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, α-amino-N-valeric acid, 4-methylisoleucine, β-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.
Highlights
Campylobacter jejuni is a Gram-negative, microaerophilic, flagellated bacterium that colonises the intestines of many wild and domestic animals, resulting in a commensal relationship between the bacterium and the host [1,2,3,4]
That study demonstrated that the Tlp3 side chains that form interactions with the ammonium and carboxylate groups of isoleucine (Lys149, Trp151, Tyr167, Asp169, and Asp196) are essential for isoleucine binding [32]. These five residues are structurally conserved among double Cache (dCache)-type chemoreceptors that are specific to amino acids [32,42,43], suggesting that the dCache-type receptors share a common mechanism of recognition of the invariant moiety of an amino acid
7.5 mL of the bacterial cell suspension in phosphate-buffered saline (PBS) was mixed with 7.5 mL of 0.6% (w/v) bacteriological agar in PBS tempered at 50 ◦C, and the mixture was poured into the Petri dish with hard agar plug (HAP)
Summary
Campylobacter jejuni is a Gram-negative, microaerophilic, flagellated bacterium that colonises the intestines of many wild and domestic animals, resulting in a commensal relationship between the bacterium and the host [1,2,3,4]. That study demonstrated that the Tlp side chains that form interactions with the ammonium and carboxylate groups of isoleucine (Lys149, Trp151, Tyr167, Asp169, and Asp196) are essential for isoleucine binding [32] These five residues are structurally conserved among dCache-type chemoreceptors that are specific to amino acids [32,42,43], suggesting that the dCache-type receptors share a common mechanism of recognition of the invariant moiety of an amino acid. The crystal structures of dCache LBDs from various species in complex with different amino acids revealed that ligands bind to their membrane-distal subdomains in a mode that is very similar to that observed for isoleucine bound. We determined the crystal structures of their complexes and performed a structure–activity relationship (SAR) study that identified molecular features recognised by this chemoreceptor
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