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. 2016 Jan 6:5:101.
doi: 10.3389/fcimb.2015.00101. eCollection 2015.

Evasion of Innate Immune Responses by the Highly Virulent Cryptococcus gattii by Altering Capsule Glucuronoxylomannan Structure

Affiliations

Evasion of Innate Immune Responses by the Highly Virulent Cryptococcus gattii by Altering Capsule Glucuronoxylomannan Structure

Makoto Urai et al. Front Cell Infect Microbiol. .

Abstract

Cryptococcus neoformans causes life-threatening diseases mainly in immunosuppressed hosts such as AIDS patients; C. gattii causes disseminated infections even in healthy hosts. To identify the possible molecular mechanisms underlying this difference in virulence, we investigated the survival and histopathology of lung tissue in wild-type and CD4-depleted mice infected with C. neoformans H99 and C. gattii JP02 (the highly virulent strain isolated in Japan); we then compared dendritic cell (DC) cytokine release responses to different cell fractions from these two strains. JP02-infected mice exhibited shorter survival and fewer inflammatory cells in the lung than H99-infected control mice. Depletion of CD4-related cellular immunity reduced survival of H99-infected mice but had no effect on the survival or inflammatory cell infiltration in JP02-infected mice, suggesting that JP02 evades immune detection. To identify the molecule(s) conferring this difference, we measured cytokine production from murine DCs co-cultured with H99 and JP02 in vitro. The levels of inflammatory cytokines from DCs treated with intact JP02 cells, the extracted capsule, secreted extracellular polysaccharides, and purified glucuronoxylomannan (GXM) were markedly lower than those induced by intact H99 cells and corresponding H99 fractions. Structural analysis of GXM indicated that JP02 altered one of two O-acetyl groups detected in the H99 GXM. Deacetylated GXM lost the ability to induce inflammatory cytokine release from DCs, implicating these O-acetyl groups in immune recognition. We conclude that the highly virulent C. gattii processes a structural alteration in GXM that allows this pathogen to evade the immune response and therefore elimination.

Keywords: Cryptococcus gattii; Cryptococcus neoformans; O-acetylation; capsule; dendritic cells; glucuronoxylomannan; innate immune responses; structure.

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Figures

Figure 1
Figure 1
Greater virulence and mortality of Cryptococcus gattii JP02 infection compared to Cryptococcus neoformans H99 infection and independence from CD4 cell-mediated immunity. (A) Shorter survival of mice infected with JP02 compared with H99-infected mice (n = 8). Some mice were depleted of CD4-positive cells by an anti-CD4 injection before infection. The survival from H99 infection was shorter in CD4-depleted mice compared with that in CD4-positive control mice, while CD4 depletion had no effect on the survival from JP02 infection. (B) Fungal burden was substantially higher in the lungs of JP02-infected mice compared with the lungs of H99-infected mice. Results are representative of two individual experiments with n = 5 for each experiment (means ± standard deviations, SD). (C) Capsule formation as evaluated by India ink staining of infected lung homogenates showing thick capsule formation by both species. Bar, 10 μm. Survival curves were compared by log-rank test and the means of fungal burden were compared by the unpaired t-test. *P < 0.01; NS, not significant.
Figure 2
Figure 2
Histopathological differences between H99- and JP02-infected lungs suggest less protective inflammation induced by JP02. Lung paraffin sections were prepared from mice on day 14 after intratracheal infection, stained with hematoxylin-eosin (HE) or alcian blue (AB), and examined at magnifications of (A) ×ばつ 40 (bar, 1 mm) and (B) ×ばつ 400 (bar, 100 μm) under a light microscope. (A) Lung sections of control and CD4-depleted mice infected with H99 showed well-demarcated nodular lesions (arrows), but these were enlarged in CD4-depleted mice. In contrast, neither the lung sections of control nor CD4-depleted mice infected with JP02 exhibited nodular lesions, but both showed eccentric pulmonary enlargement and alveolar airspace variation. (B) Lung sections of control and CD4-depleted mice infected with H99 showed numerous MGCs (arrows). Yeast cells (arrow heads) were observed only in the nodular lesions, and these surfaces were well-stained by alcian blue. In contrast, the lung sections of control and CD4-depleted JP02-infected mice exhibited no inflammatory cell infiltration. Numerous yeast cells were observed in the alveoli, and its airspaces were filled with large amounts of an acidic substance stained by alcian blue.
Figure 3
Figure 3
Lower IL-6 release from JAWSII dendritic cells in response to live or heat-killed JP02 cells compared with live or heat-killed H99 cells. JAWSII cells (2 ×ばつ 105 cells/mL) were incubated with live (MOI = 1) or heat-killed (MOI = 5) cryptococcal cells for 24 h, and supernatant was obtained for ELISA. (A) Incubation with live cells. (B) Incubation with heat-killed cells. H99 + JP02 means co-incubation with heat-killed H99 and heat-killed JP02 cells (MOI = 5, each). The experiments were performed in triplicate (means ± SD). Treatment group means were compared by the unpaired t-test, except for vs. H99 in (B), which were compared by the Mann–Whitney U-test. *P < 0.01; NS, not significant.
Figure 4
Figure 4
Lower IL-6 release from JAWSII cells in response to CPS and EPS fractions of JP02 cells compared to CPS and EPS fractions of H99 cells. JAWSII cells (2 ×ばつ 105 cells/mL) were incubated with CPS or EPS for 24 h, and supernatant obtained for ELISA. (A) CPS; (B) EPS, and (C) co-incubation with EPS from both H99 and JP02 (50 μg/mL, each). The experiments were performed in triplicate (means ± SD). Treatment group means were compared by the unpaired t-test, except for vs. CPS 500 μg/mL, which were compared by the Mann–Whitney U-test. *P < 0.01; NS, not significant.
Figure 5
Figure 5
Ion-exchange chromatography of EPS fractions from H99 and JP02 cells indicate similar compositions. (A) H99 and (B) JP02. EPSs were dissolved in 10 mM Tris–HCl buffer, applied to a DEAE-Toyopearl column (180 mm ×ばつ 25 mm φ), and eluted with a 600-mL linear gradient (0–0.5 M) of NaCl. Subfractions containing saccharides were monitored by the phenol–H2SO4 method.
Figure 6
Figure 6
500-MHz 1H NMR spectra of native and modified GXM purified from H99 and JP02 strains recorded in D2O at 70°C. (A) H99 and (B) JP02. (a) native, (b) O-deacetylated, and (c) O-deacetylated and sonicated GXM.
Figure 7
Figure 7
Predicted structures of O-deacetylated GXMs purified from H99 and JP02 strains. (A) H99 and (B) JP02.
Figure 8
Figure 8
Cytokine release from dendritic cells in response to native GXM purified from H99 and JP02 strains and the corresponding O-deacetylated products. IL-6 (A) and TNF-α (B) release from JAWSII cells (2 ×ばつ 105cells/mL) stimulated by GXMs (50 μg/mL). IL-12p40 (C) and TNF-α (D) release from BMDCs (1 ×ばつ 106cells/mL) stimulated by GXMs. The experiments were performed in triplicate (means ± SD). Data sets in (A) or (B) were compared by the unpaired t-test, except for vs. control in (A), which were compared by the Mann–Whitney U-test. Treatment group means of (C) or (D) were compared to H99 GXM by the unpaired t-test, except for vs. JP02 at 10 μg/mL in (C), which were compared by Welch's t-test. *P < 0.01; NS, not significant. Representative data from 3 independent experiments are shown.

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