Cryptococcal HSP70 homologue Ssa1 contributes to pulmonary expansion of C. neoformans during the afferent phase of the immune response by promoting macrophage M2 polarization

Numerous virulence factors expressed by C. neoformans (C. neo) modulate host defenses by promoting non-protective Th2-biased adaptive immune responses. Prior studies demonstrate that the HSP70 homologue, Ssa1, significantly contributes to serotype-D C. neo virulence through the induction of laccase, a Th2-skewing and CNS-tropic factor. In the current study, we sought to determine whether Ssa1 modulates host defenses in mice infected with a highly virulent serotype A (serA) strain of C. neo (H99). To investigate this, we assessed pulmonary fungal growth, CNS dissemination, and survival in mice infected with either H99, an SSA1-deleted H99 strain (Δssa1), and a complement strain with restored SSA1 expression (Δssa1::SSA1). Mice infected with the Δssa1 strain displayed substantial reductions in lung fungal burden during the innate phase (days 3 and 7) of the host response whereas less pronounced reductions were observed during the adaptive phase (day 14) and mouse survival increased only by 5 days. Surprisingly, laccase activity assays revealed that Δssa1 was not laccase-deficient, demonstrating that H99 does not require Ssa1 for laccase expression, which explains the CNS tropism we still observed in the Ssa1-deficient strain. Lastly, our immunophenotyping studies showed that Ssa1 directly promotes early M2 skewing of lung mononuclear phagocytes during the innate, but not the adaptive phase of the immune response. We conclude that Ssa1’s virulence mechanism in H99 is distinct and laccase-independent. Ssa1 directly interferes with early macrophage polarization, limiting innate control of C. neo, but ultimately has no effect on cryptococcal control by adaptive immunity.