CsrA interacts directly with <i>fleQ</i>-mRNA <i>in vitro</i>.

<p><b>A</b>) Electromobility shift assays (EMSA) with 200nM of biotinylated <b><i>fleQ</i>-mRNA</b> combined with varying concentrations of purified CsrA-His were undertaken in a 6% Native Tris-PAGE gel. Lane 1: no CsrA, lane 2: 0.2 μM CsrA, lane 3: 0.5 μM CsrA, lane 4: 1.0 μM CsrA, lane 5: 2.0 μM CsrA, lane 6: 5.0 μM CsrA, lane 7: 5.0 μM CsrA + 2.0 μM unlabeled RsmZ. <b>B</b>) Mfold secondary structure prediction of the <b><i>fleQ</i>-mRNA</b> fragment used for the EMSA. Red, two potential CsrA-binding sites, which are mutated in C. Blue, transcriptional start codon. <b>C</b>) EMSA with recombinant CsrA and 200 nM of non-mutated RNA (pFleQ) or mutated in the indicated regions (mFleQ). AGGA motifs were replaced by an AAAA sequence using PCR mutagenesis. Lane 1: no CsrA + non-mutated <b><i>fleQ</i>-mRNA</b>, lane 2: 5.0 μM CsrA + non-mutated <b><i>fleQ</i>-mRNA</b>, lane 3: 5.0 μM CsrA + m<b><i>fleQ</i>-mRNA</b> mutated in region 1, lane 4: 5.0 μM CsrA + m<b><i>fleQ</i>-mRNA</b> mutated in region 2, lane 5: 5.0 μM CsrA + m<b><i>fleQ</i>-mRNA</b> mutated in both region 1 and 2.</p

CsrA interacts directly with <i>lqsR</i> mRNA <i>in vitro</i>.

<p><b>A)</b> Electromobility shift assay (EMSA) with 200nM of biotinylated <i>lqsR</i> mRNA combined with varying concentrations of purified CsrA-His <i>lqsR</i> mRNA and recombinant CsrA in 6% Native Tris-PAGE. Lane 1: no CsrA, lane 2: 0.2 μM CsrA, lane 3: 0.5 μM CsrA, lane 4: 1.0 μM CsrA, lane 5: 2.0 μM CsrA, lane 6: 5.0 μM CsrA, lane 7: 5.0 μM CsrA + 2.0 μM unlabled RsmZ. <b>B)</b> Mfold secondary structure prediction of the <i>lqsR</i> mRNA fragment used for the EMSA. Red, two potential CsrA-binding sites, which are mutated in C. Blue, transcriptional start codon. <b>C</b>) EMSA with recombinant CsrA and 200 nM of non-mutated RNA (pLqsR) or mutated in the indicated regions (mLqsR). AGGA motifs were replaced by an AAAA sequence using PCR mutagenesis. Lane 1: no CsrA + non-mutated <i>lqsR</i> mRNA, lane 2: 5.0 μM CsrA + non-mutated <i>lqsR</i> mRNA, lane 2: 5.0 μM CsrA + m<i>lqsR</i> mRNA mutated in region 1, lane 3: 5.0 μM CsrA + m<i>lqsR</i> mRNA mutated in region 2, lane 4: 5.0 μM CsrA + m<i>lqsR</i> mRNA mutated in both region 1 and 2.</p

CsrA modulates the expression of a thiamine pyrophosphate (TPP) riboswitch element.

<p><b>A</b>) Schematic representation of the <i>thi</i>-operon in <i>L</i>. <i>pneumophila</i> including the transcriptional start site (TSS), the CsrA-binding region, the thi element of the predicted TPP riboswitch and a predicted transcription termination site upstream of the start codon (AUG). The CsrA-binding site is overlapping the <i>thi</i> element of the TPP riboswitch. This organization suggests that CsrA is implicated in the fine-tuning of the expression of the downstream <i>thi</i>-operon most probably due to conformational changes in the secondary RNA structure. <b>B</b>) EMSA with 200nM of biotinylated thi-element (TPP) RNA and purified CsrA: Lane 1: no CsrA, lane 2: 1.0 μM CsrA, lane 3: 2.0 μM CsrA, lane 4: 5.0 μM CsrA, lane 5: 5.0 μM CsrA + 2.0 μM unlabeled RsmZ. <b>C)</b> Beta-lactamase (BlaM) assay in minimal medium grown <i>Legionella</i> without, with 1 mM and with 2 mM of TPP. BlaM activity in 10μg total protein of wt and <i>csrA</i>^<i>-</i> strain containing the 5'UTR of the <i>thi</i>-operon in a pXDC61 plasmid was measured. Each value represents the mean +/- SD of three independent experiments. BlaM activity is significantly decreased in the mutant at the different conditions indicating a positive effect of CsrA on the <i>thi</i>-operon expression in <i>L</i>. <i>pneumophila</i>. <b>D</b>) Model of the TPP riboswitch modulated by CsrA. Mfold prediction of the secondary structure of the 5'UTR <i>thi</i>-region: When TPP is bound, the OFF state of the riboswitch is favored in which the expression of the operon is inhibited (most likely due to premature termination at the predicted termination site). The presence of CsrA in contrast might stabilize the ON state where the structure of the thi-element is dispersed, hence, higher amounts of TPP would be necessary to shift the element back to the OFF state leading to the down-regulation of the <i>thi</i>-genes expression.</p

CsrA feedback regulation and autoregulation of the stringent response.

<p><b>A</b>) Electromobility shift assay (EMSA) with 200nM of biotinylated RNA demonstrating the interaction of purified CsrA and <i>relA</i> mRNA <i>in vitro</i>. 200nM of biotinylated <i>relA</i> mRNA and increasing concentrations of recombinant CsrA in 6% Native Tris-PAGE were used. Lane 1: no CsrA, lane 2: 0.5 μM CsrA, lane 3: 1.0 μM CsrA lane 4: 2.0 μM CsrA, lane 5: 5.0 μM CsrA, lane 6: 5.0 μM CsrA + 2.0 μM unlabeled RsmZ. <b>B</b>) EMSA with 200nM of biotinylated RNA demonstrating the interaction of purified CsrA and <i>rpoS</i> mRNA <i>in vitro</i>. 200nM of biotinylated <i>rpoS</i> mRNA and increasing concentrations of recombinant CsrA in 6% Native Tris-PAGE were used. Lane 1: no CsrA, lane 2: 1.0 μM CsrA, lane 3: 2.0 μM CsrA, lane 4: 5.0 μM CsrA, lane 5: 5.0 μM CsrA + 2.0 μM unlabeled RsmZ. <b>C</b>) Model of the stringent response and quorum sensing network in <i>L</i>. <i>pneumophila</i> and the role of CsrA on their regulation. During the transmissive phase, amino acid and fatty acid starvation triggers the GTP pyrophosphokinase RelA and the ppGpp synthetase/hydrolase SpoT to produce the alarmone (p)ppGpp. Amongst others, the (p)ppGpp production results in a higher transcription rate of the small ncRNAs RsmX, RsmY and RsmZ which dissociate the RNA-binding protein CsrA from its target-RNAs. This leads to an activation of RpoS, LqsR and PmrA expression (positive feedback) that were formerly repressed by CsrA and an inhibition of RelA (negative feedback). Predicted negative effects in the regulatory cascade are represented by red lines, positive effects by black arrows.</p

CsrA acts as a positive regulator for Glyceraldehyde 3-phosphate (Gap) by preventing premature transcriptional termination of the PPP/Glycolysis-operon.

<p><b>A</b>) RNA secondary structure Mfold-prediction of the CsrA-binding region inside the <i>gap</i> gene reveals two major conformations: the left one contains a potential hairpin-terminator while the A(N)GGA-motif is covered in a double-strand region with low affinity to CsrA. The right one, shows the A(N)GGA-motif located in an open loop with high CsrA-interaction affinity and the hairpin structure is disrupted. Below, the nucleic acid sequence is shown that was used for Mfold modeling and the transcription termination assays. Red, CsrA-binding site A(N)GGA; green, the potential transcription terminator hairpin; blue, the putative auxiliary element of Rho-dependent termination. <b>B</b>) Left panel: <i>In vitro</i> transcription termination assay in presence of 1 μM of purified NusG-protein and varying concentration of Rho- and CsrA-protein (+ 0.5 μM, ++ 1μM; Lane 1: no Rho, no CsrA, lane 2: 1 μM Rho, no CsrA, lane 3: 1 μM Rho, 0.5 μM CsrA, lane 4: 1 μM Rho, 1 μM CsrA, lane 5: 1 μM Rho, no CsrA, lane 6: 1 μM Rho, 1 μM CsrA). A representative 10% urea-PAGE gel shows the formation of the truncated transcript from the Rho-dependent termination without CsrA and the full-length transcript with CsrA. Right panel: <i>In vitro</i> transcribed of the run-off fragment and the marker showing the size of the fragment. <b>C</b>) Regulatory model of the transcription of the PPP/Glycolysis operon. In absence of CsrA, Rho-dependent termination within the operon is responsible for polarity effects downstream of the transcriptional block. This leads to reduced transcript-levels of the <i>gap</i> gene whereas the <i>tkt</i> gene is not affected. When CsrA binds to the RNA, an anti-terminator structure is favored preventing that the elongation complex stalls at the hairpin structure. As a consequence, only the presence of CsrA ensures the efficient transcription of the glycolysis/gluconeogenesis genes of the operon.</p

Summary of the genes influenced by CsrA and discussed in detail.

<p>Summary of the genes influenced by CsrA and discussed in detail.</p

Glyceraldehyde 3-phosphate (Gap) and transketolase (Tkt) transcription is regulated differently by CsrA.

<p><b>A</b>) Schematic organization of the PPP/Glycolysis-operon in <i>L</i>. <i>pneumophila</i> Paris. TSS indicates the transcriptional start site under the control of an RpoD-dependent promoter. Green, bold arrows show the CsrA-binding region and black arrows highlight the region where qRT-PCR was conducted. <b>B</b>) EMSA with 200nM of biotinylated RNA demonstrating the interaction of purified CsrA and <i>gap</i> mRNA: Lane 1: no CsrA, lane 2: 0.5 μM CsrA, lane 3: 1.0 μM CsrA, lane 4: 2.0 μM CsrA, lane 5: 5.0 μM CsrA, lane 6: 5.0 μM CsrA + 2.0 μM unlabled RsmZ. Right side, run-off transcript produced under optimal <i>in vitro</i> transcription conditions performed with the MEGAshortscript Kit (ambion) to show transcript length compared to the low range ssRNA ladder (NEB). <b>C</b>) qRT-PCR results of the <i>gap</i> and the <i>tkt</i> transcripts at different growth stages (OD) between wt and <i>csrA</i>^- show lower expression levels of the <i>gap</i> gene in E-phase (OD1-3) in absence of CsrA whereas <i>tkt</i> is not affected. No differences are noticed during transition (OD3) and PE-phase. Complementation of the <i>csrA</i>^- strain restored the wt transcript levels</p

Half of the <i>L</i>. <i>pneumophila</i> proteins are differentially expressed upon CsrA deletion.

<p>Protein intensities in the wt and <i>csrA</i>^<i>-</i> strains (three biological replicates, <i>csrA</i>^<i>-</i> = Mut1-3; wt = WT1-3) were measured by differential shotgun proteomics and visualized in a heat map (left) and a profile plot (right) after non-supervised hierarchical clustering. Every row represents a quantified protein (n = 1448) for which the normalized (LFQ) intensity in each biological replicate is color indicated in the columns.</p

Mapping Proteolytic Processing in the Secretome of Gastric Cancer-Associated Myofibroblasts Reveals Activation of MMP-1, MMP-2, and MMP‑3

Cancer progression involves changes in extracellular proteolysis, but the contribution of stromal cell secretomes to the cancer degradome remains uncertain. We have now defined the secretome of a specific stromal cell type, the myofibroblast, in gastric cancer and its modification by proteolysis. SILAC labeling and COFRADIC isolation of methionine containing peptides allowed us to quantify differences in gastric cancer-derived myofibroblasts compared with myofibroblasts from adjacent tissue, revealing increased abundance of several proteases in cancer myofibroblasts including matrix metalloproteinases (MMP)-1 and -3. Moreover, N-terminal COFRADIC analysis identified cancer-restricted proteolytic cleavages, including liberation of the active forms of MMP-1, -2, and -3 from their inactive precursors. In vivo imaging confirmed increased MMP activity when gastric cancer cells were xenografted in mice together with gastric cancer myofibroblasts. Western blot and enzyme activity assays confirmed increased MMP-1, -2, and -3 activity in cancer myofibroblasts, and cancer cell migration assays indicated stimulation by MMP-1, -2, and -3 in cancer-associated myofibroblast media. Thus, cancer-derived myofibroblasts differ from their normal counterparts by increased production and activation of MMP-1, -2, and -3, and this may contribute to the remodelling of the cancer cell microenvironment

Mapping Proteolytic Processing in the Secretome of Gastric Cancer-Associated Myofibroblasts Reveals Activation of MMP-1, MMP-2, and MMP‑3

Cancer progression involves changes in extracellular proteolysis, but the contribution of stromal cell secretomes to the cancer degradome remains uncertain. We have now defined the secretome of a specific stromal cell type, the myofibroblast, in gastric cancer and its modification by proteolysis. SILAC labeling and COFRADIC isolation of methionine containing peptides allowed us to quantify differences in gastric cancer-derived myofibroblasts compared with myofibroblasts from adjacent tissue, revealing increased abundance of several proteases in cancer myofibroblasts including matrix metalloproteinases (MMP)-1 and -3. Moreover, N-terminal COFRADIC analysis identified cancer-restricted proteolytic cleavages, including liberation of the active forms of MMP-1, -2, and -3 from their inactive precursors. In vivo imaging confirmed increased MMP activity when gastric cancer cells were xenografted in mice together with gastric cancer myofibroblasts. Western blot and enzyme activity assays confirmed increased MMP-1, -2, and -3 activity in cancer myofibroblasts, and cancer cell migration assays indicated stimulation by MMP-1, -2, and -3 in cancer-associated myofibroblast media. Thus, cancer-derived myofibroblasts differ from their normal counterparts by increased production and activation of MMP-1, -2, and -3, and this may contribute to the remodelling of the cancer cell microenvironment