Global Secretome Characterization of the Pathogenic Yeast Candida glabrata

Abstract

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.

Keywords: GPI proteins; Yapsins; carbohydrate metabolism; cell wall organization; core fungal secretome; pathogenic fungi; secretory signal peptide.

Figure 1

Global secretome analysis of C. glabrata. Venn diagram illustrating overlap in proteins identified in the secretomes of wild-type and Cgyps1-11Δ strains.

Figure 2

Functional enrichment analysis of secretomes. Pie chart illustrating enriched GO terms for biological process category in secretomes of wild-type (A) and Cgyps1-11Δ (B), as determined by the FungiFun tool. The number of proteins classified in each category are depicted inside pie slices.

Figure 3

Protein–protein interaction network analysis. Networks, constructed by the STRING Protein–Protein Interaction Network (Ver 11.0) tool, depict interactions, among proteins identified in the wild-type secretome, that belonged to fungal-type cell wall organization (A) and carbohydrate metabolic process (B) categories. Since majority of C. glabrata proteins are uncharacterized, networks were constructed using the S. cerevisiae orthologs of identified C. glabrata proteins and S. cerevisiae as the reference strain. The line thickness reflects strength of the interaction.

Figure 4

Comparative analysis of the identified wild-type secretome with published global secretome and glycosylated protein secretome of C. glabrata. Venn diagram illustrating overlap among proteins identified in our wild-type secretome with those identified in Champer et al.’s secretome (Champer et al. 2016) and Stead et al.’s glycosylated protein secretome (Stead et al., 2010).

Figure 5

Comparative analysis of secretomes of C. glabrata, C. albicans, and S. cerevisiae. Venn diagram illustrating overlap among proteins identified in the C. glabratawild-type secretome with those identified in the secretomes of C. albicans (Gil-Bona et al. 2015) and S. cerevisiae (Smeekens et al. 2017). Of note, each of the three S. cerevisiae proteins, Scw11, Cwp1, and Gas1, corresponded to two different ORFs in C. glabrata, as indicated by *. Similarly, four of the S. cerevisiae proteins, Mkc7, Yps3, Cts1, and Plb3, corresponded to two, three, three, and two ORFs in C. albicans, respectively, as denoted by **.

Figure 6

Quantitative secretome analysis of C. glabrata. (A) Venn diagram illustrating overlap in proteins identified in the quantitative secretomes of wild-type and Cgyps1-11Δ strains. (B) Bar graph depicting 49 proteins with higher abundance in the Cgyps1-11Δ secretome compared to the wild-type secretome. (C) Bar graph depicting 16 proteins with lower abundance in the Cgyps1-11Δ secretome compared to the wild-type secretome. The fold-difference in levels are presented as Log2 values.

Figure 7

Comparative analysis of global and quantitative secretomes of C. glabrata. Venn diagrams illustrating overlap in proteins identified in global and quantitative secretomes of the wild-type (A) and Cgyps1-11Δ (B) strains.

Figure 8

CgYps1 and CgYps7 are secreted into the medium. (A) Representative SDS-PAGE gel image indicating increased protein secretion into the medium of the Cgyps1-11Δ mutant. The secretomes of wild-type and Cgyps1-11Δ mutants were collected after 11 doublings in the YNB medium, and 50 μL were resolved on a 12% SDS-PAGE gel. Proteins were stained with Coomassie Brilliant Blue (CBB) for visualization. (B) Representative SDS-PAGE gel image depicting the total membrane, cell lysate, and secretory protein profiles of wild-type and Cgyps1-11Δ mutants. Equal volume of secretomes (50 μL) and 100 μg of total membrane and cell lysate were resolved on a 10% SDS-PAGE gel and stained with CBB for visualization. (C) Representative western blot images of CgYps1 and CgYps7 indicating their secretion into the medium of the wild-type strain. Equal volume (50 μL) of secretomes of wild-type and Cgyps1-11Δ strains were loaded on a 10% SDS-PAGE and resolved for 4 h. Proteins were transferred to the polyvinylidene fluoride membrane and probed with anti-CgYps1 and anti-CgYps7 antibodies. CBB-stained SDS-PAGE gels were used as loading control. Of note, the red asterisk marks a nonspecific band seen in the Cgyps1-11Δ secretome.

Figure 9

C. glabrata secretome has no effect on the production of the proinflammatory cytokine IL-1β by THP-1 macrophages. PMA-differentiated THP-1 cells were either left untreated or incubated with wild-type and Cgyps1-11Δ secretome for 24 h. Levels of secreted IL-1β were measured in 100 μL of culture media using the BD OptEIA ELISA kit. Data (mean ± standard deviation (SD); n = 2) represent relative amounts of IL-1β produced by secretome-co-incubated THP-1 cells compared to the untreated THP-1 macrophages (considered as 1.0).