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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.

Cyclic ADP ribose (cADPR) isomers are signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via NAD + hydrolysis. We show that v-cADPR (2′cADPR) and v2-cADPR (3′cADPR) isomers are cyclized by O -glycosidic bond formation between the ribose moieties in ADPR. Structures of 2′cADPR-producing TIR domains reveal conformational changes leading to an active assembly that resembles those of Toll-like receptor adaptor TIR domains. Mutagenesis reveals a conserved tryptophan essential for cyclization. We show that 3′cADPR is an activator of ThsA effector proteins from bacterial anti-phage defense systems termed Thoeris, and a suppressor of plant immunity when produced by the effector HopAM1. Collectively, our results reveal the molecular basis of cADPR isomer production and establish 3′cADPR in bacteria as an antiviral and plant immunity-suppressing signaling molecule.

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.

The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histoblood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a K d of 1.88 × 10 −5 M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism.S treptococcus pneumoniae is a leading cause of morbidity and mortality worldwide. This bacterial pathogen is responsible for a range of diseases, including pneumonia, meningitis, septicemia, and otitis media. One of the major virulence factors of S. pneumoniae is the multifunctional pore-forming toxin pneumolysin (Ply). Ply is produced by virtually all clinical isolates of S. pneumoniae and is a member of the cholesterol-dependent cytolysin (CDC) family of toxins (1). The key feature of the CDCs, which are expressed by a number of pathogenic Grampositive bacteria, is the ability to form pores in cholesterolcontaining cell membranes. The pore-forming mechanism of the CDCs is a multistep process that involves recognition and binding to the cholesterol-containing membrane by domain 4 of the toxin, oligomerization of ∼34-50 soluble monomers on the target cell membrane to form a large prepore complex (2), and penetration of the prepore structure into the membrane to become a transmembrane β-barrel pore (3-5).The cytolytic mechanism of the CDCs depends on the presence of cholesterol in the target cell membrane; hence, it was thought that cholesterol served as the cellular receptor for these toxins. The first suggestion of this cholesterol serving as the receptor occurred in the 1970s, when it was found that preincubation of the CDC of Streptococcus pyogenes, streptolysin O (SL...