Date Published: January 25, 2019
Publisher: Public Library of Science
Author(s): Anastasia H. Potts, Yinping Guo, Brian M. M. Ahmer, Tony Romeo, Roy Martin Roop.
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.
Salmonella enterica serovar Typhimurium (Salmonella) is a major cause of foodborne illness and diarrheal disease worldwide [1–7]. Salmonella utilizes a variety of dedicated virulence factors, many of which are encoded within horizontally acquired genomic regions called Salmonella pathogenicity islands (SPI)[8,9]. The best characterized of these are SPI-1 and SPI-2, each of which encodes a type III secretion system (T3SS)[10–12]. 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 [13–15]. A variety of other regulators that sense diverse environmental signals converge to control the expression of HilD and thus SPI-1 [16,17]. In vitro the SPI-1 T3SS is induced by late exponential growth in rich media [18,19]. The SPI-2 T3SS is important for later stages of infection, including establishment of a modified phagosomal compartment called the Salmonella containing vacuole (SCV) and manipulation of host cells to promote intracellular growth and survival [10,12,20–23]. The two-component regulatory system (TCS) SsrA-SsrB integrates a variety of regulatory signals to control SPI-2 expression [24–29]. 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 . There is significant crosstalk between regulators of the SPIs, and models with distinct roles for SPI-1 and SPI-2 in early and later stages of infection, respectively, are likely overly simplistic [18,24]. For example, HilD regulates expression of SPI-1 but it also controls SPI-2 expression in rich media by activating the transcription of the genes encoding its master regulator SsrA-SsrB [18,30].
To our knowledge, this is the first study to examine the global effects of CsrA on gene expression under more than one growth condition. As a result, we found that the growth condition is a strong determinant of CsrA-dependent regulation. There may be multiple explanations for the observed condition-specific effects. First, differences in CsrA activity between the two conditions may contribute. CsrA may also require coregulators expressed in only one condition. In addition, transcription from alternative promoters under the two conditions may present alternative sites for CsrA-dependent regulation. On the other hand, conditionally regulated genes may be indirectly responsive to CsrA via regulator(s) that are subject to the direct effects of CsrA as well as differentially affected by the two growth conditions. Regardless of the regulatory mechanisms, it is difficult to predict the effect of CsrA on gene expression under new growth conditions based on the wealth of data reported here. Nevertheless, our findings reveal that CsrA acts as a truly flexible global regulator of gene expression.