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Non-Membrane Permeabilizing Modes of Action of Antimicrobial Peptides on Bacteria
No full textAntimicrobial peptides (AMPs) are a large class of innate immunity effectors with a remarkable capacity to inactivate microorganisms. Their ability to kill bacteria by membranolytic effects has been well established. However, a lot of evidence points to alternative, non-lytic modes of action for a number of AMPs, which operate through interactions with specific molecular targets. It has been reported that non-membrane-permeabilizing AMPs can bind to and inhibit DNA, RNA or protein synthesis processes, inactivate essential intracellular enzymes, or affect membrane septum formation and cell wall synthesis. This minireview summarizes recent findings on these alternative, non-lytic modes of antimicrobial action with an emphasis to the experimental approaches used to clarify each step of their intracellular action, i.e. the cell penetration mechanism, intracellular localization and molecular mechanisms of antibacterial action. Despite the fact that such data exists for a large number of peptides, our analysis indicates that only for a small number of AMPs sufficient data have been collected to support a mode of action with an authentic and substantial contribution by intracellular targeting. In most cases, peptides with non-lytic features have not been thoroughly analyzed, or only a single aspect of their mode of action has been taken into consideration and therefore their mechanism of action can only be hypothesized. A more detailed knowledge of this class of AMPs would be important in the design of novel antibacterial agents against unexploited targets, endowed with the capacity to penetrate into pathogen cells and kill them from within
In vitro and in vivo antimicrobial activity of two alpha-helical cathelicidin peptides and of their synthetic analogs.
No full textRapid and reliable detection of antimicrobial peptide penetration into Gram-negative bacteria based on fluorescence quenching.
No full textIn this paper, we describe a rapid flow cytometry method to identify antimicrobial peptides that are
internalized into bacterial cells and differentiate them from those that are membrane active. The method was
applied to fluorescently labeled Bac71-35 and polymyxin B, whose mechanisms of action are, respectively, based
on cell penetration and on membrane binding and permeabilization. Identification of peptides with the former
mechanism is of considerable interest for the intracellular delivery of membrane-impermeant drugs
Pro-rich antimicrobial peptides from animals: structure, biological functions and mechanism of action
No full textPEGylation of the peptide Bac7(1–35) reduces renal clearance while retaining antibacterial activity and bacterial cell penetration capacity
Full text linkThe proline-rich antibacterial peptide Bac7(1–35) protects mice against Salmonella typhimurium infection, despite its rapid clearance. To overcome this problem the peptide was linked to a polyethylene glycol (PEG) molecule either via a cleavable ester bond or via a non-hydrolysable amide bond. Both the PEGylated conjugates retained most of the in vitro activity against S. typhimurium. In addition, the ester bond was cleaved in human serum or plasma, releasing a carboxymethyl derivative of Bac7(1–35) which accounts for a higher activity of this peptide with relative to the other, non-hydrolysable form. Both PEGylated peptides maintained the capacity of the unconjugated form to kill bacteria without permeabilizing the bacterial membranes, by penetrating into cells. They exploited the same transporter as unmodified Bac7(1–35), suggesting it has the capacity to internalize quite sizeable cargo if this is linked to Bac7 fragment. PEGylation allows the peptide to have a wide distribution in mice, and a slow renal clearance, indicating that this strategy would improve the bioavailability of Bac7, and in principle of other antimicrobial peptides. This can be an equally important issue to reducing cytotoxicity for therapeutic use of these antibacterials
The mechanism of killing by the proline-rich peptide Bac7(1-35) against clinical strains of Pseudomonas aeruginosa differs from that against other gram-negative bacteria
Full text linkPseudomonas aeruginosa infections represent a serious threat to worldwide health. Proline-rich antimicrobial peptides (PR-AMPs), a particular group of peptide antibiotics, have demonstrated in vitro activity against P. aeruginosa strains.
Here we show that the mammalian PR-AMP Bac7(1–35) is active against some multidrug-resistant cystic fibrosis isolates of P. aeruginosa. By confocal microscopy and cytometric analyses, we investigated the mechanism of killing against P. aeruginosa strain PAO1 and three selected isolates, and we observed that the peptide inactivated the target cells by disrupting their cellular membranes. This effect is deeply different from that previously described for PR-AMPs in Escherichia coli and Salmonella enterica serovar Typhimurium, where these peptides act intracellularly after having been internalized by means of the transporter SbmA without membranolytic effects. The heterologous expression of SbmA in PAO1 cells enhanced the internalization of Bac7(1–35) into the cytoplasm, making the bacteria more susceptible to the peptide but at the same time more resistant to the membrane lysis, similarly to what occurs in E. coli. The results evidenced a new mechanism of action for PRAMPs
and indicate that Bac7 has multiple and variable modes of action that depend on the characteristics of the different target species and the possibility to be internalized by bacterial transporters. This feature broadens the spectrum of activity of the peptide and makes the development of peptide-resistant bacteria a more difficult process
Investigating the Mode of Action of Proline-rich Antimicrobial Peptides Using a Genetic Approach: a Tool to Identify New Bacterial Targets Amenable to the Design of Novel Antibiotics.
No full textThe proline-rich antimicrobial peptides (PRPs) are considered to act by crossing bacterial membranes without altering them and then binding to, and functionally modifying, one or more specific targets. This implies that they can be used as molecular hooks to identify the intracellular or membrane proteins that are involved in their mechanism of action and that may be subsequently used as targets for the design of novel antibiotics with mechanisms different from those now in use. The targets can be identified by using peptide-based affinity columns or via the genetic approach described here. This approach depends on chemical mutagenesis of a PRP-susceptible bacterial strain to select mutants that are either more resistant or more susceptible to the relevant peptide. The genes conferring the mutated phenotype can then be isolated and identified by subcloning and sequencing. In this manner, we have currently identified several genes that are involved in the mechanism of action of these peptides, including peptide-transport systems or potential resistance factors, which can be used or taken into account in drug design efforts
Interaction of antimicrobial peptides with bacterial polysaccharides from lung pathogens
No full textThe interaction of two cathelicidin antimicrobial peptides, LL-37 and SMAP-29, with three bacterial polysaccharides, respectively, produced
by Pseudomonas aeruginosa, Burkholderia cepacia and Klebsiella pneumoniae, was investigated to identify possible mechanisms adopted
by lung pathogens to escape the action of innate immunity effectors. In vitro assays indicated that the antibacterial activity of both peptides
was inhibited to a variable extent by the three polysaccharides. Circular dichroism experiments showed that these induced an -helical
conformation in the two peptides, with the polysaccharides from K. pneumoniae and B. cepacia showing, respectively, the highest and the
lowest effect. Fluorescence measurements also indicated the presence of peptide–polysaccharide interactions. A model is proposed in which
the binding of peptides to the polysaccharide molecules induces, at low polysaccharide to peptide ratios, a higher order of aggregation, due to
peptide–peptide interactions. Overall, these results suggest that binding of the peptides by the polysaccharides produced by lung pathogens
can contribute to the impairment of peptide-based innate defenses of airway surface
Dual mode of action of Bac7, a proline-rich antibacterial peptide
No full textProline-rich peptides are a unique group of antimicrobial peptides that exert their activity selectively against Gram-negative bacteria through an
apparently non-membranolytic mode of action that is not yet well understood. We have investigated the mechanism underlying the antibacterial
activity of the proline-rich cathelicidin Bac7 against Salmonella enterica and Escherichia coli. The killing and membrane permeabilization
kinetics as well as the cellular localization were assessed for the fully active N-terminal fragment Bac7(1–35), its all-D enantiomer and for
differentially active shortened fragments. At sub-micromolar concentrations, Bac7(1–35) rapidly killed bacteria by a non-lytic, energy-dependent
mechanism, whereas its D-enantiomer was inactive. Furthermore, while the L-enantiomer was rapidly internalized into bacterial cells, the Denantiomer
was virtually excluded. At higher concentrations (≥64 μM), both L- and D-Bac7(1–35) were instead able to kill bacteria also via a
lytic mechanism. Overall, these results suggest that Bac7 may inactivate bacteria via two different modes of action depending on its concentration:
(i) at near-MIC concentrations via a mechanism based on a stereospecificity-dependent uptake that is likely followed by its binding to an
intracellular target, and (ii) at concentrations several times the MIC value, via a non-stereoselective, membranolytic mechanism
Antimicrobial peptides: synthesis and antibacterial activity of linear and cyclic drosocin and apidaecin 1b analogues.
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