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Prof. Cynthia Sharma, Julius-Maximilians-Universität Würzburg: Small RNAs and small proteins in stress response and virulence control of pathogenic Epsilonproteobacteria

Speaker Series. Abstract: Non-coding RNAs and associated RNA-binding proteins (RBPs) play central roles in diverse physiological processes and disease development in all kingdoms of life. Research in our lab focuses on RNA-based regulation in bacterial pathogens. We are especially interested in the molecular mechanisms and physiological roles of small, regulatory RNAs (sRNAs) and RBPs in bacterial stress response and virulence control. In my talk, I will describe multiple examples of diverse mechanisms of gene regulation in bacteria. For example, we recently discovered two sRNAs in the food-borne pathogen Campylobacter jejuni involved in flagella biogenesis and control of motility, a key virulence trait of Campylobacter. While biogenesis of the complex flagellar machinery requires strict hierarchical transcriptional control of the individual components, less is known about post-transcriptional control of flagella biogenesis via sRNAs. In C. jejuni, we found two sRNAs with opposing effects on the flagellar cascade: CJnc230 sRNA is processed from the mRNA encoding the flagellar structural hook protein FlgE and acts as an enhancer of flagellar length and motility. In contrast, overexpression of CJnc170 sRNA, which is expressed downstream in the transcriptional cascade, represses flagellar length and motility. This interplay of two sRNAs fine-tunes flagellar biosynthesis through balancing of the hierarchically expressed components and such post-transcriptional regulation of flagellar biogenesis via sRNAs is emerging to be more widespread among bacteria. In addition to small RNAs, we are also studying another class of small gene products, so-called small proteins (<70aa). To globally identify small open reading frames (sORFs) that encode small proteins, we have been employing and developing diverse ribosome profiling approaches for global translatome analyses. Small proteins are an emerging, yet largely unexplored class of gene products involved in diverse cellular processes, which often interact with larger protein complexes. For example, in addition to the above mentioned sRNAs involved in flagella regulation, we identified a small protein essential for flagella biogenesis and motility of C. jejuni. To globally identify interaction partners of RNAs, we are also employing genome-wide approaches, such as RBP capture methods. We recently developed a new RNA interactome approach for bacteria based on primary transcript capture. Our pilot screens in the intestinal pathogens Salmonella and Campylobacter jejuni successfully captured known RBPs but also uncovered many new RBP candidates, including metabolic or cell division proteins that lack canonical RNA binding domains. We also identified a pair of KH-domain proteins, KhpA and KhpB, in C. jejuni. KhpA/B homologs are widespread among bacteria, but their cellular roles remain poorly understood. Based on co-immunoprecipitation assays, we validated KhpA/B interaction and identified overlapping and distinct sets of RNAs bound by both proteins as well as crucial RNA binding domains of both proteins. Overall, many players in bacterial regulatory networks remain to be discovered. Deciphering their mechanisms will provide new insights into molecular principles of gene regulation as well as virulence control of bacterial pathogens and open new ways to better treat and prevent infections.

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