Characterisation of the Candida albicans Phosphopantetheinyl Transferase Ppt2 as a Potential Antifungal Drug Target

Abstract

Antifungal drugs acting via new mechanisms of action are urgently needed to combat the increasing numbers of severe fungal infections caused by pathogens such as Candida albicans. The phosphopantetheinyl transferase of Aspergillus fumigatus, encoded by the essential gene pptB, has previously been identified as a potential antifungal target. This study investigated the function of its orthologue in C. albicans, PPT2/C1_09480W by placing one allele under the control of the regulatable MET3 promoter, and deleting the remaining allele. The phenotypes of this conditional null mutant showed that, as in A. fumigatus, the gene PPT2 is essential for growth in C. albicans, thus fulfilling one aspect of an efficient antifungal target. The catalytic activity of Ppt2 as a phosphopantetheinyl transferase and the acyl carrier protein Acp1 as a substrate were demonstrated in a fluorescence transfer assay, using recombinant Ppt2 and Acp1 produced and purified from E.coli. A fluorescence polarisation assay amenable to high-throughput screening was also developed. Therefore we have identified Ppt2 as a broad-spectrum novel antifungal target and developed tools to identify inhibitors as potentially new antifungal compounds.

Phosphopantetheinyl transferase (Ppt)-mediated biosynthesis of lysine, but not siderophores or DHN melanin, is required for virulence of Zymoseptoria tritici on wheat

“…The auxotrophy for lysine of Ppt mutants has led to its consideration as a potential antifungal target. For example, in the human pathogens Candida albicans and Aspergillus fumigatus , high throughput screens have been developed with the aim of identifying inhibitors of this enzyme 26 , 27 .…”

N‐acetylglucosamine transporter, Ngt1, undergoes sugar‐responsive endosomal trafficking inCandida albicans

“…To confirm the essential nature of RSP5 , Candida cells were engineered by replacing RSP5 promoter with Candida MET3 promoter (SHR5: rsp5∆/MET3p‐RSP5 ) (Care et al., 1999; Gerami‐Nejad et al., 2004), a conditional promoter that provides ON and OFF conditions for gene expression when cells were grown in SD medium with and without cysteine +methionine at 3.5 mM concentration. MET3 conditional promoter have been successfully used in C. albicans to study the role of several essential genes (Dobb et al., 2015; Nishikawa et al., 2002) as well as some important genes involved in cell cycle and filamentation (Dünkler and Wendland, 2007). To prepare rsp5 conditional mutant, “ URA3‐MET3 promoter” cassette was amplified as described previously (Gerami‐Nejad et al., 2004 and shown in Figure 5a) using long primers and transformed into heterozygous (SHR4: ∆ rsp5 / RSP5 ) deletion mutant.…”

Emerging New Targets for the Treatment of Resistant Fungal Infections

“…fumigatus and C. albicans are essential for their viability. , Thus, PPTases have been proposed as potential antifungal targets. Currently, no inhibitor of fungal PPTases has been reported.…”

“…Currently, no inhibitor of fungal PPTases has been reported. Recently, a fluorescence polarization assay amenable to HTS was developed, 142 which will accelerate the process of inhibitor discovery and target validation.…”