Cloning and expression of the Moraxella catarrhalis lactoferrin receptor genes

Abstract

The lactoferrin receptor genes from two strains of Moraxella catarrhalis have been cloned and sequenced. The lfr genes are arranged as lbpB followed by lbpA, a gene arrangement found in lactoferrin and transferrin receptor operons from several bacterial species. In addition, a third open reading frame, orf3, is located one nucleotide downstream of lbpA. The deduced lactoferrin binding protein A (LbpA) sequences from the two strains were found to be 99% identical, the LbpB sequences were 92% identical, and the ORF3 proteins were 98% identical. The lbpB gene was PCR amplified and sequenced from a third strain of M. catarrhalis, and the encoded protein was found to be 77% identical and 84% similar to the other LbpB proteins. Recombinant LbpA and LbpB proteins were expressed from Escherichia coli, and antisera raised to the purified proteins were used to assess antigenic conservation in a panel of M. catarrhalis strains. The recombinant proteins were tested for the ability to bind human lactoferrin following gel electrophoresis and electroblotting, and rLbpB, but not rLbpA, was found to bind lactoferrin. Bactericidal antibody activity was measured, and while the anti-rLbpA antiserum was not bactericidal, the anti-rLbpB antisera were found to be weakly bactericidal. Thus, LbpB may have potential as a vaccine candidate.

FIG. 1

Partial restriction map of the M. catarrhalis lactoferrin receptor locus. Restriction enzyme sites: Bg, BglI; Bs, BstEII; H, HindIII; R, EcoRI; S, SphI.

FIG. 2

Nucleotide sequences in the potential promoter regions of the strain Q8 lfr locus. (A) Potential promoter elements for the lbpB, lbpA, and orf3 genes. Potential −35, −10, and RBS sequences are underlined. The stop codon for lbpA is indicated by an asterisk. The italicized sequence in the promoter region of Q8 lbpB designates the 11 nucleotides missing from the 4223 lbpB sequence. (B) Comparison of Fur binding sequences in E. coli (14), N. meningitidis tbpB (19), H. influenzae tbpB (12), and M. catarrhalis lbpB. Dots indicate nucleotides identical to the E. coli consensus sequence. Underlining indicates dyad symmetry. The italicized sequence in the Fur binding region of Q8 lbpB is the 11 nucleotides missing from the 4223 lbpB sequence.

FIG. 3

Alignment of the amino acid sequences of LbpA from M. catarrhalis Q8 and 4223, N. meningitidis BNCV and H44/76 (27, 28), and N. gonorrhoeae FA19 (1). Dots indicate identical residues, and gaps have been introduced to maximize sequence alignments. The internal cyanogen bromide fragment used to design the oligonucleotide probe for cloning of the lbpA gene is underlined. The locations of the homologous peptide sequences obtained from M. catarrhalis 141 LbpA (4) are italicized and underlined.

FIG. 4

Alignment of the amino acid sequences of LbpB from M. catarrhalis Q8, 4223, and VH19 and the partial carboxyl-terminal sequences of LbpB from N. meningitidis BNCV and H44/76 (27, 28) and N. gonorrhoeae FA19 (translated from Biswas and Sparling [1]). Dots indicate identical residues, and gaps have been introduced to maximize sequence alignments. The residues conserved with TbpB proteins (26) are underlined, and the RGD sequence is italicized.

FIG. 5

Alignment of the amino acid sequences of the ORF3 protein from M. catarrhalis Q8 and 4223. Dots indicate identical residues, and gaps have been introduced to achieve maximum sequence alignment. The DGLG repeat sequence is underlined.

FIG. 6

Purification of rLbpA and rLbpB proteins. (A) SDS-PAGE of the purification of Q8 rLbpA. Panels B and C show the purification of Q8 rLbpB and 4223 rLbpB, respectively. Lane 1, molecular weight markers; lane 2, whole-cell lysates; lane 3, inclusion bodies; lane 4, purified protein.

FIG. 7

Lactoferrin binding of recombinant proteins. (A) SDS-PAGE of purified recombinant proteins. (B) Binding of recombinant proteins to human lactoferrin. (C) Binding of recombinant proteins to human transferrin. Lane 1, molecular weight markers; lane 2, Q8 rLbpA; lane 3, Q8 rLbpB; lane 4, 4223 rLbpB; lane 5, 4223 rTbpB.

FIG. 8

Immunoblot of M. catarrhalis strains reacted with anti-rLbpA and anti-LbpB antibodies. (A) Whole-cell lysates probed with anti-Q8 rLbpA plus anti-Q8 rLbpB antisera. All cells were grown in the presence of EDDA. (B) Whole-cell lysates probed with anti-Q8 rLbpA antibody. (C) Whole-cell lysates probed with anti-Q8 rLbpB antibody. Lane 1, strain Q8; lane 2, strain 4223; lane 3, strain VH19; lane 4, strain LES-1; lane 5, strain H-04; lane 6, strain 3. +, with EDDA; −, without EDDA.