Molecular genetic basis of Mycoplasma agalactiae surface antigenic variation: Role of the Xer1 recombinase system

Mycoplasma agalactiae is responsible for the `contagious agalactia` syndrome in sheep and goats, which primarily includes chronic mastitis, arthritis and conjunctivitis. Despite of a small genome size, this bacterial pathogen possesses a sophisticated genetic system that spontaneously alters its antigenic make-up with an unusual high frequency. This system, designated vpma, is composed of six distinct but related, single copy genes encoding major immunodominant surface proteins which vary in expression due to site-specific DNA rearrangements. These recombinational events occur within clonal populations and are thought to be driven by a site-specific recombinase encoded by the xer1 gene, which is located in the vicinity of the vpma gene locus. Analogous but distinct gene families have also been found in other mycoplasma species and are implicated to enable escape from the host immune response and host colonization. Common features existing between members of such gene families and their high variation in expression have hampered more refined studies to assess their exact role in infection and disease. This is also due to the lack of tools to genetically manipulate these organisms that would allow the modification of these particular loci via engineered mutations. Recently, foreign DNA has been successfully introduced into the M. agalactiae genome for the first time, a preliminary but encouraging result, since it sets up the basis to develop targeted gene disruption mutations in this agent. This project intends to further develop and optimize such tools in order to pave the way for a variety of experimental follow-up studies assessing the function of Vpmas during M. agalactiae infection. A main aim of this project will be to knock out the xer1 gene in order to abolish variation in Vpma expression and to generate mutants that are phase-locked in one Vpma configuration. Furthermore, the mode of action and the regulation of the Xer1 recombinase as potential key player in mediating surface antigenic variation and in pathogenicity of M. agalactiae will be assessed. Overall, the tools and data established in this project will open a wide range of perspectives for the study of this particular agent by providing an excellent basis for the identification of M. agalactiae virulence genes through insertional mutation following transformation. More precisely, the results obtained will define whether Xer1 acts alone or in concert with other factors, and how the vpma gene locus is precisely controlled. Finally, Xer1-negative mutants will provide phase- locked organisms for defining the role of the vpma locus and of the xer1 gene in pathogen-host interaction using in vitro and in vivo models.

Compared to other bacterial pathogens, the current knowledge of the molecular basis of pathogenicity of mycoplasmas is limited, and their strategies of infection at the molecular and cellular level remain to be elucidated. Several studies in the past years have shown that pathogenic mycoplasmas are equipped with sophisticated genetic systems, which allow these agents to spontaneously change their surface antigenic make-up. It is implicated that these variable surface components provide the wall-less mycoplasmas with a means to avoid the host immune response and promote host colonization. In Mycoplasma agalactiae, the agent of "contagious agalactia" in sheep and goats, a pathogenicity island-like locus has recently been identified that contains six distinct but related genes which encode the major immunodominant membrane proteins, the so-called Vpmas. It was shown that these surface-associated proteins vary in expression at an unusual high frequency due to DNA rearrangements, which are thought to be mediated by the site-specific Xer1 recombinase. The previous lack of tools to genetically manipulate M. agalactiae has hampered more refined studies to assess the exact function of Vpmas in M. agalactiae infection and disease. Recently, foreign DNA has been successfully introduced into the M. agalactiae genome for the first time, a preliminary but encouraging result, since it sets up the basis to develop targeted gene disruption mutations in this agent. To study the in vivo significance of Vpmas, we aimed to generate M. agalactiae variants in which Vpma switching was abolished to yield phase-locked mutants displaying stable Vpma phenotypes, and thus expressing a single, well-characterised Vpma product. It was proposed that disruption of the xer1 recombinase would prevent the site- specific recombinations within the vpma locus leading to stable `phase-locked` mutants. In this project we could optimize the transformation procedures allowing us to efficiently introduce foreign DNA into M. agalactiae, a prerequisite to obtain the desired disruption of the xer1 gene. We have achieved this aim by using M. agalactiae oriC vectors and demonstrated for the first time that homologous recombination and targeted gene disruption is possible in M. agalactiae. This represents an important breakthrough as such phase-locked mutants can be used in future to define the significance of Vpma oscillation under in vivo conditions in the natural host in experimental infection studies.