Structural Insights into the Redox-Sensing Mechanism of MarR-Type Regulator AbfR

As a master redox-sensing MarR-family transcriptional regulator, AbfR participates in oxidative stress responses and virulence regulations in <i>Staphylococcus epidermidis</i>. Here, we present structural insights into the DNA-binding mechanism of AbfR in different oxidation states by determining the X-ray crystal structures of a reduced-AbfR/DNA complex, an overoxidized (Cys13-SO₂H and Cys13-SO₃H) AbfR/DNA, and 2-disulfide cross-linked AbfR dimer. Together with biochemical analyses, our results suggest that the redox regulation of AbfR-sensing displays two novel features: (i) the reversible disulfide modification, but not the irreversible overoxidation, significantly abolishes the DNA-binding ability of the AbfR repressor; (ii) either 1-disulfide cross-linked or 2-disulfide cross-linked AbfR dimer is biologically significant. The overoxidized species of AbfR, resembling the reduced AbfR in conformation and retaining the DNA-binding ability, does not exist in biologically significant concentrations, however. The 1-disulfide cross-linked modification endows AbfR with significantly weakened capability for DNA-binding. The 2-disulfide cross-linked AbfR adopts a very “open” conformation that is incompatible with DNA-binding. Overall, the concise oxidation chemistry of the redox-active cysteine allows AbfR to sense and respond to oxidative stress correctly and efficiently

A Novel Signal Transduction Pathway that Modulates <i>rhl</i> Quorum Sensing and Bacterial Virulence in <i>Pseudomonas aeruginosa</i>

<div><p>The <i>rhl</i> quorum-sensing (QS) system plays critical roles in the pathogenesis of <i>P. aeruginosa</i>. However, the regulatory effects that occur directly upstream of the <i>rhl</i> QS system are poorly understood. Here, we show that deletion of gene encoding for the two-component sensor BfmS leads to the activation of its cognate response regulator BfmR, which in turn directly binds to the promoter and decreases the expression of the <i>rhlR</i> gene that encodes the QS regulator RhlR, causing the inhibition of the <i>rhl</i> QS system. In the absence of <i>bfmS</i>, the Acka-Pta pathway can modulate the regulatory activity of BfmR. In addition, BfmS tunes the expression of 202 genes that comprise 3.6% of the <i>P. aeruginosa</i> genome. We further demonstrate that deletion of <i>bfmS</i> causes substantially reduced virulence in lettuce leaf, reduced cytotoxicity, enhanced invasion, and reduced bacterial survival during acute mouse lung infection. Intriguingly, specific missense mutations, which occur naturally in the <i>bfmS</i> gene in <i>P. aeruginosa</i> cystic fibrosis (CF) isolates such as DK2 strains and RP73 strain, can produce BfmS variants (BfmS_L181P, BfmS_L181P/E376Q, and BfmS_R393H) that no longer repress, but instead activate BfmR. As a result, BfmS variants, but not the wild-type BfmS, inhibit the <i>rhl</i> QS system. This study thus uncovers a previously unexplored signal transduction pathway, BfmS/BfmR/RhlR, for the regulation of <i>rhl</i> QS in <i>P. aeruginosa</i>. We propose that BfmRS TCS may have an important role in the regulation and evolution of <i>P. aeruginosa</i> virulence during chronic infection in CF lungs.</p></div

Discovery of Novel Small Molecule Inhibitors of Dengue Viral NS2B-NS3 Protease Using Virtual Screening and Scaffold Hopping

By virtual screening, compound <b>1</b> was found to be active against NS2B-NS3 protease (IC₅₀ = 13.12 ± 1.03 μM). Fourteen derivatives (<b>22</b>) of compound <b>1</b> were synthesized, leading to the discovery of four new inhibitors with biological activity. In order to expand the chemical diversity of the inhibitors, small-molecule-based scaffold hopping was performed on the basis of the common scaffold of compounds <b>1</b> and <b>22</b>. Twenty-one new compounds (<b>23</b>, <b>24</b>) containing quinoline (new scaffold) were designed and synthesized. Protease inhibition assays revealed that 12 compounds with the new scaffold are inhibitors of NS2B-NS3 protease. Taken together, 17 new compounds were discovered as NS2B-NS3 protease inhibitors with IC₅₀ values of 7.46 ± 1.15 to 48.59 ± 3.46 μM, and 8 compounds belonging to two different scaffolds are active to some extent against DENV based on luciferase reporter replicon-based assays. These novel chemical entities could serve as lead structures for discovering therapies against DENV

Effect of <i>bfmS</i> deletion on bacterial virulence and the ability of <i>P. aeruginosa</i> to adapt to the host.

<p>In all panels, MPAO1 and Δ<i>bfmS</i> harbor plasmid PAK1900, respectively. <b>A</b>) Photographs show lettuce midribs after three days of infection with 1×10⁷ cfu of <i>P. aeruginosa</i>. Wild-type MPAO1 strain or Δ<i>bfmS</i>/p-<i>bfmS</i> strain shows necrosis and tissue maceration of infection. The Δ<i>bfmS</i> strain shows weak signs of infection. <b>B</b>) Constitutive expression of <i>rhlR</i> in Δ<i>bfmS</i> strain could restore the virulence to the level of the wild-type MPAO1 strain. In <b>A</b>) and <b>B</b>), lettuce leaves were inoculated with 10 mM MgSO₄ as a control. <b>C</b>) Effect of <i>P. aeruginosa</i> inoculum on the survival rate of murine lung epithelial cell line 12 (MLE-12). <b>D</b>) Internalization of <i>P. aeruginosa</i> into the murine lung epithelial cell line 12 (MLE-12). In <b>C</b>) and <b>D</b>), values represent means ± SEM. <b>E</b>) Recovery of <i>P. aeruginosa</i> derivatives in a mouse model of acute pneumonia. Results are expressed as the ratio of cfu recovered per lung (output) to cfu present in the initial inoculum (input) and represent results from n = 9–10 mice per strain; the line shows the geometric mean for each group. CFU, Colony-Forming Unit. The Mann–Whitney test was used to calculate p-values (two-tailed). * p<0.05, ** p<0.01, *** p<0.001. Results are representative of two independent experiments.</p

Direct binding of BfmR to its own promoter.

<p><b>A</b>) EMSA showing that 6His-BfmR binds to its own promoter but not to the promoter of either <i>rhlA</i> or <i>rhlC</i>. <b>B</b>) Dye primer-based DNase I footprint assays show the protection pattern of the <i>bfmR</i> promoter after digestion with DNase I following incubation in the absence or presence of different amounts of 6His-BfmR, as indicated. Three BfmR-protected regions in the <i>bfmR</i> promoter DNA were demonstrated. <b>C</b>) <i>bfmR</i> promoter sequence with a summary of the DNase I footprint assay results. The BfmR-protected regions are underlined, and the putative BfmR box is in bold and in italics. Start codon of <i>bfmR</i> is in bold.</p

Effect of <i>bfmS</i> deletion on the production of virulence-associated factors and the promoter activity of <i>bfmR</i>.

<p>In all panels, MPAO1, Δ<i>bfmS</i>, and Δ<i>bfmRS</i> harbor plasmid PAK1900, respectively. <b>A</b>) Upper panel, <i>P. aeruginosa</i> MPAO1 and its derivatives were grown in Pyocyanin production broth (PPB) medium at 37°C for 36 h with shaking (250 rpm); the presence of the blue-green pigment indicates pyocyanin production. Lower panel, bacterial strains were inoculated onto a cetyltrimethylammonium bromide (CTAB) plate and incubated at 37°C for 24 h and then for 72 h at room temperature; the presence of a blue halo surrounding the colonies indicates the production of rhamnolipids. <b>B</b>) Relative amount of C4-HSL measured by the pDO100 (pKD-<i>rhlA</i>) system. MPAO1 and its derivatives were grown in M8-glutamate minimal medium supplemented with 0.2% glucose at 37°C for 24 h with shaking (250 rpm). Supernatants were subsequently prepared and measured for their relative C4-HSL contents. Plasmid pKD-<i>rhlA</i> carries the C4-HSL-responsive <i>rhlA</i> promoter fused to <i>luxCDABE</i>, so CPS (counts per second) values become an indirect measure of supernatant C4-HSL. <b>C</b>) Upper panel, MPAO1 and its derivatives were grown in PPB medium at 37°C for 24 h with shaking (250 rpm). Lower panel, MPAO1 and its derivatives were grown on CTAB plate and incubated at 37°C for 24 h and then for 72 h at room temperature. <b>D</b>) Expression of <i>bfmR-lux</i> in MPAO1 and its derivatives. Bacteria were grown in M8-glutamate minimal medium supplemented with 0.2% glucose at 37°C for 24 h with shaking (250 rpm) and then the <i>bfmR-lux</i> activity was measured. All experiments were independently repeated at least three times and the data shown represent comparable results. Values represent means ± standard error of the mean (SEM).</p

Effect of DK2 lineage-specific amino acid substitutions in BfmS on the activation of BfmR.

<p>In all panels, MPAO1 and Δ<i>bfmS</i> harbor plasmid PAK1900, respectively. <b>A</b>) The relative expression of <i>bfmR-lux</i> in the wild-type MPAO1 and its derivatives when bacteria were grown in M8-glutamate minimal medium supplemented with 0.2% glucose at 37°C for 36 h with shaking (250 rpm), as indicated. Values are relative to MPAO1 (set to 1). <b>B</b>) Relative amount of C4-HSL measured by the pDO100 (pKD-<i>rhlA</i>) system. MPAO1 and its derivatives were grown in M8-glutamate minimal medium supplemented with 0.2% glucose at 37°C for 36 h with shaking (250 rpm). Supernatants were subsequently prepared and measured for the relative C4-HSL contents. <b>C</b>) The relative expression of <i>bfmR-lux</i> in the wild-type MPAO1 and its derivatives when bacteria were grown in M8-glutamate minimal medium supplemented with 0.082% sodium acetate at 37°C for 36 h with shaking (250 rpm). Values are relative to MPAO1 (set to 1). <b>D</b>) The <i>bfmR-lux</i> activity in Δ<i>bfmS</i>/p-<i>bfmS</i> strain (<i>bfmS</i>), Δ<i>bfmS</i>/p-<i>bfmS_H238A</i> strain (<i>H238A</i>), Δ<i>bfmS</i>/p-<i>bfmS_L181P/E376Q</i> (<i>L181P/E376Q</i>), and Δ<i>bfmS</i>/p-<i>bfmS_{L181P/E376Q/H238A}</i> strain (<i>L181P/E376Q/H238A</i>) when bacteria were grown in M8-glutamate minimal medium supplemented with 0.2% glucose at 37°C for 36 h with shaking (250 rpm). Values are relative to Δ<i>bfmS</i>/p-<i>bfmS</i> strain (set to 1). In <b>A</b>) to <b>D</b>), results are representative of three independent experiments and values represent means ± SEM. <b>E</b>) Phos-tag analysis shows that amino acid substitution L181P/E376Q, but not the L181P/E376Q/H238A, causes overproduction of phosphorylated and unphosphorylated BfmR. Western blot analysis of BfmS showing that missense mutations in <i>bfmS</i> do not affect the protein levels of BfmS, and immunoblots for RNAP (RNA polymerase) served as loading control. Cell lysates of Δ<i>bfmRS</i>::<i>BfmR</i>-<i>Flag</i>/p-<i>bfmS</i> strain (<i>bfmS</i>), Δ<i>bfmRS</i>::<i>BfmR</i>-<i>Flag</i>/p-<i>bfmS_L181P/E376Q</i> (<i>L181P/E376Q</i>), and Δ<i>bfmRS</i>::<i>BfmR</i>-<i>Flag</i>/p-<i>bfmS_{L181P/E376Q/H238A}</i> strain (<i>L181P/E376Q/H238A</i>) were used as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004340#s4" target="_blank">Materials and Methods</a> section.</p

Model of the regulatory networks involving BfmRS in <i>P. aeruginosa</i>.

<p>The lines show the interaction between the players: arrow, activation; hammerheads, repression; solid line, a direct influence or direct connection; dotted line, a putative or indirect connection. The question mark (“?”) denotes a yet-unidentified factor (factors) required for triggering the kinase or phosphatase activity of BfmS. QteE reduces LasR protein stability <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004340#ppat.1004340-Siehnel1" target="_blank">[80]</a>. QteE also blocks RhlR accumulation, and this effect is independent of QteE's action on LasR <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004340#ppat.1004340-Siehnel1" target="_blank">[80]</a>. Inactivation of RpoS causes elevated levels of <i>rhlI</i> (but not <i>rhlR</i>) transcription <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004340#ppat.1004340-Whiteley1" target="_blank">[81]</a>. See text for details. OM, outer membrane. IM, inner membrane. AcP, acetyl phosphate.</p

Effect of the Pta-AckA pathway and carbon sources availability on the activation of BfmR in the absence of BfmS.

<p>In all panels, MPAO1, Δ<i>bfmS</i>, and Δ<i>bfmS</i>Δ<i>ackA</i>-<i>pta</i> harbor plasmid PAK1900, respectively. <b>A</b>) The relative expression of <i>bfmR-lux</i> in the wild-type MPAO1, the Δ<i>bfmS</i> strain, the Δ<i>bfmS</i>Δ<i>ackA</i>-<i>pta</i> strain, and its complementary strain (Δ<i>bfmS</i>Δ<i>ackA</i>-<i>pta</i>/p-<i>ackA-pta</i>) when bacteria were grown in M8-glutamate minimal medium supplemented with 0.2% glucose at 37°C for 24 h with shaking (250 rpm). Values are relative to MPAO1 (set to 1). <b>B</b>) Relative amount of C4-HSL measured by the pDO100 (pKD-<i>rhlA</i>) system. MPAO1 and its derivatives were grown in M8-glutamate minimal medium supplemented with 0.2% glucose at 37°C for 24 h with shaking (250 rpm). Supernatants were subsequently prepared and measured for the relative C4-HSL contents. Values represent means ± SEM. The assays were independently repeated at least three times with similar results obtained, and the graphs show a set of representative data.</p

BfmR regulates the expression of <i>rhlR</i> in a direct manner.

<p><b>A</b>) EMSA shows that 6His-BfmR directly binds to the promoter DNA of <i>rhlR</i> but not to that of <i>rhlC</i>. <b>B</b>) Electropherograms show the protection pattern of the <i>rhlR</i> promoter after digestion with DNase I following incubation in the absence or the presence of 6His-BfmR. There are three BfmR-protected regions (I, II, and III) in the promoter of the <i>rhlR</i>. BfmR-protected regions I harbors a putative BfmR-binding motif, which was highlighted in bold and in italics, as indicated. <b>C</b>) The relative expression of <i>rhlR-lux</i> in the wild-type MPAO1 (harboring PAK1900), the Δ<i>bfmS</i> strain (harboring PAK1900), the Δ<i>bfmS</i>/p-<i>bfmS</i> strain, the Δ<i>bfmRS</i> strain (harboring PAK1900), and the Δ<i>bfmRS</i>/p-<i>bfmR</i> strain. The relative gene expression in the wild-type MPAO1 was set to 1, and the other values were adjusted accordingly. <b>D</b>) The expression of <i>rhlR-lux</i> and <i>rhlR-D-lux</i> in the wild-type MPAO1, the Δ<i>bfmS</i> strain and the Δ<i>bfmRS</i> strain, as indicated. Bacteria were grown in low phosphate (0.32 mM) M8-glutamate minimal medium supplemented with 2% glucose at 37°C for 48 h. Values represent means ± SEM. The assays were independently repeated at least three times and the data shown represent comparable results.</p