Streptococcus mutans strain JH1000 is a clinical isolate which has been intensively studied because its superior ability to colonize the human oral cavity makes it a logical choice for use in the replacement therapy of dental caries. Mutant analysis was used to show that this stain's colonization potential is dependent on its production of a small, highly potent bacteriocin-like inhibitory substance (BLIS) that has a broad antimicrobial spectrum of activity. Recent studies have proven that the BLIS activity is a previously unidentified lantibiotic, which has been named mutacin 1140. In the course of identifying this activity, we also identified two N-acyl-L-homoserine lactone (AHSL)-like compounds, which are molecules involved in quorum sensing in Gram negative bacteria and which have not been previously described in Gram positive bacteria. Preliminary evidence indicates that one or both of these compounds positively regulates mutacin 1140 synthesis. Certain studies proposed in this application are designed to elucidate the structure, chemistry, and genetics of mutacin 1140 for its potential application in replacement therapy and as an antibiotic and food preservative. Other studies are designed to purify the AHSL-like molecules to enable their precise and detailed structural characterization. Physiology studies and mutant analysis will be used to confirm their role in quorum sensing.
In specific aim 1 of this proposal, large scale purification methods will be used to obtain sufficient mutacin 1140 to enable complete structural identification using chemical methods and NMR spectroscopy.
In specific aim 2, the mechanism of action of mutacin 1140 on target strains will be analyzed by determining its ability to form pores in their cytoplasmic membranes.
In specific aim 3, we will clone and sequence all of the genes essential for mutacin 1140 synthesis and determine their structural organization. Isogenic mutants will be constructed and tested to determine the roles of the various genes in mutacin 1140 synthesis.
In specific aim 4, the optimum cultivation conditions for AHSL synthesis will be determined and methods will be developed to purify them to homogeneity. They will be structurally characterized using spectroscopic methods.
In specific aim 5, a reporter gene-labeled construct of JH1140 will be mutagenized with Tn917 and mutants lacking positive regulators for mutacin 1140 expression will be isolated and analyzed.
| Ghobrial, Oliver G; Derendorf, Hartmut; Hillman, Jeffery D (2009) Pharmacodynamic activity of the lantibiotic MU1140. Int J Antimicrob Agents 33:70-4 |
| Smith, Leif; Hillman, Jd (2008) Therapeutic potential of type A (I) lantibiotics, a group of cationic peptide antibiotics. Curr Opin Microbiol 11:401-8 |
| Smith, Leif; Zachariah, Cherian; Thirumoorthy, Ramanan et al. (2003) Structure and dynamics of the lantibiotic mutacin 1140. Biochemistry 42:10372-84 |
