Role of CsrA in stress responses and metabolism important for Salmonella virulence revealed by integrated transcriptomics

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.

Fig 1. Overview of experimental design.

Wild type Salmonella enterica serovar Typhimurium strain 14028s and an isogenic csrA mutant strain were grown in LB and mLPM media to mid-exponential phase. For analyses of translation, RNA abundance, and translation efficiency, cultures were treated with chloramphenicol to stall translation elongation. Samples were then quickly isolated and lysed under ribosome stabilizing conditions. Part of this lysate was treated with micrococcal nuclease, and ribosomes were isolated by ultracentrifugation through a sucrose cushion. Ribosome protected fragments (RPF) were extracted, size selected, and used to generate sequencing libraries reflecting the actively translated RNA. Another part of the lysate was used to extract total RNA. This RNA was depleted of rRNA, fragmented, and size selected. These RNA fragments were used to generate sequencing libraries reflecting steady state RNA levels. For analysis of RNA stability, cultures were treated with rifampicin to stop transcription initiation. Samples were collected at 0, 2.5, 5, 10, and 15 minutes after the addition of rifampicin and immediately stabilized with a phenol/ethanol stop solution. Total RNA was purified and depleted of rRNA. Sequencing libraries were prepared that reflect the abundance of RNA as it decays after the addition of rifampicin.

Fig 2. Integration of results from different transcriptomics methods.

Number of genes with changes in their expression in (A) mLPM, (B) LB, and (C) a comparison between mLPM and LB. Data for predicted direct CsrA targets was taken from Kulkarni et al. [53] (CSRA-TARGET) and Homlqvist et al. [52] (CLIP-seq).

Fig 3. Exposure to mLPM medium reduces CsrB levels.

(A) Levels of CsrB were analyzed during mid-exponential growth in LB medium and at several time points after transfer to several variations of mLPM medium. CsrB levels were quantified from the northern blot in (B) and displayed as arbitrary units (A.U.) after normalized to the 16s rRNA levels. Data from an additional experiment are presented in S6 Fig, showing that this response is reproducible.

Fig 4. GO terms significantly enriched in CsrA regulated genes.

Fold enrichment of GO terms in genes regulated by CsrA in both mLPM and LB (A) and in either LB or mLPM (B). Significance was determined with a Fisher’s exact test with TopGO [152]. Categories shown twice in panel B are indicative of larger lists of non-overlapping genes in mLPM and/or LB vs. genes in common. The list of significant terms (S6 Table) was shortened for visualization with REViGO [153].

Fig 5. CsrA regulates genes encoded in Salmonella pathogenicity islands.

RNA sequence and mFold [154] predicted structure of 5’-untranslated and early coding regions and qRT-PCR analysis of steady state mRNA levels of (A) sopD2 and (B) iacP. GGA motifs are shown in red and CLIP-seq peaks from Holmqvist et al. [52] are shown in bold. RNA levels were assessed at mid-exponential growth in LB (green) or mLPM (purple) and normalized to the 16S rRNA levels. Error bars show SEM of three biological replicates. Statistical significance was determined with a two-sided student’s t-test. Asterisks denote statistical significance (p<0.05: *, p<0.005: **, p<0.0005: ***).

Fig 6. CsrA regulates oxidative stress responses.

(A) qRT-PCR analysis of steady state argT and katE mRNA levels during mid-exponential growth in LB (green) or mLPM (purple) normalized to the 16S rRNA levels. Error bars show SEM of three biological replicates. Statistical significance was determined with a two-sided student’s t-test. Asterisks denote statistical significance (p<0.05: *, p<0.005: **, p<0.0005: ***). (B) Survival of wild type and csrA mutant strains upon exposure to H₂O₂. Strains in mid-exponential phase of growth in mLPM or LB were exposed to 20 μM or 5 μM of H₂O₂, respectively, for the indicated times. Washed and 10-fold serially diluted samples were plated and grown overnight on LB agar plates before imaging. Data from a biological replicate analyzed in the same experimented is presented in S7 Fig. Altogether, this experiment was repeated three times with similar results.

Fig 7. CsrA regulates metabolism important for establishing infection in the intestine.

(A) Summary of the effect of CsrA on the expression of genes involved growth in the intestine. Squares represent the effect of CsrA on translation (RPF, left), RNA abundance (RNA, top), RNA stability (stability, bottom), and translational efficiency (TE, right). Purple indicates repression by CsrA, and green indicates activation. Asterisks show gene(s) that are associated with CLIP-seq peak(s) and/or were predicted to express CsrA-binding transcripts [52,53]. Genes of SPI-1 or SPI-2 are represented as a single entity although the specific genes vary in response to CsrA effects and as direct CsrA targets. S2 Table contains a full description of the observed effects. (B) Levels of steady state appC (cyxA), cydA and narZ mRNA levels during mid-exponential growth in LB normalized to the 16S rRNA levels measured with qRT-PCR and shown in arbitrary units (A.U.). Error bars show SEM of three biological replicates. Statistical significance was determined with a two-sided student’s t-test. Asterisks denote statistical significance (p<0.05: *, p<0.005: **, p<0.0005: ***).

Fig 8. Models of Csr system in mLPM and LB.

Dotted lines represent repression and solid lines activation. These arrows represent both direct and indirect effects, as we are unable to differentiate these without further study.