\u003e, et al.\u003c/em\u003e The peptide-binding cavity is essential for Als3-mediated adhesion of Candida albicans to human cells. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e289\u003c/strong\u003e, 18401-18412 (2014).\u003c/li\u003e\n\u003cli\u003ePhan, Q.T.\u003cem\u003e, et al.\u003c/em\u003e Als3 is a Candida albicans invasin that binds to cadherins and induces endocytosis by host cells. \u003cem\u003ePLoS Biol\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, e64 (2007).\u003c/li\u003e\n\u003cli\u003eLiu, Y. \u0026amp; Filler, S.G. Candida albicans Als3, a multifunctional adhesin and invasin. \u003cem\u003eEukaryot Cell\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 168-173 (2011).\u003c/li\u003e\n\u003cli\u003eZhao, X.\u003cem\u003e, et al.\u003c/em\u003e ALS3 and ALS8 represent a single locus that encodes a Candida albicans adhesin; functional comparisons between Als3p and Als1p. \u003cem\u003eMicrobiology (Reading)\u003c/em\u003e \u003cstrong\u003e150\u003c/strong\u003e, 2415-2428 (2004).\u003c/li\u003e\n\u003cli\u003evan Putten, J.P.M. \u0026amp; Strijbis, K. Transmembrane Mucins: Signaling Receptors at the Intersection of Inflammation and Cancer. \u003cem\u003eJ Innate Immun\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 281-299 (2017).\u003c/li\u003e\n\u003cli\u003eBork, P. \u0026amp; Patthy, L. The SEA module: a new extracellular domain associated with O-glycosylation. \u003cem\u003eProtein Sci\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 1421-1425 (1995).\u003c/li\u003e\n\u003cli\u003eMaeda, T.\u003cem\u003e, et al.\u003c/em\u003e Solution structure of the SEA domain from the murine homologue of ovarian cancer antigen CA125 (MUC16). \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e279\u003c/strong\u003e, 13174-13182 (2004).\u003c/li\u003e\n\u003cli\u003eLevitin, F.\u003cem\u003e, et al.\u003c/em\u003e The MUC1 SEA module is a self-cleaving domain. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e280\u003c/strong\u003e, 33374-33386 (2005).\u003c/li\u003e\n\u003cli\u003eOgonuki, N.\u003cem\u003e, et al.\u003c/em\u003e A high-speed congenic strategy using first-wave male germ cells. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, e4943 (2009).\u003c/li\u003e\n\u003cli\u003eHuang, Y.\u003cem\u003e, et al.\u003c/em\u003e IL-25-responsive, lineage-negative KLRG1(hi) cells are multipotential \u0026apos;inflammatory\u0026apos; type 2 innate lymphoid cells. \u003cem\u003eNat Immunol\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 161-169 (2015).\u003c/li\u003e\n\u003cli\u003eUnderwood, J.A., Williams, J.W. \u0026amp; Keate, R.F. Clinical findings and risk factors for Candida esophagitis in outpatients. \u003cem\u003eDis Esophagus\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 66-69 (2003).\u003c/li\u003e\n\u003cli\u003eSolis, N.V. \u0026amp; Filler, S.G. Mouse model of oropharyngeal candidiasis. \u003cem\u003eNat Protoc\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 637-642 (2012).\u003c/li\u003e\n\u003cli\u003eHajjeh, R.A.\u003cem\u003e, et al.\u003c/em\u003e Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program. \u003cem\u003eJ Clin Microbiol\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 1519-1527 (2004).\u003c/li\u003e\n\u003cli\u003eFehm, T., Beck, E., Valerius, T., Gramatzki, M. \u0026amp; Jager, W. CA 125 elevations in patients with malignant lymphomas. \u003cem\u003eTumour Biol\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 283-289 (1998).\u003c/li\u003e\n\u003cli\u003eSteentoft, C.\u003cem\u003e, et al.\u003c/em\u003e Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology. \u003cem\u003eEMBO J\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 1478-1488 (2013).\u003c/li\u003e\n\u003cli\u003eZhang, M., Cheng, S., Jin, Y., Zhao, Y. \u0026amp; Wang, Y. Roles of CA125 in diagnosis, prediction, and oncogenesis of ovarian cancer. \u003cem\u003eBiochim Biophys Acta Rev Cancer\u003c/em\u003e \u003cstrong\u003e1875\u003c/strong\u003e, 188503 (2021).\u003c/li\u003e\n\u003cli\u003eDas, S. \u0026amp; Batra, S.K. Understanding the Unique Attributes of MUC16 (CA125): Potential Implications in Targeted Therapy. \u003cem\u003eCancer Res\u003c/em\u003e \u003cstrong\u003e75\u003c/strong\u003e, 4669-4674 (2015).\u003c/li\u003e\n\u003cli\u003eMbanefo, E.C.\u003cem\u003e, et al.\u003c/em\u003e Characterization of a gene family encoding SEA (sea-urchin sperm protein, enterokinase and agrin)-domain proteins with lectin-like and heme-binding properties from Schistosoma japonicum. \u003cem\u003ePLoS Negl Trop Dis\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, e2644 (2014).\u003c/li\u003e\n\u003cli\u003eChen, S.H., Dallas, M.R., Balzer, E.M. \u0026amp; Konstantopoulos, K. Mucin 16 is a functional selectin ligand on pancreatic cancer cells. \u003cem\u003eFASEB J\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 1349-1359 (2012).\u003c/li\u003e\n\u003cli\u003eMuniyan, S.\u003cem\u003e, et al.\u003c/em\u003e MUC16 contributes to the metastasis of pancreatic ductal adenocarcinoma through focal adhesion mediated signaling mechanism. \u003cem\u003eGenes Cancer\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 110-124 (2016).\u003c/li\u003e\n\u003cli\u003eRump, A.\u003cem\u003e, et al.\u003c/em\u003e Binding of ovarian cancer antigen CA125/MUC16 to mesothelin mediates cell adhesion. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e279\u003c/strong\u003e, 9190-9198 (2004).\u003c/li\u003e\n\u003cli\u003eEdwards, J.E., Jr.\u003cem\u003e, et al.\u003c/em\u003e A Fungal Immunotherapeutic Vaccine (NDV-3A) for Treatment of Recurrent Vulvovaginal Candidiasis-A Phase 2 Randomized, Double-Blind, Placebo-Controlled Trial. \u003cem\u003eClin Infect Dis\u003c/em\u003e \u003cstrong\u003e66\u003c/strong\u003e, 1928-1936 (2018).\u003c/li\u003e\n\u003cli\u003eTakeichi, M. Cadherin cell adhesion receptors as a morphogenetic regulator. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e251\u003c/strong\u003e, 1451-1455 (1991).\u003c/li\u003e\n\u003cli\u003eStrale, P.O.\u003cem\u003e, et al.\u003c/em\u003e The formation of ordered nanoclusters controls cadherin anchoring to actin and cell-cell contact fluidity. \u003cem\u003eJ Cell Biol\u003c/em\u003e \u003cstrong\u003e210\u003c/strong\u003e, 333-346 (2015).\u003c/li\u003e\n\u003cli\u003eDejana, E., Orsenigo, F. \u0026amp; Lampugnani, M.G. The role of adherens junctions and VE-cadherin in the control of vascular permeability. \u003cem\u003eJ Cell Sci\u003c/em\u003e \u003cstrong\u003e121\u003c/strong\u003e, 2115-2122 (2008).\u003c/li\u003e\n\u003cli\u003eZhong, X., Chen, B., Yang, L. \u0026amp; Yang, Z. Molecular and physiological roles of the adaptor protein CARD9 in immunity. \u003cem\u003eCell Death Dis\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 52 (2018).\u003c/li\u003e\n\u003cli\u003eSwidergall, M.\u003cem\u003e, et al.\u003c/em\u003e EphA2 Is a Neutrophil Receptor for Candida albicans that Stimulates Antifungal Activity during Oropharyngeal Infection. \u003cem\u003eCell Rep\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 423-433 e425 (2019).\u003c/li\u003e\n\u003cli\u003eOrozco, A.S., Zhou, X. \u0026amp; Filler, S.G. Mechanisms of the proinflammatory response of endothelial cells to Candida albicans infection. \u003cem\u003eInfect Immun\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 1134-1141 (2000).\u003c/li\u003e\n\u003cli\u003ePhan, Q.T.\u003cem\u003e, et al.\u003c/em\u003e Serum bridging molecules drive candidal invasion of human but not mouse endothelial cells. \u003cem\u003ePLoS Pathog\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, e1010681 (2022).\u003c/li\u003e\n\u003cli\u003eRosentul, D.C.\u003cem\u003e, et al.\u003c/em\u003e Genetic variation in the dectin-1/CARD9 recognition pathway and susceptibility to candidemia. \u003cem\u003eJ Infect Dis\u003c/em\u003e \u003cstrong\u003e204\u003c/strong\u003e, 1138-1145 (2011).\u003c/li\u003e\n\u003cli\u003eAmrhein, V., Greenland, S. \u0026amp; McShane, B. Scientists rise up against statistical significance. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e567\u003c/strong\u003e, 305-307 (2019).\u003c/li\u003e\n\u003cli\u003ePolley, M.C., Korn, E.L. \u0026amp; Freidlin, B. Phase III Precision Medicine Clinical Trial Designs That Integrate Treatment and Biomarker Evaluation. \u003cem\u003eJCO Precis Oncol\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e(2019).\u003c/li\u003e\n\u003cli\u003eFiller, S.G., Pfunder, A.S., Spellberg, B.J., Spellberg, J.P. \u0026amp; Edwards, J.E., Jr. Candida albicans stimulates cytokine production and leukocyte adhesion molecule expression by endothelial cells. \u003cem\u003eInfect Immun\u003c/em\u003e \u003cstrong\u003e64\u003c/strong\u003e, 2609-2617 (1996).\u003c/li\u003e\n\u003cli\u003eSoderberg, O.\u003cem\u003e, et al.\u003c/em\u003e Direct observation of individual endogenous protein complexes in situ by proximity ligation. \u003cem\u003eNat Methods\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 995-1000 (2006).\u003c/li\u003e\n\u003cli\u003eColeman, D.A.\u003cem\u003e, et al.\u003c/em\u003e Monoclonal antibodies specific for Candida albicans Als3 that immunolabel fungal cells in vitro and in vivo and block adhesion to host surfaces. \u003cem\u003eJ Microbiol Methods\u003c/em\u003e \u003cstrong\u003e78\u003c/strong\u003e, 71-78 (2009).\u003c/li\u003e\n\u003cli\u003eCzogalla, J., Schweda, F. \u0026amp; Loffing, J. The Mouse Isolated Perfused Kidney Technique. \u003cem\u003eJ Vis Exp\u003c/em\u003e (2016).\u003c/li\u003e\n\u003cli\u003eKuwajima, T.\u003cem\u003e, et al.\u003c/em\u003e ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue. \u003cem\u003eDevelopment\u003c/em\u003e \u003cstrong\u003e140\u003c/strong\u003e, 1364-1368 (2013).\u003c/li\u003e\n\u003cli\u003eAlvarez, D.F.\u003cem\u003e, et al.\u003c/em\u003e Lung microvascular endothelium is enriched with progenitor cells that exhibit vasculogenic capacity. \u003cem\u003eAm J Physiol Lung Cell Mol Physiol\u003c/em\u003e \u003cstrong\u003e294\u003c/strong\u003e, L419-430 (2008).\u003c/li\u003e\n\u003cli\u003eKarczewski, K.J.\u003cem\u003e, et al.\u003c/em\u003e The mutational constraint spectrum quantified from variation in 141,456 humans. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e581\u003c/strong\u003e, 434-443 (2020).\u003c/li\u003e\n\u003cli\u003eMcCarthy, S.\u003cem\u003e, et al.\u003c/em\u003e A reference panel of 64,976 haplotypes for genotype imputation. \u003cem\u003eNat Genet\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 1279-1283 (2016).\u003c/li\u003e\n\u003cli\u003eDas, S.\u003cem\u003e, et al.\u003c/em\u003e Next-generation genotype imputation service and methods. \u003cem\u003eNat Genet\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 1284-1287 (2016).\u003c/li\u003e\n\u003cli\u003ePurcell, S.\u003cem\u003e, et al.\u003c/em\u003e PLINK: a tool set for whole-genome association and population-based linkage analyses. \u003cem\u003eAm J Hum Genet\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 559-575 (2007).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"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":"Virulence, Candida, MUC16, CA125, Endothelium, Cancer","lastPublishedDoi":"10.21203/rs.3.rs-8810991/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8810991/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Bloodstream infection with Candida species is associated with high mortality, yet the molecular basis of vascular adhesion and invasion remains unclear. We identify the endothelial surface mucin MUC16 as a key host factor enabling fungal vascular adherence. The MUC16 SEA domain directly engages the Candida adhesin Als3p and endothelial N-cadherin to form a ternary complex that promotes endothelial attachment and vascular invasion. Genetic deletion of Muc16 in mice markedly reduced fungal vascular binding, renal fungal burden, and Ly6Ghi CD11b+ neutrophil recruitment following intravenous infection, and diminished oral colonization in an oral model. Inflammatory induction of MUC16 occurred via MINCLE-, TLR9-, and Dectin-1–dependent signaling, converging on CARD9. In clinical cohorts, a MUC16 T10155I variant and elevated circulating MUC16 were associated with increased 30-day mortality. These findings establish MUC16 as a mechanistic link between endothelial sensing and Candida vascular invasion, with prognostic relevance in candidemia.","manuscriptTitle":"MUC16-dependent Renal Vascular Adhesion of Candida Promotes Tissue Invasion and Predicts Clinical Outcome in Candidemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-04 07:03:22","doi":"10.21203/rs.3.rs-8810991/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":"430ed274-73c9-422e-95dd-20f2e184b8ee","owner":[],"postedDate":"March 4th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-11T18:46:42+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-04-30T22:04:37+00:00","index":2,"fulltext":"This content is not available."}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":63865452,"name":"Health sciences/Medical research"},{"id":63865453,"name":"Biological sciences/Microbiology/Fungi"}],"tags":[],"updatedAt":"2026-03-04T07:03:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-04 07:03:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8810991","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8810991","identity":"rs-8810991","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}