Elucidation of the Antimicrobial Mechanism of Mutacin 1140 Leif Smith,* ,‡ Hester Hasper, § Eefjan Breukink, § Jan Novak, | Jirˇı ´C ˇ erkasov, ⊥ J. D. Hillman, # Shawanda Wilson-Stanford, ‡ and Ravi S. Orugunty ∇ Department of Biological Sciences, Mississippi State UniVersity, Mississippi State, Mississippi 39762, Department Biochemistry of Membranes, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht UniVersity, Padualaan 8, 3584 CH Utrecht, The Netherlands, Department of Microbiology, UniVersity of Alabama at Birmingham, Birmingham, Alabama 35294, Department of Animal Physiology and DeVelopmental Biology, Charles UniVersity, Vinicna 7, 120 00 Prague, Czech Republic, Antimicrobial DiVision, Oragenics, Inc., Alachua, Florida 32615, and Mass Spectrometry Center, ThermoFisher Scientific, West Palm Beach, Florida 33407 ReceiVed June 27, 2007; ReVised Manuscript ReceiVed December 20, 2007 ABSTRACT: Mutacin 1140 and nisin A are peptide antibiotics that belong to the lantibiotic family. N-Terminal rings A and B of nisin A and mutacin 1140 (lipid II-binding domain) share many structural and sequence similarities. Nisin A binds lipid II and thus disrupts cell wall synthesis and also forms transmembrane pores. Very little is known about mutacin 1140 in this regard. We performed fluorescence-based studies using a bacteria-mimetic membrane system. The results indicated that lipid II monomers are arranged differently in the mutacin 1140 complex than in the nisin A complex. These differences in complex formation may be attributed to the fact that nisin A uses lipid II to form a distinct pore complex, while mutacin 1140 does not form pores in this membrane system. Further experiments demonstrated that the mutacin 1140-lipid II and nisin A-lipid II complexes are very stable and capable of withstanding competition from each other. Transmembrane electrical potential experiments using a Streptococcus rattus strain, which is sensitive to mutacin 1140, demonstrated that mutacin 1140 does not form pores in this strain even at a concentration 8 times higher than the minimum inhibitory concentration (MIC). Circular complexes of mutacin 1140 and nisin A were observed by electron microscopy, providing direct evidence for a lateral assembly mechanism for these antibiotics. Mutacin 1140 did exhibit a membrane disruptive function in another commonly used artificial bacterial membrane system, and its disruptive activity was enhanced by increasing amounts of anionic phospholipids. The increasing occurrence of multidrug-resistant bacteria has created a dramatic need for the identification and testing of new antibiotics to take the place of those that are failing. Mutacin 1140 is a posttranslationally modified peptide antibiotic produced by Streptococcus mutans JH1140 (1). It belongs to a family of antibiotics called lantibiotics because of their unique posttranslational modification involving the formation of lanthionine rings. This class of antibiotics has received considerable attention because of their broad spectrum of activity, high potency, low immunogenicity, and good structural stability. Importantly, genetically stable resistant variants of sensitive strains have not yet been found, suggesting that the structure and chemistry of mutacin 1140 may provide important information for the development of new antibiotics. Mutacin 1140 is also a key component in the development of replacement therapy for the prevention of dental caries (2). Thus, for these reasons, it is important to obtain a comprehensive understanding of mutacin 1140 and its mechanism of activity. The 3-D structure of mutacin 1140 was determined using the membrane-mimetic solvent acetonitrile-water (80:20), which is a reasonable starting point to predict the orientation of the membrane-bound molecule (3). Similar to nisin A and gallidermin, the thioether ring structures of mutacin 1140 were rigid and well-defined, and the regions not spanned by thioether linkages were quite flexible (4–6). These flexible regions may be important for bactericidal activity by allowing the lantibiotic to orient itself properly in the membrane (7–10) and for complex formation. Mutacin 1140 was determined to have an overall horseshoe-like structure and is ap- proximately 26 Å in length, 14 Å in width, and would be inserted about 17 Å into the membrane. Electrostatic interactions between the lantibiotic and the bacterial membrane are believed to be important for the initial binding. Experiments with nisin A have demonstrated that the sensitivity of the host bacterium is dependent on the charged state of its cell wall and membrane (7, 10, 11). More importantly, cell-lantibiotic interactions are enhanced by the presence of docking molecules such as lipid II (12–14). Lipid II aids in complex formation and, in the case of nisin A, also in the formation of transmembrane pores. The interaction of nisin A with lipid II involves a novel lipid II binding motif that has been characterized by NMR 1 (15). The N-terminal portion of nisin A, lanthionine rings A and B, interacts with * To whom correspondence should be addressed. E-mail: jls859@ msstate.edu. Fax: (662) 325-7939. Phone: (662) 325-1244. ‡ Mississippi State University. § Utrecht University. | University of Alabama at Birmingham. ⊥ Charles University. # Oragenics, Inc. ∇ ThermoFisher Scientific. 1 Abbreviations: MIC, minimum inhibitory concentration; DOPG, 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; DOPC, 1,2-dio- leoyl-sn-glycero-3-phosphocholine; NMR, nuclear magnetic resonance; TEM, transmission electron microscopy; LUV(s), large unilamellar vesicle(s); TPP + , tetraphenylphosphonium; CF, carboxyfluorescein. Biochemistry 2008, 47, 3308–3314 3308 10.1021/bi701262z CCC: $40.75  2008 American Chemical Society Published on Web 02/12/2008