Ssk2 Mitogen-Activated Protein Kinase Kinase Kinase Governs Divergent Patterns of the Stress-Activated Hog1 Signaling Pathway in Cryptococcus neoformans▿ †

The stress-activated p38/Hog1 mitogen-activated protein kinase (MAPK) pathway is structurally conserved in many diverse organisms, including fungi and mammals, and modulates myriad cellular functions. The Hog1 pathway is uniquely specialized to control differentiation and virulence factors in a majority of clinical Cryptococcus neoformans serotype A and D strains. Here, we identified and characterized the Ssk2 MAPKKK that functions upstream of the MAPKK Pbs2 and the MAPK Hog1 in C. neoformans. The SSK2 gene was identified as a potential component responsible for the difference in Hog1 phosphorylation between the serotype D f1 sibling strains B-3501 and B-3502 through comparative analysis of meiotic maps showing their meiotic segregation patterns of Hog1-dependent sensitivity to the antifungal drug fludioxonil. Ssk2 is the only component of the Hog1 MAPK cascade that is polymorphic between the two strains, and the B-3501 and B-3502 SSK2 alleles were distinguished by two coding sequence changes. Supporting this finding, SSK2 allele exchange completely interchanged the Hog1-controlled signaling patterns, related phenotypes, and virulence levels of strains B-3501 and JEC21. In the serotype A strain H99, disruption of the SSK2 gene enhanced capsule and melanin biosynthesis and mating efficiency, similar to pbs2 and hog1 mutations. Furthermore, ssk2Δ, pbs2Δ, and hog1Δ mutants were hypersensitive to a variety of stresses and resistant to fludioxonil. In agreement with these results, Hog1 phosphorylation was abolished in the ssk2Δ mutant, similar to what occurred in the pbs2Δ mutant. Taken together, these findings indicate that Ssk2 is a critical interface connecting the two-component system and the Pbs2-Hog1 MAPK pathway in C. neoformans

Spores as Infectious Propagules of Cryptococcus neoformans▿ †

Cryptococcus neoformans and Cryptococcus gattii are closely related pathogenic fungi that cause pneumonia and meningitis in both immunocompromised and immunocompetent hosts and are a significant global infectious disease risk. Both species are found in the environment and are acquired via inhalation, leading to an initial pulmonary infection. The infectious propagule is unknown but is hypothesized to be small desiccated yeast cells or spores produced by sexual reproduction (opposite- or same-sex mating). Here we characterize the morphology, germination properties, and virulence of spores. A comparative morphological analysis of hyphae and spores produced by opposite-sex mating, same-sex mating, and self-fertile diploid strains was conducted by scanning electron microscopy, yielding insight into hyphal/basidial morphology and spore size, structure, and surface properties. Spores isolated by microdissection were found to readily germinate even on water agarose medium. Thus, nutritional signals do not appear to be required to stimulate spore germination, and as-yet-unknown environmental factors may normally constrain germination in nature. As few as 500 CFU of a spore-enriched infectious inoculum (∼95% spores) of serotype A C. neoformans var. grubii were fully virulent (100% lethal infection) in both a murine inhalation virulence model and the invertebrate model host Galleria mellonella. In contrast to a previous report on C. neoformans var. neoformans, spores of C. neoformans var. grubii were not more infectious than yeast cells. Molecular analysis of isolates recovered from tissues of infected mice (lung, spleen, and brain) provides evidence for infection and dissemination by recombinant spore products. These studies provide a detailed morphological and physiological analysis of the spore and document that spores can serve as infectious propagules

Surfactant Protein D Increases Phagocytosis of Hypocapsular Cryptococcus neoformans by Murine Macrophages and Enhances Fungal Survival▿ †

Cryptococcus neoformans is a facultative intracellular opportunistic pathogen and the leading cause of fungal meningitis in humans. In the absence of a protective cellular immune response, the inhalation of C. neoformans cells or spores results in pulmonary infection. C. neoformans cells produce a polysaccharide capsule composed predominantly of glucuronoxylomannan, which constitutes approximately 90% of the capsular material. In the lungs, surfactant protein A (SP-A) and SP-D contribute to immune defense by facilitating the aggregation, uptake, and killing of many microorganisms by phagocytic cells. We hypothesized that SP-D plays a role in C. neoformans pathogenesis by binding to and enhancing the phagocytosis of the yeast. Here, the abilities of SP-D to bind to and facilitate the phagocytosis and survival of the wild-type encapsulated strain H99 and the cap59Δ mutant hypocapsular strain are assessed. SP-D binding to cap59Δ mutant cells was approximately sixfold greater than binding to wild-type cells. SP-D enhanced the phagocytosis of cap59Δ cells by approximately fourfold in vitro. To investigate SP-D binding in vivo, SP-D−/− mice were intranasally inoculated with Alexa Fluor 488-labeled cap59Δ or H99 cells. By confocal microscopy, a greater number of phagocytosed C. neoformans cells in wild-type mice than in SP-D−/− mice was observed, consistent with in vitro data. Interestingly, SP-D protected C. neoformans cells against macrophage-mediated defense mechanisms in vitro, as demonstrated by an analysis of fungal viability using a CFU assay. These findings provide evidence that C. neoformans subverts host defense mechanisms involving surfactant, establishing a novel virulence paradigm that may be targeted for therapy

Same-sex mating and the origin of the Vancouver Island Cryptococcus gattii outbreak

Genealogy can illuminate the evolutionary path of important human pathogens. In some microbes, strict clonal reproduction predominates, as with the worldwide dissemination of Mycobacterium leprae, the cause of leprosy(1). In other pathogens, sexual reproduction yields clones with novel attributes, for example, enabling the efficient, oral transmission of the parasite Toxoplasma gondii(2). However, the roles of clonal or sexual propagation in the origins of many other microbial pathogen outbreaks remain unknown, like the recent fungal meningoencephalitis outbreak on Vancouver Island, Canada, caused by Cryptococcus gattii(3). Here we show that the C. gattii outbreak isolates comprise two distinct genotypes. The majority of isolates are hypervirulent and have an identical genotype that is unique to the Pacific Northwest. A minority of the isolates are significantly less virulent and share an identical genotype with fertile isolates from an Australian recombining population. Genotypic analysis reveals evidence of sexual reproduction, in which the majority genotype is the predicted offspring. However, instead of the classic a - alpha sexual cycle, the majority outbreak clone appears to have descended from two alpha mating-type parents. Analysis of nuclear content revealed a diploid environmental isolate homozygous for the major genotype, an intermediate produced during same-sex mating. These studies demonstrate how cryptic same-sex reproduction can enable expansion of a human pathogen to a new geographical niche and contribute to the ongoing production of infectious spores. This has implications for the emergence of other microbial pathogens and inbreeding in host range expansion in the fungal and other kingdoms