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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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Inefficient heme transfer from Shr to apoHtsA. ApoHtsA or apoShp (75 μM) was incubated with 15 μM Shr in 0.1 ml of 20 mM Tris-HCl, pH 8.0, at room temperature for 2 min. Each sample was then loaded onto a 0.1-ml SP Sepharose column and eluted as described in the text. Presented are the normalized spectra of Shr without or with treatment with apoHtsA or apoShp. The normalization was done by setting A280 = 1.0.
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Figure 6: Inefficient heme transfer from Shr to apoHtsA. ApoHtsA or apoShp (75 μM) was incubated with 15 μM Shr in 0.1 ml of 20 mM Tris-HCl, pH 8.0, at room temperature for 2 min. Each sample was then loaded onto a 0.1-ml SP Sepharose column and eluted as described in the text. Presented are the normalized spectra of Shr without or with treatment with apoHtsA or apoShp. The normalization was done by setting A280 = 1.0.

Mentions: To test whether Shr transfers its heme to apoHtsA, 15 μM holoShr was incubated with 75 μM apoHtsA or apoShp (positive control) for 2 min, and Shr was isolated from the mixtures. The normalized spectrum of the isolated Shr from its apoHtsA mixture was compared with those of untreated Shr and Shr isolated from its mixture with apoShp. As shown in Fig. 6, the ratio of A410/A280 of Shr from the Shr/apoHtsA reaction was decreased from 1.05 to 0.91, whereas the A410/A280 ratio of Shr from the Shr/apoShp reaction was lowered from 1.05 to 0.3. According the extinction coefficients of holoShr and apoShr at 280 and 408 nm and these A410/A280 ratios, Shr lost 14% and 73% heme in its reactions with apoHtsA and apoShp, respectively. These results indicate that Shr transfers its heme to apoShp more efficiently than to apoHtsA.


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)

Inefficient heme transfer from Shr to apoHtsA. ApoHtsA or apoShp (75 μM) was incubated with 15 μM Shr in 0.1 ml of 20 mM Tris-HCl, pH 8.0, at room temperature for 2 min. Each sample was then loaded onto a 0.1-ml SP Sepharose column and eluted as described in the text. Presented are the normalized spectra of Shr without or with treatment with apoHtsA or apoShp. The normalization was done by setting A280 = 1.0.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Inefficient heme transfer from Shr to apoHtsA. ApoHtsA or apoShp (75 μM) was incubated with 15 μM Shr in 0.1 ml of 20 mM Tris-HCl, pH 8.0, at room temperature for 2 min. Each sample was then loaded onto a 0.1-ml SP Sepharose column and eluted as described in the text. Presented are the normalized spectra of Shr without or with treatment with apoHtsA or apoShp. The normalization was done by setting A280 = 1.0.
Mentions: To test whether Shr transfers its heme to apoHtsA, 15 μM holoShr was incubated with 75 μM apoHtsA or apoShp (positive control) for 2 min, and Shr was isolated from the mixtures. The normalized spectrum of the isolated Shr from its apoHtsA mixture was compared with those of untreated Shr and Shr isolated from its mixture with apoShp. As shown in Fig. 6, the ratio of A410/A280 of Shr from the Shr/apoHtsA reaction was decreased from 1.05 to 0.91, whereas the A410/A280 ratio of Shr from the Shr/apoShp reaction was lowered from 1.05 to 0.3. According the extinction coefficients of holoShr and apoShr at 280 and 408 nm and these A410/A280 ratios, Shr lost 14% and 73% heme in its reactions with apoHtsA and apoShp, respectively. These results indicate that Shr transfers its heme to apoShp more efficiently than to apoHtsA.

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