B-252. A Novel Type IV Secretion System Is Unique to the Campylobacter fetus subspecies venerealis, and Contributes both to Bacterial Virulence and Conjugative DNA Delivery

S. Kienesberger1, G. Gorkiewicz2, E. L. Zechner1;
1Inst. of Molecular Biosciences, Univ. of Graz, Graz, AUSTRIA, 2Inst. of Pathology, Med. Univ. of Graz, Graz, AUSTRIA.

The pathogen Campylobacter fetus comprises two subspecies, C. fetus subsp. fetus (Cff) and C. fetus subsp. venerealis (Cfv). Although these taxa are genetically highly related, they show distinct host and tissue preferences. Cff infects a diversity of hosts including humans colonizing the gastro-intestinal tract. By contrast Cfv is largely restricted to the bovine genital tract, causing epidemic abortion in these animals. These features make C. fetus an ideal model to study the molecular basis of host adaptation. A subtractive hybridization approach was applied to the genomes of the subspecies, which revealed a genomic island exclusively present on the Cfv chromosome. This island includes all components necessary for bacterial type IV secretion. Macromolecular transporters of this type are involved in conjugative DNA transfer and/or delivery of effector proteins to host cells, thereby contributing to virulence in various pathogens. Since little is known about C. fetus virulence mechanisms, we adapted typical in vitro approaches to evaluate host-pathogen interactions, like the standard gentamicin protection assay, differential immunofluorescence staining and an assay, which allows us to measure cytotoxic effects displayed by C. fetus on eukaryotic cells. These assays showed that both bacterial subspecies can invade a diversity of eukaryotic cells including CaCo-2 and ACH-3P cells (human placenta cells) and readily migrate underneath the cultured cells, a proposed virulence strategy of Campylobacter. Mutational inactivation of virB9, a type IV secretion system (T4SS) component, led to reduced host cell killing compared to the wild type parental strain in cytotoxicity assays. The Cfv T4SS was also shown to promote conjugational DNA delivery to other bacteria highlighting the distinct repertoire of adaptive functions conferred to this subspecies by acquisition of the genomic island. Efforts now focus on the construction of specific mutants of highly virulent C. fetus isolates. Analysis of these mutants in the established in vitro assays will improve our understanding of C. fetus virulence and the role of the Cfv T4SS.