RESUMEN
This study was concerned with the interaction between the cationic antimicrobial peptide, protamine (Ptm) and the cytoplasmic membranes of the gram-negative bacteria Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa. The objective of the study was to explain the observed paradox of internalization without permanent disruption of the cell envelope. We carried out Monte Carlo computer simulation of Ptm in an aqueous environment in the presence of ~100 mM NaCl and model membranes consisting of either (65:35) or (75:25) PE:PG molar ratios. The (75:25) model, representative of the gram-negative cytoplasmic membrane, showed that the Ptm center of mass remained at least 7 nm from the membrane surface leading to the prediction that Ptm would not internalize via disruption of the inner membrane. By using immunoelectron microscopy of Ptm-treated cells, we showed that Ptm internalization to the cytoplasm took place rapidly in the presence or absence of the outer envelope. Ultrastructural examination revealed no obvious morphological changes to cells that were treated with subinhibitory or bactericidal levels of Ptm. Reconstituted phospholipid bilayers were constructed and were unperturbed by Ptm treatment over a wide range of concentrations and applied transmembrane voltages. We conclude that in the cases of the cell envelopes of E. coli, S. typhimurium and P. aeruginosa, Ptm internalized by means independent of the phospholipid bilayer, most likely mediated by one or more membrane proteins such as cation-selective barrel-like proteins. Work is currently underway to test this hypothesis.
Asunto(s)
Antibacterianos/farmacología , Membrana Celular/efectos de los fármacos , Escherichia coli/metabolismo , Protaminas/farmacología , Pseudomonas aeruginosa/metabolismo , Salmonella typhimurium/metabolismo , Secuencia de Aminoácidos , Simulación por Computador , Farmacorresistencia Microbiana , Enlace de Hidrógeno , Membrana Dobles de Lípidos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Unión Proteica , Electricidad EstáticaRESUMEN
Grazing incidence x-ray scattering techniques and Monte Carlo (MC) simulations are combined to reveal the influence of molecular structure (genetic mutation) and divalent cations on the survival of gram negative bacteria against cationic peptides such as protamine. The former yields detailed structures of bacterial lipopolysaccharide (LPS) membranes with minimized radiation damages, while the minimal computer model based on the linearized Poisson-Boltzmann theory allows for the simulation of conformational changes of macromolecules (LPSs and peptides) that occur in the time scale of ms. The complementary combination of the structural characterizations and MC simulation demonstrates that the condensations of divalent ions (Ca2+ or Mg2+) in the negatively charged core saccharides are crucial for bacterial survival.
Asunto(s)
Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Viabilidad Microbiana/efectos de los fármacos , Método de Montecarlo , Protaminas/farmacología , Animales , Calcio/farmacología , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/fisiología , Lípido A/química , Lipopolisacáridos/genética , Mutación , Presión , Protaminas/metabolismoRESUMEN
A model of the outer membrane of Gram-negative bacteria was created by the deposition of a monolayer of purified rough mutant lipopolysaccharides at an air/water interface. The density profiles of monovalent (K(+)) and divalent (Ca(2+)) cations normal to the lipopolysaccharides (LPS) monolayers were investigated using grazing-incidence X-ray fluorescence. In the absence of Ca(2+), a K(+) concentration peak was found in the negatively charged LPS headgroup region. With the addition of CaCl(2), Ca(2+) ions almost completely displaced K(+) ions from the headgroup region. By integrating the experimentally reconstructed excess ion density profiles, we obtained an accurate measurement of the effective charge density of LPS monolayers. The experimental findings were compared to the results of Monte Carlo simulations based on a coarse-grained minimal model of LPS molecules and showed excellent agreement.
Asunto(s)
Cationes/análisis , Membrana Celular/química , Bacterias Gramnegativas/química , Lipopolisacáridos/química , Modelos Biológicos , Calcio , Simulación por Computador , Método de Montecarlo , Potasio , Espectrometría por Rayos X , Electricidad EstáticaRESUMEN
Lipopolysaccharide (LPS) monolayers deposited on planar, hydrophobic substrates were used as a defined model of outer membranes of Pseudomonas aeruginosa strain dps 89. To investigate the influence of ions on the (out-of-plane) monolayer structure, we measured specular X-ray reflectivity at high energy (22 keV) to ensure transmission through water. Electron density profiles were reconstructed from the reflectivity curves, and they indicate that the presence of Ca(2+) ions induces a significant change in the conformation of the charged polysaccharide head groups (O-side chains). Monte Carlo simulations based on a minimal computer model of LPS molecules allow for the modelling of 100 or more molecules over 10(-3) s and theoretically explained the tendency found by experiments.