Hemin-binding surface protein from Bartonella quintana

Abstract

Bartonella quintana, the agent of trench fever and a cause of endocarditis and bacillary angiomatosis in humans, has the highest reported in vitro hemin requirement for any bacterium. We determined that eight membrane-associated proteins from B. quintana bind hemin and that a approximately 25-kDa protein (HbpA) was the dominant hemin-binding protein. Like many outer membrane proteins, HbpA partitions to the detergent phase of a Triton X-114 extract of the cell and is heat modifiable, displaying an apparent molecular mass shift from approximately 25 to 30 kDa when solubilized at 100 degrees C. Immunoblots of purified outer and inner membranes and immunoelectron microscopy with whole cells show that HbpA is strictly located in the outer membrane and surface exposed, respectively. The N-terminal sequence of mature HbpA was determined and used to clone the HbpA-encoding gene (hbpA) from a lambda genomic library. The hbpA gene is 816 bp in length, encoding a predicted immature protein of approximately 29.3 kDa and a mature protein of 27.1 kDa. A Fur box homolog with 53% identity to the Escherichia coli Fur consensus is located upstream of hbpA and may be involved in regulating expression. BLAST searches indicate that the closest homologs to HbpA include the Bartonella henselae phage-associated membrane protein, Pap31 (58.4% identity), and the OMP31 porin from Brucella melitensis (31.7% identity). High-stringency Southern blots indicate that all five pathogenic Bartonella spp. possess hbpA homologs. Recombinant HbpA can bind hemin in vitro; however, it does not confer a hemin-binding phenotype upon E. coli. Intact B. quintana treated with purified anti-HbpA Fab fragments show a significant (P < 0.004) dose-dependent decrease in hemin binding relative to controls, suggesting that HbpA plays an active role in hemin acquisition and therefore pathogenesis. HbpA is the first potential virulence determinant characterized from B. quintana.

FIG. 1

Identification of hemin-binding proteins of B. quintana using hemin blots. (A) Total cell lysates. (B) Total membranes. Samples were solubilized in LSB at 24 or 100°C as indicated and then separated by SDS-PAGE, transferred to nitrocellulose, probed with hemin, and developed using ECL reagents (Amersham Pharmacia) as described in Materials and Methods. The position of the two forms of HbpA are indicated by asterisks. Molecular mass standards are given to the left in kilodaltons.

FIG. 2

Monospecificity of the anti-HbpA antibody and reactivity against both molecular mass forms of HbpA. (A) Coomassie blue-stained SDS-PAGE gel containing a B. quintana cell lysate (lane 1) and a phase-separated Triton X-114 cell extract of the bacterium (lane 2). Both samples were solubilized at 100°C in LSB. HbpA is arrowed. (B) Immunoblot corresponding to panel A but developed with rabbit anti-HbpA antiserum showing monospecificity of the anti-HbpA antibody. (C) Immunoblot showing anti-HbpA antibody reactivity to phase-separated Triton X-114 cell extracts of B. quintana solubilized at 25°C (lane 1) and 100°C (lane 2). Note that the antibody recognizes both molecular mass forms of HbpA in panel C. Molecular mass standards are given to the left in kilodaltons.

FIG. 3

Localization of HbpA in the outer membrane of B. quintana. Lanes: 1, cell lysate; 2, purified inner membrane; 3, outer membrane. (A) Coomassie blue-stained SDS-PAGE gel. (B) Corresponding immunoblot developed with anti-HbpA showing HbpA in the outer membrane (arrowed). All samples were solubilized at 100°C in LSB. Molecular mass standards are given to the left in kilodaltons.

FIG. 4

Immunoelectron microscopy showing surface localization of HbpA. Immunogold analysis and negative staining were done as previously described (50), using anti-HbpA and intact bacteria. Bar, 0.5 μm.

FIG. 5

Anti-HbpA Fab fragments inhibit hemin binding by B. quintana. The percent hemin uptake as a function of treatment volumes is shown. B. quintana was pretreated with 0, 40, or 80 μl of PBS or anti-HbpA Fab (0.2 and 0.4 mg/ml, respectively) and then assayed by a standard liquid hemin-binding assay. The values are the average of three determinations ± the SEM.

FIG. 6

Sequence of hbpA, its encoded protein, and identification of a potential Fur box in the promoter region. Nucleotides within the hbpA ORF are given in uppercase letters. The promoter, as determined by prokaryotic promoter prediction by neural network, is shown in boldface lowercase letters, with a potential transcriptional start site (TSS) indicated by a boldface uppercase “G.” The fur box homolog within the promoter region is boxed. A potential ribosome-binding site (RBS) is indicated above the sequence. The deduced amino acid sequence of HbpA is shown below the second base of its respective triplet codon. Amino acids constituting the secretory signal sequence are shown in boldface. The N-terminal sequence experimentally determined from mature HbpA is boxed. The stop codon is denoted by an asterisk. Inverted repeats constituting a potential ρ-independent terminator of transcription are indicated by opposing arrows.

FIG. 7

Multiple sequence alignment of B. quintana HbpA with B. henselae Pap31 and B. melitensis Omp31. Identical amino acid residues are noted in black, conserved residues in gray and introduced gaps by hyphens. The GenBank accession numbers for the Pap31 and Omp31 homologs are AF001274 and U39453, respectively.

FIG. 8

Expression of hbpA in E. coli and ability of recombinant HbpA to bind hemin in vitro. (A) Immunoblot developed with anti-HbpA antibody, showing lysates of E. coli XLOLR containing no plasmid (lane 1), pBK-CMV cloning vector (lane 2), or pHBP-CMV (lane 3). (B) Blots of an E. coli XLOLR(pHBP-CMV) lysate probed with hemin showing rHbpA binding hemin (indicated by an asterisk) (lane 1). The corresponding immunoblot developed with anti-HbpA antiserum and ECL reagents (Amersham), identifying the recombinant HbpA (arrowed) (lane 2), is also shown. All samples were solubilized at 100°C in LSB. Molecular mass standards are given to the left in kilodaltons.