The human microbiota consists of around 100 trillion cells, which represent at least ten times as many cells constituting the human body. The majority of these microorganisms are located in the gastrointestinal tract. The processes governing interactions between the host and its microbiota remain largely uncharacterised. Enteric bacteria are known to secrete outer membrane vesicles (OMVs), which contain RNA in addition to DNA and proteins. OMVs have the ability to be uptaken by gastric epithelial cells where they can deliver bacterial factors. Extracellular RNA has been found to be a key component for developing immunity against Listeria spp. infection, in contrast to its intracellular RNA counterpart. Moreover, we have recently demonstrated that OMVs released from Escherichia coli contain small RNAs which have immunostimulatory properties. Furthermore, we have identified bacterial small RNAs in the plasma of healthy individuals. Thus, for the present project, we hypothesize that bacterial extracellular small RNA participate in host-microbe interactions analogous to exosome-mediated crosstalk between mammalian cells. To explore this research question, we will investigate further the RNA secretome of an enteric pathogen (Salmonella enterica serovar Typhimurium) and a probiotic strain (Escherichia coli Nissle 1917) in relation to their immunostimulatory effects in humans. In particular, we will carry out an in-depth characterization of the OMV-associated RNA fraction. Therefore, we plan to challenge primary human immune cells co-cultured or not with the Caco-2 epithelial cell line – mimicking the gut epithelial barrier – with extracellular RNAs derived from either pathogenic or probiotic strains. The primary read-out will be transcriptomic data. In parallel, we will investigate the bacterial extracellular RNA fraction by deep sequencing and carry out in silico target prediction analysis against human genes or microRNAs. From our own preliminary data, we hypothesize that bacterial extracellular RNAs are not only being sensed as pathogen-associated molecular patterns but they might also act as antisense molecules in human immune cells and/or epithelial cells which may lead to suppression of host gene and/or microRNA functions. The action of such antisense exogenous RNAs has recently been described for a plant residing fungal pathogen that transfers its small RNA effector into its host cells to suppress host immunity in order to achieve infection. Our hypothesis is that this cross-kingdom RNA interference mechanism is not limited to host-fungi interactions but that it may also play a role in host-bacteria interactions. An understanding of the potential role of bacterial derived exogenous RNA in host-microbe interactions will elucidate new mechanisms of cross-talk and perhaps allow us to identify new drug targets and/or to develop RNA-based vaccines.