title: ATP-binding cassette transporters of Paracoccidiodes brasiliensis
Creators: name: Gray, Christopher H
Publisher: name: University of Glasgow
Year: 2001
Subjects: subject: Microbiology.

description: ATP-binding cassette (ABC) transporters represent one of the largest recognised superfamilies of proteins. They are ubiquitous in nature and have been implicated in a wide range of physiological functions, including multi-drug resistance (MDR). All of these transporters are structurally related, with active transport complexes exhibiting the minimal complement of twelve transmembrane helices and two nucleotide-binding domains. Despite the structural similarity, the range of functions performed by these proteins is variable. In many cases the substrate range is equally diverse with the proteins displaying the capacity to translocate substrates which appear structurally unrelated. This intensifies the problem of MDR where a number of classes of drug are susceptible to efflux. A number of ABC transporters have been identified in fungi including Saccharomyces cerevisiae and Candida albicans. An increasing wealth of information is emerging for a few of these transporters. The current knowledge on the various characteristics of the S. cerevisiae transporter PDR5 approaches that relating to P-glycoprotein (Pgp) of mammalian cells. The ABC transporter CDRl from Candida albicans has been shown to confer clinical drug resistance in a number of disease cases. Paracoccidiodes brasiliensis is a thermally dimorphic human pathogen that is exclusive to South America. Inhalation is the route of infection with disease progression originating in the lungs and progressing to other parts of the body. Paracoccidiodiomycosis is the most prevalent systemic mycosis in Latin America. Exposure to the organism is most common in rural workers, particularly those involved in deforestation. Disease progression occurs when the host is immunocompromised and tends to follow and extended period of latency which can last over ten years. Outgrowth of the organism is dependent on a morphological transition from the mycelial to the yeast form of the organism. This transition is inhibited by the female hormone oestrogen resulting in the clinical syndrome displaying a bias towards males. The drug of choice is ketoconazole, a member of the azole family of anti-fungal agents. Azoles act on an intermediate enzyme in the ergosterol biosynthesis pathway, lanosterol demethylase, resulting in the perturbation of cell membrane integrity. Resistance to azole drugs can occur in two ways. A mutation in lanosterol demethylase can alter the drug-binding site so that the target is not recognised. Alternatively, the intracellular concentration of azole can be reduced by the ABC transporter mediated active efflux of the drug. The remit of this thesis was to screen the genome of P.brasiliensis for loci encoding ABC transporters. Particular interest would be paid to proteins that may be involved with drug resistance. Initially a strategy employed by other investigators was employed. Two degenerate oligonucleotides (MDRl and MDR2), designed on the basis of conserved motifs from the nucleotide binding domains of a number of ABC transporters were used in PCR. A single amplicon was obtained which showed homology to other sequences coding for nucleotide-binding domains. This amplicon was used to screen a lambda genomic library and a single phage was isolated. This phage clone contains part of a gene with displayed considerable identity to an ABC half-transporter from Aspergillus fumigatus. The complete sequence of the gene was then obtained using genome walking SSP-PCR. The gene, pftl, spans 2627bp and is interrupted by 2 introns of 68bp and 77bp. A similar approach was employed in a search for additional genes. The amino acid sequences from a number of PDR5-like fungal ABC transporters were aligned. This allowed the identification of conserved motifs that were specific to these fungal proteins. (Abstract shortened by ProQuest.).