Role of microbial miRNAs in infections by the intravacuolar pathogens Brucella and Coxiella

• Applicants: Professor Dr. Sascha Al Dahouk; Professorin Dr. Anja Lührmann
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 529926221

The host-pathogen-interplay is decisive for disease outcome. Bacterial virulence factors are important in modulating the host to enable bacterial survival and replication. So far, mainly bacterial virulence factors belonging to the group of proteins and lipids have been studied. Recently, exciting preliminary studies identified bacterial miRNA-like sRNAs (miRNA) delivered into the host cell to modulate its function, which opens unprecedented avenues for understanding bacterial pathogenesis with countless applications. Using bioinformatics, our consortium recently identified candidate pre-miRNAs encoded in the genomes of the bacterial pathogens Brucella suis and Coxiella burnetii, responsible for worldwide zoonosis outbreaks with severe impact on health and economy. Brucella spp. and C. burnetii are considered stealth pathogens as they evade the innate immune response and establish persistent infections by protecting infected cells from apoptosis. Importantly, preliminary data allowed us to validate an initial subset of predicted miRNAs translocated into the cytoplasm of infected cells and associated with the host cell RISC. Thus, we validated 8 predicted pre-miRNA-like sequences in the genome of B. suis and 12 predicted pre-miRNA-like sequences in the genome of C. burnetii. We confirmed for two predicted pre-miRNA-like sequences, one of each pathogen, the ability to bind to host cell miRNA recognition elements, indicating that they might have miRNA properties. Importantly, for the miRNA MIR6325 of B. suis we demonstrate a role in intracellular replication. Thus, our data provide the proof of principle that the intracellular pathogen B. suis modulates the host-pathogen-interactions using the bacterial miRNA MIR6325. Of note, the use of Brucella spp. and C. burnetii as working models represents several significant advantages and complementarities for the study of bacterial miRNAs: 1) both bacteria use T4SS, and produce OMVs, which could be involved in the delivery of sRNAs; 2) both pathogens replicate to large numbers within membrane-bound niches, greatly facilitating the intracellular localization of miRNAs. In this project, we aim to dissect the role of the bacterial miRNAs in infection using NGS, cellular microbiology, microscopy and screening approaches, which are all well-established in our consortium. In detail, we will 1) identify miRNAs transported early and late during infection into the host cell; determine whether stress conditions influences the expression and transport of miRNAs; compare the miRNA repertoire of different B. suis biovars and C. burnetii isolates. 2) Identify the transport pathway and the processing mechanisms of the miRNAs. 3) Identify and validate host cell targets of the bacterial miRNAs; and we will 4) characterize the biological function of the miRNAs and their role during infection. Completing these aims will allow us to characterize the bacterial miRNAs and their role in pathogenesis of two zoonotic pathogens.

• DFG Programme: Research Grants
• International Connection: France
• Cooperation Partner: Professor Dr. Stephan Köhler