[14 Contribution of Neelaredoxin to Oxygen Tolerance by Treponema pallidum

By Karsten R. O. Hazlett, David L. Cox, Robert A. Sikkink, Françoise Auch'ere, Frank Rusnak, and Justin D. Radolf

Introduction

Treponema pallidum, the syphilis spirochete, remains one of the few major bacterial pathogens of humans that cannot be cultivated in vitro, although limited replication has been achieved by coculture with rabbit epithelial cells.1"4 During the course of infection, the bacterium encounters ambient oxygen tensions ranging from the relatively low partial pressures within peripheral tissues (approximately 40 mmHg) to the much higher partial pressures of arterial blood (100 mmHg).5 Moreover, infection of the cerebrospinal fluid (CSF), a body fluid presumed to have a relatively high level of oxygenation, occurs in a substantial proportion of syphilis patients.6,7 That T. pallidum readily disseminates hematogeneously and can survive within well-oxygenated host environments presents something of a paradox when viewed against in vitro studies demonstrating that oxygen concentrations above 5% are inhibitory, and those above 12% are lethal.8-12 Assuming that this conception of the T. pallidum's exposure to oxygen and reactive oxygen species (ROS) in vivo is correct, it follows that T. pallidum is protected from ROS during infection by mechanisms that are not accurately reproduced by current in vitro cultivation systems. Identification of these factors could provide us with a much better understanding of how T. pallidum survives within its obligate human host

1 D. L. Cox, R. A. Moeckli, and A. H. Fieldsteel, In Vitro 20, 879 (1984).

2 A. H. Fieldsteel, D. L. Cox, and R. A. Moeckli, Infect. Immun. 32, 908 (1981).

3 A. H. Fieldsteel, D. L. Cox, and R. A. Moeckli, Infect. Immun. 35,449 (1982).

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and could be instrumental for achieving continuous in vitro propagation, a goal that has eluded syphilis researchers for nearly a century.

Resolving the apparent contradiction between the oxygen sensitivity of the syphilis spirochete and its ability to withstand host environments with high redox potential will require (1) delineation of the pathways in this bacterium for oxygen detoxification and (2) a comparative analysis of their function in vivo and in vitro. The complete genomic sequence13 represents a powerful new tool with which to identify the oxidative defense enzymes of the spirochete, a line of investigation now undergoing a resurgence among syphilis researchers.14'15 Treponema pallidum lacks the oxidative defense enzymes superoxide dismutase (SOD), cata-lase, and peroxidase13 typically found in aerotolerant microorganisms. It does, however, express neelaredoxin, a superoxide reductase (SOR) that catalyzes the reduction of superoxide to hydrogen peroxide, which, presumably, compensates for the lack ofSOD.14'15 In addition, T. pallidum appears also to possess homologs for the additional constituents of a primitive, SOR-dependent, oxygen detoxification pathway (i.e., NADH oxidase, rubredoxin, thioredoxin reductase, thioredoxin, and the C subunit of alkyl hydroperoxide reductase).13 Consistent with the mi-croaerophilic nature of the spirochete in vitro, SORs have, thus far, been identified only in anaerobic sulfate-reducing archaea and microaerophilic sulfate-reducing bacteria.16-21

Although serial passage has yet to be achieved, the current in vitro cocul-tivation system allows investigators to monitor treponemal responses to various oxygen tensions. As such, syphilis researchers are now poised to compare both the transcriptional and enzymatic activities of these putative oxygen detoxification

13 C. M. Fraser, S. J. Norris, G. M. Weinstock, O. White, G. C. Sutton, R. Dodson, M. Gwinn, E. K. Hickey, R. Clayton, K. A. Ketchum, E. Sodergren, J. M. Hardham, M. R McLeod, S. Salzberg, J. Peterson, H. Khalak, D. Richardson, J. K. Howell, M. Chidambaram, T. Utterback, L. McDonald, P. Artiach, C. Bowman, M. D. Cotton, C. Fujii, S. Garland, B. Hatch, K. Horst, K. Roberts, M. Sandusky, J. Weidman, H. O. Smith, and J. C. Venter, Science 281, 375 (1998).

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genes under controlled oxygen tensions. Conversely, applying these methodologies to treponemes harvested from rabbit tissues, blood, and cerebrospinal fluid could yield clues to the ability of the treponeme to survive hematogeneous dissemination and CSF invasion. Presumably, comparing transcriptional profiles of in vitro- and in vi'vo-grown treponemes would shed light on the critical "missing ingredients" of the current in vitro cultivation techniques.

This chapter summarizes the methods of treponemal propagation both in vivo and in vitro, and the techniques employed to analyze the expression and enzymatic activity of neelaredoxin. A similar conceptual and methodological synthesis could be engaged to empirically confirm the identity of the remaining constituents of the oxygen detoxification pathway in this bacterium.

Propagation of Treponema pallidum and Effects of Oxidative Stress

Soon after the isolation of Treponema pallidum as the etiologic agent of syphilis, Noguchi8 reported in 1911 that anaerobiosis was beneficial to its survival in vitro. Over the next 70 years, many attempts were made to culture T. pallidum, the best of which prolonged viability a few days, but failed to promote treponemal replication. Starting in the mid-1970s, results from pure culture methods began to refine our understanding of treponemal oxygen tolerance. Norris et al. demonstrated that anaerobic or near anaerobic (<0.5% 02) conditions were actually detrimental to spirochete viability, and that O2 above 12.5%, even in the presence of reducing agents, caused rapid loss of viability.12 Baseman and Hayes9 reported that low oxygen tensions stimulated treponemal protein synthesis and glucose utilization. Subsequently, it was reported that T. pallidum lacked endogenous cata-lase and superoxide dismutase activities,22 was sensitive to superoxide,23 and was 10 times more sensitive to hydrogen peroxide than Escherichia coli.24 Results from early work with coculture systems supported these findings in that dissolved oxygen tensions, from 2 to 5%, were found to be most beneficial for long-term (5- to 21-day) viability of the treponemes.2,25-26

In 1980, by incorporating a reduced oxygen partial pressure, the potent reducing agent dithiothreitol (DTT), a slow-growing rabbit cell line (SflEp), and prescreened lots of fetal bovine serum (FBS), Fieldsteel and co-workers established the first in vitro coculture system that supported treponemal replication.2 DTT was used in place of cysteine and glutathione as these reducing agents are

22 F. E. Austin, J. T. Barbieri, R. E. Conn, K. E. Grigas, and C. D. Cox, Infect. Immun. 33, 372 (1981).

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24 B. M. Steiner, G. H. W. Wong, P. Sutrave, and S. R. Graves, Can. J. Microbiol. 30, 1467 (1983).

25 T. J. Fitzgerald, R. C. Johnson, J. A. Sykes, and J. N. Miller, Infect. Immun. 15,444 (1977).

26 A. H. Fieldsteel, F. A. Becker, and J. G. Stout, Infect. Immun. 18, 173 (1977).

readily inactivated in the presence of oxygen. By screening multiple lots of fetal bovine serum, it was noted that a low FBS iron content was critical for spirochetal growth. Although useful as an enzyme cofactor, iron is well recognized as a redox-active element that can generate reactive oxygen species (ROS) and participate in Fenton chemistry. Although the reason(s) that the Sf lEp cell line supports treponemal growth better than other cell lines is unknown, it has been postulated that the slow growth of these cells results in decreased generation of ROS. During the next 10 years, further improvements in the tissue culture system came from efforts to neutralize ROS. The addition of exogenous antioxidants such as SOD (25 U/ml), catalase (10 U/ml), mannitol (550 ¡xM), histidine (230 /iA/j, and vitamin E (16 nM) was reported to further increase the growth of T. pallidum in vitro.11 The rationale for adding multiple antioxidants was that each of the compounds acted on a distinct ROS. Moreover, the chemical antioxidants were hypothesized to cross the treponemal outer membrane to protect cellular constituents.

Although serial passage in vitro has yet to be achieved, the similarities between the in vitro cultivation conditions and those in vivo suggest that the cocultivation system is suitable to molecular analysis of treponemal oxygen tolerance, such as oxygen- and/or ROS-dependent transcriptional responses, in vivo, the generation time of T. pallidum is estimated to be between 30 and 33 hr27; the generation time in vitro is between 35 and 40 hr. The dissolved oxygen (d02) in both the tissue culture flasks and human tissues is near 5%.5 The following section summarizes both the rabbit model of treponemal propagation and the current in vitro coculture system that has been used to study the oxygen requirements and tolerances of T. pallidum. With minor modification, these systems should prove to be valuable tools in the molecular analysis of T. pallidum oxygen detoxicification pathways.

In Vivo Propagation of Treponema pallidum in Rabbit Testis

Preparation of Medium for Extraction of Treponema pallidum from Rabbit Testis. Usually 50 ml of T. pallidum culture medium base (TpCM base; Table I) without DTT is prepared the day before treponemes are to be harvested. The pH of the base is adjusted to pH 7.4, filter sterilized, and placed in a sterile 125-ml side-arm flask and sealed. The flask is alternately evacuated and gassed with a 5% C02-95% (v/v) N2 mixture three times and stored. The next day a fresh solution of DTT is prepared (5 mg in 2 ml of base), filter sterilized, and added to the flask. The flask is regassed as described above and stored for the extraction of treponemes. It must be noted that the lot of FBS used for extraction and cultivation must first be screened for its ability to support treponemal growth; many lots of FBS are toxic. Several samples can then be obtained from various serum companies and compared with a reference lot. In our experience, about one out of every four or

27 M. C. Cumberland and T. B. Turner, Am. J. Syph. 33, 201 (1949).

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