The RIM101 signal transduction pathway regulates Candida albicans virulence during experimental keratomycosis

Abstract

Purpose: To examine the role of the fungal RIM101 signal transduction pathway in the pathogenesis of Candida albicans keratitis.

Methods: C. albicans wild-type strain SC5314, prototrophic mutant control DAY185, and homozygous fungal mutants for the rim8, rim13, rim20, rim101, and phr1 genes were evaluated in vitro using proliferation and filamentation assays. Scarified corneas of BALB/c and C57BL/6J mice were topically inoculated and observed daily for keratitis severity. Corneal adaptation and pathogenicity were assessed ex vivo by maintaining infected porcine corneas for 3 days in an explantation culture system for histologic evaluation of hyphal penetration.

Results: All C. albicans strains had similar growth kinetics, and SC5314 and DAY185 demonstrated pH-induced filamentation. Fungal mutants had reduced hyphal formation at alkaline and neutral pH, but normal acidic assays ascertained that mutant strains did not have a generalized filamentation defect. SC5314 and DAY185 caused moderate to severe keratitis in mice, whereas fungal strains lacking constituents of the RIM101 pathway had significantly (P<0.05) attenuated severity in vivo. Three days after inoculation of porcine corneas, SC5314 and DAY185 produced hyphae that penetrated 28% and 25%, respectively, of the corneal thickness, and all five mutant strains showed significantly (P<0.05) less stromal penetration.

Conclusions: The RIM101 signal transduction pathway plays an important role in the development of C. albicans keratitis. The fungal pathway intermediates Rim8p, Rim13p, Rim20p, and Rim101p and the downstream cell-wall protein Phr1p are pivotal in the process of corneal invasion by C. albicans.

Figure 1.

In vitro growth kinetics of Candida albicans strains SC5314, DAY185, DAY25, DAY111, DAY117, GKO88, and CAS10. Triplicate samples of each strain were inoculated into M199 media at pH 6.0 (A), 7.3 (B), and 8.0 (C) and monitored by spectrophotometry over time. Mean OD₆₀₀ measurements converted to colony-forming unit equivalents (± SD) are plotted.

Figure 2.

Murine keratomycosis severity induced by C. albicans in BALB/c mice. Immunocompetent BALB/c mice were infected with 10⁵ CFU of C. albicans used in the study and were followed and scored for disease severity for 7 days. Each point represents the mean score (± SD) of five eyes.

Figure 3.

Progression of murine keratomycosis induced by C. albicans in BALB/c mice. Scarified corneas of immunocompetent BALB/c mice were infected with 10⁵ CFU of C. albicans used in the study and photographed on days 1, 3, and 7 PI. Representative images among five eyes per group are shown.

Figure 4.

Murine keratomycosis severity induced by C. albicans in C57BL/6J mice. Immunocompetent C57BL/6J mice were infected with 10⁵ CFU of C. albicans strains SC5314, GKO88, or CAS10 and were scored for disease severity for 7 days. Each point represents the mean score (± SD) of five eyes.

Figure 5.

Progression of murine keratomycosis induced by C. albicans in C57BL/6J mice. Scarified corneas of immunocompetent C57BL/6J mice were infected with 10⁵ CFU of C. albicans strains SC5314, GKO88, or CAS10 and were photographed on days 1, 3, and 7 PI. Representative images among five eyes per group are shown.

Figure 6.

Histopathology of porcine keratomycosis induced by C. albicans strains. Porcine corneas inoculated ex vivo with 10⁵ CFU of C. albicans strains SC5314 (A), DAY185 (B), DAY25 (C), DAY111 (D), DAY117 (E), GKO88 (F), or CAS10 (G) or with PBS (H) were cryosectioned at 10-μm thickness 3 days PI and stained with periodic acid-Schiff. Representative sections among three corneas per strain are shown. Original magnification ×200.