Molecular basis of Ureaplasma parvum antigenic variation

Several studies in the past years have shown that pathogenic Mycoplasma species are equipped with sophisticated genetic systems, which allow these agents to spontaneously change their surface antigenic surface make-up thereby promoting host adaptation. This surface antigenic variation involving abundant immunodominant surface membrane proteins is often associated with chromosomal rearrangement events which regulate the expression of silent genes and switching between their ON and OFF expression state. This research project investigates the genetic mechanism driving ON/OFF phase variation of two prominent surface protein antigens of U. parvum, an Ureaplasma species which is increasingly gaining recognition as an important opportunistic human pathogen implicated to play a causative role in a variety of genital disease syndromes in women and associated neonatal disease. From preliminary studies it is postulated that ON/OFF switching in expression of the major U. parvum surface membrane protein, the multiple-banded antigen (MBA), and its counterpart, the UU376 membrane protein, which are both encoded by the mba locus, is a result of DNA inversion events. The major goal of this project is to elucidate how the genetic switch to regulate expression of MBA and UU376 is taking place. To reach this goal, previously characterized clonal variants derived from strains and clinical isolates of U. parvum serovar 3 which differ in their MBA/UU376 expression state, as well as immunological reagents for monitoring MBA/UU376 phase variation will be used to establish well-defined clonal lineages in order (i) to identify the possible recombination and inversion sites within the mba locus, (ii) to analyze gene expression on the transcriptional level and characterize the expression unit of the mba locus, and (iii) to identify the recombinase involved in the postulated inversion. Unravelling the mechanism and the factors regulating ON/OFF switching in the mba locus will pave the way to further assess the role of these variable surface membrane proteins in molecular pathogenesis, with the long-term perspective to gain a deeper understanding of the pathobiology of U. parvum and its currently unknown molecular strategy by which it establishes invasive and disseminated disease patterns. In addition, this project may also lead to valuable serodiagnostic approaches in clinical microbiology and to new concepts of the interrelationship and molecular evolution of U. parvum serovars.

Several studies in the past years have shown that pathogenic Mycoplasma species are equipped with sophisticated genetic systems, which allow these agents to spontaneously change their surface antigenic surface make-up thereby promoting host adaptation. This surface antigenic variation involving abundant immunodominant surface membrane proteins is often associated with chromosomal rearrangement events which regulate the expression of silent genes and switching between their ON and OFF expression state. This research project focussed on the identification and characterization of phase variable loci in U. parvum, a species that is increasingly gaining recognition as important opportunistic human pathogens, implicated to play a causative role in a variety of genital disease syndromes in women and associated neonatal disease. During the course of this research project, we have identified and thoroughly described two phase variable loci and have taken first steps in investigating the genetic mechanism that drives ON/OFF phase variation in these two gene clusters designated `mba locus` and `UU172 phase variable element`, both encoding prominent surface protein antigens. The antigenic switch emerging from these loci is believed to be a strategy by which ureaplasmas evade host immune responses, as antibody induced stress against specific antigens selected for variants with alternating expression patterns in vitro. ON/OFF switching in expression in these two loci is a result of DNA inversion events taking place between short inverted DNA repeats located in the 5`-regions of oppositely oriented coding sequences, resulting in alternating expression of oppositely oriented reading frames. Furthermore, a potential tyrosine recombinase that interacts with these short sequences has been identified, suggesting its involvement in promoting the postulated site-specific recombination event. Preliminary results indicate, however, that other factors must be involved in the site-specific recombination process and that the proposed DNA inversion event is not mediated by the single tyrosine recombinase alone. We have also identified a binding site of a second potential recombinase that might be involved in chromosome dimer resolution. Taken collectively, the information accumulated during this project leads to novel insights into the molecular pathways of pathogenesis enabling ureaplasmas to adapt to their host environment. Our results establish the basis for future investigations related (i) to the characterization of recombination machineries, (ii) to new molecular approaches for diagnosis, and (iii) to potential therapeutic and preventive strategies.