The Bacterial Membrane

Saltón, M. R. J.

doi:10.1007/978-1-4684-3327-2_1

Cited by 23 publications

(27 citation statements)

References 219 publications

(206 reference statements)

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“…The unusual character of hydrophobic OM proteins inhibits the application of the usual immunochemical methods (Salton & Owen, 1976). Their insolubility requires the use of SDS as a solubilizing agent in methods based upon diffusion in agarose.…”

Section: Discussionmentioning

confidence: 99%

Preparation and Quantitative Determination of Antibodies Against Major Outer Membrane Proteins of Escherichia coli 026 K60

Hofstra

Dankert

1980

Microbiology

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437Antisera against isolated outer membrane (OM) proteins I and 11" of Escherichia co/i 0 2 6 K60 were elicited in rabbits. Antisera obtained after intramuscular administration with Freund's complete adjuvant showed high titres of specific antibodies. Intravenous administration of the same preparations yielded a considerable antibody response against bacterial lipopolysaccharide, a minor contaminant of the protein preparations. Antibody titres against OM proteins I and II*, lipopolysaccharide and murein-lipoprotein were determined by the enzyme-Iinked immunosorbent assay (ELISA) in these sera, and in antisera elicited against whole formaldehyde-fixed bacteria or isolated OM. Comparison of ELISA with single radial immunodiffusion and interfacial immunoprecipitation tests revealed that ELISA was not only the most uniformly applicable, but also the most specific and the most convenient method. In double diffusion tests no cross-reactivity between proteins I and II* was seen. Antibodies against proteins I and II*, lipopolysaccharide and lipoprotein could be specifically absorbed from the sera with the appropriate antigen preparations. Absorption experiments with intact E. coli 0 2 6 K60, Tris/EDTA-sheared bacteria and isolated OM revealed that antibodies against protein I were hardly absorbed at all probably because the antibody, evoked against denatured protein I, did not react with the protein in its native configuration. Antibodies against protein 11* and lipoprotein were absorbed by intact as well as by sheared bacteria, but to a much greater extent by isolated OM, which indicates that these OM components are accessible from the outside, but that they are situated relatively deep in the OM structure. I N T R O D U C T I O NThe outer membrane (OM) of Escherichia coli contains a number of major proteins in the 27000 to 41000 molecular weight range, designated Ia, Ib (Schmitges & Henning, 1976) and 11* (Garten et al., 1975) or b, c and d (Lugtenberg et al., 1975). Another major OM constituent is the murein-bound or free lipoprotein (Braun & Rehn, 1969). The function of OM proteins in the immunology of Gram-negative bacteria is not yet fully understood. Their immunological investigation requires the purification of these antigens and the development of reliable techniques to determine antibodies against them. Braun et al. ( 1 976) determined antibodies against the lipoprotein by passive haemagglutination. The enzyme-linked immunosorbent assay was applied by Sippel et al. (1978) for the detection of anti bodies against cell envelope proteins of Neisseria meningitidis and Salmonella typhi. Studying phage reception by OM proteins, Diedrich et al. (1977) H . H O F S T R A A N D J . D A N K E R Twas determined by double diffusion in agarose. Other methods applied for the detection of antibodies against OM proteins are immunoprecipitation (Inouye et al., 1976; Dankert & Hofstra, 1978) and crossed imrnunoelectrophoresis (Van To1 et a/., 1979).I n this study we report the quantitative determination of antibodies a...

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Section: Discussionmentioning

confidence: 99%

Preparation and Quantitative Determination of Antibodies Against Major Outer Membrane Proteins of Escherichia coli 026 K60

Hofstra

Dankert

1980

Microbiology

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“…The envelope mutant D22 (envA) had low barrier indexes for erythromycin, rifampin, ampicillin, and cloxacillin. For the beta-lactams, outer membrane penetration and affinity for inner membrane target site(s) triggering cell lysis were measured as independent components of the overall activity; although penetration and overall activity varied greatly, the affinities of most were within a narrow range.The limiting layers of the gram-negative cell envelope are the cytoplasmic or inner membrane (IM) and the outer membrane (OM) (29). Between the IM and OM is a murein sacculus within a tenuous space known as the periplasm, which contains a number of wall-associated enzymes (28).…”

mentioning

confidence: 99%

“…The limiting layers of the gram-negative cell envelope are the cytoplasmic or inner membrane (IM) and the outer membrane (OM) (29). Between the IM and OM is a murein sacculus within a tenuous space known as the periplasm, which contains a number of wall-associated enzymes (28).…”

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confidence: 99%

Outer-Membrane Penetration Barriers as Components of Intrinsic Resistance to Beta-Lactam and Other Antibiotics in Escherichia coli K-12

Scudamore

Beveridge

Goldner

1979

Antimicrob Agents Chemother

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A new technique has been devised to investigate the penetration of antibiotics through the gram-negative outer membrane; the application here was to study intrinsic resistance of Escherichia coli K-12. Exponential cells in broth were briefly treated with 2.5 mM ethylenediaminetetraacetic acid at 5°C to disrupt the outer membrane penetration barrier, and the response of treated and untreated cells to antibiotics was compared by turbidimetry. A barrier index was derived to describe the ability of 7 beta-lactam and 10 other antibiotics to penetrate the outer membrane of strain Y10. There was correlation between the molecular weight and logio barrier index (r = 0.59, P = 0.01). The envelope mutant D22 (envA) had low barrier indexes for erythromycin, rifampin, ampicillin, and cloxacillin. For the beta-lactams, outer membrane penetration and affinity for inner membrane target site(s) triggering cell lysis were measured as independent components of the overall activity; although penetration and overall activity varied greatly, the affinities of most were within a narrow range.The limiting layers of the gram-negative cell envelope are the cytoplasmic or inner membrane (IM) and the outer membrane (OM) (29). Between the IM and OM is a murein sacculus within a tenuous space known as the periplasm, which contains a number of wall-associated enzymes (28).The OM is a barrier to molecules above a critical size; in Escherichia coli and Salmonella typhimurium, the penetration of oligopeptides (25) and oligosaccharides (8) declines sharply as the molecular weight exceeds 600, and they are essentially excluded at 1,000. Similarly, there is at least partial exclusion of some antibiotics by the OM contributing to "intrinsic" resistance (12), although in most cases the effect has not been well quantified. According to a popular model, solutes may penetrate the OM through narrow aqueous pores which restrict entry nonspecifically by size, and so provide a molecular sieving function (8). This function involves certain OM proteins which have been called "porins" (14,17,18,19,22).In this paper, we describe a technique which was used to measure the penetration of 17 antibiotics through the OM of E. coli K-12. A preliminary report has been made (30). The OM penetrability barrier was disrupted by brief treatment with ethylenediaminetetraacetic acid (EDTA), and the turbidimetric response of treated and normal cells to antibiotics was then compared over a short period of growth. (There was no adjustment of the external osmotic pressure). In support of the pore model, we found that antibiotic penetration through the OM was limited by molecular weight, and that the limits agreed well with those for oligosaccharides (8). However, it was clear that in some cases other variables must also control penetration.Beta-lactam antibiotics were differentiated with respect to penetration ofthe OM and affinity for the IM target site(s) promoting cell lysis. This gave an insight into the basis for the wide range of activities against E. coli which are encount...

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“…47. Such a mechanism has been invoked previously to explain the formation of the enigmatic mesosomal veiscles, structures also enriched with antigens from the trans face of the cytoplasmic membrane (51).…”

Section: Downloaded Frommentioning

confidence: 85%

Identification, immunochemical characterization, and purification of a major lipoprotein antigen associated with the inner (cytoplasmic) membrane of Escherichia coli

Doherty¹,

Yamada²,

Caffrey³

et al. 1986

J Bacteriol

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A major antigenic constituent of the inner membrane of Escherichia coli ML308-225 was identified as a 28.5-kilodalton lipoprotein containing covalently bound glycerol and palmitate. This lipoprotein corresponded to antigen 47 in the crossed immunoelectrophoresis profile of membrane vesicles (P. Owen and H. R. Kaback, Proc. Natl. Acad. Sci. USA 75:3148-3152, 1978) and to new lipoprotein 4 described for E. coli B by Ichihara et al. (S. Ichihara, H. Hussain, and S. Mizushima, J. Biol. Chem. 256:3125-3129, 1980). Experiments involving isopycnic centrifugation of spheroplast envelopes indicated that antigen 47 was enriched in cytoplasmic membrane subfractions of low density. The protein did not manifest an obvious association with peptidoglycan of the types displayed by the bound form of the Braun (Lpp) lipoprotein, the 21-kilodalton peptidoglycan-associated lipoprotein, or the ompFIC gene products. Antibodies specific for antigen 47 were used to demonstrate that the molecule was immunologically distinct from both the Braun lipoprotein and the peptidoglycan-associated lipoprotein of E. coli. Antigens of similar molecular mass to and cross-reacting with antigen 47 were present in the envelopes of eight type species of the Enterobacteriaceae. A protocol for the purification of antigen 47, based upon its solubility in a chloroform-methanol-water mixture, was developed.The covalent attachment of lipid to proteins was first suggested over 30 years ago by Folch and Lees (16). Since that time, an increasing number of membrane proteins of both eucaryotic and procaryotic origin have been reported to contain covalently bound lipid (12,23,29,47,49,52). Common features of most procaryotic lipoproteins studied to date appear to be an N-acyl diglyceride cysteine at the N terminus of the molecule (18,25,35) and a posttranslational modification event involving a signal peptidase which can be selectively inhibited by the antibiotic globomycin (22,23,60). In gram-positive bacteria, the covalent association of membrane-bound penicillinases with lipid is well documented (25,34,35,37,53). In Escherichia coli, the envelope is known to contain a number of lipoproteins (4,23,30). Of these, the most dominant and best characterized are associated with the outer membrane system and include the Braun (Lpp) lipoprotein, the peptidoglycan-associated lipoprotein (PAL), and the traT gene product (4,20,30,31,48). The presence of several other (minor) envelope-associated lipoproteins (termed new lipoproteins or NLPs) has also been documented (23), and there is evidence that some of these, viz., NLP3, NLP4, and possibly NLP6, may reside in the inner membrane of E. coli (23, 32).Reports emanating from this laboratory over the course of the last 10 years (for reviews, see references 39-41) have been concerned with an analysis of the numerous antigens associated with the envelope of E. coli (50, 54). Our understanding of the complexity, identity, and topography of these antigens, many of which are proteinaceous in nature, has been facilitated by the us...

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The Bacterial Membrane

Cited by 23 publications

References 219 publications

Preparation and Quantitative Determination of Antibodies Against Major Outer Membrane Proteins of Escherichia coli 026 K60

Preparation and Quantitative Determination of Antibodies Against Major Outer Membrane Proteins of Escherichia coli 026 K60

Outer-Membrane Penetration Barriers as Components of Intrinsic Resistance to Beta-Lactam and Other Antibiotics in Escherichia coli K-12

Identification, immunochemical characterization, and purification of a major lipoprotein antigen associated with the inner (cytoplasmic) membrane of Escherichia coli

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