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Structure-Activity Relationship Study Reveals the Molecular Basis for Specific Sensing of Hydrophobic Amino Acids by the Campylobacter jejuni Chemoreceptor Tlp3.

ABSTRACT: Chemotaxis is an important virulence factor of the foodborne pathogen Campylobacter jejuni. Inactivation of chemoreceptor Tlp3 reduces the ability of C. jejuni to invade human and chicken cells and to colonise the jejunal mucosa of mice. Knowledge of the structure of the ligand-binding domain (LBD) of Tlp3 in complex with its ligands is essential for a full understanding of the molecular recognition underpinning chemotaxis. To date, the only structure in complex with a signal molecule is Tlp3 LBD bound to isoleucine. Here, we used in vitro and in silico screening to identify eight additional small molecules that signal through Tlp3 as attractants by directly binding to its LBD, and determined the crystal structures of their complexes. All new ligands (leucine, valine, ?-amino-N-valeric acid, 4-methylisoleucine, ?-methylnorleucine, 3-methylisoleucine, alanine, and phenylalanine) are nonpolar amino acids chemically and structurally similar to isoleucine. X-ray crystallographic analysis revealed the hydrophobic side-chain binding pocket and conserved protein residues that interact with the ammonium and carboxylate groups of the ligands determine the specificity of this chemoreceptor. The uptake of hydrophobic amino acids plays an important role in intestinal colonisation by C. jejuni, and our study suggests that C. jejuni seeks out hydrophobic amino acids using chemotaxis.

SUBMITTER: Khan MF

PROVIDER: S-EPMC7277094 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Structure-Activity Relationship Study Reveals the Molecular Basis for Specific Sensing of Hydrophobic Amino Acids by the Campylobacter jejuni Chemoreceptor Tlp3.

Khan Mohammad F MF Machuca Mayra A MA Rahman Mohammad M MM Koç Cengiz C Norton Raymond S RS Smith Brian J BJ Roujeinikova Anna A

Biomolecules 20200511 5

Chemotaxis is an important virulence factor of the foodborne pathogen Campylobacter jejuni. Inactivation of chemoreceptor Tlp3 reduces the ability of C. jejuni to invade human and chicken cells and to colonise the jejunal mucosa of mice. Knowledge of the structure of the ligand-binding domain (LBD) of Tlp3 in complex with its ligands is essential for a full understanding of the molecular recognition underpinning chemotaxis. To date, the only structure in complex with a signal molec ...[more]

PMID: 32403336

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Project description:Campylobacter jejuni is the leading cause of human gastroenteritis worldwide with over 500 million cases annually. Chemotaxis and motility have been identified as important virulence factors associated with C. jejuni colonisation. Group A transducer-like proteins (Tlps) are responsible for sensing the external environment for bacterial movement to or away from a chemical gradient or stimulus. In this study, we have demonstrated Cj1564 (Tlp3) to be a multi-ligand binding chemoreceptor and report direct evidence supporting the involvement of Cj1564 (Tlp3) in the chemotaxis signalling pathway via small molecule arrays, surface plasmon and nuclear magnetic resonance (SPR and NMR) as well as chemotaxis assays of wild type and isogenic mutant strains. A modified nutrient depleted chemotaxis assay was further used to determine positive or negative chemotaxis with specific ligands. Here we demonstrate the ability of Cj1564 to interact with the chemoattractants isoleucine, purine, malic acid and fumaric acid and chemorepellents lysine, glucosamine, succinic acid, arginine and thiamine. An isogenic mutant of cj1564 was shown to have altered phenotypic characteristics of C. jejuni, including loss of curvature in bacterial cell shape, reduced chemotactic motility and an increase in both autoagglutination and biofilm formation. We demonstrate Cj1564 to have a role in invasion as in in vitro assays the tlp3 isogenic mutant has a reduced ability to adhere and invade a cultured epithelial cell line; interestingly however, colonisation ability of avian caeca appears to be unaltered. Additionally, protein-protein interaction studies revealed signal transduction initiation through the scaffolding proteins CheV and CheW in the chemotaxis sensory pathway. This is the first report characterising Cj1564 as a multi-ligand receptor for C. jejuni, we therefore, propose to name this receptor CcmL, Campylobacter chemoreceptor for multiple ligands. In conclusion, this study identifies a novel multifunctional role for the C. jejuni CcmL chemoreceptor and illustrates its involvement in the chemotaxis pathway and subsequent survival of this organism in the host.

Project description:Chemotaxis is an important virulence factor in some enteric pathogens, and it is involved in the pathogenesis and colonization of the host. However, there is limited knowledge regarding the environmental signals that promote chemotactic behavior and the sensing of these signals by chemoreceptors. To date, there is no information on the ligand molecule that directly binds to and is sensed by Campylobacter jejuni Tlp1, which is a chemoreceptor with a dCache-type ligand-binding domain (LBD). dCache (double Calcium channels and chemotaxis receptor) is the largest group of sensory domains in bacteria, but the dCache-type chemoreceptor that directly binds to formate has not yet been discovered. In this study, formate was identified as a direct-binding ligand of C. jejuni Tlp1 with high sensing specificity. We used the strategy of constructing a functional hybrid receptor of C. jejuni Tlp1 and the Escherichia coli chemoreceptor Tar to screen for the potential ligand of Tlp1, with the binding of formate to Tlp1-LBD being verified using isothermal titration calorimetry. Molecular docking and experimental analyses indicated that formate binds to the membrane-proximal pocket of the dCache subdomain. Chemotaxis assays demonstrated that formate elicits robust attractant responses of the C. jejuni strain NCTC 11168, specifically via Tlp1. The chemoattraction effect of formate via Tlp1 promoted the growth of C. jejuni, especially when competing with Tlp1- or CheY-knockout strains. Our study reveals the molecular mechanisms by which C. jejuni mediates chemotaxis toward formate, and, to our knowledge, is the first report on the high-specificity binding of the dCache-type chemoreceptor to formate as well as the physiological role of chemotaxis toward formate. IMPORTANCE Chemotaxis is important for Campylobacter jejuni to colonize favorable niches in the gastrointestinal tract of its host. However, there is still a lack of knowledge about the ligand molecules for C. jejuni chemoreceptors. The dCache-type chemoreceptor, namely, Tlp1, is the most conserved chemoreceptor in C. jejuni strains; however, the direct-binding ligand(s) triggering chemotaxis has not yet been discovered. In the present study, we found that the ligand that binds directly to Tlp1-LBD with high specificity is formate. C. jejuni exhibits robust chemoattraction toward formate, primarily via Tlp1. Tlp1 is the first reported dCache-type chemoreceptor that specifically binds formate and triggers strong chemotaxis. We further demonstrated that the formate-mediated promotion of C. jejuni growth is correlated with Tlp1-mediated chemotaxis toward formate. Our work provides important insights into the mechanism and physiological function of chemotaxis toward formate and will facilitate further investigations into the involvement of microbial chemotaxis in pathogen-host interactions.

Project description:Campylobacter jejuni is one of the most prevalent causes of bacterial gastroenteritis worldwide, and it is largely associated with consumption of contaminated poultry. Current Campylobacter control measures at the poultry production level remain insufficient, and hence there is the need for alternative control strategies. We evaluated the potential of the monoterpene (-)-α-pinene for control of C. jejuni in poultry. The antibacterial and resistance-modulatory activities of (-)-α-pinene were also determined against 57 C. jejuni strains. In addition, the anti-quorum-sensing activity of (-)-α-pinene against C. jejuni NCTC 11168 was determined for three subinhibitory concentrations (125, 62.5, 31.25 mg/L) over three incubation times using an autoinducer-2 bioassay based on Vibrio harveyi BB170 bioluminescence measurements. The effects of a subinhibitory concentration of (-)-α-pinene (250 mg/L) on survival of C. jejuni, and in combination with enrofloxacin on fluoroquinolone resistance development in C. jejuni, were determined in a broiler chicken model, by addition of (-)-α-pinene to the broiler water supply. The reduction of C. jejuni numbers by (-)-α-pinene was further determined in broiler chickens that were colonized with either fluoroquinolone-susceptible or -resistant strains, by direct gavage treatment. We observed weak in vitro antimicrobial activity for (-)-α-pinene alone (MIC >500 mg/L), but strong potentiating effects on antibiotics erythromycin and ciprofloxacin against different Campylobacter strains (>512 fold change). After 24 h of treatment of C. jejuni with (-)-α-pinene, its quorum-sensing signaling was reduced by >80% compared to the untreated control. When given in the drinking water, (-)-α-pinene did not show any significant inhibitory effects on the level of C. jejuni in the colonized chickens, and did not reduce fluoroquinolone resistance development in combination with enrofloxacin. Conversely, when (-)-α-pinene was administered by direct gavage, it significantly reduced the number of fluoroquinolone susceptible C. jejuni in the colonized broiler chickens. These results demonstrate that (-)-α-pinene modulates quorum-sensing in Campylobacter, potentiates antibiotics against different Campylobacter strains, and reduces Campylobacter colonization in broiler chickens.

Dataset Information

Structure-Activity Relationship Study Reveals the Molecular Basis for Specific Sensing of Hydrophobic Amino Acids by the Campylobacter jejuni Chemoreceptor Tlp3.

Publications

Structure-Activity Relationship Study Reveals the Molecular Basis for Specific Sensing of Hydrophobic Amino Acids by the <i>Campylobacter jejuni</i> Chemoreceptor Tlp3.

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