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1.  Mnt1p and Mnt2p of Candida albicans Are Partially Redundant α-1,2-Mannosyltransferases That Participate in O-Linked Mannosylation and Are Required for Adhesion and Virulence* 
The Journal of biological chemistry  2004;280(2):1051-1060.
The MNT1 gene of the human fungal pathogen Candida albicans is involved in O-glycosylation of cell wall and secreted proteins and is important for adherence of C. albicans to host surfaces and for virulence. Here we describe the molecular analysis of CaMNT2, a second member of the MNT1-like gene family in C. albicans. Mnt2p also functions in O-glycosylation. Mnt1p and Mnt2p encode partially redundant α-1,2-mannosyltransferases that catalyze the addition of the second and third mannose residues in an O-linked mannose pentamer. Deletion of both copies of MNT1 and MNT2 resulted in reduction in the level of in vitro mannosyltransferase activity and truncation of O-mannan. Both the mnt2Δ and mnt1Δ single mutants were significantly reduced in adherence to human buccal epithelial cells and Matrigel-coated surfaces, indicating a role for O-glycosylated cell wall proteins or O-mannan itself in adhesion to host surfaces. The double mnt1Δmnt2Δ mutant formed aggregates of cells that appeared to be the result of abnormal cell separation. The double mutant was attenuated in virulence, underlining the importance of O-glycosylation in pathogenesis of C. albicans infections.
doi:10.1074/jbc.M411413200
PMCID: PMC3749086  PMID: 15519997
2.  Role of the Candida albicans MNN1 gene family in cell wall structure and virulence 
BMC Research Notes  2013;6:294.
Background
The Candida albicans cell wall is the first point of contact with the host, and its outer surface is heavily enriched in mannoproteins modified through the addition of N- and O-mannan. Previous work, using mutants with gross defects in glycosylation, has clearly identified the importance of mannan in the host-pathogen interaction, immune recognition and virulence. Here we report the first analysis of the MNN1 gene family, which contains six members predicted to act as α-1,3 mannosyltransferases in the terminal stages of glycosylation.
Findings
We generated single null mutants in all members of the C. albicans MNN1 gene family, and disruption of MNN14 led to both in vitro and in vivo defects. Null mutants in other members of the family demonstrated no phenotypic defects, suggesting that these members may display functional redundancy. The mnn14Δ null mutant displayed hypersensitivity to agents associated with cell wall and glycosylation defects, suggesting an altered cell wall structure. However, no gross changes in cell wall composition or N-glycosylation were identified in this mutant, although an extension of phosphomannan chain length was apparent. Although the cell wall defects associated with the mnn14Δ mutant were subtle, this mutant displayed a severe attenuation of virulence in a murine infection model.
Conclusion
Mnn14 plays a distinct role from other members of the MNN1 family, demonstrating that specific N-glycan outer chain epitopes are required in the host-pathogen interaction and virulence.
doi:10.1186/1756-0500-6-294
PMCID: PMC3750861  PMID: 23886038
Candida albicans; Glycosylation; Mannoproteins; Cell wall; MNN1; Virulence
3.  Reporters for the analysis of N-glycosylation in Candida albicans☆ 
Fungal Genetics and Biology  2013;56(100):107-115.
Highlights
•Reporters for dissection of N-glycosylation in Candida albicans.•Detection of glycosylation at the single site on epitope-tagged reporter.•Reporter faithfully reflects glycosylation defects in cell wall mutants.
A large proportion of Candida albicans cell surface proteins are decorated post-translationally by glycosylation. Indeed N-glycosylation is critical for cell wall biogenesis in this major fungal pathogen and for its interactions with host cells. A detailed understanding of N-glycosylation will yield deeper insights into host-pathogen interactions. However, the analysis of N-glycosylation is extremely challenging because of the complexity and heterogeneity of these structures. Therefore, in an attempt to reduce this complexity and facilitate the analysis of N-glycosylation, we have developed new synthetic C. albicans reporters that carry a single N-linked glycosylation site derived from Saccharomyces cerevisiae Suc2. These glycosylation reporters, which carry C. albicans Hex1 or Sap2 signal sequences plus carboxy-terminal FLAG3 and His6 tags, were expressed in C. albicans from the ACT1 promoter. The reporter proteins were successfully secreted and hyperglycosylated by C. albicans cells, and their outer chain glycosylation was dependent on Och1 and Pmr1, which are required for N-mannan synthesis, but not on Mnt1 and Mnt2 which are only required for O-mannosylation. These reporters are useful tools for the experimental dissection of N-glycosylation and other related processes in C. albicans, such as secretion.
doi:10.1016/j.fgb.2013.03.009
PMCID: PMC3705205  PMID: 23608318
Candida albicans; Glycosylation; Cell wall; Glycosylation reporter
4.  The Mnn2 Mannosyltransferase Family Modulates Mannoprotein Fibril Length, Immune Recognition and Virulence of Candida albicans 
PLoS Pathogens  2013;9(4):e1003276.
The fungal cell wall is the first point of interaction between an invading fungal pathogen and the host immune system. The outer layer of the cell wall is comprised of GPI anchored proteins, which are post-translationally modified by both N- and O-linked glycans. These glycans are important pathogen associated molecular patterns (PAMPs) recognised by the innate immune system. Glycan synthesis is mediated by a series of glycosyl transferases, located in the endoplasmic reticulum and Golgi apparatus. Mnn2 is responsible for the addition of the initial α1,2-mannose residue onto the α1,6-mannose backbone, forming the N-mannan outer chain branches. In Candida albicans, the MNN2 gene family is comprised of six members (MNN2, MNN21, MNN22, MNN23, MNN24 and MNN26). Using a series of single, double, triple, quintuple and sextuple mutants, we show, for the first time, that addition of α1,2-mannose is required for stabilisation of the α1,6-mannose backbone and hence regulates mannan fibril length. Sequential deletion of members of the MNN2 gene family resulted in the synthesis of lower molecular weight, less complex and more uniform N-glycans, with the sextuple mutant displaying only un-substituted α1,6-mannose. TEM images confirmed that the sextuple mutant was completely devoid of the outer mannan fibril layer, while deletion of two MNN2 orthologues resulted in short mannan fibrils. These changes in cell wall architecture correlated with decreased proinflammatory cytokine induction from monocytes and a decrease in fungal virulence in two animal models. Therefore, α1,2-mannose of N-mannan is important for both immune recognition and virulence of C. albicans.
Author Summary
Candida albicans is an opportunistic fungal pathogen of humans, which can cause superficial infections of the oral and genital epithelium or fatal systemic disease in immune compromised individuals. The cell wall of C. albicans consists of an inner layer of chitin and β-glucan and an outer fibrillar layer of highly glycosylated mannoproteins. These mannose-rich carbohydrates play crucial roles in fungal immune recognition. Deletion of genes encoding the Mnn2 family of α1,2-mannosyltransferases, which are involved in the biosynthesis of N-mannan outer chains, demonstrated that the addition of mannan side chains is necessary for the stabilisation and extension of the N-mannan backbone. Mutants of the MNN2 gene family had marked alterations in mannan microfibril length. Reductions in the degree of N-mannan branching resulted in decreased immune recognition by monocytes, whilst complete removal of the branched outer chains increased β-glucan exposure, and correlated with an attenuated virulence. Therefore, the Mnn2 mannosyltransferase family is critical for microfibril structure, immune recognition and virulence.
doi:10.1371/journal.ppat.1003276
PMCID: PMC3636026  PMID: 23633946
5.  The Evolutionary Rewiring of Ubiquitination Targets Has Reprogrammed the Regulation of Carbon Assimilation in the Pathogenic Yeast Candida albicans 
mBio  2012;3(6):e00495-12.
ABSTRACT
Microbes must assimilate carbon to grow and colonize their niches. Transcript profiling has suggested that Candida albicans, a major pathogen of humans, regulates its carbon assimilation in an analogous fashion to the model yeast Saccharomyces cerevisiae, repressing metabolic pathways required for the use of alterative nonpreferred carbon sources when sugars are available. However, we show that there is significant dislocation between the proteome and transcriptome in C. albicans. Glucose triggers the degradation of the ICL1 and PCK1 transcripts in C. albicans, yet isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are stable and are retained. Indeed, numerous enzymes required for the assimilation of carboxylic and fatty acids are not degraded in response to glucose. However, when expressed in C. albicans, S. cerevisiae Icl1 (ScIcl1) is subjected to glucose-accelerated degradation, indicating that like S. cerevisiae, this pathogen has the molecular apparatus required to execute ubiquitin-dependent catabolite inactivation. C. albicans Icl1 (CaIcl1) lacks analogous ubiquitination sites and is stable under these conditions, but the addition of a ubiquitination site programs glucose-accelerated degradation of CaIcl1. Also, catabolite inactivation is slowed in C. albicans ubi4 cells. Ubiquitination sites are present in gluconeogenic and glyoxylate cycle enzymes from S. cerevisiae but absent from their C. albicans homologues. We conclude that evolutionary rewiring of ubiquitination targets has meant that following glucose exposure, C. albicans retains key metabolic functions, allowing it to continue to assimilate alternative carbon sources. This metabolic flexibility may be critical during infection, facilitating the rapid colonization of dynamic host niches containing complex arrays of nutrients.
IMPORTANCE
Pathogenic microbes must assimilate a range of carbon sources to grow and colonize their hosts. Current views about carbon assimilation in the pathogenic yeast Candida albicans are strongly influenced by the Saccharomyces cerevisiae paradigm in which cells faced with choices of nutrients first use energetically favorable sugars, degrading enzymes required for the assimilation of less favorable alternative carbon sources. We show that this is not the case in C. albicans because there has been significant evolutionary rewiring of the molecular signals that promote enzyme degradation in response to glucose. As a result, this major pathogen of humans retains enzymes required for the utilization of physiologically relevant carbon sources such as lactic acid and fatty acids, allowing it to continue to use these host nutrients even when glucose is available. This phenomenon probably enhances efficient colonization of host niches where sugars are only transiently available.
doi:10.1128/mBio.00495-12
PMCID: PMC3520108  PMID: 23232717
6.  Elevated Cell Wall Chitin in Candida albicans Confers Echinocandin Resistance In Vivo 
Candida albicans cells with increased cell wall chitin have reduced echinocandin susceptibility in vitro. The aim of this study was to investigate whether C. albicans cells with elevated chitin levels have reduced echinocandin susceptibility in vivo. BALB/c mice were infected with C. albicans cells with normal chitin levels and compared to mice infected with high-chitin cells. Caspofungin therapy was initiated at 24 h postinfection. Mice infected with chitin-normal cells were successfully treated with caspofungin, as indicated by reduced kidney fungal burdens, reduced weight loss, and decreased C. albicans density in kidney lesions. In contrast, mice infected with high-chitin C. albicans cells were less susceptible to caspofungin, as they had higher kidney fungal burdens and greater weight loss during early infection. Cells recovered from mouse kidneys at 24 h postinfection with high-chitin cells had 1.6-fold higher chitin levels than cells from mice infected with chitin-normal cells and maintained a significantly reduced susceptibility to caspofungin when tested in vitro. At 48 h postinfection, caspofungin treatment induced a further increase in chitin content of C. albicans cells harvested from kidneys compared to saline treatment. Some of the recovered clones had acquired, at a low frequency, a point mutation in FKS1 resulting in a S645Y amino acid substitution, a mutation known to confer echinocandin resistance. This occurred even in cells that had not been exposed to caspofungin. Our results suggest that the efficacy of caspofungin against C. albicans was reduced in vivo due to either elevation of chitin levels in the cell wall or acquisition of FKS1 point mutations.
doi:10.1128/AAC.00683-11
PMCID: PMC3256049  PMID: 21986821
7.  Animal models of mucosal Candida infection 
FEMS microbiology letters  2008;283(2):129-139.
Rodent models of oral, vaginal and gastrointestinal Candida infection are described and discussed in terms of their scientific merits. The common feature of all experimental mucosal Candida infections is the need for some level of host immunocompromise or exogenous treatment to ensure quantitatively reproducible disease. A growing literature describes the contributions of such candidiasis models to our understanding of certain aspects of fungal virulence and host response to mucosal Candida albicans challenge. Evidence to date shows that T-lymphocyte responses dominate host immune defences to oral and gastrointestinal challenge, while other, highly compartmentalized responses defend vaginal surfaces. By contrast the study of C. albicans virulence factors in mucosal infection models has only begun to unravel the complex of attributes required to define the difference between strongly and weakly muco-invasive strains.
doi:10.1111/j.1574-6968.2008.01160.x
PMCID: PMC3244615  PMID: 18422625
animal; mucosal; epithelium; oral; model; vaginal
8.  MAPKKK-independent Regulation of the Hog1 Stress-activated Protein Kinase in Candida albicans* 
The Journal of Biological Chemistry  2011;286(49):42002-42016.
The Hog1 stress-activated protein kinase regulates both stress responses and morphogenesis in Candida albicans and is essential for the virulence of this major human pathogen. Stress-induced Hog1 phosphorylation is regulated by the upstream MAPKK, Pbs2, which in turn is regulated by the MAPKKK, Ssk2. Here, we have investigated the role of phosphorylation of Hog1 and Pbs2 in Hog1-mediated processes in C. albicans. Mutation of the consensus regulatory phosphorylation sites of Hog1 (Thr-174/Tyr-176) and Pbs2 (Ser-355/Thr-359), to nonphosphorylatable residues, resulted in strains that phenocopied hog1Δ and pbs2Δ cells. Consistent with this, stress-induced phosphorylation of Hog1 was abolished in cells expressing nonphosphorylatable Pbs2 (Pbs2AA). However, mutation of the consensus sites of Pbs2 to phosphomimetic residues (Pbs2DD) failed to constitutively activate Hog1. Furthermore, Ssk2-independent stress-induced Hog1 activation was observed in Pbs2DD cells. Collectively, these data reveal a previously uncharacterized MAPKKK-independent mechanism of Hog1 activation in response to stress. Although Pbs2DD cells did not exhibit high basal levels of Hog1 phosphorylation, overexpression of an N-terminal truncated form of Ssk2 did result in constitutive Hog1 activation, which was further increased upon stress. Significantly, both Pbs2AA and Pbs2DD cells displayed impaired stress resistance and attenuated virulence in a mouse model of disease, whereas only Pbs2AA cells exhibited the morphological defects associated with loss of Hog1 function. This indicates that Hog1 mediates C. albicans virulence by conferring stress resistance rather than regulating morphogenesis.
doi:10.1074/jbc.M111.265231
PMCID: PMC3234903  PMID: 21994942
Fungi; Pathogenesis; Signal Transduction; Stress Response; Yeast; Candida albicans; Stress-activated Protein Kinases
9.  Differential Regulation of Kidney and Spleen Cytokine Responses in Mice Challenged with Pathology-Standardized Doses of Candida albicans Mannosylation Mutants▿ †  
Infection and Immunity  2010;79(1):146-152.
Cell surface polysaccharides are key determinants of host responses to fungal infection. We determined the effects of alterations in Candida albicans cell surface polysaccharide composition and gross changes in the host immune response in groups of mice challenged intravenously with five C. albicans strains at doses adjusted to give equal disease progression 3 days later. The five strains used were the parental strain NGY152, two mutants with defective cell wall mannosylation, pmr1Δ mutant and mnt1/2Δ mutant, and the same two strains with a copy of PMR1 and MNT1 reintegrated, respectively. Renal and spleen levels of chemokines and cytokines previously shown to be key components of early host response to C. albicans were determined at intervals up to 3 days after challenge. By 12 h after C. albicans challenge, the levels of granulocyte colony-stimulating factor (G-CSF), keratinocyte-derived chemokine (KC), interleukin 6 (IL-6), monocyte chemotactic peptide 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and MIP-2 were higher in the kidneys of mice challenged with the pmr1Δ mutant than in animals challenged with the other strains and were lower by day 3, suggesting an earlier host response to the pmr1Δ mutant. The production of these chemokines also diminished earlier than controls in mice infected with the mnt1/2Δ strain. Although these differences were statistically significant, their magnitude was seldom great, and no unambiguous evidence was obtained for individual responses specific to any cell surface glycosylation change. We conclude that complex, multifactorial local responses offset and obscure any differences resulting from differences in surface mannosylation of C. albicans strains when infection results from pathology-standardized challenges.
doi:10.1128/IAI.01004-10
PMCID: PMC3019922  PMID: 21059878
10.  Glycosylation status of the C. albicans cell wall affects the efficiency of neutrophil phagocytosis and killing but not cytokine signaling 
Medical Mycology  2011;49(5):513-524.
The cell wall of the opportunistic human fungal pathogen, Candida albicans is a complex, layered network of rigid structural polysaccharides composed of β-glucans and chitin that is covered with a fibrillar matrix of highly glycosylated mannoproteins. Poly-morphonuclear cells (PMNs, neutrophils) are the most prevalent circulating phagocytic leukocyte in peripheral blood and they are pivotal in the clearance of invading fungal cells from tissues. The importance of cell-wall mannans for the recognition and uptake of C. albicans by human PMNs was therefore investigated. N- and O-glycosylation-deficient mutants were attenuated in binding and phagocytosis by PMNs and this was associated with reduced killing of C. albicans yeast cells. No differences were found in the production of the respiratory burst enzyme myeloperoxidase (MPO) and the neutrophil chemokine IL-8 in PMNs exposed to control and glycosylation-deficient C. albicans strains. Thus, the significant decrease in killing of glycan-deficient C. albicans strains by PMNs is a consequence of a marked reduction in phagocytosis rather than changes in the release of inflammatory mediators by PMNs.
doi:10.3109/13693786.2010.551425
PMCID: PMC3119872  PMID: 21254968
C. albicans; glycosylation; innate immunity; PMN
11.  Recognition and Blocking of Innate Immunity Cells by Candida albicans Chitin ▿ †  
Infection and Immunity  2011;79(5):1961-1970.
Chitin is a skeletal cell wall polysaccharide of the inner cell wall of fungal pathogens. As yet, little about its role during fungus-host immune cell interactions is known. We show here that ultrapurified chitin from Candida albicans cell walls did not stimulate cytokine production directly but blocked the recognition of C. albicans by human peripheral blood mononuclear cells (PBMCs) and murine macrophages, leading to significant reductions in cytokine production. Chitin did not affect the induction of cytokines stimulated by bacterial cells or lipopolysaccharide (LPS), indicating that blocking was not due to steric masking of specific receptors. Toll-like receptor 2 (TLR2), TLR4, and Mincle (the macrophage-inducible C-type lectin) were not required for interactions with chitin. Dectin-1 was required for immune blocking but did not bind chitin directly. Cytokine stimulation was significantly reduced upon stimulation of PBMCs with heat-killed chitin-deficient C. albicans cells but not with live cells. Therefore, chitin is normally not exposed to cells of the innate immune system but is capable of influencing immune recognition by blocking dectin-1-mediated engagement with fungal cell walls.
doi:10.1128/IAI.01282-10
PMCID: PMC3088140  PMID: 21357722
12.  Melanin Externalization in Candida albicans Depends on Cell Wall Chitin Structures▿ 
Eukaryotic Cell  2010;9(9):1329-1342.
The fungal pathogen Candida albicans produces dark-pigmented melanin after 3 to 4 days of incubation in medium containing l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. Expression profiling of C. albicans revealed very few genes significantly up- or downregulated by growth in l-DOPA. We were unable to determine a possible role for melanin in the virulence of C. albicans. However, we showed that melanin was externalized from the fungal cells in the form of electron-dense melanosomes that were free or often loosely bound to the cell wall exterior. Melanin production was boosted by the addition of N-acetylglucosamine to the medium, indicating a possible association between melanin production and chitin synthesis. Melanin externalization was blocked in a mutant specifically disrupted in the chitin synthase-encoding gene CHS2. Melanosomes remained within the outermost cell wall layers in chs3Δ and chs2Δ chs3Δ mutants but were fully externalized in chs8Δ and chs2Δ chs8Δ mutants. All the CHS mutants synthesized dark pigment at equivalent rates from mixed membrane fractions in vitro, suggesting it was the form of chitin structure produced by the enzymes, not the enzymes themselves, that was involved in the melanin externalization process. Mutants with single and double disruptions of the chitinase genes CHT2 and CHT3 and the chitin pathway regulator ECM33 also showed impaired melanin externalization. We hypothesize that the chitin product of Chs3 forms a scaffold essential for normal externalization of melanosomes, while the Chs8 chitin product, probably produced in cell walls in greater quantity in the absence of CHS2, impedes externalization.
doi:10.1128/EC.00051-10
PMCID: PMC2937336  PMID: 20543065
13.  Genetic Dissection of Azole Resistance Mechanisms in Candida albicans and Their Validation in a Mouse Model of Disseminated Infection▿ †  
Principal mechanisms of resistance to azole antifungals include the upregulation of multidrug transporters and the modification of the target enzyme, a cytochrome P450 (Erg11) involved in the 14α-demethylation of ergosterol. These mechanisms are often combined in azole-resistant Candida albicans isolates recovered from patients. However, the precise contributions of individual mechanisms to C. albicans resistance to specific azoles have been difficult to establish because of the technical difficulties in the genetic manipulation of this diploid species. Recent advances have made genetic manipulations easier, and we therefore undertook the genetic dissection of resistance mechanisms in an azole-resistant clinical isolate. This isolate (DSY296) upregulates the multidrug transporter genes CDR1 and CDR2 and has acquired a G464S substitution in both ERG11 alleles. In DSY296, inactivation of TAC1, a transcription factor containing a gain-of-function mutation, followed by sequential replacement of ERG11 mutant alleles with wild-type alleles, restored azole susceptibility to the levels measured for a parent azole-susceptible isolate (DSY294). These sequential genetic manipulations not only demonstrated that these two resistance mechanisms were those responsible for the development of resistance in DSY296 but also indicated that the quantitative level of resistance as measured in vitro by MIC determinations was a function of the number of genetic resistance mechanisms operating in any strain. The engineered strains were also tested for their responses to fluconazole treatment in a novel 3-day model of invasive C. albicans infection of mice. Fifty percent effective doses (ED50s) of fluconazole were highest for DSY296 and decreased proportionally with the sequential removal of each resistance mechanism. However, while the fold differences in ED50 were proportional to the fold differences in MICs, their magnitude was lower than that measured in vitro and depended on the specific resistance mechanism operating.
doi:10.1128/AAC.01645-09
PMCID: PMC2849354  PMID: 20086148
14.  A Multifunctional Mannosyltransferase Family in Candida albicans Determines Cell Wall Mannan Structure and Host-Fungus Interactions* 
The Journal of Biological Chemistry  2010;285(16):12087-12095.
The cell wall proteins of fungi are modified by N- and O-linked mannosylation and phosphomannosylation, resulting in changes to the physical and immunological properties of the cell. Glycosylation of cell wall proteins involves the activities of families of endoplasmic reticulum and Golgi-located glycosyl transferases whose activities are difficult to infer through bioinformatics. The Candida albicans MNT1/KRE2 mannosyl transferase family is represented by five members. We showed previously that Mnt1 and Mnt2 are involved in O-linked mannosylation and are required for virulence. Here, the role of C. albicans MNT3, MNT4, and MNT5 was determined by generating single and multiple MnTΔ null mutants and by functional complementation experiments in Saccharomyces cerevisiae. CaMnt3, CaMnt4, and CaMnt5 did not participate in O-linked mannosylation, but CaMnt3 and CaMnt5 had redundant activities in phosphomannosylation and were responsible for attachment of approximately half of the phosphomannan attached to N-linked mannans. CaMnt4 and CaMnt5 participated in N-mannan branching. Deletion of CaMNT3, CaMNT4, and CaMNT5 affected the growth rate and virulence of C. albicans, affected the recognition of the yeast by human monocytes and cytokine stimulation, and led to increased cell wall chitin content and exposure of β-glucan at the cell wall surface. Therefore, the MNT1/KRE2 gene family participates in three types of protein mannosylation in C. albicans, and these modifications play vital roles in fungal cell wall structure and cell surface recognition by the innate immune system.
doi:10.1074/jbc.M109.081513
PMCID: PMC2852947  PMID: 20164191
Cell Wall; Cell-Cell Interaction; Cytokine; Fungi; Glycoprotein Biosynthesis; Glycosylation; Innate Immunity; Pattern Recognition Receptor; Candida; Mannan
15.  Glucose Promotes Stress Resistance in the Fungal Pathogen Candida albicans 
Molecular Biology of the Cell  2009;20(22):4845-4855.
Metabolic adaptation, and in particular the modulation of carbon assimilatory pathways during disease progression, is thought to contribute to the pathogenicity of Candida albicans. Therefore, we have examined the global impact of glucose upon the C. albicans transcriptome, testing the sensitivity of this pathogen to wide-ranging glucose levels (0.01, 0.1, and 1.0%). We show that, like Saccharomyces cerevisiae, C. albicans is exquisitely sensitive to glucose, regulating central metabolic genes even in response to 0.01% glucose. This indicates that glucose concentrations in the bloodstream (approximate range 0.05–0.1%) have a significant impact upon C. albicans gene regulation. However, in contrast to S. cerevisiae where glucose down-regulates stress responses, some stress genes were induced by glucose in C. albicans. This was reflected in elevated resistance to oxidative and cationic stresses and resistance to an azole antifungal agent. Cap1 and Hog1 probably mediate glucose-enhanced resistance to oxidative stress, but neither is essential for this effect. However, Hog1 is phosphorylated in response to glucose and is essential for glucose-enhanced resistance to cationic stress. The data suggest that, upon entering the bloodstream, C. albicans cells respond to glucose increasing their resistance to the oxidative and cationic stresses central to the armory of immunoprotective phagocytic cells.
doi:10.1091/mbc.E09-01-0002
PMCID: PMC2777113  PMID: 19759180
16.  Pyrosequencing Analysis of 20 Nucleotides of Internal Transcribed Spacer 2 Discriminates Candida parapsilosis, Candida metapsilosis, and Candida orthopsilosis▿  
Journal of Clinical Microbiology  2009;47(7):2307-2310.
Two new cryptic sister species, Candida orthopsilosis and Candida metapsilosis, were recently identified by consistent DNA sequence differences among several genes within the genetically heterogeneous Candida parapsilosis complex. Here, we present data demonstrating that Pyrosequencing analysis of 20 nucleotides of internal transcribed spacer region 2 rapidly and robustly distinguishes between these three closely related Candida species.
doi:10.1128/JCM.00240-09
PMCID: PMC2708508  PMID: 19403763
17.  Property Differences among the Four Major Candida albicans Strain Clades ▿ †  
Eukaryotic Cell  2009;8(3):373-387.
A selection of 43 Candida albicans isolates, chosen to represent the four major strain clades of the species and also intraclade diversity, was screened for their virulence in the murine intravenous challenge model of C. albicans infection, for a range of properties measurable in vitro that might relate to virulence, and for the numbers of midrepeat sequences in genes of the ALS and HYR families. Heterozygosity at the mating type locus and low whole-cell acid phosphatase activity and growth rate at 40°C were found to be significantly positively associated with the most virulent isolates. Acid phosphatase activity and growth in 2 M NaCl were statistically significant variables between clades by univariate analysis. Isolates in different clades also differed significantly in midrepeat sequence alleles of ALS2, ALS4, ALS6, ALS7, ALS9, HYR1, and HYR2. There was no association between the midrepeat alleles of any ALS or HYR gene and the virulence of isolates to mice. Genome-wide transcript profiles of 20 isolates (5 per clade) grown under two conditions showed considerable variation between individual isolates, but only a small number of genes showed statistically significant differential gene expression between clades. Analysis of the expression profiles by overall strain virulence revealed 18 open reading frames differing significantly between isolates of high, intermediate, and low virulence. Four of these genes encoded functions related to phosphate uptake and metabolism. This finding and the significant association between whole-cell acid phosphatase activity and virulence led us to disrupt PHO100, which encodes a predicted periplasmic acid phosphatase. The pho100Δ mutant was mildly but significantly attenuated in terms of survival curves in the mouse model. The study has extended the range of properties known to differ between C. albicans clades and suggests a possible but minor role of phosphate metabolism in the virulence of the species.
doi:10.1128/EC.00387-08
PMCID: PMC2653250  PMID: 19151328
18.  Early-Expressed Chemokines Predict Kidney Immunopathology in Experimental Disseminated Candida albicans Infections 
PLoS ONE  2009;4(7):e6420.
Background
The mouse intravenous challenge model of Candida albicans infection is widely used to determine aspects of host-fungus interaction. We investigated the production of cytokines in the kidneys and spleen of animals up to 48 h after challenge with virulent and attenuated isolates and related these responses to semi-quantitative estimations of histopathological changes in the kidney.
Methodology/Principal Findings
Progression of Candida albicans infection of the kidney in response to highly virulent fungal strains was characterized by higher levels of host cellular infiltrate, higher lesion densities and greater quantities of fungal elements at 24 and 48 h, and by higher kidney concentrations of IL-1β, MCP-1, KC, IL-6, G-CSF, TNF, MIP-2 and MIP-1β, among the immune effectors measured. Levels of the chemokine KC as early as 12 h after challenge correlated significantly with all later measurements of lesion severity. Early renal IL-6 and MIP-1β concentrations also correlated with subsequent damage levels, but less significantly than for KC. All chemokines tested appeared in kidney homogenates, while most of the cytokines were undetectable in kidney and spleen homogenates. GM-CSF and IL-10 showed inverse correlations with measures of lesion severity, suggesting these alone may have exerted a defensive role. Spleen levels of KC at all times showed significant associations with kidney lesion measurements.
Conclusions/Significance
Elevated chemokine levels, including KC, represent the earliest responses to C. albicans infection in the mouse kidney. Fungal strains of low mouse virulence stimulate a lower innate response and less host infiltrate than more virulent strains. These findings are consistent with immunopathological damage to kidneys in the mouse C. albicans infection model and with growing evidence implicating some TLR pathways as the main point of interaction between fungal surface polysaccharides and leukocytes.
doi:10.1371/journal.pone.0006420
PMCID: PMC2712765  PMID: 19641609
19.  Biofilm Matrix Regulation by Candida albicans Zap1 
PLoS Biology  2009;7(6):e1000133.
The zinc-responsive transcription factor Zap1 has a striking role in fungal biofilm formation and is reported to regulate matrix formation.
A biofilm is a surface-associated population of microorganisms embedded in a matrix of extracellular polymeric substances. Biofilms are a major natural growth form of microorganisms and the cause of pervasive device-associated infection. This report focuses on the biofilm matrix of Candida albicans, the major fungal pathogen of humans. We report here that the C. albicans zinc-response transcription factor Zap1 is a negative regulator of a major matrix component, soluble β-1,3 glucan, in both in vitro and in vivo biofilm models. To understand the mechanistic relationship between Zap1 and matrix, we identified Zap1 target genes through expression profiling and full genome chromatin immunoprecipitation. On the basis of these results, we designed additional experiments showing that two glucoamylases, Gca1 and Gca2, have positive roles in matrix production and may function through hydrolysis of insoluble β-1,3 glucan chains. We also show that a group of alcohol dehydrogenases Adh5, Csh1, and Ifd6 have roles in matrix production: Adh5 acts positively, and Csh1 and Ifd6, negatively. We propose that these alcohol dehydrogenases generate quorum-sensing aryl and acyl alcohols that in turn govern multiple events in biofilm maturation. Our findings define a novel regulatory circuit and its mechanism of control of a process central to infection.
Author Summary
A biofilm is a surface-associated population of microbes that is embedded in a cement of extracellular compounds. This cement is known as matrix. The two main functions of matrix are to protect cells from their surrounding environment, preventing drugs and other stresses from penetrating the biofilm, and to maintain the architectural stability of the biofilm, acting as a glue to hold the cells together. The presence of matrix is a contributing factor to the high degree of resistance to antimicrobial drugs observed in biofilms. Because biofilms have a major impact on human health, and because matrix is such a pivotal component of biofilms, it is important to understand how the production of matrix is regulated. We have begun to address this question in the major human fungal pathogen Candida albicans. We found that the zinc-responsive regulatory protein Zap1 controls the expression of several genes important for matrix formation in C. albicans. These target genes encode glucoamylases and alcohol dehydrogenases, enzymes that probably govern the synthesis of distinct matrix constituents. The findings here offer insight into the metabolic processes that contribute to biofilm formation and indicate that Zap1 functions broadly as a negative regulator of biofilm maturation.
doi:10.1371/journal.pbio.1000133
PMCID: PMC2688839  PMID: 19529758
20.  Revised Definitions of Invasive Fungal Disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group 
Background
Invasive fungal diseases are important causes of morbidity and mortality. Clarity and uniformity in defining these infections are important factors in improving the quality of clinical studies. A standard set of definitions strengthens the consistency and reproducibility of such studies.
Methods
After the introduction of the original European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group definitions, advances in diagnostic technology and the recognition of areas in need of improvement led to a revision of this document. The revision process started with a meeting of participants in 2003, to decide on the process and to draft the proposal. This was followed by several rounds of consultation until a final draft was approved in 2005. This was made available for 6 months to allow public comment, and then the manuscript was prepared and approved.
Results
The revised definitions retain the original classifications of “proven,” “probable,” and “possible” invasive fungal disease, but the definition of “probable” has been expanded, whereas the scope of the category “possible” has been diminished. The category of proven invasive fungal disease can apply to any patient, regardless of whether the patient is immunocompromised, whereas the probable and possible categories are proposed for immunocompromised patients only.
Conclusions
These revised definitions of invasive fungal disease are intended to advance clinical and epidemiological research and may serve as a useful model for defining other infections in high-risk patients.
doi:10.1086/588660
PMCID: PMC2671227  PMID: 18462102
21.  Mixed Candida albicans strain populations in colonized and infected mucosal tissues 
Fems Yeast Research  2008;8(8):1334-1338.
Multilocus sequence typing of six Candida albicans colonies from primary isolation plates revealed instances of colony-to-colony microvariation and carriage of two strain types in single oropharyngeal and vaginal samples. Higher rates of colony variation in commensal samples suggest selection of types from mixed populations either in the shift to pathogenicity or the response to antifungal treatment.
doi:10.1111/j.1567-1364.2008.00438.x
PMCID: PMC2667310  PMID: 18795958
Candida albicans; MLST; strain typing; epidemiology
22.  Immune Recognition of Candida albicans β-glucan by Dectin-1 
The Journal of infectious diseases  2007;196(10):1565-1571.
β(1,3)-glucans represent 40% of the cell wall of the yeast Candida albicans. The dectin-1 lectin-like receptor has shown to recognize fungal β(1,3)-glucans and induce innate immune responses. The importance of β-glucan-dectin-1 pathways for the recognition of C. albicans by human primary blood cells has not been firmly established. In this study we demonstrate that cytokine production by both human peripheral blood mononuclear cells and murine macrophages is dependent on the recognition of β-glucans by dectin-1. Heat killing of C. albicans resulted in exposure of β-glucans on the surface of the cell wall and subsequent recognition by dectin-1, whereas live yeasts stimulated monocytes mainly via recognition of cell-surface mannans. Dectin-1 induced cytokine production through the following 2 pathways: Syk-dependent production of the T-helper (Th) 2-type anti-inflammatory cytokine interleukin-10 and Toll-like receptor-Myd88-dependent stimulation of monocyte-derived proinflammatory cytokines, such as tumor necrosis factor-α. In contrast, stimulation of Th1-type cytokines, such as interferon-γ, by C. albicans was independent of the recognition of β-glucans by dectin-1. In conclusion, C. albicans induces production of monocyte-derived and T cell-derived cytokines through distinct pathways dependent on or independent of dectin-1.
doi:10.1086/523110
PMCID: PMC2655640  PMID: 18008237
23.  Genome-wide analysis of Candida albicans gene expression patterns during infection of the mammalian kidney 
Fungal Genetics and Biology  2009;46(2):210-219.
Global analysis of the molecular responses of microbial pathogens to their mammalian hosts represents a major challenge. To date few microarray studies have been performed on Candida albicans cells derived from infected tissues. In this study we examined the C. albicans SC5314 transcriptome from renal infections in the rabbit. Genes involved in adhesion, stress adaptation and the assimilation of alternative carbon sources were up-regulated in these cells compared with control cells grown in RPMI 1640, whereas genes involved in morphogenesis, fermentation and translation were down-regulated. When we compared the congenic virulent C. albicans strains NGY152 and SC5314, there was minimal overlap between their transcriptomes during kidney infections. This suggests that much of the gene regulation observed during infections is not essential for virulence. Indeed, we observed a poor correlation between the transcriptome and phenome for those genes that were regulated during kidney infection and that have been virulence tested.
doi:10.1016/j.fgb.2008.10.012
PMCID: PMC2698078  PMID: 19032986
Candida albicans; Infection; Genomics; Microarrays; Transcript profiling
24.  Multilocus Sequence Typing of Pathogenic Candida Species▿  
Eukaryotic Cell  2008;7(7):1075-1084.
doi:10.1128/EC.00062-08
PMCID: PMC2446668  PMID: 18456859
25.  Carnitine-Dependent Transport of Acetyl Coenzyme A in Candida albicans Is Essential for Growth on Nonfermentable Carbon Sources and Contributes to Biofilm Formation▿  
Eukaryotic Cell  2008;7(4):610-618.
In eukaryotes, acetyl coenzyme A (acetyl-CoA) produced during peroxisomal fatty acid β-oxidation needs to be transported to mitochondria for further metabolism. Two parallel pathways for acetyl-CoA transport have been identified in Saccharomyces cerevisiae; one is dependent on peroxisomal citrate synthase (Cit), while the other requires peroxisomal and mitochondrial carnitine acetyltransferase (Cat) activities. Here we show that the human fungal pathogen Candida albicans lacks peroxisomal Cit, relying exclusively on Cat activity for transport of acetyl units. Deletion of the CAT2 gene encoding the major Cat enzyme in C. albicans resulted in a strain that had lost both peroxisomal and mitochondrion-associated Cat activities, could not grow on fatty acids or C2 carbon sources (acetate or ethanol), accumulated intracellular acetyl-CoA, and showed greatly reduced fatty acid β-oxidation activity. The cat2 null mutant was, however, not attenuated in virulence in a mouse model of systemic candidiasis. These observations support our previous results showing that peroxisomal fatty acid β-oxidation activity is not essential for C. albicans virulence. Biofilm formation by the cat2 mutant on glucose was slightly reduced compared to that by the wild type, although both strains grew at the same rate on this carbon source. Our data show that C. albicans has diverged considerably from S. cerevisiae with respect to the mechanism of intracellular acetyl-CoA transport and imply that carnitine dependence may be an important trait of this human fungal pathogen.
doi:10.1128/EC.00017-08
PMCID: PMC2292619  PMID: 18281597

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