Vol. 16, No. 1 Printed in U.S. A .
INFECTION AND IMMUNITY, Apr. 1977, p. 60-68 Copyright ©D 1977 American Society for Microbiology
Catabolism of Glucose and Fatty Acids by Virulent Treponema pallidum NEAL L. SCHILLER AND C. D. COX* Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01002
Received for publication 22 November 1976
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. ing 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 extracted in the appropriate medium (10 ml/testicle) in an atmosphere of 95% N2 and 5% CO2 by rotation at 70 rpm for 1 h at 25°C, followed by 1 h at 4°C. These extracts were adjusted for cell density in the extraction medium. In previous experiments, some of which we have reported (7), treponemes extracted from tissue by differential centrifugation consumed 02 at levels significantly higher than animal-cell controls from infected tissue. Treponeme and animal-cell preparations were established by procedures previously outlined by us (7) and later described by Nichols and Baseman (20). In the present studies tissue extracts were centrifuged at 300 x g for 10 min to remove the majority of contaminating tissue in the low-speed pellet (LSP). The supernatant fluid (LSS) was filtered through Nucleopore filters (see Results), and the filtered suspension of treponemes was centrifuged at 17,000 x g for 30 min. Treponemes in the high-speed pellet (HSP) were resuspended in a small amount of high-speed supernatant fluid (HSS) and used in incorporation and radiorespirometric experiments. Enzyme assays were performed on sonically treated treponemes from HSP. The tissue-cell controls consisted of tissue cells from LSP resuspended in HSS to a concentration of 104/ml, or about 500 times higher than detectable levels in the filtered
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 consist60
VOL. 16, 1977
CATABOLISM BY VIRULENT T. PALLIDUM
treponemal suspension or the HSP. The other control consisted of HSS to reflect soluble or membraneassociated enzyme activity resulting from the few tissue cells and/or treponemes. Tissue-cell controls were used to monitor and correct values in incorporation studies, and both tissue-cell and HSS controls were used similarly in metabolic experiments. Incorporation experiments. PBS-G was selected as the extraction medium since it was free from glucose and fatty acids, thereby enhancing the chance of detecting any uptake of "4C-labeled substrate added to this medium. After the addition of isotopically labeled compounds to treponeme suspensions, the samples were swirled on a platform rotator at 120 rpm at 25°C. Preliminary experiments in several metabolic studies indicated no substantial differences among those performed at 25, 30, or 37°C, and room temperature of 250C was chosen for these and subsequent experiments. At selected time intervals, samples of cell suspensions were removed, incubated at 56°C for 5 min, and centrifuged at 30,000 x g for 30 min. The cells were washed in PBS-G and centrifuged as before, and the pellet was suspended in absolute ethanol and transferred to scintillation vials. After evaporation, the residue was resuspended in scintillation fluid, and radioactivity was determined in a Beckman LS-100 liquid scintillation counter. The efficiency of counting in these experiments was 95%. The scintillation fluid contained 700 ml of scintillation-grade toluene, 300 ml of absolute ethanol, 0.6% 2,5-diphenyloxazole (PPO), and 0.1% p-bis-(o-methylstyryl)-benzene (bis-MSB). In some experiments uptake of radioisotopes was terminated by the addition of trichloroacetic acid. The cells were collected by centrifugation and washed once with PBS-G. They were then washed in cold 5% trichloroacetic acid, suspended, and transferred to scintillation vials as described above. Radiorespirometric experiments. The extraction fluid contained 75 mM sodium citrate and 10% (vol/ vol) heat-inactivated pooled rabbit serum. Assays were performed in a 10-ml Erlenmeyer flask sealed with a rubber serum stopper, with a plastic center well (Kontes Glass Co.) suspended from the stopper. After 1.8 ml of the appropriate solution or suspension was added, the flask was stoppered, 0.2 ml of Hyamine hydroxide was injected into the center well, and 0.2 ml of the isotope solution was added to the solution or suspension. The flasks were incubated at 250C and rotated on a platform shaker at 70 rpm. At the end of the reaction period of 2 h, 0.2 ml of 3 M H2SO4 was added to the reaction mixture to release any soluble C02, and the flasks were incubated for an additional 60 min. The flasks were then opened. The cup was immediately placed into 15 ml of scintillation fluid and radioactivity was measured. Total glucose present was determined with the Glucostat assay (Worthington Biochemicals Corp.). Enzyme assays. Testicles were extracted in PBSG, and the second extraction was performed with fresh PBS-G for 1 h at 25°C. The HSP was washed in 10 ml of 0.02 M sodium phosphate buffer (PBS), pH 7.2, resuspended in a small volume of PBS, and either frozen or used immediately.
61
Cells were disrupted by sonic treatment in a Raytheon Sonifier using the minimal exposure necessary for the disappearance of all intact treponemes as observed by dark-field microscopy. At least three 1-min bursts were usually required. Cellular debris was removed by centrifugation for 20 min at 12,000 x g, and the supernatant fraction was used as a source of soluble enzymes. The pellet was resuspended in PBS and used as a source of membraneassociated enzymes. Extracts were kept at 4°C and used immediately for enzyme assays. Protein was determined by the Folin phenol method (15). Assays were made on extracts containing 10 to 100 ,ug of protein. Most enzyme assays involved coupling the specific reaction to be determined to a pyridine nucleotide-dependent system and, unless otherwise noted, followed the general procedures described in Methods in Enzymology, vol. 1 (6). Oxidation or reduction of pyridine nucleotides was followed at 340 nm in a Gilford model 240 recording spectrophotometer. A total volume of 0.15 ml was used in each cuvette unless otherwise noted. One unit of enzyme activity was defined as the amount of enzyme resulting in the oxidation or reduction of 1 ,umol of pyridine nucleotide per min at 25°C. Specific activities are expressed in terms of units of enzyme per milligram of crude treponemal extract protein. Control cuvettes lacking various components of the specific assay, or treponemal extract, or including enzymes from other sources were included for each assay. Enzymes of the citric acid cycle were determined in the following manner. The malate dehydrogenase (EC 1.1.1.37) assay reaction mixture contained 10.0 ,umol of tris(hydroxymethyl)aminomethane (Tris) buffer (pH 8.0), 2.0 ,umol of potassium oxalacetate, 1.0 gmol of MgSO4, 0.075 ,imol of NADH, and 1.0 ,unmol of 2-mercaptoethanol. A reaction mixture for measuring citrate synthase (EC 4.1.3.7) was employed which required the presence of malate dehydrogenase in the extract. This enzyme system combined 10.0 ,umol of Tris buffer (pH 8.0), 2.0 gtmol of sodium malate, 0.3 ymol of acetyl coenzyme A (acetyl-CoA), 1.0 ,umol of MgSO4, 0.075 ,umol of nicotinamide adenine dinucleotide (NAD), 1.0 ,umol of 2mercaptoethanol, and pig heart malic dehydrogenase. Fumarate hydratase (EC 4.2.1.2) activity was estimated by replacing sodium malate in the prior assay with potassium fumarate. Fumarate hydratase was also assayed according to a procedure described by Massey (17) which was based on the high ultraviolet absorption of fumarate. This assay mixture contained 2.0 ,mol of potassium fumarate, 0.02 M sodium phosphate buffer, pH 7.2, and cell-free extract in a total volume of 0.15 ml. The consumption of fumarate was monitored by following the decrease in absorbance at 300 nm. Isocitrate dehydrogenase (EC 1.1.1.42) assay mixture contained 10.0 ,imol of Tris buffer (pH 8.0), 2.0 ,umol of sodium isocitrate, 1.0 ,umol of MgSO4, and 0.075 ,umol of nicotinamide adenine dinucleotide phosphate (NADP). Sodium citrate substituted for isocitrate in assays for aconitate hydratase (EC 4.2.1.3). Oxoglutarate dehydrogenase (EC 1.2.4.2) reaction mixture contained 10.0 ,mol of Tris buffer (pH 8.0), 2.0 ,umol of sodium oxoglutarate, 0.075 zmol of NAD, and 0.2
62
SCHILLER AND COX
,umol of coenzyme A (CoA). Succinic dehydrogenase (EC 1.3.99.1) was assayed according to the procedure described by Massey (18), with a 3.0-ml reaction mixture. Isocitrate lyase (EC 4.1.3.1) was measured essentially by the method described by Reeves and Ajl (23), with the following components in a 0.15-ml reaction mixture: 10.0 gmol of Tris buffer (pH 7.3), 1.0 umoi of MgSO4, 1.0 gmol of phenylhydrazine hydrochloride, 0.1 ,umol of cysteine hydrochloride, and 2.0 ,umol of sodium isocitrate. The malate dehydrogenase (decarboxylating) (NADP+) enzyme (EC 1.1.1.40) was assayed essentially as described by Ochoa (21) in a reaction mixture containing 10.0 ,umol of Tris buffer (pH 7.3), 1.0 ,mol of MgSO4, 0.075 ,umol of NADP, and 2.0 ,tmol of either sodium malate or potassium oxalacetate. Glutamate dehydrogenase (EC 1.4.1.2) was assayed according to a procedure described by Strecker (25). When assayed in the forward direction, the assay mixture contained 10.0 umol of Tris buffer (pH 8.0), 0.075 ,umol of NAD, and 2.0 ,tmol of potassium glutamate. For the reverse reaction, the assay mixture contained 10.0 gmol of Tris buffer (pH 8.0), 1.0 ,umol of NH4Cl, 0.075 ,tmol of NADH, and 2.0 i,mol of sodium oxoglutarate. The acetyl-CoA acetyltransferase (EC 2.3.1.9) assay followed the general procedure described by Henneberry and Cox (12), with the following components in 0.15 ml: 10.0 ,umol of Tris buffer (pH 8.0), 1.0 ,mol of MgSO4, 0.05 gmol of CoA, 0.075 ,mol of NAD, 2.0 ,umol of sodium malate, 0.12 umol of acetoacetyl-CoA, 1.0 ,umol of dithiothreitol, 0.92 unit of pig heart citrate synthase, and 1.2 units of pig heart malic dehydrogenase. Enoyl-CoA hydratase (EC 4.2.1.17) was measured according to the procedure of Henneberry and Cox (12), with a reaction mixture containing 10.0 ,umol of Tris buffer (pH 8.0), 1.0 ,umol of dithiothreitol, 1.0 ,umol of MgCl2, 0.075 ,umol of NAD, 0.6 ,umol of crotonyl-CoA, and 0.04 unit of pig heart 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35). The presence of 3-hydroxyacyl-CoA dehydrogenase was determined by employing the same assay mixture without the commercial enzyme. Enzymes of the glycolytic and pentose shunt pathways were also investigated. Glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.12) was measured in a reaction mixture containing 10.0 ,umol of Tris buffer (pH 8.0), 0.075 ,umol of NAD, 2.5 ,umol of sodium arsenate, 1.0 ,umol of 2-mercaptoethanol, and 2.0 ,umol of glyceraldehyde-3-phosphate. Attempts were made to couple other enzymatic reactions to reduction of NAD via glyceraldehyde-phosphate dehydrogenase. Fructose-biphosphate aldolase (EC 4.1.2.13) was assayed by replacing glyceraldehyde-3-phosphate with fructose-1,6-diphosphate. Triosephosphate isomerase (EC 5.3.1.1) was assayed by replacing glyceraldehyde-3-phosphate with dihydroxyacetone phosphate and by substituting triethanolamine buffer (pH 8.5) for Tris buffer. 6-Phosphofructokinase (EC 2.7.1.11) was assayed with fructose-6-phosphate as the substrate, with the addition of 0.75 ,umol of adenosine triphosphate (ATP) and 1.0 ,mol of MgSO4. Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was assayed in a reaction mixture containing 10.0
INFECT. IMMUN.
,tmol of Tris buffer (pH 8.0), 1.0 ,umol of MgSO4, 0.075 ,umol of NADP, and 2.0 /mol of glucose-6phosphate. Phosphogluconate dehydrogenase (EC 1.1.1.43) was estimated by replacing glucose-6-phosphate with 6-phosphogluconate. The presence of glucose-6-phosphate dehydrogenase enabled the measurement of two enzymes by coupling these enzymes to the dehydrogenase assay. Hexokinase (EC 2.7.1.1) was measured by replacing glucose-6-phosphate with glucose and by adding 0.75 Amol of ATP. Glucosephosphate isomerase (EC 5.3.1.9) was assayed with 2.0 ,hmol of fructose-6-phosphate used as substrate in place of glucose-6-phosphate. Determinations of lactate dehydrogenase (EC 1.1.1.27) involved a system containing 10.0 ,umol of Tris buffer (pH 8.0), 2.0 ,umol of sodium pyruvate, 0.1 ,Lmol of ZnC12, 1.0 ,umol of MgSO4, and 0.075 ,umol of NADH. Pyruvate kinase (EC 2.7.1.40) was determined by coupling pyruvate formation to lactate dehydrogenase-mediated consumption of NADH. Reaction mixtures contained 10.0 ,umol of triethanolamine buffer (pH 8.5), 2.0 umol of phosphoenolpyruvate (PEP), 0.1 ,umol of ZnCl2, 1.0 ,umol of MgSO4, 0.75 ,umol of adenosine diphosphate (ADP), 1.0 ,tmol of 2-mercaptoethanol, and 0.075 ,umol of NADH. For determination of enolase (EC 4.2.1.11) and phosphoglyceromutase (EC 2.7.5.3) activity, the pyruvate kinase assay mixture was supplemented with 2.0 umol of 3-phosphoglycerate and 2.2 units of rabbit muscle pyruvate kinase, and PEP was omitted. In all assays where NADH consumption was measured, activities were corrected for NADH dehydrogenase activity. NADH dehydrogenase (EC 1.6.99.3) was assayed in a reaction mixture c6ntaining 10.0 ,umol of Tris buffer (pH 8.0) and 0.075 ,umol of NADH. The pyruvate dehydrogenase (EC 1.2.4.1) assay was performed by a method described by Korkes (13) and contained 10.0 umol of Tris buffer (pH 8.0), 1.0 ,umol of MgSO4, 0.1 umol of CoA, 0.075 ,umol of NAD, 1.0 ,umol of reduced glutathione, 0.1 ,umol of thiamine pyrophosphate, and 2.0 ,umol of sodium pyruvate. Pyruvate oxidase (EC 1.2.3.3) was assayed as described by Hager and Lipmann (10), and acetyl phosphate formation was measured by the hydroxamic method of Lipmann and Tuttle (14). Pyruvate dehydrogenase (cytochrome) (EC 1.2.2.2) was determined by the procedure described by Williams and Hager (32). 14CO2 was trapped and measured as described earlier for the radiorespirometric experiments. Chemicals. [U-14C]oleate and [U-14C]pyruvate were purchased from ICN Radioisotope Division; all other radioactive compounds were obtained from New England Nuclear Corp. All chemicals were reagent grade or the equivalent. Sigma Chemical Co. was the source of all commercial enzymes used in this study.
RESULTS Removal of tissue cells from suspensions of T. pallidum. Although the previous extraction procedure (7) seemed adequate for 02 uptake
VOL. 16, 1977
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63
studies, the use of this preparation for sub- filtration. Filters of smaller pore sizes were not strate oxidation and enzymatic analyses was tested. considered unacceptable. Therefore, attempts The metabolic activity of the treponemes bewere made to free these treponemal preparafore and after filtration was examined by detertions from contaminating tissue cells. Several mination of Q(02) values as described by Cox investigators have examined various tech- and Barber (7). As shown in Table 1, the metaniques to obtain such preparations. These bolic activity of the treponemes was maintained methods include density gradient centrifuga- after passage through 0.8- or 1.0-,um Nucleotion (22), continuous-flow zonal centrifugation pore filters. Removal of tissue cells from trepoin cesium chloride gradients (26), discontinuous neme suspensions without decreasing Q(02) gradients of sodium and meglumine diatri- values further supports the previous conclusion zoates (4), and membrane filtration procedures (7) that 02 consumption was associated with T. (5). All of these techniques damaged the trepo- pallidum. Although the filtration procedure nemes except the filtration procedure, which was successful in removing essentially all tisremoved too many treponemes. However, J. W. sue cells from the treponeme suspension, the Foster suggested the use of Nuclepore filters for possibility remained that a few tissue cells and this purpose (personal communication), and soluble enzymes or membranes from tissue several experiments were designed to investi- cells or treponemes might contribute to the gate this possibility. metabolic activities observed. Thus, activities Nucleopore membranes ranging from 0.8- to of the tissue-cell and HSS controls continued to 5.0-,um pore sizes were examined in an effort to be routinely monitored. remove all observable tissue cells while allowSubstrate incorporation studies. Several ing full recovery of the treponemes. Cells were 14C-labeled compounds were added to restingcounted immediately before and after filtration cell suspensions of T. pallidum in an attempt to by use of a Petroff-Hausser counting chamber define compounds which might serve as a major with dark-field illumination. Either 25- or 47- source of cell carbon. At indicated times, sammm diameter filters were routinely used with ples were removed and the amount of incorpomild vacuum. The limit of cell detection, based rated radioactivity was determined. [Uon the observation of 1 or 2 cells per entire 14C]glucose was not incorporated into either counting chamber, was between 5 x 103 and 1 x medium- or trichloroacetic acid-washed cells 104 cells per ml. Both 0.8- and 1.0-,um Nucleo- during a 2-h test period. Incorporation over pore filters consistently removed all observable longer time periods was not examined because tissue cells while allowing essentially full re- the motility of the treponemes decreased after 2 covery of T. pallidum (Table 1). The sensitivity h. of the counting procedure was increased by cenNelson (19) and Weber (30) have reported trifuging the filtrate at 35,000 x g for 30 min that bovine serum albumin (BSA) prolonged and resuspending the pellet in smaller vol- treponemal survival. It seemed reasonable that umes. This procedure indicated that less than this might reflect a requirement for fatty acids 200 tissue cells per ml could have passed present in BSA, and studies were performed to through the 0.8-,um filter. Larger pore sizes of demonstrate the incorporation of fatty acids by 3.0 and 5.0 ,um allowed spermatozoa, small T. pallidum. Since free fatty acids were preciplymphocytes, and some red blood cells to pass itated with cold trichloroacetic acid, determinathrough the filters. However, prefiltration tions were performed only on medium-washed through 3.0-jum filters permitted 50 to 80 ml of cells. Incorporation of fatty acids into mediumtreponemal suspension to pass through the 0.8,um filter before plugging of the membrane oc- washed cells was observed by using treponemes curred, compared with 30 to 50 ml without pre- extracted in PBS-G and suspended in PBS-G TABLE 1. Use of Nuclepore membranes to remove tissue cells from T. pallidum suspensions Before filtration After filtration Filter size (Am) T. pallidum Tissue cells T. pallidum Tissue cellsa Q(02)b Q(O2) 1.5 x 108 0.0293
INFECTION AND IMMUNITY, Apr. 1977, p. 60-68 Copyright ©D 1977 American Society for Microbiology
Catabolism of Glucose and Fatty Acids by Virulent Treponema pallidum NEAL L. SCHILLER AND C. D. COX* Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01002
Received for publication 22 November 1976
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. ing 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 extracted in the appropriate medium (10 ml/testicle) in an atmosphere of 95% N2 and 5% CO2 by rotation at 70 rpm for 1 h at 25°C, followed by 1 h at 4°C. These extracts were adjusted for cell density in the extraction medium. In previous experiments, some of which we have reported (7), treponemes extracted from tissue by differential centrifugation consumed 02 at levels significantly higher than animal-cell controls from infected tissue. Treponeme and animal-cell preparations were established by procedures previously outlined by us (7) and later described by Nichols and Baseman (20). In the present studies tissue extracts were centrifuged at 300 x g for 10 min to remove the majority of contaminating tissue in the low-speed pellet (LSP). The supernatant fluid (LSS) was filtered through Nucleopore filters (see Results), and the filtered suspension of treponemes was centrifuged at 17,000 x g for 30 min. Treponemes in the high-speed pellet (HSP) were resuspended in a small amount of high-speed supernatant fluid (HSS) and used in incorporation and radiorespirometric experiments. Enzyme assays were performed on sonically treated treponemes from HSP. The tissue-cell controls consisted of tissue cells from LSP resuspended in HSS to a concentration of 104/ml, or about 500 times higher than detectable levels in the filtered
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 consist60
VOL. 16, 1977
CATABOLISM BY VIRULENT T. PALLIDUM
treponemal suspension or the HSP. The other control consisted of HSS to reflect soluble or membraneassociated enzyme activity resulting from the few tissue cells and/or treponemes. Tissue-cell controls were used to monitor and correct values in incorporation studies, and both tissue-cell and HSS controls were used similarly in metabolic experiments. Incorporation experiments. PBS-G was selected as the extraction medium since it was free from glucose and fatty acids, thereby enhancing the chance of detecting any uptake of "4C-labeled substrate added to this medium. After the addition of isotopically labeled compounds to treponeme suspensions, the samples were swirled on a platform rotator at 120 rpm at 25°C. Preliminary experiments in several metabolic studies indicated no substantial differences among those performed at 25, 30, or 37°C, and room temperature of 250C was chosen for these and subsequent experiments. At selected time intervals, samples of cell suspensions were removed, incubated at 56°C for 5 min, and centrifuged at 30,000 x g for 30 min. The cells were washed in PBS-G and centrifuged as before, and the pellet was suspended in absolute ethanol and transferred to scintillation vials. After evaporation, the residue was resuspended in scintillation fluid, and radioactivity was determined in a Beckman LS-100 liquid scintillation counter. The efficiency of counting in these experiments was 95%. The scintillation fluid contained 700 ml of scintillation-grade toluene, 300 ml of absolute ethanol, 0.6% 2,5-diphenyloxazole (PPO), and 0.1% p-bis-(o-methylstyryl)-benzene (bis-MSB). In some experiments uptake of radioisotopes was terminated by the addition of trichloroacetic acid. The cells were collected by centrifugation and washed once with PBS-G. They were then washed in cold 5% trichloroacetic acid, suspended, and transferred to scintillation vials as described above. Radiorespirometric experiments. The extraction fluid contained 75 mM sodium citrate and 10% (vol/ vol) heat-inactivated pooled rabbit serum. Assays were performed in a 10-ml Erlenmeyer flask sealed with a rubber serum stopper, with a plastic center well (Kontes Glass Co.) suspended from the stopper. After 1.8 ml of the appropriate solution or suspension was added, the flask was stoppered, 0.2 ml of Hyamine hydroxide was injected into the center well, and 0.2 ml of the isotope solution was added to the solution or suspension. The flasks were incubated at 250C and rotated on a platform shaker at 70 rpm. At the end of the reaction period of 2 h, 0.2 ml of 3 M H2SO4 was added to the reaction mixture to release any soluble C02, and the flasks were incubated for an additional 60 min. The flasks were then opened. The cup was immediately placed into 15 ml of scintillation fluid and radioactivity was measured. Total glucose present was determined with the Glucostat assay (Worthington Biochemicals Corp.). Enzyme assays. Testicles were extracted in PBSG, and the second extraction was performed with fresh PBS-G for 1 h at 25°C. The HSP was washed in 10 ml of 0.02 M sodium phosphate buffer (PBS), pH 7.2, resuspended in a small volume of PBS, and either frozen or used immediately.
61
Cells were disrupted by sonic treatment in a Raytheon Sonifier using the minimal exposure necessary for the disappearance of all intact treponemes as observed by dark-field microscopy. At least three 1-min bursts were usually required. Cellular debris was removed by centrifugation for 20 min at 12,000 x g, and the supernatant fraction was used as a source of soluble enzymes. The pellet was resuspended in PBS and used as a source of membraneassociated enzymes. Extracts were kept at 4°C and used immediately for enzyme assays. Protein was determined by the Folin phenol method (15). Assays were made on extracts containing 10 to 100 ,ug of protein. Most enzyme assays involved coupling the specific reaction to be determined to a pyridine nucleotide-dependent system and, unless otherwise noted, followed the general procedures described in Methods in Enzymology, vol. 1 (6). Oxidation or reduction of pyridine nucleotides was followed at 340 nm in a Gilford model 240 recording spectrophotometer. A total volume of 0.15 ml was used in each cuvette unless otherwise noted. One unit of enzyme activity was defined as the amount of enzyme resulting in the oxidation or reduction of 1 ,umol of pyridine nucleotide per min at 25°C. Specific activities are expressed in terms of units of enzyme per milligram of crude treponemal extract protein. Control cuvettes lacking various components of the specific assay, or treponemal extract, or including enzymes from other sources were included for each assay. Enzymes of the citric acid cycle were determined in the following manner. The malate dehydrogenase (EC 1.1.1.37) assay reaction mixture contained 10.0 ,umol of tris(hydroxymethyl)aminomethane (Tris) buffer (pH 8.0), 2.0 ,umol of potassium oxalacetate, 1.0 gmol of MgSO4, 0.075 ,imol of NADH, and 1.0 ,unmol of 2-mercaptoethanol. A reaction mixture for measuring citrate synthase (EC 4.1.3.7) was employed which required the presence of malate dehydrogenase in the extract. This enzyme system combined 10.0 ,umol of Tris buffer (pH 8.0), 2.0 gtmol of sodium malate, 0.3 ymol of acetyl coenzyme A (acetyl-CoA), 1.0 ,umol of MgSO4, 0.075 ,umol of nicotinamide adenine dinucleotide (NAD), 1.0 ,umol of 2mercaptoethanol, and pig heart malic dehydrogenase. Fumarate hydratase (EC 4.2.1.2) activity was estimated by replacing sodium malate in the prior assay with potassium fumarate. Fumarate hydratase was also assayed according to a procedure described by Massey (17) which was based on the high ultraviolet absorption of fumarate. This assay mixture contained 2.0 ,mol of potassium fumarate, 0.02 M sodium phosphate buffer, pH 7.2, and cell-free extract in a total volume of 0.15 ml. The consumption of fumarate was monitored by following the decrease in absorbance at 300 nm. Isocitrate dehydrogenase (EC 1.1.1.42) assay mixture contained 10.0 ,imol of Tris buffer (pH 8.0), 2.0 ,umol of sodium isocitrate, 1.0 ,umol of MgSO4, and 0.075 ,umol of nicotinamide adenine dinucleotide phosphate (NADP). Sodium citrate substituted for isocitrate in assays for aconitate hydratase (EC 4.2.1.3). Oxoglutarate dehydrogenase (EC 1.2.4.2) reaction mixture contained 10.0 ,mol of Tris buffer (pH 8.0), 2.0 ,umol of sodium oxoglutarate, 0.075 zmol of NAD, and 0.2
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SCHILLER AND COX
,umol of coenzyme A (CoA). Succinic dehydrogenase (EC 1.3.99.1) was assayed according to the procedure described by Massey (18), with a 3.0-ml reaction mixture. Isocitrate lyase (EC 4.1.3.1) was measured essentially by the method described by Reeves and Ajl (23), with the following components in a 0.15-ml reaction mixture: 10.0 gmol of Tris buffer (pH 7.3), 1.0 umoi of MgSO4, 1.0 gmol of phenylhydrazine hydrochloride, 0.1 ,umol of cysteine hydrochloride, and 2.0 ,umol of sodium isocitrate. The malate dehydrogenase (decarboxylating) (NADP+) enzyme (EC 1.1.1.40) was assayed essentially as described by Ochoa (21) in a reaction mixture containing 10.0 ,umol of Tris buffer (pH 7.3), 1.0 ,mol of MgSO4, 0.075 ,umol of NADP, and 2.0 ,tmol of either sodium malate or potassium oxalacetate. Glutamate dehydrogenase (EC 1.4.1.2) was assayed according to a procedure described by Strecker (25). When assayed in the forward direction, the assay mixture contained 10.0 umol of Tris buffer (pH 8.0), 0.075 ,umol of NAD, and 2.0 ,tmol of potassium glutamate. For the reverse reaction, the assay mixture contained 10.0 gmol of Tris buffer (pH 8.0), 1.0 ,umol of NH4Cl, 0.075 ,tmol of NADH, and 2.0 i,mol of sodium oxoglutarate. The acetyl-CoA acetyltransferase (EC 2.3.1.9) assay followed the general procedure described by Henneberry and Cox (12), with the following components in 0.15 ml: 10.0 ,umol of Tris buffer (pH 8.0), 1.0 ,mol of MgSO4, 0.05 gmol of CoA, 0.075 ,mol of NAD, 2.0 ,umol of sodium malate, 0.12 umol of acetoacetyl-CoA, 1.0 ,umol of dithiothreitol, 0.92 unit of pig heart citrate synthase, and 1.2 units of pig heart malic dehydrogenase. Enoyl-CoA hydratase (EC 4.2.1.17) was measured according to the procedure of Henneberry and Cox (12), with a reaction mixture containing 10.0 ,umol of Tris buffer (pH 8.0), 1.0 ,umol of dithiothreitol, 1.0 ,umol of MgCl2, 0.075 ,umol of NAD, 0.6 ,umol of crotonyl-CoA, and 0.04 unit of pig heart 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35). The presence of 3-hydroxyacyl-CoA dehydrogenase was determined by employing the same assay mixture without the commercial enzyme. Enzymes of the glycolytic and pentose shunt pathways were also investigated. Glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.12) was measured in a reaction mixture containing 10.0 ,umol of Tris buffer (pH 8.0), 0.075 ,umol of NAD, 2.5 ,umol of sodium arsenate, 1.0 ,umol of 2-mercaptoethanol, and 2.0 ,umol of glyceraldehyde-3-phosphate. Attempts were made to couple other enzymatic reactions to reduction of NAD via glyceraldehyde-phosphate dehydrogenase. Fructose-biphosphate aldolase (EC 4.1.2.13) was assayed by replacing glyceraldehyde-3-phosphate with fructose-1,6-diphosphate. Triosephosphate isomerase (EC 5.3.1.1) was assayed by replacing glyceraldehyde-3-phosphate with dihydroxyacetone phosphate and by substituting triethanolamine buffer (pH 8.5) for Tris buffer. 6-Phosphofructokinase (EC 2.7.1.11) was assayed with fructose-6-phosphate as the substrate, with the addition of 0.75 ,umol of adenosine triphosphate (ATP) and 1.0 ,mol of MgSO4. Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was assayed in a reaction mixture containing 10.0
INFECT. IMMUN.
,tmol of Tris buffer (pH 8.0), 1.0 ,umol of MgSO4, 0.075 ,umol of NADP, and 2.0 /mol of glucose-6phosphate. Phosphogluconate dehydrogenase (EC 1.1.1.43) was estimated by replacing glucose-6-phosphate with 6-phosphogluconate. The presence of glucose-6-phosphate dehydrogenase enabled the measurement of two enzymes by coupling these enzymes to the dehydrogenase assay. Hexokinase (EC 2.7.1.1) was measured by replacing glucose-6-phosphate with glucose and by adding 0.75 Amol of ATP. Glucosephosphate isomerase (EC 5.3.1.9) was assayed with 2.0 ,hmol of fructose-6-phosphate used as substrate in place of glucose-6-phosphate. Determinations of lactate dehydrogenase (EC 1.1.1.27) involved a system containing 10.0 ,umol of Tris buffer (pH 8.0), 2.0 ,umol of sodium pyruvate, 0.1 ,Lmol of ZnC12, 1.0 ,umol of MgSO4, and 0.075 ,umol of NADH. Pyruvate kinase (EC 2.7.1.40) was determined by coupling pyruvate formation to lactate dehydrogenase-mediated consumption of NADH. Reaction mixtures contained 10.0 ,umol of triethanolamine buffer (pH 8.5), 2.0 umol of phosphoenolpyruvate (PEP), 0.1 ,umol of ZnCl2, 1.0 ,umol of MgSO4, 0.75 ,umol of adenosine diphosphate (ADP), 1.0 ,tmol of 2-mercaptoethanol, and 0.075 ,umol of NADH. For determination of enolase (EC 4.2.1.11) and phosphoglyceromutase (EC 2.7.5.3) activity, the pyruvate kinase assay mixture was supplemented with 2.0 umol of 3-phosphoglycerate and 2.2 units of rabbit muscle pyruvate kinase, and PEP was omitted. In all assays where NADH consumption was measured, activities were corrected for NADH dehydrogenase activity. NADH dehydrogenase (EC 1.6.99.3) was assayed in a reaction mixture c6ntaining 10.0 ,umol of Tris buffer (pH 8.0) and 0.075 ,umol of NADH. The pyruvate dehydrogenase (EC 1.2.4.1) assay was performed by a method described by Korkes (13) and contained 10.0 umol of Tris buffer (pH 8.0), 1.0 ,umol of MgSO4, 0.1 umol of CoA, 0.075 ,umol of NAD, 1.0 ,umol of reduced glutathione, 0.1 ,umol of thiamine pyrophosphate, and 2.0 ,umol of sodium pyruvate. Pyruvate oxidase (EC 1.2.3.3) was assayed as described by Hager and Lipmann (10), and acetyl phosphate formation was measured by the hydroxamic method of Lipmann and Tuttle (14). Pyruvate dehydrogenase (cytochrome) (EC 1.2.2.2) was determined by the procedure described by Williams and Hager (32). 14CO2 was trapped and measured as described earlier for the radiorespirometric experiments. Chemicals. [U-14C]oleate and [U-14C]pyruvate were purchased from ICN Radioisotope Division; all other radioactive compounds were obtained from New England Nuclear Corp. All chemicals were reagent grade or the equivalent. Sigma Chemical Co. was the source of all commercial enzymes used in this study.
RESULTS Removal of tissue cells from suspensions of T. pallidum. Although the previous extraction procedure (7) seemed adequate for 02 uptake
VOL. 16, 1977
CATABOLISM BY VIRULENT T. PALLIDUM
63
studies, the use of this preparation for sub- filtration. Filters of smaller pore sizes were not strate oxidation and enzymatic analyses was tested. considered unacceptable. Therefore, attempts The metabolic activity of the treponemes bewere made to free these treponemal preparafore and after filtration was examined by detertions from contaminating tissue cells. Several mination of Q(02) values as described by Cox investigators have examined various tech- and Barber (7). As shown in Table 1, the metaniques to obtain such preparations. These bolic activity of the treponemes was maintained methods include density gradient centrifuga- after passage through 0.8- or 1.0-,um Nucleotion (22), continuous-flow zonal centrifugation pore filters. Removal of tissue cells from trepoin cesium chloride gradients (26), discontinuous neme suspensions without decreasing Q(02) gradients of sodium and meglumine diatri- values further supports the previous conclusion zoates (4), and membrane filtration procedures (7) that 02 consumption was associated with T. (5). All of these techniques damaged the trepo- pallidum. Although the filtration procedure nemes except the filtration procedure, which was successful in removing essentially all tisremoved too many treponemes. However, J. W. sue cells from the treponeme suspension, the Foster suggested the use of Nuclepore filters for possibility remained that a few tissue cells and this purpose (personal communication), and soluble enzymes or membranes from tissue several experiments were designed to investi- cells or treponemes might contribute to the gate this possibility. metabolic activities observed. Thus, activities Nucleopore membranes ranging from 0.8- to of the tissue-cell and HSS controls continued to 5.0-,um pore sizes were examined in an effort to be routinely monitored. remove all observable tissue cells while allowSubstrate incorporation studies. Several ing full recovery of the treponemes. Cells were 14C-labeled compounds were added to restingcounted immediately before and after filtration cell suspensions of T. pallidum in an attempt to by use of a Petroff-Hausser counting chamber define compounds which might serve as a major with dark-field illumination. Either 25- or 47- source of cell carbon. At indicated times, sammm diameter filters were routinely used with ples were removed and the amount of incorpomild vacuum. The limit of cell detection, based rated radioactivity was determined. [Uon the observation of 1 or 2 cells per entire 14C]glucose was not incorporated into either counting chamber, was between 5 x 103 and 1 x medium- or trichloroacetic acid-washed cells 104 cells per ml. Both 0.8- and 1.0-,um Nucleo- during a 2-h test period. Incorporation over pore filters consistently removed all observable longer time periods was not examined because tissue cells while allowing essentially full re- the motility of the treponemes decreased after 2 covery of T. pallidum (Table 1). The sensitivity h. of the counting procedure was increased by cenNelson (19) and Weber (30) have reported trifuging the filtrate at 35,000 x g for 30 min that bovine serum albumin (BSA) prolonged and resuspending the pellet in smaller vol- treponemal survival. It seemed reasonable that umes. This procedure indicated that less than this might reflect a requirement for fatty acids 200 tissue cells per ml could have passed present in BSA, and studies were performed to through the 0.8-,um filter. Larger pore sizes of demonstrate the incorporation of fatty acids by 3.0 and 5.0 ,um allowed spermatozoa, small T. pallidum. Since free fatty acids were preciplymphocytes, and some red blood cells to pass itated with cold trichloroacetic acid, determinathrough the filters. However, prefiltration tions were performed only on medium-washed through 3.0-jum filters permitted 50 to 80 ml of cells. Incorporation of fatty acids into mediumtreponemal suspension to pass through the 0.8,um filter before plugging of the membrane oc- washed cells was observed by using treponemes curred, compared with 30 to 50 ml without pre- extracted in PBS-G and suspended in PBS-G TABLE 1. Use of Nuclepore membranes to remove tissue cells from T. pallidum suspensions Before filtration After filtration Filter size (Am) T. pallidum Tissue cells T. pallidum Tissue cellsa Q(02)b Q(O2) 1.5 x 108 0.0293
