Porphyromonas gingivalis is an oral/systemic pathogen implicated in chronic conditions, although the mechanism(s) whereby it resists immune defenses and persists in the host is poorly understood. The virulence of this pathogen partially depends upon expression of fimbriae comprising polymerized fimbrillin (FimA) associated with quantitatively minor proteins (FimCDE). In this study, we show that isogenic mutants lacking FimCDE are dramatically less persistent and virulent in a mouse periodontitis model and express shorter fimbriae than the wild type. Strikingly, native fimbriae allowed P. gingivalis to exploit the TLR2/complement receptor 3 pathway for intracellular entry, inhibition of IL-12p70, and persistence in macrophages. This virulence mechanism also required FimCDE; indeed, mutant strains exhibited significantly reduced ability to inhibit IL-12p70, invade, and persist intracellularly, attributable to failure to interact with complement receptor 3, although not with TLR2. These results highlight a hitherto unknown mechanism of immune evasion by P. gingivalis that is surprisingly dependent upon minor constituents of its fimbriae, and support the concept that pathogens evolved to manipulate innate immunity for promoting adaptive fitness and thus their capacity to cause disease.

The FimA fimbriae of Porphyromonas gingivalis, the causative agent of periodontitis, have been implicated in various aspects of pathogenicity, such as colonization, adhesion and aggregation. In this study, the four open reading frames (ORF1, ORF2, ORF3 and ORF4) downstream of the fimbrilin gene (fimA) in strain ATCC 33277 were examined. ORF2, ORF3 and ORF4 were demonstrated to encode minor components of the fimbriae and were therefore renamed fimC, fimD and fimE, respectively. Immunoblotting analyses revealed that inactivation of either fimC or fimD by an ermF-ermAM insertion, but not inactivation of ORF1, was accompanied by concomitant loss of the products from the downstream genes, raising the possibility that fimC, fimD and fimE constitute a transcription unit. The fimE mutant produced FimC and FimD, but fimbriae purified from it contained neither protein, suggesting that FimE is required for the assembly of FimC and FimD onto the fimbrilin (FimA) fibre. The fimC, fimD and fimE mutants lost autoaggregation abilities. Fimbriae purified from these three mutants showed attenuated binding activities to glyceraldehyde-3-phosphate dehydrogenase of Streptococcus oralis and to two extracellular matrix proteins, fibronectin and type I collagen. These results suggest that FimE, as well as FimC and FimD, play critical roles in the adhesive activities of the mature FimA fimbriae in P. gingivalis.

The aspartate chemoreceptor Tar has a thermosensing function that is modulated by covalent modification of its four methylation sites (Gln295, Glu302, Gln309, and Glu491). Without posttranslational deamidation, Tar has no thermosensing ability. When Gln295 and Gln309 are deamidated to Glu, the unmethylated and heavily methylated forms function as warm and cold sensors, respectively. In this study, we carried out alanine-scanning mutagenesis of the methylation sites. Although alanine substitutions influenced the signaling bias and the methylation level, all of the mutants retained aspartate-sensing function. Those with single substitutions had almost normal thermosensing properties, indicating that substitutions at any particular methylation site do not seriously impair thermosensing function. In the posttranslational modification-defective background, some of the alanine substitutions restored thermosensing ability. Warm sensors were found among mutants retaining two glutamate residues, and cold sensors were found among those with one or no glutamate residue. This result suggests that the negative charge at the methylation sites is one factor that determines thermosensor phenotypes, although the size and shape of the side chain may also be important. The warm, cold, and null thermosensor phenotypes were clearly differentiated, and no intermediate phenotypes were found. Thus, the different thermosensing phenotypes that result from covalent modification of the methylation sites may reflect distinct structural states. Broader implications for the thermosensing mechanism are also discussed.Escherichia coli responds to small changes in temperature by altering its swimming behavior to migrate in spatial temperature gradients (22). This phenomenon, thermotaxis, is well suited to studies of the molecular mechanism of thermosensing. Four closely related transmembrane proteins have been identified as thermosensors (21,27,29). These "thermometer" proteins were originally identified as methyl-accepting chemotaxis proteins (MCPs) and as transducers responsible for chemotaxis (for reviews, see references 23, 32 and 40). In response to a repellent or an attractant, the receptor activates or inactivates the cytoplasmic histidine kinase CheA, which phosphorylates itself and serves as a phosphodonor for the cytoplasmic signaling protein CheY. Phospho-CheY interacts with the flagellar motor, which otherwise rotates counterclockwise (CCW), to cause clockwise (CW) rotation that results in loss of propulsive power and change of swimming direction (tumbling). A warm sensor mediates attractant and repellent responses upon increases and decreases in temperature, respectively, and a cold sensor mediates the opposite responses to the same stimuli (29).Both in the presence and in the absence of its ligand, the receptors exist as homodimers (25) that form stable complexes with the CheA homodimer and two molecules of the coupling protein CheW (14, 34). The thermosensing mechanism is thought to involve temperature-dependent changes in the str...

Vibrio cholerae, the etiological agent of cholera, was found to be attracted by taurine (2-aminoethanesulfonic acid), a major constituent of human bile. Mlp37, the closest homolog of the previously identified amino acid chemoreceptor Mlp24, was found to mediate taxis to taurine as well as L-serine, L-alanine, L-arginine, and other amino acids. Methylation of Mlp37 was enhanced upon the addition of taurine and amino acids. Isothermal titration calorimetry demonstrated that a purified periplasmic fragment of Mlp37 binds directly to taurine, L-serine, L-alanine and L-arginine. Crystal structures of the periplamic domain of Mlp37 revealed that L-serine and taurine bind to the membrane-distal PAS domain in essentially in the same way. The structural information was supported by characterising the in vivo properties of alanine-substituted mutant forms of Mlp37. The fact that the ligand-binding domain of the L-serine complex had a small opening, which would accommodate a larger R group, accounts for the broad ligand specificity of Mlp37 and allowed us to visualise ligand binding to Mlp37 with fluorescently labelled L-serine. Taken together, we conclude that Mlp37 serves as the major chemoreceptor for taurine and various amino acids.

The periodontitis-associated pathogen Porphyromonas gingivalis colonizes and forms a biofilm in gingival crevices through fimbriae. It is known that the often-used strains ATCC 33277 and 381 produce long FimA fimbriae. We found a possible nonsense mutation within fimB, immediately downstream from fimA, coding a major subunit of FimA fimbriae of the strains. Indeed, P. gingivalis strains, except for ATCC 33277 and 381, universally expressed FimB, the gene product of fimB. Electron micrographs revealed that a FimB-restored strain had short and dense, "toothbrush"-like, FimA fimbriae. FimA overexpression elongated the fimbriae, whereas FimB overexpression shortened them. FimB restoration increased production of FimA and its accessory proteins. Thus, FimB regulates the length and expression of FimA fimbriae. Additionally, FimB restoration significantly reduced the release of FimA fimbriae from the cell surface, suggesting that FimB functions as an anchor of the fimbriae. The restoration enhanced adherent activity as well.