We coupled the antimicrobial activity of two well-studied lactoferricin derivatives, LF11-215 and LF11-324, in Escherichia coli and different lipid-only mimics of its cytoplasmic membrane using a common thermodynamic framework for peptide partitioning. In particular, we combined an improved analysis of microdilution assays with ζ-potential measurements, which allowed us to discriminate between the maximum number of surface-adsorbed peptides and peptides fully partitioned into the bacteria. At the same time, we measured the partitioning of the peptides into vesicles composed of phosphatidylethanolamine (PE), phosphatidylgylcerol (PG), and cardiolipin (CL) mixtures using tryptophan fluorescence and determined their membrane activity using a dye leakage assay and small-angle X-ray scattering. We found that the vast majority of LF11-215 and LF11-324 readily enter inner bacterial compartments, whereas only 1−5% remain surface bound. We observed comparable membrane binding of both peptides in membrane mimics containing PE and different molar ratios of PG and CL. The peptides' activity caused a concentration-dependent dye leakage in all studied membrane mimics; however, it also led to the formation of large aggregates, part of which contained collapsed multibilayers with sandwiched peptides in the interstitial space between membranes. This effect was least pronounced in pure PG vesicles, requiring also the highest peptide concentration to induce membrane permeabilization. In PE-containing systems, we additionally observed an effective shielding of the fluorescent dyes from leakage even at highest peptide concentrations, suggesting a coupling of the peptide activity to vesicle fusion, being mediated by the intrinsic lipid curvatures of PE and CL. Our results thus show that LF11-215 and LF11-324 effectively target inner bacterial components, while the stored elastic stress makes membranes more vulnerable to peptide translocation.

A previously reported multi-scale model for (ultra-)small-angle X-ray (USAXS/SAXS) and (very) small-angle neutron scattering (VSANS/SANS) of live Escherichia coli was revised on the basis of compositional/metabolomic and ultrastructural constraints. The cellular body is modeled, as previously described, by an ellipsoid with multiple shells. However, scattering originating from flagella was replaced by a term accounting for the oligosaccharide cores of the lipopolysaccharide leaflet of the outer membrane including its cross-term with the cellular body. This was mainly motivated by (U)SAXS experiments showing indistinguishable scattering for bacteria in the presence and absence of flagella or fimbrae. The revised model succeeded in fitting USAXS/SAXS and differently contrasted VSANS/SANS data of E. coli ATCC 25922 over four orders of magnitude in length scale. Specifically, this approach provides detailed insight into structural features of the cellular envelope, including the distance of the inner and outer membranes, as well as the scattering length densities of all bacterial compartments. The model was also successfully applied to E. coli K12, used for the authors' original modeling, as well as for two other E. coli strains. Significant differences were detected between the different strains in terms of bacterial size, intermembrane distance and its positional fluctuations. These findings corroborate the general applicability of the approach outlined here to quantitatively study the effect of bactericidal compounds on ultrastructural features of Gram-negative bacteria without the need to resort to any invasive staining or labeling agents.

We report the real-time response of E. coli to lactoferricin-derived antimicrobial peptides (AMPs) on length-scales bridging microscopic cell-sizes to nanoscopic lipid packing using millisecond time-resolved synchrotron small-angle X-ray scattering. Coupling a multi-scale scattering data analysis to biophysical assays for peptide partitioning revealed that the AMPs rapidly permeabilize the cytosolic membrane within less than three seconds-much faster than previously considered. Final intracellular AMP concentrations of ~ 80 to 100 mM suggest an efficient obstruction of physiologically important processes as primary cause for bacterial killing. On the other hand, damage of the cell envelope and leakage occurred also at sublethal peptide concentrations, thus emerging as a collateral effect of AMP activity that does not kill the bacteria. This implies that the impairment of the membrane barrier is a necessary but not sufficient condition for microbial killing by lactoferricins. The most efficient AMP studied exceeds others in both speed of permeabilizing membranes and lowest intracellular peptide concentration needed to inhibit bacterial growth.

Antimicrobial peptides can kill bacteria by permeabilizing their membranes. One proposed mechanism is by forming a membrane-spanning toroidal pore, where peptides in a transmembrane orientation are lining the pore. It is hard to study such transient pores unless conditions to stabilize the pore are found. We have recently shown that pore formation is promoted and pores stabilized by lipids with a positive spontaneous curvature, like lyso-lipids [1,2]. Using KIA peptides with a repetitive amino acid sequence KIAGKIA of different length, we observed that the amphipathic a-helices can insert into DMPC membranes in the presence of lyso-MPC, and that the tilt angle of the peptides depend on hydrophobic matching between the peptide length and the membrane thickness, i.e., that longer peptides are more tilted for a given bilayer thickness [3,4]. We used solid-state 31 P-, 15 N-and 2 H-NMR on the cationic antimicrobial peptide MSI-103 (also called KIA21) to characterize the pore in detail. The minimum concentration of peptide and lyso-lipid for pore formation was determined, and the effect of charges on the normal lipid or the lyso-lipid was studied. Our results show that pore formation is enhanced in the presence of anionic lyso-MPG compared to neutral lyso-MPC, indicating that a toroidal wormhole pore, enriched in lyso-lipids, is indeed formed. We also found that pore formation is affected by terminal charges of the peptides: charged residues are improving activity when located at the N-terminus but reducing it at the C-terminus. This is discussed in terms of the 3D hydrophobic moment [5] of the peptides. References: [1]

No abstract

We report the real-time response of E. coli to lactoferricin-derived antimicrobial peptides (AMPs) on length-scales bridging microscopic cell-sizes to nanoscopic lipid packing using millisecond time-resolved synchrotron small-angle X-ray scattering. Coupling a multi-scale scattering data analysis to biophysical assays for peptide partitioning revealed that the AMPs rapidly saturate the bacterial envelope and reach the cytosol within less than three seconds—much faster than previously considered. Final cytosolic AMP concentrations of ~ 100 mM suggest an efficient shut-down of metabolism as primary cause for bacterial killing. On the other hand, the damage of the cell envelope is a collateral effect of AMP activity that does not kill the bacteria. This implies that the impairment of the membrane barrier is a necessary but not sufficient condition for microbial killing by lactoferricins. The most efficient AMP studied exceeds others in both speed of reaching cytoplasm and lowest cytosolic peptide concentration.

We have revised a previously reported multi-scale model for (ultra) small angle X-ray (USAXS/SAXS) and (very) small angle neutron scattering (VSANS/SANS) of live Escherichia coli based on compositional/metabolomic and ultrastructural constraints. The cellular body is modelled, as previously described, by an ellipsoid with multiple shells. However, scattering originating from flagella was substituted by a term accounting for the oligosaccharide cores of the lipopolysaccharide leaflet of the outer membrane including its cross-term with the cellular body. This was mainly motivated by (U)SAXS experiments showing indistinguishable scattering for bacteria in the presence and absence of flagella or fimbrae. The revised model succeeded in fitting USAXS/SAXS and differently contrasted VSANS/SANS data of E. coli ATCC 25922 over four orders of magnitude in length scale, providing specifically detailed insight into structural features of the cellular envelope, including the distance of the inner and outer membranes, as well as the scattering length densities of all bacterial compartments. Consecutively, the model was also successfully applied to E. coli K12, used for our original modelling, as well as for two other E. coli strains, detecting significant differences between the different strains in terms of bacterial size, intermembrane distance and its positional fluctuations. These findings corroborate the general applicability of our approach to quantitatively study the effect of bactericidal compounds on ultrastructural features of Gram-negative bacteria without the need to resort to any invasive staining or labelling agents.