Large-scale identification of pathogenicity factors in "Bartonella" by signature-tagged mutagenesis

Bartonellae are bacterial pathogens uniquely adapted to cause intraerythrocytic infection in their mammalian reservoir hosts. In the case of human-specific Bartonella bacilliformis and Bartonella quintana, the intraerythrocytic bacteremia leads to the clinical manifestations of Oroya fever and trench fever, respectively. Here, we adapted large-scale signature-tagged mutagenesis (STM) for the first time to Bartonella, allowing us to screen for pathogenicity factors required for infection of the mammalian reservoir host in vivo. A total of 3084 STM mutants of rat-specific B. tribocorum were screened in a rat infection model for these criteria. After two rounds of screening, 130 mutants showed severe attenuation compared to wild-type B. tribocorum. We mapped the transposon insertion sites of these mutants to 80 different genes, and categorized them according to their putative function. Besides already described pathogenicity factors responsible for interaction with the host, like the two type IV secretion systems VirB-D4 and Trw, we discovered factors previously unlinked to pathogenesis. These belong to diverse functional classes, like transport, gene-expression regulation, cell envelope integrity, or metabolism. A quarter of the identified genes are (conserved) hypothetical coding for novel pathogenicity factors. We have used an additional PCR-screening approach on the entire mutant library to test for the level of mutational saturation and to identify non-essential genes in a pathogenicity island encoding 18 gene products related to the process of type IV secretion