Vol. 25, No. 1

INFECTION AND IMMUNITY, July 1979, p. 157-163

0019-9567/79/07-0157/07$02.00/0

Pyruvate Oxidation by Treponema pallidum JOSEPH T. BARBIERI AND C. D. COX* Department ofMicrobiology, University of Massachusetts, Amherst, Massachusetts 01003 Received for publication 16 April 1979

Cell-free extracts of Treponema pallidum catalyzed the decarboxylation of pyruvate. This activity was suppressed at low 02 tensions and appeared to be coenzyme A independent. Pyruvate decarboxylation was inorganic phosphate dependent, and evidence suggested that acetyl phosphate was a product. Oxygen was consumed, and data indicated that H202 was produced. These results indicated that the overall oxidation of pyruvate was: pyruvate + 02 + inorganic phosphate -+ C02 + acetyl phosphate + H202. Phosphotransacetylase and acetate kinase activities were also observed in the cell-free extracts and could catalyze formation of acetyl coenzyme A and adenosine 5'-triphosphate, respectively, from

acetyl phosphate. Although our knowledge of the physiology of Treponemapallidum is still fragmentary, recent investigations have shown treponemes to be capable of both catabolic and anabolic metabolism in vitro (2, 15, 18). Glucose degradation proceeds via the hexose monophosphate and EmbdenMeyerhof-Parnas pathways (18), producing lactate, acetate, and C02 (3). Reduced nicotinamide adenine dinucleotide-dependent lactate dehydrogenase catalyzes the reduction of pyruvate to lactate, but the mechanism of pyruvate oxidation to acetate and C02 has not been determined (18). This report describes the enzymatic properties of pyruvate oxidation by T. pallidum. MATERIALS AND MERTHODS Preparation of treponeme extracts. The Nichols strain of virulent T. pallidum was used throughout this study. Procedures for the cultivation of virulent T. pallidum in rabbits have been described (4, 13). Testicles at peak orchitis were aseptically removed from the rabbits, minced, and placed into a solution (10 ml/testicle) of phosphate-buffered saline containing 2 mM reduced glutathione, pH 7.4 (PBS-G). This testicular suspension was shaken under atmospheric 02 tensions for 1 h at room temperature, followed by shaking for 1 h at 40C. The fluid was decanted, and a second 15-min extraction was performed with fresh PBS-G. Both extracts were combined and centrifuged at 300 X g for 10 min to remove host cells and debris. The supernatant fluid, containing primarily treponemes, was filtered sequentially by negative pressure through 3.0- and 0.8-um membrane filters (Nuclepore Corp.) (18). The filtered supernatant fluid was centrifuged at 17,000 x g for 20 min. High-speed pellets were rinsed but not disrupted in fresh PBS-G, recentrifuged at 17,000 x g for 20 min, and suspended in an appropriate volume (0.2 to 4.0 ml) of 20.0 mM sodium phosphate buffer, pH 7.4. The cells were disrupted by sonic treatment (18), and this suspension was labeled

the cell-free extract (CFE). Before spectrophotometric determination of enzyme activity, cellular debris was removed from the CFE by centrifugation at 30,000 x g for 20 min. Enzyme assays. Pyruvate decarboxylation was determined by measuring the release of "CO2 from [1'4C]pyruvate. The reaction mixture contained the following: [1-'4C]pyruvate, sodium salt (106 cpm/0.2 tmol), approximately 0.2 ,tmol (see below for exact concentrations); sodium phosphate buffer, pH 7.4, 125 manol; CFE; and water to a volume of 2.0 ml. Any supplements or inhibitors were incubated in this reaction mixture 15 min before the addition of pyruvate, which initiated the reaction. The procedure used for trapping and measuring "4CO2 has been described (18). Phosphotransacetylase (EC 2.3.1.8) was measured essentially by the arsenolysis procedure of Stadtman (19). The reaction mixture contained 1.0 timol of

tris(hydroxymethyl)aminomethane-hydrochloride buffer (Tris buffer) (pH 8.0), 1.2 ,mol of acetyl phosphate, 2.4 x 10-2 jtmol of coenzyme A (CoA), 0.1 Mmol of cysteine-HCl, CFE, and water to a volume of 120

arsenate was pl. After 5 min 10 FImol (20 pl) of sodium added. The reaction was terminated after 10 min by adding 100 umol (50 pl) of neutralized hydroxylamine. After a 10-min incubation to allow for the formation of acetyl hydroxamate, 100 ,.l of a solution containing

equal volumes of 15% (wt/vol) FeCl3 in 0.1 N HCl, 36% trichloroacetic acid, and 9.0 N HCl were added. Precipitated protein was removed by centrifugation, and the remaining acetyl phosphate was determined as acetyl hydroxamate spectrophotometrically at 540 nm. Specific activity was recorded as micromoles of acetyl phosphate degraded per minute per milligram of protein. Acetate kinase (EC 2.7.2.1) was assayed by measuring the production of adenosine 5'-triphosphate (ATP) from acetyl phosphate (10). ATP was determined spectrophotometrically by the Pinchot method (16), which couples the production of ATP to the reduction of nicotinamide adenine dinucleotide phosphate (NADP+), and was measured at 340 nm. The reaction

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INFECT. IMMUN.

mixture contained the following: acetyl phosphate, 2.0 was constructed by using known concentrations of umol; adenosine 5'-diphosphate, 0.5 pmol; Tris buffer acetyl phosphate. (pH 8.0), 0.2 Lmol; D-glucose, 5.0 Mmol; MgCl2, 1.2 Other procedures. CoA was removed from the pmol; NADP', 0.04 Mmol; glucose 6-phosphate dehy- CFE by the method of Stadtman et al. (20). CFE was drogenase, 0.05 U; hexokinase, 0.2 U; CFE; and water added to Dowex 1-X4 resin (Cl1; 50 to 100 mesh) in a to a volume of 200 pl. ratio of 1 to 0.5 (vol/vol). This suspension was mixed Acetate kinase was also measured by the method of for 10 min and centrifuged to separate resin from CFE. Rose (17). The reaction mixture contained 31.0 tmol CFE was then filtered through Whatman no. 1 filter of sodium acetate, 4.8 Mmol of Tris buffer (pH 7.4), 0.9 paper to remove any remaining resin. This procedure Mmol of MgCl2, 2.0 jmol of ATP, 60 tmol of neutralized was observed to remove 95 to 99% of exogenously hydroxylamine, CFE, and water to a volume of 130 p1. added CoA from the CFE. Endogenous levels of CoA CFE was added last to start the reaction. After 5 min, in the CFE were determined by the CoA-dependent 50 Ad of 25% trichloroacetic acid was added to stop the phosphotransacetylase reaction which was assayed as reaction. The acetyl hydroxamate was determined at previously described, except that 9.4 U of phospho540 nm after the addition of 100 pI of 5.0% FeCL3 in 1.0 transacetylase was used as the source of enzyme and N HCl and removal of precipitated protein. Specific the CFE was used as the source of CoA. Known activity was recorded as micromoles of acetyl hydrox- concentrations of CoA were used to construct a stanamate formed per minute per milligram of protein. dard curve. Under these assay conditions, linearity of Acetyl CoA synthetase (EC 6.2.1.1) was assayed by enzyme activity was observed between 0.1 and 9.0 measuring the production of ATP from acetyl CoA nmol of CoA. Before Dowex treatment, 150 jig of CFE (10). ATP was measured spectrophotometrically by protein contained

Pyruvate oxidation by Treponema pallidum.

Vol. 25, No. 1 INFECTION AND IMMUNITY, July 1979, p. 157-163 0019-9567/79/07-0157/07$02.00/0 Pyruvate Oxidation by Treponema pallidum JOSEPH T. BAR...
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