RNA target profiles direct the discovery of virulence functions for the cold-shock proteins CspC and CspE

Abstract

The functions of many bacterial RNA-binding proteins remain obscure because of a lack of knowledge of their cellular ligands. Although well-studied cold-shock protein A (CspA) family members are induced and function at low temperature, others are highly expressed in infection-relevant conditions. Here, we have profiled transcripts bound in vivo by the CspA family members of Salmonella enterica serovar Typhimurium to link the constitutively expressed CspC and CspE proteins with virulence pathways. Phenotypic assays in vitro demonstrated a crucial role for these proteins in membrane stress, motility, and biofilm formation. Moreover, double deletion of cspC and cspE fully attenuates Salmonella in systemic mouse infection. In other words, the RNA ligand-centric approach taken here overcomes a problematic molecular redundancy of CspC and CspE that likely explains why these proteins have evaded selection in previous virulence factor screens in animals. Our results highlight RNA-binding proteins as regulators of pathogenicity and potential targets of antimicrobial therapy. They also suggest that globally acting RNA-binding proteins are more common in bacteria than currently appreciated.RNA-binding protein | cold-shock protein | Salmonella | bacterial pathogenesis | stress response T he myriad of coding and noncoding RNAs in a cell generally do not act in isolation but rapidly associate with RNA-binding proteins (RBPs) to execute their functions. Recent methods relying on the global capture of polyadenylated transcripts in eukaryotes have dramatically expanded our knowledge of RBP activity. These approaches have revealed many previously unsuspected RBPs, suggesting a much wider scope of posttranscriptional control based on thousands of new RBP-mRNA interactions (1, 2). Nevertheless, the functions of many of these newly discovered RBPs remain unclear.In contrast to the situation in eukaryotes, there is a paucity of knowledge about RBPs in prokaryotes. This lack of knowledge is compounded further by the lack of a poly(A) tail on functional transcripts, which precludes similar global discovery studies of bacterial RBP networks. Extensive profiling of cellular targets of the small RNA (sRNA) chaperone Hfq and the translational repressor CsrA (3-7) have shown that large posttranscriptional networks also exist in bacteria. In addition, the ProQ protein has recently been identified as a previously overlooked global RBP in Salmonella enterica (8). As new methods are developed to identify RPBs in bacteria globally, it is essential to be able to analyze their functions systematically to understand their cellular and physiological roles (9).Cold-shock proteins (CSPs) constitute the largest nonribosomal RBP family in Gram-negative bacteria, including the model species Escherichia coli and Salmonella enterica. Their conserved nucleic acid-binding cold-shock domain makes them members of a widespread RBP superfamily that includes the well-investigated eukaryotic Y-box proteins (10, 11). Of the nine and six CSP paralogs present in E. c...

The Ralstonia solanacearum csp22 peptide, but not flagellin‐derived peptides, is perceived by plants from the Solanaceae family

“…However, genes encoding CSP proteins are not only expressed in cold temperatures, but also constitutively and under other stresses (Bae et al, 2000). Besides their known roles in the regulation of cellular growth and adaptation to environmental conditions, recent studies have shown that specific CSPs are important for pathogenicity in the enterobacterial pathogen Salmonella enterica, where they are induced at higher temperatures, revealing a role for CSPs in bacteria-host interaction (Michaux et al, 2017). In R. solanacearum, genes encoding proteins that contain csp22-like sequences are expressed during the infection in tomato (Jacobs et al, 2012).…”

RNA-binding proteins in bacteria

“…While the full scope of CspA activity remains to be described, CspC and CspE are now established as truly globally acting RBPs 44,45 . Their combined activities impact the expression of 20% of all genes in Salmonella and are essential for the virulence of this pathogen 44 .…”

Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

“…6c). Comparing recovered protein intensities from UVirradiated versus control cells (biological duplicates), we find 172 proteins ( Supplementary Table S3), among them 33 ribosomal proteins, components of the RNA polymerase com-plex (subunit α, σ factor RpoD, DksA) and 4 out of the 5 established mRNA-binding proteins of Salmonella (Hfq, ProQ, CspC/CspE) (63,65,66). 113 of the enriched proteins are so-far unknown to interact with RNA.…”