RNAi-Based Functional Genomics Identifies New Virulence Determinants in Mucormycosis

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.
Author Summary Mucormycosis is an infectious disease caused by organisms of the order Mucorales. It is a lethal infection that is raising the alarm in the medical and scientific community due to its high mortality rates, unusual antifungal drug resistance and its emerging character. Among the reasons explaining the nescience about this disease is the lack of knowledge on the biology of the organisms that cause mucormycosis, which is encouraged by the reluctance of these species to genetic studies. In this work, we have developed an RNAi-based functional genomic platform to study virulence in Mucorales. It is a powerful tool available for the scientific community that will contribute to solve the reluctance of Mucorales to genetic studies and will help to understand the genetic basis of virulence in these organisms. Secondly, and as a proof of concept, we have used this genetic tool to identify two new virulence determinants in Mucor circinelloides. Lack of function of these determinants delays germination and growth of spores, conceding time to macrophages for the inactivation of the pathogen. The two genes identified, mcplD and mcmyo5, represent promising targets for future development of new antifungal therapies against mucormycosis.