The yapsin family of aspartyl proteases regulate glucose homeostasis in Candida glabrata.

Invasive candidiasis poses a major healthcare threat. The human opportunistic fungal pathogen Candida glabrata, which causes mucosal and deep-seated infections, is armed with distinct virulence attributes, including a family of 11 glycosylphosphatidylinositol-linked aspartyl proteases, CgYapsins. Here, we have profiled total membrane proteomes of the C. glabrata wildtype and 11 proteases-deficient strain, Cgyps1-11Δ, by mass spectrometry analysis and uncovered a novel role for fungal yapsins in glucose sensing and homeostasis. Furthermore, through label-free quantitative membrane proteome analysis, we showed differential abundance of 42% of identified membrane proteins, with electron transport chain and glycolysis proteins displaying lower and higher abundance in Cgyps1-11Δ cells, compared with wildtype cells, respectively. We also demonstrated elevated glucose uptake and upregulation of genes that code for the low-glucose sensor CgSnf3, transcriptional regulators CgMig1 and CgRgt1, and hexose transporter CgHxt2/10 in the Cgyps1-11Δ mutant. We further elucidated a potential underlying mechanism through genetic and transcript measurement analysis under low- and high-glucose conditions and found CgSNF3 deletion to rescue high glucose uptake and attenuated growth of the Cgyps1-11Δ mutant in YPD medium, thereby linking CgYapsins with regulation of the CgSnf3-dependent low-glucose sensing pathway. Last, high ethanol production, diminished mitochondrial membrane potential, and elevated susceptibility to oxidative phosphorylation inhibitors point toward increased fermentative and decreased respiratory metabolism in the Cgyps1-11Δ mutant. Altogether, our findings revealed new possible glucose metabolism-regulatory roles for putative cell surface-associated CgYapsins and advanced our understanding of fungal carbohydrate homeostasis mechanisms.

Host-pathogen interaction in Candida glabrata infection: current knowledge and implications for antifungal therapy.

INTRODUCTION: The opportunistic fungal pathogen Candida glabrata poses a clinical challenge in the successful treatment of invasive Candida infections, owing to its low inherent susceptibility toward azole antifungals and the recent acquisition of coresistance toward azole and echinocandin drugs. Compared to other prevalent Candida bloodstream pathogens, C. glabrata neither exhibits secreted proteolytic activity nor invokes a strong immune response in a variety of host cells and is less virulent. It also does not form true hyphae, and the success of C. glabrata, therefore, as a prevalent human fungal pathogen, appears to be built upon a distinct set of virulence attributes. AREAS COVERED: The focus of this review is to outline, in brief, the interaction of C. glabrata with the host, deduced from the knowledge gained from different in vitro, ex vivo, and in vivo model systems. In addition, we briefly discuss the current antifungals, antifungal resistance mechanisms, and the development of new antifungal therapies, along with the available information on the host response. EXPERT OPINION: A detailed understanding of stresses, selection pressures and differential immune responses in the presence and absence of antifungals that C. glabrata encounters in varied niches of the host, is required to design effective antifungal therapy.

Global Secretome Characterization of the Pathogenic Yeast Candida glabrata.

Secretory proteins are key modulators of host-pathogen interaction. The human opportunistic fungal pathogen Candida glabrata lacks secreted proteolytic activity but possesses 11 glycosylphosphatidylinositol-anchored aspartyl proteases, also referred to as Yapsins (CgYps1-11), that are essential for its virulence. To delineate the role of CgYapsins in interaction with host cells, we have profiled, through liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, the total secretome of wild-type and Cgyps1-11Δ mutant. The wild-type secretome consisted of 119 proteins which were primarily involved in cell wall organization, carbohydrate metabolism, proteolysis, and translation processes. Of eight CgYapsins identified in the secretome, the release of two major CgYapsins, CgYps1 and CgYps7, to the medium was confirmed by Western analysis. Further, comparative analysis revealed 20 common proteins, probably signifying the core fungal secretome, among C. glabrata, Saccharomyces cerevisiae, and Candida albicans secretomes. Strikingly, the Cgyps1-11Δ secretome was 4.6-fold larger, and contained 65 differentially abundant proteins, as revealed by label-free quantitative profiling, with 49 and 16 being high- and low-abundant proteins, respectively, compared to the wild-type secretome. Importantly, the CgMsb2 mucin, a putative CgYapsins' substrate, was six-fold underrepresented in the mutant secretome. Altogether, we demonstrate for the first time that CgYapsins are both bona fide constituents and key modulators of the C. glabrata secretome.

Aspartyl proteases in Candida glabrata are required for suppression of the host innate immune response.

A family of 11 cell surface-associated aspartyl proteases (CgYps1-11), also referred as yapsins, is a key virulence factor in the pathogenic yeast Candida glabrata However, the mechanism by which CgYapsins modulate immune response and facilitate survival in the mammalian host remains to be identified. Here, using RNA-Seq analysis, we report that genes involved in cell wall metabolism are differentially regulated in the Cgyps1-11Δ mutant. Consistently, the mutant contained lower ß-glucan and mannan levels and exhibited increased chitin content in the cell wall. As cell wall components are known to regulate the innate immune response, we next determined the macrophage transcriptional response to C. glabrata infection and observed differential expression of genes implicated in inflammation, chemotaxis, ion transport, and the tumor necrosis factor signaling cascade. Importantly, the Cgyps1-11Δ mutant evoked a different immune response, resulting in an enhanced release of the pro-inflammatory cytokine IL-1ß in THP-1 macrophages. Further, Cgyps1-11Δ-induced IL-1ß production adversely affected intracellular proliferation of co-infected WT cells and depended on activation of spleen tyrosine kinase (Syk) signaling in the host cells. Accordingly, the Syk inhibitor R406 augmented intracellular survival of the Cgyps1-11Δ mutant. Finally, we demonstrate that C. glabrata infection triggers elevated IL-1ß production in mouse organs and that the CgYPS genes are required for organ colonization and dissemination in the murine model of systemic infection. Altogether, our results uncover the basis for macrophage-mediated killing of Cgyps1-11Δ cells and provide the first evidence that aspartyl proteases in C. glabrata are required for suppression of IL-1ß production in macrophages.

GPI (glycosylphosphatidylinositol)-linked aspartyl proteases regulate vacuole homoeostasis in Candida glabrata.

A family of 11 GPI (glycosylphosphatidylinositol)-linked cell surface-associated aspartyl proteases (yapsins) in the human opportunistic fungal pathogen Candida glabrata is required for cell wall remodelling, pH homoeostasis, survival in macrophages and virulence in a murine model of disseminated candidiasis. In the present paper, we report new roles for yapsins in C. glabrata physiology and implicate them for the first time in the regulation of vacuole homoeostasis. In the present study we show that a C. glabrata mutant lacking all 11 yapsins, Cgyps1-11∆, possesses an enlarged vacuole and displays vma- (vacuolar membrane ATPase)-like phenotypes with elevated metal ion susceptibility in an alkaline pH medium and diminished Vma activity. The results of the present study also demonstrate a singular role for CgYps1 (C. glabrata yapsin 1) in the maintenance of ion homoeostasis under normal and calcineurin-inhibited conditions. Elevated polyphosphate levels and diminished cellular CPY (carboxypeptidase Y) activity in the Cgyps1-11∆ mutant highlight the yapsin requirement for a properly functioning vacuole. Lastly, a gross perturbation of cellular homoeostasis in the Cgyps1-11∆ mutant, even in the absence of external stressors, characterized by reduced levels of ATP and stress metabolites, elevated ROS (reactive oxygen species) levels, cell surface abnormalities, and a constitutively activated PKC (protein kinase C) signalling pathway underscore diverse physiological functions of yapsins in C. glabrata.