Searching for Novel sRNA‐Binding Proteins in Chlamydia trachomatis

Abstract

Non‐coding small RNAs (sRNAs) are regulators of mRNA expression, and sRNA‐mediated gene expression plays an important role in bacterial processes such as stress responses, metabolism, and virulence, providing a promising field of study to understand bacterial behavior. In the well‐studied case of E. coli, global RNA‐binding proteins such as Hfq and ProQ are important for supporting the stability and function of sRNAs. However, many bacterial species that produce sRNAs do not encode either of these RNA‐binding proteins. This raises the intriguing possibility that there may be as‐of‐yet‐undiscovered RNA‐binding proteins in the subset of bacteria that do not possess an Hfq or ProQ protein. Among such bacteria, Chlamydia trachomatis is a human pathogen that causes a sexually transmitted infection and is the most common cause of blindness worldwide. C. trachomatis has been shown to express numerous sRNAs (Elwell et al., 2016; Klepsch, 2020), but its genome does not encode Hfq or ProQ. In this study, we are employing a bacterial three‐hybrid (B3H) assay to search for new RNA‐binding proteins encoded in the genome of this pathogenic bacterium. This B3H assay, developed by Berry and Hochschild (2018) to assess RNA‐protein interactions, was derived from a bacterial two‐hybrid (B2H) assay, which has been used with a genomic‐fragment library from C. trachomatis to identify a new protein‐protein interaction in Rao et al. (2009). To screen for new sRNA‐protein interactions in C. trachomatis, four sRNAs (ctrR1, ctrR2, ctrR0332, and IhtA), previously identified by Albrecht et al. (2010), were cloned into the B3H system’s pBait (RNA moiety) and transformed into reporter cells with a pPrey (protein moiety) genomic‐fragment library from C. trachomatis. Some candidate sRNA‐binding peptides and proteins have been found through this screening process. And, we are conducting β‐galactosidase assays to quantify the interactions of these candidates with sRNAs, along with computational analysis to map the candidate fragments back to the context of the Chlamydia genome and proteome. In the future, we hope to confirm the underlying mechanisms of interactions through site‐directed mutagenesis and to expand our screens to the genomes of additional bacterial pathogens such as Mycobacterium tuberculosis. By finding novel sRNA‐binding proteins in C. trachomatis and M. tuberculosis, we would expand our understanding of RNA‐based regulation in these bacteria and potentially provide new pharmaceutical targets in the future.