Nadh

NADH Oxidase

2HgO

^leelaredoxin 4 (tp0823) J

Rubredoxin (Rd) Thioredoxin ( 'rx)

NADH Oxidase

NADH Oxidase

2H20

2H20

FIG. 6. Tentative model for oxygen detoxification by T. pallidum. The annotation and activities of neelaredoxin and rubredoxin are based on empirically derived data reviewed in this work. The putative annotations and activities proposed for NADH oxidase, alkyl hydroperoxide reductase C (AhpC), thioredoxin, and thioredoxin reductase are based on homology with enzymes in other organisms and their empirically determined activities. The numerical designations (TP#) below each enzyme are in accordance with the nomenclature of The Institute for Genomic Research (www.tigr.org).

associated with either AhpF or a Nox-l-type NADH oxidase, thioredoxin and thioredoxin reductase have been shown to interact with AhpC in Helicobacter pylori to reduce hydrogen peroxide to water.33 Thus, we speculate that T. pallidum encodes all the activities required for the complete reduction of superoxide to water (Fig. 6). In addition, T. pallidum encodes a Nox-2-like NADH oxidase that may i) lower the potential for superoxide generation by directly reducing molecular oxygen to water, and ii) provide reducing potential to rubredoxin. Isogenic mutants of Nox-2 in other organisms, including the spirochetal pathogen Brachyspira hyodysenteriae, have been shown to be hypersensitive to oxygen and less virulent in animal models of infection.34"36

Testing this scheme for oxygen detoxification will require a multifaceted approach including biochemical analysis of recombinant proteins, complementation of appropriate heterologous mutants, and transcriptional analysis of candidate treponemal ROS detoxification genes. Ultimately, in vitro reconstitution of the

33 L. M. Baker, A. Raudonikiene, P. S. Hoffman, and L. B. Poole, J. Bacteriol. 183, 1961 (2001).

34 J. Yu, A. P. Bryant, A. Marra, M. A. Lonetto, K. A. Ingraham, A. F. Chalker, D. J. Holmes, D. Holden, M. Rosenberg, and D. McDevitt, Microbiology 147,431 (2001).

35 C. M. Gibson, T. C. Mallett, A. Claiborne, and M. G. Caparon, J. Bacteriol. 182,448 (2000).

36 T. B. Stanton, E. L. Rosey, M. J. Kennedy, N. S. Jensen, andB. T. Bosworth,Appl. Environ. Microbiol. 65, 5028 (1999).

full enzymatic pathway, in concert with detection of these activities in lysates of Percoll-purified spirochetes, will be required to conclusively delineate the components of the treponemal oxidative defense pathway(s). Moreover, comparative functional analysis of these pathways in vivo and in vitro may reveal a relationship between treponemal oxidative stress responses and syphilis pathogenesis. These lines of investigation hold the potential to yield knowledge essential to the long-sought continuous cultivation of T. pallidum in vitro.

Acknowledgments

This research was supported in part by NRS a postdoctoral fellowship award ai-10573 to K.R.O.H., U.S. Public Health Service Grant ai-26756 to J.D.R., and by support from the Mayo Foundation to F.R.

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