SummaryThe lipopolysaccharide (LPS) and fimbriae of Porphyromonas gingivalis play important roles in periodontal inflammation and pathogenesis. We investigated fimbriae and LPS from several P. gingivalis strains in terms of relative dependence on Toll-like receptor (TLR) signalling partners or accessory patternrecognition molecules mediating ligand transfer to TLRs, and determined induced assembly of receptor complexes in lipid rafts. Fimbriae could utilize TLR1 or TLR6 for cooperative TLR2-dependent activation of transfected cell lines, in contrast to LPS and a mutant version of fimbriae which displayed preference for TLR1. Whether used to activate human cell lines or mouse macrophages, fimbriae exhibited strong dependence on membrane-expressed CD14 (mCD14), which could not be substituted for by soluble CD14 (sCD14). In contrast, sCD14 efficiently substituted for mCD14 in LPS-induced cellular activation. LPSbinding protein was more important for LPS-than for fimbria-induced cell activation, whereas the converse was true for CD11b/CD18. Cell activation by LPS or fimbriae required lipid raft function and formation of heterotypic receptor complexes (TLR1-2/CD14/ CD11b/CD18), although wild-type fimbriae additionally recruited TLR6. In summary, TLR2 activation by P. gingivalis LPS or fimbriae involves differential dependence on accessory signalling or ligandbinding receptors, which may differentially influence innate immune responses.

ABSTRACTThe chemotaxis ofVibrio cholerae, the causative agent of cholera, has been implicated in pathogenicity. The bacterium has more than 40 genes for methyl-accepting chemotaxis protein (MCP)-like proteins (MLPs). In this study, we found that glycine and at least 18l-amino acids, including serine, arginine, asparagine, and proline, serve as attractants to the classical biotype strain O395N1. Based on the sequence comparison withVibrio parahaemolyticus, we speculated that at least 17 MLPs ofV. choleraemay mediate chemotactic responses. Among them, Mlp24 (previously named McpX) is required for the production of cholera toxin upon mouse infection.mlp24deletion strains of both classical and El Tor biotypes showed defects in taxis toward several amino acids, which were complemented by the expression of Mlp24. These amino acids enhanced methylation of Mlp24. Serine, arginine, asparagine, and proline were shown to bind directly to the periplasmic fragment of Mlp24. The structural information of its closest homolog, Mlp37, predicts that Mlp24 has two potential ligand-binding pockets per subunit, the membrane distal of which was suggested, by mutational analyses, to be involved in sensing of amino acids. These results suggest that Mlp24 is a chemoreceptor for multiple amino acids, including serine, arginine, and asparagine, which were previously shown to stimulate the expression of several virulence factors, implying that taxis toward a set of amino acids plays critical roles in pathogenicity ofV. cholerae.

The histidine autokinase CheA functions as the central processing unit in the Escherichia coli chemotaxis signaling machinery. CheA receives autophosphorylation control inputs from chemoreceptors and in turn regulates the flux of signaling phosphates to the CheY and CheB response regulator proteins. Phospho-CheY changes the direction of flagellar rotation; phospho-CheB covalently modifies receptor molecules during sensory adaptation. The CheA phosphorylation site, His-48, lies in the N-terminal P1 domain, which must engage the CheA ATP-binding domain, P4, to initiate an autophosphorylation reaction cycle. The docking determinants for the P1-P4 interaction have not been experimentally identified. We devised mutant screens to isolate P1 domains with impaired autophosphorylation or phosphotransfer activities. One set of P1 mutants identified amino acid replacements at surface-exposed residues distal to His-48. These lesions reduced the rate of P1 transphosphorylation by P4. However, once phosphorylated, the mutant P1 domains transferred phosphate to CheY at the wild-type rate. Thus, these P1 mutants appear to define interaction determinants for P1-P4 docking during the CheA autophosphorylation reaction.

Escherichia coli chemoreceptors can sense changes in temperature for thermotaxis. Here we found that the aerotaxis transducer Aer, a homolog of chemoreceptors lacking a periplasmic domain, mediates thermoresponses. We propose that thermosensing by the chemoreceptors is a general attribute of their highly conserved cytoplasmic domain (or their less conserved transmembrane domain).Most organisms have evolved mechanisms to sense and respond to changes in temperature since they can live only within a limited temperature range. Although studies of such thermosensing systems are generally difficult, thermotaxis of Escherichia coli is exceptionally well characterized (5, 6). Wild-type E. coli cells are attracted to warmer and repelled by colder environments. These thermotactic behaviors are mediated by the E. coli chemotaxis signaling system, which regulates the cell's direction of flagellar rotation. Attractant increases, sensed by transmembrane chemoreceptors (Tsr, Tar, Trg, and Tap), also known as methyl-accepting chemotaxis proteins (MCPs), promote counterclockwise (CCW) rotation and forward swimming, whereas repellent increases, sensed by the same receptors, promote clockwise (CW) rotation and random turns or tumbles (4). Early studies revealed that the chemoreceptors of E. coli also mediate thermotactic responses (5, 6). Tsr, Tar, and Trg function as warm sensors, which produce CCW signals upon temperature upshift and CW signals upon temperature downshift. In contrast, Tap functions as a cold sensor that produces signals of the opposite output to temperature changes (5,7,9). Tsr and Tar show altered thermosensing properties after adaptation to their attractants, serine and aspartate, respectively. Tsr loses temperature-sensing ability, whereas Tar shifts from a warm sensor to a cold sensor (7,8,(10)(11)(12). To obtain deeper molecular insights into the thermosensing mechanism of receptors, in this study we focused on Aer, a redox sensor of E. coli. Aer has a cytoplasmic kinase control module like that of the other chemoreceptors, but instead of a periplasmic domain, Aer has a large amino-terminal, cytoplasmic PAS domain that binds flavin adenine dinucleotide (FAD) ( Fig. 1; for a review, see reference 17).In this study, we developed a new temperature-control device (Fig. 2). It consisted of a chamber formed from two coverslips (18 mm ϫ 18 mm), with short pieces of glass capillaries (1.0 cm in length) fixed with epoxy adhesive serving as spacers between the two slips. Four silicon tubes were placed at the four corners of the chamber and sealed with epoxy adhesive. The two inlet tubes on the left side were connected to one larger tube (2-mm inside diameter) with a Y adapter. The two outlet tubes on the right side were connected together in the same way. Temperature changes inside the chamber were initiated by switching the water flow between two tanks maintained at different temperatures. The water flow was produced by siphon action, and the flow rate was adjusted by shifting the relative height of the tanks. Altho...