Bioluminescent Mucor circinelloides – a promising new tool to study mucormycosis and antifungal drug efficacy

Ulrike Binder 1 Maria Isabella Navarro-Mendoza 2 Francisco Nicolas 2 Verena Naschberger 1 Cornelia Lass-Flörl 1 Victoriano Garre 2
1Division of Hygiene and Medical Microbiology, Medical University Innsbruck, Innsbruck, Austria
2Fungal Genomics and Molecular Biotechnology, University of Murcia, Murcia, Spain

Invasive infections caused by members of the Mucorales (mucormycosis) have increased in the last years, making it the third most common invasive fungal infection after aspergillosis and candidiasis. Despite this increasing clinical relevance, little is known about the establishment of disease, its progression and successful therapy. New tools to study this disease in more detail are needed, therefore the objective of this work was to construct a luciferase expressing Mucor circinelloides strain, as one representative of mucormycosis causing pathogens. Here, we describe the construction and functional analysis of the strains, which will further be used as a reporter system for in vivo and in vitro models of Mucorales infections.

A leucine auxotroph M. circinelloides strain, R7B, was used as recipient strain to allow selection of transformants on selective medium. Firefly luciferase gene without the peroxisomal target sequence was cloned in the pMAT1477 vector. Expression of firefly luciferase under the control of a constitutive promoter was successful in M. circinelloides at several conditions. Light emission was detectable by imaging and with a luminometer. Data so far indicate the strain being suitable for further in vivo and in vitro studies. Phenotype, virulence potential and antifungal susceptibility are indifferent to the wild type strains

The construction of this first bioluminescent Mucor strain will allow for the visualization of temporal and spatial progression of infection by a non-invasive method in insect and murine models, and the testing of antifungal efficacy by other means than survival only. This will give valuable new insights in the pathogenesis of Mucorales infections.