Abstract
Pseudodesmin A (PSD) is a cyclic lipodepsipeptide produced by Pseudomonas that kills certain bacteria at MIC1/2 in the single micromolar range, probably by permeabilizing their cellular membranes. Synthetic PSD variants, where the native decanoic (C10) acyl chain is varied in length from C4 to C8 and C12 to C14 carbons, were described to be not or less active against a panel of gram-positive strains, as compared to native PSD-C10. Here, we test the membrane-permeabilizing activity of PSD-C4 through PSD-C14 in terms of calcein release from liposomes, which is characterized in detail by the fluorescence-lifetime based leakage assay. Antagonistic concentrations and their chain length dependence agree well for liposome leakage and antimicrobial activity. The optimal chain length is governed by a balance between membrane partitioning (favoring longer chains) and the local perturbation or “damage” inflicted by a membrane-bound molecule (weakening for longer chains). Local perturbation, in turn, may involve at least two modes of action. Asymmetry stress between outer and inner leaflet builds up as the lipopeptides enter the outer leaflet and when it reaches a system-specific stability threshold, it causes a transient membrane failure that allows for the flip of some molecules from the outer to the inner leaflet. This cracking-in may be accompanied by transient, incomplete leakage from the aqueous cores of the liposomes observed, typically, for some seconds or less. The mismatch of the lipopeptide with the lipid leaflet geometry, expressed for example in terms of a spontaneous curvature, has two effects. First, it affects the threshold for transient leakage as described. Second, it controls the rate of equilibrium leakage proceeding as the lipopeptide has reached sufficient local concentrations in both leaflets to form quasi-toroidal defects or pores. Both modes of action, transient and equilibrium leakage, synergize for intermediate chain lengths such as the native, i.e., for PSD-C10. These mechanisms may also account for the reported chain-length dependent specificities of antibiotic action against the target bacteria.
Highlights
Occurring lipopeptides and other membrane-active compounds may have great potential for a more individual, selective design of antimicrobials, insecticides, plant protection agents, and drugs and are considered a “gold mine” (Cochrane and Vederas, 2016) and a highly promising approach to resolve growing resistances and loss of biodiversity, even though their modes of action are yet still partially enigmatic (Balleza et al, 2019; Wimley and Hristova, 2019)
Besides classic antimicrobial peptides, which are often produced via ribosomal synthesis (Datta et al, 2014; Kormilets et al, 2019), the diverse collective of membrane-active biomolecules includes cyclic lipopeptides (CLiPs) (Raaijmakers et al, 2006; Kleijn and Martin, 2018) synthesized non-ribosomally by bacteria such as Bacillus (Ongena and Jacques, 2008) or Pseudomonas (Geudens et al, 2017; Geudens and Martins, 2018)
Preliminary experiments showed that Pseudodesmin A (PSD) became inactivated for membrane permeabilization when attempted to be dissolved in buffer with ≤3 v% of dimethyl sulfoxide (DMSO)
Summary
Occurring lipopeptides and other membrane-active compounds may have great potential for a more individual, selective design of antimicrobials, insecticides, plant protection agents, and drugs and are considered a “gold mine” (Cochrane and Vederas, 2016) and a highly promising approach to resolve growing resistances and loss of biodiversity, even though their modes of action are yet still partially enigmatic (Balleza et al, 2019; Wimley and Hristova, 2019). Besides classic antimicrobial peptides, which are often produced via ribosomal synthesis (Datta et al, 2014; Kormilets et al, 2019), the diverse collective of membrane-active biomolecules includes cyclic lipopeptides (CLiPs) (Raaijmakers et al, 2006; Kleijn and Martin, 2018) synthesized non-ribosomally by bacteria such as Bacillus (Ongena and Jacques, 2008) or Pseudomonas (Geudens et al, 2017; Geudens and Martins, 2018). They consist of a (partially) cyclic oligopeptide group attached to a lipid chain and are amphipathic. Since these studies employed naturally occurring variants, they were limited both in scale and in scope
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