Morphological and physiological investigations on the action of polymyxin B on Escherichia coli

Wahn, K.; Lutsch, Gudrun; Rockstroh, T; Zapf, K

doi:10.1007/bf00412165

Cited by 26 publications

(18 citation statements)

References 34 publications

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“…This is due to lysis of the bacteria (40), which includes severe degradation of nucleic acids (21,35). The resulting disappearance of, mainly, the large aggregations is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

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Action of Polymyxin B on Bacterial Membranes: Morphological Changes in the Cytoplasm and in the Outer Membrane of Salmonella typhimurium and Escherichia coli B

Schindler

Teuber

1975

Antimicrob Agents Chemother

148

119

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Though the primary action of the cationic antibiotic polymyxin B is against the membrane of susceptible bacteria, severe morphological changes are detected in the cytoplasm. Using fluorescence microscopy and a mono-N-dansylpolymyxin B derivative, we could demonstrate aggregations of the antibiotic with cellular material, possibly nucleic acids and/or ribosomes. These aggregations were only produced by minimum inhibitory or higher concentrations of the antibiotic as shown with Salmonella and Escherichia strains differing in their polymyxin susceptibility. The outer membrane of Salmonella typhimurium revealed characteristic blebs when treated with polymyxin B. This was investigated by the gentle methods of spray-freezing and freeze-etching. The obtained electron micrographs suggest that the polymyxin-induced blebs are projections of the outer monolayer of the outer membrane. A possible mechanism of penetration of polymyxin B through the cell envelope of gram-negative bacteria is presented.The basic polypeptide polymyxin B (PX) acts specifically on gram-negative bacteria by electrostatic and hydrophobic interactions with anionic cell envelope components like phospholipids (10, 11, 21, 34, 36) and lipopolysaccharides (LPS) (3, 26). These molecules are most likely the primary receptor molecules and responsible for the classification of the polymyxins as membrane-specific antibiotics. A possible penetration of the antibiotic into the cytoplasm, however, has not been directly demonstrated, although Few and Schulmann (7) supposed from binding studies that such a penetration could occur. In the same sense, Handley et al. (9) interpreted the condensation of nuclear material in PX-treated bacteria as revealed by electron maicroscopy as polymyxin-deoxyribonucleic acid complexes. These have been described in vitro (18). By the use of mono-N-dimethylaminonaphthalene-sulfonyl-polymyxin B (dansyl-PX), we were able to prove the intracellular accumulation of PX. A preliminary report has been presented (37).An additional structural change, the formation of blebs in the outer membrane of gram-negative bacteria (9, 13, 31, 40), was reinvestigated by the application of spray-freezing and freeze-etching (1, 2, 24). Freeze-etching has been described for Escherichia coli B to give a relatively close approximation to the living state (5). It could be excluded that PX-induced bleb formation is an artifact of fixation of detergent-weakened membranes. The technique used suggests these blebs to be evaginations of the cell surface monolayer of Salmonella typhimurium. MATERIALS AND METHODSBacterial strains and culture conditions. S. typhimurium SL 1135 (complete 0-antigenic side chain of LPS) and SL 1102 (chemotype Re) were obtained from S. Schlecht, Max-Planck-Institut fur Immunbiologie, Freiburg (Germany). S. typhimurium G 30 (a uridine 5'-diphosphate-galactose-4-epimerase-lacking mutant, chemotype Rc) was provided by M. J. Osbom, University of Connecticut at Farmington. S. typhimurium SL 1102-RE was a polymyxin-resistant mutant isolated in ...

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“…This is due to lysis of the bacteria (40), which includes severe degradation of nucleic acids (21,35). The resulting disappearance of, mainly, the large aggregations is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…An additional structural change, the formation of blebs in the outer membrane of gram-negative bacteria (9,13,31,40), was reinvestigated by the application of spray-freezing and freeze-etching (1,2,24). Freeze-etching has been described for Escherichia coli B to give a relatively close approximation to the living state (5).…”

mentioning

confidence: 99%

Action of Polymyxin B on Bacterial Membranes: Morphological Changes in the Cytoplasm and in the Outer Membrane of Salmonella typhimurium and Escherichia coli B

Schindler

Teuber

1975

Antimicrob Agents Chemother

148

119

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“…PXB binds anionic lipids, revealing their concentration distribution. After exposure to PXB, the outer membranes of Gram-negative bacteria erupt in protrusions (4)(5)(6) discernable by electron microscopy of thin-section, freeze-fracture, and scanning preparations (7,8); the number of protrusions is correlated with the PXB concentration (9). Moreover, PXB has a proven affinity for anionic lipids (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21).…”

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Anionic lipid domains: correlation with functional topography in a mammalian cell membrane.

Bearer¹,

Friend²

1980

Proc. Natl. Acad. Sci. U.S.A.

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Polymyxin B was used to explore distribution of anionic phospholipids in sperm plasma membranes by electron microscopy of freeze-fracture replicas. After exposure to Hepes/Tris-buffered polymyxin at 4 mM, phosphatidylcholine liposomes showed no perturbations nor didthey fluoresce with dansylated incubation. When phosphatidylethanolamine was included in the liposomes, they became perturbed and fluoresced. Plasma membranes of Drosophila larval cells, containing or lacking cholesterol, were also disrupted by polymyxin.The cell membranes of guinea pig sperm were likewise disrupted but in specific functional areas. Fusional membrane domains showed protrusions; the stable membrane of the flagellum revealed diffuse bubbling. Regions of well-defined particle arrays and the postacrosomal segment maintained smooth contours. By fluorescence microscopy, we detected the same heterogeneous binding of the polymyxin dansyl derivative.By electron microscopy of freeze-fracture preparations, the cell membrane is a tapestry of different textural domains. In the guinea pig sperm plasma membrane, particle arrays (proteins) observed in freeze-fracture replicas exhibit characteristic patterns on particle surfaces-e.g., the "quilt" of the head, and the zipper and annulus of the tail (1). Recently, labeling sperm cells with filipin, a polyene, has also demonstrated cholesterol in a heterogeneous distribution (2, 3). Like filipin, polymyxin B (PXB) disturbs the contour of membranes containing the lipids that it binds. PXB binds anionic lipids, revealing their concentration distribution. After exposure to PXB, the outer membranes of Gram-negative bacteria erupt in protrusions (4-6) discernable by electron microscopy of thin-section, freeze-fracture, and scanning preparations (7,8); the number of protrusions is correlated with the PXB concentration (9). Moreover, PXB has a proven affinity for anionic lipids (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21).In this report, we demonstrate that PXB binds to a mammalian spermatozoon and insect larval cells, producing protrusions similar to those observed in prokaryotes. Unlike the uniformly disposed eruptions of bacterial membranes, however, various membrane perturbations emboss only specific regions of the sperm membrane. Tris buffer at pH 7.4. The vesicles were stored under argon at 4VC until used. We incubated treated and untreated vesicles in 25% glycerol for 1-2 hr before freezing, or processed them without glycerol for thin-sectioning as specified (2). Treatment of liposomes consisted of incubation at 370C in 2 mM PXB (Sigma) in 0.1 M Hepes/Tris buffer (pH 7.4). Additionally, one group of PtdCho/PtdEtn/cholesterol liposomes was incubated for 1 hr in 1.25% glutaraldehyde (pH 7.4, 1% sucrose) in the same buffer and processed for freeze-fracture.Cell Suspensions. Third-instar Drosophila melanogaster larval cells, provided by C. M. Havel (Department of Biochemistry and Biophysics, University of California, San Francisco), were grown in cholesterol-free medium until depleted of the ster...

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“…[12][13][14] and subsequently, disorganization of the outer membrane occurs if the cells are susceptible to the antibiotic.…”

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Evidences for complex formation between polymyxin B and lipopolysaccharides from Serratia marcescens.

1976

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In vitro and in vivo complex formations of polymyxin B and lipopolysaccharides (LPS) from resistant and sensitive cells of Serratia marcescens were studied by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and electron microscopy. In vitro treatment of LPS from resistant cells with polymyxin B gave two populations of spherical complexes of different molecular weights as determined electrophoretically. Similar treatment of LPS from sensitive cells resulted in dissociation of the LPS-protein and subsequent complexing with the LPS moiety into stable spheres.In vivo treatment of resistant cells with polymyxin B resulted in LPS-polymyxin B complexes which were comparatively smaller and existed in two morphological forms: spheres and linear ribbons.LPS from the sensitive cells were degraded extensively into small rods and an amorphous mass by the in vivo polymyxin B treatment.In both systems, the electrophoretic results consistently matched the electron microscopic evidences for complex formation of LPS with polymyxin B. It is suggested that the disruptive effects of polymyxin B on LPS in the outer membrane of S. marcescens may be the explanation for the change in permeability barrier in the resistant cells and disorganization of the outer membrane and subsequent death in the sensitive cells. Furthermore, the ability of the LPS to complex with the polymyxin B molecules in resistant cells may be the basis of their resistance to the antibiotic.Polymyxin B, a cationic polypeptide antibiotic, interacts with various anionic cellular components of gram-negative bacteria such as lipopolysaccharide (LPS),1,1) phospholipids (PL),3-6) deoxyribonucleic acid (DNA) 7-9) and ribosomes.8-10) Polymyxin B treatment of LPS isolated from susceptible bacteria results in a breakdown of the LPS molecule (in vitro effect).11) When whole cells are treated with the antibiotic, blebs are formed on the outer membrane of the cell envelope7.12-14) and subsequently, disorganization of the outer membrane occurs if the cells are susceptible to the antibiotic.Although the formation of the blebs in the outer membrane has been suggested to be caused by an aggregation of the antibiotic with outer membrane components such as LPS and PL9) (in vivo effect), direct evidences for the formation of such aggregates or complexes have not been shown, nor was the sequence of events from the moment of complex formation to the disorganization of the outer membrane described. In this communication, we report the electrophoretic and electron microscopic evidences of the in vitro and in vivo formation of complexes between polymyxin B and LPS from cells of Serratia marcescens, which were either resistant or sensitive to the antibiotic.

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Morphological and physiological investigations on the action of polymyxin B on Escherichia coli

Cited by 26 publications

References 34 publications

Action of Polymyxin B on Bacterial Membranes: Morphological Changes in the Cytoplasm and in the Outer Membrane of Salmonella typhimurium and Escherichia coli B

Action of Polymyxin B on Bacterial Membranes: Morphological Changes in the Cytoplasm and in the Outer Membrane of Salmonella typhimurium and Escherichia coli B

Anionic lipid domains: correlation with functional topography in a mammalian cell membrane.

Evidences for complex formation between polymyxin B and lipopolysaccharides from Serratia marcescens.

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