Catabolism of glucose and fatty acids by virulent Treponema pallidum

Abstract

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

Unique lipid composition of Treponema pallidum (Nichols virulent strain)

“…Studies on the lipid composition of T. pallidum could be performed only if organisms were obtained free of rabbit tissue components. To accomplish this, we utilized an extensive purification scheme that included three of the best available tools for obtaining high yields of purified T. pallidum (2,8,25). We obtained 1010 to LIPIDS OF T. PALLIDUM 715 4 x 10's (mean = 2.3 x 1010) treponemes per rabbit, with an average recovery of 45% after purification.…”

“…Major problems in research on T. pallidum are the acquisition of sufficient treponemes and their purification from infected tissue. We have obtained high yields of purified organisms for studies of lipid composition by using several well-documented tools: Hypaque gradients (2), Nuclepore membranes (25), and corticosteroidtreated rabbits (8). Baseman et al (2) introduced the use of Hypaque gradients to obtain purified T. pallidum, but heavy bands of tissue material were present in their gradients above the treponemal fractions.…”

“…Some intestinal and oral strains, however, can utilize short-chain fatty acids (26). Recent studies suggest that T. pallidum may also have lesions in lipid metabolism similar to many cultivable treponemes (9,18,21,25).Lipids comprise 15 to 20% of the cellular dry weight of the cultivable treponemes (10, 16). The major component is a glycolipid, monoglycosyldiglyceride (MGDG), containing galactose generally as the sugar moiety.…”

“…A major impedance of research on T. pallidum is the difficulty of obtaining large yields of the organism free of rabbit testicular material. In recent years, however, the use of corticosteroid-treated rabbits (8), Nuclepore filters (25), and Hypaque gradients (2) has proven remarkably useful in reducing this problem. We have used all of these tools to obtain high numbers of purified T. pallidum for lipid analysis.…”

“…The suspension was centrifuged at 250 x g for 7 min at 220C to remove aggregated tissue material resulting from the high-speed centrifugation. The supernatant fluid was filtered through an 0.8-pm Nuclepore filter to remove tissue cells (25). The treponemes were concentrated as before, suspended in PBS-G, and further purified by sedimentation at 100,000 x g (r,,) for 45 min at 15'C through discontinuous gradients of Hypaque-M (Winthrop Laboratories, New York, N.Y.) in Beckman SW 39L or SW 41 Ti rotors (2).…”