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Robert E. W. Hancock - One of the best experts on this subject based on the ideXlab platform.
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Influence of Culture Conditions on Expression of the 40-Kilodalton Porin Protein of Vibrio anguillarum Serotype O2
Applied and environmental microbiology, 1998Co-Authors: Michelle L. Davey, Robert E. W. Hancock, Lucy M. MuthariaAbstract:Vibrio anguillarum serotype O2 strains express a 40-kDa outer membrane Porin Protein. Immunoblot analysis revealed that antigenic determinants of the V. anguillarum O2 40-kDa Porin were conserved within bacterial species of the genus Vibrio. The relative amounts of the V. anguillarum O2 40-kDa Porin were enhanced by growth of V. anguillarum O2 in CM9 medium containing 5 to 10% sucrose or 0.1 to 0.5 M NaCl. In contrast, the levels of the Porin were significantly reduced when cells were grown at 37°C, and a novel 60-kDa Protein was also observed. However, the osmolarity or ionic concentration of the growth medium did not influence expression of the 60-kDa Protein. Growth in medium containing greater than 0.6 mM EDTA reduced production of the V. anguillarum O2 40-kDa Porin and enhanced levels of a novel 19-kDa Protein. Thus, expression of the V. anguillarum O2 40-kDa Porin was osmoregulated and possibly coregulated by temperature. The N-terminal amino acid sequence of the V. anguillarum O2 40-kDa Protein and the effect of environmental factors on the cellular levels of the Porin suggested that the V. anguillarum O2 40-kDa Porin was functionally similar to the OmpC Porin of Escherichia coli. However, pore conductance assays revealed that the V. anguillarum O2 40-kDa Porin was a general diffusion Porin with a pore size in the range of that of the OmpF Porin of E. coli.
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isolation and characterization of a conserved Porin Protein from helicobacter pylori
Journal of Bacteriology, 1995Co-Authors: P Doig, Robert E. W. Hancock, M M Exner, T J TrustAbstract:Helicobacter pylori is a causative agent of gastritis in humans and is correlated with gastric ulcer formation. Infections with this bacterium have proven difficult to treat with antimicrobial agents. To better understand how this bacterium transports compounds such as antimicrobial agents across its outer membrane, identification of Porin Proteins is important. We have recently identified a family of H. pylori Porins (HopA to HopD) (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567-1572, 1995). Here, we report on an unrelated Porin species (HopE) from this bacterium. This Protein had a apparent molecular mass of 31 kDa and was seen to form 50- and 90-kDa aggregates that were designated putative dimeric and trimeric forms, respectively. The Protein was purified to homogeneity and, with a model planar lipid membrane system, was shown to act as a nonselective pore with a single channel conductance in 1.0 M KCl of 1.5 nS, similarly to other bacterial nonspecific Porins. An internal peptide sequence of HopE shared homology with the P2 Porin of Haemophilus influenzae. HopE was also shown to be antigenic in vivo as assessed by sera taken from H. pylori-infected individuals and was immunologically conserved with both patient sera and specific monoclonal antibodies. From these data, it appears that HopE is a major nonselective Porin of H. pylori. The implications of these findings are discussed.
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epitope mapping of the pseudomonas aeruginosa major outer membrane Porin Protein oprf
Infection and Immunity, 1995Co-Authors: E Rawling, N. L. Martin, Robert E. W. HancockAbstract:The Pseudomonas aeruginosa major outer membrane Protein OprF has been proposed for use as a vaccine and as a target for immunotherapeutic and diagnostic monoclonal antibodies. The well-conserved epitopes for 10 surface-reactive, OprF-specific monoclonal antibodies were localized by both overlapping peptide analysis and immunodetection of OprF peptides generated by cyanogen bromide and the protease papain. Three of the monoclonal antibodies bound to specific overlapping octapeptides, which had been synthesized on 160 pins to cover the entire 326 amino acids of OprF. The highest reactivities were as follows: MA7-1 to the pin with attached peptide GTYETGNK (amino acids 55 to 62), MA7-2 to NLADFMKQ (amino acids 237 to 244), and MA5-8 to TAEGRAIN (amino acids 307 to 314). The other monoclonal antibodies showed no reactivity, indicating that they do not recognize linear epitopes. Two polyclonal sera were also tested and demonstrated weak reactivity with discrete regions of OprF, suggesting that the majority of antibodies produced might recognize conformational epitopes. Utilizing defined peptides generated with cyanogen bromide and papain, the conformational epitopes recognized by the seven monoclonal antibodies were localized to regions that were 42 to 90 amino acids long. These regions were located on two adjacent loops in the middle of an amended structural model of OprF.
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genetic definition of the substrate selectivity of outer membrane Porin Protein oprd of pseudomonas aeruginosa
Journal of Bacteriology, 1993Co-Authors: H Huang, Robert E. W. HancockAbstract:Earlier studies proved that Pseudomonas aeruginosa OprD is a specific Porin for basic amino acids and imipenem. It was also considered to function as a nonspecific Porin that allowed the size-dependent uptake of monosaccharides and facilitation of the uptake of quinolone and other antibiotics. In the present study, we utilized P. aeruginosa strains with genetically defined levels of OprD to characterize the in vivo substrate selectivity of this Porin. An oprD::omega interposon mutant was constructed by gene replacement utilizing an in vitro mutagenized cloned oprD gene. In addition, OprD was overexpressed from the lac promoter by cloning the oprD gene into the broad-host-range plasmid pUCP19. To test the substrate selectivity, strains were grown in minimal medium with limiting concentrations of the carbon sources glucose, gluconate, or pyruvate. In minimal medium with 0.5 mM gluconate, the growth rates of the parent strain H103 and its oprD::omega mutant H729 were only 60 and 20%, respectively, of that of the OprD-overexpressing strain H103(pXH2). In contrast, no significant differences were observed in the growth rates of these three strains on glucose or pyruvate, indicating that OprD selectively facilitated the transport of gluconate. To determine the role of OprD in antibiotic uptake, nine strains representing different levels of OprD and OprF were used to determine the MICs of different antibiotics. The results clearly demonstrated that OprD could be utilized by imipenem and meropenem but that, even when substantially overexpressed, it could not be significantly utilized by other beta-lactams, quinolones, or aminoglycosides. In addition, competition experiments confirmed that imipenem had common binding sites with basic amino acids in the OprD channel, but not with gluconate or glucose. Images
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conservation of surface epitopes in pseudomonas aeruginosa outer membrane Porin Protein oprf
Fems Microbiology Letters, 1993Co-Authors: N. L. Martin, E Rawling, Rebecca S Y Wong, Mae Joanne Rosok, Robert E. W. HancockAbstract:The outer membrane Proteins of several prominent bacterial pathogens demonstrate substantial variation in their surface antigenic epitopes. To determine if this was also true for Pseudomonas aeruginosa outer membraine Protein OprF, gene sequencing of a serotype 5 isolate was performed to permit comparison with the published serotype 12 oprF gene sequence. Only 16 nucleotide substitutions in the 1053 nucleotide coding region were observed; none of these changed the amino acid sequence. A panel of 10 monoclonal antibodies (mAbs) reacted with each of 46 P. aeruginosa strains representing all 17 serotype strains, 12 clinical isolates, 15 environmental isolates and 2 laboratory isolates. Between two and eight of these mAbs also reacted with Proteins from representatives of the rRNA homology group I of the Pseudomonadaceae. Nine of the ten mAbs recognized surface antigenic epitopes as determined by indirect immunofluorescence techniques and their ability to opsonize P. aeuroginosa for phagocytosis. These epitopes were partially masked by lipopolysacharide side chains as revealed using a side chain-deficient mutant. It is concluded that OprF is a highly conserved Protein with several conserved surface antigenic epitopes.
E Rawling - One of the best experts on this subject based on the ideXlab platform.
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epitope mapping of the pseudomonas aeruginosa major outer membrane Porin Protein oprf
Infection and Immunity, 1995Co-Authors: E Rawling, N. L. Martin, Robert E. W. HancockAbstract:The Pseudomonas aeruginosa major outer membrane Protein OprF has been proposed for use as a vaccine and as a target for immunotherapeutic and diagnostic monoclonal antibodies. The well-conserved epitopes for 10 surface-reactive, OprF-specific monoclonal antibodies were localized by both overlapping peptide analysis and immunodetection of OprF peptides generated by cyanogen bromide and the protease papain. Three of the monoclonal antibodies bound to specific overlapping octapeptides, which had been synthesized on 160 pins to cover the entire 326 amino acids of OprF. The highest reactivities were as follows: MA7-1 to the pin with attached peptide GTYETGNK (amino acids 55 to 62), MA7-2 to NLADFMKQ (amino acids 237 to 244), and MA5-8 to TAEGRAIN (amino acids 307 to 314). The other monoclonal antibodies showed no reactivity, indicating that they do not recognize linear epitopes. Two polyclonal sera were also tested and demonstrated weak reactivity with discrete regions of OprF, suggesting that the majority of antibodies produced might recognize conformational epitopes. Utilizing defined peptides generated with cyanogen bromide and papain, the conformational epitopes recognized by the seven monoclonal antibodies were localized to regions that were 42 to 90 amino acids long. These regions were located on two adjacent loops in the middle of an amended structural model of OprF.
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conservation of surface epitopes in pseudomonas aeruginosa outer membrane Porin Protein oprf
Fems Microbiology Letters, 1993Co-Authors: N. L. Martin, E Rawling, Rebecca S Y Wong, Mae Joanne Rosok, Robert E. W. HancockAbstract:The outer membrane Proteins of several prominent bacterial pathogens demonstrate substantial variation in their surface antigenic epitopes. To determine if this was also true for Pseudomonas aeruginosa outer membraine Protein OprF, gene sequencing of a serotype 5 isolate was performed to permit comparison with the published serotype 12 oprF gene sequence. Only 16 nucleotide substitutions in the 1053 nucleotide coding region were observed; none of these changed the amino acid sequence. A panel of 10 monoclonal antibodies (mAbs) reacted with each of 46 P. aeruginosa strains representing all 17 serotype strains, 12 clinical isolates, 15 environmental isolates and 2 laboratory isolates. Between two and eight of these mAbs also reacted with Proteins from representatives of the rRNA homology group I of the Pseudomonadaceae. Nine of the ten mAbs recognized surface antigenic epitopes as determined by indirect immunofluorescence techniques and their ability to opsonize P. aeuroginosa for phagocytosis. These epitopes were partially masked by lipopolysacharide side chains as revealed using a side chain-deficient mutant. It is concluded that OprF is a highly conserved Protein with several conserved surface antigenic epitopes.
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Analysis of two gene regions involved in the expression of the imipenem-specific, outer membrane Porin Protein OprD of Pseudomonas aeruginosa.
FEMS microbiology letters, 1992Co-Authors: H Huang, R J Siehnel, F Bellido, E Rawling, R E HancockAbstract:A Tn501 mutant of Pseudomonas aeruginosa resistant to imipenem and lacking the imipenem-specific outer membrane Porin Protein OprD was isolated. The mutation could be complemented to imipenem susceptibility and OprD-sufficiency by a cloned 6-kb EcoRI-PstI fragment of DNA from the region of chromosome of the wild-type strain surrounding the site of Tn501 insertion. However, this fragment did not contain the oprD structural gene as judged by its inability to hybridize with an oligonucleotide corresponding to the N-terminal amino acid sequence of OprD. DNA sequencing of 3.9 kb of the region surrounding the Tn501 insertion site revealed three large open reading frames, one of which would be interrupted by the Tn501 insertion in the mutant. This latter open reading frame, named opdE (for putative regulator of oprD expression), predicted a hydrophobic Protein of M(r) 41,592. Using the above-mentioned oligonucleotide, the oprD structural gene was cloned and expressed in Escherichia coli on a 2.1-kb Bam HI-KpnI fragment. DNA sequencing predicted a 420 amino acid mature OprD Protein with a 23 amino acid signal sequence.
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Analysis of two gene regions involved in the expression of the imipenem-specific, outer membrane Porin Protein OprD of Pseudomonas aeruginosa
Fems Microbiology Letters, 1992Co-Authors: H Huang, Richard Siehnel, F Bellido, E Rawling, Robert E. W. HancockAbstract:Abstract A Tn501 mutant of Pseudomonas aeruginosa resistant to imipenem and lacking the imipenemspecific outer membrane Porin Protein OprD was isolated. The mutation could be complemented to imipenem susceptibility and OprD-sufficiency by a cloned 6-kb Eco RI- Pst I fragment of DNA from the region of chromosome of the wild-type strain surrounding the site of Tn501 insertion. However, this fragment did not contain the oprD structural gene as judged by its inability to hybridize with an oligonucleotide corresponding to the N-terminal amino acid sequence of OprD. DNA sequencing of 3.9 kb of the region surrounding the Tn501 insertion site revealed three large open reading frames, one of which would be interrupted by the Tn501 insertion in the mutant. This latter open reading frame, named opdE (for putative regulator of oprD ), predicted a hydrophobic Protein of M T 41 592. Using the above-mentioned oligonucleotide, the oprD structural gene was cloned and expressed in Escherichia coli on a 2.1-kb Bam HI- Kpn I fragment. DNA sequencing predicted a 420 amino acid mature OprD Protein with a 23 amino acid signal sequence.
H Huang - One of the best experts on this subject based on the ideXlab platform.
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genetic definition of the substrate selectivity of outer membrane Porin Protein oprd of pseudomonas aeruginosa
Journal of Bacteriology, 1993Co-Authors: H Huang, Robert E. W. HancockAbstract:Earlier studies proved that Pseudomonas aeruginosa OprD is a specific Porin for basic amino acids and imipenem. It was also considered to function as a nonspecific Porin that allowed the size-dependent uptake of monosaccharides and facilitation of the uptake of quinolone and other antibiotics. In the present study, we utilized P. aeruginosa strains with genetically defined levels of OprD to characterize the in vivo substrate selectivity of this Porin. An oprD::omega interposon mutant was constructed by gene replacement utilizing an in vitro mutagenized cloned oprD gene. In addition, OprD was overexpressed from the lac promoter by cloning the oprD gene into the broad-host-range plasmid pUCP19. To test the substrate selectivity, strains were grown in minimal medium with limiting concentrations of the carbon sources glucose, gluconate, or pyruvate. In minimal medium with 0.5 mM gluconate, the growth rates of the parent strain H103 and its oprD::omega mutant H729 were only 60 and 20%, respectively, of that of the OprD-overexpressing strain H103(pXH2). In contrast, no significant differences were observed in the growth rates of these three strains on glucose or pyruvate, indicating that OprD selectively facilitated the transport of gluconate. To determine the role of OprD in antibiotic uptake, nine strains representing different levels of OprD and OprF were used to determine the MICs of different antibiotics. The results clearly demonstrated that OprD could be utilized by imipenem and meropenem but that, even when substantially overexpressed, it could not be significantly utilized by other beta-lactams, quinolones, or aminoglycosides. In addition, competition experiments confirmed that imipenem had common binding sites with basic amino acids in the OprD channel, but not with gluconate or glucose. Images
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Analysis of two gene regions involved in the expression of the imipenem-specific, outer membrane Porin Protein OprD of Pseudomonas aeruginosa.
FEMS microbiology letters, 1992Co-Authors: H Huang, R J Siehnel, F Bellido, E Rawling, R E HancockAbstract:A Tn501 mutant of Pseudomonas aeruginosa resistant to imipenem and lacking the imipenem-specific outer membrane Porin Protein OprD was isolated. The mutation could be complemented to imipenem susceptibility and OprD-sufficiency by a cloned 6-kb EcoRI-PstI fragment of DNA from the region of chromosome of the wild-type strain surrounding the site of Tn501 insertion. However, this fragment did not contain the oprD structural gene as judged by its inability to hybridize with an oligonucleotide corresponding to the N-terminal amino acid sequence of OprD. DNA sequencing of 3.9 kb of the region surrounding the Tn501 insertion site revealed three large open reading frames, one of which would be interrupted by the Tn501 insertion in the mutant. This latter open reading frame, named opdE (for putative regulator of oprD expression), predicted a hydrophobic Protein of M(r) 41,592. Using the above-mentioned oligonucleotide, the oprD structural gene was cloned and expressed in Escherichia coli on a 2.1-kb Bam HI-KpnI fragment. DNA sequencing predicted a 420 amino acid mature OprD Protein with a 23 amino acid signal sequence.
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Analysis of two gene regions involved in the expression of the imipenem-specific, outer membrane Porin Protein OprD of Pseudomonas aeruginosa
Fems Microbiology Letters, 1992Co-Authors: H Huang, Richard Siehnel, F Bellido, E Rawling, Robert E. W. HancockAbstract:Abstract A Tn501 mutant of Pseudomonas aeruginosa resistant to imipenem and lacking the imipenemspecific outer membrane Porin Protein OprD was isolated. The mutation could be complemented to imipenem susceptibility and OprD-sufficiency by a cloned 6-kb Eco RI- Pst I fragment of DNA from the region of chromosome of the wild-type strain surrounding the site of Tn501 insertion. However, this fragment did not contain the oprD structural gene as judged by its inability to hybridize with an oligonucleotide corresponding to the N-terminal amino acid sequence of OprD. DNA sequencing of 3.9 kb of the region surrounding the Tn501 insertion site revealed three large open reading frames, one of which would be interrupted by the Tn501 insertion in the mutant. This latter open reading frame, named opdE (for putative regulator of oprD ), predicted a hydrophobic Protein of M T 41 592. Using the above-mentioned oligonucleotide, the oprD structural gene was cloned and expressed in Escherichia coli on a 2.1-kb Bam HI- Kpn I fragment. DNA sequencing predicted a 420 amino acid mature OprD Protein with a 23 amino acid signal sequence.
F Bellido - One of the best experts on this subject based on the ideXlab platform.
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Analysis of two gene regions involved in the expression of the imipenem-specific, outer membrane Porin Protein OprD of Pseudomonas aeruginosa.
FEMS microbiology letters, 1992Co-Authors: H Huang, R J Siehnel, F Bellido, E Rawling, R E HancockAbstract:A Tn501 mutant of Pseudomonas aeruginosa resistant to imipenem and lacking the imipenem-specific outer membrane Porin Protein OprD was isolated. The mutation could be complemented to imipenem susceptibility and OprD-sufficiency by a cloned 6-kb EcoRI-PstI fragment of DNA from the region of chromosome of the wild-type strain surrounding the site of Tn501 insertion. However, this fragment did not contain the oprD structural gene as judged by its inability to hybridize with an oligonucleotide corresponding to the N-terminal amino acid sequence of OprD. DNA sequencing of 3.9 kb of the region surrounding the Tn501 insertion site revealed three large open reading frames, one of which would be interrupted by the Tn501 insertion in the mutant. This latter open reading frame, named opdE (for putative regulator of oprD expression), predicted a hydrophobic Protein of M(r) 41,592. Using the above-mentioned oligonucleotide, the oprD structural gene was cloned and expressed in Escherichia coli on a 2.1-kb Bam HI-KpnI fragment. DNA sequencing predicted a 420 amino acid mature OprD Protein with a 23 amino acid signal sequence.
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Analysis of two gene regions involved in the expression of the imipenem-specific, outer membrane Porin Protein OprD of Pseudomonas aeruginosa
Fems Microbiology Letters, 1992Co-Authors: H Huang, Richard Siehnel, F Bellido, E Rawling, Robert E. W. HancockAbstract:Abstract A Tn501 mutant of Pseudomonas aeruginosa resistant to imipenem and lacking the imipenemspecific outer membrane Porin Protein OprD was isolated. The mutation could be complemented to imipenem susceptibility and OprD-sufficiency by a cloned 6-kb Eco RI- Pst I fragment of DNA from the region of chromosome of the wild-type strain surrounding the site of Tn501 insertion. However, this fragment did not contain the oprD structural gene as judged by its inability to hybridize with an oligonucleotide corresponding to the N-terminal amino acid sequence of OprD. DNA sequencing of 3.9 kb of the region surrounding the Tn501 insertion site revealed three large open reading frames, one of which would be interrupted by the Tn501 insertion in the mutant. This latter open reading frame, named opdE (for putative regulator of oprD ), predicted a hydrophobic Protein of M T 41 592. Using the above-mentioned oligonucleotide, the oprD structural gene was cloned and expressed in Escherichia coli on a 2.1-kb Bam HI- Kpn I fragment. DNA sequencing predicted a 420 amino acid mature OprD Protein with a 23 amino acid signal sequence.
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a pleiotropic posttherapy enoxacin resistant mutant of pseudomonas aeruginosa
Antimicrobial Agents and Chemotherapy, 1992Co-Authors: Laura J V Piddock, F Bellido, M C Hall, M Bains, Robert E. W. HancockAbstract:An enoxacin-resistant Pseudomonas aeruginosa mutant (G49) isolated during patient therapy was characterized in detail. The G49 mutant was cross resistant to several classes of antibiotics including quinolones, beta-lactams, chloramphenicol, and tetracycline, but not imipenem or aminoglycosides. Compared with its paired pretherapy isolate G48, this mutant had several alterations in outer membrane Proteins including a complete loss of the major Porin Protein OprF and a substantially altered lipopolysaccharide profile. Revertants were selected at a frequency of approximately 1% after enrichment for OprF+ cells on low-salt proteose peptone no. 2 medium. Ninety-seven of these OprF+ revertants were as susceptible to carbenicillin and norfloxacin as the pretherapy isolate. One of these revertants was characterized in more detail and shown to be indistinguishable in all properties from the pretherapy isolate. It is proposed that the multiple-antibiotic-resistance (Mar) phenotype of this mutant resulted from a single pleiotropic mutation. Images
Timothy F. Murphy - One of the best experts on this subject based on the ideXlab platform.
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Antibodies to Loop 6 of the P2 Porin Protein of Nontypeable Haemophilus influenzae Are Bactericidal against Multiple Strains
Infection and immunity, 2001Co-Authors: John M. Neary, Richard J. Karalus, Timothy F. MurphyAbstract:The P2 Porin Protein is the most abundant Protein in the outer membrane of nontypeable Haemophilus influenzae (NTHI). Analysis of sequences of P2 from different strains reveals the presence of both heterogeneous and conserved surface-exposed loops of the P2 molecule among strains. The present study was undertaken to test the hypothesis that antibodies to a conserved surface-exposed loop are bactericidal for multiple strains of NTHI and could thus form the basis of vaccines to prevent infection due to NTHI. Polyclonal antiserum to a peptide corresponding to loop 6 was raised and was immunopurified over a loop 6 peptide column. Analysis of the antibodies to whole organisms and peptides corresponding to each of the eight loops of P2 by immunoassays revealed that the antibodies were highly specific for loop 6 of P2. The immunopurified antibodies bound to P2 of 14 of 15 strains in immunoblot assays. These antibodies to loop 6 demonstrated complement-mediated bactericidal killing of 8 of 15 strains. These results support the concept of using conserved regions of the P2 Protein as a vaccine antigen.
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Mapping of bactericidal epitopes on the P2 Porin Protein of nontypeable Haemophilus influenzae.
Infection and immunity, 1994Co-Authors: Elaine M. Haase, Gene D. Morse, Timothy F. MurphyAbstract:Abstract The P2 Porin Protein is the major outer membrane Protein of nontypeable Haemophilus influenzae and is a potential target of a protective immune response. Nine monoclonal antibodies (MAbs) to P2 were developed by immunizing mice with nontypeable H. influenzae whole organisms. Each MAb reacted exclusively with the homologous strain in a whole-cell immunodot assay demonstrating exquisite strain specificity. All nine MAbs recognized abundantly expressed surface-exposed epitopes on the intact bacterium by immunofluorescence and immunoelectron microscopy. Each MAb was bactericidal to the homologous strain in an in vitro complement-mediated killing assay. Immunoblot assay of cyanogen bromide cleavage products of purified P2 indicated that MAb 5F2 recognized the 10-kDa fragment, and the other eight MAbs recognized the 32-kDa fragment. Competitive ELISAs confirmed that 5F2 recognized an epitope that is different from the other eight MAbs. To further localize epitopes, MAbs 5F2 and 6G3 were studied in Protein footprinting by using reversed-phase high-performance liquid chromatography. Three potential epitope-containing peptides which were reactive in an enzyme-linked immunosorbent assay with both 5F2 and 6G3 were isolated. These peptides were identified by N-terminal amino acid sequence and localized to loops 5 and 8 of the proposed model for P2. Fusion Proteins consisting of glutathione S-transferase fused with variable-length peptides from loops 5 and 8 were expressed in the pGEX-2T vector. Immunoblot assay of fusion peptides of loops 5 and 8 confirmed that 5F2 recognized an epitope within residues 338 to 354 of loop 8; 6G3 and the remaining MAbs recognized an epitope within residues 213 to 229 of loop 5. These studies indicate that nontypeable H. influenzae contains bactericidal epitopes which have been mapped to two different surface-exposed loops of the P2 molecule. These potentially protective epitopes are strain specific and abundantly expressed on the surface of the intact bacterium.
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Molecular analysis of the P2 Porin Protein of nontypeable Haemophilus influenzae
Infection and immunity, 1992Co-Authors: D J Sikkema, Timothy F. MurphyAbstract:The P2 Porin Protein is the most abundant outer membrane Protein (OMP) of nontypeable Haemophilus influenzae (NTHI) and shows extensive antigenic heterogeneity among strains. To study the molecular basis of this heterogeneity, the DNA sequences of the genes encoding the P2 Proteins of three unrelated strains of NTHI were determined, and restriction fragment length polymorphisms around the P2 genes of 35 strains were analyzed. The deduced amino acid sequences of the P2 genes from the three strains of NTHI revealed four major (12 to 35 amino acids long) and several smaller (2 to 7 amino acids) hypervariable regions in each Protein. The major variations occurred in identical portions of the genes, and these regions showed a high antigenic index and surface exposure probability in computer modeling analysis. Differences in the molecular mass of the P2 Protein correlate with differences in the size of the variable region in each strain. Oligonucleotide primers suitable for amplification of the P2 genes by polymerase chain reaction were developed. Restriction fragment length polymorphism analysis showed marked heterogeneity in and around the ompP2 locus of 35 NTHI strains. These results contrast with the high degree of conservation of the P2 genes in H. influenzae type b strains. We conclude that the molecular mass and antigenic heterogeneity of the P2 molecule of NTHI is due to variations in gene sequence that are clustered primarily in four large hypervariable regions of the gene.
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Strain-specific and immunodominant surface epitopes of the P2 Porin Protein of nontypeable Haemophilus influenzae.
Infection and immunity, 1991Co-Authors: Elaine M. Haase, Anthony A. Campagnari, J Sarwar, M Shero, M. A. Wirth, C U Cumming, Timothy F. MurphyAbstract:The P2 Porin Protein is the major outer membrane Protein of nontypeable Haemophilus influenzae. Five monoclonal antibodies to P2 of four strains of nontypeable H. influenzae were developed by immunizing mice with whole bacterial cells. All five antibodies recognized epitopes on P2 in immunoblot assays of whole organism lysates, purified outer membrane, and purified P2. Competitive enzyme-linked immunosorbent assays and immunoblot assays of cyanogen bromide-digested P2 showed that two antibodies to the P2 Protein of strain 1479 recognized different epitopes on the molecule. Immunofluorescence and immunoelectron microscopy demonstrated that each of the five antibodies recognized epitopes that were abundantly expressed on the bacterial surface. Analysis of 120 H. influenzae strains indicated that three of the five antibodies were reactive exclusively with the homologous strain. The remaining two antibodies were reactive with less than 3% of the strains. These studies indicate that the P2 Protein expresses a highly strain-specific and immunodominant epitope on the bacterial surface. The expression of strain-specific and immunodominant epitopes on the bacterial surface may represent a mechanism by which the bacterium induces antibodies that will protect against recurrent infection by the homologous strain but will not protect against infection by heterologous strains. Images