Lights, Camera, Action! Antimicrobial Peptide Mechanisms Imaged in Space and Time

Choi, Heejun; Rangarajan, N.; Weisshaar, James C.

doi:10.1016/j.tim.2015.11.004

Cited by 72 publications

(67 citation statements)

References 43 publications

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“…Similar observations have been reported for other antimicrobial peptides, such as human β -defensins (hBDs), LL-37, and cecropin A (21, 53). In Enterococcus feacalis , the translocase SecA and several sortases are concentrated in membrane foci in division septa and in the cell envelope along a helical pattern.…”

Section: Discussionsupporting

confidence: 85%

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Daptomycin–Phosphatidylglycerol Domains in Lipid Membranes

2017

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Daptomycin is an acidic, 13-amino acid, cyclic polypeptide that contains a number of non-proteinogenic residues and is modified at its N-terminus with a decanoyl chain. It has been in clinical use since 2003 against selected drug-resistant Staphylococcus aureus and Enterococcus spp infections. In vitro, daptomycin is active against Gram-positive pathogens at low concentrations but its antibiotic activity depends critically on the presence of calcium ions. This dependence has been thought to arise from binding of one or two Ca2+ ions to daptomycin as a required step in its interaction with the bacterial membrane. Here, we investigated the interaction of daptomycin with giant unilamellar vesicles (GUVs) composed 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG). We used fluorescence confocal microscopy to monitor binding of the peptide to GUVs and follow its effect on the membrane of the vesicle. We found that, in the absence of POPG or Ca2+, daptomycin does not bind measurably to the lipid membrane. In the presence of 20–30% PG in the membrane and 2 mM Ca2+, daptomycin induces the formation of membrane domains rich in acidic lipids. This effect is not induced by Ca2+ alone. In addition, daptomycin causes GUV collapse, but it does not translocate across the membrane to the inside of intact POPC:POPG vesicles. We conclude that pore formation is probably not the mechanism by which the peptide functions. On the other hand, we found that daptomycin coclusters with the anionic phospholipid POPG and the fluorescent probes used, leading to extensive formation of daptomycin–POPG domains in the membrane.

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Section: Discussionsupporting

confidence: 85%

“…These observations led to the suggestion that daptomycin disrupts peptidoglycan synthesis by causing mislocalization of proteins involved in cell division (20). Interestingly, PG is found at higher concentration in the membrane in those locations (21). …”

Section: Discussionmentioning

confidence: 99%

Daptomycin–Phosphatidylglycerol Domains in Lipid Membranes

2017

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“…This model involves AMP association, via ionic interactions, with bacterial membranes, and when sufficient local concentrations of the antimicrobial are reached, patches of membrane are disrupted, allowing ion flux [18,[38][39][40][41]. Morphological changes in Gram-negative bacteria in response to AMPs include blebbing of the outer membranes, [42][43][44] and the same type of blebbing has occurs upon treatment of Gram-negative bacteria with ceragenins [33,45] Bacterial endotoxins are constituents of bacterial membranes, [46] and the affinity of ceragenins for bacterial membranes translates into the ability of ceragenins to sequester bacterial endotoxins and inhibit innate immune recognition. Bucki et al [47] demonstrated that a lead ceragenin, CSA-13, binds to lipopolysaccharide (LPS) and inhibits its ability to activate TLR-4, preventing NF-kB translocation to the nucleus; NF-kB translocation leads to inflammatory cytokine production and release.…”

Section: Mechanism Of Actionmentioning

confidence: 99%

Ceragenins as Mimics of Endogenous Antimicrobial Peptides

Hashemi¹,

Holden²,

DurnaÅ³

et al. 2017

J Antimicrob Agents

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Ceragenins are small molecule mimics of endogenous antimicrobial peptides (AMPs), and as such display broadspectrum antimicrobial activity. These molecules are derived from a common bile acid and can be prepared at a large scale. Because ceragenins are not peptide based, they are not substrates for proteases. Gram-negative and positive bacteria are susceptible to ceragenins, including drug resistant organisms. Although ceragenins and colistin have common features, ceragenins retain full antibacterial activity against colistin-resistant Gram-negative bacteria. Bactericidal activity of ceragenins involves selective association with bacterial membranes followed by membrane depolarization. Due to this mechanism of action, which provides bactericidal activity against sessile bacteria, ceragenins eradicate established biofilms. Lipid-enveloped viruses (e.g. vaccinia) are deactivated by ceragenins, and topical application of a lead ceragenin decreases transmission of the virus in skin in a murine model. More recently, the activities of ceragenins against fungal pathogens have been reported, with minimum inhibition concentrations comparable to clinically used anti-fungal agents. In addition to antimicrobial activities, ceragenins have been shown to display some of the "secondary" activities attributed to AMPs. In vivo use of ceragenins to eradicate biofilms, prevent infection and accelerate bone growth demonstrate some of the types of applications in which ceragenins may be used to augment or replace activities of endogenous AMPs.

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“…3, 4 A wide variety of bulk culture and single-cell diagnostic assays have revealed numerous physical and biochemical effects of AMPs on cultured bacteria. 5, 6 Our lab has been developing single-cell, time resolved, fluorescence-based assays that monitor a variety of AMP-induced “symptoms”. 7–15 We have focused primarily on the Gram negative species E. coli under attack by the polycationic AMPs LL-37, 7, 14 Cecropin A, 8 CM15, 11 and Melittin, 15 as well as by the synthetic cationic random β-peptide copolymer MM 63 :CHx 37 (Table 1).…”

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confidence: 99%

HaloTag Assay Suggests Common Mechanism of E. coli Membrane Permeabilization Induced by Cationic Peptides

Yang

Weisshaar

2018

ACS Chem. Biol.

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Permeabilization of the Gram negative bacterial outer membrane (OM) by antimicrobial peptides (AMPs) is the initial step enabling access of the AMP to the cytoplasmic membrane. We present a new single-cell, time-resolved fluorescence microscopy assay that reports on the permeabilization of the E. coli OM to small molecules, with time resolution of 3 sec or better. When the profluorophore JF646 (702 Da) crosses the outer membrane (OM) and gains access to the periplasm, it binds to localized HaloTag protein (34 kDa) and fluoresces in a characteristic hollow spatial pattern. Previous work used the much larger periplasmic GFP (27 kDa) probe, which reports on OM permeabilization to globular proteins. We test the assay on three cationic agents: the Gellman random β-peptide copolymer MM63:CHx37, the human AMP LL-37, and the synthetic hybrid AMP CM15. These results combined with previous work suggest a unifying sequence of OM and cytoplasmic membrane (CM) events that may prove commonplace in the attack of cationic peptides on Gram negative bacteria. The peptide initially induces gradual OM permeabilization to small molecules, likely including the peptide itself. After a lag time, abrupt permeabilization of the OM, abrupt re-sealing of the OM, and abrupt permeabilization of the CM (all to globular proteins) occur in rapid sequence. We propose a mechanism based on membrane curvature stress induced by time-dependent differential binding of peptide to the outer leaflet of the OM and CM. The results provide fresh insight into the critical OM permeabilization step leading to a variety of damaging downstream events.

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Lights, Camera, Action! Antimicrobial Peptide Mechanisms Imaged in Space and Time

Cited by 72 publications

References 43 publications

Daptomycin–Phosphatidylglycerol Domains in Lipid Membranes

Daptomycin–Phosphatidylglycerol Domains in Lipid Membranes

Ceragenins as Mimics of Endogenous Antimicrobial Peptides

HaloTag Assay Suggests Common Mechanism of E. coli Membrane Permeabilization Induced by Cationic Peptides

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