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The surface protein Shr of Streptococcus pyogenes binds heme and transfers it to the streptococcal heme-binding protein Shp.

Zhu H, Liu M, Lei B - BMC Microbiol. (2008)

Bottom Line: These results suggest that Shr directly transfers its heme to Shp.In addition, the rates of heme transfer from human hemoglobin to apoShp are close to those of simple ferric heme dissociation from hemoglobin, suggesting that methemoglobin does not directly transfer its heme to apoShp.These results suggest the possibility that Shr is a source of heme for Shp and that the Shr-to-Shp heme transfer is a step of the heme acquisition process in S. pyogenes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717, USA. dzhuhui@yahoo.com.cn

ABSTRACT

Background: The heme acquisition machinery in Streptococcus pyogenes is believed to consist of the surface proteins, Shr and Shp, and heme-specific ATP-binding cassette transporter HtsABC. Shp has been shown to rapidly transfer its heme to the lipoprotein component, HtsA, of HtsABC. The function of Shr and the heme source of Shp have not been established.

Results: The objective of this study was to determine whether Shr binds heme and is a heme source of Shp. To achieve the objective, recombinant Shr protein was prepared. The purified Shr displays a spectrum typical of hemoproteins, indicating that Shr binds heme and acquires heme from Escherichia coli hemoproteins in vivo. Spectral analysis of Shr and Shp isolated from a mixture of Shr and heme-free Shp (apoShp) indicates that Shr and apoShp lost and gained heme, respectively; whereas Shr did not efficiently lose its heme in incubation with apoHtsA under the identical conditions. These results suggest that Shr directly transfers its heme to Shp. In addition, the rates of heme transfer from human hemoglobin to apoShp are close to those of simple ferric heme dissociation from hemoglobin, suggesting that methemoglobin does not directly transfer its heme to apoShp.

Conclusion: We have demonstrated that recombinant Shr can acquire heme from E. coli hemoproteins in vivo and appears to directly transfer its heme to Shp and that Shp appears not to directly acquire heme from human methemoglobin. These results suggest the possibility that Shr is a source of heme for Shp and that the Shr-to-Shp heme transfer is a step of the heme acquisition process in S. pyogenes. Further characterization of the Shr/Shp/HtsA system would advance our understanding of the mechanism of heme acquisition in S. pyogenes.

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Western blots showing the presence of Shr-specific antibodies in convalescent sera from patients of streptococcal pharyngitis. Purified Shr (50 ng/lane) was resolved by SDS PAGE and analyzed by Western blotting using convalescent sera from three pharyngitis patients (lanes 1–3) and a person without S. pyogenes exposure as negative control (lane 4).
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Figure 3: Western blots showing the presence of Shr-specific antibodies in convalescent sera from patients of streptococcal pharyngitis. Purified Shr (50 ng/lane) was resolved by SDS PAGE and analyzed by Western blotting using convalescent sera from three pharyngitis patients (lanes 1–3) and a person without S. pyogenes exposure as negative control (lane 4).

Mentions: Since Shp appears not to directly acquire heme from methemoglobin, the heme source of Shp may be a S. pyogenes protein. We speculated that this protein was Shr. If this is true, Shr should bind heme and transfer it to Shp. To test this idea, we first prepared recombinant Shr protein. In comparison with Escherichia coli containing the empty vector, E. coli containing plasmid pET-His2-shr with the cloned shr gene displayed an extra band in SDS PAGE, which had approximately the expected molecular weight of Shr (theoretical molecular weight = 139,406) (Fig. 2). The band was obvious but not dominant, indicating moderate expression of Shr. The deduced amino acid sequence of the cloned shr gene has a 6xHis tag and predicted net charge of +7.6 at pH 7.0. As expected, the expressed protein bound to a cation exchange column of SP Sepharose at pH 8.0 and Ni+-NTA column. Shr obtained had a purity of 80% according to the densities of the bands in SDS-PAGE analysis (lane 4 in Fig. 2). To confirm that the protein purified was not an E. coli protein, the proteins of E. coli/pET-His2 (vector control) obtained using SP Sepharose chromatography, the first step in the Shr purification, was analyzed by SDS PAGE, and no protein with the size of Shr was detected (lane 5 in Fig. 2). The purified protein sample was also probed with convalescent sera from 3 patients of streptococcal pharyngitis and an individual without S. pyogenes exposure by Western blotting analysis. All three patients, but not the control individual, had antibodies specific for the purified protein (Fig. 3), further supporting that the purified protein was Shr. Thus, recombinant Shr can be expressed at moderate levels in E. coli.


The surface protein Shr of Streptococcus pyogenes binds heme and transfers it to the streptococcal heme-binding protein Shp.

Zhu H, Liu M, Lei B - BMC Microbiol. (2008)

Western blots showing the presence of Shr-specific antibodies in convalescent sera from patients of streptococcal pharyngitis. Purified Shr (50 ng/lane) was resolved by SDS PAGE and analyzed by Western blotting using convalescent sera from three pharyngitis patients (lanes 1–3) and a person without S. pyogenes exposure as negative control (lane 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2266757&req=5

Figure 3: Western blots showing the presence of Shr-specific antibodies in convalescent sera from patients of streptococcal pharyngitis. Purified Shr (50 ng/lane) was resolved by SDS PAGE and analyzed by Western blotting using convalescent sera from three pharyngitis patients (lanes 1–3) and a person without S. pyogenes exposure as negative control (lane 4).
Mentions: Since Shp appears not to directly acquire heme from methemoglobin, the heme source of Shp may be a S. pyogenes protein. We speculated that this protein was Shr. If this is true, Shr should bind heme and transfer it to Shp. To test this idea, we first prepared recombinant Shr protein. In comparison with Escherichia coli containing the empty vector, E. coli containing plasmid pET-His2-shr with the cloned shr gene displayed an extra band in SDS PAGE, which had approximately the expected molecular weight of Shr (theoretical molecular weight = 139,406) (Fig. 2). The band was obvious but not dominant, indicating moderate expression of Shr. The deduced amino acid sequence of the cloned shr gene has a 6xHis tag and predicted net charge of +7.6 at pH 7.0. As expected, the expressed protein bound to a cation exchange column of SP Sepharose at pH 8.0 and Ni+-NTA column. Shr obtained had a purity of 80% according to the densities of the bands in SDS-PAGE analysis (lane 4 in Fig. 2). To confirm that the protein purified was not an E. coli protein, the proteins of E. coli/pET-His2 (vector control) obtained using SP Sepharose chromatography, the first step in the Shr purification, was analyzed by SDS PAGE, and no protein with the size of Shr was detected (lane 5 in Fig. 2). The purified protein sample was also probed with convalescent sera from 3 patients of streptococcal pharyngitis and an individual without S. pyogenes exposure by Western blotting analysis. All three patients, but not the control individual, had antibodies specific for the purified protein (Fig. 3), further supporting that the purified protein was Shr. Thus, recombinant Shr can be expressed at moderate levels in E. coli.

Bottom Line: These results suggest that Shr directly transfers its heme to Shp.In addition, the rates of heme transfer from human hemoglobin to apoShp are close to those of simple ferric heme dissociation from hemoglobin, suggesting that methemoglobin does not directly transfer its heme to apoShp.These results suggest the possibility that Shr is a source of heme for Shp and that the Shr-to-Shp heme transfer is a step of the heme acquisition process in S. pyogenes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717, USA. dzhuhui@yahoo.com.cn

ABSTRACT

Background: The heme acquisition machinery in Streptococcus pyogenes is believed to consist of the surface proteins, Shr and Shp, and heme-specific ATP-binding cassette transporter HtsABC. Shp has been shown to rapidly transfer its heme to the lipoprotein component, HtsA, of HtsABC. The function of Shr and the heme source of Shp have not been established.

Results: The objective of this study was to determine whether Shr binds heme and is a heme source of Shp. To achieve the objective, recombinant Shr protein was prepared. The purified Shr displays a spectrum typical of hemoproteins, indicating that Shr binds heme and acquires heme from Escherichia coli hemoproteins in vivo. Spectral analysis of Shr and Shp isolated from a mixture of Shr and heme-free Shp (apoShp) indicates that Shr and apoShp lost and gained heme, respectively; whereas Shr did not efficiently lose its heme in incubation with apoHtsA under the identical conditions. These results suggest that Shr directly transfers its heme to Shp. In addition, the rates of heme transfer from human hemoglobin to apoShp are close to those of simple ferric heme dissociation from hemoglobin, suggesting that methemoglobin does not directly transfer its heme to apoShp.

Conclusion: We have demonstrated that recombinant Shr can acquire heme from E. coli hemoproteins in vivo and appears to directly transfer its heme to Shp and that Shp appears not to directly acquire heme from human methemoglobin. These results suggest the possibility that Shr is a source of heme for Shp and that the Shr-to-Shp heme transfer is a step of the heme acquisition process in S. pyogenes. Further characterization of the Shr/Shp/HtsA system would advance our understanding of the mechanism of heme acquisition in S. pyogenes.

Show MeSH
Related in: MedlinePlus