scholarly journals Serum Resistance in Haemophilus ducreyiRequires Outer Membrane Protein DsrA

2000 ◽  
Vol 68 (3) ◽  
pp. 1608-1619 ◽  
Author(s):  
Christopher Elkins ◽  
K. John Morrow ◽  
Bonnie Olsen
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Serum Antibody ◽  
Amino Acid Sequences ◽  
Serum Resistance ◽  
Naturally Occurring

ABSTRACT Haemophilus ducreyi is resistant to killing by normal serum antibody and complement. We discovered an H. ducreyiouter membrane protein required for expression of serum resistance and termed it DsrA (for “ducreyi serum resistance A”). ThedsrA locus was cloned, sequenced, and mutagenized. An isogenic mutant (FX517) of parent strain 35000 was constructed and characterized, and it was found to no longer express dsrA. FX517 was at least 10-fold more serum susceptible than 35000. DsrA was expressed by all strains of H. ducreyi tested, except three naturally occurring, avirulent, serum-sensitive strains. FX517 and the three naturally occurring dsrA-nonexpressing strains were complemented in trans with a plasmid expressingdsrA. All four strains were converted to a serum-resistant phenotype, including two that contained truncated lipooligosaccharide (LOS). Therefore, serum resistance in H. ducreyi does not require expression of full-length LOS but does require expression ofdsrA. The dsrA locus from eight additionalH. ducreyi strains was sequenced, and the deduced amino acid sequences were more than 85% identical. The major difference between the DsrA proteins was due to the presence of one, two, or three copies of the heptameric amino acid repeat NTHNINK. These repeats account for the variability in apparent molecular mass of the monomeric form of DsrA (28 to 35 kDa) observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since DsrA is present in virulent strains, is highly conserved, and is required for serum resistance, we speculate that it may be a virulence factor and a potential vaccine candidate.

scholarly journals Sequence Polymorphism, Predicted Secondary Structures, and Surface-Exposed Conformational Epitopes of Campylobacter Major Outer Membrane Protein

2000 ◽  
Vol 68 (10) ◽  
pp. 5679-5689 ◽  
Author(s):  
Qijing Zhang ◽  
Jerrel C. Meitzler ◽  
Shouxiong Huang ◽  
Teresa Morishita
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Outer Membrane Proteins ◽  
Major Outer Membrane Protein ◽  
Amino Acid Sequences ◽  
Variable Regions ◽  
Conserved Sequences ◽  
Conformational Epitopes

ABSTRACT The major outer membrane protein (MOMP), a putative porin and a multifunction surface protein of Campylobacter jejuni, may play an important role in the adaptation of the organism to various host environments. To begin to dissect the biological functions and antigenic features of this protein, the gene (designatedcmp) encoding MOMP was identified and characterized from 22 strains of C. jejuni and one strain of C. coli. It was shown that the single-copy cmp locus encoded a protein with characteristics of bacterial outer membrane proteins. Prediction from deduced amino acid sequences suggested that each MOMP subunit consisted of 18 β-strands connected by short periplasmic turns and long irregular external loops. Alignment of the amino acid sequences of MOMP from different strains indicated that there were seven localized variable regions dispersed among highly conserved sequences. The variable regions were located in the putative external loop structures, while the predicted β-strands were formed by conserved sequences. The sequence homology of cmp appeared to reflect the phylogenetic proximity of C. jejuni strains, since strains with identical cmp sequences had indistinguishable or closely related macrorestriction fragment patterns. Using recombinant MOMP and antibodies recognizing linear or conformational epitopes of the protein, it was demonstrated that the surface-exposed epitopes of MOMP were predominantly conformational in nature. These findings are instrumental in the design of MOMP-based diagnostic tools and vaccines.

The porin OmpC ofSalmonella typhimuriummediates adherence to macrophages

1999 ◽  
Vol 45 (8) ◽  
pp. 658-669 ◽  
Author(s):  
Robert S Negm ◽  
Thomas G Pistole
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Virulent Strain ◽  
Salmonella Typhi ◽  
Amino Acid Sequences ◽  
Wild Type ◽  
E Coli ◽  
Host Cell Recognition

Macrophages recognize, adhere to, and phagocytose Salmonella typhimurium. The major outer membrane protein OmpC is a candidate ligand for macrophage recognition. To confirm this we used transposon mutagenesis to develop an ompC-deficient mutant in a known virulent strain of S. typhimurium; mutant and wild type were compared in macrophage adherence and association assays. Radiolabeled wild type S. typhimurium bound to macrophages at five-fold higher levels than did the ompC mutant. In association assays, macrophages in monolayers bound and internalized three-fold more wild type than mutant, while macrophages in suspension bound and internalized 40-fold more wild type than mutant. The ompC gene of our test strain of S. typhimurium contains several discrete differences compared with the ompC genes of Salmonella typhi and Escherichia coli. The deduced OmpC amino acid sequence of S. typhimurium shares 77 and 98% identity with OmpC amino acid sequences of E. coli and S. typhi, respectively. Evidence from this study supports a role for the OmpC protein in initial recognition by macrophages and distinguishes regions of this protein that potentially participate in host-cell recognition of bacteria by phagocytic cells.Key words: Salmonella, porin, macrophage, outer membrane protein, DNA sequencing.

scholarly journals Molecular Cloning, Sequencing, and Expression ofomp-40, the Gene Coding for the Major Outer Membrane Protein from the Acidophilic Bacterium Thiobacillus ferrooxidans

2000 ◽  
Vol 66 (6) ◽  
pp. 2318-2324 ◽  
Author(s):  
Nicolas Guiliani ◽  
Carlos A. Jerez
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Amino Acid Sequence ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Thiobacillus Ferrooxidans ◽  
Sodium Dodecyl ◽  
Major Outer Membrane Protein ◽  
Gene Coding ◽  
Chemolithoautotrophic Bacteria

ABSTRACT Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. Some of the surface components of this microorganism are probably involved in adaptation to their acidic environment and in bacterium-mineral interactions. We have isolated and characterizedomp40, the gene coding for the major outer membrane protein from T. ferrooxidans. The deduced amino acid sequence of the Omp40 protein has 382 amino acids and a calculated molecular weight of 40,095.7. Omp40 forms an oligomeric structure of about 120 kDa that dissociates into the monomer (40 kDa) by heating in the presence of sodium dodecyl sulfate. The degree of identity of Omp40 amino acid sequence to porins from enterobacteria was only 22%. Nevertheless, multiple alignments of this sequence with those from several OmpC porins showed several important features conserved in the T. ferrooxidans surface protein, such as the approximate locations of 16 transmembrane beta strands, eight loops, including a large external L3 loop, and eight turns which allowed us to propose a putative 16-stranded beta-barrel porin structure for the protein. These results together with the previously known capacity of Omp40 to form ion channels in planar lipid bilayers strongly support its role as a porin in this chemolithoautotrophic acidophilic microorganism. Some characteristics of the Omp40 protein, such as the presence of a putative L3 loop with an estimated isoelectric point of 7.21 allow us to speculate that this can be the result of an adaptation of the acidophilic T. ferrooxidans to prevent free movement of protons across its outer membrane.

scholarly journals Mapping of a Protective Epitope of the CopB Outer Membrane Protein of Moraxella catarrhalis

1998 ◽  
Vol 66 (2) ◽  
pp. 540-548 ◽  
Author(s):  
Christoph Aebi ◽  
Leslie D. Cope ◽  
Jo L. Latimer ◽  
Sharon E. Thomas ◽  
Clive A. Slaughter ◽  
...  
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Moraxella Catarrhalis ◽  
Amino Acid Sequences ◽  
Nucleotide Sequence Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protective Epitope

ABSTRACT A monoclonal antibody (MAb) (MAb 10F3) directed against the CopB outer membrane protein of Moraxella catarrhalis previously was found to enhance pulmonary clearance of M. catarrhalisin an animal model (M. Helminen, I. Maciver, J. L. Latimer, L. D. Cope, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 61:2003–2010, 1993). In the present study, this same MAb was shown to exert complement-dependent bactericidal activity against this pathogen in vitro. Nucleotide sequence analysis of thecopB gene from two MAb 10F3-reactive and two MAb 10F3-unreactive strains of M. catarrhalis revealed that the deduced amino acid sequences of these four CopB proteins were at least 90% identical. Comparison of the amino acid sequences of these proteins allowed localization of possible MAb 10F3 binding sites to five relatively small regions of the CopB protein from M. catarrhalis O35E. When five synthetic peptides representing these regions were tested for their ability to bind MAb 10F3 in a direct enzyme-linked immunosorbent assay system, an oligopeptide containing 26 amino acids was shown to bind this MAb. The actual binding region for MAb 10F3 was localized further through the use of overlapping decapeptides that spanned this 26-mer. A fusion protein containing the same 26-mer readily bound MAb 10F3 and was used to immunize mice. The resultant antiserum contained antibodies that reacted with the CopB protein of the homologous M. catarrhalis strain in Western blot analysis and bound to the surface of both homologous and heterologous strains of M. catarrhalis.

scholarly journals Genetic Diversity of the 28-Kilodalton Outer Membrane Protein Gene in Human Isolates of Ehrlichia chaffeensis

1999 ◽  
Vol 37 (4) ◽  
pp. 1137-1143 ◽  
Author(s):  
Xue-Jie Yu ◽  
Jere W. McBride ◽  
David H. Walker
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Dna Sequence ◽  
Outer Membrane Protein ◽  
Signal Sequence ◽  
Outer Membrane Proteins ◽  
Protein A ◽  
Amino Acid Sequences ◽  
Ehrlichia Chaffeensis

The Ehrlichia chaffeensis 28-kDa outer membrane protein (p28) gene was sequenced completely by genomic walking with adapter PCR. The DNA sequence of the p28 gene was nearly identical to the previously reported sequence (N. Ohashi, N. Zhi, Y. Zhang, and Y. Rikihisa, Infect. Immun. 66:132–139, 1998), but analysis of a further 75 bp on the 5′ end of the gene revealed DNA that encoded a 25-amino-acid signal sequence. The leader sequence was removed from the N terminus of a 30-kDa precursor to generate the mature p28 protein. A monoclonal antibody (MAb), 1A9, recognizing four outer membrane proteins of E. chaffeensis (Arkansas strain) including the 25-, 26-, 27-, and 29-kDa proteins (X.-J. Yu, P. Brouqui, J. S. Dumler, and D. Raoult, J. Clin. Microbiol. 31:3284–3288, 1993) reacted with the recombinant p28 protein. This result indicated that the four proteins recognized by MAb 1A9 were encoded by the multiple genes of the 28-kDa protein family. DNA sequence alignment analysis revealed divergence of p28 among all five human isolates of E. chaffeensis. The E. chaffeensis strains could be divided into three genetic groups on the basis of the p28 gene. The first group consisted of the Sapulpa and St. Vincent strains. They had predicted amino acid sequences identical to each other. The second group contained strain 91HE17 and strain Jax, which only showed 0.4% divergence from each other. The third group contained the Arkansas strain only. The amino acid sequences of p28 differed by 11% between the first two groups, by 13.3% between the first and third groups, and by 13.1% between the second and third groups. The presence of antigenic variants of p28 among the strains of E. chaffeensis and the presence of multiple copies of heterogeneous genes suggest a possible mechanism by which E. chaffeensismight evade the host immune defenses. Whether or not immunization with the p28 of one strain of E. chaffeensis would confer cross-protection against other strains needs to be investigated.

scholarly journals Characterization of Outer Membrane Protein OmpU of Vibrio cholerae O1

1998 ◽  
Vol 66 (10) ◽  
pp. 4726-4728 ◽  
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.

scholarly journals Structural and Functional Analyses of the Major Outer Membrane Protein of Chlamydia trachomatis

2007 ◽  
Vol 189 (17) ◽  
pp. 6222-6235 ◽  
Author(s):  
Guifeng Sun ◽  
Sukumar Pal ◽  
Annahita K. Sarcon ◽  
Soyoun Kim ◽  
Etsuko Sugawara ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Disulfide Bonds ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Spatial Relationship ◽  
Major Outer Membrane Protein ◽  
Cross Linking

ABSTRACT Chlamydia trachomatis is a major pathogen throughout the world, and preventive measures have focused on the production of a vaccine using the major outer membrane protein (MOMP). Here, in elementary bodies and in preparations of the outer membrane, we identified native trimers of the MOMP. The trimers were stable under reducing conditions, although disulfide bonds appear to be present between the monomers of a trimer and between trimers. Cross-linking of the outer membrane complex demonstrated that the MOMP is most likely not in a close spatial relationship with the 60- and 12-kDa cysteine-rich proteins. Extraction of the MOMP from Chlamydia isolates under nondenaturing conditions yielded the trimeric conformation of this protein as shown by cross-linking and analysis by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with different concentrations of acrylamide. Using circular dichroism spectroscopy, we determined that the trimers were formed mainly of β-pleated sheet structures in detergent micelles. Using a liposomal swelling assay, the MOMP was found to have porin activity, and the size of the pore was estimated to be approximately 2 nm in diameter. The trimers were found to be stable in SDS at temperatures ranging from 4 to 37°C and over a pH range of 5.0 to 8.0. In addition, the trimers of MOMP were found to be resistant to digestion with trypsin. In conclusion, these results show that the native conformation of the MOMP of C. trachomatis is a trimer with predominantly a β-sheet structure and porin function.

scholarly journals The Regulated Outer Membrane Protein Omp21 from Comamonas acidovorans Is Identified as a Member of a New Family of Eight-Stranded β-Sheet Proteins by Its Sequence and Properties

1998 ◽  
Vol 180 (15) ◽  
pp. 3741-3749 ◽  
Author(s):  
C. Baldermann ◽  
A. Lupas ◽  
J. Lubieniecki ◽  
H. Engelhardt
Keyword(s):  
Amino Acids ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Spontaneous Mutant ◽  
Sequence Comparisons ◽  
Comamonas Acidovorans

ABSTRACT Omp21, a minor outer membrane protein of the soil bacteriumComamonas acidovorans, was purified from a spontaneous mutant lacking a surface layer and long-chain lipopolysaccharide. Omp21 synthesis is enhanced by oxygen depletion, and the protein has a variable electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to its heat-modifiable behavior. The structural gene omp21 encodes a precursor of 204 amino acids with a putative signal peptide of 21 amino acids. Mature Omp21 is a typical outer membrane protein with a high content of β structure as determined by infrared spectroscopy. Sequence comparisons show that it belongs to a new outer membrane protein family, characterized by eight amphipathic β strands, which includes virulence proteins, such as the neisserial opacity proteins,Salmonella typhimurium Rck, and Yersinia enterocolitica Ail, as well as the major outer membrane proteins OmpA from Escherichia coli and OprF fromPseudomonas aeruginosa.

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

2016 ◽  
Vol 8 (3) ◽  
pp. 269-283 ◽  
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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