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 ZNF2oe cells are immunoprotective. Here, we discovered that serum from protected animals vaccinated with inactivated ZNF2oe cells recognizes cryptococcal antigens that reside within the capsule. Consistently, capsule is required for immunoprotection offered by ZNF2oe cells. Interestingly, the serum from protective animals recognizes antigens in both wild-type yeast cells and ZNF2oe 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 ZNF2oe cells. Deletion of BRF1 drastically reduces the protective effect of ZNF2oe cells in both live and heat-killed forms even though the ZNF2oebrf1Δ 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 ZNF2oe 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 ZNF2oe cells. We discovered increased accumulation of antigens located within the caspusle of ZNF2oe cells and consequently the requirement of the capsule for ZNF2oe strain-elicited host protection. Furthermore, genetically blocking the ability of ZNF2oe cells to grow in the hyphal form significantly reduces antigen accumulation and impairs the ability of ZNF2oe strain to provide host protection. Our findings highlight the importance of identifying the Znf2-regulated capsular surface factors that are fundamental in host protection.

Legionella pneumophila LegC7 effector protein drives aberrant endoplasmic reticulum:endosome contacts in yeast.

Legionella pneumophila is a facultative intracellular bacterial pathogen, causing the severe form of pneumonia known as Legionnaires' disease. Legionella actively alters host organelle trafficking through the activities of "effector" proteins secreted via a type-IVB secretion system, in order to construct the bacteria-laden Legionella-containing vacuole (LCV) and prevent lysosomal degradation. The LCV is created with membrane derived from host endoplasmic reticulum (ER), secretory vesicles and phagosomes, although the precise molecular mechanisms that drive its synthesis remain poorly understood. In an effort to characterize the in vivo activity of the LegC7/YlfA SNARE-like effector protein from Legionella in the context of eukaryotic membrane trafficking in yeast, we find that LegC7 interacts with the Emp46p/Emp47p ER-to-Golgi glycoprotein cargo adapter complex, alters ER morphology and induces aberrant ER:endosome interactions, as measured by visualization of ER cargo degradation, reconstitution of split-GFP proteins and enhanced oxidation of the ER lumen. LegC7-dependent toxicity, disruption of ER morphology and ER:endosome fusion events were dependent upon endosomal VPS class C tethering complexes and the endosomal t-SNARE, Pep12p. This work establishes a model in which LegC7 functions to recruit host ER material to the bacterial phagosome during infection by driving ER:endosome contacts, potentially through interaction with host membrane tethering complexes and/or cargo adapters.