31 research outputs found

    Screening and Evaluation of Small Organic Molecules as ClpB Inhibitors and Potential Antimicrobials

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    Inhibition of ClpB, the bacterial representative of the heat-shock protein 100 family that is associated with virulence of several pathogens, could be an effective strategy to develop new antimicrobial agents. Using a high-throughput screening method, we have identified several compounds that bind to different conformations of ClpB and analyzed their effect on the ATPase and chaperone activities of the protein. Two of them inhibit these functional properties as well as the growth of Gram negative bacteria (<i>E. coli</i>), displaying antimicrobial activity under thermal or oxidative stress conditions. This activity is abolished upon deletion of ClpB, indicating that the action of these compounds is related to the stress cellular response in which ClpB is involved. Moreover, their moderate toxicity in human cell lines suggests that they might provide promising leads against bacterial growth

    Phenylalanine Hydroxylase from <em>Legionella pneumophila</em> Is a Thermostable Enzyme with a Major Functional Role in Pyomelanin Synthesis

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    <div><h3>Background</h3><p><em>Legionella pneumophila</em> is a pathogenic bacterium that can cause Legionnaires’ disease and other non-pneumonic infections in humans. This bacterium produces a pyomelanin pigment, a potential virulence factor with ferric reductase activity. In this work, we have investigated the role of phenylalanine hydroxylase from <em>L. pneumophila</em> (lpPAH), the product of the <em>phhA</em> gene, in the synthesis of the pyomelanin pigment and the growth of the bacterium in defined compositions.</p> <h3>Methodology/Principal Findings</h3><p>Comparative studies of wild-type and <em>phhA</em> mutant corroborate that lpPAH provides the excess tyrosine for pigment synthesis. <em>phhA</em> and <em>letA</em> (<em>gacA</em>) appear transcriptionally linked when bacteria were grown in buffered yeast extract medium at 37°C. <em>phhA</em> is expressed in <em>L. pneumophila</em> growing in macrophages. We also cloned and characterized lpPAH, which showed many characteristics of other PAHs studied so far, including Fe(II) requirement for activity. However, it also showed many particular properties such as dimerization, a high conformational thermal stability, with a midpoint denaturation temperature (<em>T</em><sub>m</sub>) = 79±0.5°C, a high specific activity at 37°C (10.2±0.3 µmol L-Tyr/mg/min) and low affinity for the substrate (<em>K</em><sub>m</sub> (L-Phe) = 735±50 µM.</p> <h3>Conclusions/Significance</h3><p>lpPAH has a major functional role in the synthesis of pyomelanin and promotes growth in low-tyrosine media. The high thermal stability of lpPAH might reflect the adaptation of the enzyme to withstand relatively high survival temperatures.</p> </div

    Conformational stability of lpPAH.

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    <p>(A) Far-UV CD spectrum of lpPAH (6 µM in 50 mM Na-phosphate buffer, pH 6.5) at 37°C (<b><sup>____</sup></b>), at 85°C (––) and at 37°C after heating the sample to 100°C (⋅⋅⋅⋅⋅). [θ], mean residual ellipticity. (B) CD-monitored (at 222 nm) thermal denaturation lpPAH (6 µM in 20 mM Na-Hepes, 200 mM NaCl, pH 7.0) without (•) or with (○) 6 µM Fe(II) (added as ferrous ammonium sulphate) and 6 µM Fe(II) and 5 mM L-Phe (▾). The lines show a fitting of the data to a two-state unfolding equation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046209#pone.0046209-Swint1" target="_blank">[79]</a> and points are averaged over ten data points after conversion to fraction unfolded <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046209#pone.0046209-Agashe1" target="_blank">[80]</a>. (C) DSC-monitored thermal denaturation of lpPAH (30 µM) in 20 mM Na-Hepes, pH 7.0. The scan rate was 1°C/min.</p

    Pathway for phenylalanine/tyrosine catabolism and pyomelanin synthesis in <i>L. pneumophila.</i>

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    <p>The stippled arrow illustrates the polymerization of autoxidated homogentisate to make pyomelanin. PAH, phenylalanine hydroxylase; AT, amino acid transferase; HPPD/Lly, 4-hydroxyphenylpyruvate dioxygenase/legiolysin; HmgA, homogentisate-1,2-dioxygenase; HmgC, maleylacetoacetate isomerase; HmgB, fumarylacetoacetate hydrolase. The two <i>L. pneumophila</i> mutants used in this work are marked by asterisks and their gene names are shown (in italics).</p

    Transcriptional linkage between <i>phhA</i> and <i>letA</i>.

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    <p>(A) The gray horizontal arrows denote the <i>phhA</i> and <i>letA</i> genes. The thin horizontal lines below the genes signify the approximate size and location of transcripts identified by RT-PCR analysis, including an intergenic transcript. (B) Wild-type strain 130b was grown in BYE at 37°C, and then RNA was analyzed by RT-PCR utilizing primer pairs specific to either <i>phhA, letA,</i> or the intergenic region spanning <i>phhA</i> and <i>letA</i>. That the PCR products obtained resulted from mRNA templates was confirmed by the lack of product obtained when the PCR did not incorporate RT (-RT). PCR products obtained from genomic DNA appear in the left-most lanes, indicating that the mRNAs observed are full-length. The results presented are representative of at least three independent experiments.</p

    Growth of wild-type and <i>phhA</i> mutant <i>L. pneumophila</i> in CDM containing different amounts of tyrosine.

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    <p><i>L. pneumophila</i> 130b wild-type (WT) and <i>phhA</i> mutant strains NU406 and NU407 were inoculated into either standard CDM containing tyrosine (A) or CDM lacking tyrosine (B) and at the indicated time points, the extent of bacterial growth was determined by recording the optical density (OD) of the cultures. In panel (B), the ODs of the mutant cultures were significantly less than that of the WT cultures at 18, 24, and 40 h post-inoculation, as indicated by the asterisks (<i>P</i><0.05, Student’s <i>t</i>-test). (C) WT and mutant NU406 carrying the empty vector (pMMB2002) and NU406 carrying the <i>phhA</i> gene cloned into pMMB2002 (i.e., pPhhA) were cultured in CDM without the tyrosine supplement and then at 24 h post-inoculation the OD of the cultures were determined. The OD of the NU406 (pMMB2002) culture was significantly less than that of both the WT and the complemented mutant (<i>P</i><0.05, Student’s <i>t</i>-test). All experiments are representative of three independent trials.</p

    Location and expression of <i>phhA</i>.

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    <p>(A) Depiction of the region of the <i>L. pneumophila</i> chromosome containing <i>phhA</i>. The white horizontal arrows denote the relative size and orientation of <i>phhA</i> and its neighboring genes. The thin horizontal line below the gene map signifies the approximate size and location of the <i>phhA</i>-specific transcript identified by RT-PCR analysis. (B) Expression of <i>phhA</i> transcripts. Wild-type strain 130b was grown in BYE or CDM broth at the indicated temperatures, and then RNA was analyzed by RT-PCR utilizing primers specific to <i>phhA</i>. That the PCR products obtained resulted from mRNA templates was confirmed by the lack of product obtained when the PCR did not incorporate RT. PCR products obtained from genomic DNA appear in the left-most lane, indicating that the mRNAs observed are full-length. RT-PCR analysis of 16S rRNA served as a positive control. The results presented are representative of at least three independent experiments.</p

    Pigmentation of wild-type and <i>phhA</i> mutant <i>L</i>. <b><i>pneumophila</i></b>.

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    <p>(A) Wild-type (WT) <i>L. pneumophila</i> 130b was inoculated into either standard CDM, CDM lacking tyrosine (CDMˆno Tyr), or CDM containing twice the normal amount of tyrosine (CDMˆ2X Tyr), and then at 24 and 48 h post-inoculation the levels of pigment in the cultures were determined by measuring the OD<sub>400</sub> of the cultures supernatants. (B) <i>L. pneumophila</i> 130b WT, <i>phhA</i> mutants NU406 and NU407, and <i>lly</i> mutant NU408 were inoculated into standard CDM containing tyrosine, and then at 24 and 48 h, the amount of pigment produced was determined by assessing the OD<sub>400</sub> of culture supernatants. (C) WT and mutant NU406 carrying the vector pMMB2002 and NU406 carrying the <i>phhA</i> gene cloned into pMMB2002 (i.e., pPhhA) were cultured in either standard CDM containing tyrosine (gray bars) or CDM containing twice the normal amount of tyrosine (black bars), and then at 24 h post-inoculation the OD<sub>400</sub> of the cultures were determined. (D) Photographs of cell-free supernatants from 24-h CDM cultures (with the standard amount of tyrosine) of WT and <i>phhA</i> mutant strain NU406 carrying vector and NU406 carrying the cloned <i>phhA</i>. In panels (A – C), the asterisks indicate a <i>P</i> value of <0.05 compared to the WT control (Student’s <i>t</i>-test). All experiments are representative of three independent trials.</p

    PAH activity in lysates of <i>L. pneumophila</i> 130b.

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    <p>PAH activity measured without (A) and with (B) the addition of 100 µM Fe(II) in the assay in lysates from wild-type (WT) cultures grown in BYE medium with (BYÊhigh Fe; white bars) and without (BYÊlow Fe; black bars) standard (1.3 mM) FeCl<sub>3</sub> supplement (see text for details). The data presented are the means and SD from triplicate measurements of duplicate cultures for each condition. The activity of a lysate of a culture of <i>phhA</i> mutant grown in BYÊhigh Fe is also shown in (B). Differences between conditions were found to be significant (P<0.01) with respect to WT in BYÊhigh Fe, in both (A) and (B).</p

    Steady-state kinetic characterization of lpPAH.

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    <p>(A) Residual activity at 37°C after incubation for 10 min at the indicated temperatures. (B) Temperature dependence of lpPAH activity. Inset, Arrhenius plot (providing an activation energy (<i>E</i><sub>a</sub>) of 15.8±2.3 kcal/mol). (C) Effect of L-Phe concentration on lpPAH activity, measured with 0.2 mM BH<sub>4</sub> and 100 µM Fe(II). The solid line is a fitting to the Michaelis-Menten equation. (D) Effect of Fe(II)-concentration on lpPAH activity, measured with 1 mM L-Phe and 0.2 mM BH<sub>4</sub>. One enzyme unit (U) corresponds to the amount of enzyme that catalyzes the production of 1 µmol L-Tyr per minute.</p
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