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Hemin uptake in Porphyromonas gingivalis: Omp26 is a hemin-binding surface protein
1Department of Periodontics, University of Texas Health Science Center at San Antonio 78284-7894.
Abstract:
A 26-kDa outer membrane protein (Omp26) has been proposed to play a role in hemin acquisition by Porphyromonas gingivalis (T. E. Bramanti and S. C. Holt, J. Bacteriol. 174:5827-5839, 1992). We studied [55Fe]hemin uptake in P. gingivalis grown under conditions of hemin starvation (Omp26 expressed on the outer membrane surface) and hemin excess (Omp26 not expressed on surface). [55Fe]hemin uptake occurred rapidly in hemin-starved cells which incorporated up to 70% of total [55Fe]hemin within 3 min. P. gingivalis grown under hemin-starved conditions or treated with the iron chelator 2,2'-bipyridyl to induce an iron stress took up six times more [55Fe]hemin than hemin-excess-grown cells. Polyclonal monospecific anti-Omp26 antibody added to hemin-starved cells inhibited [55Fe]hemin uptake by more than 50%, whereas preimmune serum had no effect. [55Fe]hemin uptake in hemin-starved P. gingivalis was inhibited (36 to 67%) in the presence of equimolar amounts of unlabeled hemin, protoporphyrin IX, zinz protoporphyrin, and Congo red dye but was not inhibited in the presence of non-hemin-containing iron sources. Heat shock treatment (45 degrees C) of hemin-excess-grown P. gingivalis (which cases translocation of Omp26 to the surface) increased [55Fe]hemin uptake by threefold after 3 min in comparison with cells grown at 37 degrees C. However, no [55Fe] hemin uptake beyond 3 min was observed in either hemin-excess-grown or hemin-starved cells exposed to heat shock. In experiments using heterobifunctional cross-linker analysis, hemin and selected porphyrins were cross-linked to Omp26 in hemin-starved P. gingivalis, but no cross-linking was seen with hemin-excess-grown cells. However, cross-linking of hemin to Omp26 was observed after heat shock treatment of hemin-excess-grown cells. Finally, anti-Omp26 antibody inhibited cross-linked of hemin to Omp26. These findings indicate that hemin binding and transport into P.gingivalis cell mediated by Omp26.
Insights
Porphyromonas gingivalis utilizes the outer membrane protein Omp26 for hemin uptake, especially under iron-scarce conditions. This study demonstrates Omp26
Area of Science:
- Microbiology
- Bacterial Physiology
- Protein Function
Background:
- Porphyromonas gingivalis is a key pathogen in periodontitis.
- Hemin acquisition is crucial for P. gingivalis virulence.
- The role of outer membrane protein Omp26 in hemin uptake was previously suggested.
Purpose of the Study:
- To investigate the role of Omp26 in [55Fe]hemin uptake by P. gingivalis.
- To determine the conditions under which Omp26 mediates hemin transport.
- To elucidate the mechanism of Omp26-mediated hemin binding and transport.
Main Methods:
- Studied [55Fe]hemin uptake in P. gingivalis under hemin starvation and excess conditions.
- Utilized iron chelator 2,2'-bipyridyl to induce iron stress.
- Employed polyclonal monospecific anti-Omp26 antibody to assess Omp26's role.
- Investigated the effect of unlabeled hemin, protoporphyrin IX, zinc protoporphyrin, and Congo red on hemin uptake.
- Analyzed heat shock effects on Omp26 surface expression and hemin uptake.
- Used heterobifunctional cross-linker analysis to study hemin-Omp26 interactions.
Main Results:
- Hemin-starved cells showed rapid [55Fe]hemin uptake (70% within 3 min).
- Iron-stressed cells exhibited six times higher [55Fe]hemin uptake than hemin-excess cells.
- Anti-Omp26 antibody inhibited [55Fe]hemin uptake by over 50%.
- Hemin and related porphyrins competed for Omp26 binding.
- Heat shock induced Omp26 translocation and increased initial hemin uptake.
- Cross-linking experiments confirmed direct binding of hemin to Omp26.
Conclusions:
- Omp26 is essential for hemin binding and transport into P. gingivalis.
- Omp26-mediated hemin acquisition is regulated by iron availability.
- Omp26 functions as a hemin receptor on the P. gingivalis outer membrane.