Trehalase as novel antifungal target in Aspergillus fumigatus

Abstract

Aspergillus fumigatus is a filamentous ascomycetous fungus that leads a saprotrophic lifestyle. Due to its airborne spores, this fungus can spread easily and colonize different environments such as plants, soil, and animals. In humans, these spores can end up in the respiratory track, where it is usually cleared by the host’s immune system. However, this is not always the case for patients suffering from either immunological problems, for example due to chemotherapy, or genetic make up and/or pulmonary diseases like cystic fibrosis. In these patients, Aspergillus fumigatus thrives and causes health complications in the lungs, such as the deadly manifestation of aspergillosis: invasive pulmonary aspergillosis. As the available antifungals for aspergillosis treatment and management are limited, and antifungal resistant Aspergilli are emerging, there is an urgent need for novel antifungals. Previously, trehalase has been coined as a novel antifungal target due to its role in germination of conidiospores. Hence, this study focusses on multiple aspects related to trehalase inhibition to advance potential drug development. One of the goals was to express TreB in E. coli BL21 and purify it for selection of a novel peptide inhibitor via the RaPID Bioaffinity assay. In addition, chemical inhibition of TreB was performed using validamycin A in a EUCAST-like protocol, and the germination kinetics were followed and compared to other (clinically relevant) Aspergilli. The last goal of this research was to create a ΔtreB mutant through a protoplast mediated CRISPR/Cas9 protocol to simulate complete inhibition of the enzyme. Using the E. coli BL21 system, trehalase initially seemed to be produced sufficiently. Unfortunately, protein extraction and purification remained unfruitful. This could be due to the low expression or stability of the TreB protein since it lacked 4 amino acids, as was revealed after DNA sequencing. Furthermore, chemical inhibition of conidiospores illustrate the versatility of the spores, as the lack of intracellular trehalose digestion does not seem to limit the germination process when other carbon sources than trehalose are available. Interestingly, Aspergillus nidulans and Aspergillus niger were affected by validamycin when grown in AMM containing trehalose, but Aspergillus fumigatus was not. Although not proven in this study, this might be due to either a different three-dimensional configuration of the trehalose-specific binding site in A. fumigatus trehalase, or an alternative system that does rescue the germination of A. fumigatus, regardless of validamycin A induced TreB inhibition. This does highlight the importance of creating a ΔtreB mutant strain, as these hypothesises could then be tested. Finally, a knock-out strain of the treB gene has not been created successfully. However, it was demonstrated that the spores exposed to the CRISPR/Cas9 procedure did inherit change in micromorphology, as hyphae were found to be thinner in AMM containing trehalose and germlings were less developed in AMM containing proline. These results highlight the necessary caution upon these kind of experiments as the used CEA10Δku80 strain has an overall higher mutation rate, thus increasing the odds of observing finding unexpected changes in phenotype.