Cryptococcus strains with different pathogenic potentials have diverse protein secretomes

Abstract

Secreted proteins are the frontline between the host and pathogen. In mammalian hosts, secreted proteins enable invasive infection and can modulate the host immune response. Cryptococcosis, caused by pathogenic Cryptococcus species, begins when inhaled infectious propagules establish to produce pulmonary infection, which, if not resolved, can disseminate to the central nervous system to cause meningoencephalitis. Strains of Cryptococcus species differ in their capacity to cause disease, and the mechanisms underlying this are not well understood. To investigate the role of secreted proteins in disease, we determined the secretome for three genome strains of Cryptococcus species, including a hypovirulent and a hypervirulent strain of C. gattii and a virulent strain of C. neoformans. Sixty-seven unique proteins were identified, with different numbers and types of proteins secreted by each strain. The secretomes of the virulent strains were largely limited to proteolytic and hydrolytic enzymes, while the hypovirulent strain had a diverse secretome, including non-conventionally secreted canonical cytosolic and immunogenic proteins that have been implicated in virulence. The hypovirulent strain cannot establish pulmonary infection in a mouse model, but strains of this genotype have caused human meningitis. To directly test brain infection, we used intracranial inoculation and found that the hypovirulent strain was substantially more invasive than its hypervirulent counterpart. We suggest that immunogenic proteins secreted by this strain invoke a host response that limits pulmonary infection but that there can be invasive growth and damage if infection reaches the brain. Given their known role in virulence, it is possible that non-conventionally secreted proteins mediate this process.

FIG 1

Predicted functional groups and secretory pathways for the secreted Cryptococcus proteins. (A) The secretomes of high-virulence C. neoformans and C. gattii VGIIa are dominated by hydrolytic and proteolytic proteins, while the secretome of C. gattii VGIIb contains a large number of proteins involved in metabolism. (B) C. gattii VGIIb has a much greater proportion of proteins secreted by nonclassical pathways than C. neoformans and C. gattii VGIIa.

FIG 2

Survival curve, histochemistry, and immunohistochemistry for C57BL/6 and SCID mice infected intracranially with Cryptococcus strains. (A) Intracranial infection results in rapid death of mice infected with C. neoformans and C. gattii strain R272, which is considered hypovirulent in a pulmonary infection model. In contrast, most mice survive infection with the “hypervirulent” C. gattii VGIIa strain. (B) Histochemistry and immunohistochemistry for representative infected C57BL/6 mice. (Top panel) alcian blue counterstaining showing cryptococcal cells in the mice meninges (left-pointing arrowhead). The fungal burden is considerably lower in mice infected with C. gattii VGIIa (see also Fig. S2 in the supplemental material for a zoomed-out field of view). (Middle panel) Anti-GFAP antibody staining showing astrocytes (right-pointing arrowhead). (Bottom panel) Anti-Iba1 visualizing microglial cells (upward-pointing arrowhead). The immune response is extremely limited, particularly for C. neoformans infection, despite the significant fungal burden. There was a greater microglial response in mice infected with C. gattii VGIIb than in those infected with C. neoformans and C. gattii VGIIa. Bar, 50 μm. H&E, hematoxylin and eosin.