Peptide transport in Candida albicans and synthetic antifungal agents

Abstract

These studies have characterized the peptide transport systems of Candida albicans, with a view to the rational design of peptide antifungal agents exploiting the 'smugglin' concept. In initial studies, a series of polyoxin complexes (peptide-nucleoside antibiotics) and individual components, were isolated from a batch of agricultural fungicide (Polyoxin Z). Isolated fractions were toxic to a particulate chitin synthetase preparation from Candida albicans. Different strains of Candida albicans exhibited varied sensitivities to a series of peptide analogues. From a sensitive strain, B2630, spontaneous mutants were selected for resistance to each analogue; certain mutants showed cross-resistance to other analogues and associated defects in peptide transport. A bacilysin-resistant mutant was cross-resistant to the other analogues and to m- fluorophenylalanylalanylalanine a but retained sensitivity to m- fluorophenylalanylalanylalanine. This mutant showed defective dipeptide transport but normal oligopeptide transport, and was unable to utilize Ala-Ala as a sole nitrogen source. Thus, Candida albicans has distinguishable mechanisms for dipeptide and oligopeptide transport which can be exploited for uptake of peptide-drug adducts. Peptide transport was shown to be stimulated by the presence of peptides (peptone) in the growth medium. On transferring cells from minimal to peptone medium, this stimulatory effect was shown to be rapid, independent of protein synthesis and to override ammonia regulation of peptide transport. The reduction of transport activity on transferring cells from peptone to minimal medium was also rapid. It was speculated that regulation of peptide transport is achieved by a rapid, reversible activation of preformed transport components, or a mechanism of exocytotic insertion and endocytic retrieval of preformed transporters. The effect of protein-modification reagents on transport activity was also examined. Dipeptide transport was specifically inhibited by N-ethyl-5-phenylisoxazolium-3'-sulphonate (Woodwards Reagent K), offerring potential for the specific labelling of the component(s) of this system. Peptide transport was shown not to be sensitive to osmotic shock though a series of uncharacterized polypeptides was released by the shock treatment.