Understanding stability, regulation, and recognition within the cryptococcus neoformans capsule

Cryptococcus neoformans tolerates the host environment using virulence factors including a polysaccharide capsule. In this dissertation, we investigated mechanisms behind capsular stability, regulation, and recognition through three aims. To investigate stability, we generated a deletion mutant lacking the overproducer of inositol (OPI3) gene, which encodes an enzyme for phosphatidylcholine synthesis. To understand Opi3's role in membrane stability, capsule formation, and virulence, we hypothesized that Opi3 would play a critical role in membrane and capsular stability. We show that Opi3 is localized to the endoplasmic reticulum and is involved in maintaining membrane integrity, as shown by changes in lipid profiles. We are the first to show that capsule attachment is impaired in an opi3∆ deletion mutant, suggesting Opi3 indirectly influences capsule formation through membrane and cell wall stability. However, Opi3 was not required in a mouse model of cryptococcosis, likely due to host choline availability. To examine regulation, we investigated the influence of nicotinamide adenine dinucleotide (NAD⁺) synthesis on capsule formation and virulence by generating deletion strains for Bna2, Bna5, Bna3, and Bna1. We hypothesized that NAD⁺-dependent mitochondrial activity would impact processes related to capsule formation, such as iron homeostasis and thermotolerance. bna1∆, bna2∆, and bna5∆ mutants had impaired mitochondrial activity and were capsule deficient. These mutants also failed to regulate oxidative stress response, thermotolerance, and iron homeostasis. Furthermore, bna5∆ was unable to cause disease in a mouse model. We are the first to show that the Kynurenine pathway is essential for capsule formation and virulence. In the last part of our study, we determined which C-type lectin receptors (CLRs) are involved in capsule-mediated recognition. We hypothesized that host CLRs can interact with the capsule despite its non-immunogenic properties. Using a library of recombinant C-type lectin receptors (lectibodies), we screened capsule-induced cryptococcus cells, identifying four lectibodies—MBL2, FRERM1, CLEC11A, and CLEC12A—that localized to capsular edges. Two lectibodies induced macrophage phagocytosis, and one, FREM1, increased mouse survival in a cryptococcosis model. Together, this work reveals novel aspects of capsular stability, regulation, and recognition in C. neoformans and highlights lectibodies as a potential therapeutic strategy against cryptococcosis.