INFECTION AND IMMUNITY, Apr. 1979, 0019-9567/79/04-0291/03$02.0/0
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Vol. 24, No. 1
291-293
Glucose Incorporation by Treponema pallidum JOSEPH T. BARBIERI AND C. D. COX* Department ofMicrobiology, University of Massachusetts, Amherst, Massachusetts 01003 Received for publication 12 January 1979
Treponema pallidum incorporated glucose into trichloroacetic acid-precipitable material. The amount of incorporation was proportional to the number of treponemes and was estimated to equal 3% of the glucose oxidized. Treponema pallidum has been observed to incorporate amino acids (1) and uridine (11) into trichloroacetic acid-precipitable material. Although this organism has not been cultivated in the laboratory, these results indicate that this treponeme performs anabolic metabolism in vitro. However, the incorporation of glucose into cell material has not been observed (3, 12). The inability of these treponemes to utilize glucose as a carbon source could be of considerable importance. This report describes our reinvestigation of glucose incorporation by T. pallidum using high-specific-activity [U-'4C]glucose. Extraction, purification, and counting of the Nichols strain of virulent T. pallidum from infected testicles have been described previously (8, 12). Extraction was performed for 1 h at room temperature in isotonic phosphate-buffered saline containing 2.0 mM reduced glutathione at pH 7.4 (PBS-G). After the addition of [U-'4C]glucose (Amersham/Searle; specific activity, 333 ItCi/ttmol), treponemes were incubated at 350C under atmospheric conditions. Radiorespirometry was performed as previously described (12). Incorporation experiments were terminated at appropriate times by adding 2 ml of cold PBS-G containing 5% glucose to the treponemal suspensions. The treponemes were centrifuged at 30,000 x g for 20 min, and the resulting pellets were resuspended in 1 ml of PBS-G containing 5% glucose. Pellet suspensions were placed on 0.45-nm membrane filters (Millipore Corp.) which had been prewashed with 1 ml of PBS-G containing 5% glucose. Glucose uptake by whole cells was determined after washing the filters three times with 5 ml of 20.0 mM phosphate buffer, pH 7.4. Glucose incorporation into trichloroacetic acid-precipitable material was determined after washing the filters three times with 5 ml of 5% cold trichloroacetic acid, followed by one wash with 5 ml of 20.0 mM phosphate buffer, pH 7.4. Filters were air dried, and radioactivity was determined by liquid scintillation counting. Treponemes were diluted in the high-speed
supernatant fluid resulting from centrifuging treponemal suspensions at 17,000 x g for 30 min. The high-speed supernatant fluid contained less than 106 treponemes per ml, and its use assured constant amounts of glucose in dilutions of the treponeme suspensions. Glucose concentrations in the treponeme suspensions were determined by the Statzyme glucose test (Worthington Diagnostics), and 02 levels were measured as previously described (12). Over 95% of the treponemes remained motile throughout these experiments. Tabular data are from one set of at least three repeated experiments showing similar results. Glucose decarboxylation and incorporation into trichloroacetic acid-precipitable material were observed during the incubation periods (Table 1). Uptake of glucose into whole cells was greater than incorporation into trichloroacetic acid precipitate, indicating that the treponemes accumulated pools of trichloroacetic acid-soluble material derived from glucose. Reducing the numbers of treponemes in the reaction mixtures resulted in proportionate decreases in the amount of glucose incorporated into trichloroacetic acid-precipitable material (Table 2). Heating the treponemes for 30 min at 560C reduced incorporation of glucose after 90 min to
p.
Vol. 24, No. 1
291-293
Glucose Incorporation by Treponema pallidum JOSEPH T. BARBIERI AND C. D. COX* Department ofMicrobiology, University of Massachusetts, Amherst, Massachusetts 01003 Received for publication 12 January 1979
Treponema pallidum incorporated glucose into trichloroacetic acid-precipitable material. The amount of incorporation was proportional to the number of treponemes and was estimated to equal 3% of the glucose oxidized. Treponema pallidum has been observed to incorporate amino acids (1) and uridine (11) into trichloroacetic acid-precipitable material. Although this organism has not been cultivated in the laboratory, these results indicate that this treponeme performs anabolic metabolism in vitro. However, the incorporation of glucose into cell material has not been observed (3, 12). The inability of these treponemes to utilize glucose as a carbon source could be of considerable importance. This report describes our reinvestigation of glucose incorporation by T. pallidum using high-specific-activity [U-'4C]glucose. Extraction, purification, and counting of the Nichols strain of virulent T. pallidum from infected testicles have been described previously (8, 12). Extraction was performed for 1 h at room temperature in isotonic phosphate-buffered saline containing 2.0 mM reduced glutathione at pH 7.4 (PBS-G). After the addition of [U-'4C]glucose (Amersham/Searle; specific activity, 333 ItCi/ttmol), treponemes were incubated at 350C under atmospheric conditions. Radiorespirometry was performed as previously described (12). Incorporation experiments were terminated at appropriate times by adding 2 ml of cold PBS-G containing 5% glucose to the treponemal suspensions. The treponemes were centrifuged at 30,000 x g for 20 min, and the resulting pellets were resuspended in 1 ml of PBS-G containing 5% glucose. Pellet suspensions were placed on 0.45-nm membrane filters (Millipore Corp.) which had been prewashed with 1 ml of PBS-G containing 5% glucose. Glucose uptake by whole cells was determined after washing the filters three times with 5 ml of 20.0 mM phosphate buffer, pH 7.4. Glucose incorporation into trichloroacetic acid-precipitable material was determined after washing the filters three times with 5 ml of 5% cold trichloroacetic acid, followed by one wash with 5 ml of 20.0 mM phosphate buffer, pH 7.4. Filters were air dried, and radioactivity was determined by liquid scintillation counting. Treponemes were diluted in the high-speed
supernatant fluid resulting from centrifuging treponemal suspensions at 17,000 x g for 30 min. The high-speed supernatant fluid contained less than 106 treponemes per ml, and its use assured constant amounts of glucose in dilutions of the treponeme suspensions. Glucose concentrations in the treponeme suspensions were determined by the Statzyme glucose test (Worthington Diagnostics), and 02 levels were measured as previously described (12). Over 95% of the treponemes remained motile throughout these experiments. Tabular data are from one set of at least three repeated experiments showing similar results. Glucose decarboxylation and incorporation into trichloroacetic acid-precipitable material were observed during the incubation periods (Table 1). Uptake of glucose into whole cells was greater than incorporation into trichloroacetic acid precipitate, indicating that the treponemes accumulated pools of trichloroacetic acid-soluble material derived from glucose. Reducing the numbers of treponemes in the reaction mixtures resulted in proportionate decreases in the amount of glucose incorporated into trichloroacetic acid-precipitable material (Table 2). Heating the treponemes for 30 min at 560C reduced incorporation of glucose after 90 min to
