Autophagy Regulates Fungal Virulence and Sexual Reproduction in Cryptococcus neoformans.

Su-Ting Jiang,Tong-Bao Liu,Lian-Tao Han,An-Ni Chang,Yuan-Hong Li,Jie-Shu Guo

doi:10.3389/fcell.2020.00374

Su-Ting Jiang, Tong-Bao Liu + Show 4 more

Open Access

https://doi.org/10.3389/fcell.2020.00374

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Abstract

Autophagy (macroautophagy) is an evolutionarily conserved degradation pathway involved in bulk degradation of cytoplasmic organelles, old protein, and other macromolecules and nutrient recycling during starvation. Extensive studies on functions of autophagy-related genes have revealed that autophagy plays a role in cell differentiation and pathogenesis of pathogenic fungi. In this study, we identified and characterized 14 core autophagy machinery genes (ATGs) in C. neoformans. To understand the function of autophagy in virulence and fungal development in C. neoformans, we knocked out the 14 ATGs in both α and a mating type strain backgrounds in C. neoformans, respectively, by using biolistic transformation and in vivo homologous recombination. Fungal virulence assay showed that virulence of each atgΔ mutants was attenuated in a murine inhalation systemic-infection model, although virulence factor production was not dramatically impaired in vitro. Fungal mating assays showed that all the 14 ATGs are essential for fungal sexual reproduction as basidiospore production was blocked in bilateral mating between each atgΔ mutants. Fungal nuclei development assay showed that nuclei in the bilateral mating of each atgΔ mutants failed to undergo meiosis after fusion, indicating autophagy is essential for regulating meiosis during mating. Overall, our study showed that autophagy is essential for fungal virulence and sexual reproduction in C. neoformans, which likely represents a conserved novel virulence and sexual reproduction control mechanism that involves the autophagy-mediated proteolysis pathway.

Highlights

Cryptococcus neoformans is ubiquitous encapsulated yeast pathogen causing life-threating meningoencephalitis predominantly in the immunocompromised population (Casadevall and Perfect, 1998)
To test whether the core autophagy genes we identified in C. neoformans are required for autophagosome formation, the 14 atg mutants were first cultured overnight in YPD and washed and switched to starvation conditions (SD-N) medium supplemented with 2 mM PMSF and further incubated for 4 or 5 h
Our results showed that the expression levels of all the 14 candidate genes increased under nitrogen starvation conditions (Figure 1), implying that the 14 candidate genes are autophagy machinery genes (ATGs) genes in C. neoformans

Summary

Introduction

Cryptococcus neoformans is ubiquitous encapsulated yeast pathogen causing life-threating meningoencephalitis predominantly in the immunocompromised population (Casadevall and Perfect, 1998). As a heterothallic basidiomycetous fungus, C. neoformans has two mating types, α and a, and can undergo a dimorphic transition to a filamentous growth by mating and monokaryotic fruiting. During mating in C. neoformans, haploid cells of α and a mating types fused to form dikaryotic hyphae leading to the formation of a basidium, and four chains of haploid basidiospores were eventually produced on top of the basidium following the completion of meiosis inside the basidium (Figure 9; Lin and Heitman, 2006; Zhao et al, 2019b). Cryptococcal cells of a single mating type, e.g., α, can fuse and undergo monokaryotic fruiting to produce filaments and basidiospores under laboratory conditions (Zhao et al, 2019b)

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