# A conserved enzymatic toolkit targeting host cell metabolism is associated with Cryptococcus neoformans intracellular survival in protozoal and mammalian phagocytic cells

## TL;DR

This study finds that Cryptococcus neoformans uses a shared set of enzymes to survive inside different types of phagocytic cells, including amoebae and human cells.

## Contribution

The discovery of a conserved enzymatic toolkit in C. neoformans that targets host cell metabolism across diverse phagocytic hosts.

## Key findings

- Cryptococcal phagosomes were successfully isolated and characterized from murine and human phagocytes.
- A shared set of fungal proteins was identified across amoeba, mouse, and human phagocytes, indicating a conserved response to ingestion.
- The fungal toolkit includes metabolic enzymes that disrupt host cell metabolism to enhance intracellular survival.

## Abstract

The outcome of the interaction between Cryptococcus neoformans and infected hosts can be determined by whether the fungal cell survives ingestion by phagocytic cells. This applies to both unicellular and multicellular hosts such as amoeba and animals, respectively. Ingestion by phagocytic cells results in the formation of the cryptococcal phagosome but this structure has proved difficult to isolate. In this study, we report the successful isolation of cryptococcal phagosomes from murine and human phagocytes, followed by their characterization using proteomic and transcriptional analysis. Comparison of cryptococcal proteins from Acanthamoeba castellanii, Mus musculus, and Homo sapiens phagocytes revealed the existence of a shared set suggesting a conserved fungal response to ingestion by phagocytic cells. Given that the cryptococcal intracellular pathogenic strategy is ancient, dating to at least to the cretaceous epoch, these results are consistent with the notion that the fungal response to ingestion reflects the result of selection pressures by environmental ameboid predators over eons of evolutionary time. We propose the existence of a conserved cryptococcal toolkit for intracellular survival that includes metabolic enzymes, which disrupt host cell metabolic function, thus providing a common strategy for cryptococcal survival after ingestion by phylogenetically distant phagocytic hosts.

A fundamental problem in the field of microbial pathogenesis is the mechanism by which microbes can cause disease in diverse hosts. In contrast to specialized pathogenic microbes that have an association with single or few hosts, generalist-type pathogens must survive in diverse hosts and thus must have non-specialized strategies to subvert cellular defenses, such as phagocytic cells. In this study we analyzed the interaction of the human pathogenic fungus Cryptococcus neoformans with amoeba and mouse and human macrophages, each phagocytic cell, comparing fungal proteins released into these cells after ingestion as well as transcriptional changes. C. neoformans is a generalist-type pathogenic fungus capable of causing diseases in mammals, fish, reptiles and insects. Despite the phylogenetic differences between these three types of phagocytic cells, their interaction with C. neoformans was remarkably similar. The results are consistent with the hypothesis that for C. neoformans the capacity for mammalian virulence emerged accidentally from selection pressures driven by phagocytic predators in soils such as amoeba. Furthermore, the results imply the existence of a conserved cryptococcal toolkit in the form of released proteins that enhance intracellular fungal cell survival by altering host phagocytic cell metabolism.