title: 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.
authors.affiliation: F2G Ltd., Lankro Way, Eccles, Manchester, M30 0LX, United Kingdom.
authors.forename: Katharine S
authors.initials: KS
authors.lastname: Dobb
authors.affiliation: F2G Ltd., Lankro Way, Eccles, Manchester, M30 0LX, United Kingdom.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Kaye
authors.affiliation: F2G Ltd., Lankro Way, Eccles, Manchester, M30 0LX, United Kingdom.
authors.forename: Nicola
authors.initials: N
authors.lastname: Beckmann
authors.affiliation: F2G Ltd., Lankro Way, Eccles, Manchester, M30 0LX, United Kingdom.
authors.forename: John L
authors.initials: JL
authors.lastname: Thain
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, United Kingdom.
authors.forename: Lubomira
authors.initials: L
authors.lastname: Stateva
authors.affiliation: F2G Ltd., Lankro Way, Eccles, Manchester, M30 0LX, United Kingdom.
authors.forename: Mike
authors.initials: M
authors.lastname: Birch
authors.affiliation: F2G Ltd., Lankro Way, Eccles, Manchester, M30 0LX, United Kingdom.
authors.forename: Jason D
authors.initials: JD
authors.lastname: Oliver
mesh_terms: D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002176:Candida albicans / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D002352:Carrier Proteins; D019295:Computational Biology; D004789:Enzyme Activation; D015870:Gene Expression; D008969:Molecular Sequence Data; D010641:Phenotype; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D017384:Sequence Deletion; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; C082662:phosphopantetheinyl transferase; D017855:Transferases (Other Substituted Phosphate Groups)