Brucella abortushijacks the host protein Slc2a1via the SepA effector to promote intracellular survival in macrophages

Brucella abortushijacks the host protein Slc2a1via the SepA effector to promote intracellular survival in macrophages | 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 Research Article Brucella abortus hijacks the host protein Slc2a1via the SepA effector to promote intracellular survival in macrophages Yuanhao Yang, Yaping Zu, Xin Wang, Hui Wang, Xiaofang Liu, Ting Tang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5800583/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Brucella spp. are facultative intracellular bacteria that infect and induce brucellosis in a diverse range of mammalian hosts. The disease causes major global economic losses and also is a worldwide threat to public health security. Characterization of bacterial and host factors that promote intracellular survival of Brucella is key for the prevention and control of brucellosis. In this study, we identified proteins involved in intracellular survival of Brucella abortus A19 in RAW264.7 macrophage cells by liquid chromatography-mass spectrometry of macrophages with or without B. abortus infection. The functions of these proteins, the signaling pathways in which the proteins participate, the domain entries enriched by the proteins, and the subcellular localization of the differentially-expressed proteins were deciphered. Differential protein expression revealed that Slc2a1, which is a key Glycolytic protein, was significantly upregulated in infected macrophage cells. This observation was confirmed by qRT-PCR and Western blotting studies. The role of Slc2a1 in the intracellular survival of B. abortus was probed by overexpressing and knocking down SLC2A1 in RAW264.7 cells. Overproduction of the protein promoted intracellular proliferation of B. abortus whereas knockdown of SLC2A1 inhibited proliferation of the bacterium. Finally, we determined that the Secreted Effector Protein A (SepA) effector of B. abortus enhanced SLC2A1 expression in macrophage cells. Thus, B. abortus stimulates host SLC2A1 expression via the SepA effector protein to aid bacterial survival in the macrophage environment which suggests that SepA may be a novel antibacterial target to combat Brucella infection. B. abortus A19 LC-MS/MS Slc2a1 SepA intracellular survival Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Brucella is a facultative intracellular bacterium that infects and causes brucellosis in a wide range of mammalian hosts [ 1 ]. Brucella spp. infect goats, sheep, cattle, pigs, and dogs, and cause abortion and infertility in these hosts [ 2 ]. Twelve Brucella genotypes have been described among which Brucella melitensis , B. abortus , B. suis , B. canis , and B. ovis are the most prominent genera. The bacterium is disseminated widely in more than 170 countries, causes major economic losses, and also is a significant global public health threat [ 3 ]. For example, recent studies suggest 1.6 to 2.1 million new cases occur annually in humans [ 4 ]. Macrophages are the natural host cells of Brucella . The bacterium adopts numerous strategies to resist attack during infection of macrophages which causes host cells to undergo diverse physiological and biochemical responses. Brucella modulates autophagy and apoptosis pathways, the immune response, and metabolic processes in macrophages, thereby evading clearance by the host cell. The VceA and BtpB proteins are important effectors that are secreted by the type IV secretion system (T4SS) in Brucella . Deletion of the vceA and btpB genes leads to an increase in levels of the host LC3-II protein which is a key factor in the autophagy pathway and a decrease in p62 protein that is part of the nuclear pore complex of the nuclear envelope, as well as an increase in autophagic lysosomes [ 5 , 6 ]. T4SS effector proteins BspJ and BspG are nucleus-regulating proteins secreted by Brucella that enter and function in the host cell nucleus. Deletion of the corresponding loci leads to enhanced apoptosis of macrophages which reduces Brucella survival and increases the risk of bacterial killing by abnormal surges in inflammatory factor secretion [ 7 – 9 ]. Macrophages are a critical part of the host immune system and Brucella infection leads to macrophage M1 polarization [ 10 – 12 ]. Activated M1 macrophages release numerous pro-inflammatory factors to combat pathogen invasion through the pro-inflammatory immune response [ 13 ]. M1 macrophages have a high demand for energy and biosynthetic raw materials and mainly complete biochemical processes via glycolysis. Although this process is less efficient than oxidative phosphorylation in generating energy, glycolysis nevertheless rapidly generates energy to meet the needs of macrophages. Glycolysis is important for maintaining macrophage immune function, and hexokinase-1 and PKM2 enzymes in this pathway are involved in the activation of inflammasomes [ 14 ]. Glycolysis is inhibited in M1 macrophages which affects the production of both reactive oxygen species and cytokines [ 15 ]. Although glycolysis provides energy for macrophages to combat invading microbial pathogens, Brucella takes advantage of host cell glycolysis to facilitate survival [ 12 ]. The role of host cell glycolysis in Brucella infection and the mechanism by which glycolysis regulates intracellular survival of the bacterium require further analysis. In this work, we determined that Brucella infection modulates expression of the SLC2A1 gene in host macrophages. Slc2a1 is an important and widely-distributed glucose transporter in mammalian cells [ 16 ], and is implicated in both cellular glycolysis and glucose metabolism [ 17 , 18 ]. The protein also exerts an important role in the immune response of macrophages and participates in the production of inflammatory factors [ 15 , 16 , 19 , 20 ]. The contribution of Slc2a1 to intracellular survival of Brucella was previously uncertain, but we show here that Slc2a1 plays an important role in regulating bacterial persistence in RAW264.7 macrophages. Moreover, the Secreted Effector Protein A (SepA) of the Brucella T4SS promotes SLC2A1 expression in these cells which suggests that SepA may be a novel antibacterial target to combat Brucella infection. 2. Materials and Methods 2.1 Bacterial strains and RAW264.7 cell line The B. abortus A19 vaccine strain was provided by Shaanxi Veterinary Drug Supervision Institute (shaanxi province) and was grown on tryptic soy agar or tryptic soy broth at 37°C. B. abortus A19 colonies were selected from agar medium and inoculated in tryptic soy broth. For cell enumeration, 100 µl of the cell suspension was diluted in a ten-fold gradient after 24 hours of growth and plated on tryptic soy agar medium. Colony numbers were counted after 72 h at 37°C. RAW264.7 macrophage cells were cultured in DMEM containing 10% fetal bovine serum (FBS) at 5% CO 2 at 37°C. 2.2 Liquid chromatography-mass spectrometry in data-independent acquisition mode and sequencing The RAW264.7 cells were seeded in 100 mm cell culture dishes in DMEM containing 10% FBS. When the cell density reached 70%, cells were infected with B. abortus A19 (MOI = 200) for 4 h, and the cell medium then was changed to DMEM containing 10% FBS with gentamicin (50 ng/µl) for 1 h. The medium subsequently was changed to DMEM containing 10% FBS with gentamicin (25 ng/µl) for 24 h at which time cells were harvested and placed at -80°C. Subsequently, the samples were analyzed by protein liquid chromatography-mass spectrometry (LC-MS/MS) (Novogene Bioinformatics Technology [Beijing, China]). Gene Ontology (GO) and InterPro (IPR) functional analyses were conducted using the InterProScan program against non-redundant protein databases, including Pfam, PRINTS, ProDom, SMART, ProSite, and PANTHER. The Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to analyze protein families and pathways. Differentially expressed proteins were used for Volcanic map analysis, cluster heat map analysis, and enrichment analysis of GO, IPR and KEGG. 2.3 Western blotting The RAW264.7 cells were lysed using RIPA Lysis Buffer (Beyotime, Shanghai, China), and total protein concentration subsequently was measured using the BCA kit (Solarbio, Beijing, China). Ten micrograms of each sample were analyzed by 12.5% SDS-PAGE and then transferred to a PVDF membrane. The membrane was incubated with TBST buffer containing 10% skim milk for 2 h at room temperature and subsequently transferred to TBST containing Slc2a1 antibody (1:1000) or β-actin (1:1000) antibody (both from Proteintech, Wuhan, China) for 2 h at room temperature. Unbound antibody was washed off with TBST followed by incubation with HRP-labeled rabbit secondary antibody (1:5000) (Beijing CWBIO, Beijing, China) in TBST for 2 h at room temperature. Visualization was performed by enhanced chemiluminescence after an additional TBST wash. 2.4 RNA interference The siSlc2a1-526, siSlc2a1-820, siSlc2a1-1121, and siSlc2a1-1470 small interfering RNA (siRNA) molecules were designed and synthesized by GenePharma (Shanghai, China) (Table 1 ). The relevant siRNA was transfected into RAW264.7 cells using Advanced DNA RNA Transfection Reagent (Zeta Life, USA) followed by infection with B. abortus A19 (MOI = 200). The cell culture medium was discarded after 4 h incubation and the cells were washed with PBS in triplicate. The medium was replaced with DMEM containing 10% FBS with gentamicin (50 ng/µl) for 1 h, followed by replacement with medium containing 25 ng/µl gentamicin. Cells were harvested 24 h later and total RNA or cell lysates were prepared. Table 1 Primers or siRNA used in this work Gene forward reverse siSlc2a1-526 5'-GCUUCAUCAUCGGUGUGUATT-3' 5'-UACACACCGAUGAUGAAGCTT-3' siSlc2a1-820 5'-CCAAGAGUGUGCUGAAGAATT-3' 5'-UUCUUCAGCACACUCUUGGTT-3' siSlc2a1-1121 5'-GCUGUUUGUUGUAGAGCGATT-3' 5'-UCGCUCUACAACAAACAGCTT-3' siSlc2a1-1470 5'-CUCGUGCUCUUCUUCAUCUTT-3' 5'-AGAUGAAGAAGAGCACGAGTT-3' q-Slc2a1 5'-GCTGTGCTTATGGGCTTCTC-3' 5'-CACATACATGGGCACAAAGC-3' q-β-actin 5'-GGCTGTATTCCCCTCCATCG-3' 5'-CCAGTTGGTAACAATGCCATGT-3' Slc2a1 5'-AAGCTTATGTTGGCTGTGGGAGGAGCAGTGC-3' 5'-GGATCCTCACACTTGGGAGTCCGCCCCCAGA − 3 ' SepA 5'-AAGCTTATGATGCCCGTGATTAGACT − 3' 5'-GGATCCTTAGGCGGACGCCGGGCCAG − 3 ' BspE 5'-GCGGCCGCGATGACGTTATCGACGCGTAT-3' 5'-GGTACCTCAGGCAGCAACTTGCGATG-3' NyxA 5'-AAGCTTATGAACGCTCACACAAACAT-3' 5'-GGATCCTCAAAGCTCCAAGCATCTAA-3' NyxB 5'-AAGCTTATGAACACGCAAGCAACAAT − 3' 5'-GGATCCTCAAGGCATCTCGATAAGGC − 3' 2.5 RNA extraction and Quantitative Real-Time PCR Total RNA was extracted from RAW264.7 cells using TRIzol reagent (Invitrogen, California, USA), and was reverse transcribed to cDNA using the PrimeScript RT Reagent Kit (TaKaRa, Tokyo, Japan). Quantitative Real-Time PCR (qRT-PCR) was performed with the ChamQ SYBR qPCR Master-Mix Kit (Vazyme, Nanjing, China) in the Bio-Rad CFX96 real-time PCR System (Bio-Rad, Hercules, USA). The qRT-PCR primers were q-Slc2a1 and q-β-actin for the relevant genes (Table 1 ). Relative gene expression was calculated using the 2 −ΔΔCT method. 2.6 Plasmids The cDNA of SLC2A1 , and the sepA , bspE , nyxA and nyxB genes were cloned into the p3xFLAG-CMV vector for production of proteins that are tagged with 3xFLAG at the N-terminus in mammalian cells. HindIII and EcoRI restriction enzymes were used for all plasmid constructions. Primers used are listed in Table 1 . 2.7 Enumeration of B. abortus in infected macrophages RAW264.7 cells were seeded in 24-well cell culture plates at a density of 2x10 5 cells/well and were infected with B. abortus A19 (MOI = 200). The culture medium was discarded and cells were washed in triplicate with PBS after 4 h. The culture medium was changed to DMEM containing 10% FBS with gentamicin (50 ng/µl) for 1 h, and then was changed to DMEM containing 10% FBS with gentamicin (25 ng/µl). Cells were harvested after 24 h and lysed with 0.5% Triton X-100 at room temperature for 10 min. The lysate was diluted in a ten-fold gradient, inoculated on tryptic soy agar, and colony numbers were counted after 72 h at 37°C. 2.8 Statistical analysis Bar graphs were plotted to show the mean ± standard deviation (SD) of at least three independent experiments. All statistical analyses were performed by two-sided Student's t -test. P < 0.05 and P < 0.01 indicate significant and highly significant differences, respectively. 3. Results 3.1 Identifcation of differentially expressed proteins in B. abortus -infected and uninfected macrophages Differentially expressed proteins in RAW264.7 macrophages infected with B. abortus A19 compared to uninfected cells were identified by LC-MS/MS in data-independent acquisition mode. A total of 2702 proteins were distinguished, of which 1339 and 1363 proteins were significantly upregulated and downregulated, respectively (Fig. 1 A). These differentially expressed proteins are presented in a Volcano plot (Fig. 1 B) and were analyzed further by cluster analysis. Genes that were upregulated and downregulated were observed by cluster heatmaps (Fig. 1 C). 3.2 GO and KEGG enrichment analysis in B. abortus -infected and uninfected macrophages GO functional significant enrichment analysis determines the GO functional items that are enriched significantly in a set of differentially expressed proteins compared with all identified proteins, thereby ascribing biological functions to the former. GO enrichment analysis was performed on differentially expressed proteins in B. abortus -infected and uninfected RAW264.7 macrophages, and Biological Process, Cellular Component, and Molecular Function aspects were assessed. Differentially expressed proteins in Biological Process mainly were implicated in protein dephosphorylation, microtubule-based processes, ribosome biogenesis, ribonucleoprotein complex biogenesis, immune system processes, and small molecule biosynthesis. In terms of Cellular Component, differentially expressed proteins were found principally in the extracellular region, extracellular space, preribosome, small-subunit processome, microtuble organizing center, and spindle. Differentially expressed proteins in Molecular Function were involved mainly in phosphatase activity, receptor activity, phosphoprotein phosphatase activity, protein tyrosine phosphatase activity, acyl-CoA dehydrogenase activity, and regulatory region DNA binding (Fig. 2 A and 2 B). KEGG enrichment analysis was performed by applying the hypergeometric test to identify pathways that were enriched significantly in the set of differentially expressed proteins compared with the background panel of all identified proteins. This analysis showed that differentially expressed proteins were involved mainly in signaling pathways, including pathways in cancer, pathways in influenza A, Kaposi's sarcoma-associated herpesvirus infection, hepatitis C infection, IL-17 signaling pathway, ribosome biogenesis in eukaryotes, p53 signaling pathway, pathways in small cell lung cancer, and fatty acid elongation (Fig. 2 C and 2 D). 3.3 Domain enrichment analysis and subcellular localization studies of differentially expressed proteins in B. abortus -infected and uninfected macrophages Domain enrichment analysis identifies domain entries that are statistically enriched in a defined set of proteins. Domain enrichment analysis here showed that the domains of differentially expressed proteins between B. abortus -infected and uninfected macrophages mainly included the helicase superfamily 1/2 ATP-binding domain, Death-like domain, immunoglobulin V-set, protein-tyrosine phosphatase, receptor/non-receptor type, RUN, tetraspanin/peripherin, and transcription factor jumonji JmiN domains (Fig. 3 A). In addition, subcellular localization analyses showed that the differentially expressed proteins were distributed mainly in proteins of the nucleus (36.28%), cytoplasm (18.47%), plasma membrane (10.21%), endoplasmic reticulum (7.06%), mitochondrion (7.01%), Golgi apparatus (5.11%), as well as extracellular proteins (3.75%) (Fig. 3 B). 3.4 B. abortus infection promotes the expression of Slc2a1 in macrophage cells Lactate is the end product of glycolysis and lactate levels may reflect changes in glycolytic activity. Interestingly, B. abortus infection significantly promoted the production of lactate in RAW264.7 macrophages (Fig. 4 A). LC-MS/MS analysis was performed to explore how infection induced changes in host glycolysis activity which revealed that the Slc2a1 protein was significantly upregulated in pathways in cancer signaling obtained in KEGG enrichment analysis. One hundred differentially expressed proteins were identified in pathways in cancer among which only Slc2a1 was involved in glycolysis (Fig. 2 B and 4 B). The Slc2a1 protein is an important and widely-distributed glucose transporter in mammalian cells [ 16 ], and exerts a key role in cellular glycolysis and glucose metabolism [ 17 , 18 ]. Uninfected and infected cell samples were harvested and analyzed by qRT-PCR and Western blotting to assess further the expression of SLC2A1 . The qRT-PCR assays showed that SLC2A1 mRNA levels were upregulated significantly in B. abortus -infected cells (Fig. 4 C) and Western blotting also indicated that the level of Slc2a1 protein in infected cells was significantly higher than in uninfected cells (Fig. 4 D and 4 E). Thus, the enhanced expression of SLC2A1 in macrophages following B. abortus infection may underpin the elevated levels of lactate that were detected in these cells. 3.5 Slc2a1 is involved in regulation of B. abortus intracellular proliferation in macrophages Although Slc2a1 expression was upregulated in RAW264.7 cells following B. abortus infection, it is unknown whether the protein plays a role subsequent to infection. The role of Slc2a1 in infected cells was explored by using an overexpression plasmid to overproduce the protein following bacterial infection. Overexpression of SLC2A1 significantly promoted the intracellular proliferation of B. abortus in RAW264.7 cells (Fig. 5 A and 5 B). Subsequently, we designed and synthesized four siRNA (siSlc2a1) to knock down SLC2A1 mRNA levels. The qRT-PCR and Western blotting results revealed that siSlc2a1-1121 and siSlc2a1-1470 significantly reduced both mRNA and protein levels of Slc2a1 compared with the non-interfering siRNA (siNC) (Fig. 5 C and 5 D). Knockdown assays subsequently were performed on B. abortus -infected cells: intracellular proliferation of the bacterium was inhibited significantly by using siSlc2a1-1470 (Fig. 5 E). As Slc2a1 may be involved in regulating the expression of inflammatory factors [ 16 , 19 – 21 ], we hypothesized that the protein may control the intracellular proliferation of B. abortus by modulating the expression of inflammatory factors that are involved in combatting infection. However, knockdown of SLC2A1 did not affect the elevated levels of either IL-1β and IL-6 cytokines that occur after B. abortus infection of RAW264.7 macrophages (Fig. 5 F and 5 G). The preceding data suggest that Slc2a1 is involved in counteracting B. abortus infection of macrophages, but that this effect is not mediated via IL-1β or IL-6 cytokines. 3.6 B. abortus effector protein SepA promotes Slc2a1 expression The T4SS is a critical Brucella virulence factor that is essential for intracellular survival of the bacterium [ 22 , 23 ]. We speculated that B. abortus may promote post-infection expression of SLC2A1 via effector proteins secreted by the T4SS. Therefore, plasmids that overexpress the sepA, bspE, nyxA , or nyxB effector genes of B. abortus were constructed (Fig. 6 A). Macrophage expression of Slc2a1 increased significantly when SepA was overexpressed, but not when BspE, NyxA, or NyxB were overproduced (Fig. 6 B). SepA sequences of B. abortus A19, B. abortus 2308, B. suis 1330, and B. canis ATCC 23365 were aligned. The sequences were completely consistent in the B. abortus homologs, with only a few substitutions in the SepA proteins in the other genera (Fig. 6 C). In summary, the preceding data suggest that the conserved SepA effector protein enhances expression of host Slc2a1 to facilitate survival of B. abortus in the macrophage environment. 4. Discussion Brucella spp. are facultative intracellular bacterial parasites that induce a series of physiological perturbations in host cells which activate the host immune response. Therefore, avoidance of elimination by the host is a key survival strategy for Brucella . Brucella deploys diverse stratagems and also subverts host functions to circumvent killing and to adapt to the intracellular environment. Identification of the bacterial and host factors that affect the intracellular proliferation of Brucella is critical for prevention and control of Brucella infections. Brucella infection causes numerous physiological and biochemical fluctuations in host cells, including initiation of autophagy, apoptosis, mitophagy, and inflammation [ 5 , 6 , 24 – 27 ]. Brucella infection of macrophages alters the expression of key macrophage proteins. Certain proteins are used by host cells to resist Brucella invasion, whereas other factors are hijacked by the bacterium for intracellular survival. In the present study, we performed LC-MS/MS analysis of uninfected RAW264.7 macrophage cells and of cells infected with B. abortus A19 with the aim of detecting differentially expressed proteins that are hallmarks of infection. This analysis revealed changed production of 2702 proteins in infected cells compared with uninfected macrophages, among which 1339 and 1363 proteins were upregulated and downregulated, respectively (Fig. 1 ). GO functional significant enrichment analysis indicated the functional entries that were enriched in the differentially expressed proteins, thereby revealing the biological functions with which the differential proteins were associated. This analysis indicated that the pertinent proteins were involved mainly in microtubule-based processes, protein dephosphorylation, immune system processes, and pathways in cancer. In addition, the hypergeometric distribution was used in KEGG enrichment analysis to assess whether the set of differential proteins in a pathway was higher than the set of proteins external to the pathway. The differentially expressed proteins were implicated principally in signaling pathways, including pathways in cancer, pathways in influenza A, Kaposi's sarcoma-associated herpesvirus infection, hepatitis C, and the IL-17 signaling pathway (Fig. 2 ). Furthermore, domain enrichment analysis showed that the domains of differentially expressed proteins between uninfected and B. abortus -infected cells mainly included the helicase superfamily 1/2 ATP-binding domain, Death-like domain, immunoglobulin V-set, protein-tyrosine phosphatase, receptor/non-receptor type, RUN, tetraspanin/peripherin, and transcription factor jumonji JmiN domains. Finally, subcellular localization enrichment analysis showed that the differentially expressed proteins were mainly distributed in the nucleus, followed by the cytoplasm, plasma membrane, and endoplasmic reticulum (Fig. 3 ). LC-MS/MS analysis demonstrated that the internal environment of macrophage cells underwent major changes after B. abortus infection, and that the expression of proteins related to metabolism, the immune system, material transport, and protein modification was altered significantly. Proteins localized in the nucleus accounted for a large proportion of the differentially expressed proteins. We hypothesize that the expression of nuclear proteins is modified first following macrophage infection by B. abortus , and that these regulatory perturbations subsequently modulate expression of cytoplasmic proteins, membrane factors, and other proteins to achieve clearance of Brucella . Conversely, B. abortus may hijack nuclear proteins to regulate cytoplasmic factors, membrane proteins, and other cellular components to promote intracellular survival. Therefore, determining the functions of differentially expressed proteins is crucial to uncovering the mechanisms of Brucella infection and intracellular persistence. Host cells respond quickly to invasion by pathogenic microorganisms by synthesizing and releasing diverse inflammatory factors. Cells rapidly initiate glycolysis in order to meet the accompanying energy requirements. Although glycolysis provides energy for host immune responses to resist invasion by pathogens, glycolysis may also help pathogen survival [ 28 – 32 ]. Accordingly, Brucella infection upregulates host cell glycolysis which aids intracellular survival of the bacterium [ 12 ]. However, the mechanism by which Brucella upregulates glycolysis is unknown. Here, we found that lactate concentrations in macrophage cells increased following B. abortus infection. Lactate is the end product of glycolysis for which the molecule also is an indicator. LC-MS/MS, qRT-PCR and Western blotting analysis showed that expression of the Slc2a1 protein increased in macrophages infected with B. abortus (Fig. 4 C- 4 E). Slc2a1 is an important and widely distributed glucose transporter in mammalian cells [ 16 – 18 ]. We hypothesized that B. abortus upregulates glycolytic levels in macrophages via the Slc2a1 protein. The role of Slc2a1 in macrophage infection by B. abortus was explored further with an Slc2a1 overexpression plasmid which revealed that overproduction of the protein promoted intracellular proliferation of the bacterium (Fig. 5 A and 5 B), whereas knockdown of Slc2a1 by siRNA inhibited proliferation (Fig. 5 C- 5 E). Thus, Slc2a1 indeed plays an important role in the intracellular persistence of Brucella . As the Slc2a1 protein participates in regulating expression of host inflammatory factors [ 16 , 19 – 21 ], we hypothesized that the protein promoted the proliferation of B. abortus by modifying expression of host inflammatory factors. However, Slc2a1 was not involved in the expression of IL-1β and IL-6 cytokines following B. abortus infection of macrophages (Fig. 5 F and 5 G). Thus, the molecular mechanism by Slc2a1 enhances intracellular proliferation of B. abortus requires further exploration. The T4SS is a key virulence factor that is essential for the intracellular survival of Brucella . The T4SS in Brucella is a 12 protein complex that is encoded by the VirB operon [ 33 ]. The complex acts through 15 secreted effector proteins which impinge on vital host signaling pathways, thereby helping the bacterium to survive and replicate in the host [ 34 ]. We speculated that the T4SS in B. abortus may play an important role in promoting the expression of host Slc2a1 protein. In agreement with this hypothesis, the T4SS effector protein SepA stimulated Slc2a1 expression which accords with the finding that SepA is involved in regulating Brucella survival within host cells [ 35 ]. SepA appears to be highly conserved in Brucella spp. (Fig. 6 ) which suggests that the protein may perform equivalent functions in different mammalian hosts. In summary, the data here suggest that B. abortus secretes the effector protein SepA via the T4SS which promotes the expression of host Slc2a1 to assist the intracellular persistence of this pathogen. These observations also suggest that SepA may be a promising antibacterial target to combat infection by Brucella . Declarations Funding This work was supported by funds from the National Natural Science Foundation of China (Grant No. 32373016, 31672584) Authors' contributions Aihua Wang: Supervision, Funding acquisition, Data curation, Conceptualization. Yaping Jin: Supervision, Funding acquisition, Data curation, Conceptualization. Yuanhao Yang: Writing – original draft, Validation, Methodology, Investigation, Formal analysis. Yaping Zu: Writing – original draft, Validation, Methodology, Investigation, Formal analysis. Dong Zhou: Supervision, Conceptualization. Hui Wang: Supervision. Xiaofang Liu: Supervision, Software. Gaowa Wudong: Supervision, Software. 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Jiang L., Wang P., Song X., Zhang H., Ma S., Wang J., Li W., Lv R., Liu X., Ma S., Yan J., Zhou H., Huang D., Cheng Z., Yang C., Feng L., Wang L., Salmonella Typhimurium reprograms macrophage metabolism via T3SS effector SopE2 to promote intracellular replication and virulence, Nature Communications. (2021) 12. Zhou L., He R., Fang P., Li M., Yu H., Wang Q., Yu Y., Wang F., Zhang Y., Chen A., Peng N., Lin Y., Zhang R., Trilling M., Broering R., Lu M., Zhu Y., Liu S., Hepatitis B virus rigs the cellular metabolome to avoid innate immune recognition, Nature Communications. (2021) 12. Li H., Lin C., Qi W., Sun Z., Xie Z., Jia W., Ning Z., Senecavirus A-induced glycolysis facilitates virus replication by promoting lactate production that attenuates the interaction between MAVS and RIG-I, PLOS Pathogens. (2023) 19. Chen L.F., Cai J.X., Zhang J.J., Tang Y.J., Chen J.Y., Xiong S., Li Y.L., Zhang H., Liu Z., Li M.M., Respiratory syncytial virus co-opts hypoxia-inducible factor-1α-mediated glycolysis to favor the production of infectious virus, mBio. (2023) 14:e0211023. Alvarez-Martinez C.E., Christie P.J., Biological Diversity of Prokaryotic Type IV Secretion Systems, Microbiology and Molecular Biology Reviews. (2009) 73:775-808. Zheng M L.R., Zhu J D.Q., Chen J J.P., Zhang H L.J., Z. C., Effector Proteins of Type IV Secretion System: Weapons of Brucella Used to Fight Against Host Immunity., Curr Stem Cell Res Ther. (2024) 19:145-153. Döhmer P.H., Valguarnera E., Czibener C., Ugalde J.E., Identification of a type IV secretion substrate ofBrucella abortusthat participates in the early stages of intracellular survival, Cellular Microbiology. (2014) 16:396-410. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5800583","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":403675073,"identity":"2e68b0f0-a0b8-4180-a3ed-599029540d4c","order_by":0,"name":"Yuanhao Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYDADAwYG5sd/fvyTY2NvP0C0FjYD3p4Dxnw8ZxKI1sIgwcN2IHGehIMBfpXHzx5+zdtml2fOfviAgQTPnfQ2CYYEhh8V23BrOZOXZs3bllxs2ZOW8MDA4llum3TjAcaeM7dxazmQY2bM28acuOFAjoFBAg9zbpvMgQRmxjY8Ws6/AWmpT9xw/o2BxAE25nQ2iQQD/Fpu5Bg/5m07nLjhRo6BZAPb4QSCWiRvvDFjnHPuOFDLszRjxp40wzZgIB/E5xe+8znGH96UVQMdlnz4McMPG3n59vaDD35U4NaicICBTYoHXfQATvVAIN/AwPzxBz4Vo2AUjIJRMAoA+8lfmzjns0sAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0005-2232-5193","institution":"Northwest A\u0026F University","correspondingAuthor":true,"prefix":"","firstName":"Yuanhao","middleName":"","lastName":"Yang","suffix":""},{"id":403675074,"identity":"d5125d25-bcaa-4f2a-b6dc-b40bb28865a7","order_by":1,"name":"Yaping Zu","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Yaping","middleName":"","lastName":"Zu","suffix":""},{"id":403675075,"identity":"797f76c3-36b2-4262-bc2b-776e1d9e1b82","order_by":2,"name":"Xin Wang","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Wang","suffix":""},{"id":403675076,"identity":"a783c521-2f47-4952-82ee-2ec38fbd9647","order_by":3,"name":"Hui Wang","email":"","orcid":"","institution":"Jigedaqi District Livestock and Aquaculture Service Center","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Wang","suffix":""},{"id":403675077,"identity":"e51a3d0a-0353-42ae-bc0b-943b1f923ef2","order_by":4,"name":"Xiaofang Liu","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Xiaofang","middleName":"","lastName":"Liu","suffix":""},{"id":403675078,"identity":"68e6240f-9ac5-4397-8896-784d42e838eb","order_by":5,"name":"Ting Tang","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Tang","suffix":""},{"id":403675079,"identity":"fd73048b-d5b8-4e98-a4aa-aec591bba8b2","order_by":6,"name":"Yunyi Zhai","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Yunyi","middleName":"","lastName":"Zhai","suffix":""},{"id":403675080,"identity":"9809ca34-5b7e-441f-8296-2db375ad0732","order_by":7,"name":"Gaowa Wudong","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Gaowa","middleName":"","lastName":"Wudong","suffix":""},{"id":403675081,"identity":"82c79748-acde-4389-84ab-62be214513f2","order_by":8,"name":"Pingping Wang","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Pingping","middleName":"","lastName":"Wang","suffix":""},{"id":403675082,"identity":"6d3a6dd8-9ab5-43ef-ab8b-12aafac56784","order_by":9,"name":"Ningqiu Yuan","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Ningqiu","middleName":"","lastName":"Yuan","suffix":""},{"id":403675083,"identity":"88f5d5be-63a0-4762-ad5d-f1ee26040477","order_by":10,"name":"Dong Zhou","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Dong","middleName":"","lastName":"Zhou","suffix":""},{"id":403675084,"identity":"844dcd1e-624d-4cf4-b4fc-903a19a71f91","order_by":11,"name":"Yaping Jin","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Yaping","middleName":"","lastName":"Jin","suffix":""},{"id":403675085,"identity":"23df497e-adb1-4025-a549-625dde3bb4b8","order_by":12,"name":"Aihua Wang","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Aihua","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-01-10 05:06:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5800583/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5800583/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":74268867,"identity":"37c886d2-3991-4d5f-b35c-85b2f61cc735","added_by":"auto","created_at":"2025-01-20 13:27:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":508750,"visible":true,"origin":"","legend":"Differentially expressed proteins in uninfected and A19-infected RAW264.7 macrophage cells. (A) Numbers of differentially expressed proteins in uninfected and infected cells. (B) Scatter plot of proteins co-expressed in uninfected and infected macrophages. (C) Heat map showing levels of differentially expressed proteins in uninfected and infected cells. Experiments were repeated at least three times with similar results. *, \u0026thinsp;\u0026lt;\u0026thinsp;0.05; **, \u0026thinsp;\u0026lt;\u0026thinsp;0.01.","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5800583/v1/3fee0afac291f456b1cded8f.jpg"},{"id":74267292,"identity":"473a4fbf-dad7-4419-8cbf-c43d11517895","added_by":"auto","created_at":"2025-01-20 13:11:19","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":903095,"visible":true,"origin":"","legend":"\u003cp\u003eGO and KEGG enrichment analyses of differentially expressed proteins. (A, B) GO enrichment analysis of the biological functions that were associated significantly with differentially expressed proteins in uninfected and infected cells. BP, Biological Process; CC, Cellular Component; and, MF, Molecular Function. (C, D) KEGG enrichment analysis revealed major biochemical metabolic pathways and signal transduction pathways that involved differentially expressedproteins in uninfected and infected macrophages. Experiments were repeated at least three times with similar results. *, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5800583/v1/c73a7805e90848b34524b176.jpg"},{"id":74269018,"identity":"9e3538ba-2abd-4dfc-a016-3ac813c968a6","added_by":"auto","created_at":"2025-01-20 13:35:19","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":653209,"visible":true,"origin":"","legend":"\u003cp\u003eDomain enrichment analysis and subcellular localization analysis of differentially expressed proteins. (A) Domain enrichment showed domain entries that were enriched significantly for differentially expressedproteins in uninfected and infected cells. (B) Subcellular localization of differentially expressedproteins. Cellular localization of these proteins in uninfected and infected cells was analyzed. Experiments were repeated at least three times with similar results. *, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5800583/v1/945c0f0ad993e27e8c45008d.jpg"},{"id":74267527,"identity":"a3e3cee8-865c-4d14-b6b5-4366dd9e2687","added_by":"auto","created_at":"2025-01-20 13:19:19","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":610365,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eB. abortus\u003c/em\u003eA19 infection promotes expression of Slc2a1 in RAW264.7 macrophage cells. (A) RAW264.7 cells were infected or not infected with \u003cem\u003eB. abortus \u003c/em\u003efor 24 h. Supernatants of RAW264.7 cells were harvested and the L-lactate content was detected. (B) Heat map showing Slc2a1 protein levels in uninfected and infected cells in pathways in cancer (C-E). qRT-PCR and western blotting were used to assess mRNA and protein levels, respectively, of Slc2a1 in RAW264.7 cells uninfected and infected with \u003cem\u003eB. abortus\u003c/em\u003e A19 for 24 h. Experiments were repeated at least three times with similar results. *, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5800583/v1/c95501a8cc5d5f4426dd6d7c.jpg"},{"id":74267296,"identity":"068051a7-e316-4370-aad4-07e70293ce7e","added_by":"auto","created_at":"2025-01-20 13:11:19","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":522502,"visible":true,"origin":"","legend":"\u003cp\u003eSlc2a1 is involved in intracellular proliferation of \u003cem\u003eB. abortus\u003c/em\u003e in RAW264.7 macrophage cells. (A) RAW264.7 cells were transfected with p3xFLAG-CMV empty vector or p3xFLAG-CMV-Slc2a1 and were harvested after 48 h. Expression of Slc2a1 was detected by Western blotting and the difference in expression was determined by gray scale analysis. (B) RAW264.7 cells were transfected with p3xFLAG-CMV or p3xFLAG-CMV-Slc2a1 and infected with \u003cem\u003eB. abortus\u003c/em\u003e A19 (MOI=200) after 24 h. Cells were harvested after an additional 24 h and bacterial CFUs were determined. (C,D) RAW264.7 cells were transfected with siSlc2a1 or siNC and cells were harvested after 24 h. The mRNA and protein expression of Slc2a1 were detected by qRT-PCR and Western blotting, respectively, and differences in expression were determined by gray scale analysis. (E) RAW264.7 cells were transfected with siSlc2a1 or siNC and infected with \u003cem\u003eB. abortus\u003c/em\u003eA19 (MOI=200) after 24 h. Cells were harvested after an additional 24 h and bacterial CFUs were determined. (F,G) RAW264.7 cells were transfected with siSlc2a1 or siNC, infected with \u003cem\u003eB. abortus\u003c/em\u003e A19 (MOI=200) after 24 h, and IL-1β and IL-6 mRNA levels were measured by qRT-PCR after harvesting the cells after an additional 24 h. Experiments were repeated at least three times with similar results. *, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5800583/v1/2ca404bc0166ca43ee4902dd.jpg"},{"id":74267298,"identity":"087ebaa2-96bb-4dc1-b071-2089c75ed611","added_by":"auto","created_at":"2025-01-20 13:11:19","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1040054,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eB. abortus\u003c/em\u003eA19 effector protein SepA promotes Slc2a1 expression. (A,B) RAW264.7 cells were transfected with p3xFLAG-CMV, p3xFLAG-CMV-SepA, p3xFLAG-CMV-BspE, p3xFLAG-CMV-NyxA, or p3xFLAG-CMV-NyxB for 48 h and levels of the FLAG tag and Slc2a1 were determined by Western blotting. Experiments were repeated at least three times with similar results. *, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01. (C) Conservation of SepA amino acid sequences of \u003cem\u003eB. abortus\u003c/em\u003e A19, \u003cem\u003eB. abortus \u003c/em\u003e2308,\u003cem\u003e B. suis\u003c/em\u003e 1330, and \u003cem\u003eB. canis\u003c/em\u003eATCC 23365 using DNAMAN alignment.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5800583/v1/cd0061befa0eb2447b837821.jpg"},{"id":78061989,"identity":"88c3dc3e-09df-466e-9431-540d8f66f774","added_by":"auto","created_at":"2025-03-08 21:42:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5195192,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5800583/v1/9a172138-9684-40e8-943e-cafb34438679.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBrucella\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eabortus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ehijacks the host protein Slc2a1via the SepA effector to promote intracellular survival in macrophages\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eBrucella\u003c/em\u003e is a facultative intracellular bacterium that infects and causes brucellosis in a wide range of mammalian hosts [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. \u003cem\u003eBrucella\u003c/em\u003e spp. infect goats, sheep, cattle, pigs, and dogs, and cause abortion and infertility in these hosts [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Twelve \u003cem\u003eBrucella\u003c/em\u003e genotypes have been described among which \u003cem\u003eBrucella melitensis\u003c/em\u003e, \u003cem\u003eB. abortus\u003c/em\u003e, \u003cem\u003eB. suis\u003c/em\u003e, \u003cem\u003eB. canis\u003c/em\u003e, and \u003cem\u003eB. ovis\u003c/em\u003e are the most prominent genera. The bacterium is disseminated widely in more than 170 countries, causes major economic losses, and also is a significant global public health threat [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. For example, recent studies suggest 1.6 to 2.1\u0026nbsp;million new cases occur annually in humans [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMacrophages are the natural host cells of \u003cem\u003eBrucella\u003c/em\u003e. The bacterium adopts numerous strategies to resist attack during infection of macrophages which causes host cells to undergo diverse physiological and biochemical responses. \u003cem\u003eBrucella\u003c/em\u003e modulates autophagy and apoptosis pathways, the immune response, and metabolic processes in macrophages, thereby evading clearance by the host cell. The VceA and BtpB proteins are important effectors that are secreted by the type IV secretion system (T4SS) in \u003cem\u003eBrucella\u003c/em\u003e. Deletion of the \u003cem\u003evceA\u003c/em\u003e and \u003cem\u003ebtpB\u003c/em\u003e genes leads to an increase in levels of the host LC3-II protein which is a key factor in the autophagy pathway and a decrease in p62 protein that is part of the nuclear pore complex of the nuclear envelope, as well as an increase in autophagic lysosomes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. T4SS effector proteins BspJ and BspG are nucleus-regulating proteins secreted by \u003cem\u003eBrucella\u003c/em\u003e that enter and function in the host cell nucleus. Deletion of the corresponding loci leads to enhanced apoptosis of macrophages which reduces \u003cem\u003eBrucella\u003c/em\u003e survival and increases the risk of bacterial killing by abnormal surges in inflammatory factor secretion [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Macrophages are a critical part of the host immune system and \u003cem\u003eBrucella\u003c/em\u003e infection leads to macrophage M1 polarization [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Activated M1 macrophages release numerous pro-inflammatory factors to combat pathogen invasion through the pro-inflammatory immune response [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. M1 macrophages have a high demand for energy and biosynthetic raw materials and mainly complete biochemical processes via glycolysis. Although this process is less efficient than oxidative phosphorylation in generating energy, glycolysis nevertheless rapidly generates energy to meet the needs of macrophages. Glycolysis is important for maintaining macrophage immune function, and hexokinase-1 and PKM2 enzymes in this pathway are involved in the activation of inflammasomes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Glycolysis is inhibited in M1 macrophages which affects the production of both reactive oxygen species and cytokines [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Although glycolysis provides energy for macrophages to combat invading microbial pathogens, \u003cem\u003eBrucella\u003c/em\u003e takes advantage of host cell glycolysis to facilitate survival [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The role of host cell glycolysis in \u003cem\u003eBrucella\u003c/em\u003e infection and the mechanism by which glycolysis regulates intracellular survival of the bacterium require further analysis.\u003c/p\u003e \u003cp\u003eIn this work, we determined that \u003cem\u003eBrucella\u003c/em\u003e infection modulates expression of the \u003cem\u003eSLC2A1\u003c/em\u003e gene in host macrophages. Slc2a1 is an important and widely-distributed glucose transporter in mammalian cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and is implicated in both cellular glycolysis and glucose metabolism [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The protein also exerts an important role in the immune response of macrophages and participates in the production of inflammatory factors [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The contribution of Slc2a1 to intracellular survival of \u003cem\u003eBrucella\u003c/em\u003e was previously uncertain, but we show here that Slc2a1 plays an important role in regulating bacterial persistence in RAW264.7 macrophages. Moreover, the Secreted Effector Protein A (SepA) of the \u003cem\u003eBrucella\u003c/em\u003e T4SS promotes \u003cem\u003eSLC2A1\u003c/em\u003e expression in these cells which suggests that SepA may be a novel antibacterial target to combat \u003cem\u003eBrucella\u003c/em\u003e infection.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Bacterial strains and RAW264.7 cell line\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eB. abortus\u003c/em\u003e A19 vaccine strain was provided by Shaanxi Veterinary Drug Supervision Institute (shaanxi province) and was grown on tryptic soy agar or tryptic soy broth at 37\u0026deg;C. \u003cem\u003eB. abortus\u003c/em\u003e A19 colonies were selected from agar medium and inoculated in tryptic soy broth. For cell enumeration, 100 \u0026micro;l of the cell suspension was diluted in a ten-fold gradient after 24 hours of growth and plated on tryptic soy agar medium. Colony numbers were counted after 72 h at 37\u0026deg;C. RAW264.7 macrophage cells were cultured in DMEM containing 10% fetal bovine serum (FBS) at 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Liquid chromatography-mass spectrometry in data-independent acquisition mode and sequencing\u003c/h2\u003e \u003cp\u003eThe RAW264.7 cells were seeded in 100 mm cell culture dishes in DMEM containing 10% FBS. When the cell density reached 70%, cells were infected with \u003cem\u003eB. abortus\u003c/em\u003e A19 (MOI\u0026thinsp;=\u0026thinsp;200) for 4 h, and the cell medium then was changed to DMEM containing 10% FBS with gentamicin (50 ng/\u0026micro;l) for 1 h. The medium subsequently was changed to DMEM containing 10% FBS with gentamicin (25 ng/\u0026micro;l) for 24 h at which time cells were harvested and placed at -80\u0026deg;C. Subsequently, the samples were analyzed by protein liquid chromatography-mass spectrometry (LC-MS/MS) (Novogene Bioinformatics Technology [Beijing, China]).\u003c/p\u003e \u003cp\u003eGene Ontology (GO) and InterPro (IPR) functional analyses were conducted using the InterProScan program against non-redundant protein databases, including Pfam, PRINTS, ProDom, SMART, ProSite, and PANTHER. The Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to analyze protein families and pathways. Differentially expressed proteins were used for Volcanic map analysis, cluster heat map analysis, and enrichment analysis of GO, IPR and KEGG.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Western blotting\u003c/h2\u003e \u003cp\u003eThe RAW264.7 cells were lysed using RIPA Lysis Buffer (Beyotime, Shanghai, China), and total protein concentration subsequently was measured using the BCA kit (Solarbio, Beijing, China). Ten micrograms of each sample were analyzed by 12.5% SDS-PAGE and then transferred to a PVDF membrane. The membrane was incubated with TBST buffer containing 10% skim milk for 2 h at room temperature and subsequently transferred to TBST containing Slc2a1 antibody (1:1000) or β-actin (1:1000) antibody (both from Proteintech, Wuhan, China) for 2 h at room temperature. Unbound antibody was washed off with TBST followed by incubation with HRP-labeled rabbit secondary antibody (1:5000) (Beijing CWBIO, Beijing, China) in TBST for 2 h at room temperature. Visualization was performed by enhanced chemiluminescence after an additional TBST wash.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 RNA interference\u003c/h2\u003e \u003cp\u003eThe siSlc2a1-526, siSlc2a1-820, siSlc2a1-1121, and siSlc2a1-1470 small interfering RNA (siRNA) molecules were designed and synthesized by GenePharma (Shanghai, China) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The relevant siRNA was transfected into RAW264.7 cells using Advanced DNA RNA Transfection Reagent (Zeta Life, USA) followed by infection with \u003cem\u003eB. abortus\u003c/em\u003e A19 (MOI\u0026thinsp;=\u0026thinsp;200). The cell culture medium was discarded after 4 h incubation and the cells were washed with PBS in triplicate. The medium was replaced with DMEM containing 10% FBS with gentamicin (50 ng/\u0026micro;l) for 1 h, followed by replacement with medium containing 25 ng/\u0026micro;l gentamicin. Cells were harvested 24 h later and total RNA or cell lysates were prepared.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers or siRNA used in this work\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eforward\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ereverse\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiSlc2a1-526\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-GCUUCAUCAUCGGUGUGUATT-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-UACACACCGAUGAUGAAGCTT-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiSlc2a1-820\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CCAAGAGUGUGCUGAAGAATT-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-UUCUUCAGCACACUCUUGGTT-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiSlc2a1-1121\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-GCUGUUUGUUGUAGAGCGATT-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-UCGCUCUACAACAAACAGCTT-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiSlc2a1-1470\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CUCGUGCUCUUCUUCAUCUTT-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-AGAUGAAGAAGAGCACGAGTT-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eq-Slc2a1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-GCTGTGCTTATGGGCTTCTC-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-CACATACATGGGCACAAAGC-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eq-β-actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-GGCTGTATTCCCCTCCATCG-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-CCAGTTGGTAACAATGCCATGT-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSlc2a1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-AAGCTTATGTTGGCTGTGGGAGGAGCAGTGC-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-GGATCCTCACACTTGGGAGTCCGCCCCCAGA \u0026minus;\u0026thinsp;3 '\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSepA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-AAGCTTATGATGCCCGTGATTAGACT \u0026minus;\u0026thinsp;3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-GGATCCTTAGGCGGACGCCGGGCCAG \u0026minus;\u0026thinsp;3 '\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBspE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-GCGGCCGCGATGACGTTATCGACGCGTAT-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-GGTACCTCAGGCAGCAACTTGCGATG-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNyxA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-AAGCTTATGAACGCTCACACAAACAT-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-GGATCCTCAAAGCTCCAAGCATCTAA-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNyxB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-AAGCTTATGAACACGCAAGCAACAAT \u0026minus;\u0026thinsp;3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-GGATCCTCAAGGCATCTCGATAAGGC \u0026minus;\u0026thinsp;3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 RNA extraction and Quantitative Real-Time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from RAW264.7 cells using TRIzol reagent (Invitrogen, California, USA), and was reverse transcribed to cDNA using the PrimeScript RT Reagent Kit (TaKaRa, Tokyo, Japan). Quantitative Real-Time PCR (qRT-PCR) was performed with the ChamQ SYBR qPCR Master-Mix Kit (Vazyme, Nanjing, China) in the Bio-Rad CFX96 real-time PCR System (Bio-Rad, Hercules, USA). The qRT-PCR primers were q-Slc2a1 and q-β-actin for the relevant genes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Relative gene expression was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Plasmids\u003c/h2\u003e \u003cp\u003eThe cDNA of \u003cem\u003eSLC2A1\u003c/em\u003e, and the \u003cem\u003esepA\u003c/em\u003e, \u003cem\u003ebspE\u003c/em\u003e, \u003cem\u003enyxA\u003c/em\u003e and \u003cem\u003enyxB\u003c/em\u003e genes were cloned into the p3xFLAG-CMV vector for production of proteins that are tagged with 3xFLAG at the N-terminus in mammalian cells. HindIII and EcoRI restriction enzymes were used for all plasmid constructions. Primers used are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Enumeration of \u003cem\u003eB. abortus\u003c/em\u003e in infected macrophages\u003c/h2\u003e \u003cp\u003eRAW264.7 cells were seeded in 24-well cell culture plates at a density of 2x10\u003csup\u003e5\u003c/sup\u003e cells/well and were infected with \u003cem\u003eB. abortus\u003c/em\u003e A19 (MOI\u0026thinsp;=\u0026thinsp;200). The culture medium was discarded and cells were washed in triplicate with PBS after 4 h. The culture medium was changed to DMEM containing 10% FBS with gentamicin (50 ng/\u0026micro;l) for 1 h, and then was changed to DMEM containing 10% FBS with gentamicin (25 ng/\u0026micro;l). Cells were harvested after 24 h and lysed with 0.5% Triton X-100 at room temperature for 10 min. The lysate was diluted in a ten-fold gradient, inoculated on tryptic soy agar, and colony numbers were counted after 72 h at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eBar graphs were plotted to show the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of at least three independent experiments. All statistical analyses were performed by two-sided Student's \u003cem\u003et\u003c/em\u003e-test. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 indicate significant and highly significant differences, respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Identifcation of differentially expressed proteins in \u003cem\u003eB. abortus\u003c/em\u003e-infected and uninfected macrophages\u003c/h2\u003e \u003cp\u003eDifferentially expressed proteins in RAW264.7 macrophages infected with \u003cem\u003eB. abortus\u003c/em\u003e A19 compared to uninfected cells were identified by LC-MS/MS in data-independent acquisition mode. A total of 2702 proteins were distinguished, of which 1339 and 1363 proteins were significantly upregulated and downregulated, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). These differentially expressed proteins are presented in a Volcano plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and were analyzed further by cluster analysis. Genes that were upregulated and downregulated were observed by cluster heatmaps (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 GO and KEGG enrichment analysis in \u003cem\u003eB. abortus\u003c/em\u003e-infected and uninfected macrophages\u003c/h2\u003e \u003cp\u003eGO functional significant enrichment analysis determines the GO functional items that are enriched significantly in a set of differentially expressed proteins compared with all identified proteins, thereby ascribing biological functions to the former. GO enrichment analysis was performed on differentially expressed proteins in \u003cem\u003eB. abortus\u003c/em\u003e-infected and uninfected RAW264.7 macrophages, and Biological Process, Cellular Component, and Molecular Function aspects were assessed. Differentially expressed proteins in Biological Process mainly were implicated in protein dephosphorylation, microtubule-based processes, ribosome biogenesis, ribonucleoprotein complex biogenesis, immune system processes, and small molecule biosynthesis. In terms of Cellular Component, differentially expressed proteins were found principally in the extracellular region, extracellular space, preribosome, small-subunit processome, microtuble organizing center, and spindle. Differentially expressed proteins in Molecular Function were involved mainly in phosphatase activity, receptor activity, phosphoprotein phosphatase activity, protein tyrosine phosphatase activity, acyl-CoA dehydrogenase activity, and regulatory region DNA binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKEGG enrichment analysis was performed by applying the hypergeometric test to identify pathways that were enriched significantly in the set of differentially expressed proteins compared with the background panel of all identified proteins. This analysis showed that differentially expressed proteins were involved mainly in signaling pathways, including pathways in cancer, pathways in influenza A, Kaposi's sarcoma-associated herpesvirus infection, hepatitis C infection, IL-17 signaling pathway, ribosome biogenesis in eukaryotes, p53 signaling pathway, pathways in small cell lung cancer, and fatty acid elongation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e3.3 Domain enrichment analysis and subcellular localization studies of differentially expressed proteins in \u003cem\u003eB. abortus\u003c/em\u003e-infected and uninfected macrophages\u003c/p\u003e \u003cp\u003eDomain enrichment analysis identifies domain entries that are statistically enriched in a defined set of proteins. Domain enrichment analysis here showed that the domains of differentially expressed proteins between \u003cem\u003eB. abortus\u003c/em\u003e-infected and uninfected macrophages mainly included the helicase superfamily 1/2 ATP-binding domain, Death-like domain, immunoglobulin V-set, protein-tyrosine phosphatase, receptor/non-receptor type, RUN, tetraspanin/peripherin, and transcription factor jumonji JmiN domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In addition, subcellular localization analyses showed that the differentially expressed proteins were distributed mainly in proteins of the nucleus (36.28%), cytoplasm (18.47%), plasma membrane (10.21%), endoplasmic reticulum (7.06%), mitochondrion (7.01%), Golgi apparatus (5.11%), as well as extracellular proteins (3.75%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 \u003cem\u003eB. abortus\u003c/em\u003e infection promotes the expression of Slc2a1 in macrophage cells\u003c/h2\u003e \u003cp\u003eLactate is the end product of glycolysis and lactate levels may reflect changes in glycolytic activity. Interestingly, \u003cem\u003eB. abortus\u003c/em\u003e infection significantly promoted the production of lactate in RAW264.7 macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). LC-MS/MS analysis was performed to explore how infection induced changes in host glycolysis activity which revealed that the Slc2a1 protein was significantly upregulated in pathways in cancer signaling obtained in KEGG enrichment analysis. One hundred differentially expressed proteins were identified in pathways in cancer among which only Slc2a1 was involved in glycolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The Slc2a1 protein is an important and widely-distributed glucose transporter in mammalian cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and exerts a key role in cellular glycolysis and glucose metabolism [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Uninfected and infected cell samples were harvested and analyzed by qRT-PCR and Western blotting to assess further the expression of \u003cem\u003eSLC2A1\u003c/em\u003e. The qRT-PCR assays showed that \u003cem\u003eSLC2A1\u003c/em\u003e mRNA levels were upregulated significantly in \u003cem\u003eB. abortus\u003c/em\u003e-infected cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) and Western blotting also indicated that the level of Slc2a1 protein in infected cells was significantly higher than in uninfected cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Thus, the enhanced expression of \u003cem\u003eSLC2A1\u003c/em\u003e in macrophages following \u003cem\u003eB. abortus\u003c/em\u003e infection may underpin the elevated levels of lactate that were detected in these cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Slc2a1 is involved in regulation of \u003cem\u003eB. abortus\u003c/em\u003e intracellular proliferation in macrophages\u003c/h2\u003e \u003cp\u003eAlthough Slc2a1 expression was upregulated in RAW264.7 cells following \u003cem\u003eB. abortus\u003c/em\u003e infection, it is unknown whether the protein plays a role subsequent to infection. The role of Slc2a1 in infected cells was explored by using an overexpression plasmid to overproduce the protein following bacterial infection. Overexpression of \u003cem\u003eSLC2A1\u003c/em\u003e significantly promoted the intracellular proliferation of \u003cem\u003eB. abortus\u003c/em\u003e in RAW264.7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Subsequently, we designed and synthesized four siRNA (siSlc2a1) to knock down \u003cem\u003eSLC2A1\u003c/em\u003e mRNA levels. The qRT-PCR and Western blotting results revealed that siSlc2a1-1121 and siSlc2a1-1470 significantly reduced both mRNA and protein levels of Slc2a1 compared with the non-interfering siRNA (siNC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Knockdown assays subsequently were performed on \u003cem\u003eB. abortus\u003c/em\u003e-infected cells: intracellular proliferation of the bacterium was inhibited significantly by using siSlc2a1-1470 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). As Slc2a1 may be involved in regulating the expression of inflammatory factors [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], we hypothesized that the protein may control the intracellular proliferation of \u003cem\u003eB. abortus\u003c/em\u003e by modulating the expression of inflammatory factors that are involved in combatting infection. However, knockdown of \u003cem\u003eSLC2A1\u003c/em\u003e did not affect the elevated levels of either IL-1β and IL-6 cytokines that occur after \u003cem\u003eB. abortus\u003c/em\u003e infection of RAW264.7 macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). The preceding data suggest that Slc2a1 is involved in counteracting \u003cem\u003eB. abortus\u003c/em\u003e infection of macrophages, but that this effect is not mediated via IL-1β or IL-6 cytokines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6 \u003cem\u003eB. abortus\u003c/em\u003e effector protein SepA promotes Slc2a1 expression\u003c/h2\u003e \u003cp\u003eThe T4SS is a critical \u003cem\u003eBrucella\u003c/em\u003e virulence factor that is essential for intracellular survival of the bacterium [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. We speculated that \u003cem\u003eB. abortus\u003c/em\u003e may promote post-infection expression of \u003cem\u003eSLC2A1\u003c/em\u003e via effector proteins secreted by the T4SS. Therefore, plasmids that overexpress the \u003cem\u003esepA, bspE, nyxA\u003c/em\u003e, or \u003cem\u003enyxB\u003c/em\u003e effector genes of \u003cem\u003eB. abortus\u003c/em\u003e were constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Macrophage expression of Slc2a1 increased significantly when SepA was overexpressed, but not when BspE, NyxA, or NyxB were overproduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). SepA sequences of \u003cem\u003eB. abortus\u003c/em\u003e A19, \u003cem\u003eB. abortus\u003c/em\u003e 2308, \u003cem\u003eB. suis\u003c/em\u003e 1330, and \u003cem\u003eB. canis\u003c/em\u003e ATCC 23365 were aligned. The sequences were completely consistent in the \u003cem\u003eB. abortus\u003c/em\u003e homologs, with only a few substitutions in the SepA proteins in the other genera (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). In summary, the preceding data suggest that the conserved SepA effector protein enhances expression of host Slc2a1 to facilitate survival of \u003cem\u003eB. abortus\u003c/em\u003e in the macrophage environment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cem\u003eBrucella\u003c/em\u003e spp. are facultative intracellular bacterial parasites that induce a series of physiological perturbations in host cells which activate the host immune response. Therefore, avoidance of elimination by the host is a key survival strategy for \u003cem\u003eBrucella\u003c/em\u003e. \u003cem\u003eBrucella\u003c/em\u003e deploys diverse stratagems and also subverts host functions to circumvent killing and to adapt to the intracellular environment. Identification of the bacterial and host factors that affect the intracellular proliferation of \u003cem\u003eBrucella\u003c/em\u003e is critical for prevention and control of \u003cem\u003eBrucella\u003c/em\u003e infections.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBrucella\u003c/em\u003e infection causes numerous physiological and biochemical fluctuations in host cells, including initiation of autophagy, apoptosis, mitophagy, and inflammation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. \u003cem\u003eBrucella\u003c/em\u003e infection of macrophages alters the expression of key macrophage proteins. Certain proteins are used by host cells to resist \u003cem\u003eBrucella\u003c/em\u003e invasion, whereas other factors are hijacked by the bacterium for intracellular survival. In the present study, we performed LC-MS/MS analysis of uninfected RAW264.7 macrophage cells and of cells infected with \u003cem\u003eB. abortus\u003c/em\u003e A19 with the aim of detecting differentially expressed proteins that are hallmarks of infection. This analysis revealed changed production of 2702 proteins in infected cells compared with uninfected macrophages, among which 1339 and 1363 proteins were upregulated and downregulated, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). GO functional significant enrichment analysis indicated the functional entries that were enriched in the differentially expressed proteins, thereby revealing the biological functions with which the differential proteins were associated. This analysis indicated that the pertinent proteins were involved mainly in microtubule-based processes, protein dephosphorylation, immune system processes, and pathways in cancer. In addition, the hypergeometric distribution was used in KEGG enrichment analysis to assess whether the set of differential proteins in a pathway was higher than the set of proteins external to the pathway. The differentially expressed proteins were implicated principally in signaling pathways, including pathways in cancer, pathways in influenza A, Kaposi's sarcoma-associated herpesvirus infection, hepatitis C, and the IL-17 signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Furthermore, domain enrichment analysis showed that the domains of differentially expressed proteins between uninfected and \u003cem\u003eB. abortus\u003c/em\u003e-infected cells mainly included the helicase superfamily 1/2 ATP-binding domain, Death-like domain, immunoglobulin V-set, protein-tyrosine phosphatase, receptor/non-receptor type, RUN, tetraspanin/peripherin, and transcription factor jumonji JmiN domains. Finally, subcellular localization enrichment analysis showed that the differentially expressed proteins were mainly distributed in the nucleus, followed by the cytoplasm, plasma membrane, and endoplasmic reticulum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLC-MS/MS analysis demonstrated that the internal environment of macrophage cells underwent major changes after \u003cem\u003eB. abortus\u003c/em\u003e infection, and that the expression of proteins related to metabolism, the immune system, material transport, and protein modification was altered significantly. Proteins localized in the nucleus accounted for a large proportion of the differentially expressed proteins. We hypothesize that the expression of nuclear proteins is modified first following macrophage infection by \u003cem\u003eB. abortus\u003c/em\u003e, and that these regulatory perturbations subsequently modulate expression of cytoplasmic proteins, membrane factors, and other proteins to achieve clearance of \u003cem\u003eBrucella\u003c/em\u003e. Conversely, \u003cem\u003eB. abortus\u003c/em\u003e may hijack nuclear proteins to regulate cytoplasmic factors, membrane proteins, and other cellular components to promote intracellular survival. Therefore, determining the functions of differentially expressed proteins is crucial to uncovering the mechanisms of \u003cem\u003eBrucella\u003c/em\u003e infection and intracellular persistence.\u003c/p\u003e \u003cp\u003eHost cells respond quickly to invasion by pathogenic microorganisms by synthesizing and releasing diverse inflammatory factors. Cells rapidly initiate glycolysis in order to meet the accompanying energy requirements. Although glycolysis provides energy for host immune responses to resist invasion by pathogens, glycolysis may also help pathogen survival [\u003cspan additionalcitationids=\"CR29 CR30 CR31\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Accordingly, \u003cem\u003eBrucella\u003c/em\u003e infection upregulates host cell glycolysis which aids intracellular survival of the bacterium [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the mechanism by which \u003cem\u003eBrucella\u003c/em\u003e upregulates glycolysis is unknown. Here, we found that lactate concentrations in macrophage cells increased following \u003cem\u003eB. abortus\u003c/em\u003e infection. Lactate is the end product of glycolysis for which the molecule also is an indicator. LC-MS/MS, qRT-PCR and Western blotting analysis showed that expression of the Slc2a1 protein increased in macrophages infected with \u003cem\u003eB. abortus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Slc2a1 is an important and widely distributed glucose transporter in mammalian cells [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. We hypothesized that \u003cem\u003eB. abortus\u003c/em\u003e upregulates glycolytic levels in macrophages via the Slc2a1 protein.\u003c/p\u003e \u003cp\u003eThe role of Slc2a1 in macrophage infection by \u003cem\u003eB. abortus\u003c/em\u003e was explored further with an Slc2a1 overexpression plasmid which revealed that overproduction of the protein promoted intracellular proliferation of the bacterium (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), whereas knockdown of Slc2a1 by siRNA inhibited proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Thus, Slc2a1 indeed plays an important role in the intracellular persistence of \u003cem\u003eBrucella\u003c/em\u003e. As the Slc2a1 protein participates in regulating expression of host inflammatory factors [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], we hypothesized that the protein promoted the proliferation of \u003cem\u003eB. abortus\u003c/em\u003e by modifying expression of host inflammatory factors. However, Slc2a1 was not involved in the expression of IL-1β and IL-6 cytokines following \u003cem\u003eB. abortus\u003c/em\u003e infection of macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Thus, the molecular mechanism by Slc2a1 enhances intracellular proliferation of \u003cem\u003eB. abortus\u003c/em\u003e requires further exploration.\u003c/p\u003e \u003cp\u003eThe T4SS is a key virulence factor that is essential for the intracellular survival of \u003cem\u003eBrucella\u003c/em\u003e. The T4SS in \u003cem\u003eBrucella\u003c/em\u003e is a 12 protein complex that is encoded by the VirB operon [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The complex acts through 15 secreted effector proteins which impinge on vital host signaling pathways, thereby helping the bacterium to survive and replicate in the host [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. We speculated that the T4SS in \u003cem\u003eB. abortus\u003c/em\u003e may play an important role in promoting the expression of host Slc2a1 protein. In agreement with this hypothesis, the T4SS effector protein SepA stimulated Slc2a1 expression which accords with the finding that SepA is involved in regulating \u003cem\u003eBrucella\u003c/em\u003e survival within host cells [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. SepA appears to be highly conserved in \u003cem\u003eBrucella\u003c/em\u003e spp. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) which suggests that the protein may perform equivalent functions in different mammalian hosts. In summary, the data here suggest that \u003cem\u003eB. abortus\u003c/em\u003e secretes the effector protein SepA via the T4SS which promotes the expression of host Slc2a1 to assist the intracellular persistence of this pathogen. These observations also suggest that SepA may be a promising antibacterial target to combat infection by \u003cem\u003eBrucella\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by funds from the National Natural Science Foundation of China (Grant No. 32373016, 31672584)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAihua Wang:\u0026nbsp;\u003c/strong\u003eSupervision, Funding acquisition, Data curation, Conceptualization.\u003cstrong\u003e\u0026nbsp;Yaping Jin:\u003c/strong\u003e Supervision, Funding acquisition, Data curation, Conceptualization.\u003cstrong\u003e\u0026nbsp;Yuanhao Yang:\u0026nbsp;\u003c/strong\u003eWriting – original draft, Validation, Methodology, Investigation, Formal analysis.\u0026nbsp;\u003cstrong\u003eYaping Zu:\u0026nbsp;\u003c/strong\u003eWriting – original draft, Validation, Methodology, Investigation, Formal analysis.\u0026nbsp;\u003cstrong\u003eDong Zhou:\u0026nbsp;\u003c/strong\u003eSupervision, Conceptualization.\u0026nbsp;\u003cstrong\u003eHui Wang:\u0026nbsp;\u003c/strong\u003eSupervision.\u0026nbsp;\u003cstrong\u003eXiaofang Liu:\u003c/strong\u003e Supervision, Software.\u0026nbsp;\u003cstrong\u003eGaowa Wudong:\u0026nbsp;\u003c/strong\u003eSupervision, Software.\u0026nbsp;\u003cstrong\u003eYunyi Zhai:\u003c/strong\u003e Supervision, Software.\u0026nbsp;\u003cstrong\u003eXin Wang:\u0026nbsp;\u003c/strong\u003eSupervision, Investigation.\u0026nbsp;\u003cstrong\u003eTing Tang:\u003c/strong\u003e Supervision.\u0026nbsp;\u003cstrong\u003ePingping Wang:\u003c/strong\u003e Supervision.\u0026nbsp;\u003cstrong\u003eNingqiu Yuan:\u003c/strong\u003e Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGaneshan K., Chawla A., Metabolic Regulation of Immune Responses, Annual Review of Immunology. 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(2014) 16:396-410.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"B. abortus A19, LC-MS/MS, Slc2a1, SepA, intracellular survival","lastPublishedDoi":"10.21203/rs.3.rs-5800583/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5800583/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eBrucella\u003c/em\u003e spp. are facultative intracellular bacteria that infect and induce brucellosis in a diverse range of mammalian hosts. The disease causes major global economic losses and also is a worldwide threat to public health security. Characterization of bacterial and host factors that promote intracellular survival of \u003cem\u003eBrucella\u003c/em\u003e is key for the prevention and control of brucellosis. In this study, we identified proteins involved in intracellular survival of \u003cem\u003eBrucella abortus\u003c/em\u003e A19 in RAW264.7 macrophage cells by liquid chromatography-mass spectrometry of macrophages with or without \u003cem\u003eB. abortus\u003c/em\u003e infection. The functions of these proteins, the signaling pathways in which the proteins participate, the domain entries enriched by the proteins, and the subcellular localization of the differentially-expressed proteins were deciphered. Differential protein expression revealed that Slc2a1, which is a key Glycolytic protein, was significantly upregulated in infected macrophage cells. This observation was confirmed by qRT-PCR and Western blotting studies. The role of Slc2a1 in the intracellular survival of \u003cem\u003eB. abortus\u003c/em\u003e was probed by overexpressing and knocking down \u003cem\u003eSLC2A1\u003c/em\u003e in RAW264.7 cells. Overproduction of the protein promoted intracellular proliferation of \u003cem\u003eB. abortus\u003c/em\u003e whereas knockdown of \u003cem\u003eSLC2A1\u003c/em\u003e inhibited proliferation of the bacterium. Finally, we determined that the Secreted Effector Protein A (SepA) effector of \u003cem\u003eB. abortus\u003c/em\u003e enhanced \u003cem\u003eSLC2A1\u003c/em\u003e expression in macrophage cells. Thus, \u003cem\u003eB. abortus\u003c/em\u003e stimulates host \u003cem\u003eSLC2A1\u003c/em\u003e expression via the SepA effector protein to aid bacterial survival in the macrophage environment which suggests that SepA may be a novel antibacterial target to combat \u003cem\u003eBrucella\u003c/em\u003e infection.\u003c/p\u003e","manuscriptTitle":"Brucella abortushijacks the host protein Slc2a1via the SepA effector to promote intracellular survival in macrophages","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-20 13:11:14","doi":"10.21203/rs.3.rs-5800583/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ca1d683d-2dc2-4d0b-a743-5516c8a832fd","owner":[],"postedDate":"January 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-08T21:34:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-20 13:11:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5800583","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5800583","identity":"rs-5800583","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}