Aspergillus fumigatus is a human pathogen and the principal etiologic agent of invasive and chronic aspergillosis leading to several hundreds of thousands of deaths every year. Very few antifungals are available to treat infections caused by A. fumigatus, and resistance is developing to those we have. Our understanding of the molecular mechanisms that drive pathogenicity and drug resistance have been hampered by the lack of large mutant collections, which limits our ability to perform functional genomics analysis. Here we present a high-throughput gene knockout method that combines a highly reproducible fusion PCR method to enable generation of gene replacement cassettes with a multiwell format transformation procedure. This process can be used to generate 96 null mutants within 5 days by a single person at a cost of less than £18 ($24) per mutant and is being employed in our laboratory to generate a barcoded genome-wide knockout library in A. fumigatus. C 2019 The Authors.A deeper insight of the pathogen and host factors at the molecular level is vastly important for us to be able to overcome the burden of such diseases; however, our understanding is actually very limited. Genome-wide knockout libraries have been used to great effect to establish an in-depth understanding of microbial functional genomics.In this protocol, we describe a high-throughput generic process for the generation of gene replacement cassettes by adapting the aforementioned PCR approach to incorporate common-fusion sequences that improve reproducibility of amplification. In addition, our cassette generation method allows for the inclusion of randomly generated barcodes that can support the use of competitive fitness profiling methods. We also describe how replacement cassettes can be used to generate null mutants in A. fumigatus in a multiwell format in a rapid and inexpensive way. The fusion PCR and transformation procedures are integrated here into a single protocol that allows one person to generate 96 knockout mutants in <5 days. This protocol was established to facilitate the generation of an A. fumigatus genome-wide knockout library, which is an ongoing project in our laboratory. With a few modifications, this method is also applicable to other filamentous fungal species, such as Aspergillus niger or Aspergillus nidulans.

Aspergillus fumigatus is an opportunistic human pathogenic mold. DNA extraction from this fungus is usually performed by mechanical perturbation of cells, as it possesses a rigid and complex cell wall. While this is not problematic for single isolates, it can be time consuming for large numbers of strains if using traditional DNA extraction procedures. Therefore, in this article we describe a fast and efficient thermal‐shock method to release DNA from spores of A. fumigatus and other filamentous fungi without the need for complex extraction methods. This is especially important for high‐throughput PCR analyses of mutants in 96‐ or 384‐well formats in a very short period of time without any concern about sample cross‐contamination. This method is currently being used to validate the protein‐coding gene and non‐coding RNA knockout libraries in A. fumigatus generated in our laboratory, and could be used in the future for diagnostics purposes. © 2019 The Authors.

Olorofim is a new antifungal in clinical development which has a novel mechanism of action against dihydroorotate dehydrogenase (DHODH). DHODH form a ubiquitous family of enzymes in the de novo pyrimidine biosynthetic pathway and are split into class 1A, class 1B and class 2. Olorofim specifically targets the fungal class 2 DHODH present in a range of pathogenic moulds. The nature and number of DHODH present in many fungal species have not been addressed for large clades of this kingdom. Mucorales species do not respond to olorofim; previous work suggests they have only class 1A DHODH and so lack the class 2 target that olorofim inhibits. The dematiaceous moulds have mixed susceptibility to olorofim, yet previous analyses imply that they have class 2 DHODH. As this is at odds with their intermediate susceptibility to olorofim, we hypothesised that these pathogens may maintain a second class of DHODH, facilitating pyrimidine biosynthesis in the presence of olorofim. The aim of this study was to investigate the DHODH repertoire of clinically relevant species of Mucorales and dematiaceous moulds to further characterise these pathogens and understand variations in olorofim susceptibility. Using bioinformatic analysis, S. cerevisiae complementation and biochemical assays of recombinant protein, we provide the first evidence that two representative members of the Mucorales have only class 1A DHODH, substantiating a lack of olorofim susceptibility. In contrast, bioinformatic analyses initially suggested that seven dematiaceous species appeared to harbour both class 1A-like and class 2-like DHODH genes. However, further experimental investigation of the putative class 1A-like genes through yeast complementation and biochemical assays characterised them as dihydrouracil oxidases rather than DHODHs. These data demonstrate variation in dematiaceous mould olorofim susceptibility is not due to a secondary DHODH and builds on the growing picture of fungal dihydrouracil oxidases as an example of horizontal gene transfer.