Abstract
Cryptococcus gattii is a capsular fungal pathogen, which causes life-threatening cryptococcosis in immunocompetent individuals. This emerging pathogen is less likely to be recognized by innate immunity compared to traditional Cryptococcus neoformans strains. Previous studies indicate that C-type lectin receptors (CLRs), including dectin-1 and dectin-2, play a role in recognizing cryptococcal cells; however, it remains to be elucidated whether the receptors physically associate with C. gattii yeast cell surfaces. Based on the previous findings, we hypothesized that culture conditions influence the expression or exposure of CLR ligands on C. gattii. Therefore, in the present study, we first investigated the culture conditions that induce exposure of CLR ligands on C. gattii yeast cells via the binding assay using recombinant fusion proteins of mouse CLR and IgG Fc, Fc dectin-1 and Fc dectin-2. Common fungal culture media, such as yeast extract–peptone–dextrose (YPD) broth, Sabouraud broth, and potato dextrose agar, did not induce the exposure of dectin-1 ligands, including β-1,3-glucan, on both capsular and acapsular C. gattii strains, in contrast to Fc dectin-1 and Fc dectin-2 bound to C. gattii cells growing in the conventional synthetic dextrose (SD) medium [may also be referred to as a yeast nitrogen base with glucose medium]. The medium also induced the exposure of dectin-1 ligands on C. neoformans, whereas all tested media induced dectin-1 and dectin-2 ligands in a control fungus Candida albicans. Notably, C. gattii did not expose dectin-1 ligands in SD medium supplemented with yeast extract or neutral buffer. In addition, compared to YPD medium-induced C. gattii, SD medium-induced C. gattii more efficiently induced the phosphorylation of Syk, Akt, and Erk1/2 in murine dendritic cells (DCs). Afterwards, the cells were considerably engulfed by DCs and remarkably induced DCs to secrete the inflammatory cytokines. Overall, the findings suggest that C. gattii alters its immunostimulatory potential in response to the environment.
Highlights
Cryptococcus gattii is an encapsulated fungal pathogen which infects to immunocompetent individuals and causes cryptococcosis
C. albicans SC5314, C. neoformans H99, C. gattii R265, C. gattii PNG18, and derivative strains were cultivated for 2 days at 30 ̊C in the following medium or agar plate: yeast extract–peptone–dextrose (YPD) broth [1% (w/v) yeast extract, 2% (w/v) Bacto peptone, and 2% (w/v) dextrose, premix powder purchased from BD Difco], synthetic dextrose (SD) medium [0.67% (w/v) yeast nitrogen base (YNB) with amino acids and ammonium sulfate (BD Difco), and 2% (w/v) dextrose], Sabouraud broth [1% (w/v) polypeptone (BD Difco) and 2% (w/v) dextrose], and potato dextrose agar [0.4% (w/v) potato starch, 2% (w/v) dextrose, and 1.5% (w/v) agar, premix powder purchased from BD Difco] [2,5,35]
Fc dectin-1 and anti-β-1,3-glucan monoclonal antibody (mAb) bound to the cell surface of live and heat-killed C. albicans cultivated in any medium (Fig 1A–1C), and the binding of Fc dectin-1 to C. albicans was decreased in the presence of competitive soluble β-glucan SPG (Fig 1D)
Summary
Cryptococcus gattii is an encapsulated fungal pathogen which infects to immunocompetent individuals and causes cryptococcosis. Following a C. gattii infection outbreak in North America since 1999, several highly virulent C. gattii strains, including R265 and JP02, have been isolated globally, and the relationship between their antigenic potential and their virulence has been investigated [1,2,3,4]. In contrast to encapsulated wild type strains, capsular strain CAP60Δ strongly stimulated DCs to produce inflammatory cytokines and the costimulatory molecules and were efficiently phagocytized by DCs [5]. C. gattii is less likely to be recognized compared to Cryptococcus neoformans under in vivo situations because C. gattii induces lower cytokines production and leukocyte recruitment in the lungs compared to C. neoformans [2,3,4]. The findings above suggest that C. gattii wild type strains have lower antigenic potential in vitro and in vivo
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