Interplay of cold shock protein E with an uncharacterized protein, YciF, lowers porin expression and enhances bile resistance in Salmonella Typhimurium

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 (⌬cspE) 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 ⌬cspE strain. Complementation with WT CspE, but not with a F30V CspE variant, abrogated the bile sensitivity of ⌬cspE 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 ⌬cspE 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 saltsstressed ⌬cspE 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.

RpoS-regulated SEN1538 gene promotes resistance to stress and influences Salmonella enterica serovar enteritidis virulence

“…This explained Δ1538 showing a sensitized phenotype to several stress factors, which are likely to be encountered during host infection. Δ1538 displayed reduced survival under bile stress, corroborating with the previously reported role of the KGG family protein YciG [16,51,52].…”

Salmonella Typhimurium encoded cold shock protein E is essential for motility and biofilm formation

“…Typhimurium 14028 s (WT) was used as the parent strain for all experiments. The ΔcspE strain was reported in an earlier study [15] and the ΔcspA strain was generated similarly and confirmed using primers that are listed in Table S2. The double deletion of cspE and cspA were generated by amplifying the region flanking cspA and electroporating the amplicon into ΔcspE cells harboring pKD46 plasmid (Table S2).…”

“…Antibiotics were used at the following concentrations: Ampicillin-100µg ml -1 (HiMedia Laboratories), Chloramphenicol-30 µg ml −1 (HiMedia Laboratories), and Kanamycin-50 µg ml −1 (Sigma Aldrich, Missouri, USA). Arabinose (HiMedia Laboratories) was used at 40 mM for induction of Red recombinase by pKD46 [15].…”

“…Several members of the CSP family are non-cold inducible and their expression is induced during different stresses [8]. A variety of functions have been attributed to the CspA homologs such as adaptation to stationary phase and nutrient deprivation [9], antibiotic synthesis [10], UV sensitivity [11], regulation of expression of several stress response proteins [12], camphor resistance and chromosome condensation [13], resistance to toluene [14] and bile [15].…”

“…Several other additive factors add to the final outcome of a sturdy biofilm, namely, flagella [51], O-antigen capsule [52], type I fimbriae [53] and other uncharacterised polysaccharides [54]. In an earlier study [15], we had observed that CspE lacking S. Typhimurium 14028 s was sensitive to bile stress. In order to implicate an in vivo scenario of attenuated host colonisation, we investigated the possible regulation of biofilm by CspE.…”