Expression and Characterization of an Iron-Regulated Hemin-Binding Protein, HbpA, fromLeptospira interrogansSerovar Lai

Abstract

In an earlier study, based on the ferric enterobactin receptor FepA of Escherichia coli, we identified and modeled a TonB-dependent outer membrane receptor protein (LB191) from the genome of Leptospira interrogans serovar Lai. Based on in silico analysis, we hypothesized that this protein was an iron-dependent hemin-binding protein. In this study, we provide experimental evidence to prove that this protein, termed HbpA (hemin-binding protein A), is indeed an iron-regulated hemin-binding protein. We cloned and expressed the full-length 81-kDa recombinant rHbpA protein and a truncated 55-kDa protein from L. interrogans serovar Lai, both of which bind hemin-agarose. Assay of hemin-associated peroxidase activity and spectrofluorimetric analysis provided confirmatory evidence of hemin binding by HbpA. Immunofluorescence studies by confocal microscopy and the microscopic agglutination test demonstrated the surface localization and the iron-regulated expression of HbpA in L. interrogans. Southern blot analysis confirmed our earlier observation that the hbpA gene was present only in some of the pathogenic serovars and was absent in Leptospira biflexa. Hemin-agarose affinity studies showed another hemin-binding protein with a molecular mass of approximately 44 kDa, whose expression was independent of iron levels. This protein was seen in several serovars, including nonpathogenic L. biflexa. Sequence analysis and immunoreactivity with specific antibodies showed this protein to be LipL41.

Serological diagnosis of leptospiral uveitis by HbpA IgG ELISA

“…HbpA was first reported as an iron-regulated, haeminbinding protein in serovar Lai (Sritharan et al, 2005;Asuthkar et al, 2007). Its absence from non-pathogenic Leptospira species and the in vivo expression of the protein, coupled to its usefulness in screening of clinical isolates by PCR (Sridhar et al, 2008) and ELISA (Sivakolundu et al, 2011), led us to extend its usefulness to the diagnosis of leptospiral uveitis.…”

“…Its absence from non-pathogenic Leptospira species and the in vivo expression of the protein, coupled to its usefulness in screening of clinical isolates by PCR (Sridhar et al, 2008) and ELISA (Sivakolundu et al, 2011), led us to extend its usefulness to the diagnosis of leptospiral uveitis. Recombinant HbpA was prepared as described previously (Asuthkar et al, 2007) and we showed that there are no epitopes in the protein that are shared with other bacterial proteins. There was low crossreactivity with other TonB-dependent proteins within the same organism, as reflected by the low reactivity of the leptospiral extract of L. interrogans serovar Manilae grown under high-iron conditions; a threefold increase was seen in extracts of low-iron-grown organisms as a result of increased expression of HbpA.…”

“…The alkaline phosphatase substrate p-nitrophenyl phosphate (100 ml; BioRad) was added and incubated for 15 min and the absorbance was read at 405 nm in an ELISA reader (model 680XR; Bio-Rad). Purified rHbpA (100 ng) and whole-cell sonicates of Leptospira interrogans serovar Manilae, grown under high-and low-iron conditions (Asuthkar et al, 2007), were included as controls. The experiment was repeated three times with each reaction set up in duplicate.…”

“…Recombinant HbpA (rHbpA) and anti-HbpA antibodies were prepared as reported previously (Asuthkar et al, 2007). The polyclonal antibodies were tested for cross-reactivity against whole-cell sonicates of Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris, Klebsiella pneumoniae (collection of the L. V. Prasad Eye Institute, Hyderabad, India) and Mycobacterium tuberculosis ATCC 27294.…”

“…In a previous study, we identified a haemin-binding, TonBdependent outer-membrane protein, HbpA (Sritharan et al, 2005;Asuthkar et al, 2007), in pathogenic Leptospira species. HbpA is an 81 kDa protein expressed upon limitation of iron.…”