You are what you secrete: extracellular proteins and virulence in Cryptococcus

Fungal organisms secrete a wide range of biomolecules, including degradative enzymes that are essential for nutrition, toxins, effectors and secondary compounds that modulate interactions with host animals and plants, and a variety of signaling and stress-related proteins¹. As these are likely to be key determinants of virulence and may also be useful diagnostic and therapeutic targets, we investigated the secretome of different strains of the fungal pathogen Cryptococcus. Virulent strains secreted predominantly hydrolytic and proteolytic enzymes, while the least virulent strain secreted a range of additional non-degradative proteins including many that lacked secretion signals, some that appear to be ‘moonlighting’, and a number that are known to be allergenic. It appears that in Cryptococcus, the secretome may influence virulence both through the presence of harmful enzymes and through the absence of proteins that alert the host defence mechanisms.

Cryptococcus is an encapsulated yeast with two predominant pathogenic species: Cryptococcus neoformans and Cryptococcus gattii. These cause cryptococcosis in animals and humans, with disease ranging from asymptomatic to severe, fatal meningitis. There are a number of differences between C. gattii and C. neoformans including their preferred environmental niche, basidiospore morphology, drug susceptibility, epidemiology, the clinical manifestations of associated disease, and host susceptibility². In addition, there are significant differences among stains within each species. In C. gattii, a hypervirulent sub-genotype designated VGIIa has caused a recent significant outbreak of cryptococcosis on Vancouver Island in British Columbia, Canada and in the Pacific Northwest of the United States. In contrast, a closely related sub-genotype designated VGIIb is globally distributed and hypovirulent³. These differences between Cryptococcus species and sub-genotypes provide an opportunity for understanding pathogenicity and disease progression by what are otherwise very genetically similar fungal organisms.

Our laboratory has been using ‘omics approaches to understand virulence in Cryptococcus, and used proteomic analysis to characterize the secretome produced by three Cryptococcus strains. Two strains were of high virulence (C. neoformans and C. gattii sub-genotype VGIIa) and the third of low virulence (C. gattii sub-genotype VGIIb). In our previous work on Cryptococcus proteomics, we found conditions optimized to simulate those encountered in the host induced the production of large amounts of shed capsular material, which interfered with the isolation and identification of proteins⁴. Therefore, we developed a novel method of protein capture using BioRad ProteoMiner™ beads, followed by mass spectroscopy. Sixty-seven cryptococcal proteins were identified and only one was common to all three strains. The secretomes of the high virulence C. neoformans and C. gattii VGIIa strains were similar and mostly consisted of a hydrolytic and proteolytic proteins. In contrast the lower virulence C. gattii VGIIb strain had a larger number of proteins with a greater diversity of functions (Figure 1). A significant proportion of these proteins are known to have roles in metabolism, signaling/transport, glycolysis and redox processes, and are considered to be canonical intracellular proteins. Published studies have reported very similar proteins in the extracellular milieu of various cell types from other organisms, and there is growing evidence that these may have ‘moonlighting’ functions, where they participate in completely different processes in alternative environments⁵. An additional subset of proteins found only in the C. gattii VGIIb secretome were orthologous to proteins known to elicit an immune response in the host, including the glycolytic proteins enolase and glyceraldehyde-3-phosphate dehydrogenase^6,7. Most of these unusual secreted proteins lack secretion signals and are likely to be exported via alternative secretion pathways such as inside microvesicles, which have previously been isolated from Cryptococcus⁸; indeed the regulatory 14-3-3 protein, which is considered a biomarker of microvesicles⁹, was present exclusively in the VGIIb secretome.

Figure 1. The secretome of high and low virulence strains of Cryptococcus. Red triangles: hydrolytic and proteolytic enzymes; Green circles: proteins involved in metabolism, signaling/transport, redox, stress responses or with unknown function.

Mammals have a high level of innate immunity to most fungi, and the ability to infect immunocompetent hosts remains a rare trait. Cryptococcus is an environmental fungus, and as it cannot be spread from host to host, mammalian infection is likely to be accidental¹⁰. The questions of what determines virulence, and what processes underlie the evolution of strains that cause significant outbreaks in a dead-end host, are therefore intriguing. Comparative genomic studies have identified genes that are particular to the high virulence strains but their role in virulence is yet to be verified^11,12. As secreted biomolecules are mediators of contact between the host and the pathogen, differences in these are likely to influence whether a pathogen will be rapidly recognized and eliminated, or will be able to bypass the host response and use host resources to establish an active infection. The results of our secretome analysis suggest that virulence in Cryptococcus may in part be determined by restricted secretion of proteins likely to elicit an immune response, and that in the absence of these the production and secretion of degradative enzymes enables host invasion.