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The activity of the C4-dicarboxylic acid chemoreceptor of Pseudomonas aeruginosa is controlled by chemoattractants and antagonists.

ABSTRACT: Chemotaxis toward organic acids has been associated with colonization fitness and virulence and the opportunistic pathogen Pseudomonas aeruginosa exhibits taxis toward several tricarboxylic acid intermediates. In this study, we used high-throughput ligand screening and isothermal titration calorimetry to demonstrate that the ligand binding domain (LBD) of the chemoreceptor PA2652 directly recognizes five C4-dicarboxylic acids with KD values ranging from 23?µM to 1.24?mM. In vivo experimentation showed that three of the identified ligands act as chemoattractants whereas two of them behave as antagonists by inhibiting the downstream chemotaxis signalling cascade. In vitro and in vivo competition assays showed that antagonists compete with chemoattractants for binding to PA2652-LBD, thereby decreasing the affinity for chemoattractants and the subsequent chemotactic response. Two chemosensory pathways encoded in the genome of P. aeruginosa, che and che2, have been associated to chemotaxis but we found that only the che pathway is involved in PA2652-mediated taxis. The receptor PA2652 is predicted to contain a sCACHE LBD and analytical ultracentrifugation analyses showed that PA2652-LBD is dimeric in the presence and the absence of ligands. Our results indicate the feasibility of using antagonists to interfere specifically with chemotaxis, which may be an alternative strategy to fight bacterial pathogens.

SUBMITTER: Martin-Mora D

PROVIDER: S-EPMC5795001 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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The activity of the C4-dicarboxylic acid chemoreceptor of Pseudomonas aeruginosa is controlled by chemoattractants and antagonists.

Martín-Mora David D Ortega Álvaro Á Pérez-Maldonado Francisco J FJ Krell Tino T Matilla Miguel A MA

Scientific reports 20180201 1

Chemotaxis toward organic acids has been associated with colonization fitness and virulence and the opportunistic pathogen Pseudomonas aeruginosa exhibits taxis toward several tricarboxylic acid intermediates. In this study, we used high-throughput ligand screening and isothermal titration calorimetry to demonstrate that the ligand binding domain (LBD) of the chemoreceptor PA2652 directly recognizes five C4-dicarboxylic acids with K<sub>D</sub> values ranging from 23 µM to 1.24 mM. In vivo exper ...[more]

PMID: 29391435

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Project description:Bacteria have evolved a wide range of chemoreceptors with different ligand specificities. Typically, chemoreceptors bind ligands with elevated specificity and ligands serve as growth substrates. However, there is a chemoreceptor family that has a broad ligand specificity including many compounds that are not of metabolic value. To advance the understanding of this family, we have used the PcaY_PP (PP2643) chemoreceptor of Pseudomonas putida KT2440 as a model. Using Isothermal Titration Calorimetry we showed here that the recombinant ligand binding domain (LBD) of PcaY_PP recognizes 17 different C6-ring containing carboxylic acids with KD values between 3.7 and 138 ?M and chemoeffector affinity correlated with the magnitude of the chemotactic response. Mutation of the pcaY_PP gene abolished chemotaxis to these compounds; phenotype that was restored following gene complementation. Growth experiments using PcaY_PP ligands as sole C-sources revealed functional relationships between their metabolic potential and affinity for the chemoreceptor. Thus, only 7 PcaY_PP ligands supported growth and their KD values correlated with the length of the bacterial lag phase. Furthermore, PcaY_PP ligands that did not support growth had significantly higher KD values than those that did. The receptor has thus binds preferentially compounds that serve as C-sources and amongst them those that rapidly promote growth. Tightest binding compounds were quinate, shikimate, 3-dehydroshikimate and protocatechuate, which are at the interception of the biosynthetic shikimate and catabolic quinate pathways. Analytical ultracentrifugation studies showed that ligand free PcaY_PP-LBD is present in a monomer-dimer equilibrium (KD = 57.5 ?M). Ligand binding caused a complete shift to the dimeric state, which appears to be a general feature of four-helix bundle LBDs. This study indicates that the metabolic potential of compounds is an important parameter in the molecular recognition by broad ligand range chemoreceptors.

Project description:The chemotaxis network, one of the most prominent prokaryotic sensory systems, is present in most motile bacteria and archaea. Although the conserved signaling core of this network is well characterized, ligand specificities of a large majority of diverse chemoreceptors encoded in bacterial genomes remain unknown. Here, we performed a systematic identification and characterization of new chemoeffectors for the opportunistic pathogen Pseudomonas aeruginosa, which has 26 chemoreceptors possessing most of the common types of ligand binding domains. By performing capillary chemotaxis assays for a library of growth-promoting compounds, we first identified a number of novel chemoattractants of varying strength. We subsequently mapped specificities of these ligands by performing Förster resonance energy transfer and microfluidic measurements for 16 hybrid chemoreceptors that combine the periplasmic ligand binding domains of P. aeruginosa receptors and the cytoplasmic signaling domain of the Escherichia coli Tar receptor. Direct binding of putative ligands to chemoreceptors was further confirmed using thermal shift assay and microcalorimetry. Altogether, the combination of methods enabled us to assign several new attractants, including methyl 4-aminobutyrate, 5-aminovalerate, L-ornithine, 2-phenylethylamine, and tyramine, to previously characterized chemoreceptors and to annotate a novel purine-specific receptor PctP. Responses of hybrid receptors to changes in pH further revealed a complex bidirectional pH sensing mechanism in P. aeruginosa, which involves at least four chemoreceptors PctA, PctC, TlpQ, and PctP. Our screening strategy could be applied for the systematic characterization of unknown sensory domains in a wide range of bacterial species. IMPORTANCE Chemotaxis of motile bacteria has multiple physiological functions. It enables bacteria to locate optimal ecological niches, mediates collective behaviors, and can play an important role in infection. These multiple functions largely depend on ligand specificities of chemoreceptors, and the number and identities of chemoreceptors show high diversity between organisms. Similar diversity is observed for the spectra of chemoeffectors, which include not only chemicals of high metabolic value but also bacterial, plant, and animal signaling molecules. However, the systematic identification of chemoeffectors and their mapping to specific chemoreceptors remains a challenge. Here, we combined several in vivo and in vitro approaches to establish a systematic screening strategy for the identification of receptor ligands and we applied it to identify a number of new physiologically relevant chemoeffectors for the important opportunistic human pathogen P. aeruginosa. This strategy can be equally applicable to map specificities of sensory domains from a wide variety of receptor types and bacteria.

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The activity of the C4-dicarboxylic acid chemoreceptor of Pseudomonas aeruginosa is controlled by chemoattractants and antagonists.

Publications

The activity of the C4-dicarboxylic acid chemoreceptor of Pseudomonas aeruginosa is controlled by chemoattractants and antagonists.

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