Comparative gene mapping of Trichomonas vaginalis strains varying in metronidazole susceptibility

Abstract

Trichomonas vaginalis is an anaerobic protozoan parasite of the human urogenital tract, and is sexually transmitted to over 170 million people worldwide each year. Although most infections go unnoticed, serious health implications include the increased transmission and acquisition rate of human immunodeficiency virus (HIV) and low infant birth weight. Drug resistance in T. vaginalis is an emerging and alarming predicament as only one drug, metronidazole, has been approved for treatment. Several gene families have been implicated in the development of derived drug resistance in T. vaginalis, however, very little is known about how this phenomenon arises in clinical samples. Genome plasticity is common in protists, and specific rearrangements, changes in ploidy and chromosome size are frequently correlated with drug-resistant phenotypes in other protozoan parasites. Limited genomic information is available for T. vaginalis due to an unusually large genome size and highly potent nucleases which confound modern molecular technologies. The present study aimed to investigate genome polymorphism using modified, traditional mammalian karyotyping techniques and a collection of five T. vaginalis strains varying in metronidazole susceptibility (including clinical and laboratory-derived resistance) and geographic sampling origin (Australia, United States of America and United Kingdom). My study produced metaphase-arrested chromosome spreads to confirm a chromosome number of six for T. vaginalis, which was conserved across all strains examined. This study also observed chromosome length polymorphism in the form of a duplication event on chromosome C1 in one clinically metronidazole resistant strain. Due to the limited information available from restriction fragment length polymorphism (RFLP) and Southern hybridisation profiling, this study aimed to develop a fluorescence in situ hybridization (FISH) technique that would allow gene localisation to whole chromosomes. Using this technique, major rDNA arrays were mapped to a small chromosome (C4) and additional incomplete 28S rDNA sites were observed on the subtelomeric regions of all other chromosomes, suggesting a centralised transcription centre on C4 and possible structural roles for incomplete rDNA units during mitosis and nuclear division. Collectively, these arrays served as molecular markers for the identification of individual chromosomes for subsequent mapping experiments. As very little information is known about the mechanisms of drug resistance in clinically resistant strains, a genome-wide mining project was undertaken to identify and compile gene families involved in resistance. Gene families included metabolic proteins pyruvate:ferredoxin oxidoreductases (PFO), ferredoxins (Fd), alternative metabolism proteins aldo-keto oxidoreductases (AKR), antioxidant proteins superoxide dismutastes (SOD), nitroimidazole resistance proteins (nim), extrusion proteins ABC transporters (ABC), multi-drug and toxin extrusion proteins (MatE), and major facilitator superfamily transporters (MFS). This study further identified five novel PFO genes, three novel Fd genes, 19 novel AKR genes, two novel nim genes never before identified in a eukaryote, 109 ABC genes (including 38 MDR genes), and 12 novel MatE genes. Candidate genes for mapping studies were chosen through extensive bioinformatics analysis including the matching of conserved domains with known drug-resistance proteins and cross-referencing with organelle targeting sequences and expressed sequence tag (EST) libraries. To evaluate differences in gene copy number and chromosomal location of these candidate genes, probes were generated and verified via polymerase chain reaction (PCR) and then used in multi-colour FISH assays in conjunction with the chromosomal marker probe (rDNA). Hybridisation profiles were generated for all strains and compared to identify any areas of unique and conserved similarity between drug resistant strains. Results revealed an extensively replicated genome well beyond current estimates, and confirmed the plastic nature and genetic diversity of T. vaginalis genomes but yielded no definitive markers for drug resistance. These results support the heterogenic nature of drug-resistance in naturally occurring asexual populations of T. vaginalis parasites, and highlights the difficulties in predicting successful drug resistant genotypes under significant selective pressure, particularly in various geographic locations. This study also revealed the apparent importance of subtelomeric regions in the genetic diversity of T. vaginalis strains, as almost all genes studied localised to these regions. The duplication and distribution of genes across almost all chromosomes suggests an important role for ectopic and non-homologous recombination in T. vaginalis diversity, and supports growing evidence for sex in these parasites. In summary, this study established a novel karyotyping technique for Trichomonas vaginalis (a protozoan parasite notoriously refractory to contemporary electrophoretic karyotyping techniques); used bioinformatics to identify a range of genes putatively involved in drug resistance; and utilized FISH assays to map a selection of genes to specific chromosomes in five different parasite strains. The expansion of this study to include larger sample sizes from different geographic locations and a larger candidate probe subset would be invaluable in further elucidating any common themes between drug resistant strains, and may illuminate key mechanisms that can then be mined for future drug targets.