Cryptococcal Heat Shock Protein 70 Homolog Ssa1 Contributes to Pulmonary Expansion of Cryptococcus neoformans during the Afferent Phase of the Immune Response by Promoting Macrophage M2 Polarization

Abstract

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.

Systemic Approach to Virulence Gene Network Analysis for Gaining New Insight into Cryptococcal Virulence

“…These results indicate that SHO1 expression is not required for the immediate adaptation of C. neoformans to lung environment but it supports optimal growth rate of the pathogen at time points consistent with the innate fungal control (Eastman et al, 2015) and the development of the polarized adaptive immunity (Osterholzer et al, 2009; Eastman et al, 2015), suggesting that SHO1 may have a role in promoting immunomodulatory effects of C. neoformans .…”

IL-10 is required for polarization of macrophages to M2-like phenotype by mycobacterial DnaK (heat shock protein 70)

“…Mice infected with a Cryptococcus neoformans strain lacking Ssa1 -Hsp70 ortholog -presented decreased lung fungal burden compared to controls [24]. It was also proposed that Ssa1 directly promoted the polarization of macrophages to an M2 phenotype in order to evade host protective immune responses [24]. Ssa1-lacking Candida albicans strain showed a reduced virulence in murine model of candidiasis due to a reduced capacity to be endocytosed by endothelial and oral epithelial cells [25].…”

“…Mice infected with a Cryptococcus neoformans strain lacking Ssa1 -Hsp70 ortholog -presented decreased lung fungal burden compared to controls [24]. It was also proposed that Ssa1 directly promoted the polarization of macrophages to an M2 phenotype in order to evade host protective immune responses [24].…”

Anti-Immune Strategies of Pathogenic Fungi

“…Eastman et al (2015) determined the effect of Ssa1 in mice infected with a highly virulent serotype A (serA) strain of C. neoformans (H99—Ssa1 deleted) and, surprisingly, noted that, unlike serotype D, H99-serA does not require Ssa1 for laccase expression. The authors further showed that Ssa1 directly promotes early M2 macrophage polarization to improve fungal growth during the innate phase of the immune response.…”