Genetic switches and pathogenicity of Mycoplasma agalactiae

Mycoplasma agalactiae is an economically important pathogen of sheep and goats capable of inducing severe clinical manifestations - known as the Contagious Agalactia syndrome - by as yet unknown mechanisms. Vpmas are the major immunodominant membrane proteins of M. agalactiae that vary in expression at an unusually high frequency due to DNA inversions mediated by the site-specific Xer1 recombinase encoded on a pathogenicity island-like locus. The targeted disruption of the xer1 gene resulting in Vpma phase-locked mutants (PLMs) each expressing a well-characterized single Vpma protein was an important breakthrough as this provided the basis to assess the role of Vpmas in the natural host in vivo. The results obtained with a sheep infection model demonstrated that the PLMs which lack the Xer1 recombinase and are unable to switch their Vpma phenotype in vitro, undergo Vpma phase variation in vivo via a Xer1-independent alternative switching mechanism, most likely under the selection pressure of the host immune response. This indicated the significance of Vpma surface antigenic variation for the adaptation and persistence of M. agalactiae in ist natural host. In this project these Xer1- independent molecular switches which are activated inside the immunocompetent host and lead to Vpma phase variation, as well as the host factors triggering these events will be defined and characterized, thereby proving the essential role of Vpma phase variation in the survival and spread of M. agalactiae during infection and disease. The results will not only identify novel features of Vpma surface antigenic variation and ist regulation in response to host factors, but will also contribute to a better understanding of the role of the different Vpmas in pathogen-host interaction. Together with in vivo studies on the relative fitness, pathogenicity potential and tissue tropism of the different Vpma PLMs, the project will increase our understanding of the precise functions of the Vpma proteins in the host and will provide new insights into the infection biology and pathogenic mechanisms of M. agalactiae at the genetic and molecular level which will be important for the future development of new approaches for the immunological intervention or prevention of Contagious Agalactia.

Mycoplasma agalactiae is an economically important pathogen of sheep and goats that causes serious and difficult-to-eradicate infections by as yet unknown mechanisms. Like many other successful pathogens, it possesses a family of six different surface lipoproteins called Vpmas that vary in expression at an unusually high frequency due to DNA inversions mediated by the Xer1 recombinase encoded on the same pathogenicity island-like locus. Although it is believed that Vpmas play a role in avoiding the host immune responses, the exact role of Vpmas in infection and disease remained to be studied. Knocking-out the xer1 gene resulted in Vpma phase-locked mutants (PLMs), each expressing a single well-characterized Vpma protein without further switching. This project aimed to study the significance of Vpma phase variations and the relative pathogenicity of the six different PLMs by comparing them with each other and with the wild type phase variable (PG2) strain in an experimental sheep infection trial. Results indicated that although individual PLMs are capable of initiating and establishing an infection, there are significant differences in the colonization and infection potential of different Vpmas. Using in vitro experiments we could attribute the better pathogenicity of specific Vpmas to their higher adhesion rates. Not only did we demonstrate a novel function of adhesion for Vpmas, we also showed for the first time that M. agalactiae is not a strict extracellular pathogen but can also invade host cells, the relative invasiveness of the six PLMs strongly correlating with their adherence and in vivo fitness. Another interesting finding was that all six PLMs, which were phase-invariable in vitro, could change their Vpma expression in vivo. Complex intergenic and intragenic vpma recombinations that were so far unknown had now occurred in the absence of the Xer1 recombinase creating new chimeric vpma genes indicating the significance of Vpma switching for the adaptation and persistence of M. agalactiae in its host. These newly observed Xer1-independent mechanisms of Vpma phase variation were shown to be triggered by Vpma-specific antibodies highlighting new features of regulation in response to host factors. Furthermore, the dynamics of M. agalactiae induced host responses in the mastitic sheep mammary gland were studied for the first time to show the robust up and down regulation of several host factors, especially the innate and adaptive immune responses. Altogether, this research has furthered our understanding of M. agalactiae pathogenesis in many ways, and is anticipated to establish the basis for development and exploitation of new concepts for the rational design of intervention strategies against mycoplasma pathogens.