MOLECULAR AND BIOINFORMATICS APPROACHES TO REDEFINE OUR UNDERSTANDING OF UREAPLASMAS: MOVING BEYOND SEROVARS
Glass, John I
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<italic>Ureaplasma parvum</italic> and <italic>Ureaplasma urealyticum</italic> are sexually transmitted, opportunistic pathogens of the human urogenital tract. There are 14 known serovars of the two species. For decades, it has been postulated that virulence is related to serotype specificity. Understanding of the role of ureaplasmas in human diseases has been thwarted due to two major barriers: (1) lack of suitable diagnostic tests and (2) lack of genetic manipulation tools for the creation of mutants to study the role of potential pathogenicity factors. To address the first barrier we developed real-time quantitative PCRs (RT-qPCR) for the reliable differentiation of the two species and 14 serovars. We typed 1,061 ureaplasma clinical isolates and observed about 40% of isolates to be genetic mosaics, arising from the recombination of multiple serovars. Furthermore, comparative genome analysis of the 14 serovars and 5 clinical isolates showed that the mba gene, used for serotyping ureaplasmas was part of a large, phase variable gene system, and some serovars shown to express different MBA proteins also encode <italic>mba</italic> genes associated with other serovars. Together these data suggests that differential pathogenicity and clinical outcome of an ureaplasmal infection is most likely due to the presence or absence of potential pathogenicity factors in individual ureaplasma clinical isolates and/or patient to patient differences in terms of autoimmunity and microbiome. To address the second barrier we are adapting the traditional molecular biology and novel synthetic biology tools to Ureaplasma, such as creation of oriC plasmids, use of transposons, and most prominently the engineering bacterial genomes cloned as yeast centromeric plasmids followed by genome transplantation to make ureaplasma mutants programmed by the genomes manipulated in yeast. This will allow for the creation of targeted single or multiple mutants that will greatly increase the understanding of ureaplasma pathogenicity. Efforts to transplant the genomes of bacteria, outside the<italic>mycoides</italic> group have been thwarted due to recombination between the donor and recipient cell genomes. We are exploring the use of the DNA cross-linking drug, Mitomycin C, to inactivate the recipient cell genomes and thus prevent false positive transplantation results and potentially increase the genome transplantation efficiency.