Malassezia sympodialis Mala s 1 allergen is a potential KELCH protein that cross reacts with human skin.

Malassezia are the dominant commensal yeast species of the human skin microbiota and are associated with inflammatory skin diseases, such as atopic eczema (AE). The Mala s 1 allergen of Malassezia sympodialis is a ß-propeller protein, inducing both IgE and T-cell reactivity in AE patients. We demonstrate by immuno-electron microscopy that Mala s 1 is mainly located in the M. sympodialis yeast cell wall. An anti-Mala s 1 antibody did not inhibit M. sympodialis growth suggesting Mala s 1 may not be an antifungal target. In silico analysis of the predicted Mala s 1 protein sequence identified a motif indicative of a KELCH protein, a subgroup of ß-propeller proteins. To test the hypothesis that antibodies against Mala s 1 cross-react with human skin (KELCH) proteins we examined the binding of the anti-Mala s 1 antibody to human skin explants and visualized binding in the epidermal skin layer. Putative human targets recognized by the anti-Mala s 1 antibody were identified by immunoblotting and proteomics. We propose that Mala s 1 is a KELCH-like ß-propeller protein with similarity to human skin proteins. Mala s 1 recognition may trigger cross-reactive responses that contribute to skin diseases associated with M. sympodialis.

A Human Ex Vivo Skin Model to Study Candida auris Biofilms.

Candida auris can persist for long periods on hospital surfaces and on the skin. C. auris has the ability to form drug-resistant biofilms, which can substantially impact on patient outcome. In comparison to Candida albicans, C. auris has a lower capacity to form biofilms in in vitro models and a higher capacity when tested on animal skin models. Intraspecies variation is shown to exist, with some clinical isolates having greater biofilm capabilities than others. There is a need for models that closely mimic the real niches where infection occurs on human patients. This protocol describes, in detail, a human skin model to study C. auris biofilm formation using catheterized and non-catheterized skin.

Mouse Gastrointestinal Colonization Model for Candida auris.

Animal colonization and infection models are frequently used to investigate host-pathogen interactions and disease progression. Here, we describe an effective model to investigate the ability of the newly emerged fungal pathogen Candida auris to persistently colonize the gut of immunocompetent mice. In our model, mice are inoculated by gavage and are subsequently monitored for colonization by determining daily fungal stool burdens. At the end of the experiment, mice are culled, and their spleen, liver, kidneys, lungs, heart, and caecum harvested to determine the fungal burdens in order to investigate colonization and potentially dissemination of C. auris to other host organs.

Blocking Polyphosphate Mobilization Inhibits Pho4 Activation and Virulence in the Pathogen Candida albicans.

The ability of pathogenic fungi to obtain essential nutrients from the host is vital for virulence. In Candida albicans, acquisition of the macronutrient phosphate is regulated by the Pho4 transcription factor and is important for both virulence and resistance to host-encountered stresses. All cells store phosphate in the form of polyphosphate (polyP), a ubiquitous polymer comprising tens to hundreds of phosphate residues. Release of phosphate from polyP is one of the first responses evoked in response to phosphate starvation, and here, we sought to explore the importance of polyP mobilization in the pathobiology of C. albicans. We found that two polyphosphatases, Ppn1 and Ppx1, function redundantly to release phosphate from polyP in C. albicans. Strikingly, we reveal that blocking polyP mobilization prevents the activation of the Pho4 transcription factor: following Pi starvation, Pho4 fails to accumulate in the nucleus and induce Pi acquisition genes in ppn1Δ ppx1Δ cells. Consequently, ppn1Δ ppx1Δ cells display impaired resistance to the same range of stresses that require Pho4 for survival. In addition, cells lacking both polyphosphatases are exquisitely sensitive to DNA replication stress, indicating that polyP mobilization is needed to support the phosphate-demanding process of DNA replication. Blocking polyP mobilization also results in significant morphological defects, as ppn1Δ ppx1Δ cells form large pseudohypha-like cells that are resistant to serum-induced hypha formation. Thus, polyP mobilization impacts key processes important for the pathobiology of C. albicans, and consistent with this, we found that blocking this process attenuates the virulence of this important human fungal pathogen. IMPORTANCE Acquisition of the essential macronutrient phosphate is important for the virulence of Candida albicans, a major human fungal pathogen. All cells store phosphate as polyphosphate (polyP), which is rapidly mobilized when phosphate is limiting. Here, we identified the major phosphatases involved in releasing phosphate from polyP in C. albicans. By blocking this process, we found that polyP mobilization impacts many process that contribute to C. albicans pathogenesis. Notably, we found that blocking polyP mobilization inhibits activation of the Pho4 transcription factor, the master regulator of phosphate acquisition. In addition, cell cycle progression, stress resistance, morphogenetic switching, and virulence are all impaired in cells that cannot mobilize polyP. This study therefore provides new insight into the importance of polyP mobilization in promoting the virulence of C. albicans. As phosphate homeostasis strategies differ between fungal pathogen and host, this offers promise for the future development of antifungals.

Monoclonal Antibodies Targeting Surface-Exposed Epitopes of Candida albicans Cell Wall Proteins Confer In Vivo Protection in an Infection Model.

Monoclonal antibody (mAb)-based immunotherapies targeting systemic and deep-seated fungal infections are still in their early stages of development, with no licensed antifungal mAbs currently being available for patients at risk. The cell wall glycoproteins of Candida albicans are of particular interest as potential targets for therapeutic antibody generation due to their extracellular location and key involvement in fungal pathogenesis. Here, we describe the generation of recombinant human antibodies specifically targeting two key cell wall proteins (CWPs) in C. albicans: Utr2 and Pga31. These antibodies were isolated from a phage display antibody library using peptide antigens representing the surface-exposed regions of CWPs expressed at elevated levels during in vivo infection. Reformatted human-mouse chimeric mAbs preferentially recognized C. albicans hyphal forms compared to yeast cells, and increased binding was observed when the cells were grown in the presence of the antifungal agent caspofungin. In J774.1 macrophage interaction assays, mAb pretreatment resulted in the faster engulfment of C. albicans cells, suggesting a role of the CWP antibodies as opsonizing agents during phagocyte recruitment. Finally, in a series of clinically predictive mouse models of systemic candidiasis, our lead mAb achieved improved survival (83%) and a several-log reduction of the fungal burden in the kidneys, similar to the levels achieved for the fungicidal drug caspofungin and superior to the therapeutic efficacy of any anti-Candida mAb reported to date.

General hospital outbreak of invasive candidiasis due to azole-resistant Candida parapsilosis associated with an Erg11 Y132F mutation.

An increasing number of outbreaks due to resistant non-albicans Candida species have been reported worldwide. Between 2014 and 2016, Candida isolates causing invasive candidiasis were recovered in a Mexican hospital. Isolates were identified to species level and antifungal susceptibility was determined. In the time period studied, 74 invasive candidiasis cases were identified, with 38% (28/74) caused by Candida parapsilosis, out of which 54% (15/28) were fluconazole resistant. The ERG11 gene was sequenced for 12 recoverable fluconazole-resistant C. parapsilosis isolates and SNPs identified. The 12 isolates had one common silent point mutation in ERG11 (T591C) and seven isolates had an additional (A395T) mutation, which corresponded to Y132F. Four of the isolates carrying this mutation were closely related within the same cluster by microsatellite typing. This is the first report of an invasive candidiasis outbreak in Mexico due to azole-resistant C. parapsilosis associated with the Y132F substitution.

Three Related Enzymes in Candida albicans Achieve Arginine- and Agmatine-Dependent Metabolism That Is Essential for Growth and Fungal Virulence.

Amino acid metabolism is crucial for fungal growth and development. Ureohydrolases produce amines when acting on l-arginine, agmatine, and guanidinobutyrate (GB), and these enzymes generate ornithine (by arginase), putrescine (by agmatinase), or GABA (by 4-guanidinobutyrase or GBase). Candida albicans can metabolize and grow on arginine, agmatine, or guanidinobutyrate as the sole nitrogen source. Three related C. albicans genes whose sequences suggested that they were putative arginase or arginase-like genes were examined for their role in these metabolic pathways. Of these, Car1 encoded the only bona fide arginase, whereas we provide evidence that the other two open reading frames, orf19.5862 and orf19.3418, encode agmatinase and guanidinobutyrase (Gbase), respectively. Analysis of strains with single and multiple mutations suggested the presence of arginase-dependent and arginase-independent routes for polyamine production. CAR1 played a role in hyphal morphogenesis in response to arginine, and the virulence of a triple mutant was reduced in both Galleria mellonella and Mus musculus infection models. In the bloodstream, arginine is an essential amino acid that is required by phagocytes to synthesize nitric oxide (NO). However, none of the single or multiple mutants affected host NO production, suggesting that they did not influence the oxidative burst of phagocytes.IMPORTANCE We show that the C. albicans ureohydrolases arginase (Car1), agmatinase (Agt1), and guanidinobutyrase (Gbu1) can orchestrate an arginase-independent route for polyamine production and that this is important for C. albicans growth and survival in microenvironments of the mammalian host.

The environmental stress sensitivities of pathogenic Candida species, including Candida auris, and implications for their spread in the hospital setting.

Candida auris is an emerging pathogenic yeast of significant clinical concern because of its frequent intrinsic resistance to fluconazole and often other antifungal drugs and the high mortality rates associated with systemic infections. Furthermore, C. auris has a propensity for persistence and transmission in health care environments. The reasons for this efficient transmission are not well understood, and therefore we tested whether enhanced resistance to environmental stresses might contribute to the ability of C. auris to spread in health care environments. We compared C. auris to other pathogenic Candida species with respect to their resistance to individual stresses and combinations of stresses. Stress resistance was examined using in vitro assays on laboratory media and also on hospital linen. In general, the 17 C. auris isolates examined displayed similar degrees of resistance to oxidative, nitrosative, cationic and cell wall stresses as clinical isolates of C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii, C. lusitaniae and C. kefyr. All of the C. auris isolates examined were more sensitive to low pH (pH 2, but not pH 4) compared to C. albicans, but were more resistant to high pH (pH 13). C. auris was also sensitive to low pH, when tested on contaminated hospital linen. Most C. auris isolates were relatively thermotolerant, displaying significant growth at 47°C. Furthermore, C. auris was relatively resistant to certain combinations of combinatorial stress (e.g., pH 13 plus 47°C). Significantly, C. auris was sensitive to the stress combinations imposed by hospital laundering protocol (pH > 12 plus heat shock at >80°C), suggesting that current laundering procedures are sufficient to limit the transmission of this fungal pathogen via hospital linen.

Host Responses in an Ex Vivo Human Skin Model Challenged With Malassezia sympodialis.

Malassezia species are a major part of the normal mycobiota and colonize mainly sebum-rich skin regions of the body. This group of fungi cause a variety of infections such as pityriasis versicolor, folliculitis, and fungaemia. In particular, Malassezia sympodialis and its allergens have been associated with non-infective inflammatory diseases such as seborrheic dermatitis and atopic eczema. The aim of this study was to investigate the host response to M. sympodialis on oily skin (supplemented with oleic acid) and non-oily skin using an ex vivo human skin model. Host-pathogen interactions were analyzed by SEM, histology, gene expression, immunoassays and dual species proteomics. The skin response to M. sympodialis was characterized by increased expression of the genes encoding ß-defensin 3 and RNase7, and by high levels of S100 proteins in tissue. Supplementation of oleic acid onto skin was associated with direct contact of yeasts with keratinocytes and epidermal damage. In oily conditions, there was increased expression of IL18 but no expression of antimicrobial peptide genes in the skin's response to M. sympodialis. In supernatants from inoculated skin plus oleic acid, TNFα, IL-6, and IL1-ß levels were decreased and IL-18 levels were significantly increased.

An ex vivo Human Skin Model to Study Superficial Fungal Infections.

Human skin fungal infections (SFIs) affect 25% of the world's population. Most of these infections are superficial. The main limitation of current animal models of human superficial SFIs is that clinical presentation is different between the different species and animal models do not accurately reflect the human skin environment. An ex vivo human skin model was therefore developed and standardised to accurately model SFIs. In this manuscript, we report our protocol for setting up ex vivo human skin infections and report results from a primary superficial skin infection with Trichophyton rubrum, an anthropophilic fungus. The protocol includes a detailed description of the methodology to prepare the skin explants, establish infection, avoid contamination, and obtain high quality samples for further downstream analyses. Scanning electronic microscopy (SEM), histology and fluorescent microscopy were applied to evaluate skin cell viability and fungal morphology. Furthermore, we describe a broad range of assays, such as RNA extraction and qRT-PCR for human gene expression, and protein extraction from tissue and supernatants for proteomic analysis by liquid chromatography-mass spectrometry (LC-MS/MS). Non-infected skin was viable after 14 days of incubation, expressed genes and contained proteins associated with proliferative, immune and differentiation functions. The macroscopic damage caused by T. rubrum had a similar appearance to the one expected in clinical settings. Finally, using this model, the host response to T. rubrum infection can be evaluated at different levels.

A Bright Future for Fluorescence Imaging of Fungi in Living Hosts.

Traditional in vivo investigation of fungal infection and new antifungal therapies in mouse models is usually carried out using post mortem methodologies. However, biomedical imaging techniques focusing on non-invasive techniques using bioluminescent and fluorescent proteins have become valuable tools. These new techniques address ethical concerns as they allow reduction in the number of animals required to evaluate new antifungal therapies. They also allow better understanding of the growth and spread of the pathogen during infection. In this review, we concentrate on imaging technologies using different fungal reporter proteins. We discuss the advantages and limitations of these different reporters and compare the efficacy of bioluminescent and fluorescent proteins for fungal research.

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.

Single human B cell-derived monoclonal anti-Candida antibodies enhance phagocytosis and protect against disseminated candidiasis.

The high global burden of over one million annual lethal fungal infections reflects a lack of protective vaccines, late diagnosis and inadequate chemotherapy. Here, we have generated a unique set of fully human anti-Candida monoclonal antibodies (mAbs) with diagnostic and therapeutic potential by expressing recombinant antibodies from genes cloned from the B cells of patients suffering from candidiasis. Single class switched memory B cells isolated from donors serum-positive for anti-Candida IgG were differentiated in vitro and screened against recombinant Candida albicans Hyr1 cell wall protein and whole fungal cell wall preparations. Antibody genes from Candida-reactive B cell cultures were cloned and expressed in Expi293F human embryonic kidney cells to generate a panel of human recombinant anti-Candida mAbs that demonstrate morphology-specific, high avidity binding to the cell wall. The species-specific and pan-Candida mAbs generated through this technology display favourable properties for diagnostics, strong opsono-phagocytic activity of macrophages in vitro, and protection in a murine model of disseminated candidiasis.

The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis.

Fungal cells change shape in response to environmental stimuli, and these morphogenic transitions drive pathogenesis and niche adaptation. For example, dimorphic fungi switch between yeast and hyphae in response to changing temperature. The basidiomycete Cryptococcus neoformans undergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproducible in vitro induction system. Here we demonstrate reproducible in vitro Titan cell induction in response to environmental stimuli consistent with the host lung. In vitro Titan cells exhibit all the properties of in vivo generated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify the bacterial peptidoglycan subunit Muramyl Dipeptide as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid and bacterial co-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established (cAMP/PKA) and previously undescribed (USV101) regulators of Titanisation in vitro. Finally, we investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-Titan transition and establish an essential in vitro model for the future characterization of this important morphotype.

Stress-induced nuclear accumulation is dispensable for Hog1-dependent gene expression and virulence in a fungal pathogen.

Stress-activated protein kinase (SAPK) pathways are evolutionarily conserved eukaryotic signalling modules that are essential for the virulence of human pathogenic fungi. The Hog1 SAPK in Candida albicans is robustly phosphorylated in response to a number of host-imposed stresses, and is essential for virulence. The current dogma is that stress-induced phosphorylation activates the SAPK, and promotes its nuclear accumulation that is necessary for the expression of SAPK-dependent stress-protective genes. Here we challenge this dogma. C. albicans strains were constructed in which Hog1 was either tethered to the plasma membrane or constitutively nuclear. Strikingly, tethering Hog1 to the plasma membrane did not abrogate stress resistance or stress-induced gene expression. Furthermore, preventing the nuclear accumulation of Hog1 had no impact on C. albicans virulence in two distinct models of systemic infection. However, tethering Hog1 to the plasma membrane did impact on signal fidelity, and on the magnitude and kinetics of the stress-induced phosphorylation of this SAPK. Taken together, these findings challenge the dogma that nuclear accumulation of SAPKs is a pre-requisite for SAPK-dependent gene expression, and reveal that stress-induced nuclear accumulation of Hog1 is dispensable for the virulence of a major human fungal pathogen.

Adaptation of Candida albicans to environmental pH induces cell wall remodelling and enhances innate immune recognition.

Candida albicans is able to proliferate in environments that vary dramatically in ambient pH, a trait required for colonising niches such as the stomach, vaginal mucosal and the GI tract. Here we show that growth in acidic environments involves cell wall remodelling which results in enhanced chitin and ß-glucan exposure at the cell wall periphery. Unmasking of the underlying immuno-stimulatory ß-glucan in acidic environments enhanced innate immune recognition of C. albicans by macrophages and neutrophils, and induced a stronger proinflammatory cytokine response, driven through the C-type lectin-like receptor, Dectin-1. This enhanced inflammatory response resulted in significant recruitment of neutrophils in an intraperitoneal model of infection, a hallmark of symptomatic vaginal colonisation. Enhanced chitin exposure resulted from reduced expression of the cell wall chitinase Cht2, via a Bcr1-Rim101 dependent signalling cascade, while increased ß-glucan exposure was regulated via a non-canonical signalling pathway. We propose that this "unmasking" of the cell wall may induce non-protective hyper activation of the immune system during growth in acidic niches, and may attribute to symptomatic vaginal infection.

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.

Blocking two-component signalling enhances Candida albicans virulence and reveals adaptive mechanisms that counteract sustained SAPK activation.

The Ypd1 phosphorelay protein is a central constituent of fungal two-component signal transduction pathways. Inhibition of Ypd1 in Saccharomyces cerevisiae and Cryptococcus neoformans is lethal due to the sustained activation of the 'p38-related' Hog1 stress-activated protein kinase (SAPK). As two-component signalling proteins are not found in animals, Ypd1 is considered to be a prime antifungal target. However, a major fungal pathogen of humans, Candida albicans, can survive the concomitant sustained activation of Hog1 that occurs in cells lacking YPD1. Here we show that the sustained activation of Hog1 upon Ypd1 loss is mediated through the Ssk1 response regulator. Moreover, we present evidence that C. albicans survives SAPK activation in the short-term, following Ypd1 loss, by triggering the induction of protein tyrosine phosphatase-encoding genes which prevent the accumulation of lethal levels of phosphorylated Hog1. In addition, our studies reveal an unpredicted, reversible, mechanism that acts to substantially reduce the levels of phosphorylated Hog1 in ypd1Δ cells following long-term sustained SAPK activation. Indeed, over time, ypd1Δ cells become phenotypically indistinguishable from wild-type cells. Importantly, we also find that drug-induced down-regulation of YPD1 expression actually enhances the virulence of C. albicans in two distinct animal infection models. Investigating the underlying causes of this increased virulence, revealed that drug-mediated repression of YPD1 expression promotes hyphal growth both within murine kidneys, and following phagocytosis, thus increasing the efficacy by which C. albicans kills macrophages. Taken together, these findings challenge the targeting of Ypd1 proteins as a general antifungal strategy and reveal novel cellular adaptation mechanisms to sustained SAPK activation.

Candida albicans Chitin Increases Arginase-1 Activity in Human Macrophages, with an Impact on Macrophage Antimicrobial Functions.

The opportunistic human fungal pathogen Candida albicans can cause a variety of diseases, ranging from superficial mucosal infections to life-threatening systemic infections. Phagocytic cells of the innate immune response, such as neutrophils and macrophages, are important first-line responders to an infection and generate reactive oxygen and nitrogen species as part of their protective antimicrobial response. During an infection, host cells generate nitric oxide through the enzyme inducible nitric oxide synthase (iNOS) to kill the invading pathogen. Inside the phagocyte, iNOS competes with the enzyme arginase-1 for a common substrate, the amino acid l-arginine. Several pathogenic species, including bacteria and parasitic protozoans, actively modulate the production of nitric oxide by inducing their own arginases or the host's arginase activity to prevent the conversion of l-arginine to nitric oxide. We report here that C. albicans blocks nitric oxide production in human-monocyte-derived macrophages by induction of host arginase activity. We further determined that purified chitin (a fungal cell wall polysaccharide) and increased chitin exposure at the fungal cell wall surface induces this host arginase activity. Blocking the C. albicans-induced arginase activity with the arginase-specific substrate inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) or the chitinase inhibitor bisdionin F restored nitric oxide production and increased the efficiency of fungal killing. Moreover, we determined that C. albicans influences macrophage polarization from a classically activated phenotype toward an alternatively activated phenotype, thereby reducing antimicrobial functions and mediating fungal survival. Therefore, C. albicans modulates l-arginine metabolism in macrophages during an infection, potentiating its own survival. IMPORTANCE: The availability and metabolism of amino acids are increasingly recognized as crucial regulators of immune functions. In acute infections, the conversion of the "conditionally essential" amino acid l-arginine by the inducible nitric oxide synthase to nitric oxide is a resistance factor that is produced by the host to fight pathogens. Manipulation of these host defense mechanisms by the pathogen can be key to successful host invasion. We show here that the human opportunistic fungal pathogen Candida albicans influences l-arginine availability for nitric oxide production by induction of the substrate-competing host enzyme arginase-1. This led to a reduced production of nitric oxide and, moreover, reduced eradication of the fungus by human macrophages. We demonstrate that blocking of host arginase-1 activity restored nitric oxide production and increased the killing potential of macrophages. These results highlight the therapeutic potential of l-arginine metabolism in fungal diseases.

Lactate signalling regulates fungal ß-glucan masking and immune evasion.

As they proliferate, fungi expose antigens at their cell surface that are potent stimulators of the innate immune response, and yet the commensal fungus Candida albicans is able to colonize immuno competent individuals. We show that C. albicans may evade immune detection by presenting a moving immunological target. We report that the exposure of ß-glucan, a key pathogen-associated molecular pattern (PAMP) located at the cell surface of C. albicans and other pathogenic Candida species, is modulated in response to changes in the carbon source. Exposure to lactate induces ß-glucan masking in C. albicans via a signalling pathway that has recruited an evolutionarily conserved receptor (Gpr1) and transcriptional factor (Crz1) from other well-characterized pathways. In response to lactate, these regulators control the expression of cell-wall-related genes that contribute to ß-glucan masking. This represents the first description of active PAMP masking by a Candida species, a process that reduces the visibility of the fungus to the immune system.