Chitotriosidase Activity Is Counterproductive in a Mouse Model of Systemic Candidiasis.

Mammalian cells do not produce chitin, an insoluble polymer of N-acetyl-D-glucosamine (GlcNAc), although chitin is a structural component of the cell wall of pathogenic microorganisms such as Candida albicans. Mammalian cells, including cells of the innate immune system elaborate chitinases, including chitotriosidase (Chit1), which may play a role in the anti-fungal immune response. In the current study, using knockout mice, we determined the role of Chit1 against systemic candidiasis. Chit1-deficient mice showed significant decrease in kidney fungal burden compared to mice expressing the functional enzyme. Using in vitro anti-candidal neutrophil functional assays, the introduction of the Chit1:chitin digestion end-product, chitobiose (N-acetyl-D-glucosamine dimer, GlcNAc2), decreased fungal-induced neutrophil swarming and Candida killing in vitro. Also, a role for the lectin-like binding site on the neutrophil integrin CR3 (Mac-1, CD11b/CD18) was found through physiological competitive interference by chitobiose. Furthermore, chitobiose treatment of wild type mice during systemic candidiasis resulted in the significant increase in fungal burden in the kidney. These data suggest a counterproductive role of Chit1 in mounting an efficient anti-fungal defense against systemic candidiasis.

Candida albicans SC5314 inhibits NLRP3/NLRP6 inflammasome expression and dampens human intestinal barrier activity in Caco-2 cell monolayer model.

Candida albicans is an opportunistic fungal pathogen that colonizes human gastro-intestinal mucosal tissues. Its effect on the immune response in intestinal epithelial cells and on the intestinal mucosal barrier are not yet fully understood. In this study, we investigated Caco-2 cells, a monolayer model of intestinal epithelial cells, with or without treatment with C. albicans SC5314 (CA) or heat-inactivated CA (CA-inact). RNA sequencing was conducted, and the mRNA and protein levels of NOD-like receptor pyrin domain-containing protein 3 (NLRP3) or NLRP6/ASC/caspase-1 inflammasome signaling pathway components, inflammatory cytokines (interleukin-18 [IL-18] and IL-1ß), anti-microbial peptides (AMPs; ß-defensin-2 [BD-2], BD-3, and LL-37), and tight junction proteins (occludin and zona occludens-1 [ZO-1]) were examined by real-time PCR, western blotting, and/or immunofluorescence microscopy. Lactase dehydrogenase (LDH) activity in the Caco-2 cell supernatant were measured by enzyme kinetics analysis. Our results showed that the NOD-like receptor signaling pathway participates in the CA- and CA-inact-infected Caco-2 cells, as shown by microarray analysis of total mRNA expression. The expression of NLRP3, NLRP6, ASC, BD-2, BD-3, occludin, and ZO-1 were significantly decreased in Caco-2 cells infected with CA and CA-inact compared to that in the untreated control. IL-1ß expression was decreased in the Caco-2 cells in both the CA- and CA-inact-infected groups compared to that in the control. Caspase-1 and IL-18 levels were not markedly affected by CA or CA-inact in Caco-2 cells. Our findings indicate that CA can inhibit the NLRP3 and NLRP6 pathways and dampen human intestinal mucosal barrier activity by decreasing the production of AMPs and tight junction proteins, independent of CA activity.

Endothelial nitric oxide synthase limits host immunity to control disseminated Candida albicans infections in mice.

Three isoforms of nitric oxide synthase (NOS) occur in mammals. High levels of NO produced by NOS2/iNOS can protect against bacterial and parasitic infections, but the role of NOS in fungal innate immunity is less clear. Compared to wild type mice, Nos3-/- mice showed significantly higher survival of candidemia caused by Candida albicans SC5314. NOS3/eNOS is expressed by endothelial cells in the kidney, and colonization of this organ was decreased during the sub-acute stage of disseminated candidiasis. Nos3-/- mice more rapidly eliminated Candida from the renal cortex and exhibited more balanced local inflammatory reactions, with similar macrophage but less neutrophil infiltration than in infected wild type. Levels of the serum cytokines IL-9, IL-12, IL-17 and chemokines GM-CSF, MIP1α, and MIP1ß were significantly elevated, and IL-15 was significantly lower in infected Nos3-/- mice. Spleens of infected Nos3-/- mice had significantly more Th2 and Th9 but not other CD4+ T cells compared with wild type. Inflammatory genes associated with leukocyte chemotaxis, IL-1 signaling, TLR signaling and Th1 and Th2 cell differentiation pathways were significantly overexpressed in infected Nos3-/- kidneys, with Nos2 being the most strongly induced. Conversely, the general NOS inhibitor NG-nitro-L-arginine methyl ester increased virulence in the mouse candidemia model, suggesting that iNOS contributes to the protective mechanism in infected Nos3-/- mice. By moderating neutrophil infiltration, the absence of eNOS may reduce the collateral damage to kidney cortex, and Th-9 CD4+ cells may enhance clearance of the infection. These data suggest that selective eNOS inhibition could mitigate candidemia by a combination of systemic and local responses that promote a more effective host immune response.

Targeting SHIP-1 in Myeloid Cells Enhances Trained Immunity and Boosts Response to Infection.

ß-Glucan-induced trained immunity in myeloid cells leads to long-term protection against secondary infections. Although previous studies have characterized this phenomenon, strategies to boost trained immunity remain undefined. We found that ß-glucan-trained macrophages from mice with a myeloid-specific deletion of the phosphatase SHIP-1 (LysMΔSHIP-1) showed enhanced proinflammatory cytokine production in response to lipopolysaccharide. Following ß-glucan training, SHIP-1-deficient macrophages exhibited increased phosphorylation of Akt and mTOR targets, correlating with augmented glycolytic metabolism. Enhanced training in the absence of SHIP-1 relied on histone methylation and acetylation. Trained LysMΔSHIP-1 mice produced increased amounts of proinflammatory cytokines upon rechallenge in vivo and were better protected against Candida albicans infection compared with control littermates. Pharmacological inhibition of SHIP-1 enhanced trained immunity against Candida infection in mouse macrophages and human peripheral blood mononuclear cells. Our data establish proof of concept for improvement of trained immunity and a strategy to achieve it by targeting SHIP-1.

Commercial AHAS-inhibiting herbicides are promising drug leads for the treatment of human fungal pathogenic infections.

The increased prevalence of drug-resistant human pathogenic fungal diseases poses a major threat to global human health. Thus, new drugs are urgently required to combat these infections. Here, we demonstrate that acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway, is a promising new target for antifungal drug discovery. First, we show that several AHAS inhibitors developed as commercial herbicides are powerful accumulative inhibitors of Candida albicans AHAS (Ki values as low as 800 pM) and have determined high-resolution crystal structures of this enzyme in complex with several of these herbicides. In addition, we have demonstrated that chlorimuron ethyl (CE), a member of the sulfonylurea herbicide family, has potent antifungal activity against five different Candida species and Cryptococcus neoformans (with minimum inhibitory concentration, 50% values as low as 7 nM). Furthermore, in these assays, we have shown CE and itraconazole (a P450 inhibitor) can act synergistically to further improve potency. Finally, we show in Candida albicans-infected mice that CE is highly effective in clearing pathogenic fungal burden in the lungs, liver, and spleen, thus reducing overall mortality rates. Therefore, in view of their low toxicity to human cells, AHAS inhibitors represent a new class of antifungal drug candidates.

Fungus-derived hydroxyl radicals kill hepatic cells by enhancing nuclear transglutaminase.

We previously reported the importance of induced nuclear transglutaminase (TG) 2 activity, which results in hepatic cell death, in ethanol-induced liver injury. Here, we show that co-incubation of either human hepatic cells or mouse primary hepatocytes derived from wild-type but not TG2-/- mice with pathogenic fungi Candida albicans and C. glabrata, but not baker's yeast Saccharomyces cerevisiae, induced cell death in host cells by enhancing cellular, particularly nuclear, TG activity. Further pharmacological and genetic approaches demonstrated that this phenomenon was mediated partly by the production of reactive oxygen species (ROS) such as hydroxyl radicals, as detected by a fluorescent probe and electron spin resonance. A ROS scavenger, N-acetyl cysteine, blocked enhanced TG activity primarily in the nuclei and inhibited cell death. In contrast, deletion of C. glabrata nox-1, which encodes a ROS-generating enzyme, resulted in a strain that failed to induce the same phenomena. A similar induction of hepatic ROS and TG activities was observed in C. albicans-infected mice. An antioxidant corn peptide fraction inhibited these phenomena in hepatic cells. These results address the impact of ROS-generating pathogens in inducing nuclear TG2-related liver injuries, which provides novel therapeutic targets for preventing and curing alcoholic liver disease.

Trehalose-6-Phosphate as a Potential Lead Candidate for the Development of Tps1 Inhibitors: Insights from the Trehalose Biosynthesis Pathway in Diverse Yeast Species.

In some pathogens, trehalose biosynthesis is induced in response to stress as a protection mechanism. This pathway is an attractive target for antimicrobials as neither the enzymes, Tps1, and Tps2, nor is trehalose present in humans. Accumulation of T6P in Candida albicans, achieved by deletion of TPS2, resulted in strong reduction of fungal virulence. In this work, the effect of T6P on Tps1 activity was evaluated. Saccharomyces cerevisiae, C. albicans, and Candida tropicalis were used as experimental models. As expected, a heat stress induced both trehalose accumulation and increased Tps1 activity. However, the addition of 125 µM T6P to extracts obtained from stressed cells totally abolished or reduced in 50 and 60 % the induction of Tps1 activity in S. cerevisiae, C. tropicalis, and C. albicans, respectively. According to our results, T6P is an uncompetitive inhibitor of S. cerevisiae Tps1. This kind of inhibitor is able to decrease the rate of reaction to zero at increased concentrations. Based on the similarities found in sequence and function between Tps1 of S. cerevisiae and some pathogens and on the inhibitory effect of T6P on Tps1 activity observed in vitro, novel drugs can be developed for the treatment of infectious diseases caused by organisms whose infectivity and survival on the host depend on trehalose.

Identification of Components of the SUMOylation Machinery in Candida glabrata: ROLE OF THE DESUMOYLATION PEPTIDASE CgUlp2 IN VIRULENCE.

Regulation of protein function by reversible post-translational modification, SUMOylation, is widely conserved in the eukaryotic kingdom. SUMOylation is essential for cell growth, division, and adaptation to stress in most organisms, including fungi. As these are key factors in determination of fungal virulence, in this study, we have investigated the importance of SUMOylation in the human pathogen, Candida glabrata We identified the enzymes involved in small ubiquitin-like modifier conjugation and show that there is strong conservation between Saccharomyces cerevisiae and C. glabrata We demonstrate that SUMOylation is an essential process and that adaptation to stress involves changes in global SUMOylation in C. glabrata Importantly, loss of the deSUMOylating enzyme CgUlp2 leads to highly reduced small ubiquitin-like modifier protein levels, and impaired growth, sensitivity to multiple stress conditions, reduced adherence to epithelial cells, and poor colonization of specific tissues in mice. Our study thus demonstrates a key role for protein SUMOylation in the life cycle and pathobiology of C. glabrata.

Chitinase activation in patients with fungus-associated cystic fibrosis lung disease.

BACKGROUND: Chitinases have recently gained attention in the field of pulmonary diseases, particularly in asthma and chronic obstructive pulmonary disease, but their potential role in patients with cystic fibrosis (CF)-associated lung disease remains unclear. OBJECTIVE: The aim of this study was to assess chitinase activity systemically and in the airways of patients with CF and asthma compared with healthy subjects. Additionally, we assessed factors that regulate chitinase activity within the lungs of patients with CF. METHODS: Chitinase activities were quantified in serum and bronchoalveolar lavage fluid from patients with CF, asthmatic patients, and healthy control subjects. Mechanistically, the role of CF airway proteases and genetic chitinase deficiency was assessed. RESULTS: Chitinase activity was systemically increased in patients with CF compared with that in healthy control subjects and asthmatic patients. Further stratification showed that chitinase activity was enhanced in patients with CF colonized with Candida albicans compared with that in noncolonized patients. CF proteases degraded chitinases in the airway microenvironment of patients with CF. Genetic chitinase deficiency was associated with C albicans colonization in patients with CF. CONCLUSION: Patients with CF have enhanced chitinase activation associated with C albicans colonization. Therefore chitinases might represent a novel biomarker and therapeutic target for CF-associated fungal disease.

Chitinase 3-Like 1 Promotes Candida albicans Killing and Preserves Corneal Structure and Function by Controlling Host Antifungal Responses.

Chitinase 3-like 1 (CHI3L1) has been shown to play a role in promoting antibacterial responses, decreasing tissue injury, and enhancing pulmonary repair. This study sought to elucidate the role of CHI3L1 in augmenting the corneal innate immune response to Candida albicans infection in an animal model of fungal keratitis. Flagellin applied topically 24 h prior to C. albicans inoculation significantly protected the corneal from C. albicans and induced CHI3L1 expression in C57BL/6 mouse corneas. CHI3L1, however, played a detectable but minor role in flagellin-induced protection. While C. albicans keratitis was more severe in the corneas treated with Chi3l1 small interfering RNA (siRNA), corneas treated with recombinant CHI3L1 before C. albicans inoculation had markedly ameliorated keratitis, reduced fungal load, and decreased polymorphonucleocyte (PMN) infiltration in an interleukin 13 receptor α2 (IL-13Rα2)-dependent manner. CHI3L1 treatment resulted in the induction of the antimicrobial peptides ß-defensin 3, CRAMP, and chemokine CXCL10 and its receptor CXCR3 in corneal epithelial cells. Importantly, CHI3L1 administered after C. albicans inoculation also had strong protection against fungal keratitis, suggesting a therapeutic window. This is the first report demonstrating that CHI3L1 is induced during fungal infection, where it acts as an immunomodulator to promote fungal clearance and to regulate antifungal innate immune responses in the cornea.

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.

Novel mechanism coupling cyclic AMP-protein kinase A signaling and golgi trafficking via Gyp1 phosphorylation in polarized growth.

The cyclic AMP (cAMP)-protein kinase A (PKA) signaling activates virulence expression during hyphal development in the fungal human pathogen Candida albicans. The hyphal growth is characterized by Golgi polarization toward the hyphal tips, which is thought to enhance directional vesicle transport. However, how the hypha-induction signal regulates Golgi polarization is unknown. Gyp1, a Golgi-associated protein and the first GTPase-activating protein (GAP) in the Rab GAP cascade, critically regulates membrane trafficking from the endoplasmic reticulum to the plasma membrane. Here, we report a novel pathway by which the cAMP-PKA signaling triggers Golgi polarization during hyphal growth. We demonstrate that Gyp1 plays a crucial role in actin-dependent Golgi polarization. Hyphal induction activates PKA, which in turn phosphorylates Gyp1. Phosphomimetic mutation of four PKA sites identified by mass spectrometry (Gyp1(4E)) caused strong Gyp1 polarization to hyphal tips, whereas nonphosphorylatable mutations (Gyp1(4A)) abolished it. Gyp1(4E) exhibited enhanced association with the actin motor Myo2, while Gyp1(4A) showed the opposite effect, providing a possible mechanism for Golgi polarization. A GAP-dead Gyp1 (Gyp1(R292K)) showed strong polarization similar to that seen with Gyp1(4E), indicating a role for the GAP activity. Mutating the PKA sites on Gyp1 also impaired the recruitment of a late Golgi marker, Sec7. Furthermore, proper PKA phosphorylation and GAP activity of Gyp1 are required for virulence in mice. We propose that the cAMP-PKA signaling directly targets Gyp1 to promote Golgi polarization in the yeast-to-hypha transition, an event crucial for C. albicans infection.

Functional characterization of chitinase-3 reveals involvement of chitinases in early embryo immunity in zebrafish.

The function and mechanism of chitinases in early embryonic development remain largely unknown. We show here that recombinant chitinase-3 (rChi3) is able to hydrolyze the artificial chitin substrate, 4-methylumbelliferyl-ß-D-N,N',N″-triacetylchitotrioside, and to bind to and inhibit the growth of the fungus Candida albicans, implicating that Chi3 plays a dual function in innate immunity and chitin-bearing food digestion in zebrafish. This is further corroborated by the expression profile of Chi3 in the liver and gut, which are both immune- and digestion-relevant organs. Compared with rChi3, rChi3-CD lacking CBD still retains partial capacity to bind to C. albicans, but its enzymatic and antifungal activities are significantly reduced. By contrast, rChi3-E140N with the putative catalytic residue E140 mutated shows little affinity to chitin, and its enzymatic and antifungal activities are nearly completely lost. These suggest that both enzymatic and antifungal activities of Chi3 are dependent on the presence of CBD and E140. We also clearly demonstrate that in zebrafish, both the embryo extract and the developing embryo display antifungal activity against C. albicans, and all the findings point to chitinase-3 (Chi3) being a newly-identified factor involved in the antifungal activity. Taken together, a dual function in both innate immunity and food digestion in embryo is proposed for zebrafish Chi3. It also provides a new angle to understand the immune role of chitinases in early embryonic development of animals.

Niche-specific requirement for hyphal wall protein 1 in virulence of Candida albicans.

Specialized Candida albicans cell surface proteins called adhesins mediate binding of the fungus to host cells. The mammalian transglutaminase (TG) substrate and adhesin, Hyphal wall protein 1 (Hwp1), is expressed on the hyphal form of C. albicans where it mediates fungal adhesion to epithelial cells. Hwp1 is also required for biofilm formation and mating thus the protein functions in both fungal-host and self-interactions. Hwp1 is required for full virulence of C. albicans in murine models of disseminated candidiasis and of esophageal candidiasis. Previous studies correlated TG activity on the surface of oral epithelial cells, produced by epithelial TG (TG1), with tight binding of C. albicans via Hwp1 to the host cell surfaces. However, the contribution of other Tgs, specifically tissue TG (TG2), to disseminated candidiasis mediated by Hwp1 was not known. A newly created hwp1 null strain in the wild type SC5314 background was as virulent as the parental strain in C57BL/6 mice, and virulence was retained in C57BL/6 mice deleted for Tgm2 (TG2). Further, the hwp1 null strains displayed modestly reduced virulence in BALB/c mice as did strain DD27-U1, an independently created hwp1Δ/Δ in CAI4 corrected for its ura3Δ defect at the URA3 locus. Hwp1 was still needed to produce wild type biofilms, and persist on murine tongues in an oral model of oropharyngeal candidiasis consistent with previous studies by us and others. Finally, lack of Hwp1 affected the translocation of C. albicans from the mouse intestine into the bloodstream of mice. Together, Hwp1 appears to have a minor role in disseminated candidiasis, independent of tissue TG, but a key function in host- and self-association to the surface of oral mucosa.

NADPH oxidase-driven phagocyte recruitment controls Candida albicans filamentous growth and prevents mortality.

Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis.

Release of biologically active kinin peptides, Met-Lys-bradykinin and Leu-Met-Lys-bradykinin from human kininogens by two major secreted aspartic proteases of Candida parapsilosis.

In terms of infection incidence, the yeast Candida parapsilosis is the second after Candida albicans as causative agent of candidiases in humans. The major virulence factors of C. parapsilosis are secreted aspartic proteases (SAPPs) which help the pathogen to disseminate, acquire nutrients and dysregulate the mechanisms of innate immunity of the host. In the current work we characterized the action of two major extracellular proteases of C. parapsilosis, SAPP1 and SAPP2, on human kininogens, proteinaceous precursors of vasoactive and proinflammatory bradykinin-related peptides, collectively called the kinins. The kininogens, preferably the form with lower molecular mass, were effectively cleaved by SAPPs, with the release of two uncommon kinins, Met-Lys-bradykinin and Leu-Met-Lys-bradykinin. While optimal at acidic pH (4-5), the kinin release yield was only 2-3-fold lower at neutral pH. These peptides were able to interact with cellular kinin receptors of B2 subtype and to stimulate the human endothelial cells HMEC-1 to increased secretion of proinflammatory interleukins (ILs), IL-1ß and IL-6. The analysis of the stability of SAPP-generated kinins in plasma suggested that they are biologically equivalent to bradykinin, the best agonist of B2 receptor subtype and can be quickly converted to des-Arg(9)-bradykinin, the agonist of inflammation-inducible B1 receptors.

Candida albicans secreted aspartic proteases 4-6 induce apoptosis of epithelial cells by a novel Trojan horse mechanism.

Systemic infection by the pathogenic yeast Candida albicans produces high mortality in immune-compromised people. Such infection starts with the penetration of the organism at the mucosal surfaces, facilitated by the secreted aspartic proteases (Saps) 4, 5, and 6. The functional mechanism of these virulence factors is unclear. We discovered that Saps 4-6 each contains amino acid motifs RGD/KGD to bind integrins on epithelial cell A549 and are internalized to endosomes and lysosomes. These processes are inhibited by RGD-containing peptides or by substituting RGD motifs of these Saps. The internalization of Saps 4-6 results in partial permeabilization of lysosomal membranes, measured by the redistribution of the lysosomal tropic dye acridine orange to the cytosol, and the triggering of apoptosis via caspase activation. Sap 2 and mutated Saps 4-6 contain no RGD motif, are ineffective in these processes, and a proteolytic inhibitor abolished Sap 4 activity in lysosome permeabilization. Same results were also seen for human tongue keratinocyte SCC-15 cells. Mucosal lesions from this fundamental new mechanism may permit C. albicans to enter the body and may be used to attack cells in immune defense during systemic infections. RGD-motif may also be incorporated in Sap inhibitors for Candidiasis drugs targeting to lysosomes.

Secreted aspartic protease cleavage of Candida albicans Msb2 activates Cek1 MAPK signaling affecting biofilm formation and oropharyngeal candidiasis.

Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK) pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1) phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA), a selective inhibitor of aspartic proteases (Saps). Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ) or Sap8 (sap8Δ/Δ) resulted in higher C. albicans surface ß-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) and TGFß-activated kinase 1 (TAK1) play essential roles in the C-type lectin receptor signaling in response to Candida albicans infection.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) and TGFß-activated kinase 1 (TAK1) are considered as key intermediates in Toll-like receptor (TLR) signaling. However, the role of TRAF6 and TAK1 in C-type lectin receptors (CLRs) in response to fungal infection has not been studied. In this study, we have utilized macrophages derived from TRAF6 knock-out mice and myeloid-specific TAK1-deficient mice and determined the role of TRAF6 and TAK1 in CLR-induced signal transduction events. We demonstrate that TRAF6 and TAK1 are required for NF-κB and JNK activation, and expression of proinflammatory cytokines in response to Candida albicans infection. Our results highlight TRAF6 and TAK1 as key components in the signaling cascade downstream of C-type lectin receptors and as critical mediators of the anti-fungal immune response. Therefore, our studies provide a mechanistic understanding of the host immune response to C. albicans, which has a significant impact for the development of anti-fungal therapeutics and in understanding risk-factors and determining susceptibility to C. albicans infection.