Analysis of Campylobacter fetus virulence mechanisms

The bacterial species Campylobacter fetus is a significant animal pathogen, causing epidemic abortion and infertility in cattle. It is also an important human pathogen causing septicaemia, putting predominantly immunocompromised people at risk. Very little is understood about C. fetus virulence mechanisms and the interaction with its hosts. C. fetus consists of two subspecies, C. fetus subsp. fetus und C. fetus subsp. venerealis, which are extraordinarily closely related on the genetic level but exhibit striking differences in tissue and host specificity. Comparative analysis of these subspecies thus provides an excellent model to understand the molecular nature of this pathogen`s host and tissue tropism and to elucidate the molecular interactions with its host cells. We have compared the genomes of the subspecies and identified genome segments unique to each with a putative role in pathogenicity. These include a type IV macromolecular transport system (T4SS) typically involved in secretion of bacterial virulence proteins to eukaryotic host cells, and an enzyme linking LPS biosynthesis and structure to environmental cues. This research aims to determine the contribution of specific genes to the host tropism and virulence of C. fetus subspecies. To do so we require much more detailed understanding of the natural appearance of the C. fetus infection process. We propose a thorough histopathological analysis of infected tissues derived from the veterinary field. Insights gained here will be enhanced by fine resolution of host-pathogen interactions on the cellular level. C.fetus - susceptible in vitro models of cultured animal and human cells will be developed to assess the functional contribution of putative virulence genes to stages of the C. fetus infection process. Techniques developed in this project should differentiate involvement of the virulence genes in bacterial adherence, cellular entry, proinflammatory activation of host cells, chemokine secretion and cytotoxicity. The important applications of these models will be not only to compare the divergent virulence properties of the C. fetus subspecies but to evaluate phenotypic differences in host-pathogen interactions following mutagenesis of putative virulence genes. We have established a new series of genetics tools for this organism which enable us to perform mutational analyses readily. These studies will provide much clearer insights into the function of specific genes in determining host preference and the molecular basis of C. fetus pathogenicity.

Campylobacter fetus represents a Gram-negative bacterial pathogen of humans and animals highly adapted to colonize mucous membranes (e.g. the gastrointestinal- or the genital tract) because of its cell structure (spiral morphology & motility) and special physiology (microaerophilic growth under reduced oxygen conditions). C. fetus is divided into two different groups of bacteria (subspecies) with different host preferences and disease associations. C. fetus subsp. fetus preferentially infects the gastrointestinal tract of a variety of hosts, including humans, sheep, cattle and also reptiles. Human infection results often in remitting septicemia due to the inability of the host to eradicate the pathogen. C. fetus subsp. venerealis, on the other hand, exclusively colonizes the genital tract of cattle, leading to the epidemic disease called bovine venereal campylobacteriosis (BVC). BVC is characterized by epidemic abortion and infertility of cattle, posing a huge economic burden especially in countries where large cattle herds exist (e.g. Argentina, Australia). The dichotomy in the host preferences and disease associations of C. fetus subspecies makes them ideal models to identify virulence factors (genes) by comparative genetic analyses. In that regard we performed genome analyses and comparisons and identified several important virulence traits displayed differentially by the subspecies. interestingly, the genomes of the two subspecies share a high level of identity (93%), yet differences are clustered in specific genetic islands acquired by horizontal gene transfer (i.e. acquisition of genetic information from other, not or just distantly related bacteria). For instance, we were able to identify a bacterial secretion system (a so called type 4 secretion system), which enables C. fetus subsp. venerealis to confer cytotoxicity to and invade eukaryotic host cells. Another virulence attribute, located in one of these genomic islands, was represented by a gene important for surface sugar (LPS) biosynthesis of the bacterial cell (GDP-mannose 4,6-dehydratase). This enables C. fetus subsp. fetus to survive the acidic stomach passage during oral infection ahead of intestinal colonization. A related enzyme also involved in LPS biosythesis (UDP- galactopyranose mutase) renders C. fetus subsp. fetus resistant against the bactericidal action of (human) serum. These and other identified and molecularly characterized virulence factors give for the first time insights into the molecular virulence strategies employed by the pathogen C. fetus and pose valuable targets for the development of diagnostic assays (e.g. by means of the polymerase chain-reaction) and for vaccine development. The latter issue has huge economic relevance due to BVC.