Generation of A Mucor circinelloides Reporter Strain-A Promising New Tool to Study Antifungal Drug Efficacy and Mucormycosis.

Ingo Bauer,Maria Isabel Navarro-Mendoza,Francisco E Nicolas,Ulrike Binder,Verena Naschberger,Cornelia Lass-Flörl,Victoriano Garre,Johannes D Pallua

doi:10.3390/genes9120613

Ingo Bauer, Maria Isabel Navarro-Mendoza + Show 6 more

Open Access

https://doi.org/10.3390/genes9120613

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Journal: Genes	Publication Date: Dec 7, 2018
Citations: 17	License type: CC BY 4.0

Affiliation: Innsbruck Medical University, Universität Innsbruck

Abstract

Invasive fungal infections caused by Mucorales (mucormycosis) have increased worldwide. These life-threatening infections affect mainly, but not exclusively, immunocompromised patients, and are characterized by rapid progression, severe tissue damage and an unacceptably high rate of mortality. Still, little is known about this disease and its successful therapy. New tools to understand mucormycosis and a screening method for novel antimycotics are required. Bioluminescent imaging is a powerful tool for in vitro and in vivo approaches. Hence, the objective of this work was to generate and functionally analyze bioluminescent reporter strains of Mucor circinelloides, one mucormycosis-causing pathogen. Reporter strains were constructed by targeted integration of the firefly luciferase gene under control of the M. circinelloides promoter Pzrt1. The luciferase gene was sufficiently expressed, and light emission was detected under several conditions. Phenotypic characteristics, virulence potential and antifungal susceptibility were indifferent to the wild-type strains. Light intensity was dependent on growth conditions and biomass, being suitable to determine antifungal efficacy in vitro. This work describes for the first time the generation of reporter strains in a basal fungus that will allow real-time, non-invasive infection monitoring in insect and murine models, and the testing of antifungal efficacy by means other than survival.

Highlights

IntroductionA member of the Mucoromycota, is ubiquitously found in the environment
Mucor circinelloides, a member of the Mucoromycota, is ubiquitously found in the environment
For the generation of luciferase-producing M. circinelloides strains, we cloned the firefly (P. pyralis) luciferase gene, optimized for use in mammalian cells, under the control of a strong M. circinelloides zrt1 promoter into plasmid pMAT1477 that contained the leuA gene, which was used as a selective marker in transformations

Summary

Introduction

A member of the Mucoromycota, is ubiquitously found in the environment. It is thermotolerant, able to grow on a wide range of organic substrates and sporulates fast and abundantly [1,2]. Able to grow on a wide range of organic substrates and sporulates fast and abundantly [1,2] It can cause mucormycosis—a severe animal and human disease. The rapid progression, linked with shortcomings in diagnosis and therapy, results in high mortality rates which are estimated to range between 40–>90%, depending on the site of infection, the condition of the host and the therapeutic interventions [3,5,6,7].

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