Immunoprotection against Cryptococcosis Offered by Znf2 Depends on Capsule and the Hyphal Morphology

Systemic cryptococcosis is fatal without treatment. Globally, this disease kills 180,000 of the 225,000 infected people each year, even with the use of antifungal therapies. Currently, there is no vaccine to prevent cryptococcosis. Previously, we discovered that Znf2, a morphogenesis regulator that directs Cryptococcus yeast-to-hyphal transition, profoundly affects cryptococcal interaction with the host-overexpression of ZNF2 drives filamentous growth, attenuates cryptococcal virulence, and elicits protective host immune responses. Importantly, immunization with cryptococcal cells overexpressing ZNF2, either in live or heat-inactivated form, offers significant protection to the host from a subsequent challenge by the otherwise lethal wild-type H99 strain. We hypothesize that cellular components enriched in ZNF2^oe cells are immunoprotective. Here, we discovered that serum from protected animals vaccinated with inactivated ZNF2^oe cells recognizes cryptococcal antigens that reside within the capsule. Consistently, capsule is required for immunoprotection offered by ZNF2^oe cells. Interestingly, the serum from protective animals recognizes antigens in both wild-type yeast cells and ZNF2^oe cells, with higher abundance in the latter. Consequently, even the heat-inactivated wild-type cells become immunoprotective with an increased vaccination dose. We also found that disruption of a chromatin remodeling factor Brf1, which is important for initiation of filamentation by Znf2, reduces the antigen level in ZNF2^oe cells. Deletion of BRF1 drastically reduces the protective effect of ZNF2^oe cells in both live and heat-killed forms even though the ZNF2^oebrf1Δ strain itself is avirulent. Collectively, our findings underscore the importance of identifying the subset of cryptococcal surface factors that are beneficial in host protection. IMPORTANCE Cryptococcosis claims close to 200,000 lives annually. There is no vaccine clinically available for this fungal disease. Many avirulent mutant strains do not provide protection against cryptococcosis. We previously discovered that hyphal ZNF2^oe strains elicit protective host immune responses both in the live and heat-inactivated forms. Here we seek to understand the mechanism underlying the host protection provided by ZNF2^oe cells. We discovered increased accumulation of antigens located within the caspusle of ZNF2^oe cells and consequently the requirement of the capsule for ZNF2^oe strain-elicited host protection. Furthermore, genetically blocking the ability of ZNF2^oe cells to grow in the hyphal form significantly reduces antigen accumulation and impairs the ability of ZNF2^oe strain to provide host protection. Our findings highlight the importance of identifying the Znf2-regulated capsular surface factors that are fundamental in host protection.