Hemin-binding surface protein from Bartonella quintana

Bartonella quintana, the agent of trench fever and a cause of endocarditis and bacillary angiomatosis in humans, has the highest reported in vitro hem in requirement for any bacterium. We determined that eight membrane-associa ted proteins from B. quintana bind hemin and that a similar to 25-kDa prote in (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 s hift from approximately 25 to 30 kDa when solubilized at 100 degreesC. Immu noblots 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 w as 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 i mmature 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. B LAST searches indicate that the closest homologs to HbpA include the Barton ella henselae phage associated membrane protein, Pap31 (58.4% identity), an d the OMP31 porin from Brucella melitensis (31.7% identity). High-stringenc y Southern blots indicate that all five pathogenic Bartonella spp. possess hbpA homologs. Recombinant HbpA can bind hemin in vitro; however, it does n ot confer a hemin-binding phenotype upon E. coli. Intact B. quintana treate d 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. qui ntana.