Antimicrobial lipopeptide tridecaptin A1 selectively binds to Gram-negative lipid II Stephen A. Cochranea,1, Brandon Findlaya,1, Alireza Bakhtiarya, Jeella Z. Acedoa, Eva M. Rodriguez-Lopeza, Pascal Mercierb, and John C. Vederasa,2 aDepartment of Chemistry, University of Alberta, Edmonton, AB, Canada T6G 2G2; and bNational High Field NMR Centre, University of Alberta, Edmonton, AB, Canada T6G 2E1 Edited by Samuel H. Gellman, University of Wisconsin, Madison, WI, and approved August 19, 2016 (received for review May 27, 2016) Tridecaptin A1 (TriA1) is a nonribosomal lipopeptide with selective Results antimicrobial activity against Gram-negative bacteria. Here we show Tridecaptin A1 Targets the Cell Membrane. Our initial efforts focused that TriA exerts its bactericidal effect by binding to the bacterial cell- 1 on identifying the mode of action of TriA1. Measuring the time wall precursor lipid II on the inner membrane, disrupting the proton taken for an antibiotic to exert its antimicrobial effect can provide motive force. Biochemical and biophysical assays show that binding valuable information on the cellular process targeted by that to the Gram-negative variant of lipid II is required for membrane compound. Bacteriostatic agents halt cell division but do not re- disruption and that only the proton gradient is dispersed. The NMR duce the number of viable cells, and antibiotics that target protein solution structure of TriA1 in dodecylphosphocholine micelles with synthesis and nucleic acid synthesis typically fall within this cate- lipid II has been determined, and molecular modeling was used to gory. Bactericidal agents reduce the population of viable bacterial provide a structural model of the TriA –lipid II complex. These results 1 cells, and the time taken for a bactericidal agent to kill bacteria suggest that TriA kills Gram-negative bacteria by a mechanism of 1 provides further information on its target. Therefore, we monitored action using a lipid-II–binding motif. the growth kinetics of E. coli cells exposed to TriA1 and a number of other antibiotics (Fig. 2). Lipopeptides like polymyxin B are antibiotic | peptide | lipid II | peptidoglycan | membrane pore generally bactericidal within minutes of exposure due to the for- mation of large, nonspecific pores in the bacterial membrane, MICROBIOLOGY ecently, a lot of media coverage has been focused on the whereas many other antibiotics (like ampicillin) exert their killing Rproblem of antimicrobial resistance. A report commissioned by effect over several hours. Bacteriostatic agents (like chloramphen- the UK government predicts that by 2050 antimicrobial resistance icol) halt cell growth, but do not kill cells. Optical density mea- will have caused 300 million premature deaths and cost the global surements showed that cells exposed to TriA1 grew at a reduced economy over $100 trillion (1). Even more worrying is the lack of rate for 20 min, at which point a steady decrease in cell count was new classes of antibiotics active against Gram-negative bacteria. In observed (Fig. 2A). Polymyxin B reduced the cell count immedi- the past 50 y, only a few structurally and mechanistically distinct ately, whereas ampicillin displayed an expected 3-h lag and chlor- CHEMISTRY classes of antibiotics have been clinically approved to treat systemic amphenicol did not reduce optical density. Complementary results infections (including fidaxomicin, bedaquiline, linezolid, and dap- were obtained from a time-kill assay (Fig. 2B), which revealed that tomycin), yet none of these are active against Gram-negative bacteria (2, 3). Two new classes of Gram-negative targeting anti- Significance biotics in the clinical pipeline are POL7080 and brilacidin (4, 5). Both of these compounds are modeled on antimicrobial peptides, which are becoming increasingly important in the fight against The increasing development of antimicrobial resistance is a major global concern, and there is an urgent need for the development antibiotic resistance (6). Bacteria produce a wealth of antimicrobial of new antibiotics. We show that the antimicrobial lipopeptide peptides, both ribosomally, including the lantibiotics (7, 8), and tridecaptin A selectively binds to the Gram-negative analogue nonribosomally, including lipopeptides (9). In particular, lip- 1 of peptidoglycan precursor lipid II, disrupting the proton motive opeptides are a rich source of antimicrobial compounds, and sev- force and killing Gram-negative bacteria. We present an example eral examples with activity against Gram-positive (10, 11) and/or of the selective targeting of Gram-negative lipid II and a binding Gram-negative bacteria (12) have been recently characterized. mode to this peptidoglycan precursor. No persistent resistance Tridecaptin A1 (TriA1) is a member of the tridecaptin family, a develops against tridecaptin A1 in Escherichia coli cells exposed group of nonribosomal lipopeptides produced by Bacillus and to subinhibitory concentrations of this peptide during a 1-mo – Paenibacillus species (Fig. 1) (13 15). This acylated tridecapeptide period. This study showcases the excellent antibiotic properties displays strong and selective antimicrobial activity against Gram- of the tridecaptins in an age where new antibiotics that target negative bacteria, including multidrug-resistant strains of Klebsiella Gram-negative bacteria are desperately needed. pneumoniae, Acinetobacter baumannii,andEscherichia coli (16). TriA1 analogs have low cytotoxicity and have been shown to treat Author contributions: S.A.C., B.F., A.B., J.Z.A., and J.C.V. designed research; S.A.C., B.F., A.B., K. pneumoniae infections in mice (16, 17). Therefore, we believe J.Z.A., E.M.R.-L., and P.M. performed research; P.M. contributed new reagents/analytic tools; S.A.C., B.F., A.B., J.Z.A., E.M.R.-L., and J.C.V. analyzed data; and S.A.C. and J.C.V. wrote that tridecaptin A1 could be an excellent antibiotic candidate. the paper. However, before our investigations little was known about how The authors declare no conflict of interest. TriA exerts its selective bactericidal effect against Gram-negative 1 This article is a PNAS Direct Submission. bacteria. A previous structure–activity relationship study by our Freely available online through the PNAS open access option. group suggested that TriA1, akin to many other lipopeptides, is a Data deposition: The NMR, atomic coordinates, chemical shifts, and restraints have been membrane-targeting agent. We found that removal of the N-ter- deposited in the Biological Magnetic Resonance Bank, www.bmrb.wisc.edu (BMRB codes minal lipid tail abolishes antimicrobial activity; however, the chiral 25737 and 25741) and the Protein Data Bank, www.pdb.org (PDB ID codes 2n5w and 2n5y). lipid tail could be replaced with an octanoyl chain to give Oct-TriA1 1S.A.C. and B.F. contributed equally to this work. (Fig. 1), which retains full activity (16). We therefore sought to 2To whom correspondence should be addressed. Email: [email protected]. identify the precise mode and mechanism of action by which TriA1 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. kills Gram-negative bacteria. 1073/pnas.1608623113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1608623113 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 have also found that the enantiomer of TriA1 (Ent-TriA1)is NH fourfold less active than the natural peptide, suggesting that TriA1 interacts with a chiral target (19). Ent-TriA1 contains the same O O H H OH O R N N O amino acids and lipid tail as TriA1 and therefore retains the same N N N R = H H H amphiphilic properties. It is likely that the observed activity at O O NH HO higher concentrations is due to membrane lysis by a nonspecific HO TriA1 NH 2 HN O O detergent-like effect, akin to the mode of action of the polymyxins. O This observation that both TriA and its enantiomer bind to LPS O O O NH 1 H H 2 HO N N NH with similar affinities suggested that another chiral receptor is in- N N N Oct-TriA H H H 1 volved in the mode of action. O O O NH2 O OH TriA1 Disrupts the Proton Motive Force. With a rationale for how TriA1 crosses the outer membrane, we next sought to uncover the Fig. 1. Structures of the tridecaptin analogs TriA1 and Oct-TriA1. mechanism by which it acts on the inner membrane. To this end, a series of experiments were performed using fluorescent dyes (Fig. 3). DiBAC4 is a common dye used to monitor membrane cellsexposedtoTriA1 showed slightly reduced cell viability after depolarization (20). It enters depolarized cells and binds to pro- 15 min of exposure, a significant reduction after 30 min, and com- teins or membrane components, exhibiting enhanced fluorescence. plete killing after 60 min. Polymyxin B acted faster, significantly Addition of TriA1 to DiBAC4-treated E. coli cells resulted in no reducing the viable cell population after 5 min and killing all cells membrane depolarization, whereas application of polymyxin B led by 30 min. These results suggested that TriA1 is a membrane- to a rapid increase in fluorescence (Fig. 3A). To further assess the targeting peptide, but does not act by a generic membrane lysis effect of TriA1 on the inner membrane, its ability to form large mechanism like polymyxin B. This is further supported by the pores was assessed using the dye SYTOX Green (Fig. 3B). Upon lysis of the inner membrane, this dye binds to nucleic acids in the selectivity of TriA1 against Gram-negative bacteria because a peptide that operates through a generic lysis mechanism would cytoplasm, causing an increase in fluorescence (21). Immediate also target Gram-positive organisms. pore formation was not observed when E. coli cells pretreated with SYTOX Green were exposed to TriA1, whereas addition of the TriA1 Binds to Lipopolysaccharide on the Outer Membrane. Gram- surfactant Triton X-100 caused an instant fluorescence increase.