Abstract
To cause infection, Salmonella must survive and replicate in host niches that present dramatically different environmental conditions. This requires a flexible metabolism and physiology, responsive to conditions of the local milieu. The sequence specific RNA binding protein CsrA serves as a global regulator that governs gene expression required for pathogenicity, metabolism, biofilm formation, and motility in response to nutritional conditions. Its activity is determined by two noncoding small RNAs (sRNA), CsrB and CsrC, which sequester and antagonize this protein. Here, we used ribosome profiling and RNA-seq analysis to comprehensively examine the effects of CsrA on mRNA occupancy with ribosomes, a measure of translation, transcript stability, and the steady state levels of transcripts under in vitro SPI-1 inducing conditions, to simulate growth in the intestinal lumen, and under in vitro SPI-2-inducing conditions, to simulate growth in the Salmonella containing vacuole (SCV) of the macrophage. Our findings uncovered new roles for CsrA in controlling the expression of structural and regulatory genes involved in stress responses, metabolism, and virulence systems required for infection. We observed substantial variation in the CsrA regulon under the two growth conditions. In addition, CsrB/C sRNA levels were greatly reduced under the simulated intracellular conditions and were responsive to nutritional factors that distinguish the intracellular and luminal environments. Altogether, our results reveal CsrA to be a flexible regulator, which is inferred to be intimately involved in maintaining the distinct gene expression patterns associated with growth in the intestine and the macrophage.
Highlights
Salmonella enterica serovar Typhimurium (Salmonella) is a major cause of foodborne illness and diarrheal disease worldwide [1,2,3,4,5,6,7]
As we expect that CsrA will indirectly affect the transcription of many genes, this will result in changes in RNA abundance and translation of indirect targets
We used ribosome profiling and paired RNA-seq in an integrated analysis of differential translation (RPF levels), steady state RNA abundance (RNA levels), and translation efficiency (RPF and RNA levels) of protein coding genes in the wild type and csrA mutant strains grown in LB and mLPM media (Fig 1)
Summary
Salmonella enterica serovar Typhimurium (Salmonella) is a major cause of foodborne illness and diarrheal disease worldwide [1,2,3,4,5,6,7]. The SPI-1 T3SS is required for initial invasion of the intestinal epithelium, and its expression is controlled by the transcriptional regulators HilD and HilA [13,14,15]. A variety of other regulators that sense diverse environmental signals converge to control the expression of HilD and SPI-1 [16,17]. In vitro the SPI-1 T3SS is induced by late exponential growth in rich media [18,19]. SPI-2 expression is induced in vitro by growth in acidic minimal media with limiting concentrations of magnesium and phosphate or in late stationary phase in rich media [18]. HilD regulates expression of SPI-1 but it controls SPI-2 expression in rich media by activating the transcription of the genes encoding its master regulator SsrA-SsrB [18,30]
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