Identification and characterization of virulence-associated factors of Campylobacter jejuni by using a high throughput negative selection method

• Applicant: Professor Dr. Uwe Groß
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: from 2009 to 2013
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 104754421

Campylobacter jejuni is one of the most frequent bacterial causes of foodborneillness in the industrial world including Germany. Whereas human infection may result in symptomatic disease, intestinal colonization of chicken usually is asymptomatic. Together with a German Campylobacter consortium and by using different in-vitro and in-vivo models, we aim to identify bacterial factors that determine the so-called fitness of C. jejuni thereby allowing the different outcome of infection in chicken and humans (or analogous in the mouse model). Despite its well known importance as a human pathogen, remarkably little is known about the pathogenesis of the disease caused by this bacterium. Different factors are reported to be contributing in this lack of understanding likewise, the unavailability of efficient systems of experimental genetics, the lack of appropriate animal models for the disease, and the genetic diversity of Campylobacter strains. We established a transposon-based system for random mutagenesis of C. jejuni: using an in-vitro screen, some of the transposon insertions were found in several motility-modulating genes of C. jejuni, including Cj0977 (initiation factor eIF-2B like protein) and Cj0793 (signal transduction histidine kinase). In addition, two other mutants (Cj01508c, FdhD protein; and Cj0954c, DnaJ-like protein), showed significant reduction in their swarming ability on motility agar. The exact role of these two genes in association with the motility phenotype has not yet been determined. Similarly, a mutant with an insertion in the gene Cj0009 (gltD), encoding the NADPH-dependent glutamate synthase small subunit showed reduced growth under high-osmolar conditions. Furthermore, we identified a clinical isolate of C. jejuni that was able to survive in the liver of infected BALB/c mice for up to seven days post infection. Combining our transposon-based mutagenesis system and the BALB/c mice as an in-vivo screening model proved the principal feasibility of this system for the identification of putative virulence factors of C. jejuni. In the proposed project, we first aim to further characterize the previously mutated genes of C. jejuni to determine their role in motility and osmoregulation. Osmoregulatory mechanisms to cope with osmotic stress have not yet been described in C. jejuni. We therefore are particularly interested to characterize the _gltD mutant (NADPH-dependent glutamate synthase small subunit) to gain further insight for the osmoregulatory mechanism(s) used by C. jejuni. The second aim of our proposed project is to combine (i) the identified clinical isolate, (ii) the established transposon mutagenesis system and (iii) the BALB/c mice as an in-vivo screening model to develop an extended and improved high throughput screening strategy based on negative selection of random mutants which will facilitate the identification of putative virulence factors of C. jejuni. To confirm their attenuation, the identified mutants will be retested in the screening model and an inverse PCR strategy will be used to recover the disrupted genes. The identified mutants will then be characterized by DNA sequencing, gene complementation, and - together with partners of the Campylobacter consortium - also by additional in-vivo and in-vitro assays to determine the respective phenotype.

• DFG Programme: Research Grants