Characterisation of a Multi-ligand Binding Chemoreceptor CcmL (Tlp3) of Campylobacter jejuni

Abstract

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.

Cooperation of two distinct coupling proteins creates chemosensory network connections

“…TlpA and TlpB are integral membrane proteins in H. pylori, and due to the challenging nature of their purification, we were only able to test their interaction with CheV1 and CheW using the BACTH assay. However, it is possible that multiple coupling proteins in a single organism do provide some chemoreceptor specificity, as seen in Campylobacter jejuni, where the aspartate chemoreceptor prefers CheV over CheW (16,42).…”

Chemotaxis Control of Transient Cell Aggregation

“…One possibility is that adhesins, which are present in the polar region of the cells or secreted at this location, stabilize the cell-cell contacts in clumps, akin to a similar adhesion described in closely related alphaproteobacteria (59)(60)(61). Flocculation is widespread in many motile bacteria capable of chemotaxis, and chemotaxis defects have been linked to increased cell aggregation in many bacterial species (62)(63)(64). It is thus likely that chemotaxis-dependent clumping occurs in other bacterial species.…”

Cloning, refolding, purification and preliminary crystallographic analysis of the sensory domain of theCampylobacterchemoreceptor for multiple ligands (CcmL)

“…It has been shown to play an important role in initial colonization and development of the disease in animal models (Josenhans & Suerbaum, 2002). C. jejuni is attracted to amino acids, organic acids and carbohydrates (Rahman et al, 2014;Hartley-Tassell et al, 2010;Hugdahl et al, 1988). The bacteria sense these chemicals via transducer-like proteins (Tlps) located in the bacterial membrane or cytoplasm (Zautner et al, 2012).…”

“…The aspartate receptor Tlp1, also termed CcaA (Campylobacter chemoreceptor for aspartate A; Hartley-Tassell et al, 2010), the multiligand-binding receptor Tlp3, also termed CcmL (Campylobacter chemoreceptor for multiple ligands; Rahman et al, 2014), and the formic acid receptor Tlp7 (Tareen et al, 2010) are present in most sequenced strains of C. jejuni, whereas the galactose receptor Tlp11 is the least common and has only been found in clinical isolates from patients hospitalized owing to C. jejuni infection (Day et al, 2012.…”

“…The sensory domain of CcmL, which has been shown to bind multiple ligands including isoleucine, lysine, glucosamine, aspartate, succinic acid, arginine, malic acid, thiamine and sodium deoxycholate, shares no significant sequence similarity to chemoreceptors from non-Epsilonproteobacteria (Rahman et al, 2014;Li et al, 2014). This suggests that the mechanism of ligand recognition by CcmL may be unique to Campylobacter spp.…”