Structural features, properties and regulation of the outer-membrane protein W (OmpW) of Vibrio cholerae

The outer-membrane protein OmpW of Vibrio cholerae was studied with respect to its structure, functional properties and regulation of expression. On SDS-PAGE, the membrane-associated form of OmpW protein (solubilized by either 0·1 % or 2 % SDS at 25 °C) migrated as a monomer of 19 kDa that changed to 21 kDa on boiling. The protein was hyperexpressed in Escherichia coli in the histidine-tagged form and the purified His6-OmpW (heated or unheated) migrated as a 23 kDa protein on SDS-PAGE. Circular dichroism and Fourier-transform infrared spectroscopic analyses of the recombinant protein showed the presence of β-structures (∼40 %) with minor amounts (8–15 %) of α-helix. These results were consistent with those obtained by computational analysis of the sequence data of the protein using the secondary structure prediction program Jnet. The recombinant protein did not exhibit any porin-like property in a liposome-swelling assay. An antiserum to the purified protein induced a moderate level (66·6 % and 33·3 % at 1 : 50 and 1 : 100 dilutions, respectively) of passive protection against live vibrio challenge in a suckling mouse model. OmpW-deficient mutants of V. cholerae strains were generated by insertion mutagenesis. In a competitive assay in mice, the intestinal colonization activities of these mutants were found to be either only marginally diminished (for O1 strains) or 10-fold less (for an O139 strain) as compared to those of the corresponding wild-type strains. The OmpW protein was expressed in vivo as well as in vitro in liquid culture medium devoid of glucose. Interestingly, the glucose-dependent regulation of OmpW expression was less prominent in a ToxR− mutant of V. cholerae. Further, the expression of OmpW protein was found to be dependent on in vitro cultural conditions such as temperature, salinity, and availability of nutrients or oxygen. These results suggest that the modulation of OmpW expression by environmental factors may be linked to the adaptive response of the organism under stress conditions.

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Characterization of Outer Membrane Protein OmpU of Vibrio cholerae O1

Infection and Immunity ◽

10.1128/iai.66.10.4726-4728.1998 ◽

1998 ◽

Vol 66 (10) ◽

pp. 4726-4728 ◽

Cited By ~ 21

Author(s):

Noboru Nakasone ◽

Masaaki Iwanaga

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Vibrio Cholerae ◽

Outer Membrane Protein ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Rabbit Small Intestine ◽

Vibrio Cholerae O1

ABSTRACT The outer membrane protein OmpU of Vibrio cholerae O1 strain 86B3 was characterized with reference to colonization of the intestine by the organism. The purified OmpU exhibited a pI of 3.6. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it migrated to 38, 32, and 110 kDa when the sample was heated at 100°C for 2 min, 50°C for 15 min, and room temperature for 30 min, respectively. The purified OmpU was not hemagglutinative. Anti-OmpU serum did not agglutinate strain 86B3 or other V. cholerae organisms. OmpU adhered to the brush border of the rabbit small intestine; adhesion of the organisms to the intestine treated in advance with OmpU was not inhibited. Treating the organisms in advance with anti-OmpU Fab did not inhibit adhesion to the intestine. These results obtained in vitro suggest that OmpU is not involved in the adhesion of V. cholerae to the intestinal epithelium.

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An In Vitro Assay for Outer Membrane Protein Assembly by the BAM Complex

Methods in Molecular Biology - The BAM Complex ◽

10.1007/978-1-4939-2871-2_16 ◽

2015 ◽

pp. 203-213 ◽

Cited By ~ 2

Author(s):

Giselle Roman-Hernandez ◽

Harris D. Bernstein

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Protein Assembly ◽

In Vitro Assay ◽

Vitro Assay ◽

Bam Complex

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Outer Membrane Protein (OMP) Profiles of Pasteurella multocida Isolates Associated with Haemorrhagic Septicaemia by SDS-PAGE and Western Blot Analysis

Asian Journal of Animal and Veterinary Advances ◽

10.3923/ajava.2014.513.518 ◽

2014 ◽

Vol 9 (8) ◽

pp. 513-518 ◽

Cited By ~ 1

Author(s):

S.H. Somshekhar ◽

B.M. Veeregowda ◽

V.V.S. Suryanaray ◽

G. Leena ◽

K. Dhama ◽

...

Keyword(s):

Membrane Protein ◽

Western Blot ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Blot Analysis ◽

Western Blot Analysis ◽

Pasteurella Multocida ◽

Haemorrhagic Septicaemia ◽

Sds Page

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Purification of the Major Outer Membrane Protein of Azospirillum brasilense, Its Affinity to Plant Roots, and Its Involvement in Cell Aggregation

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.4.555 ◽

2001 ◽

Vol 14 (4) ◽

pp. 555-561 ◽

Cited By ~ 44

Author(s):

Saul Burdman ◽

Gabriella Dulguerova ◽

Yaacov Okon ◽

Edouard Jurkevitch

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Azospirillum Brasilense ◽

Outer Membrane Proteins ◽

Cell Aggregation ◽

Major Outer Membrane Protein ◽

Adhesion Assay ◽

Fab Fragments

The major outer membrane protein (MOMP) of the nitrogen-fixing rhizobacterium Azospirillum brasilense strain Cd was purified and isolated by gel filtration, and antiserum against this protein was obtained. A screening of the binding of outer membrane proteins (OMPs) of A. brasilense to membrane-immobilized root extracts of various plant species revealed different affinities for the MOMP, with a stronger adhesion to extracts of cereals in comparison with legumes and tomatoes. Moreover, this protein was shown to bind to roots of different cereal seedlings in an in vitro adhesion assay. Incubation of A. brasilense cells with MOMP-antiserum led to fast agglutination, indicating that the MOMP is a surface-exposed protein. Cells incubated with Fab fragments obtained from purified MOMP-antiserum immunoglobulin G exhibited significant inhibition of bacterial aggregation as compared with controls. Bacteria preincubated with Fab fragments showed weaker adhesion to corn roots in comparison to controls without Fab fragments. These findings suggest that the A. brasilense MOMP acts as an adhesin involved in root adsorption and cell aggregation of this bacterium.

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Naturally Occurring IgG Antibodies Provide Innate Protection against Vibrio cholerae Bacteremia by Recognition of the Outer Membrane Protein U

Journal of Innate Immunity ◽

10.1159/000443646 ◽

2016 ◽

Vol 8 (3) ◽

pp. 269-283 ◽

Cited By ~ 17

Author(s):

Kyaw Min Aung ◽

Annika E. Sjöström ◽

Ulrich von Pawel-Rammingen ◽

Kristian Riesbeck ◽

Bernt Eric Uhlin ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Vibrio Cholerae ◽

Outer Membrane Protein ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Immune Defense ◽

Dependent Manner ◽

Naturally Occurring ◽

Serum Killing

Cholera epidemics are caused by Vibrio cholerae serogroups O1 and O139, whereas strains collectively known as non-O1/non-O139 V. cholerae are found in cases of extraintestinal infections and bacteremia. The mechanisms and factors influencing the occurrence of bacteremia and survival of V. cholerae in normal human serum have remained unclear. We found that naturally occurring IgG recognizing V. cholerae outer membrane protein U (OmpU) mediates a serum-killing effect in a complement C1q-dependent manner. Moreover, outer membrane vesicles (OMVs) containing OmpU caused enhanced survival of highly serum-sensitive classical V. cholerae in a dose-dependent manner. OMVs from wild-type and ompU mutant V. cholerae thereby provided a novel means to verify by extracellular transcomplementation the involvement of OmpU. Our data conclusively indicate that loss, or reduced expression, of OmpU imparts resistance to V. cholerae towards serum killing. We propose that the difference in OmpU protein levels is a plausible reason for differences in serum resistance and the ability to cause bacteremia observed among V. cholerae biotypes. Our findings provide a new perspective on how naturally occurring antibodies, perhaps induced by members of the microbiome, may play a role in the recognition of pathogens and the provocation of innate immune defense against bacteremia.

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A novel O -linked glycan modulates Campylobacter jejuni major outer membrane protein-mediated adhesion to human histo-blood group antigens and chicken colonization

Open Biology ◽

10.1098/rsob.130202 ◽

2014 ◽

Vol 4 (1) ◽

pp. 130202 ◽

Cited By ~ 33

Author(s):

Jafar Mahdavi ◽

Necmettin Pirinccioglu ◽

Neil J. Oldfield ◽

Elisabet Carlsohn ◽

Jeroen Stoof ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Blood Group ◽

Campylobacter Jejuni ◽

Outer Membrane Protein ◽

Ex Vivo ◽

Binding Capacity ◽

Major Outer Membrane Protein ◽

Blood Group Antigens

Campylobacter jejuni is an important cause of human foodborne gastroenteritis; strategies to prevent infection are hampered by a poor understanding of the complex interactions between host and pathogen. Previous work showed that C. jejuni could bind human histo-blood group antigens (BgAgs) in vitro and that BgAgs could inhibit the binding of C. jejuni to human intestinal mucosa ex vivo. Here, the major flagella subunit protein (FlaA) and the major outer membrane protein (MOMP) were identified as BgAg-binding adhesins in C. jejuni NCTC11168 . Significantly, the MOMP was shown to be O- glycosylated at Thr 268 ; previously only flagellin proteins were known to be O- glycosylated in C. jejuni . Substitution of MOMP Thr 268 led to significantly reduced binding to BgAgs. The O- glycan moiety was characterized as Gal(β1–3)-GalNAc(β1–4)-GalNAc(β1–4)-GalNAcα1-Thr 268 ; modelling suggested that O- glycosylation has a notable effect on the conformation of MOMP and this modulates BgAg-binding capacity. Glycosylation of MOMP at Thr 268 promoted cell-to-cell binding, biofilm formation and adhesion to Caco-2 cells, and was required for the optimal colonization of chickens by C. jejuni , confirming the significance of this O- glycosylation in pathogenesis.

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Heat-Inducible Surface Stress Protein (Hsp70) Mediates Sulfatide Recognition of the Respiratory Pathogen Haemophilus influenzae

Infection and Immunity ◽

10.1128/iai.69.5.3438-3441.2001 ◽

2001 ◽

Vol 69 (5) ◽

pp. 3438-3441 ◽

Cited By ~ 17

Author(s):

Evamarie Hartmann ◽

Clifford A. Lingwood ◽

Joachim Reidl

Keyword(s):

Heat Shock ◽

Membrane Protein ◽

Recombinant Protein ◽

Haemophilus Influenzae ◽

Outer Membrane Protein ◽

Surface Stress ◽

Specific Binding ◽

Stress Protein ◽

Respiratory Pathogen

ABSTRACT The in vitro glycolipid binding specificity of clinical strains of nontypeable Haemophilus influenzae is altered to include sulfated glycolipids following a brief heat shock. We have constructed, expressed, and purified a recombinant protein of H. influenzae Hsp70, which showed significant specific binding to sulfated galactolipids in vitro. Furthermore, indirect immunofluorescence demonstrates that Hsp70 proteins are surface exposed in H. influenzae only after heat shock and are contained in the outer membrane protein fractions.

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