the role of replicative aging in the pathogenesis of Cryptococcus neoformans

Abstract

Cryptococcus neoformans (Cn) is a fungal pathogen that predominantly causes chronic meningoencephalitis (CME) in immunocompromised individuals despite antifungal therapy. Hence, Cn is able to persist and replicate in the host. Replicating cells undergo divisions that result in a distinct phenotype while producing a finite number of daughter cells (replicative life span). This work examines replicative aging in a pathogen, and establishes that old Cn cells constitute the persistent population in chronic cryptococcosis.;Cn strains demonstrated a reproducible and wide range of RLSs. Compared to young cells, older cells demonstrated increased resistance to hydrogen peroxide (H202), macrophage-mediated killing, and antifungal agents. Cell size and RLS served as biomarkers to establish that older cells accumulate in rat and human CME, and mathematical modeling showed that this accumulation emerges from in vivo selection pressures. Further investigation of RLS revealed that it was either shortened or extended under relevant calorie restriction (CR) conditions. Extension was not seen when S1R2 was deleted in two serotypes. Both mutants were hypovirulent in Galleria mellonella, and in intratracheal, but not intravenous murine infection, likely due to CR differences. RNA sequencing of the mutant showed that metabolism- and transport-related genes were downregulated. RLS was manipulated such that when isonicotinamide (INAM) extended RLS, Cn was hypovirulent, and when it shortened RLS, Cn was hypervirulent. Thus, a shortened RLS could help the pathogen attain age-related resilience earlier and contribute to its persistence.Replicative aging was also shown to be relevant to Candida glabrata (Cg). Cg exhibited a range of RLSs similar to those seen in Cn , but replicated twice as fast. Older cells showed an increased phenotypic switching rate and resistance to H202, and neutrophil-mediated killing. Older cells killed Galleria significantly faster than young cells. Thus, similar to Cn, older Cg cells could constitute the persistent population.;In conclusion, we demonstrate that replicative aging is an unanticipated virulence trait that emerges during chronic cryptococcosis. Aging could confer a benefit to Cn and facilitate its persistence in the host. This emergent trait could be highly relevant to eukaryotic pathogenesis, and help introduce novel drug targets against chronic infections.