Iron alters the cell wall composition and intracellular lactate to affect Candida albicans susceptibility to antifungals and host immune response
2020; Elsevier BV; Volume: 295; Issue: 29 Linguagem: Inglês
10.1074/jbc.ra120.013413
ISSN1083-351X
AutoresAparna Tripathi, Elisabetta Liverani, Alexander Y. Tsygankov, Sumant Puri,
Tópico(s)Gut microbiota and health
ResumoFungal pathogen Candida albicans has a complex cell wall consisting of an outer layer of mannans and an inner layer of β-glucans and chitin. The fungal cell wall is the primary target for antifungals and is recognized by host immune cells. Environmental conditions such as carbon sources, pH, temperature, and oxygen tension can modulate the fungal cell wall architecture. Cellular signaling pathways, including the mitogen-activated protein kinase (MAPK) pathways, are responsible for sensing environmental cues and mediating cell wall alterations. Although iron has recently been shown to affect β-1,3-glucan exposure on the cell wall, we report here that iron changes the composition of all major C. albicans cell wall components. Specifically, high iron decreased the levels of mannans (including phosphomannans) and chitin; and increased β-1,3-glucan levels. These changes increased the resistance of C. albicans to cell wall-perturbing antifungals. Moreover, high iron cells exhibited adequate mitochondrial functioning; leading to a reduction in accumulation of lactate that signals through the transcription factor Crz1 to induce β-1,3-glucan masking in C. albicans. We show here that iron-induced changes in β-1,3-glucan exposure are lactate-dependent; and high iron causes β-1,3-glucan exposure by preventing lactate-induced, Crz1-mediated inhibition of activation of the fungal MAPK Cek1. Furthermore, despite exhibiting enhanced antifungal resistance, high iron C. albicans cells had reduced survival upon phagocytosis by macrophages. Our results underscore the role of iron as an environmental signal in multiple signaling pathways that alter cell wall architecture in C. albicans, thereby affecting its survival upon exposure to antifungals and host immune response. Fungal pathogen Candida albicans has a complex cell wall consisting of an outer layer of mannans and an inner layer of β-glucans and chitin. The fungal cell wall is the primary target for antifungals and is recognized by host immune cells. Environmental conditions such as carbon sources, pH, temperature, and oxygen tension can modulate the fungal cell wall architecture. Cellular signaling pathways, including the mitogen-activated protein kinase (MAPK) pathways, are responsible for sensing environmental cues and mediating cell wall alterations. Although iron has recently been shown to affect β-1,3-glucan exposure on the cell wall, we report here that iron changes the composition of all major C. albicans cell wall components. Specifically, high iron decreased the levels of mannans (including phosphomannans) and chitin; and increased β-1,3-glucan levels. These changes increased the resistance of C. albicans to cell wall-perturbing antifungals. Moreover, high iron cells exhibited adequate mitochondrial functioning; leading to a reduction in accumulation of lactate that signals through the transcription factor Crz1 to induce β-1,3-glucan masking in C. albicans. We show here that iron-induced changes in β-1,3-glucan exposure are lactate-dependent; and high iron causes β-1,3-glucan exposure by preventing lactate-induced, Crz1-mediated inhibition of activation of the fungal MAPK Cek1. Furthermore, despite exhibiting enhanced antifungal resistance, high iron C. albicans cells had reduced survival upon phagocytosis by macrophages. Our results underscore the role of iron as an environmental signal in multiple signaling pathways that alter cell wall architecture in C. albicans, thereby affecting its survival upon exposure to antifungals and host immune response. Iron is an essential transition metal that is required for several cellular processes including synthesis of heme and iron-sulfur (Fe-S) cluster-containing proteins, amino acids, DNA, lipids, and sterols (1Schaible U.E. Kaufmann S.H. Iron and microbial infection.Nat. Rev. Microbiol. 2004; 2 (15550940): 946-95310.1038/nrmicro1046Crossref PubMed Scopus (660) Google Scholar, 2Misslinger M. Lechner B.E. Bacher K. Haas H. Iron-sensing is governed by mitochondrial, not by cytosolic iron-sulfur cluster biogenesis in Aspergillus fumigatus.Metallomics. 2018; 10 (30395137): 1687-170010.1039/c8mt00263kCrossref PubMed Google Scholar). In excess, however, iron can generate reactive oxygen species (ROS) as a biproduct of metabolic processes via Haber–Weiss/Fenton reaction (3Halliwell B. Gutteridge J.M. Role of iron in oxygen radical reactions.Methods Enzymol. 1984; 105 (6203010): 47-5610.1016/s0076-6879(84)05007-2Crossref PubMed Scopus (158) Google Scholar). 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Cell. 2015; 14 (26453650): 1165-117210.1128/EC.00142-15Crossref PubMed Scopus (14) Google Scholar). C. albicans cell wall consists of an outer layer of mannans, covalently associated with N-, O-, and ether-linked glycosylated cell wall proteins (mannoproteins), comprising 35–40% of the cell wall and an inner layer of β-glucans and chitin. β-Glucans are the major components of the inner cell wall (50–60% of the cell wall) and consist of a core structure that is largely made of β-1,3-glucans, along with a minor amount of β-1,6-glucan (14Arana D.M. Prieto D. Roman E. Nombela C. Alonso-Monge R. Pla J. The role of the cell wall in fungal pathogenesis.Microb. Biotechnol. 2009; 2 (21261926): 308-32010.1111/j.1751-7915.2008.00070.xCrossref PubMed Scopus (29) Google Scholar). Changes to C. albicans cell wall architecture have been observed in response to different environmental conditions. Acidic environments such as lower pH or lactate reduce mannan levels in the outer cell wall layer, whereas limitation of zinc, another transition metal like iron, increases mannan levels (15Sherrington S.L. Sorsby E. Mahtey N. Kumwenda P. Lenardon M.D. Brown I. Ballou E.R. MacCallum D.M. Hall R.A. Adaptation of Candida albicans to environmental pH induces cell wall remodelling and enhances innate immune recognition.PLoS Pathog. 2017; 13 (28542528): e100640310.1371/journal.ppat.1006403Crossref PubMed Scopus (74) Google Scholar, 16Ballou E.R. Avelar G.M. Childers D.S. Mackie J. Bain J.M. Wagener J. Kastora S.L. Panea M.D. Hardison S.E. Walker L.A. Erwig L.P. Munro C.A. Gow N.A. Brown G.D. MacCallum D.M. et al.Lactate signalling regulates fungal β-glucan masking and immune evasion.Nat. Microbiol. 2016; 2 (27941860): 1623810.1038/nmicrobiol.2016.238Crossref PubMed Scopus (98) Google Scholar, 17Malavia D. Lehtovirta-Morley L.E. Alamir O. Weiß E. Gow N.A.R. Hube B. Wilson D. 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However, zinc limitation altered chitin exposure on the cell surface, without affecting β-1,3-glucan levels (17Malavia D. Lehtovirta-Morley L.E. Alamir O. Weiß E. Gow N.A.R. Hube B. Wilson D. Zinc limitation induces a hyper-adherent goliath phenotype in Candida albicans.Front. Microbiol. 2017; 8 (29184547): 223810.3389/fmicb.2017.02238Crossref PubMed Scopus (23) Google Scholar). Iron was been recently shown to affect exposure of β-1,3-glucan; however, overall effects of iron on the complex C. albicans cell wall structure remain unknown. Cell wall remodeling affects the susceptibility of C. albicans toward various antifungals, including cell wall-perturbing (CWP) agents (18Ene I.V. Adya A.K. Wehmeier S. Brand A.C. MacCallum D.M. Gow N.A. Brown A.J. 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Loss of mannosylphosphate from Candida albicans cell wall proteins results in enhanced resistance to the inhibitory effect of a cationic antimicrobial peptide via reduced peptide binding to the cell surface.Microbiology. 2009; 155 (19332808): 1058-107010.1099/mic.0.026120-0Crossref PubMed Scopus (46) Google Scholar). CWP agents inhibit fungal growth by targeting cell wall components to disrupt cell wall integrity. Tunicamycin binds to an enzyme UDP-GlcNAc:dolichyl-phosphate N-acetylglucosamine phosphotransferase, which catalyzes the primary step of N-linked mannan synthesis (22Yoo J. Mashalidis E.H. Kuk A.C.Y. Yamamoto K. Kaeser B. Ichikawa S. Lee S.Y. GlcNAc-1-P-transferase-tunicamycin complex structure reveals basis for inhibition of N-glycosylation.Nat. Struct. Mol. Biol. 2018; 25 (29459785): 217-22410.1038/s41594-018-0031-yCrossref PubMed Scopus (48) Google Scholar). Interestingly, antifungal activity of tunicamycin depends on the Cek1 MAPK pathway (23Roman E. Cottier F. Ernst J.F. Pla J. Msb2 signaling mucin controls activation of Cek1 mitogen-activated protein kinase in Candida albicans.Eukaryot. Cell. 2009; 8 (19542310): 1235-124910.1128/EC.00081-09Crossref PubMed Scopus (86) Google Scholar) that we have previously shown to be responsive to environmental iron (10Puri S. Lai W.K. Rizzo J.M. Buck M.J. Edgerton M. Iron-responsive chromatin remodelling and MAPK signalling enhance adhesion in Candida albicans.Mol. Microbiol. 2014; 93 (24889932): 291-30510.1111/mmi.12659Crossref PubMed Scopus (15) Google Scholar). Furthermore, zymolyase, a mixture of β-1,3-glucanases and proteases, lyses fungal cell by inhibiting β-1,3-glucan synthesis. In Saccharomyces cerevisiae, the activation of Slt2p (a homolog of C. albicans cell wall integrity MAPK Mkc1) makes cells resistant to glucanase digestion (24Boorsma A. de Nobel H. ter Riet B. Bargmann B. Brul S. Hellingwerf K.J. Klis F.M. Characterization of the transcriptional response to cell wall stress in Saccharomyces cerevisiae.Yeast. 2004; 21 (15116342): 413-42710.1002/yea.1109Crossref PubMed Scopus (113) Google Scholar), whereas high iron increases Slt2p-activation in nonalbicans Candida species (25Srivastava V.K. Suneetha K.J. Kaur R. The mitogen-activated protein kinase CgHog1 is required for iron homeostasis, adherence and virulence in Candida glabrata.FEBS J. 2015; 282 (25772226): 2142-216610.1111/febs.13264Crossref PubMed Scopus (16) Google Scholar). Also, Mkc1 and Cek1 MAPK pathways play a cooperative role in regulation of cell wall architecture in C. albicans (26Monge R.A. Roman E. Nombela C. Pla J. The MAP kinase signal transduction network in Candida albicans.Microbiology. 2006; 152 (16549655): 905-91210.1099/mic.0.28616-0Crossref PubMed Scopus (176) Google Scholar). Changes to C. albicans cell wall in response in host iron can therefore be consequential for the outcome of antifungal therapy. In addition, cell wall components are also crucial for host-pathogen interaction. These components act as pathogen-associated molecular patterns (PAMPs) and stimulate immune signaling, when recognized by host pattern recognition receptors (14Arana D.M. Prieto D. Roman E. Nombela C. Alonso-Monge R. Pla J. The role of the cell wall in fungal pathogenesis.Microb. Biotechnol. 2009; 2 (21261926): 308-32010.1111/j.1751-7915.2008.00070.xCrossref PubMed Scopus (29) Google Scholar). β-1,3-Glucan and chitin are normally shielded by the superficial mannans to limit pathogen recognition (14Arana D.M. Prieto D. Roman E. Nombela C. Alonso-Monge R. Pla J. The role of the cell wall in fungal pathogenesis.Microb. Biotechnol. 2009; 2 (21261926): 308-32010.1111/j.1751-7915.2008.00070.xCrossref PubMed Scopus (29) Google Scholar). Therefore, changes in cell wall mannans can contribute to exposure of underlying β-1,3-glucan and chitin. Various environmental cues including iron can induce β-1,3-glucan masking to limit recognition of fungal cells and stimulation of cytokine production by host immune cells. However, MAPK Cek1 activation by phosphorylation (Cek1-P) is crucial for β-1,3-glucan unmasking or exposure. Despite this, numerous studies focusing on the effect of environmental conditions on β-1,3-glucan masking have not examined the status of Cek1 activation under those respective conditions and how it may contribute to β-1,3-glucan exposure levels. Here we reveal for the first time that changes in environmental iron modulate the levels of all major cell wall components of C. albicans. High iron-mediated changes to the cell wall, in turn, led to an enhanced resistance to CWP agents. We further define a novel mechanism by which iron influences β-1,3-glucan exposure. We show that iron communicates with a noncanonical pathway involving lactic acid signaling through Crz1; whereby high iron limits intracellular lactate accumulation to reduce Crz1-mediated inhibition of MAPK Cek1-activation, leading to β-1,3-glucan exposure. To examine whether iron can modulate fungal cell wall components, C. albicans cells were grown in YNB-based minimal medium without added iron or with 100 μm added iron, representing comparatively limited iron medium (LIM) and higher or replete iron medium (RIM). We next compared the intracellular iron levels in C. albicans cells grown in these respective medium with intracellular iron levels of cells grown in yeast-extract peptone dextrose (YPD) medium, the most commonly used medium for fungal growth. C. albicans cells grown in our LIM had iron levels similar to YPD-grown cells, whereas cells grown in RIM showed a 97.83% increase in intracellular iron content, compared with YPD-grown cells (Fig. S1A). All three growth conditions maintained similar growth rates (Fig. S1B). LIM- and RIM-grown C. albicans cells were then used to measure the levels of all key components of the cell wall. The cells were stained with concanavalin A (ConA, for mannan), aniline blue (AB, for β-1,3-glucan), or CFW (for chitin), and mean fluorescence intensities (MFI) were measured. We also determined the levels of acid-labile mannans (phosphomannans) by measuring the levels of alcian blue binding to the cell surface. In response to changes in environmental iron, major differences in the levels of all key cell wall components were observed (Fig. 1). RIM-grown cells with higher intracellular iron (Fig. S1A) showed significantly lower fluorescence levels for ConA binding, indicating the presence of a much thinner layer of mannans, when compared with LIM cells (Fig. 1A). Similarly, the incidence of phosphomannans was significantly lower in RIM cells (Fig. S2). The levels of β-1,3-glucan, the dominant structure of the inner cell wall, were significantly higher in RIM cells (Fig. 1B); however, like mannans, levels of chitin were significantly lower in RIM-grown cells, compared with LIM cells (Fig. 1C). Because β-1,3-glucan levels in the β-glucan layer that is directly above the chitin layer were increased under high iron conditions, we also examined whether this led to a masking of chitin. Using wheat germ agglutinin (WGA) stain, a lectin that specifically binds to surface-exposed chitin, we determined that high iron RIM cells showed significantly lower exposure of chitin, compared with cells grown in LIM (Fig. S3). Thus, iron modulates all major components of the fungal cell wall, and increase in intracellular iron potentially induces reduction in synthesis of mannan and chitin, along with reduced chitin exposure, whereas increasing β-1,3-glucan synthesis. Finally, to corroborate iron-mediated cell wall changes at a cellular structure level, we performed transmission EM to examine the ultrastructure of the cell wall of LIM and RIM cells. Micrographs of RIM cells showed a significantly thinner outer layer of mannans with a scattered fibrillar arrangement and a thicker inner layer (Fig. 2). Thus, iron changed the levels of individual cell wall components to drastically affect the cell wall at the structural level. We hypothesized that changes in C. albicans cell wall structure may influence its susceptibility to CWP agents. We thus compared the sensitivity of RIM and LIM cells to tunicamycin (inhibitor of N-linked mannosylation), zymolyase (β-1,3-glucanase), and CFW (that interferes with cross-linking of chitin). Our spot assay results revealed that compared with LIM cells, RIM cells were more resistant to tunicamycin (Fig. 3A) and CFW (Fig. 3C), and similarly, also more resistant to zymolyase, with a significant increase (14.24 ± 2.76%) in cell growth (Fig. 3B). Taken together with the effect of iron on the C. albicans cell wall (Fig. 1), these results show that high iron-induced changes to the cell wall made these cells more resistant to CWP antifungals. The observed changes in cell wall components (Fig. 1), particularly mannans, are known to cause exposure of β-1,3-glucan on the cell surface (27Hall R.A. Gow N.A. Mannosylation in Candida albicans: role in cell wall function and immune recognition.Mol. Microbiol. 2013; 90 (24125554): 1147-116110.1111/mmi.12426Crossref PubMed Scopus (106) Google Scholar, 28Graus M.S. Wester M.J. Lowman D.W. Williams D.L. Kruppa M.D. Martinez C.M. Young J.M. Pappas H.C. Lidke K.A. Neumann A.K. Mannan molecular substructures control nanoscale glucan exposure in Candida.Cell Rep. 2018; 24: 2432-2442.e243510.1016/j.celrep.2018.07.088Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). We next tested the effect of iron on β-1,3-glucan exposure, using a monoclonal anti-β-1,3-glucan antibody and Cy3 labeling that allows for detection of exposed β-1,3-glucan in fungal cells. RIM cells displayed significantly higher levels of exposed β-1,3-glucans (1.7-fold increase), compared with LIM cells (Fig. 4A). This suggests that high iron conditions induce exposure of the β-1,3-glucan layer, most likely due to the thinner and scattered outer layer of mannan fibrils observed under high iron (Fig. 1A and 2Misslinger M. Lechner B.E. Bacher K. Haas H. Iron-sensing is governed by mitochondrial, not by cytosolic iron-sulfur cluster biogenesis in Aspergillus fumigatus.Metallomics. 2018; 10 (30395137): 1687-170010.1039/c8mt00263kCrossref PubMed Google Scholar). A previous study showed triggering of β-1,3-glucan masking (thus reducing its exposure) through a lactate-mediated signaling pathway, when C. albicans cells were grown in presence of lactate as carbon source (16Ballou E.R. Avelar G.M. Childers D.S. Mackie J. Bain J.M. Wagener J. Kastora S.L. Panea M.D. Hardison S.E. Walker L.A. Erwig L.P. Munro C.A. Gow N.A. Brown G.D. MacCallum D.M. et al.Lactate signalling regulates fungal β-glucan masking and immune evasion.Nat. Microbiol. 2016; 2 (27941860): 1623810.1038/nmicrobiol.2016.238Crossref PubMed Scopus (98) Google Scholar). In this study, we used glucose as the sole carbon source. However, cells with comparatively lower iron can exhibit a reduction in mitochondrial function (29Grahl N. Demers E.G. Lindsay A.K. Harty C.E. Willger S.D. Piispanen A.E. Hogan D.A. Mitochondrial activity and Cyr1 are key regulators of Ras1 activation of C. albicans virulence pathways.PLoS Pathog. 2015; 11 (26317337): e100513310.1371/journal.ppat.1005133Crossref PubMed Scopus (63) Google Scholar) because many of the proteins involved in oxidative phosphorylation are Fe-S cluster or heme iron-containing proteins. This, in turn, can shift cellular metabolism in these cells toward a fermentation-like process leading to enhanced lactic acid accumulation (30Finch C.A. Gollnick P.D. Hlastala M.P. Miller L.R. Dillmann E. Mackler B. Lactic acidosis as a result of iron deficiency.J. Clin. Invest. 1979; 64: 129-13710.1172/JCI109431Crossref PubMed Scopus (80) Google Scholar); whereas RIM cells with higher iron would instead show a reduction in lactate accumulation. We thus hypothesized that our glucose-grown RIM cells might have enhanced mitochondrial functioning that can limit lactate-mediated β-1,3-glucan masking. To examine this, we first tested mitochondrial function by measuring mitochondrial membrane potential and ATP levels in our glucose-grown C. albicans cells. High iron cells showed a 1.34-fold increase in mitochondrial membrane potential (Fig. 4B) and a 1.6-fold increase in cellular ATP levels (Fig. 4C), indicating enhanced mitochondrial activity. To confirm if this, in turn, led to reduction in l-lactate (a dominant form of lactic acid) accumulation intracellularly, we also measured the levels of intracellular l-lactate, as well as extracellular levels (to account for release from the cells, if any). l-Lactate concentration was significantly lower under high iron RIM cells, compared with LIM-grown cells. Intracellular and extracellular concentrations were 2.7-fold (82.45 versus 226.5 μg/ml) and 28.8-fold (1.64 versus 47.35 μg/ml) lower, respectively, in RIM cells, compared with LIM cells (Fig. 4D). Additionally, changes in environmental iron can also alter cellular ROS production. We examined ROS levels in low and high iron C. albicans cells. As expected, we found that ROS production was higher in high iron RIM cells, as compared LIM-grown cells (Fig. 4E). We next sought to conform if reduction in l-lactate accumulation is indeed a contributing factor in the effect of high iron on enhanced β-1,3-glucan exposure. To understand this, we tested the effect of iron on β-1,3-glucan exposure in the presence of glucose or lactate as a sole carbon source, in parallel experiments. Interestingly, levels of β-1,3-glucan exposure were similar in lactate-grown LIM and RIM cells, with a clear loss of enhanced β-1,3-glucan exposure that was observed under high iron conditions (Fig. 4F), when glucose served as the sole carbon source (Fig. 4, A and F). As expected, both LIM and RIM cells grown on lactate also showed much higher levels of intracellular l-lactate (919.7 ± 134.7 and 791.7 ± 147.4, respectively; Fig. S4). Thus, enhanced intracellular l-lactate accumulation in LIM-grown cells with glucose as the sole carbon source (Fig. 4D), as well as exogenous lactate regardless of iron levels, both led to β-1,3-glucan masking (Fig. 4, A and F, respectively). This showed that lactate dominates over the effect of high iron on β-1,3-glucan exposure, and thus high iron-induced exposure in glucose-grown cells is a result of reduction in intracellular l-lactate accumulation. A role for lactate sensor (Gpr1) and transcriptional factor Crz1 in exogenous lactate-induced β-1,3-glucan masking has been demonstrated before (16Ballou E.R. Avelar G.M. Childers D.S. Mackie J. Bain J.M. Wagener J. Kastora S.L. Panea M.D. Hardison S.E. Walker L.A. Erwig L.P. Munro C.A. Gow N.A. Brown G.D. MacCallum D.M. et al.Lactate signalling regulates fungal β-glucan masking and immune evasion.Nat. Microbiol. 2016; 2 (27941860): 1623810.1038/nmicrobiol.2016.238Crossref PubMed Scopus (98) Google Scholar). High iron in glucose-grown RIM C. albicans cells led to a decrease in intracellular as well as extracellular lactate, whereas glucose-grown LIM cells showed comparatively higher levels of both intra- and extracellular lactate (Fig. 4D). Hence, we next tested if the released extracellular l-lactate in LIM cells signals through Gpr1 to induce the observed β-1,3-glucan masking. We first examined β-1,3-glucan exposure levels in glucose-grown C. albicans gpr1Δ/gpr1Δ LIM and RIM cells, along with their respective parental WT and re-constituted strains (strains with reintegration of a single copy of the respective gene into the genome of the corresponding null mutant). Glucose-grown C. albicans cells lacking Gpr1 retained the effect of iron, with significant β-1,3-glucan masking observed in LIM cells (Fig. 5A). This showed that the effect of iron on levels of β-1,3-glucan exposure, in glucose-grown cells, is independent of Gpr1, and therefore, solely mediated by intracellular l-lactate. Furthermore, to examine the role of Crz1 on the effect of iron on β-1,3-glucan masking, we measured β-1,3-glucan exposure levels in glucose-grown crz1Δ/crz1Δ LIM and RIM cells, along with their respective parental WT and re-constituted strains. β-1,3-Glucan masking was blocked, resulting in β-1,3-glucan exposure, in glucose-grown LIM cells lacking Crz1; whereas the iron effect was restored to WT levels in the re-constituted strain (Fig. 5B). This suggests that enhanced intracellular l-lactate accumulation in LIM cells (Fig. 4D) mediates decrease in β-1,3-glucan exposure levels (causing masking instead), through Crz1 activation, independent of the lactate sensor Gpr1. Also, in C. albicans, the calcineurin pathway activates Crz1 in response to calcium and cell wall stress (31Karababa M. Valentino E. Pardini G. Coste A.T. Bille J. Sanglard D. CRZ1, a target of the calcineurin pathway in Candida albicans.Mol. Microbiol. 2006; 59 (16468987): 1429-145110.1111/j.1365-2958.2005.05037.xCrossref PubMed Scopus (155) Google Scholar). Using cyclosporine A (CsA), an inhibitor of the calcineurin pathway (32Bruno V.M. Mitchell A.P. Regulation of azole drug susceptibility by Candida albicans protein kinase CK2.Mol. Microbiol. 2005; 56 (15813744): 559-57310.1111/j.1365-2958.2005.04562.xCrossref PubMed Scopus (35) Google Scholar), we next examined β-1,3-glucan exposure levels in LIM or RIM WT C. albicans cells treated with 1 or 25 μg ml−1 CsA. l-Lactate–mediated β-1,3-glucan masking was
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