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

• Published in: PloS one 2015-11, Vol.10 (11), p.e0143770-e0143770
• Main Authors: Dobb, Katharine S, Kaye, Sarah J, Beckmann, Nicola, Thain, John L, Stateva, Lubomira, Birch, Mike, Oliver, Jason D
• Format: Article
• Language: English
• Subjects: Acyl carrier protein; Alleles; Amino Acid Sequence; Antifungal agents; Antifungal Agents - pharmacology; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biosynthesis; Candida; Candida albicans; Candida albicans - drug effects; Candida albicans - enzymology; Candida albicans - genetics; Care and treatment; Carrier Proteins; Catalysis; Catalytic activity; Computational Biology; Deoxyribonucleic acid; DNA; Drugs; E coli; Enzyme Activation; Enzymes; Fatty acids; Fluorescence; Fungal infections; Fungicides; Gene Expression; Genomes; Health aspects; High-throughput screening; Molecular Sequence Data; Mycoses; Phenotype; Promoter Regions, Genetic; Proteins; Saccharomyces cerevisiae; Sequence Alignment; Sequence Deletion; Substrate Specificity; Substrates; Transferases (Other Substituted Phosphate Groups) - antagonists & inhibitors; Transferases (Other Substituted Phosphate Groups) - chemistry; Transferases (Other Substituted Phosphate Groups) - genetics; Transferases (Other Substituted Phosphate Groups) - metabolism; Yeast
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0143770

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.