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ABSTRACTBacterially induced cell death in human lymphocytes is an important virulence factor for pathogenic bacteria. Previously discovered mechanisms of bacterially induced cell death are predominantly based on the transfer of bacterial proteins to the target host cell, such as the toxins secreted through type I, II, and VI secretion systems or effector proteins injected through type III, IV, and Vb secretion systems. Here, we report a mechanism employed by the Gram-negative oral pathogen Fusobacterium nucleatumfor cell death induction of human lymphocytes via two outer membrane proteins (OMPs), Fap2 and RadD, which share regions homologous to autotransporter secretion systems (type Va secretion systems). Genetic and physiological studies established that inactivation of the two OMPs led to significantly reduced ability to trigger cell death in Jurkat cells, while the corresponding double mutant was almost completely attenuated. Additional biochemical and molecular analyses demonstrated that cell-free F. nucleatummembranes are sufficient to induce cell death in Jurkat cells, suggesting that no active process or effector protein transfer was necessary to induce eukaryotic cell death.

ABSTRACTMany clinical isolates of Streptococcus mutansproduce peptide antibiotics called mutacins. Mutacin production may play an important role in the ecology of S. mutansin dental plaque. In this study, inactivation of a histidine kinase gene, ciaH, abolished mutacin production. Surprisingly, the same mutation also diminished competence development, stress tolerance, and sucrose-dependent biofilm formation.

ABSTRACTQuorum sensing is a bacterial mechanism for regulating gene expression in response to changes in population density. Many bacteria are capable of acyl-homoserine lactone-based or peptide-based intraspecies quorum sensing and luxS-dependent interspecies quorum sensing. While there is good evidence about the involvement of intraspecies quorum sensing in bacterial biofilm, little is known about the role of luxSin biofilm formation. In this study, we report for the first time that luxS-dependent quorum sensing is involved in biofilm formation of Streptococcus mutans. S. mutansis a major cariogenic bacterium in the multispecies bacterial biofilm commonly known as dental plaque. An ortholog of luxSfor S. mutanswas identified using the data available in the S. mutansgenome project (http://www.genome.ou.edu/smutans.html). Using an assay developed for the detection of the LuxS-associated quorum sensing signal autoinducer 2 (AI-2), it was demonstrated that this ortholog was able to complement the luxSnegative phenotype of Escherichia coliDH5α. It was also shown that AI-2 is indeed produced by S. mutans. AI-2 production is maximal during mid- to late-log growth in batch culture. Mutant strains devoid of the luxSgene were constructed and found to be defective in producing the AI-2 signal. There are also marked phenotypic differences between the wild type and the luxSmutants. Microscopic analysis of in vitro-grown biofilm structure revealed that the luxSmutant biofilms adopted a much more granular appearance, rather than the relatively smooth, confluent layer normally seen in the wild type. These results suggest that LuxS-dependent signal may play an important role in biofilm formation of S. mutans.

ABSTRACTThe ability to penetrate tissue is an important virulence factor for pathogenic spirochetes. Previous studies have recognized the role of motility in allowing pathogenic spirochetes to invade tissues and migrate to sites favorable for bacterial proliferation. However, the nature of the movements, whether they are random or controlled by chemotaxis systems, has yet to be established. In this study, we addressed the role of motility and chemotaxis in tissue penetration by the periodontal disease-associated oral spirochete Treponema denticolausing an oral epithelial cell line-based experimental approach. Wild-type T. denticolaATCC 35405 was found to penetrate the tissue layers effectively, whereas a nonmotile mutant was unable to overcome the tissue barrier. Interestingly, the chemotaxis mutants also showed impaired tissue penetration. AcheAmutant that is motile but lacks the central kinase of the chemotaxis pathway showed only about 2 to 3% of the wild-type penetration rate. The two known chemoreceptors of T. denticola, DmcA and DmcB, also appear to be involved in the invasion process. The dmcmutants were actively motile but exhibited reduced tissue penetration of about 30 and 10% of the wild-type behavior, respectively. These data suggest that not only motility but also chemotaxis is involved in the tissue penetration byT. denticola.

ABSTRACTBacteria are causative agents of periodontal diseases. Interactions between oral bacteria and gingival epithelial cells are essential aspects of periodontal infections. Using an in vitro tissue culture model, a selected group of gram-negative anaerobic bacteria frequently associated with periodontal diseases, includingBacteroides forsythus, Campylobacter curvus,Eikenella corrodens, Fusobacterium nucleatum,Porphyromonas gingivalis, and Prevotella intermedia, were examined for their ability to adhere to and invade primary cultures of human gingival epithelial cells (HGEC). The effects of these bacteria on the production of interleukin-8 (IL-8), a proinflammatory chemokine, were also measured. These studies provided an initial demonstration that F. nucleatumadhered to and invaded HGEC and that this was accompanied by high levels of IL-8 secretion from the epithelial cells. The attachment and invasion characteristics of F. nucleatumwere also tested using KB cells, an oral epithelial cell line. The invasion was verified by transmission electron microscopy and with metabolic inhibitors. Invasion appeared to occur via a “zipping” mechanism and required the involvement of actins, microtubules, signal transduction, protein synthesis, and energy metabolism of the epithelial cell, as well as protein synthesis by F. nucleatum. A spontaneous mutant, lam, of F. nucleatum, isolated as defective in autoagglutination, was unable to attach to or invade HGEC or KB cells, further indicating the requirement of bacterial components in these processes. Sugar inhibition assays indicated that lectin-like interactions were involved in the attachment of F. nucleatumto KB cells. Investigation of these new virulence phenotypes should improve our understanding of the role of F. nucleatumin periodontal infections.