Abstract
Bacterial cold shock proteins (CSPs) function as RNA chaperones. To assess CSP's roles in the intracellular human pathogen Salmonella Typhimurium, we analyzed their expression in varied stress conditions. We found that cold shock protein E (cspE or STM14_0732) is up-regulated during bile salt-induced stress and that an S. Typhimurium strain lacking cspE (ΔcspE) displays dose-dependent sensitivity to bile salts, specifically to deoxycholate. We also found that an uncharacterized gene, yciF (STM14_2092), is up-regulated in response to bile stress in WT but not in the ΔcspE strain. Complementation with WT CspE, but not with a F30V CspE variant, abrogated the bile sensitivity of ΔcspE as did multicopy overexpression of yciF. Northern blotting experiments with rifampicin disclosed that the regulation of yciF expression is, most likely, due to the RNA-stabilizing activity of CspE. Importantly, electrophoretic mobility shift assays indicated that purified CspE, but not the F30V variant, directly binds yciF mRNA. We also observed that the extra-cytoplasmic stress-response (ESR) pathway is augmented in the bile-treated ΔcspE strain, as judged by induction of RpoE regulon genes (rpoE, degP, and rybB) and downstream ESR genes (hfq, rne, and PNPase). Moreover, the transcript levels of the porin genes, ompD, ompF, and ompC, were higher in bile salts-stressed ΔcspE and correlated with higher intracellular accumulation of the fluorescent DNA stain bisBenzimide H 33258, indicating greater cell permeability. In conclusion, our study has identified YciF, a CspE target involved in the regulation of porins and in countering bile stress in S. Typhimurium.
Highlights
Bacterial cold shock proteins (CSPs) function as RNA chaperones
A family of genes involved in this response are the cold shock proteins (CSPs)3 consisting of 67–73 amino acids that are evolutionarily conserved across all three domains of life and are implicated to function as RNA chaperones [3]
Upon cspE complementation and yciF-mediated phenotype suppression, there was a lesser induction of the extra-cytoplasmic stress-response (ESR) pathway components, rpoE and rybB (Fig. S12), and the porin transcripts were significantly lower during bile stress (Fig. 6B)
Summary
Bacterial cold shock proteins (CSPs) function as RNA chaperones. To assess CSP’s roles in the intracellular human pathogen Salmonella Typhimurium, we analyzed their expression in varied stress conditions. We found that cold shock protein E (cspE or STM14_0732) is up-regulated during bile salt-induced stress and that an S. Salmonella Typhimurium CspE regulates bile resistance chaperone” under general stress conditions [18], enhances translation of several mRNAs [9], and is important in imparting camphor resistance [19]. It interferes with bacteriophage Q-mediated transcription anti-termination [20], regulation of the poly(A)-mediated 3Ј–5Ј-exonuclease activity of polynucleotide phosphorylase, and cleavage and poly(A) tail removal by RNase E [21]. The identification of YciF (STM14_2092) as a CspEregulated protein and its role in regulating the outer membrane porins and cell permeability during bile stress are the highlights of this study
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