Congenic Strains for Genetic Analysis of Virulence Traits in Cryptococcus gattii

Abstract

cCryptococcus gattii is responsible for a large outbreak of potentially fatal disease that started in the late 1990s on Vancouver Island, Canada. How this fungus and the outbreak isolates in particular cause disease in immunocompetent people is unknown, with differing hypotheses. To explore genetic contributions, a pair of congenic a and ␣ mating type strains was generated by a series of 11 backcrosses to introgress the MAT locus from a nonoutbreak strain into the background of strain R265, isolated from a Vancouver Island patient. The congenic pair was used to investigate three traits: mitochondrial inheritance, the effect of the MAT alleles on virulence, and the impact of a predicted virulence factor on pathogenicity. The two congenic strains show the same virulence in different models of cryptococcosis and equivalent levels of competition in coinfection assays. These results rule out a role of the MAT locus and mitochondrial genotype as major virulence factors in the outbreak strains. Disruption of Bwc2, a light-dependent transcription factor, resulted in reduced virulence, consistent with a similar function in the related species Cryptococcus neoformans. The C. gattii congenic strains represent a new resource for exploring the evolution of virulence in the C. neoformans-C. gattii clade.

Congenic Strains of the Filamentous Form of Cryptococcus neoformans for Studies of Fungal Morphogenesis and Virulence

“…No a-␣ coinfection experiment has been performed for other congenic pairs, except the recently constructed C. gattii AIR265␣ and AIR265a strains. AIR265␣ and AIR265a are similar in virulence when tested individually in both the inhalation and the intravenous infection models of murine cryptococcosis (55). Furthermore, neither the ␣ nor the a mating type allele confers any apparent advantages in terms of mortality or neurotropism in the AIR265 background during a-␣ coinfection in either model of murine cryptococcosis (55).…”

“…KN99␣ and KN99a exhibit no difference in virulence when they infect mice individually in the inhalation model of cryptococcosis, but KN99␣ appears to be more efficient in colonizing the brain during mixed a-␣ coinfection (33,37). In contrast, the recently constructed C. gattii congenic pair strains AIR265␣ and AIR265a are similarly virulent in mouse models, and neither of the mating types confers any competitive advantages during in vitro coculture or during a-␣ coinfection in mice, regardless of the route of inoculation (intranasal or intravenous), as noted in the accompanying article by Zhu et al (55). One plausible reason to account for the observed differences between strains is that factors located outside the mating type locus are involved in controlling both virulence and mating behavior (27).…”

“…6B and 9B). For comparison, the Cryptococcus gattii reference strain R265 predominantly amplifies in the lungs and very little in the brain (55,80). Further investigation into the molecular mechanisms underlying the strain or serotype-specific differences in neurotropism is warranted.…”

Cryptococcus gattii Infections

“…Unlike the study by Ma et al (149), the expression of the mitochondrial genes was not different in R265 and R272. The differences in the results from the two studies can be explained by the similarity in mitochondrial genomes of strains R265 and R272, since creation of congenic a and ␣ strains by backcrossing of R265 with a nonoutbreak VGII strain indicated that neither the mating type nor the mitochondrial genotype was a major virulence determinant (407).…”

Cryptococcus neoformans and Cryptococcus gattii, the Etiologic Agents of Cryptococcosis

“…Deletion of genes that encode a blue-light-sensing complex reduces virulence Zhu et al 2013). It is unknown whether darkness represents a specific signal and the relevant genes are also unknown.…”