Importance of an RTX adhesin for pathogenesis of bronchopneumonia: Rodentibacter heylii immunogen A (RhiA)-depending biofilm formation and its control by RhiA specific immunity

• Applicant: Dr. Sophie Kähl
• Subject Area: Veterinary Medical Science ; Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 524629637

Rodentibacter (R.) heylii is one of the most frequently isolated bacterial pathogens in laboratory mice. It causes bronchopneumonia, conjunctivitis and abscesses, but can also influence animal experiments unnoticed by subclinical infections. The pathogenesis is largely unexplained, only three potential virulence factors (PnxI, PnxII, PnxIII) from the RTX (repeats in toxin) toxin family have been identified. These proteins are described as cytotoxic, hemolytic and adhesive. In contrast to RTX toxins in other species, these three have not yet been functionally investigated in vivo. Another recently described RTX protein is RhiA which we identified as an immunogen using immunoproteomics. By ELISA, immunohistology and Western blot, we showed that RhiA is expressed in vivo and in vitro. Flow cytometric data proved the localization on the bacterial surface. Due to specific RTX consensus- (IXGGXGNX) and RTX-like sequences (IXGXGNX) of the primary structure as well as 17 large tandem repeats, it has been assigned to the RTX adhesins. Adhesins can mediate the binding of bacteria to surfaces and thus initially lead to invasion and biofilm formation. Biofilms protect bacteria against the host immune defense and other influences such as antibiotics. This project aims to investigate the hypothesis that RhiA is an adhesin and plays an important role in the biofilm formation and pathogenesis of R. heylii. Therefore, a deletion mutant of a RhiA-bearing R. heylii strain will be generated, which will not express RhiA. Wild type and mutant will then be compared in vitro using Western blot, flow cytometry, adherence and biofilm assays. In addition, in our previous study we have developed an animal model for R. heylii in the natural host, the mouse. Using this model, both morbidity and mortality as well as biofilm formation in the lungs can be investigated. This will be used to analyze the differences in virulence between wild type and mutant. Furthermore, the protective effect of specific antibodies after immunization with RhiA will be investigated. Both homologous protection against R. heylii infection and protection against the close relative R. pneumotropicus by experimental infection will be analyzed. All animal experiments will be evaluated immunologically, pathohistologically and bacteriologically to make precise conclusions about the RhiA-dependent pathogenesis of R. heylii. In addition to the first establishment of mutagenesis in R. heylii, the infection model for the analysis of the pathogenesis of bronchopneumonia in the natural host is a unique selling point of this project.

• DFG Programme: Research Grants