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. 1998 Sep;66(9):4183-92.
doi: 10.1128/IAI.66.9.4183-4192.1998.

The transferrin binding protein B of Moraxella catarrhalis elicits bactericidal antibodies and is a potential vaccine antigen

L E Myers  1 , Y P Yang, R P Du, Q Wang, R E Harkness, A B Schryvers, M H Klein, S M Loosmore
Affiliations

The transferrin binding protein B of Moraxella catarrhalis elicits bactericidal antibodies and is a potential vaccine antigen

L E Myers et al. Infect Immun. 1998 Sep.

Abstract

The transferrin binding protein genes (tbpA and tbpB) from two strains of Moraxella catarrhalis have been cloned and sequenced. The genomic organization of the M. catarrhalis transferrin binding protein genes is unique among known bacteria in that tbpA precedes tbpB and there is a third gene located between them. The deduced sequences of the M. catarrhalis TbpA proteins from two strains were 98% identical, while those of the TbpB proteins from the same strains were 63% identical and 70% similar. The third gene, tentatively called orf3, encodes a protein of approximately 58 kDa that is 98% identical between the two strains. The tbpB genes from four additional strains of M. catarrhalis were cloned and sequenced, and two potential families of TbpB proteins were identified based on sequence similarities. Recombinant TbpA (rTbpA), rTbpB, and rORF3 proteins were expressed in Escherichia coli and purified. rTbpB was shown to retain its ability to bind human transferrin after transfer to a membrane, but neither rTbpA nor rORF3 did. Monospecific anti-rTbpA and anti-rTbpB antibodies were generated and used for immunoblot analysis, which demonstrated that epitopes of M. catarrhalis TbpA and TbpB were antigenically conserved and that there was constitutive expression of the tbp genes. In the absence of an appropriate animal model, anti-rTbpA and anti-rTbpB antibodies were tested for their bactericidal activities. The anti-rTbpA antiserum was not bactericidal, but anti-rTbpB antisera were found to kill heterologous strains within the same family. Thus, if bactericidal ability is clinically relevant, a vaccine comprising multiple rTbpB antigens may protect against M. catarrhalis disease.

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Figures

FIG. 1
FIG. 1
M. catarrhalis tbp locus and subclones of the 4223 (A) and Q8 (B) genomic clones. Restriction enzymes: Av, AvaII; Ar, AvrI; B, BglI; C; ClaI; H, HindIII; N, NheI; S, SalI.
FIG. 2
FIG. 2
Nucleotide sequences of the promoter regions of M. catarrhalis 4223 tbpA, orf3, and tbpB genes. (A) Putative promoter elements for tbpA, orf3, and tbpB. RBS, ribosome-binding site. (B) Comparison of Fur-binding sites from E. coli (13), N. meningitidis tbpB (20), H. influenzae tbpB (11), and M. catarrhalis 4223 tbpB. Dots indicate residues identical to the E. coli consensus sequence, and underlining indicates dyad symmetry.
FIG. 3
FIG. 3
Alignment of the deduced TbpA protein sequences for M. catarrhalis 4223 and Q8, N. meningitidis B16B6 and M982 (20), N. gonorrhoeae FA19 (6), and H. influenzae type b Eagan (22). Dots indicate identical residues, and dashes have been inserted to maximize sequence alignment.
FIG. 4
FIG. 4
Alignment of the deduced TbpB sequences from M. catarrhalis 4223, Q8, 3, LES-1, M35, and R1. Dots indicate identical residues, and dashes have been inserted for maximum alignment of the sequences. Residues conserved among TbpB proteins from A. pleuropneumoniae, H. influenzae, M. catarrhalis, N. gonorrhoeae, N. meningitidis, and P. haemolytica are underlined.
FIG. 5
FIG. 5
Purification of the recombinant TbpA and TbpB proteins (A) and transferrin binding of rTbpB proteins (B). Lane 1, E. coli cells expressing 4223 rTbpA; lane 2, purified rTbpA; lane 3, E. coli cells expressing 4223 rTbpB; lane 4, purified 4223 rTbpB; E. coli cells expressing Q8 rTbpB; lane 5, purified Q8 rTbpB. Proteins were transferred to a PVDF membrane and incubated with human transferrin conjugated to HRP.
FIG. 6
FIG. 6
Antigenic conservation of TbpA and TbpB in M. catarrhalis strains. Bacteria were grown in BHI medium with (+) or without (−) EDDA, and whole-cell lysates were probed with anti-4223 rTbpA or anti-4223 rTbpB antibody. (A) Immunoblot with anti-4223 rTbpA; (B) immunoblot with anti-4223 rTbpB. Lanes represent the following strains: 1, 4223; 2, VH-9; 3, M35; 4, H-04; 5, ATCC 25240; 6, R1; 7, Q8; 8, LES-1; and 9, 3.
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