Pathogenicity determinants of Mycoplasma agalactiae

Despite their small genomes, pathogenic species of the bacterial genus Mycoplasma possess unique biological properties and complex pathogenicity determinants, and apart from rare exceptions, their infection biology and virulence mechanisms at the molecular and cellular level are still largely unexplored. This research project investigates the molecular genetic basis of pathogenicity of Mycoplasma agalactiae, an economically important agent of small ruminants causing Contagious Agalactiae and associated diseases by as yet unknown mechanisms. The major goal is to identify the infection-related genes of this agent. To reach this goal, we have previously succeeded to develop tools to genetically manipulate M. agalactiae and to generate a large pool of transposon mutants. In this project, a number of sequenced mutants selected from this pool will be simultaneously screened as mixed population in a recently established sheep infection model to identify putative pathogenesis-related genes using (i) signature sequence mutagenesis as a genetic footprinting strategy, and (ii) a novel quantitative target display (QTD) technique capable of identifying mutations that confer negative, positive as well as subtle quantitative phenotypes inside the host. To confirm the function of mutated genes and their precise role during infection, complementation and mutation reconstruction studies will be performed. The most promising attenuated mutant will be tested individually in a challenge experiment to verify and determine the degree of attenuation caused by transposon insertion. Altogether, the results of this project are not only expected to identify virulence factors of M. agalactiae which may in the future lead to valuable new approaches for prevention and control of disease caused by this agent, but would also improve our understanding of the infection biology and pathogenic mechanisms of mycoplasmas in general. In addition, QTD could emerge as a unique new powerful tool for functional analysis of hypothetical genes present in the genomes of other important pathogenic mycoplasma species and other known bacterial pathogens for which no function can be assigned but which might play subtle, yet significant roles in pathogenesis.

Despite their small genomes, pathogenic species of the bacterial genus Mycoplasma possess unique biological properties and complex pathogenicity determinants, and apart from rare exceptions, their infection biology and virulence mechanisms at the molecular and cellular level are still largely unexplored. This research project investigates the molecular genetic basis of pathogenicity of Mycoplasma agalactiae, an economically important agent of small ruminants causing Contagious Agalactiae and associated diseases by as yet unknown mechanisms. The major goal is to identify the infection-related genes of this agent. To reach this goal, we have previously succeeded to develop tools to genetically manipulate M. agalactiae and to generate a large pool of transposon mutants. The main aim of this research project was to identify genes involved in the pathogenicity of M. agalactiae, which could also serve as an important reference for deciphering the infection and virulence mechanisms of other significant ruminant mycoplasmas. In this context, we tested a pool of several different transposon mutants in an intramammary sheep infection study and identified many different genes likely to be involved in M. agalactiae infections. The shortlisted mutants were further analysed in various in vitro assays, and the most promising ones were further tested for attenuation in a second round of confirmatory screening in sheep. We have successfully identified several genes to be essential for colonizing the local infection sites (udders) and few others playing an important role in the systemic spread of the pathogen to distant host niches. Furthermore, we demonstrated for the first time M. agalactiaes ability to invade host cells in vitro and to spread systemically to distant inner organs and body sites during an experimental sheep intramammary infection, thereby proposing cell internalization as a potential strategy for its survival and persistence. Molecular cloning and complementation studies clearly proved the role of genes like pdhB in its cell invasive phenotype. Overall, the results provide new insights into the infection biology of M. agalactiae, which might be invaluable for the rational design of intervention strategies against it.