Purification of the Pathogenic Treponema Pallidum by Density Gradient Centrifugation

We describe a procedure which permits essentially full recovery of physiologically active Treponema pallidum from rabbit testicular extracts and greatly reduces contaminating tissue material. Such preparations were employed for investigations of the ability of T. pallidum to catabolize glucose and fatty acids. Radiorespirometric studies revealed that glucose and pyruvate, but not oleate or palmitate, could be degraded to CO2. The use of differentially labeled glucose, in conjunction with enzymatic analyses, indicated that glucose was catabolized by a combination of the Embden-Meyerhoff-Parnas and hexose monophosphate pathways. Pyruvate was degraded to CO2 from only the carboxyl position, suggesting the absence of a functioning Krebs cycle; this was substantiated by additional enzyme analyses and radiorespirometric experiments. Oleate and palmitate were incorporated but not catabolized by /3-oxidation. Glucose, although catabolized, was not incorporated. The potential significance of these findings is discussed. Numerous attempts have been made to cultivate virulent Treponema pallidum in pure culture, but a review of the literature indicates that this goal has not been achieved (24, 31). Although it is considered an anaerobe, Cox and Barber (7) have shown that T. pallidum consumes 02 at a rate similar to that of the aerobic spirochete Leptospira. Such 02 uptake was cyanide sensitive, indicating a functioning cytochrome oxidase. Inhibition of this 02 uptake by azide, chlorpromazine, and amytal further suggested a functioning electron transport system for the oxidation of reduced nicotinamide adenine dinucleotide (NADH) to 02. The coupling of this system to oxidative phosphorylation was suggested. The possible aerobic nature of T. pallidum was recently supported by Baseman et al. (3), who found that glucose degradation and protein synthesis proceeded optimally in 02 concentrations of 10 to 20% and were inhibited under anaerobic conditions. The purpose of this investigation was to continue to examine the physiology of this spirochete and, in particular, to determine the possible catabolic activities of virulent T. pallidum for glucose and fatty acids as potential oxidizable substrates. MATERIALS AND METHODS Bacteria. The virulent Nichols strain of T. pallidum was used throughout this study. Cultivation of this organism in rabbits has been previously described (7). Testicles from exsanguinated, infected rabbits were extracted either in a medium consisting of 0.075 M sodium citrate containing 10% (vol/ vol) inactivated pooled rabbit serum (Pel-Freez, Inc., Rodgers, Ark.) (7) or in an aqueous solution of 0.01 M Na2HPO4, 0.14 M NaCl, and 0.06% reduced glutathione, adjusted to pH 7.3 with 0.1 N NaOH (PBS-G) (4). The extraction procedure was similar to that previously described (11). Each testicle was trimmed of fat and of the dorsal vein, cut lengthwise, and the edges of each half were snipped several times with scissors. The testicular tissue was then

Section: Resultsmentioning

confidence: 99%

Catabolism of glucose and fatty acids by virulent Treponema pallidum

Schiller¹,

Cox²

1977

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confidence: 99%

“…Antigenic characterization of Treponema pallidum has been hampered by the inability to obtain large quantities of pure, motile, and virulent organisms (1,9,10,21,22,(28)(29)(30). Nonpathogenic treponemes, however, can be grown in vitro in pure culture.…”

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confidence: 99%

Molecular characterization of common treponemal antigens

Hanff¹,

Miller²,

Lovett³

1983

A molecular characterization of cross-reactive antigens of Treponema pallidum Nichols and Treponema phagedenis biotype Reiter that are reactive with normal and syphilitic human sera is described. At least 8 common polypeptides, 14 T. pallidum-specific antigens, and 2 T. phagedenis biotype Reiter-specific antigens were identified.Antigenic characterization of Treponema pallidum has been hampered by the inability to obtain large quantities of pure, motile, and virulent organisms (1,9,10,21,22,(28)(29)(30). Nonpathogenic treponemes, however, can be grown in vitro in pure culture. Thus, in an attempt to obtain information about treponemal antigenic interrelationships, investigators have concentrated their efforts toward the examination of cross-reactive antigens obtained from the nonpathogens. Most of the studies have identified antigens common to both T. pallidum Nichols and the host-indigenous, nonpathogenic Treponema phagedenis biotype Reiter (referred to here as Reiter). Complement fixation (3-5, 7, 8, 12, 18, 20), gel diffusion (12,18,20), fluorescentantibody (6,13,17,27), crossed immunoelectrophoresis (20,(23)(24)(25)(26) and Western blot (15) techniques have revealed the presence of shared protein, polysaccharide, and/or protein-lipopolysaccharide complex antigens. Furthermore, absorption studies have shown that the presence of treponemicidal activity in normal human serum against T. pallidum and Reiter is stimulated by common T. pallidum and Reiter immunogens (P. A. Hanif and J. N. Miller, submitted for publication). However, with the exception of axial filament proteins (12, 21), the common treponemal antigens have not been well characterized in terms of their cellular location or molecular weight. As a prelude to isolation and purification of these particular cell components, we have characterized the common polypeptide antigens of T. pallidum and Reiter with normal and syphilitic human sera by using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by electrophoretic transfer of the t Present address:

“…Rathlev and Pfau (8) were the first to describe treponeme purification attempts by density gradient centrifugation. They separated T. pallidum from both soluble and particulate components by sedimentation through a potas-sium tartrate gradient, but the organisms were killed by the high salt concentration.…”

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confidence: 99%

Isolation and Purification of Treponema pallidum from Syphilitic Lesions in Rabbits

Hardy

Nell

1975

Treponema pallidum were extracted from testicular syphilomas of corticosteroid-treated rabbits and purified by differential centrifugation. The steroid therapy allowed a longer holding time for infected rabbits, which produced greater treponeme yields, averaging 1.58 × 10 10 treponemes per rabbit. The treatment, which also diminished cellular infiltration and increased the extracellular mucoid material in lesions, produced much cleaner suspensions than preparations from nontreated animals. Most of the treponemes in the purified suspensions were still motile, and none carried demonstrable host immunoglobulin. The preparations were free of recognizable host tissue debris and they contained, on the average, 1.9 × 10 −7 μg of protein per treponeme.