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

Katharine S Dobb,Jason D Oliver,Lubomira Stateva,John L Thain,Sarah J Kaye,Nicola Beckmann,Mike Birch,Alix Therese Coste

doi:10.1371/journal.pone.0143770

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.

Highlights

The human burden of fungal disease is immense with approximately 1.7 billion people affected by fungal infections worldwide per annum
BLASTP analysis of the C. albicans genome was used to identify the orthologue of the phosphopantetheinyl transferase Ppt2/PptB using S. cerevisiae Ppt2 and A. fumigatus PptB as probes, and the homologues of S. cerevisae Acp1 and A. fumigatus AcpA
The mitochondrial acyl carrier proteins ScAcp1 and AcpA have been identified as the targets of ScPpt2p and PptB, respectively

Summary

Introduction

The human burden of fungal disease is immense with approximately 1.7 billion people affected by fungal infections worldwide per annum. Invasive fungal infections are estimated to be the cause of approximately 1.5 million deaths per year making this a significant health problem [1]. Candida spp. are opportunistic pathogens and the most common cause of all the human mycoses. They are commensal organisms that form part of the normal human flora and reside in the mucous membranes of the mouth, vagina or digestive tract in a majority of the population. An increase in vulnerable patients including the immunocompromised, transplant recipients and intensive care patients has caused a significant increase in the incidences of invasive fungal infections caused by Candida spp.

Methods

Results

Conclusion