Biology subjects

Lohner, K.

Publications and source records attributed to Lohner, K..

4 recordsLinked to original sources

bioRxiv2021

Lactoferricins access the cytosol of Escherichia coli within few seconds

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.

biophysics↗

bioRxiv2020

Evolution of the Analytical Scattering Model of Live Escherichia Coli

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.

biophysics↗

bioRxiv2019

Magainin 2 and PGLa in Bacterial Membrane Mimics II: Membrane Fusion and Sponge Phase Formation

We studied the synergistic mechanism of equimolar mixtures of magainin 2 (MG2a) and PGLa in phosphatidylethanolamine/phosphatidylglycerol mimics of Gram-negative cytoplasmic membranes. In a preceding paper [Pachler et al., Biophys. J. 2019 xxx], we reported on the early onset of parallel heterodimer formation of the two antimicrobial peptides already at low concentrations and the resulting defect formation in membranes. Here, we focus on the structures of the peptide/lipid aggregates occurring in the synergistic regime at elevated peptide concentrations. Using a combination of calorimetric, scattering, electron microscopic and in silico techniques, we demonstrate that the two peptides, even if applied individually, transform originally large unilamellar vesicles into multilamellar vesicles, with a collapsed interbilayer spacing resulting from peptide induced adhesion. Interestingly, the adhesion does not lead to a peptide induced lipid separation of charged and charge neutral species. In addition to this behavior, equimolar mixtures of MG2a and PGLa formed surface-aligned fibril-like structures, which induced adhesion zones between the membranes and the formation of transient fusion stalks in molecular dynamics simulations and a coexisting sponge phase observed by small-angle X-ray scattering. The previously reported increased leakage of lipid vesicles of identical composition in the presence of MG2a/PGLa mixtures is therefore related to a peptide-induced cross-linking of bilayers.\n\nSTATEMENT OF SIGNIFICANCEWe demonstrate that the synergistic activity of the antimicrobial peptides MG2a and PGLa correlates to the formation of surface-aligned fibril-like peptide aggregates, which cause membrane adhesion, fusion and finally the formation of a sponge phase.

biophysics↗

bioRxiv2019

Magainin 2 and PGLa in Bacterial Membrane Mimics I: Peptide-Peptide and Lipid-Peptide Interactions

We addressed the onset of synergistic activity of the two well-studied antimicrobial peptides magainin 2 (MG2a) and PGLa using lipid-only mimics of Gram-negative cytoplasmic membranes. Specifically, we coupled a joint analysis of small-angle X-ray and neutron scattering experiments on fully hydrated lipid vesicles in the presence of MG2a and L18W-PGLa to all-atom and coarse-grained molecular dynamics simulations. In agreement with previous studies both peptides, as well as their equimolar mixture, were found to remain in a surface-aligned topology upon membrane insertion and to induce significant membrane perturbation as evidenced by membrane thinning and hydrocarbon order parameter changes in the vicinity of the inserted peptide. These effects were particularly pronounced for the so called synergistic mixture of 1:1 (mol/mol) L18W-PGLa/MG2a and cannot be accounted for by a linear combination of the membrane perturbations of two peptides individually. Our data are consistent with parallel heterodimers forming at much lower concentrations than previously considered, but which do not induce a synergistic leakage of dyes. Our simulations further show that the heterodimers interact via salt bridges and hydrophobic forces, which apparently makes them more stable than putatively formed antiparallel L18W-PGLa and MG2a homodimers. Moreover, dimerization of L18W-PGLa and MG2a leads to a relocation of the peptides within the lipid headgroup regime as compared to the individual peptides. The early onset of dimerization of L18W-PGLa and MG2a at low peptide concentrations consequently appears to be key to their synergistic dye-releasing activity from lipid vesicles at high concentrations.\n\nSTATEMENT OF SIGNIFICANCEWe demonstrate that specific interactions of the antimicrobial peptides MG2a and PGLa with each other in POPE/POPG bilayers lead to the formation of surface-aligned parallel dimers, which provide already at low peptide concentrations the nucleus for the peptides well-known synergistic activity.

biophysics↗