Abolishing Cell Wall Glycosylphosphatidylinositol-Anchored Proteins in Candida albicans Enhances Recognition by Host Dectin-1

Abstract

Fungi can shield surface pathogen-associated molecular patterns (PAMPs) for evading host immune attack. The most common and opportunistic human pathogen, Candida albicans, can shield β-(1 3)-glucan on the cell wall, one of the major PAMPs, to avoid host phagocyte Dectin-1 recognition. The way to interfere in the shielding process for more effective antifungal defense is not well established. In this study, we found that deletion of the C. albicans GPI7 gene, which was responsible for adding ethanolaminephosphate to the second mannose in glycosylphosphatidylinositol (GPI) biosynthesis, could block the attachment of most GPI-anchored cell wall proteins (GPI-CWPs) to the cell wall and subsequently unmask the concealed β-(1,3)-glucan. Neutrophils could kill the uncloaked gpi7 mutant more efficiently with an augmented respiratory burst. The gpi7 mutant also stimulated Dectin-1-dependent immune responses of macrophages, including activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways and secretion of specific cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and IL-12p40. Furthermore, the gpi7 null mutant could induce an enhanced inflammatory response through promoting significant recruitment of neutrophils and monocytes and could stimulate stronger Th1 and Th17 cell responses to fungal infections in vivo. These in vivo phenotypes also were Dectin-1 dependent. Thus, we assume that GPI-CWPs are involved in the immune mechanism of C. albicans escaping from host recognition by Dectin-1. Our studies also indicate that the blockage of GPI anchor synthesis is a strategy to inhibit C. albicans evading host recognition.

FIG 1

Deletion of GPI7 gene in C. albicans could block GPI-anchored protein biosynthesis and anchorage on cell wall. (A) Representative immunoblotting with peroxidase-labeled ConA staining of HF-pyridine-released C. albicans GPI-CWPs (lane 1, SN152; lane 2, gpi7Δ/Δ::GPI7; lane 3, gpi7Δ/Δ) and the ratio of gpi7Δ/Δ and gpi7Δ/Δ::GPI7 to SN152. (B) Representative immunoblotting with peroxidase-labeled ConA staining of C. albicans cytosol proteins (lane 1, SN152; lane 2, gpi7Δ/Δ::GPI7; lane 3, gpi7Δ/Δ) and the ratio of gpi7Δ/Δ and gpi7Δ/Δ::GPI7 to SN152. The mannosylated protein level of C. albicans SN152 was set to 100% as a control (A and B). (C) Representative observation of HA tag-fused Als1p, a well-characterized GPI-anchor protein, on the cell surface of the indicated strains (SN152 and gpi7Δ/Δ) by confocal laser scanning microscopy and fluorescence intensity analysis. The scale bar represents 10 μm. Data shown in panels A and B were quantified by ImageJ. Data shown in panel C were quantified by the Leica LAS AF Lite program. Data represent means (± standard errors of the means [SEM]) from three independent experiments. *, P < 0.05; **, P < 0.01 (one-way ANOVA with Bonferroni's posttest [A and B] or by Student's t test [C]).

FIG 2

Cell wall β-(1,3)-glucan was highly exposed in the C. albicans gpi7 mutant in both yeast and hyphal forms. Yeast-form growing cells (A and B) and hyphal growing cells (C) were stained with anti-β-(1,3)-glucan primary antibody to visualize β-(1,3)-glucan. The fluorescence intensity was quantified by flow cytometry (B, right) and the Leica LAS AF Lite program (C, right). The scale bar represents 10 μm. Data represent means (± SD) from triplicates of one representative experiment of three. **, P < 0.01 (one-way ANOVA with Bonferroni's posttest).

FIG 3

Neutrophils killed the uncloaked gpi7 mutant more efficiently with an augmented respiratory burst. Thioglycolate-elicited peritoneal neutrophils (0.6 × 10⁶ cells) were infected with viable C. albicans (1 × 10⁴ cells) for 60 min. (A) C. albicans then was plated on YPD agar for 48 h to measure viable CFU. (B) The respiratory burst was measured by assessing the conversion of dihydrorhodamine 123 to rhodamine. Data represent means (± SD) from triplicates of one representative experiment of three. *, P < 0.05 (one-way ANOVA with Bonferroni's posttest).

FIG 4

C. albicans gpi7 mutant could be recognized by host macrophages. gpi7Δ/Δ could induce NF-κB and MAPK activation and inflammatory responses in macrophages. Thioglycolate-elicited peritoneal macrophages were stimulated by UV-inactivated C. albicans yeast SN152, gpi7Δ/Δ::GPI7, and gpi7Δ/Δ (MOI of 5) for the indicated times. The nuclear extracts (A) and cell lysates (B and C) were analyzed by immunoblotting with the indicated antibodies. (D) Enzyme-linked immunosorbent assay (ELISA) results for TNF-α, IL-6, IL-12p40, and IL-10 in supernatants of thioglycolate-elicited peritoneal macrophages, which were stimulated with the indicated C. albicans strains (MOI of 5) for 6 h. Mock, unstimulated macrophages. Data represent means (± SD) from triplicates of one representative experiment of three. *, P < 0.05 (one-way ANOVA with Bonferroni's posttest).

FIG 5

Inflammatory responses in macrophage stimulated by C. albicans gpi7Δ/Δ were Dectin-1 dependent. Thioglycolate-elicited peritoneal macrophages from wild-type or Dectin-1-deficient mice were stimulated with UV-inactivated C. albicans yeast SN152, gpi7Δ/Δ::GPI7, or gpi7Δ/Δ (MOI of 5) for 60 min (for nuclear extracts analysis) or 30 min (for total cell lysate analysis). The nuclear extracts (A) and total cell lysates (B) were analyzed by immunoblotting with the indicated antibodies. (C) ELISA results for TNF-α, IL-6, IL-12p40, and IL-10 in supernatants of thioglycolate-elicited peritoneal macrophages from wild-type mice or Dectin-1-deficient mice, which were stimulated with the indicated C. albicans yeast strains (MOI of 5) for 6 h. Mock, unstimulated macrophages. Data represent means (± SD) from triplicates of one representative experiment of three. **, P < 0.01 (two-way ANOVA with Bonferroni's posttest).

FIG 6

Host increased inflammatory responses in vivo against C. albicans gpi7Δ/Δ. C57BL/6 or Dectin-1-deficient mice were intraperitoneally infected with 2 × 10⁵ UV-inactivated C. albicans organisms of SN152, gpi7Δ/Δ::GPI7, or gpi7Δ/Δ for 4 h. (A) Flow cytometry for Gr-1^hi7/4^hi neutrophils and Gr-1⁺7/4^hi F4/80⁺ inflammatory monocytes in the peritoneum. (B) Scatter plots of myeloid cell subsets in the peritoneum. Each symbol represents an individual mouse. Neut, neutrophil; Mono, monocyte; Eos, eosinophil. (C) ELISAs for cytokines, chemokines, and growth factors in lavage fluid from the peritoneal cavities. MCP-1, chemokine CCL2; GM-CSF, granulocyte-monocyte colony-stimulating factor. Data are representative of three independent experiments. P < 0.05 (*) and P < 0.01 (**) (Mann-Whitney nonparametric t test [B] and Kruskal-Wallis nonparametric one-way ANOVA with Dunn's posttest [C]).

FIG 7

C. albicans gpi7 mutant could stimulate enhanced host Th cell responses. (A) Splenocyte recall assay. C57BL/6 and Dectin-1-deficient mice were infected with 3 × 10⁴ viable C. albicans organisms (SN152, gpi7Δ/Δ::GPI7, or gpi7Δ/Δ) for 7 days. Splenocytes were restimulated with UV-killed C. albicans SC5314 for 48 h, and IFN-γ and IL-17 accumulation in the supernatants was measured by ELISA (n = 10). Data represent means (± SD) from triplicates of one representative experiment of three. (B) ELISAs for cytokines in kidneys from C57BL/6 mice intravenously infected with 2 × 10⁵ viable C. albicans for 7 days (n = 12). Data are representative of three independent experiments. *, P < 0.05; **, P < 0.01 (Kruskal-Wallis nonparametric one-way ANOVA with Dunn's posttest).