The e (P4) outer membrane protein of Haemophilus influenzae: biologic activity of anti-e serum and cloning and sequencing of the structural gene

Abstract

Outer membrane proteins of nontypeable (NT) Haemophilus influenzae are among the major candidates for inclusion in vaccines against these organisms. This article reports the purification of the e (P4) lipoprotein of H. influenzae and the subsequent production of antiserum directed against this protein. The anti-e polyclonal serum cross-reacted with e protein in multiple clinical NT H. influenzae isolates. Monoclonal antibody analysis of e protein showed at least one surface-exposed epitope to be conserved among NT H. influenzae strains. Anti-e serum also had bactericidal activity against multiple clinical isolates of NT H. influenzae. These results are in contrast to previous reports in the literature that purified P4 protein did not elicit biologically active antibodies. Anti-e antibodies exhibited synergistic bactericidal activity directed against NT H. influenzae when mixed with antibodies directed against another Haemophilus lipoprotein, PCP. This bactericidal synergy was observed against a variety of NT clinical isolates. We also report the cloning of the Haemophilus e lipoprotein, or hel, gene encoding the e protein and its expression and processing in Escherichia coli. The nucleotide sequence of the gene and deduced amino acid sequence of the protein are given. These results demonstrate that e protein is a viable candidate to be a component of a vaccine against NT H. influenzae. * Corresponding author. tective against Hib meningitis. To further investigate this protein and to determine the degree of antigenic conservation among NT H. influenzae and the biologic activity of anti-e serum against NT H. influenzae, we purified the e protein and produced an anti-e polyclonal antiserum. This article reports that the purified protein elicited biologically active antibodies against NT H. influenzae and that anti-e OMP serum showed synergistic BC activity when mixed with antiserum against the recombinant PCP protein. We also report the cloning and sequencing of the gene encoding this protein and its expression in Escherichia coli as a lipoprotein. These results show that the e protein is a vaccine candidate against NT H. influenzae. MATERIALS AND METHODS Bacteria. Clinical isolates of NT H. influenzae P860295,

Lipoprotein e(P4) is essential for hemin uptake by Haemophilus influenzae.

“…The following specific primers were used for the amplification of the hel gene DNA fragment: AR15 (26' mer): 5'-ATTGGATCCGAAT-TCTTAAAAGGAAT-3'; and AR16 (30' mer): 5'-ATTAA-ATATTGGATCCAGTAAAAACTGAGC-3'. These oligonucleotides were designed to anneal to the flanking DNA sequences of the hel gene at bp position 1-18 for AR15, and 1032-1047 for AR16, according to the DNA sequence published by Green et al (17). BamHI restriction sites were designed into the 5' ends of primers AR15 and AR16.…”

Environmental and genetic regulation of the phosphorylcholine epitope of Haemophilus influenzae lipooligosaccharide

“…We postulated that if the lower abundance of lic1 mRNA in aerobic cells accounts for the aerobic decrease in PC epitope levels, then generating elevated amounts of lic1 mRNA aerobically will cause elevated aerobic PC epitope levels. We engineered a strain Rhel-licA, in which the native licA gene is under the transcriptional control of a strong promoter from the hel gene (The Institute for Genomic Research, TIGR locus HI0693) encoding an outer-membrane lipoprotein involved in NAD and NMN uptake (Green et al, 1991;Reilly et al, 1999;Kemmer et al, 2001), which we had found by Northern analysis to be highly expressed (data not shown). Rhel-licA contains the hel promoter immediately 5¢ of three in-frame potential licA initiation codons and an in-frame deletion of the CAAT repeat units (Experimental procedures).…”

Mutation of the Conserved Cys165 Outside of the CuA Domain Destabilizes Nitrous Oxide Reductase but Maintains Its Catalytic Activity

“…NosL has a presequence of 24 amino acids that features the cleavage site, -3 LTGTCGE -1-3, of a lipoprotein [45]. The signal sequence shows similarity to that of an outer membrane lipoprotein from Huernophilus influenzae [46]. Acylation of the terminal cysteine residue might anchor NosL in the outer membrane.…”