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.