BBA - Biomembranes 1862 (2020) 183395 Contents lists available at ScienceDirect BBA - Biomembranes journal homepage: www.elsevier.com/locate/bbamem Review DAPTOMYCIN, its membrane-active mechanism vs. that of other antimicrobial peptides T Huey W. Huang Department of Physics and Astronomy, Rice University, Houston, TX, USA ARTICLE INFO ABSTRACT Keywords: Over 3000 membrane-active antimicrobial peptides (AMPs) have been discovered, but only three of them have Daptomycin been approved by the U.S. Food and Drug Administration (FDA) for therapeutic applications, i.e., gramicidin, Membrane-active daptomycin and colistin. Of the three approved AMPs, daptomycin is a last-line-of-defense antibiotic for treating Antimicrobial-peptides Gram-positive infections. However its use has already created bacterial resistance. To search for its substitutes Ion-channel that might counter the resistance, we need to understand its molecular mechanism. The mode of action of Ionophore daptomycin appears to be causing bacterial membrane depolarization through ion leakage. Daptomycin forms a Pore-formation unique complex with calcium ions and phosphatidylglycerol molecules in membrane at a specific stoichiometric ratio: Dap2Ca3PG2. How does this complex promote ion conduction across the membrane? We hope that bio- physics of peptide-membrane interaction can answer this question. This review summarizes the biophysical works that have been done on membrane-active AMPs to understand their mechanisms of action, including gramicidin, daptomycin, and underdeveloped pore-forming AMPs. The analysis suggests that daptomycin forms transient ionophores in the target membranes. We discuss questions that remain to be answered. 1. Introduction their AMP review.) The three approved AMPs are gramicidin, dapto- mycin, and colistin. All were discovered in or derived from Gram-po- Daptomycin is one of a few membrane-active antimicrobial peptides sitive bacteria found in the soil. In contrast, the great majority of AMPs (AMPs) that have been approved by the U.S. Food and Drug listed in the APD are the components of innate immunity found in Administration (FDA) for clinical use [1,2]. Following increased clinical varieties of animals. So far none of them have been approved for usage, bacterial pathogens began to develop resistance to daptomycin therapeutic applications [11]. [3]. The recent transition of daptomycin to generic status is projected to The most important distinction between the AMPs and the con- dramatically increase availability, use, and clinical failure [4]. Many ventional antibiotics is that the former target the lipid domains of the daptomycin analogues are being developed, aiming to improve its po- cell membranes whereas the latter target specific receptors that include tency or to counter the bacterial resistance [5–9]. However, there is no proteins, DNA, RNA, ribosomes or specific lipids such as lipid II [12]. guiding principle for developing the daptomycin substitutes, because Compared with receptor-specific antibiotics, the mechanisms of mem- the molecular mechanism of how daptomycin permeabilizes bacterial brane-active AMPs are much less clear; that is, what happens after the membranes has not been clarified. What happens when molecules of AMPs reach the lipid domains is often ambiguous. Generally speaking daptomycin bind to a lipid domain? We believe that the answer is the actions of conventional antibiotics disrupt the bacterial metabolism within the domain of biophysics governing peptide-membrane inter- that limits the growth of bacteria; i.e., antibiotics are bacteriostats. In actions. This review will provide a summary of what we know about the contrast, AMPs are bacteriocidal, often described as causing membrane molecular mechanisms of membrane-active AMPs and suggest further lysis. But in fact, at the concentrations comparable to minimum in- research on daptomycin. hibition concentrations (MICs), AMPs do not cause lysis of lipid mem- AMPs for therapeutic applications were recently reviewed by Chen branes (for reasons see below). It has been pointed out [13,14] that cell and Lu [2]. Among the 3156 AMPs listed in the Antimicrobial Peptide membrane lysis is likely a secondary effect due to activation of auto- Database (APD) [10], which includes peptides and lipopeptides, only digestive enzymes. At least, this appears to be the case for daptomycin three have been approved by FDA. (Chen and Lu also included four as will be described below. Direct studies of AMPs with live bacteria lipoglycopeptide antibiotics, i.e., vancomycin and three derivatives, in [15–19] are also complicated by the presence of an outer membrane or E-mail address: [email protected]. https://doi.org/10.1016/j.bbamem.2020.183395 Received 27 February 2020; Received in revised form 4 June 2020; Accepted 7 June 2020 Available online 09 June 2020 0005-2736/ © 2020 Published by Elsevier B.V. H.W. Huang BBA - Biomembranes 1862 (2020) 183395 a cell wall. As a result, dispute on the membrane-active AMP me- In the presence of a minute amount of gramicidin, in many types of chanisms has been common. lipid bilayers, the membrane current under fixed applied potential To find ways to clarify the membrane-active mechanisms of AMPs, fluctuates in a single-channel step-like manner [32]. All three grami- we have studied the action of AMPs on live bacteria spheroplasts and cidins have closely similar ion conducting properties [33]. The ion directly compared with their action on giant unilamellar vesicles conduction mediated by gramicidin in bilayer membranes occurs due to (GUVs) similar in size [20–22]. A spheroplast is a bacterium (for ex- the formation of a cylindrical transmembrane channel by two mono- ample, E. coli) with its outer membrane removed; as a result, its shape is mers, each a single-stranded β6.3 helix, linked head-to-head spherical, comparable to a giant lipid vesicle. (These spheroplasts could (head = formyl end) by six hydrogen bonds at their N termini [34,35]. revert back to normal form of E. coli when returned to a growth medium The gramicidin channel conducts monovalent cations only. The [20].) First, we studied the physical properties of spheroplast mem- monovalent cation binding sites are inside the channel at 9.6 Å from the branes in comparison with GUVs [20]. We then used this knowledge to channel midpoint as determined by X-ray diffraction [36]. Divalent investigate the phenomena of pore-forming AMPs directly attacking the cations bind at the mouth of the channel on each end, 13Å from the cytoplasmic membranes of E. coli spheroplasts. We developed a pro- channel midpoint, that block the ion conduction [36]. cedure of fluorescence recovery after photobleaching (FRAP) to ex- To determine the dynamics of gramicidin channel formation in amine the dye leakage through the bacterial membranes as the AMPs in membranes, we used a modified gramicidin with its formyl group re- solution attacked the membranes. The permeability through the bac- placed by a tert-butoxycarbonyl (BOC) group, such that the channel is 5- terial membrane increased in a sigmoidal fashion as the AMP binding order-of-magnitude destabilized relative to the native channel [37]. X- increased in time, exhibiting a cooperative behavior of AMPs. The ray in-plane diffraction found that the signal was dominated by gra- analysis of FRAP showed that the fluxes of dye molecules into and out micidin in monomeric β helix form [37]. This showed that in mem- of the cell were consistent with diffusion of molecules through a branes, gramicidin exists as both dimers and monomers in kinetic number of pores that increased with binding of AMPs and then satu- equilibrium. Interestingly the equilibrium constant of the monomer- rated to a steady level. The study confirms that the pore forming ac- dimer kinetics depends on the membrane thickness, with the thicker tivities by LL37, melittin and alamethicin observed in GUVs are re- membranes favoring the monomers [38,39]. This was explained by produced in spheroplasts. Indeed the steady-state membrane assuming that the membrane thickness is locally hydrophobic-matching permeability induced by these three pore-forming AMPs is quantita- the dimeric channel length (Fig. 1). This local membrane deformation tively the same in spheroplasts as in GUVs. due to the hydrophobic-matching incurs an increase in free energy that However, this pattern of pore formation was not observed with affects the dimeric channel's lifetime [39]. That in turn explains the daptomycin or with the metabolic inhibitor carbonyl cyanide m-chlor- membrane-thickness dependence of the gramicidin ion conductivity ophenylhydrazone (CCCP) [23–25]. CCCP and daptomycin are each [38,39]. The clarification of the molecular mechanism of gramicidin known to cause ion leakage. The ion leakage by CCCP and daptomycin demonstrates that ion conduction is indeed an antibiotic mechanism. would lead to a dye leakage pattern in E. coli spheroplast different from We know that the presence of cholesterol in mammalian cell mem- the pore-formation pattern. Also both did not cause any dye leakage branes has a membrane-thickening effect [40]. This could be a pro- through the membrane of a GUV. There are however interesting dis- tective factor for the animal cells against the attack of gramicidin. similarities in details that reveal differences between bacterial and lipid membranes. Spheroplast membranes were permeabilized by a wide 3. Pore-forming AMPs range of AMP concentrations to the same steady-state membrane per- meability. In contrast, only a narrow range of AMP concentrations Examples include alamethicin, magainin, LL37, melittin, protegrin permeabilized GUVs to a steady state level. Tension in GUVs also in- and many others. They are by far the most extensively studied AMPs fl uences the action of AMPs, whereas the spheroplast membranes are [41,42]. However, therapeutic applications of magainin and protegrin tensionless. With this understanding, our results provide a strong sup- derivatives have failed to win the FDA approval so far [11]. In terms of port for using model membranes to study the action of AMPs on bac- molecular mechanism, the easiest to understand are the linear peptides terial membranes [21,22].