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

The host-pathogen-interplay is decisive for the establishment of infection and, thus, 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 delivered into the host cell to modulate its function. The evidence that host-pathogen-interactions are also regulated by bacterial miRNAs opens unprecedented avenues for understanding bacterial pathogenesis with countless applications. Indeed, the identification of the main hubs of host-pathogen-interaction might allow the development of novel antimicrobials and biomarkers. 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 the predicted pre-miRNA-like sequences might have miRNA properties. Importantly, for the miRNA-like sRNA MIR6325 of B. suis we demonstrate a role in intracellular replication. Taken together, our preliminary data provide the proof of principle that the intracellular pathogen B. suis modulates the host-pathogen-interactions using the bacterial miRNA-like sRNA MIR6325. In addition, we provide evidence that C. burnetii encodes predicted miRNA-like sRNAs, which might also influence the outcome of infection. 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 Type IV Secretion Systems (T4SS), and produce Outer Membrane Vesicles (OMVs), which could be involved in the delivery of sRNAs alongside (or complexed with) bacterial proteins during infections; 2) both pathogens replicate to large numbers within membrane-bound replicative niches, greatly facilitating the intracellular localisation of bacterial components (proteins or miRNAs) in the course of infection, using either biochemical or fluorescence-based approaches.
In this project, we aim to dissect the function of the bacterial miRNAs and their role in infection using Next Generation Sequencing, cellular microbiology, high-resolution microscopy and screening approaches, which are all well-established in our consortium. In detail, we will 1) identify miRNAs transported during infection into the host cell at early and late time points of infection; determine whether stress conditions influence the expression and transport of miRNAs; compare the miRNA repertoire of different B. suis biovars and C. burnetii isolates. 2) Identify the transport pathway (OMVs or T4SS) and the processing mechanisms of the predicted miRNA-like sRNAs. 3) Identify and validate host cell targets of the bacterial miRNA-like sRNAs; and finally, we will 4) characterize the biological function of the miRNA-like sRNAs and their role during infection. Completing these aims will allow us to characterize the bacterial miRNAs and their potential role as essential virulence factors influencing the outcome of infection with the zoonotic pathogens Brucella spp. and C. burnetii.