Molecular cloning, expression, and sequence of the pilin gene from nontypeable Haemophilus influenzae M37

Abstract

Nontypeable Haemophilus influenzae M37 adheres to human buccal epithelial cells and exhibits mannose-resistant hemagglutination of human erythrocytes. An isogenic variant of this strain which was deficient in hemagglutination was isolated. A protein with an apparent molecular weight of 22,000 was present in the sodium dodecyl sulfate-polyacrylamide gel profile of sarcosyl-insoluble proteins from the hemagglutination-proficient strain but was absent from the profile of the isogenic hemagglutination-deficient variant. A monoclonal antibody which reacts with the hemagglutination-proficient isolate but not with the hemagglutination-deficient isolate has been characterized. This monoclonal antibody was employed in an affinity column for purification of the protein as well as to screen a genomic library for recombinant clones expressing the gene. Several clones which contained overlapping genomic fragments were identified by reaction with the monoclonal antibody. The gene for the 22-kDa protein was subcloned and sequenced. The gene for the type b pilin from H. influenzae type b strain MinnA was also cloned and sequenced. The DNA sequence of the strain MinnA gene was identical to that reported previously for two other type b strains. The DNA sequence of the strain M37 gene is 77% identical to that of the type b pilin gene, and the derived amino acid sequence is 68% identical to that of the type b pilin.

Comparison of hemagglutinating pili of Haemophilus influenzae type b with similar structures of nontypeable H. influenzae

“…Analysis of 20 N-terminal amino acids of pilin from a Brazilian purpuric fever strain of H. influenzae (NT) showed nearly complete homology to the N terminus of pilin from Hib strain M43 (26). Recently, Coleman et al have cloned and sequenced the entire pilin gene from NT strain M37 (5), and this gene showed 77% DNA homology and 68% derived amino acid sequence homology with the pilin gene of their Hib strain Minn A, whose pilin gene is identical to that of strain M43. By Southern analysis, Forney et al (9) showed that the DNA from two NT strains, M37 and 1128, hybridized to an intragenic probe from the pilin gene of Hib strain Eagan.…”

“…After 1 h of incubation, the wells were again washed with PBS-T. Goat anti-rabbit immunoglobulin (100 ,ul) labeled with horseradish peroxidase (Cappel Laboratories, Malvern, Pa.) diluted 1:4,000 in PBS-T was added, and the plates were incubated for 1 h. Following washing with PBS-T, 100 5.01] with 10 ,ul of 30% H202) was added to each well. The reaction was allowed to proceed for 15 min in the dark and was then terminated by adding 50 ,I of 4 N H2SO4.…”

Isolation, expression, and nucleotide sequencing of the pilin structural gene of the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius

“…E. coli XL-1 Blue and the plasmid pKSwere obtained from Stratagene. Plasmid pT7-7 and phage mGP1-2 were provided by S. Tabor MinnA (the sequences of the pilin genes from these three strains are identical) (6,9,26). The sequence of the 5' oligonucleotide is 5'AACGAATTCTGCTGTITTATTAAG GCGTITTAG and contains an EcoRI site to facilitate subcloning.…”

“…The nucleotide sequences have been determined for the structural pilin genes from four different H. influenzae type b strains (M43p+ [= A02], 770235f+b°, MinnA, and Eagan) (6,8,9,13,26) and one nontypeable strain (M37) (6). The M43p+, 770235f+b°, and MinnA genes have the identical sequence, while the Eagan and M37 gene sequences differ somewhat.…”

“…sponding to these regions elicit an native pili from the divergent BPF cl Southern blotting results reporte indicate that type b strains contain a structural gene (8,9,13). The obser strain M37 are ambiguous, with on dence for a single copy (8) and a secc possibility of multiple copies (6). I found that two distinct EcoRI fragi DNA from BPF clone strains hybri pilin gene (22).…”