Dissecting interactomes and functions of the RNA binding proteins KhpA and KhpB in the gastric pathogen Helicobacter pylori

RNA binding proteins (RBPs) play central roles in gene regulation in all kingdoms of life. In bacteria, very little is known about globally acting RBPs beyond the well-studied RBPs Hfq, ProQ, and CsrA. Many bacteria, including the gastric pathogen Helicobacter pylori, lack homologs of Hfq and ProQ. This raises the question: Are there other RBPs with global regulatory roles in bacteria lacking these well-studied RBPs? Recently, two KH domain-containing proteins, KhpA and KhpB, which are widely conserved across many bacterial species, have been suggested as a new class of globally acting RBPs. RNA-immunoprecipitation and sequencing (RIP-seq) in Streptococcus pneumoniae and Clostridiodes difficile revealed that KhpA/B bind to a large number of RNAs, including sRNAs and mRNAs. KhpA/B are associated with cell division, virulence, and toxin production in these bacteria as well. In H. pylori, KhpB was shown to interact with and modulate the activity of the ATPase of the type 4 secretion system (T4SS), a key virulence factor required to inject the effector protein CagA into the host cell. The RNA binding activity of KhpA/B and their potential roles in regulating the cellular processes of H. pylori, including T4SS activity, remain unexplored. To understand the roles of H. pylori KhpA/B, we identified their RNA interaction partners using RIP-seq. Our data indicate that H. pylori KhpA/B interact with 5"UTRs and coding sequences of genes, several of which can be validated by electrophoretic mobility shift assays (EMSA). Protein interactome capture of KhpA/B by co-immunoprecipitation of FLAG-tagged proteins followed by mass spectrometry indicates potential interactions of KhpA/B with a larger number of proteins associated with different layers of bacterial physiology. Our preliminary results show that the deletion of khpA/B impacts H. pylori stress responses and gene expression, suggesting that they could play important roles in H. pylori cellular processes and potentially, infections.