HOG1 has an essential role in the stress response, virulence and pathogenicity of Cryptococcus gattii

Abstract

Cryptococcus gattii (C. gattii) is a lethal pathogen that causes the majority of cryptococcosis cases in previously healthy individuals. This pathogen poses an increasing threat to global public health, but the mechanisms underlying the pathogenesis have remained to be fully elucidated. In the present study, the role of high-osmolarity glycerol (HOG)1 in the stress reaction and virulence control of C. gattii was characterized by deleting the HOG1 gene using the clinical isolate strain CZ2012, and finally, the virulence and pathogenic traits of the deletion strain were defined. Deletion of the HOG1 gene resulted in notable growth defects under stress conditions (high salt and antifungal drugs), but different traits were observed under oxidative stress conditions (hydrogen peroxide). Similarly, the C. gattii hog1Δ strains (deletion of HOG1) also displayed decreased capsule production and melanin synthesis. Furthermore, mice infected with the hog1Δ strain had longer survival times than those infected with the wild-type strain and the reconstituted strain. The hog1Δ strain recovered from infected organs exhibited significant growth defects in terms of decreased colony count and size. The present results suggested that HOG1 has a significant role in the virulence of C. gattii and these results may help to elucidate the pathogenesis of C. gattii.

Keywords: Cryptococcus gattii; high-osmolarity glycerol 1; pathogenicity; stress response; virulence.

Figure 1

HOG1 is required for growth under certain stress conditions. Each strain (WT strain, hog1Δ strain or hog1Δ+HOG1 reconstitution strain) was grown to the midlogarithmic phase in YPD medium, 10-fold serially diluted (1-10⁴ dilutions) and 2 µl of each diluted cell suspension was spotted on YPD medium containing 1 or 1.5 mol/l NaCl, 1 or 1.5 mol/l KCl for hyperosmotic stress and 2.5 or 3 mM H₂O₂ for oxidative stress. Following incubation for 3 days, images were acquired. All tests are for C.gattii, the top panel is shows the KCl stress, the middle shows the NaCl stress, the bottom shows the H₂O₂ stress. All tests were repeated three times, with representative images being shown. WT, wild-type; HOG, high-osmolarity glycerol; YPD, yeast extract peptone dextrose.

Figure 2

HOG1 mutation reduces capsule production by C. gattii. Strains (WT strain, hog1Δ strain or hog1Δ+HOG1 reconstitution strain) were grown on solid DME medium at (A) 30˚C or (B) 37˚C for 48 h and were stained by India ink and visualized at 40x magnification. (C) The relative capsule size (%) was quantitatively measured by calculating the ratio of the length of the packed cell volume phase per length of total volume phase. All tests were repeated three times. ^*P<0.01. (D and E) Deletion of HOG1 attenuates melanin synthesis but enhances urease excretion in C. gattii. Each strain (WT strain, hog1Δ strain or hog1Δ+HOG1 reconstitution strain) was grown to the midlogarithmic phase in yeast extract peptone dextrose medium for 24 h, 10-fold serially diluted (1-10⁴ dilutions) and 2 µl of each diluted cell suspension was spotted on (D) caffeic acid agar medium for melanin production and (E) Christianson's urea agar medium for urease excretion. Following incubation for 48 h, images were acquired. All tests were repeated three times, with representative images being shown. WT, wild-type; HOG, high-osmolarity glycerol.

Figure 3

HOG1 is essential for the virulence of C. gattii in a murine model. C57bl/6 mice were intravenously inoculated with 2.5x10⁵ cells of each stain (WT strain, hog1Δ strain or the hog1Δ+HOG1 reconstitution strain). (A) immunocompetent groups and (B) immunocompromised groups. The percentage of survival was monitored for 60 days after inoculation. Mice infected with the hog1Δ strain survived the entire period. WT, wild-type; HOG, high-osmolarity glycerol.

Figure 4

HOG1 has a role in the propagation of C. gattii. After inoculation for 3 days, each strain (WT strain, hog1Δ strain or the hog1Δ+HOG1 reconstitution strain) were isolated from the lungs of infected mice and spread on yeast extract peptone dextrose agar medium. Following incubation for 3 days at 30˚C, images were acquired. The hog1Δ strains exhibited an obviously decreased growth. WT, wild-type; HOG, high-osmolarity glycerol.