Role of Hog1-mediated stress tolerance in biofilm formation by the pathogenic fungus Trichosporon asahii

Abstract

Trichosporon asahii, a dimorphic fungus, causes bloodstream infections in immunocompromised patients with neutropenia. Biofilms are formed on the surfaces of medical devices such as catheters as T. asahii transitions morphologically from yeast to hyphae in the host environment. Oxidative stress tolerance and morphological changes of T. asahii are regulated by Hog1, a mitogen-activated protein kinase. The role of Hog1 in the biofilm formation by T. asahii, however, has remained unknown. In the present study, we demonstrated that a hog1 gene-deficient T. asahii mutant formed excess biofilm under a rich medium in vitro, but did not form biofilm in an in vivo evaluation system using silkworms. The hog1 gene-deficient T. asahii mutant formed a greater amount of biofilm than the parent strain in vitro. Under an oxidative stress condition in vitro, however, lower amounts of biofilm were formed by the hog1 gene-deficient T. asahii mutant than by the parent strain. In an in vivo evaluation system using silkworms, lower amounts of biofilm were formed by the hog1 gene-deficient T. asahii mutant than by the parent strain. Our findings suggest that Hog1 regulates biofilm formation by T. asahii in response to host environmental conditions, including oxidative stress.

Keywords: Trichosporon asahii; Biofilm; Infection; Mitogen-activated protein kinase; Stress tolerance.

Fig. 1

Excess biofilm formation by the hog1 gene-deficient mutant of T. asahii in vitro. (a, b) Biofilm formation by T. asahii in Sabouraud dextrose medium in vitro was determined by crystal violet (CV) staining. The amounts of biofilm formed by the parent strain (Parent), hog1 gene-deficient mutant (∆hog1), and its complemented strain (Comp.) in Sabouraud dextrose medium were determined by CV staining. (c, d) Biofilm formation by T. asahii in RPMI medium in vitro. The amounts of biofilm formation by the parent strain (Parent), hog1 gene-deficient mutant (∆hog1), and its complemented strain (Comp.) in the RPMI medium were determined by CV staining. (a, c) Pictures of biofilm stained with CV are shown. (b, d) The amounts of CV were determined by measuring the absorbance at 550 nm (A₅₅₀). Data are shown as means ± standard deviation (SD). Statistically significant differences between groups were evaluated using Tukey’s test. * P < 0.05. n = 5/group.

Fig. 2

Increase in cell viability in the biofilm of the hog1 gene-deficient mutant of T. asahii in vitro. Cell viability in T. asahii biofilm was determined by the XTT assay. Cell viabilities in the biofilm of the parent strain (Parent), hog1 gene-deficient mutant (∆hog1), and its complemented strain (Comp.) in Sabouraud dextrose medium (a) or RPMI medium (b) were determined. The amounts of XTT were determined by measuring the absorbance at 490 (A₄₉₀) and 630 nm (A₆₃₀). Data are shown as means ± standard deviation (SD). Statistically significant differences between groups were evaluated using Tukey’s test. *P < 0.05. n = 3–5/group.

Fig. 3

Hyphal formation of the hog1 gene-deficient mutant in biofilm. The morphology of T. asahii stained with CFW was observed using fluorescence microscopy. The parent strain (Parent), the hog1 gene-deficient mutant (∆hog1), and the complemented strain of ∆hog1 (Comp.) in the biofilm were observed. CFW: calcofluor white stain. (a) Observed under a microscope at 40-fold magnification (×40). (b) Measurement of CFW fluorescence. Statistically significant differences between groups were evaluated using Tukey’s test. *P < 0.05. n = 6/group. (c) Observed under a microscope at 400-fold magnification (×400). White scale bar = 20 µm. Yellow arrows indicate long hyphae.

Fig. 4

Effect of H₂O₂ on biofilm formation by the hog1 gene-deficient mutant of T. asahii in vitro. (a, b) Biofilm formation by T. asahii in Sabouraud dextrose medium in vitro was determined by crystal violet (CV) staining. The amounts of biofilm formed by the parent strain (Parent), hog1 gene-deficient mutant (∆hog1), and its complemented strain (Comp.) in Sabouraud dextrose medium were determined by CV staining. (a) Pictures of biofilm stained with CV are shown. (b) The amounts of CV were determined by measuring the absorbance at 550 nm (A₅₅₀). Data are shown as means ± standard deviation (SD). Statistically significant differences between groups were evaluated using Tukey’s test. *P < 0.05. n = 9/group.

Fig. 5

Effect of H₂O₂ on biofilm formation by the hog1 gene-deficient mutant of T. asahii in vitro. Morphology of T. asahii stained with CFW was observed using fluorescence microscopy. The parent strain (Parent), the hog1 gene-deficient mutant (∆hog1), and the complemented strain of ∆hog1 (Comp.) in the biofilm were observed. CFW: calcofluor white stain. (a) Observed under a microscope at 40-fold magnification (×40). (b) Measurement of CFW fluorescence. Statistically significant differences between groups were evaluated using Tukey’s test. *P < 0.05. n = 3/group. (c) Observed under a microscope at 400-fold magnification (×400). White scale bar = 20 µm.

Fig. 6

Effect of hog1 gene-deficiency on T. asahii biofilm formation in vivo. (a) Experimental scheme of the in vivo biofilm assay using silkworms. Polyurethane fiber (PF)-inserted silkworms were prepared. Cell suspensions (A₆₃₀ = 2) (50 µL) of the parent strain (Parent), the hog1 gene-deficient mutant (∆hog1), and the complemented strain of ∆hog1 (Comp.) were injected into the PF-inserted silkworms. The PF-inserted silkworms were incubated at 27 °C for 24 h. After incubation, PFs were isolated from the silkworms, stained with crystal violet, and observed under a microscope (b). The absorbance of the eluted dye was measured at 590 nm (c). Statistically significant differences between groups were evaluated using Tukey’s test. *P < 0.05. n = 18/group.

Fig. 7

Model of the role of Hog1 in biofilm formation by T. asahii according to environmental conditions. Under rich medium conditions in vitro, Hog1 negatively regulates hyphal elongation and biofilm formation. In host environments in vivo, T. asahii cells are subjected to several stimuli including oxidative stress, and Hog1-mediated stress tolerance is required for biofilm formation.