Li, Yung-Hua
Permanent URI for this collectionhttps://hdl.handle.net/10222/38679
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Item Open Access Structure-activity analysis of quorum-sensing signaling peptides from Streptococcus mutans(2007-02) Syvitski, R. T.; Tian, X. L.; Sampara, K.; Salman, A.; Lee, S. F.; Jakeman, D. L.; Li, Y. H.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 alpha-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 alpha-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 alpha-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.Item Open Access Detachment of Streptococcus mutans biofilm cells by an endogenous enzymatic activity(1996-03) Lee, SF; Li, YH; Bowden, GHPrevious studies have shown that Streptococcus mutans NG8 possesses an endogenous surface protein-releasing enzyme (SPRE) activity that liberates its own surface proteins (S. P. Lee, Infect. Immun. 60:4032-4039, 1992). The present study was initiated to investigate the possible role of the release of surface proteins by SPRE in the detachment of biofilm cells in vitro. Initially, the characteristics of surface protein release by the strain (S. mutans BM71) used in this study were shown to be the same as those previously described for S. mutans NG8, BM71 displayed characteristics identical to those of NG8 in terms of pH optima and inhibitor sensitivity for protein release. Monolayer biofilms of S. mutans BM71 were formed on hydroxylapatite rods in a modified chemostat. Detachment of the biofilm cells was measured by viable cell counts of bacteria liberated after incubation of the biofilms in buffers, Results showed that biofilm cells were detached in a pH-dependent manner with a maximum rate at pH 5 (P = 0.016) to 6 (P = 0.002), a range similar to that for optimal surface protein release. The detachment of the biofilm cells was found to be inhibited by ZnCl2 (P = 0.002 to 0.023), which also inhibited surface protein release. Detachment was not inhibited significantly by CaCl2 (P = 0.525 to 0.784), precluding an ionic effect on inhibition by ZnCl2. The extent of detachment could be increased (P = 0.046) by the addition of an SPRE preparation from S. mutans but not heat-inactivated SPRE (P = 0.665) or SPRE in the presence of ZnCl2 (P = 0.199). Detachment was also studied by using biofilms of resting (viable but not dividing) cells. Results similar to those for biofilms formed from growing cells were obtained, indicating that cells detached from biofilms were not daughter cells. The results presented above show that monolayer biofilm cells of S. mutans under conditions of minimal shear force have the ability to detach from a surface and suggest that this detachment was mediated by an endogenous SPRE activity.Item Open Access Quorum sensing and bacterial social interactions in biofilms(2012) Li, Y. H.; Tian, X.Many bacteria are known to regulate their cooperative activities and physiological processes through a mechanism called quorum sensing (QS), in which bacterial cells communicate with each other by releasing, sensing and responding to small diffusible signal molecules. The ability of bacteria to communicate and behave as a group for social interactions like a multi-cellular organism has provided significant benefits to bacteria in host colonization, formation of biofilms, defense against competitors, and adaptation to changing environments. Importantly, many QS-controlled activities have been involved in the virulence and pathogenic potential of bacteria. Therefore, understanding the molecular details of quorum sensing mechanisms and their controlled social activities may open a new avenue for controlling bacterial infections.Item Open Access The BceABRS four-component system regulates the bacitracin-induced cell envelope stress response in Streptococcus mutans(2010-09) Ouyang, J.; Tian, X. L.; Versey, J.; Wishart, A.; Li, Y. H.Streptococcus mutans is known to be resistant to bacitracin, a cyclic polypeptide antibiotic produced by certain species of the genus Bacillus. This property is often exploited in the isolation of S. mutans strains from highly heterogeneous oral microflora. A genetic locus consisting of a four-gene operon, bceABRS (formerly mbrABCD), the component genes of which are homologous to Bacillus subtilis bceRS-bceAB (encoding a two-component system and an ABC transporter), is required for bacitracin resistance in S. mutans. Here we describe the identification of a DNA binding site for the BceR response regulator and its transcriptional control of the bceABRS operon in response to the presence of bacitracin. We provide evidence indicating that phosphorylated BceR binds directly to a conserved invert repeat located between bp -120 and -78 of the bceABRS promoter region and positively regulates expression of the bceABRS operon. We also demonstrate that sensing of bacitracin by the BceS histidine kinase requires the presence of an intact BceAB transporter, since deletion of either bceA or bceB abolishes BceRS-mediated bacitracin sensing. The results suggest that the BceAB transporter acts as a cosensor, together with the BceRS two-component system, for bacitracin perception in S. mutans. By searching the S. mutans genome databases, we have identified three additional genes that share the consensus BceR binding motif at their promoter regions. Our initial work has confirmed that expression of these genes is directly controlled by BceRS, indicating that the bceABRS operon, along with the three additional genes, forms the BceRS regulon in S. mutans. Taking these findings together, we conclude that BceABRS comprises a four-component system that plays an important role in stimulus sensing, signal transduction, the gene regulatory network, and substrate transport for the cell envelope stress response in S. mutans.Item Open Access A novel target-specific, salt-resistant antimicrobial peptide against the cariogenic pathogen Streptococcus mutans(2011-11) Mai, J.; Tian, X. L.; Gallant, J. W.; Merkley, N.; Biswas, Z.; Syvitski, R.; Douglas, S. E.; Ling, J.; Li, Y. H.In this study, we constructed and evaluated a target-specific, salt-resistant antimicrobial peptide (AMP) that selectively targeted Streptococcus mutans, a leading cariogenic pathogen. The rationale for creating such a peptide was based on the addition of a targeting domain of S. mutans ComC signaling peptide pheromone (CSP) to a killing domain consisting of a portion of the marine-derived, broad-spectrum AMP pleurocidin to generate a target-specific AMP. Here, we report the results of our assessment of such fusion peptides against S. mutans and two closely related species. The results showed that nearly 95% of S. mutans cells lost viability following exposure to fusion peptide IMB-2 (5.65 muM) for 15 min. In contrast, only 20% of S. sanguinis or S. gordonii cells were killed following the same exposure. Similar results were also observed in dual-species mixed cultures of S. mutans with S. sanguinis or S. gordonii. The peptide-guided killing was further confirmed in S. mutans biofilms and was shown to be dose dependent. An S. mutans mutant defective in the CSP receptor retained 60% survival following exposure to IMB-2, suggesting that the targeted peptide predominantly bound to the CSP receptor to mediate killing in the wild-type strain. Our work confirmed that IMB-2 retained its activity in the presence of physiological or higher salt concentrations. In particular, the fusion peptide showed a synergistic killing effect on S. mutans with a preventive dose of NaF. In addition, IMB-2 was relatively stable in the presence of saliva containing 1 mM EDTA and did not cause any hemolysis. We also found that replacement of serine-14 by histidine improved its activity at lower pH. Because of its effectiveness, salt resistance, and minimal toxicity to host cells, this novel target-specific peptide shows promise for future development as an anticaries agent.