No abstract

Streptococcus mutans secretes and utilizes a 21-amino-acid signaling peptide pheromone to initiate quorum sensing for genetic competence, biofilm formation, stress responses, and bacteriocin production. In this study, we designed and synthesized a series of truncated peptides and peptides with amino acid substitutions to investigate their structure-activity relationships based on the three-dimensional structures of S. mutans wild-type signaling peptide UA159sp and C-terminally truncated peptide TPC3 from mutant JH1005 defective in genetic competence. By analyzing these peptides, we demonstrated that the signaling peptide of S. mutans has at least two functional domains. The C-terminal structural motif consisting of a sequence of polar hydrophobic charged residues is crucial for activation of the signal transduction pathway, while the core ␣-helical structure extending from residue 5 to the end of the peptide is required for receptor binding. Peptides in which three or more residues were deleted from the C terminus did not induce genetic competence but competitively inhibited quorum sensing activated by UA159sp. Disruption of the amphipathic ␣-helix by replacing the Phe-7, Phe-11, or Phe-15 residue with a hydrophilic residue resulted in a significant reduction in or complete loss of the activity of the peptide. In contrast to the C-terminally truncated peptides, these peptides with amino acid substitutions did not compete with UA159sp to activate quorum sensing, suggesting that disruption of the hydrophobic face of the ␣-helical structure results in a peptide that is not able to bind to the receptor. This study is the first study to recognize the importance of the signaling peptide C-terminal residues in streptococcal quorum sensing.

Sortase is a newly discovered transpeptidase that covalently links LPXTGX-containing surface proteins to the gram-positive bacterial cell wall. In this study, the sortase gene (srtA) was isolated from Streptococcus mutans NG8 by PCR. The gene encoded a 246-amino-acid protein, including a 40-amino-acid signal peptide. The srtA gene was insertionally inactivated by a tetracycline resistance cassette. P1, a major surface protein adhesin previously shown to anchor to the peptidoglycan by the LPXTGX motif, was secreted into the culture medium by the srtA mutant. In contrast, the wild-type P1 remained cell wall associated. Complementation of the mutant with srtA restored the P1 surface expression phenotype. P1 produced by the mutant, but not that produced by the wild type and the srtA-complemented mutant, was recognized by an antibody raised against the hydrophobic domain and charged tail C terminal to the LPXTGX motif. These results suggest that the failure to anchor P1 to the cell wall is due to the lack of cleavage of P1 at the LPXTGX motif. The srtA mutant was markedly less hydrophobic than the wild type and the complemented mutant. The srtA mutant failed to aggregate in the presence of saliva or salivary agglutinin and adhered poorly to saliva-or salivary agglutinincoated hydroxylapatite. In rats, the srtA mutant colonized the teeth poorly when sucrose was absent. When sucrose was present, the srtA mutant colonized the teeth but less effectively and induced significantly less caries (P < 0.05) than the wild-type strain. In conclusion, the sortase enzyme in S. mutans is responsible for anchoring P1 to the cell surface and plays a role in modulating the surface properties and cariogenicity of S. mutans.The major cell surface protein P1, also known as antigen I/II (ca. 185 kDa), from Streptococcus mutans is an adhesin that interacts with a high-molecular-weight salivary agglutinin and has been implicated in the adherence of the bacterium to the tooth surface (16). Sequence analysis of the P1 gene reveals common features at the C terminus of the protein, as found in several hundreds of surface proteins from many gram-positive coccal bacteria (17). These features include a hydrophilic (wall-associated) region, a highly conserved hexapeptide LPXTGX, a hydrophobic (membrane-spanning) domain, and a charged tail (22,23).In our previous studies, we have demonstrated the requirement of the C-terminal domains in P1 surface localization in S. mutans (10, 11). Site-directed mutagenesis analysis showed that the Thr residue within the LPXTGX motif plays a crucial role in enzymatic cleavage and anchoring of P1 to the cell wall (15).A transpeptidase termed sortase (SrtA) was identified initially in Staphylococcus aureus (27) and recently in Streptococcus gordonii (5), Streptococcus suis (20), Streptococcus pyogenes (2), and Listeria monocytogenes (4, 9). The enzyme is responsible for covalently anchoring protein A to the cell wall (27) and plays a role the pathogenesis of S. aureus (12, 18). Recently, the role of SrtA in virulence...

No abstract

No abstract

No abstract

Treponema denticola is considered to be an agent strongly associated with periodontal disease. The lack of an animal infection model has hampered the understanding of T. denticola pathogenesis and the host's immune response to infection. In this study, we have established an oral infection model in mice, demonstrating that infection by oral inoculation is feasible. The presence of T. denticola in the oral cavities of the animals was confirmed by PCR. Mice given T. denticola developed a specific immune response to the bacterium. The antibodies generated from the infection were mainly of the immunoglobulin G1 subclass, indicating a Th2-tilted response. The antibodies recognized 11 T. denticola proteins, of which a 62-kDa and a 53-kDa protein were deemed immunodominant. The two proteins were identified, respectively, as dentilisin and the major outer sheath protein by mass spectrometry. Splenocytes cultured from the infected mice no longer produced interleukin-10 and produced markedly reduced levels of gamma interferon relative to those produced by naïve splenocytes upon stimulation with T. denticola. Mandibles of infected mice showed significantly greater bone resorption (P < 0.01) than those of mock-infected controls.

Using a bioassay-directed purification scheme, the active antibacterial principle from Caesalpina sappan was isolated and identified to be brasilin. This compound showed potent activity against antibiotic-resistant bacteria, notably methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), multi-drug resistant Burkholderia cepacia as well as a number of other bacteria. The minimal inhibitory concentrations ranged from 4 to 32 microg/mL. The results from time-kill studies showed that brasilin is bactericidal against MRSA. The addition of brasilin to growing MRSA cells resulted in a rapid inhibition of incorporation of [(3)H] thymidine or [(3)H] serine into DNA and proteins, respectively. Exposure of MRSA to a sub-MIC level of brasilin for ten consecutive subcultures did not induce resistance to the compound. The Trypan blue dye exclusion test showed that brasilin lacked cytotoxicity against Vero cells. In conclusion, brasilin is an antibacterial principle from C. sappan and it has the potential to be developed into an antibiotic.