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The ability of the human fungal pathogen Candida albicans to disseminate into tissues is promoted by a switch from budding to invasive hyphal growth. This morphological transition is stimulated by multiple environmental factors that can vary at different sites of infection. To identify genes that promote invasive growth, C. albicans mutants can be screened for defects in growing invasively into solid agar medium as a substitute for studying tissue invasion. This in vitro approach has advantages in that it permits the media conditions to be varied to mimic different host environments. In addition, the concentration of agar can be varied to determine the effects of altering the rigidity of the matrix into which the cells invade, as this provides a better indicator of invasive growth than the ability to form hyphae in a liquid culture. Testing under multiple conditions can be used to identify mutant cells with the strongest defects. Therefore, protocols and media for analyzing invasive growth of C. albicans under different conditions will be described that are appropriate for testing a single strain or high-throughput analysis of a collection of mutant C. albicans strains.

The amino sugar N-acetylglucosamine (GlcNAc) is increasingly recognized as an important signaling molecule in addition to its well-known structural roles at the cell surface. In the human fungal pathogen Candida albicans, GlcNAc stimulates several responses including the induction of the genes needed for its catabolism and a switch from budding to filamentous hyphal growth. We identified two genes needed for growth on GlcNAc (RON1 and NGS1) and found that mutants lacking these genes fail to induce the genes needed for GlcNAc catabolism. NGS1 was also important for growth on other sugars, such as maltose, but RON1 appeared to be specific for GlcNAc. Both mutants could grow on nonfermentable carbon sources indicating that they do not affect mitochondrial function, which we show is important for growth on GlcNAc but not for GlcNAc induction of hyphal morphogenesis. Interestingly, both the ron1Δ and ngs1Δ mutants were defective in forming hyphae in response to GlcNAc, even though GlcNAc catabolism is not required for induction of hyphal morphogenesis. The ron1Δ mutant showed a partial defect in forming hyphae, which was surprising since it displayed an elevated level of filamentous cells under noninducing conditions. The ron1Δ mutant also displayed an elevated basal level of expression of genes that are normally upregulated during hyphal growth. Consistent with this, Ron1 contains an Ndt80-like DNA-binding domain, indicating that it regulates gene expression. Thus, Ron1 is a key new component of the GlcNAc response pathway that acts as both an activator and a repressor of hyphal morphogenesis.

N-acetylglucosamine (GlcNAc) is being increasingly recognized for its ability to stimulate cell signaling. This amino sugar is best known as a component of cell wall peptidoglycan in bacteria, cell wall chitin in fungi and parasites, exoskeletons of arthropods, and the extracellular matrix of animal cells. In addition to these structural roles, GlcNAc is now known to stimulate morphological and stress responses in a wide range of organisms. In fungi, the model organisms Saccharomyces cerevisiae and Schizosaccharomyces pombe lack the ability to respond to GlcNAc or catabolize it, so studies with the human pathogen Candida albicans have been providing new insights into the ability of GlcNAc to stimulate cellular responses. GlcNAc potently induces C. albicans to transition from budding to filamentous hyphal growth. It also promotes an epigenetic switch from White to Opaque cells, which differ in morphology, metabolism, and virulence properties. These studies have led to new discoveries, such as the identification of the first eukaryotic GlcNAc transporter. Other results have shown that GlcNAc can induce signaling in C. albicans in two ways. One is to act as a signaling molecule independent of its catabolism, and the other is that its catabolism can cause the alkalinization of the extracellular environment, which provides an additional stimulus to form hyphae. GlcNAc also induces the expression of virulence genes in the C. albicans, indicating it can influence pathogenesis. Therefore, this review will describe the recent advances in understanding the role of GlcNAc signaling pathways in regulating C. albicans morphogenesis and virulence.

Oropharyngeal candidiasis (OPC) is a common fungal infection that is associated with severe morbidity. Another concern is that patients at risk for developing OPC often take long courses of antifungal drugs, which can lead to the emergence of drug-resistant C. albicans strains. We therefore identified nine mutants with defects in undergoing invasive hyphal growth in the oral cavity, increasing the number of genes known to be involved in OPC by more than 30%. The two strongest mutants, rvs161Δ and rvs167Δ, have defects in endocytosis. The rvsΔ mutants appear to have a specific defect in initiating invasive growth, as preinducing the cells to form hyphae prior to infection restored their ability to cause OPC. These results indicate that blocking endocytosis could have therapeutic value in preventing the initiation of OPC without leading to development of resistance against drugs currently used to treat fungal infections., Invasive growth in tissues by the human fungal pathogen Candida albicans is promoted by a switch from budding to hyphal morphogenesis that is stimulated by multiple environmental factors that can vary at different sites of infection. To identify genes that promote invasive growth in the oral cavity to cause oropharyngeal candidiasis (OPC), we first identified C. albicans mutants that failed to invade agar medium. Analysis of nine severely defective mutants in a mouse model of OPC revealed that the strongest defects were seen for the rvs161Δ and rvs167Δ mutants, which lack amphiphysin proteins needed for endocytosis. The rvsΔ mutants initially adhered to the tongue but failed to invade efficiently and were lost from the oral cavity. Previous studies indicated that rvsΔ mutants formed filamentous hyphae in the kidney albeit with morphological abnormalities, suggesting that the rvsΔ mutants were influenced by factors that vary at different sites of infection. Consistent with this, increasing concentrations of CO2, an inducer of hyphal growth that is more abundant in internal organs than air, partially rescued the invasive-growth defects of the rvsΔ mutants in vitro. Interestingly, preinduction of the rvsΔ mutants to form hyphae prior to introduction into the oral cavity restored their ability to cause OPC, identifying a key role for endocytosis in initiating invasive hyphal growth. These results highlight the influence of distinct environmental factors in promoting invasive hyphal growth in the oral cavity and indicate that blocking endocytosis could have therapeutic value in preventing the initiation of OPC.

Summary The fungal pathogen Candida albicans can transition from budding to hyphal growth, which promotes biofilm formation and invasive growth into tissues. Stimulation of adenylyl cyclase to form cAMP induces hyphal morphogenesis. The failure of cells lacking adenylyl cyclase (cyr1Δ) to form hyphae has suggested that cAMP signaling is essential for hyphal growth. However, cyr1Δ mutants also grow slowly and have defects in morphogenesis, making it unclear whether hyphal inducers must stimulate cAMP, or if normal basal levels of cAMP are required to maintain cellular health needed for hyphal growth. Interestingly, supplementation of cyr1Δ cells with low levels of cAMP enabled them to form hyphae in response to the inducer N-acetylglucosamine (GlcNAc), suggesting that a basal level of cAMP is sufficient for stimulation. Furthermore, we isolated faster-growing cyr1Δ pseudorevertant strains that can be induced to form hyphae even though they lack cAMP. The pseudorevertant strains were not induced by CO2, consistent with reports that CO2 directly stimulates adenylyl cyclase. Mutational analysis showed that induction of hyphae in a pseudorevertant strain was independent of RAS1, but was dependent on the EFG1 transcription factor that acts downstream of protein kinase A. Thus, cAMP-independent signals contribute to the induction of hyphal responses.

Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM., IMPORTANCE Fungal infections are a significant cause of morbidity and mortality worldwide. Current antifungal drugs suffer from various drawbacks, including toxicity, drug resistance, and narrow spectrum of activity. In this study, we have demonstrated that pharmaceutical inhibition of fungal glucosylceramide presents a new opportunity to treat cryptococcosis and various other fungal infections. In addition to being effective against pathogenic fungi, the compounds discovered in this study were well tolerated by animals and additive to current antifungals. These findings suggest that these drugs might pave the way for the development of a new class of antifungals.

The fungus Candida albicans is both a pathogen and a commensal in humans. The ability to utilize different carbon sources available in diverse host niches is vital for both commensalism and pathogenicity. N-acetylglucosamine (GlcNAc) is an important signaling molecule as well as a carbon source in C. albicans. Here, we report the discovery of a novel gene MCU1 essential for GlcNAc utilization. Mcu1 is located in mitochondria and associated with multiple energy- and metabolism-related proteins including Por1, Atp1, Pet9, and Mdh1. Consistently, inactivating Por1 impaired GlcNAc utilization as well. Deletion of MCU1 also caused defects in utilizing non-fermentable carbon sources and amino acids. Furthermore, MCU1 is required for filamentation in several inducing conditions and virulence in a mouse systemic infection model. We also deleted TGL99 and GUP1, two genes adjacent to MCU1, and found that the gup1/gup1 mutant exhibited mild defects in the utilization of several carbon sources including GlcNAc, maltose, galactose, amino acids, and ethanol. Our results indicate that MCU1 exists in a cluster of genes involved in the metabolism of carbon sources. Given its importance in metabolism and lack of a homolog in humans, Mcu1 could be a potential target for developing antifungal agents.

Mice lacking expression of the homologous phosphatases Sts-1 and Sts-2 (Sts−/− mice) are resistant to disseminated candidiasis caused by the fungal pathogen Candida albicans. To better understand the immunological mechanisms underlying the enhanced resistance of Sts−/− mice, we examined the kinetics of fungal clearance at early time points. In contrast to the rapid C. albicans growth seen in normal kidneys during the first 24 h postinfection, we observed a reduction in kidney fungal CFU within Sts−/− mice beginning at 12 to 18 h postinfection. This corresponds to the time period when large numbers of innate leukocytes enter the renal environment to counter the infection. Because phagocytes of the innate immune system are important for host protection against pathogenic fungi, we evaluated responses of bone marrow leukocytes. Relative to wild-type cells, Sts−/− marrow monocytes and bone marrow-derived dendritic cells (BMDCs) displayed a heightened ability to inhibit C. albicans growth ex vivo. This correlated with significantly enhanced production of reactive oxygen species (ROS) by Sts−/− BMDCs downstream of Dectin-1, a C-type lectin receptor that plays a critical role in stimulating host responses to fungi. We observed no visible differences in the responses of other antifungal effector pathways, including cytokine production and inflammasome activation, despite enhanced activation of the Syk tyrosine kinase downstream of Dectin-1 in Sts−/− cells. Our results highlight a novel mechanism regulating the immune response to fungal infections. Further understanding of this regulatory pathway could aid the development of therapeutic approaches to enhance protection against invasive candidiasis., IMPORTANCE Systemic candidiasis caused by fungal Candida species is becoming an increasingly serious medical problem for which current treatment is inadequate. Recently, the Sts phosphatases were established as key regulators of the host antifungal immune response. In particular, genetic inactivation of Sts significantly enhanced survival of mice infected intravenously with Candida albicans. The Sts−/− in vivo resistance phenotype is associated with reduced fungal burden and an absence of inflammatory lesions. To understand the underlying mechanisms, we studied phagocyte responses. Here, we demonstrate that Sts−/− phagocytes have heightened responsiveness to C. albicans challenge relative to wild-type cells. Our data indicate the Sts proteins negatively regulate phagocyte activation via regulating selective elements of the Dectin-1–Syk tyrosine kinase signaling axis. These results suggest that phagocytes lacking Sts respond to fungal challenge more effectively and that this enhanced responsiveness partially underlies the profound resistance of Sts−/− mice to systemic fungal challenge.

GlcNAc activates two pathways in the fungal pathogen Candida albicans. In one pathway, GlcNAc induces hyphal morphology. In the other, GlcNAc metabolism raises the ambient pH, which activates pH signaling pathways to induce gene expression. The dual roles are likely important in other organisms in which GlcNAc is emerging as a key signaling molecule., Various stimuli, including N-acetylglucosamine (GlcNAc), induce the fungal pathogen Candida albicans to switch from budding to hyphal growth. Previous studies suggested that hyphal morphogenesis is stimulated by transcriptional induction of a set of genes that includes known virulence factors. To better understand hyphal development, we examined the role of GlcNAc metabolism using a triple mutant lacking the genes required to metabolize exogenous GlcNAc (hxk1Δ nag1Δ dac1Δ). Surprisingly, at low ambient pH (∼pH 4), GlcNAc stimulated this mutant to form hyphae without obvious induction of hyphal genes. This indicates that GlcNAc can stimulate a separate signal to induce hyphae that is independent of transcriptional responses. Of interest, GlcNAc could induce the triple mutant to express hyphal genes when the medium was buffered to a higher pH (>pH 5), which normally occurs after GlcNAc catabolism. Catabolism of GlcNAc raises the ambient pH rather than acidifying it, as occurs after dextrose catabolism. This synergy between alkalinization and GlcNAc to induce hyphal genes involves the Rim101 pH-sensing pathway; GlcNAc induced rim101Δ and dfg16Δ mutants to form hyphae, but hyphal gene expression was partially defective. These results demonstrate that hyphal morphogenesis and gene expression can be regulated independently, which likely contributes to pathogenesis at different host sites.

Candida albicans is a common human fungal pathogen capable of causing serious systemic infections that can progress to become lethal. Current therapeutic approaches have limited effectiveness, especially once a systemic infection is established, in part due to the lack of an effective immune response. Boosting the immune response to C. albicans has been the goal of immunotherapy, but it has to be done selectively to prevent deleterious hyperinflammation (sepsis). Although an efficient inflammatory response is necessary to fight infection, the typical response to C. albicans results in collateral damage to tissues thereby exacerbating the pathological effects of infection. For this reason, identifying specific ways of modulating the immune system holds promise for development of new improved therapeutic approaches. This review will focus on recent studies that provide insight using mutant strains of mice that are more resistant to bloodstream infection by C. albicans. These mice are deficient in signal transduction proteins including the Jnk1 MAP kinase, the Cbl-b E3 ubiquitin ligase, or the Sts phosphatases. Interestingly, the mutant mice display a different response to C. albicans that results in faster clearance of infection without hyper-inflammation and collateral damage. A common underlying theme between the resistant mouse strains is loss of negative regulatory proteins that are known to restrain activation of cell surface receptor-initiated signaling cascades. Understanding the cellular and molecular mechanisms that promote resistance to C. albicans in mice will help to identify new approaches for improving antifungal therapy.

Most terrestrial plants, including crops, engage in beneficial interactions with arbuscular mycorrhizal fungi. Vital to the association is mutual recognition involving the release of diffusible signals into the rhizosphere. Previously, we identified the maize no perception 1 (nope1) mutant to be defective in early signalling. Here, we report cloning of ZmNope1 on the basis of synteny with rice. NOPE1 encodes a functional homologue of the Candida albicans N-acetylglucosamine (GlcNAc) transporter NGT1, and represents the first plasma membrane GlcNAc transporter identified from plants. In C. albicans, exposure to GlcNAc activates cell signalling and virulence. Similarly, in Rhizophagus irregularis treatment with rice wild-type but not nope1 root exudates induced transcriptome changes associated with signalling function, suggesting a requirement of NOPE1 function for presymbiotic fungal reprogramming.

N-acetylglucosamine (GlcNAc) has long been known to play important roles in cell surface structure. Recent studies are now revealing new functions for GlcNAc in cell signaling. Exposure to GlcNAc regulates virulence functions in the human fungal pathogen Candida albicans and in pathogenic bacteria. These signaling pathways sense exogenous GlcNAc and are distinct from the O-GlcNAc signaling pathways in mammalian cells in which increased levels of intracellular GlcNAc synthesis leads to post-translational modification of proteins by attachment of O-GlcNAc. The novel roles of GlcNAc in cell signaling will be the subject of this mini-review.

Contagious Bovine Pleuropneumonia caused by Mycoplasma mycoides subsp. mycoides small colony type is a respiratory disease of considerable economic importance in sub-Saharan Africa; control of the disease in Africa is hampered by diagnostic tests which are suited for herd-level but not for individual animal diagnostics. In the work presented we identified 22 potential immunogenic antigens of the Kenyan outbreak strain B237 by using phage display technology. We determined the relative strength of immunogenicity, the discriminatory capacity between bovine positive and negative sera, and the cross-reactivity with rabbit hyperimmune sera directed against 15 different mycoplasmal species. The three best-performing antigens, a conserved hypothetical protein (MSC_0636), a glycosyl transferase (MSC_0108), and an acyl carrier protein phosphodiesterase (MSC_0029) were considered candidate diagnostic proteins. They were expressed as GST-fusion proteins in Escherichia coli, purified, and used in an ELISA as solid phase antigens. The diagnostic potential of the recombinant antigens was tested using the sera of ten experimentally infected animals and six control animals. This prototype test resulted in 100% diagnostic sensitivity and specificity. In comparison, the complement fixation test and the competitive ELISA performed with a diagnostic sensitivity of 70% and 60%, respectively.

A balanced immune response to infection is essential to prevent the pathology and tissue damage that can occur from an unregulated or hyperactive host defense. Interferons (IFNs) are critical mediators of the innate defense to infection, and in this study we evaluated the contribution of a specific gene coding for IFIT2 induced by type I IFNs in a murine model of disseminated Candida albicans. Invasive candidiasis is a frequent challenge during immunosuppression or surgical medical interventions, and C. albicans is a common culprit that leads to high rates of mortality. When IFIT2 knockout mice were infected systemically with C. albicans, they were found to have improved survival and reduced fungal burden compared to wild-type mice. One of the mechanisms by which IFIT2 increases the pathological effects of invasive C. albicans appears to be suppression of NADPH oxidase activation. Loss of IFIT2 increases production of reactive oxygen species by leukocytes, and we demonstrate that IFIT2 is a binding partner of a critical regulatory subunit of NADPH oxidase, p67phox. Since the administration of IFN has been used therapeutically to combat viral infections, cancer, and multiple sclerosis, we evaluated administration of IFN-β to mice prior to C. albicans infection. IFN-β treatment promoted pathology and death from C. albicans infection. We provide evidence that IFIT2 increases the pathological effects of invasive C. albicans and that administration of IFN-β has deleterious effects during infection., IMPORTANCE The attributable mortality associated with systemic C. albicans infections in health care settings is significant, with estimates greater than 40%. This life-threatening disease is common in patients with weakened immune systems, either due to disease or as a result of therapies. Type I interferons (IFN) are cytokines of the innate defense response that are used as immune modulators in the treatment of specific cancers, viral infections, and multiple sclerosis. In this study, we show using a murine model that the loss of a specific IFN-stimulated gene coding for IFIT2 improves survival following systemic C. albicans infection. This result infers a harmful effect of IFN during C. albicans infection and is supported by our finding that administration of IFN-β prior to invasive infection promotes fatal pathology. The findings contribute to our understanding of the innate immune response to C. albicans, and they suggest that IFN therapies present a risk factor for disseminated candidiasis.

The fungal pathogen Candida albicans causes lethal systemic infections in humans. To better define how pathogens resist oxidative attack by the immune system, we examined a family of four Flavodoxin-Like Proteins (FLPs) in C. albicans. In agreement with previous studies showing that FLPs in bacteria and plants act as NAD(P)H quinone oxidoreductases, a C. albicans quadruple mutant lacking all four FLPs (pst1Δ, pst2Δ, pst3Δ, ycp4Δ) was more sensitive to benzoquinone. Interestingly, the quadruple mutant was also more sensitive to a variety of oxidants. Quinone reductase activity confers important antioxidant effects because resistance to oxidation was restored in the quadruple mutant by expressing either Escherichia coli wrbA or mammalian NQO1, two distinct types of quinone reductases. FLPs were detected at the plasma membrane in C. albicans, and the quadruple mutant was more sensitive to linolenic acid, a polyunsaturated fatty acid that can auto-oxidize and promote lipid peroxidation. These observations suggested that FLPs reduce ubiquinone (coenzyme Q), enabling it to serve as an antioxidant in the membrane. In support of this, a C. albicans coq3Δ mutant that fails to synthesize ubiquinone was also highly sensitive to oxidative stress. FLPs are critical for survival in the host, as the quadruple mutant was avirulent in a mouse model of systemic candidiasis under conditions where infection with wild type C. albicans was lethal. The quadruple mutant cells initially grew well in kidneys, the major site of C. albicans growth in mice, but then declined after the influx of neutrophils and by day 4 post-infection 33% of the mice cleared the infection. Thus, FLPs and ubiquinone are important new antioxidant mechanisms that are critical for fungal virulence. The potential of FLPs as novel targets for antifungal therapy is further underscored by their absence in mammalian cells., Author Summary Oxidative damage is a fundamental problem for cells and a particular challenge for microbial pathogens, which require special mechanisms to resist the oxidative attack by the host immune system. We identified four proteins in the human fungal pathogen Candida albicans that belong to a large family of enzymes in bacteria and plants that reduce quinone molecules to detoxify them. Interestingly, mutational studies in C. albicans showed that these enzymes also confer resistance to a wide range of oxidants, suggesting they may have broader impact by reducing the major quinone present in cells (ubiquinone or coenzyme Q). In support of this, we found that mutating the COQ3 gene to block ubiquinone synthesis rendered cells highly sensitive to oxidative stress, revealing that it plays a very important antioxidant function in addition to its well known role in energy metabolism. These quinone reductases play a critical role in vivo, as they were required for virulence in mouse infections studies. Since mammalian cells lack this type of quinone reductase, this difference could be exploited to develop much needed novel therapeutic approaches for fungal and bacterial pathogens.

Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [ N ′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo- N ′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5 , COS111 , MKK1 , and STE2 , which are involved in vesicular transport and cell cycle progression, are targeted by BHBM. IMPORTANCE Fungal infections are a significant cause of morbidity and mortality worldwide. Current antifungal drugs suffer from various drawbacks, including toxicity, drug resistance, and narrow spectrum of activity. In this study, we have demonstrated that pharmaceutical inhibition of fungal glucosylceramide presents a new opportunity to treat cryptococcosis and various other fungal infections. In addition to being effective against pathogenic fungi, the compounds discovered in this study were well tolerated by animals and additive to current antifungals. These findings suggest that these drugs might pave the way for the development of a new class of antifungals.

This study was undertaken to evaluate the effects of probiotics (Protexin) on the immune response of broiler chicks. The parameters of investigation were hemagglutination inhibition HI titer of antibodie s against avian influenza virus and post field AIV challenge. The findings were compared with the cyclophosphamide treated AIV-vaccinated; untreated and AIV-vaccinated and unvaccinated and control birds this investigation revealed that protexin treated chicks have higher AIV-HI antibody and no AIV post challenge mortality compared to the cyclophosphamide treated and untreated chicks the overall finding of this study clearly demonstrate that the use of this multistrain probiotics has good effect on immune response of broiler chicks.

The human fungal pathogen Candida albicans causes invasive candidiasis, characterized by fatal organ failure due to disseminated fungal growth and inflammatory damage. The s uppressor of T CR s ignaling 1 (Sts-1) and Sts-2 are two homologous phosphatases that negatively regulate signaling pathways in a number of hematopoietic cell lineages, including T lymphocytes, mast cells, and platelets. Functional inactivation of both Sts enzymes leads to profound resistance to systemic infection by C. albicans , such that greater than 80% of mice lacking Sts-1 and -2 survive a dose of C. albicans (2.5 × 10 5 CFU/mouse) that is uniformly lethal to wild-type mice within 10 days. Restriction of fungal growth within the kidney occurs by 24 h postinfection in the mutant mice. This occurs without induction of a hyperinflammatory response, as evidenced by the decreased presence of leukocytes and inflammatory cytokines that normally accompany the antifungal immune response. Instead, the absence of the Sts phosphatases leads to the rapid induction of a unique immunological environment within the kidney, as indicated by the early induction of a proinflammatory cytokine (CXL10). Mice lacking either Sts enzyme individually display an intermediate lethality phenotype. These observations identify an opportunity to optimize host immune responses toward a deadly fungal pathogen.

The monosaccharide N-acetylglucosamine (GlcNAc) is a major component of microbial cell walls and is ubiquitous in the environment. GlcNAc stimulates developmental pathways in the fungal pathogen Candida albicans, which is a commensal organism that colonizes the mammalian gut and causes disease in the setting of host immunodeficiency. Here we investigate GlcNAc signaling in thermally dimorphic human fungal pathogens, a group of fungi that are highly evolutionarily diverged from C. albicans and cause disease even in healthy individuals. These soil organisms grow as polarized, multicellular hyphal filaments that transition into a unicellular, pathogenic yeast form when inhaled by a human host. Temperature is the primary environmental cue that promotes reversible cellular differentiation into either yeast or filaments; however, a shift to a lower temperature in vitro induces filamentous growth in an inefficient and asynchronous manner. We found GlcNAc to be a potent and specific inducer of the yeast-to-filament transition in two thermally dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. In addition to increasing the rate of filamentous growth, micromolar concentrations of GlcNAc induced a robust morphological transition of H. capsulatum after temperature shift that was independent of GlcNAc catabolism, indicating that fungal cells sense GlcNAc to promote filamentation. Whole-genome expression profiling to identify candidate genes involved in establishing the filamentous growth program uncovered two genes encoding GlcNAc transporters, NGT1 and NGT2, that were necessary for H. capsulatum cells to robustly filament in response to GlcNAc. Unexpectedly, NGT1 and NGT2 were important for efficient H. capsulatum yeast-to-filament conversion in standard glucose medium, suggesting that Ngt1 and Ngt2 monitor endogenous levels of GlcNAc to control multicellular filamentous growth in response to temperature. Overall, our work indicates that GlcNAc functions as a highly conserved cue of morphogenesis in fungi, which further enhances the significance of this ubiquitous sugar in cellular signaling in eukaryotes., Author Summary In stark contrast to most fungal pathogens, thermally dimorphic fungal pathogens cause systemic infections in immunocompetent humans. Thermally dimorphic fungi grow in the soil as a multicellular filamentous form specialized for replication in this particular environmental niche. Upon infection of a human, these fungi transition to a parasitic cell type that is adapted for replication and pathogenesis within a mammalian host. In this work, we examined factors that are important for growth of the infectious, environmental form of thermally dimorphic fungi. We discovered that N-acetylglucosamine (GlcNAc), a ubiquitous carbohydrate with cellular roles across all kingdoms of life, stimulated a switch to the environmental form for two thermally dimorphic fungal pathogens, Histoplasma capsulatum and Blastomyces dermatitidis. Analysis of how fungal cells respond to GlcNAc revealed that these fungi possess two GlcNAc transporters that are important for controlling their ability to switch between infectious and parasitic states. Overall, our work begins to elucidate the pathways that promote growth in the infectious form of these organisms, which is critical to our understanding of environmental signals that promote disease transmission of thermally dimorphic fungi.

N-acetylglucosamine (GlcNAc) stimulates important signaling pathways in a wide range of organisms. In the human fungal pathogen Candida albicans, GlcNAc stimulates hyphal cell morphogenesis, virulence genes, and the genes needed to catabolize GlcNAc. Previous studies on the GlcNAc transporter (NGT1) indicated that GlcNAc has to be internalized to induce signaling. Therefore, the role of GlcNAc catabolism was examined by deleting the genes required to phosphorylate, deacetylate, and deaminate GlcNAc to convert it to fructose-6-PO(4) (HXK1, NAG1, and DAC1). As expected, the mutants failed to utilize GlcNAc. Surprisingly, GlcNAc inhibited the growth of the nag1Δ and dac1Δ mutants in the presence of other sugars, suggesting that excess GlcNAc-6-PO(4) is deleterious. Interestingly, both hxk1Δ and an hxk1Δ nag1Δ dac1Δ triple mutant could be efficiently stimulated by GlcNAc to form hyphae. These mutants could also be stimulated to express GlcNAc-regulated genes. Because GlcNAc must be phosphorylated by Hxk1 to be catabolized, and also for it to enter the anabolic pathways that form chitin, N-linked glycosylation, and glycosylphosphatidylinositol anchors, the mutant phenotypes indicate that GlcNAc metabolism is not needed to induce signaling in C. albicans. Thus, these studies in C. albicans reveal a novel role for GlcNAc in cell signaling that may also regulate critical pathways in other organisms.

There is growing evidence that the sugar N-acetylglucosamine (GlcNAc) plays diverse roles in cell signaling pathways that impact the virulence properties of microbes and host cells. GlcNAc is already well known as a ubiquitous structural component at the cell surface that forms part of bacterial cell wall peptidoglycan, cell wall chitin in fungi and parasites, and extracellular matrix glycosaminoglycans of animal cells. Chitin and peptidoglycan have been previously linked to cell signaling as they can stimulate responses in plant and animal host cells [1–3]. Recent studies now indicate that GlcNAc released from these polymers can also activate cell signaling via several different mechanisms [4–6]. The role of these new GlcNAc signaling pathways in the regulation of virulence factors will be the focus of this review.