Human bactericidal antibody response to outer membrane protein P2 of nontypeable Haemophilus influenzae

Murphy, Timothy F.; Bartos, L C

doi:10.1128/iai.56.10.2673-2679.1988

Cited by 74 publications

(46 citation statements)

References 32 publications

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“…4). The first 16 [3][4][5][6][7][8][9][10][11][12][13][14]. The column was washed with the same buffer and the bound proteins were eluted in 1.2-ml fractions with a linear gradient of 0 to 0.5 M NaCl.…”

Section: Resultsmentioning

confidence: 99%

“…The sheet was rinsed with PBS-0.05% Tween 20 and then with Tris-buffered saline (pH 7.5). The sheet was immersed in Tris-buffered saline containing 0.05% (wt/vol) 4-chloro-1naphthol (Bio-Rad Laboratories), 16.6% (vol/vol) methanol, and 0.015% (vol/vol) hydrogen peroxide. Color developed within a few minutes, and the reaction was stopped by rinsing the sheet extensively with deionized water.…”

Section: Methodsmentioning

confidence: 99%

“…The cells were grown in RPMI 1640 medium (GIBCO Laboratories) containing 50 ,ug of penicillin per ml, 50 ,ug of streptomycin per ml, 1 mM sodium pyruvate, 2 mM L-glutamine, 30 ,ug of endothelial cell growth supplement (Sigma) per ml, 100 ,uM hypoxanthine, 16 ,M thymidine, 0.4 ,uM aminopterin, and 15% (vol/vol) fetal bovine serum. On day 7 after fusion, the medium was replaced with medium without aminopterin.…”

Section: Methodsmentioning

confidence: 99%

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Purification and partial characterization of the major outer membrane protein of Haemophilus somnus

1993

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We purified the major outer membrane protein (MOMP), which is the most abundant OMP (with an apparent molecular mass of 40 kDa), from Haemophilus somnus strain 8025. The method involves solubilization of the MOMP with Zwittergent 3-14 and further purification accomplished by ion-exchange and molecular-sieve chromatographies. The amino-terminal sequence of the MOMP showed considerable similarity to those of porin proteins from other gram-negative bacteria. The MOMP of H. somnus is immunogenic to rabbits and calves. Hyperimmune sera from rabbits and calves reacted with both the MOMP and lipopolysaccharides in enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. The rabbit antiserum to the MOMP was cross-reactive with whole-cell preparations from strains 8025, D1238, NT2301, and 540 at a band with a molecular mass of 40 kDa in immunoblot analysis, although the reactivity of the rabbit antiserum with strain 540 was lower than those with the other strains tested. Two murine monoclonal antibodies (MAbs) to the MOMP were developed. ELISA with the OMP fractions as the antigens showed that one MAb was cross-reactive with the four strains but that the other MAb was reactive with the three strains other than strain 540. These results indicate that the MOMP of H. somnus possesses at least two antigenic determinants and that the MOMP of strain 540 is antigenically different from those of the other strains. The antigenic heterogeneity of the H. somnus MOMP has implications regarding the development of a serotyping system with MAbs that is based on the MOMP epitopes.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Purification and partial characterization of the major outer membrane protein of Haemophilus somnus

1993

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“…Non-typeable H. in£uenzae strains 3198 and 1479 were recovered from the sputum of adults with chronic bronchitis in Bu¡alo (NY, USA) and have been described previously [19,20].…”

Section: Bacterial Strainsmentioning

confidence: 99%

Purification and characterization of outer membrane protein P6, a vaccine antigen of non-typeable Haemophilus influenzae

Karalus¹

1999

FEMS Immunology and Medical Microbiology

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Outer membrane protein P6 is a promising vaccine antigen with potential to prevent infections caused by non-typeable Haemophilus influenzae. A convenient and reliable method for the purification of P6 and an assessment of the purity, yield, protein structure, antigenicity and immunogenicity of the purified protein are described. The method begins with intact H. influenzae and utilizes a series of incubations and centrifugations using a single buffer to remove all cell components with the exception of the peptidoglycan to which the P6 is associated. P6 is dissociated from the complex with heat and the insoluble peptidoglycan is removed by centrifugation. The procedure yields highly purified P6. Contamination with lipooligosaccharide is less than 0.025 endotoxin U per microgr P6. The yield of P6 is approximately 2 mg of P6 per l H. influenzae culture. The purified P6 retains both the secondary and tertiary structure as measured by circular dichroism and analysis with monoclonal antibodies. The purified P6 is immunogenic in animals. A convenient method for purifying P6 which retains antigenicity and immunogenicity will be an important tool for future studies of the vaccine potential of P6.

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“…Efforts to develop a successful vaccine against NTHi are ongoing, with several antigenic virulence factors that contribute to the early stages of bacterial colonization under consideration as potential vaccine candidates (11)(12)(13). Several proteins are under investigation as vaccine candidates (11,14), including fimbriae (OMP P5-homologous fimbriae (15)), outer membrane proteins (OMPs) (P2, P6) (16,17), transferrin binding proteins (TbpB) (18), protein D (19), PilA (major subunit of type IV pili) (20), Hia (21), Hap (22), and HMW1/2 (14,23). However, vaccine development is impeded by the high variability and heterogeneity of NTHi, especially of their surface molecules (13,24).…”

Section: Introductionmentioning

confidence: 99%

Phase variable glycosylation in non-typeableHaemophilus influenzae

Elango

Schulz

2019

Preprint

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Non-typeable Haemophilus influenzae (NTHi) is a leading cause of respiratory tract infections worldwide and continues to be a global health burden. Adhesion and colonisation of host cells are crucial steps in bacterial pathogenesis, and in many strains of NTHi interaction with the host is mediated by the high molecular weight adhesins HMW1A and HMW2A. These adhesins are N-glycoproteins which are modified by cytoplasmic glycosyltransferases HMW1C and HMW2C. Phase variation in the number of short sequence repeats in the promoters of hmw1A and hmw2A directly affects their expression. Here, we report the presence of similar variable repeat elements in the promoters of hmw1C and hmw2C in diverse NTHi isolates. In an ex vivo assay, we systematically altered substrate and glycosyltransferase expression and showed that both of these factors affected the site-specific efficiency of glycosylation on HMW-A. Glycosylation occupancy was incomplete at many sites, variable between sites, and generally lower close to the C-terminus of HMW-A. We investigated the causes of this variability. As HMW-C glycosylates HMW-A in the cytoplasm, we tested how secretion affected glycosylation on HMW-A and showed that retaining HMW-A in the cytoplasm indeed increased glycosylation occupancy across the full length of the protein. Sitedirected mutagenesis showed that HMW-C had no inherent preference for glycosylating asparagines in NxS or NxT sequons. This work provides key insights into factors contributing to the heterogenous modifications of NTHi HMW-A adhesins, expands knowledge of NTHi population diversity and pathogenic capability, and is relevant to vaccine design for NTHi and related pathogens.

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Human bactericidal antibody response to outer membrane protein P2 of nontypeable Haemophilus influenzae

Abstract: The human bactericidal antibody response to the major outer membrane protein, P2, of nontypeable Haemophilus influenzae was studied. P2 was isolated from two strains of hontypeable H. influenzae and coupled VOL. 56, 1988 on July 15, 2020 by guest http://iai.asm.org/ Downloaded from

Cited by 74 publications

References 32 publications

Purification and partial characterization of the major outer membrane protein of Haemophilus somnus

Purification and partial characterization of the major outer membrane protein of Haemophilus somnus

Purification and characterization of outer membrane protein P6, a vaccine antigen of non-typeable Haemophilus influenzae

Phase variable glycosylation in non-typeableHaemophilus influenzae

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