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Identification of a Chemoreceptor in Pseudomonas aeruginosa That Specifically Mediates Chemotaxis Toward ?-Ketoglutarate.

ABSTRACT: Pseudomonas aeruginosa is an ubiquitous pathogen able to infect humans, animals, and plants. Chemotaxis was found to be associated with the virulence of this and other pathogens. Although established as a model for chemotaxis research, the majority of the 26 P. aeruginosa chemoreceptors remain functionally un-annotated. We report here the identification of PA5072 (named McpK) as chemoreceptor for ?-ketoglutarate (?KG). High-throughput thermal shift assays and isothermal titration calorimetry studies (ITC) of the recombinant McpK ligand binding domain (LBD) showed that it recognizes exclusively ?-ketoglutarate. The ITC analysis indicated that the ligand bound with positive cooperativity (Kd1 = 301 ?M, Kd2 = 81 ?M). McpK is predicted to possess a helical bimodular (HBM) type of LBD and this and other studies suggest that this domain type may be associated with the recognition of organic acids. Analytical ultracentrifugation (AUC) studies revealed that McpK-LBD is present in monomer-dimer equilibrium. Alpha-KG binding stabilized the dimer and dimer self-dissociation constants of 55 ?M and 5.9 ?M were derived for ligand-free and ?KG-bound forms of McpK-LBD, respectively. Ligand-induced LBD dimer stabilization has been observed for other HBM domain containing receptors and may correspond to a general mechanism of this protein family. Quantitative capillary chemotaxis assays demonstrated that P. aeruginosa showed chemotaxis to a broad range of ?KG concentrations with maximal responses at 500 ?M. Deletion of the mcpK gene reduced chemotaxis over the entire concentration range to close to background levels and wild type like chemotaxis was recovered following complementation. Real-time PCR studies indicated that the presence of ?KG does not modulate mcpK expression. Since ?KG is present in plant root exudates it was investigated whether the deletion of mcpK altered maize root colonization. However, no significant changes with respect to the wild type strain were observed. The existence of a chemoreceptor specific for ?KG may be due to its central metabolic role as well as to its function as signaling molecule. This work expands the range of known chemoreceptor types and underlines the important physiological role of chemotaxis toward tricarboxylic acid cycle intermediates.

SUBMITTER: Martin-Mora D

PROVIDER: S-EPMC5126104 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Identification of a Chemoreceptor in Pseudomonas aeruginosa That Specifically Mediates Chemotaxis Toward α-Ketoglutarate.

Martín-Mora David D Ortega Alvaro A Reyes-Darias José A JA García Vanina V López-Farfán Diana D Matilla Miguel A MA Krell Tino T

Frontiers in microbiology 20161129

Pseudomonas aeruginosa is an ubiquitous pathogen able to infect humans, animals, and plants. Chemotaxis was found to be associated with the virulence of this and other pathogens. Although established as a model for chemotaxis research, the majority of the 26 P. aeruginosa chemoreceptors remain functionally un-annotated. We report here the identification of PA5072 (named McpK) as chemoreceptor for α-ketoglutarate (αKG). High-throughput thermal shift assays and isothermal titration c ...[more]

PMID: 27965656

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Project description:Histamine is a key biological signaling molecule. It acts as a neurotransmitter in the central and peripheral nervous systems and coordinates local inflammatory responses by modulating the activity of different immune cells. During inflammatory processes, including bacterial infections, neutrophils stimulate the production and release of histamine. Here, we report that the opportunistic human pathogen Pseudomonas aeruginosa exhibits chemotaxis toward histamine. This chemotactic response is mediated by the concerted action of the TlpQ, PctA, and PctC chemoreceptors, which display differing sensitivities to histamine. Low concentrations of histamine were sufficient to activate TlpQ, which binds histamine with an affinity of 639?nM. To explore this binding, we resolved the high-resolution structure of the TlpQ ligand binding domain in complex with histamine. It has an unusually large dCACHE domain and binds histamine through a highly negatively charged pocket at its membrane distal module. Chemotaxis to histamine may play a role in the virulence of P. aeruginosa by recruiting cells at the infection site and consequently modulating the expression of quorum-sensing-dependent virulence genes. TlpQ is the first bacterial histamine receptor to be described and greatly differs from human histamine receptors, indicating that eukaryotes and bacteria have pursued different strategies for histamine recognition.IMPORTANCE Genome analyses indicate that many bacteria possess an elevated number of chemoreceptors, suggesting that these species are able to perform chemotaxis to a wide variety of compounds. The scientific community is now only beginning to explore this diversity and to elucidate the corresponding physiological relevance. The discovery of histamine chemotaxis in the human pathogen Pseudomonas aeruginosa provides insight into tactic movements that occur within the host. Since histamine is released in response to bacterial pathogens, histamine chemotaxis may permit bacterial migration and accumulation at infection sites, potentially modulating, in turn, quorum-sensing-mediated processes and the expression of virulence genes. As a consequence, the modulation of histamine chemotaxis by signal analogues may result in alterations of the bacterial virulence. As the first report of bacterial histamine chemotaxis, this study lays the foundation for the exploration of the physiological relevance of histamine chemotaxis and its role in pathogenicity.

Project description:Pseudomonas aeruginosa is a Gram-negative, metabolically versatile opportunistic pathogen that elaborates a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability, in part, is mediated by two-component regulatory systems (TCS) that play a crucial role in modulating virulence mechanisms and metabolism. MifS (PA5512) and MifR (PA5511) form one such TCS implicated in biofilm formation. MifS is a sensor kinase whereas MifR belongs to the NtrC superfamily of transcriptional regulators that interact with RpoN (?54). In this study we demonstrate that the mifS and mifR genes form a two-gene operon. The close proximity of mifSR operon to poxB (PA5514) encoding a ß-lactamase hinted at the role of MifSR TCS in regulating antibiotic resistance. To better understand this TCS, clean in-frame deletions were made in P. aeruginosa PAO1 creating PAO?mifS, PAO?mifR and PAO?mifSR. The loss of mifSR had no effect on the antibiotic resistance profile. Phenotypic microarray (BioLOG) analyses of PAO?mifS and PAO?mifR revealed that these mutants were unable to utilize C5-dicarboxylate ?-ketoglutarate (?-KG), a key tricarboxylic acid cycle intermediate. This finding was confirmed using growth analyses, and the defect can be rescued by mifR or mifSR expressed in trans. These mifSR mutants were able to utilize all the other TCA cycle intermediates (citrate, succinate, fumarate, oxaloacetate or malate) and sugars (glucose or sucrose) except ?-KG as the sole carbon source. We confirmed that the mifSR mutants have functional dehydrogenase complex suggesting a possible defect in ?-KG transport. The inability of the mutants to utilize ?-KG was rescued by expressing PA5530, encoding C5-dicarboxylate transporter, under a regulatable promoter. In addition, we demonstrate that besides MifSR and PA5530, ?-KG utilization requires functional RpoN. These data clearly suggests that P. aeruginosa MifSR TCS is involved in sensing ?-KG and regulating its transport and subsequent metabolism.

Dataset Information

Identification of a Chemoreceptor in Pseudomonas aeruginosa That Specifically Mediates Chemotaxis Toward ?-Ketoglutarate.

Publications

Identification of a Chemoreceptor in <i>Pseudomonas aeruginosa</i> That Specifically Mediates Chemotaxis Toward α-Ketoglutarate.

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