Inducible Selectable Marker Genes to Improve Aspergillus fumigatus Genetic Manipulation.

Clara Baldin,Alexander Kühbacher,Petra Merschak,Anna-Maria Dietl,Hubertus Haas,Beate Abt,Fabio Gsaller,Luis Enrique Sastré-Velásquez

doi:10.3390/jof7070506

Clara Baldin, Alexander Kühbacher + Show 6 more

Open Access

https://doi.org/10.3390/jof7070506

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Abstract

The hygromycin B phosphotransferase gene from Escherichia coli and the pyrithiamine resistance gene from Aspergillus oryzae are two dominant selectable marker genes widely used to genetically manipulate several fungal species. Despite the recent development of CRISPR/Cas9 and marker-free systems, in vitro molecular tools to study Aspergillus fumigatus, which is a saprophytic fungus causing life-threatening diseases in immunocompromised hosts, still rely extensively on the use of dominant selectable markers. The limited number of drug selectable markers is already a critical aspect, but the possibility that their introduction into a microorganism could induce enhanced virulence or undesired effects on metabolic behavior constitutes another problem. In this context, here, we demonstrate that the use of ptrA in A. fumigatus leads to the secretion of a compound that allows the recovery of thiamine auxotrophy. In this study, we developed a simple modification of the two commonly used dominant markers in which the development of resistance can be controlled by the xylose-inducible promoter PxylP from Penicillium chrysogenum. This strategy provides an easy solution to avoid undesired side effects, since the marker expression can be readily silenced when not required.

Highlights

Aspergillus fumigatus is a ubiquitous saprophytic fungus that represents the primary cause of life-threatening invasive aspergillosis in immunocompromised hosts
The characterization of A. fumigatus still extensively depends on the use of dominant selectable markers, the hygromycin B phosphotransferase gene and the pyrithiamine resistance gene. hph, which confers resistance to hygromycin, encodes a phosphotransferase from Escherichia coli, and is one of the most commonly utilized dominant selectable markers to genetically modify organisms, from bacteria to mammalian cells [2,3,4,5,6,7,8,9]
When spotted on Aspergillus minimal medium (AMM) in proximity to the wt strain, A52 was not able to grow, but when in proximity of ∆fcyB-pyrithiamine resistance (ptrA), A52 showed a partial recovery of its growth phenotype on the side closer to the ptrA-carrying strain, generating a crescent moon shape (Figure 1c). These results suggested that expression of the ptrA marker cassette in A. fumigatus, in our example in ∆fcyB-ptrA, causes the release of a metabolite that promotes the growth of wt strains in the presence of pyrithiamine and allows the recovery of the thiamine auxotrophic A52 strain

Summary

Introduction

Aspergillus fumigatus is a ubiquitous saprophytic fungus that represents the primary cause of life-threatening invasive aspergillosis in immunocompromised hosts. In the case of A. fumigatus, which is not used in production processes and is studied for its effect on human health as pathogen, drug resistance markers have the advantages of not needing a specific host strain, allowing the stable integration of constructs with high efficiency and generating mutant strains that are easy to select and purify [7,16] Another important aspect to consider is that the constant expression of exogenous constructs inserted in the microbial genome can affect the overall metabolism of the studied species. We were able to prove that A. fumigatus strains genetically modified with the help of the ptrA marker cassette were unexpectedly secreting metabolites in the medium, affecting the growth of other strains plated in their proximity In light of this discovery, we decided to upgrade the commonly used dominant markers hph and ptrA to their respective inducible versions by expressing these resistance genes under the control of the xylanase promoter PxylP derived from Penicillium chrysogenum [21]. PxylP has been shown to be inactive during standard murine infection conditions unless xylose is supplied in the drinking water; i.e., it enables in vivo (fine) tuning of A. fumigatus gene activity [22]

Materials and Methods

Generation of Transformation Constructs

Fungal Transformation

Microtiter-Plate Assays

Results and Discussion

Conclusions