ABSTRACT
The htrA gene from two strains of nontypeable Haemophilus influenzae has been cloned and sequenced, and the encoded approximately 46-kDa HtrA proteins were found to be highly conserved. H. influenzae HtrA has approximately 55% identity with the Escherichia coli and Salmonella typhimurium HtrA stress response proteins, and expression of the H. influenzae htrA gene was inducible by high temperature. Recombinant HtrA (rHtrA) was expressed from E. coli, and the purified protein was found to have serine protease activity. rHtrA was found to be very immunogenic and partially protective in both the passive infant rat model of bacteremia and the active chinchilla model of otitis media. Immunoblot analysis indicated that HtrA is antigenically conserved in encapsulated and nontypeable H. influenzae species. Site-directed mutagenesis was performed on the htrA gene to ablate the endogenous serine protease activity of wild-type HtrA, and it was found that eight of nine recombinant mutant proteins had no measurable residual proteolytic activity. Two mutant proteins were tested in the animal protection models, and one, H91A, was found to be partially protective in both models. H91A HtrA may be a good candidate antigen for a vaccine against invasive H. influenzae type b disease and otitis media and is currently in phase I clinical trials.
Subject(s)
Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Haemophilus influenzae/immunology , Heat-Shock Proteins , Periplasmic Proteins , Serine Endopeptidases/immunology , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cloning, Molecular , Guinea Pigs , Immune Sera/immunology , Immunization , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Rats , Recombinant Proteins/immunology , Serine Endopeptidases/genetics , Vaccines, Synthetic/immunologyABSTRACT
We have cloned and sequenced the d15 gene from two strains of Haemophilus influenzae type b (Hib) and two strains of nontypeable H. influenzae (NTHI). The nucleotide and deduced protein sequences of d15 are highly conserved, with only a small variable region identified near the carboxyl terminus of the protein. Analysis of upstream sequences revealed that the H. influenzae d15 gene may be part of a large potential operon of closely spaced open reading frames, including one with significant homology to the Escherichia coli cds gene encoding CDP-diglyceride synthetase. Southern blot analysis demonstrated that the d15 gene is also present in H. influenzae types a, c, d, e, and f and in Haemophilus parainfluenzae. A recombinant D15 (rD15) protein was expressed in good quantity in E. coli from the inducible T7 promoter, and monospecific anti-rD15 antibodies were raised. Immunoblot analysis of H. influenzae serotypes a, b, c, d, e, and f, NTHI, and H. parainfluenzae lysates revealed that they all expressed a cross-reactive D15-like protein. Purified rD15 was found to be highly immunogenic in mice, guinea pigs, and rabbits, and passive transfer of anti-rD15 antibodies protected infant rats from challenge with H. influenzae type b or type a in infant rat models of bacteremia. Thus, D15 is a highly conserved antigen that is protective in animal models and it may be a useful component of a universal subunit vaccine against Haemophilus infection and disease.
Subject(s)
Antigens, Bacterial/chemistry , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/immunology , Haemophilus influenzae/immunology , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Cloning, Molecular , Escherichia coli , Guinea Pigs , Haemophilus influenzae/chemistry , Haemophilus influenzae/genetics , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Rabbits , Recombinant Proteins/immunology , Sequence Alignment , Sequence Homology, Amino Acid , SerotypingABSTRACT
The genomic transferrin receptor genes (tbpA and tbpB) from two strains of Haemophilus influenzae type b (Hib) and two strains of non-typable H. influenzae (NTHi) have been cloned and sequenced. The deduced protein sequences of the H. influenzae tbpA genes were 95-100% conserved and those of the tbpB genes were 66-100% conserved. The tbpB gene from one strain of NTHi was found to encode a truncated Tbp2 protein. The tbpB genes from four additional NTHi strains were amplified by the polymerase chain reaction (PCR) utilizing primers derived from the conserved N-terminal sequences of Tbp1 and Tbp2 and were found to encode full-length proteins. Although several bacterial species express transferrin receptors, when the Tbp1 and Tbp2 sequences from different organisms were compared, there was only limited homology. Recombinant Tbp1 and Tbp2 proteins were expressed from Escherichia coli and antisera were raised to the purified proteins. There was significant antigenic conservation of both Tbp1 and Tbp2 amongst H. influenzae strains, as determined by Western blot analysis. In a passive model of bacteraemia, infant rats were protected from challenge with Hib after transfer of anti-rTbp2 antiserum, but not after anti-rTbp1 antiserum.
Subject(s)
Carrier Proteins/genetics , Genes, Bacterial , Haemophilus influenzae/genetics , Receptors, Transferrin/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Carrier Proteins/chemistry , Carrier Proteins/immunology , Cloning, Molecular , Conserved Sequence , Cross Reactions , Escherichia coli/genetics , Haemophilus Infections/immunology , Haemophilus Infections/metabolism , Haemophilus influenzae/chemistry , Immune Sera/immunology , Immunization, Passive , Iron-Binding Proteins , Molecular Sequence Data , Plasmids , Rats , Receptors, Transferrin/chemistry , Receptors, Transferrin/immunology , Recombinant Proteins/chemistry , Restriction Mapping , Sequence Homology, Amino Acid , Transferrin-Binding ProteinsABSTRACT
The genes (dms) encoding the dimethylsulfoxide reductase protein complex have been cloned and sequenced from Haemophilus influenzae (Hi) type b (Hib) strain Eagan. The Hib dms genes are arranged as an operon whose genomic organization is similar to that of the Escherichia coli (Ec) dmsABC operon. The deduced Hib DmsA, and DmsB and DmsC amino-acid sequences are highly homologous to their Ec counterparts and nearly identical to the recently published sequences of the Hi type-d strain Rd Dms proteins. Hi dimethylsulfoxide reductase appears to be a new member of the superfamily of oxidoreductase enzymes.
Subject(s)
Genes, Bacterial , Haemophilus influenzae/genetics , Iron-Sulfur Proteins , Oxidoreductases/genetics , Base Sequence , Cloning, Molecular , DNA, Bacterial/genetics , Haemophilus influenzae/enzymology , Molecular Sequence DataABSTRACT
Pertactin is a surface adhesin of Bordetella pertussis which is produced in small quantities when expressed from the native prn promoter. Hybrid genes were constructed in which the prn promoter was replaced by either the fha or tox promoter. Recombinant B. pertussis strains containing chromosomally integrated hybrid tox promoter/prn (toxpprn) or fha promoter/prn (fhapprn) genes expressed pertactin at approximately 5- and 8-fold the wild-type level, respectively. The pertactin was correctly processed and secreted and was biochemically and antigenically comparable to its wild-type counterpart, as determined by N-terminal sequence analysis, immunoblotting, peptide mapping, circular dichroism and antigenicity studies. In an adherence assay, a strain over-expressing pertactin was no more adherent than the wild-type strain, but a pertactin-deficient strain was less adherent.
