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FlG. 5. Expression of neelaredoxin in T. pallidum (Tp). (A) Detection of transcripts for neelaredoxin and flaA by RT-PCR (+RT). Controls consisted of PCR performed on 50 ng of RNA template (-RT), RT-PCR of water (H2O), and PCR of 3 ng of DNA. (B) Detection of protein expression by immunoblot analysis of T. pallidum (5 x 107) and purified recombinant protein, using rat antiserum generated against purified recombinant protein. Numbers above the neelaredoxin lanes indicate the amount of protein (in nanograms). Numbers to the left of (A) and (B) indicate standards in base pairs (A) and kilodaltons (B). [Figure used with permission from T. Jovanovic, C. Ascenso, K. R. O. Hazlett, R. Sikkink, C. Krebs, R. Litwiller, L. M. Benson, I. Moura, J. J. G. Moura, J. D. Radolf, B. H. Huynh, and F. Rusnak, J. Biol. Chem. 275, 28439 (2000). Copyright © 2000, The American Society for Biochemistry and Molecular Biology.]

followed by exposure to chemiluminescence film. Using these parameters and densitometric analysis of immunoblots, we detected 11 ng of TpNlr in lysates of 5 x 107 freshly harvested testicular T. pallidum (Fig. 5B). By using these detection methods in concert with the in vitro cultivation system described above, it should be possible to analyze the effects of oxygen tension on the regulation of TpNlr in T. pallidum.

Summary and Future Directions

The syphilis spirochete expresses an active SOR, suggesting that T. pallidum has retained a primitive mechanism for coping with oxidative stress that appears to be unique among human pathogens.1415 The findings that Rd can reduce TpNlr to restore SOR activity, and that T. pallidum has an Rd homolog, suggests that the spirochete encodes enzymes for the reduction of superoxide to hydrogen peroxide. By scanning the genome, we have identified treponemal homologs for the alkyl hydroperoxide reductase protein subunit C, thioredoxin, and thioredoxin reductase. Although the reduction of hydrogen peroxide by AhpC has been most frequently neelaredoxin flaA

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