Regulation of bacterial adaptive behavior by the second messenger cyclic-di-GMP and host components

To optimize survival and growth, bacteria have evolved adaptive behaviors that respond to relevant environmental signals. A switch from the motile to the sessile lifestyle is probably the most ancient behavioral transition of microorganisms. Gram-negative bacteria such as Salmonella species and Pseudomonas aeruginosa have a set of extracellular appendages involved in motility and biofilm formation, but also in interaction with the host. These appendages can be regulated by the bacterial second messenger cyclic di-GMP, which allows a millisecond fast response. The bacterial second messenger c-di-GMP regulates the transition between sessility and motility and between acute and chronic infection. In this work, the signaling pathway involved in motility in Salmonella enterica serovar Typhimurium has been investigated in detail. The phosphodiesterase YhjH specifically downregulates motility by interfering with the flagellar functionality. Three diguanylate cyclases inhibit motility in the yhjH background and interact specifically with one of two c-di-GMP receptors affecting motility (Paper 1). Also non-canonical EAL domain proteins such as STM1697 unconventionally inhibit motility by post-transcriptionally interfering with the major flagellar regulator FlhD4C2 (Paper 2), which downregulates flagellin expression as one final outcome (Paper 2 and Paper 3). STM1697 has also an unconventional phenotype compared to EAL phosphodiesterases with respect to biofilm formation and invasion of the colon adenocarcinoma cell line HT-29 (Paper 2, Paper 4) and affects virulence mediated through the FlhD4C2 interaction (Paper 2). In general, c-di-GMP metabolizing proteins regulate virulence properties of S. Typhimurium such as invasion and production of the pro-inflammatory cytokine interleukin 8 by HT-29, but also secretion of the effector protein SipA from the invasion related type three secretion system and colonization of gut and organs in the streptomycin treated mouse (Paper 4). Surprisingly, c-di-GMP signaling inhibits virulence properties through biofilm components such as the major biofilm regulator CsgD and the cellulose synthase BcsA (Paper 4). These studies show that the c-di-GMP signaling network is involved in virulence in S. Typhimurium. In the last study, the human surfactant protein C an innate immune component of the lung, did not have an effect on bacterial growth, but affected biofilm formation and swarming motility of P. aeruginosa PAO1 (Paper 5). In conclusion, this thesis sheds light on how the c-di-GMP signaling network and the surfactant protein C regulate the adaptive behavior of S. Typhimurium and P. aeruginosa, respectively

Gre factors-mediated control of hilD transcription is essential for the invasion of epithelial cells by Salmonella enterica serovar Typhimurium

The invasion of epithelial cells by Salmonella enterica serovar Typhimurium is a very tightly regulated process. Signaling cascades triggered by different environmental and physiological signals converge to control HilD, an AraC regulator that coordinates the expression of several virulence factors. The expression of hilD is modulated at several steps of the expression process. Here, we report that the invasion of epithelial cells by S. Typhimurium strains lacking the Gre factors, GreA and GreB, is impaired. By interacting with the RNA polymerase secondary channel, the Gre factors prevent backtracking of paused complexes to avoid arrest during transcriptional elongation. Our results indicate that the Gre factors are required for the expression of the bacterial factors needed for epithelial cell invasion by modulating expression of HilD. This regulation does not occur at transcription initiation and depends on the capacity of the Gre factors to prevent backtracking of the RNA polymerase. Remarkably, genetic analyses indicate that the 3'-untranslated region (UTR) of hilD is required for Gre-mediated regulation of hilD expression. Our data provide new insight into the complex regulation of S. Typhimurium virulence and highlight the role of the hilD 3'-UTR as a regulatory motif

Complex c-di-GMP Signaling Networks Mediate Transition between Virulence Properties and Biofilm Formation in Salmonella enterica Serovar Typhimurium

Upon Salmonella enterica serovar Typhimurium infection of the gut, an early line of defense is the gastrointestinal epithelium which senses the pathogen and intrusion along the epithelial barrier is one of the first events towards disease. Recently, we showed that high intracellular amounts of the secondary messenger c-di-GMP in S. typhimurium inhibited invasion and abolished induction of a pro-inflammatory immune response in the colonic epithelial cell line HT-29 suggesting regulation of transition between biofilm formation and virulence by c-di-GMP in the intestine. Here we show that highly complex c-di-GMP signaling networks consisting of distinct groups of c-di-GMP synthesizing and degrading proteins modulate the virulence phenotypes invasion, IL-8 production and in vivo colonization in the streptomycin-treated mouse model implying a spatial and timely modulation of virulence properties in S. typhimurium by c-di-GMP signaling. Inhibition of the invasion and IL-8 induction phenotype by c-di-GMP (partially) requires the major biofilm activator CsgD and/or BcsA, the synthase for the extracellular matrix component cellulose. Inhibition of the invasion phenotype is associated with inhibition of secretion of the type three secretion system effector protein SipA, which requires c-di-GMP metabolizing proteins, but not their catalytic activity. Our findings show that c-di-GMP signaling is at least equally important in the regulation of Salmonella-host interaction as in the regulation of biofilm formation at ambient temperature

The Effects of Codon Context on <i>In Vivo</i> Translation Speed

<div><p>We developed a bacterial genetic system based on translation of the <i>his</i> operon leader peptide gene to determine the relative speed at which the ribosome reads single or multiple codons <i>in vivo</i>. Low frequency effects of so-called “silent” codon changes and codon neighbor (context) effects could be measured using this assay. An advantage of this system is that translation speed is unaffected by the primary sequence of the His leader peptide. We show that the apparent speed at which ribosomes translate synonymous codons can vary substantially even for synonymous codons read by the same tRNA species. Assaying translation through codon pairs for the 5′- and 3′- side positioning of the 64 codons relative to a specific codon revealed that the codon-pair orientation significantly affected <i>in vivo</i> translation speed. Codon pairs with rare arginine codons and successive proline codons were among the slowest codon pairs translated <i>in vivo</i>. This system allowed us to determine the effects of different factors on <i>in vivo</i> translation speed including Shine-Dalgarno sequence, rate of dipeptide bond formation, codon context, and charged tRNA levels.</p></div

Codon pairs at His4-His5 that result in a Tz-Lac⁺ phenotype.

<p>*Calculated Anti-SD free energies <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004392#pgen.1004392-Li1" target="_blank">[27]</a> are shown in parentheses.</p

The His leader with UCA(Ser)-UCA(Ser) at His4-His5 responds to either histidine or serine starvation for loss of attenuation.

<p>Two strains were tested for the effects of either histidine or serine starvation on loss of <i>his</i> operon attenuation: a strain with a wild type His leader with His codons at His4-His5 (CAU-CAC) and a strain with UCA(Ser)-UCA(Ser) codons at His4-His5. Both strains carried a <i>hisD-lac</i> operon fusion and a <i>serB</i>::Tn<i>10</i> insertion resulting in auxotrophy for both histidine and serine. A 0.1 ml portion containing 10⁸ cells for each strain was plated on minimal glucose X-gal medium supplemented with either serine or histidine. For plates supplemented with histidine, serine was added to a filter disc placed at the center of the plate. For plates supplemented with serine, histidine was added to a filter disc placed at the center of the plate. The plates were incubated overnight at 37°C. Confluent growth occurred near the filter discs containing histidine or serine supplements and growth was inhibited as the concentration of supplements became limiting as indicated by the dashed line. For the strain with histidine codons at His4 and His5 of the <i>his</i> leader peptide starvation for histidine resulted in de-attenuation and expression of the <i>hisD-lac</i> operon fusion indicated by the production of the X-gal blue color at the position in the plates where the cells are starved for histidine, while starvation for serine did not result in de-attenuation of <i>his</i> operon expression (no blue color at the position in the plates where the cells are starved for serine). For the strain with serine codons at His4 and His5 of the <i>his</i> leader peptide, starvation for either serine or histidine resulted in de-attenuation and expression of the <i>hisD-lac</i> operon fusion.</p

Attenuation mechanism for the regulation of the histidine biosynthetic operon of <i>Salmonella enterica</i>[28].

<p>The 5' regulatory region of the <i>his</i> operon encodes a 16 amino acid leader peptide, including 7 consecutive His codons, followed by a transcription terminator. <b>A</b>. Under conditions where histidyl-tRNA levels are high ribosomal translation proceeds through the leader peptide to its stop codon. This results in the formation of the E:F attenuator stem-loop and inhibition of further transcription into the <i>his</i> structural genes. <b>B</b>. Under conditions of limited histidyl-tRNA, the translation through the His codons is slowed and the stalled ribosome allows for the formation of an alternative RNA secondary structure that occludes attenuator formation. Inhibition of attenuator formation allows RNA polymerase to continue transcription into the <i>his</i> operon structural genes. <b>C</b>. Under conditions where transcription from the <i>his</i> promoter is not coupled to translation the E:F attenuator is predicted to form and inhibition of further transcription into the <i>his</i> structural genes.</p

Histidine operon expression with His5 of the leader substituted by all 64 codons.

<p>All constructs carry a <i>hisD-lac</i> operon fusion that places the <i>lac</i> operon under control of the <i>his</i> operon promoter-attenuator regulatory system. The number in each box is the β-galactosidase activity of each mutant construct divided by the activity of the wild-type construct that contains the CAC His codon at His5. This figure also shows all tRNA species represented by a single or multiple solid black circles connected by lines according to Glenn Bjork <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004392#pgen.1004392-Bjork1" target="_blank">[61]</a>. If a tRNA reads a single codon, it is represented by a single black dot (ie. UGG Trp). If a given tRNA reads multiple codons the codons it reads are represented by solid black dots connected by lines.</p

Histidine operon expression phenotypes of UCA-NNN and NNN-UCA substitutions at His4-His5 of the His leader peptide.

<p>The <i>his</i> operon leader peptide was altered with either NNN-UCA (3′-effect) substituted at positions His4-His5, or UCA-NNN (5′-effect) substituted at positions His4-His5. All constructs carry a <i>hisD-lac</i> operon fusion that places the <i>lac</i> operon under control of the <i>his</i> operon promoter-attenuator regulatory system. Levels of <i>hisD-lac</i> transcription are qualitative as determined by a color phenotype on MacConkey lactose indicator medium where white is Lac⁻ and the redder the Lac⁺ colonies, the greater the levels of β-galactosidase expressed from <i>hisD-lac</i>.</p

Effect of codon substitutions in the <i>his</i> leader region on derepression of <i>his</i> operon transcription.

<p>(<b>A</b> & <b>B</b>) Each of the seven His codons in the His leader peptide were replaced with UAG stop codons and the effect of <i>hisD-lac</i> (<b>A</b>) or <i>hisG</i> transcription was determined by either β-galactosidase assay for <i>hisD-lac</i> (<b>A</b>) or quantitative real time-PCR for <i>hisG</i> mRNA (<b>B</b>). Errors bars represent the standard deviation of the means. One-way analysis of variance followed by Turkey test showed which data set were statistically different (<i>ns</i> (not significant); * (P<0.05); ** (P<0.01); *** (P<0.001)). For panel A, means were significantly different at least at 95% confidence interval levels except for the means of His1::UAG vs His2::UAG; His1::UAG vs His7::UAG; His2::UAG vs His3::UAG; His2::UAG vs His7::UAG; His3::UAG vs His6::UAG; His4::UAG vs His6::UAG), that were not significantly different. For panel B, means were significantly different at least at 95% confidence interval levels except for the means of His leader WT vs His1::UAG; His leader WT vs His7::UAG; His1::UAG vs His7::UAG; His2::UAG vs His3::UAG; His3::UAG vs His4::UAG; His3::UAG vs His6::UAG and His4::UAG vs His6::UAG that were not significantly different. (<b>C</b>, <b>D</b> & <b>E</b>) The mRNA levels of <i>hisG</i> were determined using quantitative RT-PCR. For all the strains analyzed, the natural <i>his</i> promoter was replaced with the P<i>_tetA</i> promoter, to allow induction of <i>his</i> operon transcription with addition of the P<i>_tetA</i> inducer anhydrotetracycline (ATc). Strains were grown to OD ∼0.3, at which time spectinomycin was added to inhibit translation. When OD reached 0.4, anhydrotetracycline (ATc) was added. <b>C</b>: mRNA from P<i>_tetA</i> His5::UAG was extracted at different time points after induction of <i>his</i> operon transcription with and without added spectinomycin to inhibit translation and <i>hisG</i> mRNA levels were determined by qRT-PCR. <b>D</b>: mRNA from the <i>his</i> E-F attenuator stem-loop deletion strain under control of P<i>_tetA</i> was collected at different time points after induction of <i>his</i> operon transcription with and without added spectinomycin to inhibit translation and <i>hisG</i> mRNA levels were determined by qRT-PCR. <b>E</b>: The <i>hisG</i> mRNA levels were determined by qRT-PCR from different His5 single codon substitution and His4-His5 double codon substitution mutants at one-minute time points after <i>his</i> operon induction from P<i>_tetA</i> by ATc in the presence and absence of spectinomycin.</p