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. 2007 Sep;75(9):4582-91.
doi: 10.1128/IAI.00324-07. Epub 2007 Jun 18.

Expression and Characterization of an Iron-Regulated Hemin-Binding Protein, HbpA, From Leptospira Interrogans Serovar Lai

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Free PMC article

Expression and Characterization of an Iron-Regulated Hemin-Binding Protein, HbpA, From Leptospira Interrogans Serovar Lai

Swapna Asuthkar et al. Infect Immun. .
Free PMC article

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.

Figures

FIG. 1.
FIG. 1.
Hemin-agarose binding of rHbpA and rHbpA55. The 81-kDa rHbpA and the 55-kDa rHbpA55 bound by hemin-agarose beads are shown as Coomassie blue-stained proteins in panels a and b, respectively. (a) Two independent experiments with hemin-agarose beads, first with incubation with the Ni-NTA column-purified rHbpA (lane 1) and second with incubation with the whole-cell sonicate of the IPTG-induced recombinant clone (lane 2). The 81-kDa rHbpA is indicated by the arrow. (b) Hemin-agarose-bound 55-kDa rHbpA55 from whole-cell sonicate of IPTG-induced recombinant clone (lane 2) and uninduced E. coli sonicate (lane 1).
FIG. 2.
FIG. 2.
Peroxidase activity of the hemin bound by HbpA. A microtiter plate was coated with various protein concentrations of HbpA and incubated with 20 μg of hemin in a total volume of 100 μl in a microtiter plate. After suitable washes, the peroxidase activity of the bound hemin was assayed by the addition of tetramethylbenzidine and reading the absorbance at 450 nm. The inset shows the standard graph of peroxidase activity of hemin.
FIG. 3.
FIG. 3.
Spectrofluorimetric analysis of hemin binding by HbpA. (a) Emission spectrum of HbpA upon excitation with light with a wavelength of 295 nm. Hemin (1.5 mM stock) was added at increasing concentrations and the spectrum recorded after each addition. (b) Scatchard plot for the determination of the association constant for hemin binding by HbpA. The inset shows the decrease of fluorescence intensity as a function of hemin concentration. ΔF represents the difference in the values of the emitted spectra upon addition of various amounts of hemin.
FIG. 4.
FIG. 4.
Expression of HbpA in L. interrogans serovar Lai. (a and b) L. interrogans serovar Lai strain Lai (a) and L. borgpetersenii serovar Tarassovi strain Perepelicin (b). Each panel shows the SDS-PAGE profile and the corresponding immunoblot developed with rabbit anti-HbpA antibodies. Lane M, protein marker; lane 1, high-iron cells maintained at 37°C; lanes 2 and 3, low-iron cells maintained at 30°C and 37°C, respectively (see Materials and Methods for a detailed protocol). The lanes in the corresponding immunoblots are represented as 1′, 2′, and 3′, respectively. (c) Southern blot analysis. Chromosomal DNA was digested with HindIII and probed with the 2,148-bp hbpA PCR-amplified product. Lanes 1 to 6, L. interrogans serovars Lai and Pomona, L. biflexa serovar Patoc, L. borgpetersenii serovar Tarassovi, L. interrogans serovar Autumnalis, and L. kirschneri serovar Grippotyphosa, respectively.
FIG. 5.
FIG. 5.
Agglutination of iron-limited organisms of L. interrogans serovar Lai by anti-HbpA antibodies. (A and B) High- and low-iron organisms, respectively. Live organisms (a), live organisms incubated with preimmune serum (b), and live organisms incubated with anti-HbpA antibodies added at dilutions of 1:50 (c) and 1:100 (d) are shown.
FIG. 6.
FIG. 6.
Demonstration of iron-regulated expression of HbpA in L. interrogans by confocal microscopy. Smears of low-iron (A1 to A3) and high-iron (B1 to B3) L. interrogans organisms were fixed onto slides, anti-HbpA immunoglobulins were added, and the immunoreactivity was detected using FITC-conjugated secondary antibody. The locations of bacteria were identified by using the DNA counterstain propidium iodide. In both panels A and B, panels 1, 2, and 3 refer to FITC, propidium iodide, and composite images, respectively. The constitutive expression of LipL41 in high-iron (C1) and low-iron (C2) cells from the same batch of organisms, detected using FITC-conjugated secondary antibody, is also shown.
FIG. 7.
FIG. 7.
SDS-PAGE and immunoblotting show that the 44-kDa hemin-binding protein is LipL41. (a) SDS-PAGE and immunoblotting analysis of L. interrogans serovar Lai. Lanes developed with Coomassie blue include outer membrane proteins of high-iron (lane 1) and low-iron (lane 2) cells, the 44-kDa protein bound to hemin-agarose preincubated with outer membrane proteins from high-iron cells (lane 3), and molecular weight markers (lane M). Lanes 4 and 5 show the immunoreactivity of anti-LipL41 antibodies with the outer membrane proteins of high-iron cells and hemin-agarose purified 44-kDa protein, respectively. The arrow indicates the 44-kDa band. (b) The 44-kDa proteins from the outer membrane proteins of other serovars. Lanes developed with Coomassie blue include L. borgpetersenii serovar Ballum strain MUS 127 (lane 1), L. biflexa serovar Patoc strain Patoc I (lane 2), L. interrogans serovar Pomona strain Pomona (lane 3), and L. kirschneri serovar Grippotyphosa strain Moskva V (lane 4). Lanes 5 and 6 show the immunoreactivity of anti LipL41 with the 44-kDa proteins from L. interrogans serovar Lai and L. biflexa serovar Patoc, respectively. The arrow indicates the 44-kDa band.
FIG. 8.
FIG. 8.
Phylogenetic analysis of HbpA and other bacterial virulence proteins. The phylogenies generated by neighborhood joining with 400 bootstrap replicates, rooted at midpoint and bootstrap values, are shown as percentages. The numbers refer to the divergence between the sequences.

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