The increased prevalence of drug-resistant human pathogenic fungal diseases poses a major threat to global human health. Thus, new drugs are urgently required to combat these infections. Here, we demonstrate that acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway, is a promising new target for antifungal drug discovery. First, we show that several AHAS inhibitors developed as commercial herbicides are powerful accumulative inhibitors of Candida albicans AHAS (Ki values as low as 800 pM) and have determined high-resolution crystal structures of this enzyme in complex with several of these herbicides. In addition, we have demonstrated that chlorimuron ethyl (CE), a member of the sulfonylurea herbicide family, has potent antifungal activity against five different Candida species and Cryptococcus neoformans (with minimum inhibitory concentration, 50% values as low as 7 nM). Furthermore, in these assays, we have shown CE and itraconazole (a P450 inhibitor) can act synergistically to further improve potency. Finally, we show in Candida albicans-infected mice that CE is highly effective in clearing pathogenic fungal burden in the lungs, liver, and spleen, thus reducing overall mortality rates. Therefore, in view of their low toxicity to human cells, AHAS inhibitors represent a new class of antifungal drug candidates.

Low rates of homologous integration have hindered molecular genetic studies in Cryptococcus neoformans over the past 20 years, and new tools that facilitate genome manipulation in this important pathogen are greatly needed. To this end, we have investigated the use of a Class 2 CRISPR system in C. neoformans (formerly C. neoformans var. grubii). We first expressed a derivative of the Streptococcus pyogenes Cas9 nuclease in C. neoformans, and showed that it has no effect on growth, production of virulence factors in vitro, or virulence in a murine inhalation model. In proof of principle experiments, we tested the CAS9 construct in combination with multiple self-cleaving guide RNAs targeting the well-characterized phosphoribosylaminoamidazole carboxylase-encoding ADE2 gene. Utilizing combinations of transient and stable expression of our constructs, we revealed that functionality of our CRISPR constructs in C. neoformans is dependent upon the CAS9 construct being stably integrated into the genome, whilst transient expression of the guide RNA is sufficient to enhance rates of homologous recombination in the CAS9 genetic background. Given that the presence of the CRISPR nuclease does not influence virulence in a murine inhalation model, we have successfully demonstrated that this system is compatible with studies of C. neoformans pathogenesis and represents a powerful tool that can be exploited by researchers in the field.

Purines play an integral role in cellular processes such as energy metabolism, cell signaling and encoding the genetic makeup of all living organisms—ensuring that the purine metabolic pathway is maintained across all domains of life. To gain a deeper understanding of purine biosynthesis via the de novo biosynthetic pathway, the genes encoding purine metabolic enzymes from 35 archaean, 69 bacterial and 99 eukaryotic species were investigated. While the classic elements of the canonical purine metabolic pathway were utilized in all domains, a subset of familiar biochemical roles was found to be performed by unrelated proteins in some members of the Archaea and Bacteria. In the Bacteria, a major differentiating feature of de novo purine biosynthesis is the increasing prevalence of gene fusions, where two or more purine biosynthesis enzymes that perform consecutive biochemical functions in the pathway are encoded by a single gene. All species in the Eukaryota exhibited the most common fusions seen in the Bacteria, in addition to new gene fusions to potentially increase metabolic flux. This complexity is taken further in humans, where a reversible biomolecular assembly of enzymes known as the purinosome has been identified, allowing short‐term regulation in response to metabolic cues while expanding on the benefits that can come from gene fusion. By surveying purine metabolism across all domains of life, we have identified important features of the purine biosynthetic pathway that can potentially be exploited as prospective drug targets.

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