Oligopeptide transport : cloning and characterization of a new gene family

Peptide transport is a widely observed phenomenon defined as the translocation of peptides 2-6 residues in length across the plasma membrane in an energy-dependent manner. Internalized peptides are rapidly hydrolyzed by peptidases, and the resulting amino acids are used for protein synthesis or alternatively as a source of nitrogen or carbon. Physiological evidence suggested that the pathogenic fungus Candida albicans has at least two different peptide transporters: a di-/tripeptide transporter named CaPtr2p and an oligopeptide transporter system which accomodates peptides of 3-5 residues. The purpose of this study was to 1) clone and characterize the gene(s) responsible for oligopeptide transporter in C. albicans and 2) explore the role of oligopeptide transport in virulence. Part II of this dissertation describes how the oligopeptide transport gene OPT1 was cloned from C. albicans. Using growth conditions under which Saccharomyces cerevisiae strain PB1X-9B does not transport tetra- and pentapeptides, we were able to identify OPT1 as a gene that allowed PB1X-9B to utilize tetra- and pentapeptides as a sole source of an auxotrophic supplement. OPT1 bestowed oligopeptide transport activity to PB1X-9B as measured by sensitivity to toxic oligopeptides and the ability to accumulate a radiolabled tetrapeptide. Part III of this dissertation details how we identified the OPT1 homolog isp4 from S. pombe and how subsequent characterization of a deletion strain revealed that isp4 encodes an oligopeptide transporter. Furthermore, based upon the four criteria of protein length, function, topology, and conserved functional domains, we proposed that OPT1 from C. albicans, isp4 from S. pombe, and YPR194C and YJL212C from S. cerevisiae comprise the first identified members of a novel family of transport proteins. Part IV of this dissertation summarizes the construction and characterization of several OPT1 disruptant strains. Analyses of the disruptants revealed that Opt1p is a high-affinity/low-capacity transporter and that another oligopeptide transport system exists in C. albicans. The role of OPT1 in one murine model of systemic candidiasis was explored and the possible roles for OPT1 in virulence are discussed.