Complement-Mediated Differential Immune Response of Human Macrophages to Sporothrix Species Through Interaction With Their Cell Wall Peptidorhamnomannans.

In this study, the human immune response mechanisms against Sporothrix brasiliensis and Sporothrix schenckii, two causative agents of human and animal sporotrichosis, were investigated. The interaction of S. brasiliensis and S. schenckii with human monocyte-derived macrophages (hMDMs) was shown to be dependent on the thermolabile serum complement protein C3, which facilitated the phagocytosis of Sporothrix yeast cells through opsonization. The peptidorhamnomannan (PRM) component of the cell walls of these two Sporothrix yeasts was found to be one of their surfaces exposed pathogen-associated molecular pattern (PAMP), leading to activation of the complement system and deposition of C3b on the Sporothrix yeast surfaces. PRM also showed direct interaction with CD11b, the specific component of the complement receptor-3 (CR3). Furthermore, the blockade of CR3 specifically impacted the interleukin (IL)-1ß secretion by hMDM in response to both S. brasiliensis and S. schenckii, suggesting that the host complement system plays an essential role in the inflammatory immune response against these Sporothrix species. Nevertheless, the structural differences in the PRMs of the two Sporothrix species, as revealed by NMR, were related to the differences observed in the host complement activation pathways. Together, this work reports a new PAMP of the cell surface of pathogenic fungi playing a role through the activation of complement system and via CR3 receptor mediating an inflammatory response to Sporothrix species.

Caspofungin Induced Cell Wall Changes of Candida Species Influences Macrophage Interactions.

Candida species are known to differ in their ability to cause infection and have been shown to display varied susceptibilities to antifungal drugs. Treatment with the echinocandin, caspofungin, leads to compensatory alterations in the fungal cell wall. This study was performed to compare the structure and composition of the cell walls of different Candida species alone and in response to caspofungin treatment, and to evaluate how changes at the fungal cell surface affects interactions with macrophages. We demonstrated that the length of the outer fibrillar layer varied between Candida species and that, in most cases, reduced fibril length correlated with increased exposure of ß-1,3-glucan on the cell surface. Candida glabrata and Candida guilliermondii, which had naturally more ß-1,3-glucan exposed on the cell surface, were phagocytosed significantly more efficiently by J774 macrophages. Treatment with caspofungin resulted in increased exposure of chitin and ß-1,3-glucan on the surface of the majority of Candida species isolates that were tested, with the exception of C. glabrata and Candida parapsilosis isolates. This increase in exposure of the inner cell wall polysaccharides, in most cases, correlated with reduced uptake by macrophages and in turn, a decrease in production of TNFα. Here we show that differences in the exposure of cell wall carbohydrates and variations in the repertoire of covalently attached surface proteins of different Candida species contributes to their recognition by immune cells.

Inhibition of Classical and Alternative Modes of Respiration in Candida albicans Leads to Cell Wall Remodeling and Increased Macrophage Recognition.

The human fungal pathogen Candida albicans requires respiratory function for normal growth, morphogenesis, and virulence. Mitochondria therefore represent an enticing target for the development of new antifungal strategies. This possibility is bolstered by the presence of characteristics specific to fungi. However, respiration in C. albicans, as in many fungal organisms, is facilitated by redundant electron transport mechanisms, making direct inhibition a challenge. In addition, many chemicals known to target the electron transport chain are highly toxic. Here we made use of chemicals with low toxicity to efficiently inhibit respiration in C. albicans We found that use of the nitric oxide donor sodium nitroprusside (SNP) and of the alternative oxidase inhibitor salicylhydroxamic acid (SHAM) prevents respiration and leads to a loss of viability and to cell wall rearrangements that increase the rate of uptake by macrophages in vitro and in vivo We propose that treatment with SNP plus SHAM (SNP+SHAM) leads to transcriptional changes that drive cell wall rearrangement but which also prime cells to activate the transition to hyphal growth. In line with this, we found that pretreatment of C. albicans with SNP+SHAM led to an increase in virulence. Our data reveal strong links between respiration, cell wall remodeling, and activation of virulence factors. Our findings demonstrate that respiration in C. albicans can be efficiently inhibited with chemicals that are not damaging to the mammalian host but that we need to develop a deeper understanding of the roles of mitochondria in cellular signaling if they are to be developed successfully as a target for new antifungals.IMPORTANCE Current approaches to tackling fungal infections are limited, and new targets must be identified to protect against the emergence of resistant strains. We investigated the potential of targeting mitochondria, which are organelles required for energy production, growth, and virulence, in the human fungal pathogen Candida albicans Our findings suggest that mitochondria can be targeted using drugs that can be tolerated by humans and that this treatment enhances their recognition by immune cells. However, release of C. albicans cells from respiratory inhibition appears to activate a stress response that increases the levels of traits associated with virulence. Our results make it clear that mitochondria represent a valid target for the development of antifungal strategies but that we must determine the mechanisms by which they regulate stress signaling and virulence ahead of successful therapeutic advance.

Hypoxia Promotes Immune Evasion by Triggering ß-Glucan Masking on the Candida albicans Cell Surface via Mitochondrial and cAMP-Protein Kinase A Signaling.

Organisms must adapt to changes in oxygen tension if they are to exploit the energetic benefits of reducing oxygen while minimizing the potentially damaging effects of oxidation. Consequently, organisms in all eukaryotic kingdoms display robust adaptation to hypoxia (low oxygen levels). This is particularly important for fungal pathogens that colonize hypoxic niches in the host. We show that adaptation to hypoxia in the major fungal pathogen of humans Candida albicans includes changes in cell wall structure and reduced exposure, at the cell surface, of ß-glucan, a key pathogen-associated molecular pattern (PAMP). This leads to reduced phagocytosis by murine bone marrow-derived macrophages and decreased production of IL-10, RANTES, and TNF-α by peripheral blood mononuclear cells, suggesting that hypoxia-induced ß-glucan masking has a significant effect upon C. albicans-host interactions. We show that hypoxia-induced ß-glucan masking is dependent upon both mitochondrial and cAMP-protein kinase A (PKA) signaling. The decrease in ß-glucan exposure is blocked by mutations that affect mitochondrial functionality (goa1Δ and upc2Δ) or that decrease production of hydrogen peroxide in the inner membrane space (sod1Δ). Furthermore, ß-glucan masking is enhanced by mutations that elevate mitochondrial reactive oxygen species (aox1Δ). The ß-glucan masking defects displayed by goa1Δ and upc2Δ cells are suppressed by exogenous dibutyryl-cAMP. Also, mutations that inactivate cAMP synthesis (cyr1Δ) or PKA (tpk1Δ tpk2Δ) block the masking phenotype. Our data suggest that C. albicans responds to hypoxic niches by inducing ß-glucan masking via a mitochondrial cAMP-PKA signaling pathway, thereby modulating local immune responses and promoting fungal colonization.IMPORTANCE Animal, plant, and fungal cells occupy environments that impose changes in oxygen tension. Consequently, many species have evolved mechanisms that permit robust adaptation to these changes. The fungal pathogen Candida albicans can colonize hypoxic (low oxygen) niches in its human host, such as the lower gastrointestinal tract and inflamed tissues, but to colonize its host, the fungus must also evade local immune defenses. We reveal, for the first time, a defined link between hypoxic adaptation and immune evasion in C. albicans As this pathogen adapts to hypoxia, it undergoes changes in cell wall structure that include masking of ß-glucan at its cell surface, and it becomes better able to evade phagocytosis by innate immune cells. We also define the signaling mechanisms that mediate hypoxia-induced ß-glucan masking, showing that they are dependent on mitochondrial signaling and the cAMP-protein kinase pathway. Therefore, hypoxia appears to trigger immune evasion in this fungal pathogen.

Cell walls of the dimorphic fungal pathogens Sporothrix schenckii and Sporothrix brasiliensis exhibit bilaminate structures and sloughing of extensive and intact layers.

Sporotrichosis is a subcutaneous mycosis caused by pathogenic species of the Sporothrix genus. A new emerging species, Sporothrix brasiliensis, is related to cat-transmitted sporotrichosis and has severe clinical manifestations. The cell wall of pathogenic fungi is a unique structure and impacts directly on the host immune response. We reveal and compare the cell wall structures of Sporothrix schenckii and S. brasiliensis using high-pressure freezing electron microscopy to study the cell wall organization of both species. To analyze the components of the cell wall, we also used infrared and 13C and 1H NMR spectroscopy and the sugar composition was determined by quantitative high-performance anion-exchange chromatography. Our ultrastructural data revealed a bi-layered cell wall structure for both species, including an external microfibrillar layer and an inner electron-dense layer. The inner and outer layers of the S. brasiliensis cell wall were thicker than those of S. schenckii, correlating with an increase in the chitin and rhamnose contents. Moreover, the outer microfibrillar layer of the S. brasiliensis cell wall had longer microfibrils interconnecting yeast cells. Distinct from those of other dimorphic fungi, the cell wall of Sporothrix spp. lacked α-glucan component. Interestingly, glycogen α-particles were identified in the cytoplasm close to the cell wall and the plasma membrane. The cell wall structure as well as the presence of glycogen α-particles varied over time during cell culture. The structural differences observed in the cell wall of these Sporothrix species seemed to impact its uptake by monocyte-derived human macrophages. The data presented here show a unique cell wall structure of S. brasiliensis and S. schenckii during the yeast parasitic phase. A new cell wall model for Sporothrix spp. is therefore proposed that suggests that these fungi molt sheets of intact cell wall layers. This observation may have significant effects on localized and disseminated immunopathology.

Mitogen activated protein kinases (MAPK) and protein phosphatases are involved in Aspergillus fumigatus adhesion and biofilm formation.

The main characteristic of biofilm formation is extracellular matrix (ECM) production. The cells within the biofilm are surrounded by ECM which provides structural integrity and protection. During an infection, this protection is mainly against cells of the immune system and antifungal drugs. A. fumigatus forms biofilms during static growth on a solid substratum and in chronic aspergillosis infections. It is important to understand how, and which, A. fumigatus signal transduction pathways are important for the adhesion and biofilm formation in a host during infection. Here we investigated the role of MAP kinases and protein phosphatases in biofilm formation. The loss of the MAP kinases MpkA, MpkC and SakA had an impact on the cell surface and the ECM during biofilm formation and reduced the adherence of A. fumigatus to polystyrene and fibronectin-coated plates. The phosphatase null mutants ΔsitA and ΔptcB, involved in regulation of MpkA and SakA phosphorylation, influenced cell wall carbohydrate exposure. Moreover, we characterized the A. fumigatus protein phosphatase PphA. The ΔpphA strain was more sensitive to cell wall-damaging agents, had increased ß-(1,3)-glucan and reduced chitin, decreased conidia phagocytosis by Dictyostelium discoideum and reduced adhesion and biofilm formation. Finally, ΔpphA strain was avirulent in a murine model of invasive pulmonary aspergillosis and increased the released of tumor necrosis factor alpha (TNF-α) from bone marrow derived macrophages (BMDMs). These results show that MAP kinases and phosphatases play an important role in signaling pathways that regulate the composition of the cell wall, extracellular matrix production as well as adhesion and biofilm formation in A. fumigatus.

Elevated catalase expression in a fungal pathogen is a double-edged sword of iron.

Most fungal pathogens of humans display robust protective oxidative stress responses that contribute to their pathogenicity. The induction of enzymes that detoxify reactive oxygen species (ROS) is an essential component of these responses. We showed previously that ectopic expression of the heme-containing catalase enzyme in Candida albicans enhances resistance to oxidative stress, combinatorial oxidative plus cationic stress, and phagocytic killing. Clearly ectopic catalase expression confers fitness advantages in the presence of stress, and therefore in this study we tested whether it enhances fitness in the absence of stress. We addressed this using a set of congenic barcoded C. albicans strains that include doxycycline-conditional tetON-CAT1 expressors. We show that high basal catalase levels, rather than CAT1 induction following stress imposition, reduce ROS accumulation and cell death, thereby promoting resistance to acute peroxide or combinatorial stress. This conclusion is reinforced by our analyses of phenotypically diverse clinical isolates and the impact of stochastic variation in catalase expression upon stress resistance in genetically homogeneous C. albicans populations. Accordingly, cat1Δ cells are more sensitive to neutrophil killing. However, we find that catalase inactivation does not attenuate C. albicans virulence in mouse or invertebrate models of systemic candidiasis. Furthermore, our direct comparisons of fitness in vitro using isogenic barcoded CAT1, cat1Δ and tetON-CAT1 strains show that, while ectopic catalase expression confers a fitness advantage during peroxide stress, it confers a fitness defect in the absence of stress. This fitness defect is suppressed by iron supplementation. Also high basal catalase levels induce key iron assimilatory functions (CFL5, FET3, FRP1, FTR1). We conclude that while high basal catalase levels enhance peroxide stress resistance, they place pressure on iron homeostasis through an elevated cellular demand for iron, thereby reducing the fitness of C. albicans in iron-limiting tissues within the host.

Contribution of Fdh3 and Glr1 to Glutathione Redox State, Stress Adaptation and Virulence in Candida albicans.

The major fungal pathogen of humans, Candida albicans, is exposed to reactive nitrogen and oxygen species following phagocytosis by host immune cells. In response to these toxins, this fungus activates potent anti-stress responses that include scavenging of reactive nitrosative and oxidative species via the glutathione system. Here we examine the differential roles of two glutathione recycling enzymes in redox homeostasis, stress adaptation and virulence in C. albicans: glutathione reductase (Glr1) and the S-nitrosoglutathione reductase (GSNOR), Fdh3. We show that the NADPH-dependent Glr1 recycles GSSG to GSH, is induced in response to oxidative stress and is required for resistance to macrophage killing. GLR1 deletion increases the sensitivity of C. albicans cells to H2O2, but not to formaldehyde or NO. In contrast, Fdh3 detoxifies GSNO to GSSG and NH3, and FDH3 inactivation delays NO adaptation and increases NO sensitivity. C. albicans fdh3⎔ cells are also sensitive to formaldehyde, suggesting that Fdh3 also contributes to formaldehyde detoxification. FDH3 is induced in response to nitrosative, oxidative and formaldehyde stress, and fdh3Δ cells are more sensitive to killing by macrophages. Both Glr1 and Fdh3 contribute to virulence in the Galleria mellonella and mouse models of systemic infection. We conclude that Glr1 and Fdh3 play differential roles during the adaptation of C. albicans cells to oxidative, nitrosative and formaldehyde stress, and hence during the colonisation of the host. Our findings emphasise the importance of the glutathione system and the maintenance of intracellular redox homeostasis in this major pathogen.

The Aspergillus fumigatus sitA Phosphatase Homologue Is Important for Adhesion, Cell Wall Integrity, Biofilm Formation, and Virulence.

Aspergillus fumigatus is an opportunistic pathogenic fungus able to infect immunocompromised patients, eventually causing disseminated infections that are difficult to control and lead to high mortality rates. It is important to understand how the signaling pathways that regulate these factors involved in virulence are orchestrated. Protein phosphatases are central to numerous signal transduction pathways. Here, we characterize the A. fumigatus protein phosphatase 2A SitA, the Saccharomyces cerevisiae Sit4p homologue. The sitA gene is not an essential gene, and we were able to construct an A. fumigatus null mutant. The ΔsitA strain had decreased MpkA phosphorylation levels, was more sensitive to cell wall-damaging agents, had increased ß-(1,3)-glucan and chitin, was impaired in biofilm formation, and had decreased protein kinase C activity. The ΔsitA strain is more sensitive to several metals and ions, such as MnCl2, CaCl2, and LiCl, but it is more resistant to ZnSO4. The ΔsitA strain was avirulent in a murine model of invasive pulmonary aspergillosis and induces an augmented tumor necrosis factor alpha (TNF-α) response in mouse macrophages. These results stress the importance of A. fumigatus SitA as a possible modulator of PkcA/MpkA activity and its involvement in the cell wall integrity pathway.

Candida albicans hypha formation and mannan masking of ß-glucan inhibit macrophage phagosome maturation.

UNLABELLED: Candida albicans is a major life-threatening human fungal pathogen in the immunocompromised host. Host defense against systemic Candida infection relies heavily on the capacity of professional phagocytes of the innate immune system to ingest and destroy fungal cells. A number of pathogens, including C. albicans, have evolved mechanisms that attenuate the efficiency of phagosome-mediated inactivation, promoting their survival and replication within the host. Here we visualize host-pathogen interactions using live-cell imaging and show that viable, but not heat- or UV-killed C. albicans cells profoundly delay phagosome maturation in macrophage cell lines and primary macrophages. The ability of C. albicans to delay phagosome maturation is dependent on cell wall composition and fungal morphology. Loss of cell wall O-mannan is associated with enhanced acquisition of phagosome maturation markers, distinct changes in Rab GTPase acquisition by the maturing phagosome, impaired hyphal growth within macrophage phagosomes, profound changes in macrophage actin dynamics, and ultimately a reduced ability of fungal cells to escape from macrophage phagosomes. The loss of cell wall O-mannan leads to exposure of ß-glucan in the inner cell wall, facilitating recognition by Dectin-1, which is associated with enhanced phagosome maturation. IMPORTANCE: Innate cells engulf and destroy invading organisms by phagocytosis, which is essential for the elimination of fungal cells to protect against systemic life-threatening infections. Yet comparatively little is known about what controls the maturation of phagosomes following ingestion of fungal cells. We used live-cell microscopy and fluorescent protein reporter macrophages to understand how C. albicans viability, filamentous growth, and cell wall composition affect phagosome maturation and the survival of the pathogen within host macrophages. We have demonstrated that cell wall glycosylation and yeast-hypha morphogenesis are required for disruption of host processes that function to inactivate pathogens, leading to survival and escape of this fungal pathogen from within host phagocytes. The methods employed here are applicable to study interactions of other pathogens with phagocytic cells to dissect how specific microbial features impact different stages of phagosome maturation and the survival of the pathogen or host.

The pH-responsive PacC transcription factor of Aspergillus fumigatus governs epithelial entry and tissue invasion during pulmonary aspergillosis.

Destruction of the pulmonary epithelium is a major feature of lung diseases caused by the mould pathogen Aspergillus fumigatus. Although it is widely postulated that tissue invasion is governed by fungal proteases, A. fumigatus mutants lacking individual or multiple enzymes remain fully invasive, suggesting a concomitant requirement for other pathogenic activities during host invasion. In this study we discovered, and exploited, a novel, tissue non-invasive, phenotype in A. fumigatus mutants lacking the pH-responsive transcription factor PacC. Our study revealed a novel mode of epithelial entry, occurring in a cell wall-dependent manner prior to protease production, and via the Dectin-1 ß-glucan receptor. ΔpacC mutants are defective in both contact-mediated epithelial entry and protease expression, and significantly attenuated for pathogenicity in leukopenic mice. We combined murine infection modelling, in vivo transcriptomics, and in vitro infections of human alveolar epithelia, to delineate two major, and sequentially acting, PacC-dependent processes impacting epithelial integrity in vitro and tissue invasion in the whole animal. We demonstrate that A. fumigatus spores and germlings are internalised by epithelial cells in a contact-, actin-, cell wall- and Dectin-1 dependent manner and ΔpacC mutants, which aberrantly remodel the cell wall during germinative growth, are unable to gain entry into epithelial cells, both in vitro and in vivo. We further show that PacC acts as a global transcriptional regulator of secreted molecules during growth in the leukopenic mammalian lung, and profile the full cohort of secreted gene products expressed during invasive infection. Our study reveals a combinatorial mode of tissue entry dependent upon sequential, and mechanistically distinct, perturbations of the pulmonary epithelium and demonstrates, for the first time a protective role for Dectin-1 blockade in epithelial defences. Infecting ΔpacC mutants are hypersensitive to cell wall-active antifungal agents highlighting the value of PacC signalling as a target for antifungal therapy.

Recognition and blocking of innate immunity cells by Candida albicans chitin.

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.

Property differences among the four major Candida albicans strain clades.

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 degrees 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 pho100Delta 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.

Immune recognition of Candida albicans beta-glucan by dectin-1.

Beta (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 beta (1,3)-glucans and induce innate immune responses. The importance of beta-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 beta-glucans by dectin-1. Heat killing of C. albicans resulted in exposure of beta-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-alpha . In contrast, stimulation of Th1-type cytokines, such as interferon-gamma , by C. albicans was independent of the recognition of beta-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.

Candida albicans Pmr1p, a secretory pathway P-type Ca2+/Mn2+-ATPase, is required for glycosylation and virulence.

The cell surface of Candida albicans is the immediate point of contact with the host. The outer layer of the cell wall is enriched in highly glycosylated mannoproteins that are implicated in many aspects of the host-fungus interaction. Glycosylation of cell wall proteins is initiated in the endoplasmic reticulum and then elaborated in the Golgi as the protein passes through the secretory pathway. Golgi-bound mannosyltransferases require Mn(2+) as an essential cofactor. In Saccharomyces cerevisiae, the P-type ATPase Pmr1p transports Ca(2+) and Mn(2+) ions into the Golgi. To determine the effect of a gross defect in glycosylation on host-fungus interactions of C. albicans, we disrupted the PMR1 homolog, CaPMR1. This mutation would simultaneously inhibit many Golgi-located, Mn(2+)-dependent mannosyltransferases. The Capmr1Delta null mutant was viable in vitro and had no growth defect even on media containing low Ca(2+)/Mn(2+) ion concentrations. However, cells grown in these media progressively lost viability upon entering stationary phase. Phosphomannan was almost completely absent, and O-mannan was severely truncated in the null mutant. A defect in N-linked outer chain glycosylation was also apparent, demonstrated by the underglycosylation of surface acid phosphatase. Consistent with the glycosylation defect, the null mutant had a weakened cell wall, exemplified by hypersensitivity to Calcofluor white, Congo red, and hygromycin B and constitutive activation of the cell integrity pathway. In a murine model of systemic infection, the null mutant was severely attenuated in virulence. These results demonstrate the importance of glycosylation for cell wall structure and virulence of C. albicans.

Loss of cell wall mannosylphosphate in Candida albicans does not influence macrophage recognition.

The outer layer of the cell wall of the human pathogenic fungus Candida albicans is enriched with heavily mannosylated glycoproteins that are the immediate point of contact between the fungus and cells of the host, including phagocytes. Previous work had identified components of the acid-labile fraction of N-linked mannan, comprising beta-1,2-linked mannose residues attached via a phosphodiester bond, as potential ligands for macrophage receptors and modulators of macrophage function. We therefore isolated and disrupted the CaMNN4 gene, which is required for mannosyl phosphate transfer and hence the attachment of beta-1,2 mannose oligosaccharides to the acid-labile N-mannan side chains. With the mannosylphosphate eliminated, the mnn4Delta null mutant was unable to bind the charged cationic dye Alcian Blue and was devoid of acid-labile beta-1,2-linked oligomannosaccharides. The mnn4Delta mutant was unaffected in cell growth and morphogenesis in vitro and in virulence in a murine model of systemic C. albicans infection. The null mutant was also not affected in its interaction with macrophages. Mannosylphosphate is therefore not required for macrophage interactions or for virulence of C. albicans.