RNAi-Based Functional Genomics Identifies New Virulence Determinants in Mucormycosis

Abstract

Mucorales are an emerging group of human pathogens that are responsible for the lethal disease mucormycosis. Unfortunately, functional studies on the genetic factors behind the virulence of these organisms are hampered by their limited genetic tractability, since they are reluctant to classical genetic tools like transposable elements or gene mapping. Here, we describe an RNAi-based functional genomic platform that allows the identification of new virulence factors through a forward genetic approach firstly described in Mucorales. This platform contains a whole-genome collection of Mucor circinelloides silenced transformants that presented a broad assortment of phenotypes related to the main physiological processes in fungi, including virulence, hyphae morphology, mycelial and yeast growth, carotenogenesis and asexual sporulation. Selection of transformants with reduced virulence allowed the identification of mcplD, which encodes a Phospholipase D, and mcmyo5, encoding a probably essential cargo transporter of the Myosin V family, as required for a fully virulent phenotype of M. circinelloides. Knock-out mutants for those genes showed reduced virulence in both Galleria mellonella and Mus musculus models, probably due to a delayed germination and polarized growth within macrophages. This study provides a robust approach to study virulence in Mucorales and as a proof of concept identified new virulence determinants in M. circinelloides that could represent promising targets for future antifungal therapies.

A non-canonical RNA degradation pathway suppresses RNAi-dependent epimutations in the human fungal pathogen Mucor circinelloides

“…Detection of sRNAs in Northern blot experiments was carried out as described [5, 35]. sRNAs (25–35 μg) were separated by electrophoresis on 15% TBE-Urea gels (Invitrogen), electrotransferred to Hybond N1 filters at 400 mA for 1 h in 0.5X TBE, and cross-linked by ultraviolet irradiation (2 pulses at 1.2x10 5 μJ per cm 2 ).…”

Mucor circinelloides: Growth, Maintenance, and Genetic Manipulation

“…This strategy has allowed the identification of new virulence factors in the life-threatening fungal infection known as mucormycosis (Trieu et al, 2017). …”

“…B) Diagram of the strategy used to build a genome-wide RNAi library: 1) Purified genomic DNA is digested with restriction enzymes and repaired to make compatible ends and avoid self-ligation, 2) Digested and repaired genomic DNA fragments are cloned into the digested vector (adapted from Trieu et al, (2017) RNAi-based functional genomics identifies new virulence determinants in mucormycosis. PLOS pathogens ).…”

The mucormycete–host interface

“…In addition, targeted gene attenuation in Rhizopus can reliably be achieved using RNAi techniques [47,51,52]. Finally, the community will benefit from a recently published RNAi-based knock out library of M. circinelloides enabling screens for genes involved in germination and virulence [54]. …”