Purpose: 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 the construction luciferase expressing Mucor circinelloides strains, 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.
Methods: 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 under the control of a constitutive promoter. Linear plasmid was used to transfect M. circinelloides protoplasts. The targeted integration of the whole construct in the carRP gene resulted in easy identification of transformants, appearing as white colonies.
Homokaryons were obtained by sequential plating on selective media and checked for light emission under various conditions in in vitro assays.
Results: Expression of firefly luciferase was successful in M. circinelloides at several conditions and light emission was detectable by imaging and with a luminometer. Phenotype, virulence potential in G. mellonella and antifungal susceptibility are indifferent to the wild-type strains. Data so far indicate the strains being suitable for further in vivo and in vitro studies.
Conclusion: 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.
Full conference title:
- AAA 8th (2018)