2024BBSRC-DFG: Dissecting the RNA-centric landscape of microbiota-pathogen interactions in the gut (MiPa-RNA)

The microbiota plays a critical role in maintaining human and animal health. This is achieved in a multitude of ways such as ‘training’ of the host immune system during early mammalian development, and occupation of spatial and metabolic niches within the physical gut environment. A key function of these traits is to antagonize invasion by foreign enteric pathogens – a process collectively known as colonisation resistance. However, diverse pathogens can exploit the natural function of anaerobic symbionts (such as Bacteroidesthetaiotaomicron), for example during the breakdown of complex polysaccharides contained within dietary fibre, to establish an infectious niche in the gut or a replicative reservoir for transmission and dissemination. This is achieved through the scavenging and sensing of dietary metabolites, liberated during digestion, not only as nutrients for growth but also as regulatory effectors that stimulate the expression of key pathogen genes, including those encoding virulence and fitness-enhancing factors. This molecular crosstalk between commensal and pathogenic species in the gut underlies enteric infection by many priority pathogens such as pathogenic Escherichia coli. However, our understanding of the molecular mechanisms driving these interactions is based largely on a wealth of in vitro studies, leaving the in vivo relevance of such mechanisms poorly understood. To adapt to dynamic environments and stressful conditions, pathogenic bacteria rely on post-transcriptional control mechanisms brought about by regulatory RNA. The best understood and most prevalent class of bacterial RNA regulators comprises the so-called small RNAs (sRNAs). These ~50-250 nt-long RNA molecules typically regulate target gene expression by mediating base-pair interactions with complementary stretches within target mRNAs and control a wide range of cellular processes. For example, target genes under the control of regulatory RNAs are often involved in metabolism or stress response, as well as those encoding pathogenic virulence factors that are essential for breaking the colonisation resistance barrier. Furthermore, sRNA-mediated gene expression control likely grants a substantial evolutionary benefit in commensal species inhabiting the variable ecosystem of the mammalian gut, yet represents an understudied branch of current microbiota research. The majority of existing RNA biology research has focused on deep mechanistic in vitro studies of regulatory elements, leaving their relevance and impact within the host environment largely unknown. We will combine innovative cross-species transcriptomic and CRIPSR-based functional genomics with relevant in vivo models (using B. thetaiotaomicron, E. coli and Citrobacter rodentium) and classical genetics/biochemistry to dissect the role sRNAs play in vivo. Our work will reveal crucial new insights into the underlying molecular mechanisms of how RNA-based regulation contributes to colonisation resistance from the perspective of the microbiota and invading pathogens. A deeper knowledge of RNA regulatory interactions would pave the way for RNA-based therapeutics as a promising alternative to traditional antibiotics.