ARTICLES pubs.acs.org/acschemicalbiology Mechanism of Inhibition of Bacillus anthracis Spore Outgrowth by the Lantibiotic Nisin Ian M. Gut,†,‡ Steven R. Blanke,†,§,* and Wilfred A. van der Donk‡,§,||,* †Department of Microbiology, ‡Howard Hughes Medical Institute, §Institute for Genomic Biology, and D)epartment of Chemistry, University of Illinois, Urbana, Illinois 61801, United States bS Supporting Information ABSTRACT: The lantibiotic nisin inhibits growth of vegetative Gram-positive bacteria by binding to lipid II, which disrupts cell wall biosynthesis and facilitates pore formation. Nisin also inhibits the outgrowth of bacterial spores, including spores of Bacillus anthracis, whose structural and biochemical properties are fundamentally diﬀerent from those of vegetative bacteria. The molecular basis of nisin inhibition of spore outgrowth had not been identiﬁed, as previous studies suggested that inhibition of spore outgrowth involved either covalent binding to a spore target or loss of membrane integrity; disruption of cell wall biosynthesis via binding to lipid II had not been investigated. To provide insights into the latter possibility, the eﬀects of nisin were compared with those of vancomycin, another lipid II binding antibiotic that inhibits cell wall biosynthesis but does not form pores. Nisin and vancomycin both inhibited the replication of vegetative cells, but only nisin inhibited the transition from a germinated spore to a vegetative cell. Moreover, vancomycin prevented nisin’s activity in competition studies, suggesting that the nisin-lipid II interaction is important for inhibition of spore outgrowth. In experiments with ﬂuorescently labeled nisin, no evidence was found for a covalent mechanism for inhibition of spore outgrowth. Interestingly, mutants in the hinge region (N20P/M21P and M21P/K22P) that still bind lipid II but cannot form pores had potent antimicrobial activity against vegetative B. anthracis cells but did not inhibit spore outgrowth. Therefore, pore formation is essential for the latter activity but not the former. Collectively, these studies suggest that nisin utilizes lipid II as the germinated spore target during outgrowth inhibition and that nisin-mediated membrane disruption is essential to inhibit spore development into vegetative cells. Lpeptides produced by a range of bacteria.1 Nisin A is a 34- antibiotics are (methyl)lanthionine-containing antimicrobial amino-acid polycyclic peptide produced by Lactococcus lactis sbsp. lactis ATCC 11454 (Figure 1A). It has emerged as the prototype for studying the antibacterial properties and structure activity relationships of the lantibiotics.2,3 Nisin is ribosomally synthesized as a linear precursor peptide and then post-transla- tionally modiﬁed, generating two dehydroalanines, one dehy- drobutyrine, and ﬁve (methyl)lanthionine cross-links.1,3 Nisin acts upon Gram-positive bacteria by two distinct mechanisms.2 The compound forms pores in cell membranes4 and inhibits cell wall biosynthesis by disruption of transglycosy- lation via binding to and mislocalization of lipid II.5,6 Lipid II (Figure 1B) is an essential intermediate for cell wall biosynthesis. With two mechanisms of action, nisin has been relatively unaﬀected by the emergence of microbial resistance, despite widespread and persistent use as a food preservative.3,7 Nisin also inhibits the outgrowth of germinated bacterial spores.8 12 Organisms from several families of bacteria, includ- ing the Bacillacaea, form small endospores in nutrient-deprived conditions, allowing survival over extended periods of time, which would not be possible as vegetative cells.13 15 The endospore structure provides protection in a wide range of environments including protection against oxidation, radiation, desiccation, heat, and extremes in pH.16 The endospore consists of a dual membrane separated by a thick peptidoglycan cortex and two proteinaceous layers, the inner and outer spore coats, all sur- rounding an inner core containing the DNA (Figure 1C).13 15 Upon encountering more favorable conditions, dormant spores germinate and undergo sequential developmental changes re- sulting in outgrowth to vegetative cells.13 15 For pathogenic spore-forming bacteria, inhibition of spore outgrowth immedi- ately after germination initiation is an ideal time for antibiotic intervention, because the spore has lost its dormancy and pro- tective properties but has not yet started to produce the virulence factors that contribute to subversion of the host’s immune response and the progression of disease.14 19 A recent study with Bacillus anthracis suggested that mem- brane disruption by nisin prevents the establishment of a mem- brane potential and oxidative metabolism to inhibit outgrowth of germinated spores.9 Whether these processes are mediated by lipid II was not established, nor could inhibition of cell wall biosynthesis be ruled out as a mode of action. At present, little is known regarding lipid II content and accessibility on spores. Moreover, several studies have suggested that covalent binding to a protein target mediates nisin’s inhibitory action on spore Received: December 19, 2010 Accepted: April 25, 2011 Published: April 26, 2011 r 2011 American Chemical Society 744 dx.doi.org/10.1021/cb1004178 | ACS Chem. Biol. 2011, 6, 744–752