Gram-negative outer and inner membrane models: insertion of cyclic cationic lipopeptides.
Most Gram-negative bacteria are susceptible to polymyxin B (PxB), and development of resistance to this cationic lipopeptide is very rare. PxB mechanism of action involves interaction with both the outer membrane (OM) and the inner membrane (IM) of bacteria. For the design of new antibiotics based on the structure of PxB and with improved therapeutic indexes, it is essential to establish the key features of PxB that are important for activity. We have used an approach based on mimicking the outer layers of the OM and the IM of Gram-negative bacteria using monolayers of lipopolysaccharide (LPS) or anionic 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG), respectively, and using a combination of penetration assay, analysis of pressure/area curves, and Brewster angle microscopy to monitor surface morphology changes. Synthetic analogue sp-B maintains the basic structural characteristics of the natural compound and interacts with the OM and the IM in a similar way. Analogue sp-C, with a mutation of the sequence [d-Phe6-Leu7] into [d-Phe6-Dab7], shows that this hydrophobic domain is involved in LPS binding. The significant role of the positive charges is demonstrated with sp-Dap analogue, where l-alpha,gamma-diaminobutyric acid residues Dab1 and Dab8 are replaced by l-alpha,gamma-diaminopropionic acid (Dap), resulting in lower degrees of insertion in both LPS and PG monolayers. The importance of the N-terminal acyl chain is demonstrated with polymyxin B nonapeptide (PxB-np). PxB-np shows lower affinity for LPS compared to PxB, sp-B, or sp-C, but it does not insert into PG monolayers, although it binds superficially to the anionic film. Since PxB microbial killing appears to be mediated by osmotic instability due to OM-IM phospholipid exchange, the ability of the different peptides to induce membrane-membrane lipid exchange has been studied by use of phospholipid unilamellar vesicles. Results indicate that cationic amphipathicity determines peptide activity.
Clausell
Adrià
A
Physical Chemistry Department and Institute of Nanoscience and Nanotechnology, University of Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain.
Garcia-Subirats
Maria
M
Pujol
Montserrat
M
Busquets
M Antonia
MA
Rabanal
Francesc
F
Cajal
Yolanda
Y
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D006090:Gram-Negative Bacteria / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D007700:Kinetics; D008050:Lipid A / Q000737:chemistry; D008074:Lipoproteins / Q000737:chemistry*; D008567:Membranes, Artificial; D008956:Models, Chemical; D009842:Oligopeptides / Q000737:chemistry*; D010455:Peptides / Q000138:chemical synthesis / Q000302:isolation & purification; D010743:Phospholipids / Q000737:chemistry; D011112:Polymyxin B / Q000031:analogs & derivatives / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D000900:Anti-Bacterial Agents; D008050:Lipid A; D008074:Lipoproteins; D008567:Membranes, Artificial; D009842:Oligopeptides; D010455:Peptides; D010743:Phospholipids; D011112:Polymyxin B