Characterization of hgpA, a gene encoding a haemoglobin/haemoglobin-haptoglobin-binding protein of Haemophilus influenzae

Department of Pediatrics, CHO 2308, 940 Northeast 13th Street, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA Department of Microbiology/Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA Author for correspondence: Terrence L. Stull. Tel: +1 405 271 4401. Fax: +1 405 271 8710. e-mail: Terrence-Stull@ouhsc.edu ABSTRACT Summary: Haemophilus influenzae binds haemoglobin and the haemoglobin-haptoglobin complex and utilizes either as a sole source of haem. Previously, a DNA fragment was cloned from H. influenzae that encodes an approximately 120 kDa protein (HgpA) expressing haemoglobin-binding activity in Escherichia coli. Partial sequence analysis revealed significant homology of HgpA with other bacterial haem- and iron-utilization proteins, and a length of CCAA repeating units immediately following the nucleotide sequence encoding the putative leader peptide. In the present study, the complete nucleotide sequence of the cloned DNA fragment was determined and the sequence was analysed. In addition to homology with other haem- and iron-utilization proteins, seven regions typical of TonB-dependent proteins were identified. The transcript of hgpA was determined to be monocistronic by RT-PCR. PCR performed with different colonies of a single H. influenzae strain at one CCAA-repeat-containing locus indicated varying lengths of CCAA repeats, suggesting that haemoglobin and haemoglobin-haptoglobin binding in H. influenzae is regulated by strand slippage across CCAA repeats, as well as by haem repression. E. coli containing cloned hgpA bound both haemoglobin and the haemoglobin-haptoglobin complex. A deletion/insertion mutation of hgpA was constructed in H. influenzae strain HI689. Mutation of hgpA did not affect the ability of H. influenzae either to bind or to utilize haemoglobin or haemoglobin-haptoglobin following growth in haem-deplete media. Affinity purification of haemoglobin-binding proteins from the mutant strain revealed loss of the 120 kDa protein and an increased amount of a 115 kDa protein, suggesting that at least one additional haemoglobin-binding protein exists. Keywords: Haemophilus influenzae , haemoglobin, haemoglobin-binding protein, haem acquisition, outer-membrane protein