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 β-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-1β in THP-1 macrophages. Further, Cgyps1–11Δ–induced IL-1β 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-1β 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-1β production in macrophages.
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.
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.
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