Identification and characterization of the aspartate chemosensory receptor of Campylobacter jejuni

Abstract

SummaryCampylobacter jejuni is a highly motile bacterium that responds via chemotaxis to environmental stimuli to migrate towards favourable conditions. Previous in silico analysis of the C. jejuni strain NCTC11168 genome sequence identified 10 open reading frames, tlp1-10, that encode putative chemosensory receptors. We describe the characterization of the role and specificity of the Tlp1 chemoreceptor (Cj1506c). In vitro and in vivo models were used to determine if Tlp1 had a role in host colonization. The tlp1 -isogenic mutant was more adherent in cell culture, however, showed reduced colonization ability in chickens. Specific interactions between the purified sensory domain of Tlp1 and L-aspartate were identified using an amino acid array and saturation transfer difference nuclear magnetic resonance spectroscopy. Chemotaxis assays showed differences between migration of wild-type C. jejuni cells and that of a tlp1 -isogenic mutant, specifically towards aspartate. Furthermore, using yeast twohybrid and three-hybrid systems for analysis of protein-protein interactions, the cytoplasmic signalling domain of Tlp1 was found to preferentially interact with CheV, rather than the CheW homologue of the chemotaxis signalling pathway; this interaction was confirmed using immune precipitation assays. This is the first identification of an aspartate receptor in bacteria other than Escherichia coli and Salmonella enterica serovar Typhimurium.

Chemotaxis inCampylobacter Jejuni

“…Thus, CheW preferentially binds Tlp4, whereas CheV demonstrates a high affinity to Tlp1, Tlp4, Tlp6 as well as Tlp8 [65,66]. It is possible that this competition is part of a kind of sensory adaptation.…”

“…Tlp1 (cj1506c), Tlp4 (cj0262c) and Tlp10 (cj0019c) could be demonstrated, in contrast to Tlp7 (cj0951c/ cj0952c or cjj81176-0975) and Tlp9 (cj1189), to be relevant for chicken intestinal colonization since a mutation within the corresponding genes significantly decreased the number of bacteria in the faeces. Interestingly, a transposon insertion in gene Tlp10 (cj0019) did not affect the in vitro motility of the bacterium indicating that the lack of a chemoreceptor is not mandatorily linked to the motility of C. jejuni [65,87]. The in vivo examination of the intestinal pathology induced by C. jejuni was not feasible for decades since an appropriate vertebrate model was not available.…”

“…However, it could be shown that Tlp9 (CetA, CetB) triggers the response of C. jejuni towards pyruvate and fumarate, whereby the former serves as an electron donor and the latter is known to be a respiratory electron acceptor [84]. In addition, Tlp7 (cj0952c/cj0951c, cjj81176-0975) could be linked to the sensing of electron donor formate, and Tlp1 was identified to represent the chemoreceptor for aspartate [65,77]. Despite the progress that has been achieved to match the chemoreceptors of C. jejuni to its particular ligand, the overall picture of receptors and their respective chemoeffectors is still incomplete and awaits further examinations.…”

“…CheV and CheW are expressed in parallel in C. jejuni and other Epsilonproteobacteria. The noticeable differences between both proteins are the higher affinity to the Tlp-receptors signalling domains (at least for these of Tlp1) [65] and the additional RR-domain of CheV [30].…”

Aspartate chemosensory receptor signalling inCampylobacter jejuni

“…15 A protein-ligand interaction between Tlp1 peri and L-aspartate, but no other amino acid, was thus identified and was further confirmed by STD NMR spectroscopy. 16 In order to further analyse the role of Tlp1 in chemotactic signal transduction, an isogenic deletion mutant and complemented isogenic strains were constructed using the C. jejuni 11168-O reference strain. Chemotaxis assays (capillary and nutrient depleted) demonstrated that Tlp1 is indeed involved sensing aspartate, as the mutation in the Tlp1 sensory domain coding region resulted in a decreased chemotaxis response towards L-aspartate, while migration of the mutant bacteria towards other known chemoattractants was not affected.…”