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.

Porphyromonas gingivalis, one of the causative agents of adult periodontitis, attaches and forms biofilms on substrata of Streptococcus gordonii. Coadhesion and biofilm development between these organisms requires the interaction of the short fimbriae of P. gingivalis with the SspB streptococcal surface polypeptide. In this study we investigated the structure and binding activities of the short fimbriae of P. gingivalis. Electron microscopy showed that isolated short fimbriae have an average length of 103 nm and exhibit a helical structure with a pitch of ca. 27 nm. Mfa1, the major protein subunit of the short fimbriae, bound to SspB protein, and this reaction was inhibited by purified recombinant Mfa1 and monospecifc anti-Mfa1 serum in a dose-dependent manner. Complementation of a polar Mfa1 mutant with the mfa1 gene restored the coadhesion phenotype of P. gingivalis. Hence, the Mfa1 structural fimbrial subunit does not require accessory proteins for binding to SspB. Furthermore, the interaction of Mfa1 with SspB is necessary for optimal coadhesion between P. gingivalis and S. gordonii.

Porphyromonas gingivalis is a periodontal pathogen whose primary niche is the anaerobic environment of subgingival dental plaque, but initial colonization of the oral cavity is likely to occur on supragingival surfaces that already support robust biofilm communities. Our studies have shown that P. gingivalis adheres to Streptococcus gordonii through interaction of the minor fimbrial antigen Mfa1 with a specific region of the streptococcal SspB polypeptide (residues 1167 to 1193) designated BAR. We show that a synthetic peptide comprising the BAR sequence potently inhibits P. gingivalis adherence to S. gordonii (50% inhibitory concentration ‫؍‬ 1.3 M) and prevents the development of P. gingivalis biofilms. However, a retroinverso peptide that possessed the same side chain topology as that of BAR was inactive, suggesting that interactions of Mfa1 with the peptide backbone of BAR are important for binding. A conformationally constrained analog of BAR inhibited P. gingivalis adherence and biofilm formation but at a lower specific activity than that of BAR. Therefore, to further define the structural features of the Mfa1-BAR interaction, we functionally screened combinatorial libraries of BAR in which active site residues (Asn 1182 , Thr 1184, and Val 1185) were replaced with each of the 19 common amino acids. Peptides containing positively charged amino acids at position 1182 or hydrophobic residues at position 1185 bound P. gingivalis more efficiently than did control peptides containing Asn and Val at these positions, suggesting that electrostatic and hydrophobic interactions may contribute to Mfa1-SspB binding. In contrast, replacement of Pro or Gly at these positions was detrimental to adherence, suggesting that perturbation of the BAR secondary structure influences activity. The net effect of substitutions for Thr 1184 was less pronounced either positively or negatively than that at the other sites. These results define physicochemical characteristics of the interacting interface of Mfa1 and SspB and suggest that peptides or peptidomimetics with greater specific inhibitory activity than that of BAR can be developed. These compounds may represent potential therapeutics that target some of the first molecular interactions that allow P. gingivalis to colonize the oral cavity.Dental plaque is a complex and dynamic biofilm that accumulates through the sequential and ordered colonization of over 700 species of bacteria (13,24,25,28). An oral biofilm comprised predominantly of gram-positive commensals such as the oral streptococci and Actinomyces spp. can exist in the oral cavity in the absence of overt disease (21, 25). However, populational shifts in the biofilm that lead to overrepresentation of acidophiles or of gram-negative obligate anaerobes may contribute to the onset and progression of the most common oral diseases, caries and periodontal disease (5). Indeed, adult periodontitis is associated with elevated levels of several gramnegative anaerobes in subgingival plaque, including the asaccharolytic, obligate a...

Autoinducer 2 (AI-2) produced by the oral pathogen Actinobacillus actinomycetemcomitans influences growth of the organism under iron limitation and regulates the expression of iron uptake genes. However, the cellular components that mediate the response of A. actinomycetemcomitans to AI-2 have not been fully characterized. Analysis of the complete genome sequence of A. actinomycetemcomitans (www.oralgen.lanl.gov) indicated that the RbsB protein was related to LuxP, the AI-2 receptor of Vibrio harveyi. To determine if RbsB interacts with AI-2, the bioluminescence of the reporter strain V. harveyi BB170 (sensor 1؊, sensor 2؉) was determined after stimulation with partially purified AI-2 from A. actinomycetemcomitans or conditioned medium from V. harveyi cultures in the presence and absence of purified six-His-tagged RbsB. RbsB efficiently inhibited V. harveyi bioluminescence induced by both A. actinomycetemcomitans AI-2 and V. harveyi AI-2 in a dose-dependent manner, suggesting that RbsB competes with LuxP for AI-2. Fifty percent inhibition occurred with approximately 0.3 nM RbsB for A. actinomycetemcomitans AI-2 and 15 nM RbsB for V. harveyi AI-2. RbsB-mediated inhibition of V. harveyi bioluminescence was reversed by the addition of 50 mM ribose, suggesting that A. actinomycetemcomitans AI-2 and ribose bind at the same site of RbsB. The RbsB/AI-2 complex was thermostable since A. actinomycetemcomitans AI-2 could not be recovered by heating. This was not due to heat inactivation of A. actinomycetemcomitans AI-2 since signal activity was unaffected by heating in the absence of RbsB. Furthermore, an isogenic A. actinomycetemcomitans mutant that was unable to express rbsB was deficient in depleting A. actinomycetemcomitans AI-2 from solution relative to the wild-type organism. Inactivation of rbsB also influenced the ability of the organism to grow under iron-limiting conditions. The mutant strain attained a cell density of approximately 30% that of the wild-type organism under iron limitation. In addition, real-time PCR showed that the expression of afuABC, encoding a major ferric ion transporter, was reduced by approximately eightfold in the rbsB mutant. This phenotype was similar to that of a LuxS-deficient mutant of A. actinomycetemcomitans that is unable to produce AI-2. Together, our results suggest that RbsB may play a role in the response of A. actinomycetemcomitans to AI-2.

Our previous studies showed that the Aggregatibacter actinomycetemcomitans RbsB protein interacts with cognate and heterologous autoinducer 2 (AI-2) signals and suggested that the rbsDABCK operon encodes a transporter that may internalize AI-2 (D. James et al., Infect. Immun. 74:4021-4029, 2006.). However, A. actinomycetemcomitans also possesses genes related to the lsr operon of Salmonella enterica serovar Typhimurium which function to import AI-2. Here, we show that A. actinomycetemcomitans LsrB protein competitively inhibits the interaction of the Vibrio harveyi AI-2 receptor (LuxP) with AI-2 from either A. actinomycetemcomitans or V. harveyi. Interestingly, LsrB was a more potent inhibitor of LuxP interaction with AI-2 from V. harveyi whereas RbsB competed more effectively with LuxP for A. actinomycetemcomitans AI-2. Inactivation of lsrB in wild-type A. actinomycetemcomitans or in an isogenic RbsB-deficient strain reduced the rate by which intact bacteria depleted A. actinomycetemcomitans AI-2 from solution. Consistent with the results from the LuxP competition experiments, the LsrB-deficient strain depleted AI-2 to a lesser extent than the RbsB-deficient organism. Inactivation of both lsrB and rbsB virtually eliminated the ability of the organism to remove AI-2 from the extracellular environment. These results suggest that A. actinomycetemcomitans possesses two proteins that differentially interact with AI-2 and may function to inactivate or facilitate internalization of AI-2.Autoinducer 2 (AI-2) is a quorum-sensing signal that was initially identified in Vibrio harveyi (2) and is produced by the luxS gene (28, 29). AI-2 is produced by LuxS-catalyzed cleavage of S-ribosylhomocysteine to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione (36), which in turn undergoes further rearrangement to produce AI-2. However, two distinct structural forms of AI-2 have been identified. Salmonella enterica serovar Typhimurium produces 2R,4S-2,3,3,4-methyltetrahydroxytetrahydrofuran (R-THMF) whereas Vibrio subsp. produce the borate diester form of S-THMF (6, 22). AI-2-dependent quorum sensing is quite complex and is involved in the regulation of a variety of genes in Vibrio species, including the lux operon of V. harveyi. Detection of AI-2 by V. harveyi is mediated by LuxP, a periplasmic AI-2 receptor (6) that associates with the LuxQ sensor kinase-phosphatase (23) and initiates a phosphotransfer cascade involving LuxU (11) and the response regulator LuxO (10). LuxO in turn influences the expression of multiple small regulatory RNAs (Qrr regulators) that influence the expression of LuxR (15, 16), the master regulator of the lux operon (16). Recently, an additional twocomponent system encoded by varSA in Vibrio cholerae has been shown to converge on the quorum-sensing circuit by regulating the expression of the small RNAs encoded by csrBCD which control the expression of Qrr via CsrA (15).LuxS is highly conserved in a wide range of gram-positive and gram-negative bacteria, and many, if not all, of these organisms produ...