Processing of Candida albicans Ece1p Is Critical for Candidalysin Maturation and Fungal Virulence

Abstract

Candida albicans is an opportunistic fungal pathogen responsible for superficial and life-threatening infections in humans. During mucosal infection, C. albicans undergoes a morphological transition from yeast to invasive filamentous hyphae that secrete candidalysin, a 31-amino-acid peptide toxin required for virulence. Candidalysin damages epithelial cell plasma membranes and stimulates the activating protein 1 (AP-1) transcription factor c-Fos (via p38-mitogen-activated protein kinase [MAPK]), and the MAPK phosphatase MKP1 (via extracellular signal-regulated kinases 1 and 2 [ERK1/2]-MAPK), which trigger and regulate proinflammatory cytokine responses, respectively. The candidalysin toxin resides as a discrete cryptic sequence within a larger 271-amino-acid parental preproprotein, Ece1p. Here, we demonstrate that kexin-like proteinases, but not secreted aspartyl proteinases, initiate a two-step posttranslational processing of Ece1p to produce candidalysin. Kex2p-mediated proteolysis of Ece1p after Arg61 and Arg93, but not after other processing sites within Ece1p, is required to generate immature candidalysin from Ece1p, followed by Kex1p-mediated removal of a carboxyl arginine residue to generate mature candidalysin. C. albicans strains harboring mutations of Arg61 and/or Arg93 did not secrete candidalysin, were unable to induce epithelial damage and inflammatory responses in vitro, and showed attenuated virulence in vivo in a murine model of oropharyngeal candidiasis. These observations identify enzymatic processing of C. albicans Ece1p by kexin-like proteinases as crucial steps required for candidalysin production and fungal pathogenicity.IMPORTANCECandida albicans is an opportunistic fungal pathogen that causes mucosal infection in millions of individuals worldwide. Successful infection requires the secretion of candidalysin, the first cytolytic peptide toxin identified in any human fungal pathogen. Candidalysin is derived from its parent protein Ece1p. Here, we identify two key amino acids within Ece1p vital for processing and production of candidalysin. Mutations of these residues render C. albicans incapable of causing epithelial damage and markedly reduce mucosal infection in vivo Importantly, candidalysin production requires two individual enzymatic events. The first involves processing of Ece1p by Kex2p, yielding immature candidalysin, which is then further processed by Kex1p to produce the mature toxin. These observations identify important steps for C. albicans pathogenicity at mucosal surfaces.

Keywords: Candida albicans; candidalysin; fungal infection; kexin; mucosal immunity.

FIG 1

Alanine substitutions at positions 61 and 93 of Ece1p render C. albicans incapable of damaging or activating TR146 oral epithelial cells in vitro. (A) Epithelial cell damage induced by C. albicans Ece1p alanine substitution mutants. Epithelial cells were exposed to Ece1p alanine substitution mutants for 24 h, and levels of cell damage were assessed by LDH assay. Statistics are applied relative to the ece1Δ/Δ+ECE1 parental control (n = 5 biological repeats). (B) Western blot analysis of epithelial cells infected with C. albicans alanine substitution mutants. Epithelial cell lysates (20 μg total protein) were probed with anti-c-Fos and anti-p-MKP1 antibodies. One representative blot presented (from n = 3 biological repeats). (C) Analysis of c-Fos DNA binding activity from epithelial cells infected with C. albicans alanine substitution mutants. Statistics are applied relative to the vehicle control (n = 3 biological repeats). (D to H) Quantification of cytokines (IL-1α, IL-1β, IL-6, G-CSF, and GM-CSF) secreted from epithelial cells in response to alanine substitution mutants of C. albicans Ece1p. Statistics are applied relative to ece1Δ/Δ+ECE1 parental control (n = 3 biological repeats). (A and C to H) Data are presented as fold change relative to vehicle control (dashed line) + standard deviation (SD). Statistical significance was calculated using one-way ANOVA with a post hoc Dunnett comparison test. ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05.

FIG 2

Ece1p62–93_KA damages epithelial cells and activates c-Fos/p-MKP1 signaling and cytokine secretion in vitro. (A) Epithelial cell damage induced by Ece1p62–93_KA. Epithelial cells were exposed to Ece1p62–93_KA peptide (70, 15, and 1.5 μM) for 24 h, and levels of cell damage were assessed by LDH assay. (B) Western blot analysis of epithelial cells treated with Ece1p62–93_KA peptide (15 μM) for 2 h. Epithelial cell lysates (20 μg total protein) were probed with anti-c-Fos and anti-p-MKP1 antibodies. One representative blot is presented (from n = 3 biological repeats). (C to G) Quantification of cytokines (IL-1α, IL-1β, IL-6, G-CSF, and GM-CSF) secreted from epithelial cells in response to Ece1p62–93_KA peptide at 70, 15, and 1.5 μM. (A and C to G) Data and statistical analysis are presented relative to vehicle control (dashed line; n = 3 biological repeats) + standard deviation (SD). Statistical significance was calculated using one-way ANOVA with a post hoc Dunnett comparison test. ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05.

FIG 3

Enzymatic processing of C. albicans Ece1p is required for efficient host recognition of infecting fungus in vivo. Nonimmunosuppressed mice were infected sublingually with selected C. albicans Ece1p alanine substitution mutants and control strains for 24 h, and reverse transcriptase qPCR was performed on tongue tissue RNA to quantify expression of Ccl20 (A), Il1b (B), Il6 (C), and Csf3 transcripts (D). Data are presented as the mean of two biological repeats + standard deviation (SD). Statistical analysis is presented relative to the ece1Δ/Δ+ECE1 parental control (total numbers of animals: sham, n = 4; wild type, n = 4; ece1Δ/Δ+ECE1 strain, n = 6; R31A, n = 6; R61A, n = 5; R93A, n = 3; R61A + R93A, n = 7; ALL KA, n = 7; ece1Δ/Δ strain, n = 5; ece1Δ/Δ+ECE1_Δ184–279 strain, n = 5). Statistical significance was calculated using one-way ANOVA with a post hoc Dunnett comparison test. ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05.

FIG 4

Enzymatic processing of C. albicans Ece1p is required for fungal pathogenesis in vivo. An immunosuppressed murine model of oropharyngeal candidiasis (OPC) was infected with selected C. albicans Ece1p alanine substitution mutants and control strains, and the number of CFU per gram of tissue was enumerated after 24 h (A) and 48 h (B). Data represent two biological repeats. Total numbers of animals in panel A: sham, n = 4; wild type, n = 12; ece1Δ/Δ strain, n = 12; ece1Δ/Δ+ECE1 strain, n = 16; R61A + R93A, n = 21; ALL KA, n = 11. Total numbers of animals in panel B: wild type, n = 11; ece1Δ/Δ strain, n = 12; ece1Δ/Δ+ECE1 strain, n = 11; ALL KA, n = 12. Statistical analysis is presented relative to the ece1Δ/Δ+ECE1 parental control. Statistical significance was calculated using one-way ANOVA with a post hoc Dunnett comparison test. Geometric mean is indicated. ****, P ≤ 0.0001; ***, P ≤ 0.001; *, P ≤ 0.05.