Role of Sterylglucosidase 1 (Sgl1) on the pathogenicity of Cryptococcus neoformans: potential applications for vaccine development

Abstract

Cryptococcosis caused by Cryptococcus neoformans and Cryptococcus gattii affects a large population and is a cause of significant morbidity and mortality. Despite its public health burden, there are currently no vaccines against cryptococcosis and new strategies against such infections are needed. In this study, we demonstrate that C. neoformans has the biochemical ability to metabolize sterylglucosides (SGs), a class of immunomodulatory glycolipids. Genetic manipulations that eliminate cryptococccal sterylglucosidase lead to the accumulation of SGs and generate a mutant strain (Δsgl1) that is non-pathogenic in the mouse models of cryptococcosis. Interestingly, this mutant strain acts as a vaccine strain and protects mice against cryptococcosis following infection with C. neoformans or C. gattii. The immunity induced by the Δsgl1 strain is not CD4(+) T-cells dependent. Immunocompromised mice, which lack CD4(+) T-cells, are able to control the infection by Δsgl1 and acquire immunity against the challenge by wild-type C. neoformans following vaccination with the Δsgl1 strain. These findings are particularly important in the context of HIV/AIDS immune deficiency and suggest that the Δsgl1 strain might provide a potential vaccination strategy against cryptococcosis.

Keywords: gene expression; glycolipid; immunosuppression; vaccine development; yeast genetics.

FIGURE 1

CNAG_05607 enzyme has sterylglucosidase and not glucosylceramidase activity. (A) Cryptococcus neoformans CNAG_05607 (Cn 5607) metabolizes plants SGs in a dose dependent manner and (B) metabolizes SGs extracted from Cn cells (Cn SGs). (C) pH dependence of Cn 5607 activity, as measured based on its ability to cleave plants SGs and produce free sterols, showed maximal activity at pH 4.5. Results are based on the measurement of free sterols performed by liquid chromatography–mass spectrometry (LC–MS). Cn 5607 does not metabolize C. neoformans GlcCer (Cn GlcCer) as analyzed by (D) thin layer chromatography (TLC) or by (E) LC–MS (Cerezyme is an analog of the human enzyme β-glucocerebrosidases, and was used as a positive control). Cn 5607 is the endoglycoceramidase-related protein 2 (EGCrP2) also identified by Watanabe et al. (2015). Empty V, empty vector.

FIGURE 2

Deletion of SGL1 causes accumulation of SGs and not GlcCer. Analysis of sterylglucosides (SGs) and glucosylceramide (GlcCer) was performed by (A) TLC and (B,C) gas chromatography-mass spectrometry. Results show that the Δsgl1 mutant dramatically accumulates SGs (A,B), which are normally undetectable in wild-type (WT) or reconstituted strain (Δsgl1+ SGL1). In contrast, the level of GlcCer (A,C) in the Δsgl1 mutant is identical to the one observed in the WT or Δsgl1+SGL1 reconstituted strain. Chromatograms are representatives of three separate experiments showing similar results. Peaks denote: (1) Dehydroergosteryl-β-D-glucoside^∗; (2) Ergosteryl-β-D-glucoside; (3) Ergosta-7,22-dien-3-oyl-β-D-glucoside^∗; (4) Fecosteryl-β-D-glucoside^∗; (5) Episteryl-β-D-glucoside^∗; (6) Lanosteryl-β-D-glucoside^∗ (^∗: putative structures). (D) Structure and electron-impact (EI) mass spectrum of peak number 2.

FIGURE 3

Deletion of the SGL1 gene in C. neoformans abolishes virulence. (A) Virulence studies showed that 100% of mice infected with Δsgl1 survived the infection whereas mice infected with C. neoformans WT H99 or with Δsgl1+SGL1 reconstituted strain succumbed to infection within 24 ± 6 and 21 ± 7 days, respectively; n = 8 mice in each group. (B) Lung tissue burden analysis showed that the Δsgl1 mutant is eliminated from the lungs after 14 days of inoculation; n = 3 mice each at time point. (C) Brain tissue burden analysis showed that Δsgl1 is not found in the brain at anytime during the course of experiment; n = 3 mice at each time point.

FIGURE 4

Vaccination studies. (A) Mice were “pre-treated” with Δsgl1 and, after 30 days (time 0), challenged with a lethal dose of C. neoformans wild-type H99 (Cn WT) or Cryptococcus gattii WT R265 (Cg 265). Mice exposed to Δsgl1 remained alive during the course of experiment (80 days post-infection) whereas all mice that were not exposed to Δsgl1 but to vehicle (PBS) or to the Δgcs1 mutant strain and then challenged with Cn WT succumbed to infection within 35 days; n = 8 mice in each group. (B) Pre-treatment with Δsgl1 completely protected CD4⁺ T-cell depleted mice from a subsequent lethal challenge with C. neoformans wild-type H99 (WT); n = 8 mice in each group. Depletion of CD4⁺ was achieved by administering anti-CD4 (Ab) weekly during the entire course of the experiment. The depletion was confirmed by flow cytometry performed at the day of Cn challenge (C). Results are representative of three separate experiments showing similar results.