Concurrent assessment of inner and outer membrane permeabilization and bacteriolysis in E. coli by multiple-wavelength spectrophotometry

Lehrer, Robert I.; Barton, A. P.; Ganz, Tomas

doi:10.1016/0022-1759(88)90414-0

Cited by 196 publications

(209 citation statements)

References 14 publications

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“…Both the LPS-rich outer membrane and the cytoplasmic membrane must be crossed and disrupted for AMPs to kill Gram-negative bacteria. 57,58 In addition to phospholipid membranes, other targets of bacteria have also been found. These include bacterial cell wall precursors such as lipid II 59,60 and outer-membrane proteins for cell wall biogenesis.…”

Section: Solid-state Nmr Studies Of Ampsmentioning

confidence: 99%

Structure and dynamics of cationic membrane peptides and proteins: Insights from solid‐state NMR

Hong¹,

Su²

2011

Protein Science

130

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Many membrane peptides and protein domains contain functionally important cationic Arg and Lys residues, whose insertion into the hydrophobic interior of the lipid bilayer encounters significant energy barriers. To understand how these cationic molecules overcome the free energy barrier to insert into the lipid membrane, we have used solid-state NMR spectroscopy to determine the membrane-bound topology of these peptides. A versatile array of solid-state NMR experiments now readily yields the conformation, dynamics, orientation, depth of insertion, and site-specific protein-lipid interactions of these molecules. We summarize key findings of several Arg-rich membrane peptides, including b-sheet antimicrobial peptides, unstructured cell-penetrating peptides, and the voltage-sensing helix of voltage-gated potassium channels. Our results indicate the central role of guanidinium-phosphate and guanidinium-water interactions in dictating the structural topology of these cationic molecules in the lipid membrane, which in turn account for the mechanisms of this functionally diverse class of membrane peptides.

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Section: Solid-state Nmr Studies Of Ampsmentioning

confidence: 99%

Structure and dynamics of cationic membrane peptides and proteins: Insights from solid‐state NMR

Hong¹,

Su²

2011

Protein Science

130

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“…The effect of rCg-BPI and rhBPI 21 on integrity of the bacterial membranes was assessed by spectrophotometric assays as described in ref. 44 using E. coli ML-35 pBR322. Briefly, bacteria at OD 600 ϭ 0.4-0.6 were washed in 10 mM sodium phosphate buffer (pH 7.4), resuspended in this buffer to Ϸ5 ϫ 10 7 cfu/ml (OD 600 ϭ 0.2), and kept on ice until used.…”

Section: Production and Purification Of Recombinant Cg-bpi (Rcg-bpi)mentioning

confidence: 99%

Evidence of a bactericidal permeability increasing protein in an invertebrate, theCrassostrea gigas Cg-BPI

González-Aravena

Gueguen

Destoumieux-Garzón

et al. 2007

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

123

124

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A cDNA sequence with homologies to members of the LPS-binding protein and bactericidal/permeability-increasing protein (BPI) family was identified in the oyster Crassostrea gigas. The recombinant protein was found to bind LPS, to display bactericidal activity against Escherichia coli, and to increase the permeability of the bacterial cytoplasmic membrane. This indicated that it is a BPI rather than an LPS-binding protein. By in situ hybridization, the expression of the C. gigas BPI (Cg-bpi) was found to be induced in hemocytes after oyster bacterial challenge and to be constitutive in various epithelia of unchallenged oysters. Thus, Cg-bpi transcripts were detected in the epithelial cells of tissues/organs in contact with the external environment (mantle, gills, digestive tract, digestive gland diverticula, and gonad follicles). Therefore, Cg-BPI, whose expression profile and biological properties are reminiscent of mammalian BPIs, may provide a first line of defense against potential bacterial invasion. To our knowledge, this is the first characterization of a BPI in an invertebrate.antimicrobial ͉ epithelia ͉ hemocyte ͉ mollusk ͉ oyster innate immunity M arine invertebrates including bivalve mollusks have evolved in the continuous presence of microorganisms. The oysters, such as Crassostrea gigas, harbor a diverse microflora both on their surface (epibiosis) and inside the body cavities and hemolymph (endobiosis). They have developed an efficient immune system for maintaining balance with commensal and pathogenic bacteria, in particular with the Gram-negative Vibrio spp. abundant in their tissues/organs. LPS from Gram-negative bacteria play an important role in the interaction and activation of the innate immune system including the antimicrobial defense (1, 2). In invertebrates, LPSbinding proteins (LBP) participate in the transduction of cellular signals from LPS. LBPs have been characterized in the freshwater crayfish Pacifastacus leniusculus (3), the shrimp Litopenaeus stylirostris (4), the earthworm Eisenia foetida (5), and the silkworm Bombyx mori (6). In mammals, LBP is an acute phase plasma protein constitutively secreted by liver that induces cellular responses (7). In particular, LBP participates in the acute mobilization of circulating neutrophils to sites of tissue injury. Stored in the mobilized neutrophils, antimicrobial peptides and the bactericidal/ permeability-increasing protein (BPI) contribute to the elimination of bacteria (8, 9). BPI, another LBP, is a 55-kDa cationic protein specifically active against Gram-negative bacteria. It increases the permeability of the bacterial membranes (10). Accumulated extracellularly, BPI opsonizes bacteria, which enhances phagocytosis by neutrophils (11). LBP and BPI are structurally related, with 45% sequence identity. They have a coordinated function in the response to invading bacteria. The antibacterial BPI displays LPSneutralizing properties and suppresses LPS inflammatory activity whereas LBP is an acute-phase reactant (8, 12) that displays a concen...

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“…Bactericidal Peptide Assays-Recombinant peptides were tested for microbicidal activity against E. coli ML35, wild-type serovar Typhimurium strains CS022, JSG210, and 14082 (from Dr. Samuel I. Miller, University of Washington), Vibrio cholerae, Staphylococcus aureus 710a, and Listeria monocytogenes 104035 as described (12). Bacteria (ϳ5 ϫ 10 6 colony forming units (CFU) per milliliter), resuspended in 10 mM PIPES (pH 7.4) supplemented with 0.01 volume of trypticase soy broth, were incubated with test peptides in 50 l for 1 h at 37°C, and surviving bacteria were counted (CFU/ml) after overnight growth on semi-solid media (8,9).…”

Section: Preparation Of Recombinant Crp4 Peptide Variants-recombinantmentioning

confidence: 99%

Functional Analysis of the α-Defensin Disulfide Array in Mouse Cryptdin-4

Maemoto¹,

Qu²,

Rosengren

et al. 2004

Journal of Biological Chemistry

124

166

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The ␣-defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines ␣-defensin bactericidal activity, mouse cryptdin-4 (Crp4) tertiary structure was disrupted by pairs of site-directed Ala for Cys substitutions. In a series of Crp4 disulfide variants whose cysteine connectivities were confirmed using NMR spectroscopy and mass spectrometry, mutagenesis did not induce loss of function. To the contrary, the in vitro bactericidal activities of several Crp4 disulfide variants were equivalent to or greater than those of native Crp4. Mouse Paneth cell ␣-defensins require the proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7), prompting an analysis of the relative sensitivities of native and mutant Crp4 and proCrp4 molecules to degradation by MMP-7. Although native Crp4 and the ␣-defensin moiety of proCrp4 resisted proteolysis completely, all disulfide variants were degraded extensively by MMP-7. Crp4 bactericidal activity was eliminated by MMP-7 cleavage. Thus, rather than determining ␣-defensin bactericidal activity, the Crp4 disulfide arrangement confers essential protection from degradation by this critical activating proteinase.

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Concurrent assessment of inner and outer membrane permeabilization and bacteriolysis in E. coli by multiple-wavelength spectrophotometry

Cited by 196 publications

References 14 publications

Structure and dynamics of cationic membrane peptides and proteins: Insights from solid‐state NMR

Structure and dynamics of cationic membrane peptides and proteins: Insights from solid‐state NMR

Evidence of a bactericidal permeability increasing protein in an invertebrate, theCrassostrea gigas Cg-BPI

Functional Analysis of the α-Defensin Disulfide Array in Mouse Cryptdin-4

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