Embden-Meyerhof Pathway

Transcription

1 JOURNAL OF BACTERIOLOGY, Sept. 1971, p Vol. 17, No. 3 Copyright 1971 American Society for Microbiology Printed in U.S.A. Biochemistry of Coxiella burnetii: Embden-Meyerhof Pathway THOMAS L. McDONALD AND L. MALLAVIA Department of Bacteriology and Public Health, Washington State University, Pullman, Washington Received for publication 8 February 1971 Purified preparations of Coxiella burnetii were examined for enymes of the glycolytic pathway. Glucose-phosphate isomerase, fructose-1,6-diphosphatase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase were shown to be present in C. burnetii extracts. Heat-killed C. burnetii purified with normal yolk sacs demonstrated no activity after disruption. Aldolase was shown to be of the class II type by complete inhibition of activity in the presence of 8 x 1-3 M ethylenediaminetetraacetic acid. The host enyme activity (normal and infected yolk sacs) was not affected by the same treatment. When cellulose acetate electrophoresis was performed on the extracts, aldolase from both normal and infected yolk sacs exhibited five isoyme bands, whereas aldolase from the C. burnetii extract appeared as a single band. Studies in recent years have described a number of host-independent reactions carried out by Coxiella burnetii. These capabilities have been the subject of several reviews (1, 12). Although a variety of enymatic capabilities have been found, attempts to demonstrate exogenous glucose utiliation by whole cell suspensions of rickettsiae have been uniformly unsuccessful. The evidence that rickettsiae possess some mechanism of glucose catabolism was first provided by the demonstration of glucose-6-phosphate oxidation by cell-free extracts of C. burnetii (4). Additional evidence was recently published from this laboratory (8) which demonstrated 6-phosphogluconic acid dehydrogenase activity in this organism. The presence of these enymes has suggested that the organism can catabolie glucose via the hexose monophosphate shunt. There have been, however, no reports as to the presence or absence of enymes of the Embden-Meyerhof pathway in rickettsiae. In this paper, evidence is presented which demonstrates several glycolytic enymes in cellfree extracts of C. burnetii. MATERIALS AND METHODS Organism. C. burnetii, Nine Mile strain, phase I, was propagated in embryonated eggs, and rickettsial suspensions were prepared from infected yolk sacs by a procedure previously described (8). Extract preparation. The organisms obtained from three purifications were resuspended in 15 ml of.5 M tris(hydroxymethyl )aminomethane (Tris )-glycine buffer, ph 8.3 (28.8 g of glycine, 6. g of Tris/liter) containing acid-washed No. 13 Ballotini beads previously cooled to C (1.5 g of wet beads/ml of suspen- 864 sion). This concentrated rickettsial sispension was disrupted by using a carbon dioxide-cooled Braun model MSK homogenier (Quigley-Rochester, Inc., Rochester, N.Y.) for 1.5 min. After treatment, the glass beads were removed by centrifugation at 2 x g for I min. The preparation was then centrifuged at 21, x g for 3 min at 4 C, and the pellet was discarded. Nucleic acids were removed from the supernatant fraction by the method of Merrick and Doudoroff (9), and the cell-free extract was assayed for enymatic activity. Normal and infected yolk sac extracts were prepared by adding 15 g of 16-day-old yolk sacs and 15 ml of sucrose phosphate buffer (ph 7.4) in a ground-glass homogenier at 4 C. After homogeniation, the material was centrifuged at 12, x g for 15 min, and the liquid between the upper lipid layer and the precipitate was carefully aspirated. This material was centrifuged (model B6, International Equipment Co., Needham Hts., Mass.) at 12, x g in a swinging bucket rotor at 4 C for I hr, and the clear supernatant fluid was assayed for enymatic activity. Protein determinations.the protein concentration of all extracts was determined by the method of Lowry et al. (7). The protein standard consisted of purified, recrystallied bovine serum albumin. Assay procedures. All chemicals and commercial enymes were obtained from Sigma Chemical Co., St. Louis, Mo. In all assays, auxiliary enymes were added in excess, and the reactions were carried out at 25 C in a model DU quart spectrophotometer (Beckman Instruments, Fullerton, Calif.). The final volume of all reaction mixtures was 2.5 ml. Glucose phosphate isomerase was assayed by enymatic coupling to glucose-6-phosphate dehydrogenase and observing the reduction of nicotinamide adenine dinucleotide phosphate (NADP) at 34 nm. The mixture consisted of 1. umoles of fructose-6-phosphate;.65 gmole of NADP, glucose 6-phosphate dehydrogenase;.1 ml of extract; and 2. ml of.5 M Tris-

2 VOL. 17, 1971 EMBDEN-MEYERHOF PATHWAY IN C. BURNETII glycine buffer (ph 8.3). Fructose-1,6-diphosphatase assay was based on the procedure of Pontremoli (15). The reaction mixture consisted of 15. Mmoles of reduced glutathione;.65 umole of NADP; 1.,umoles of MgCI2; 1. mmoles of fructose- I, 6-diphosphate, glucose-6-phosphate dehydrogenase, glucose-phosphate isomerase;.1 ml of extract; and 2. ml of.5 M Tris-glycine buffer (ph 8.3). Phosphofructokinase activity was determined by a method adapted from Sols and Salas (19). The cuvette contained.5 tsmole of adenosine-5-triphosphate (ATP);.3 gmole of reduced nicotinamide adenine dinucleotide (NADH); 1. mmoles of fructose-6- phosphate, aldolase, a-glycerophosphate dehydrogenase, and triosephosphate isomerase;.1 ml of extract; and 2. ml of.1 M glycylglycine buffer (ph 7.5). Aldolase was assayed as described by Richards and Rutter (17). The cuvette contained.3 grmole of NADH; 1. mmoles of fructose-1,6-diphosphate, a- glycerophosphate dehydrogenase, and triosephosphate isomerase;.1 ml of extract; and 2.2 ml of.1 M glycylglycine buffer (ph 7.5). Glyceraldehyde-3-phosphate dehydrogenase was determined essentially as described by Velick (21). The cuvette contained 1.5 umoles of NAD, 1. gmole of sodium arsenate, 3.4 umoles of glyceraldehyde-3-phosphate,.1 ml of extract, and 2. ml of.5 M Tris-glycine buffer (ph 8.3). For this assay, the extract was treated with 24 urmoles of reduced glutathione immediately after nucleic acid precipitation. Pyruvate kinase was measured essentially as described by Valentine and Tanaka (2). The reaction mixture contained 6.,umoles of ADP;.3 grmole of NADH; 15.6,umoles of phosphoenolpyruvate, lactic acid dehydrogenase;.1 ml of extract, and 2 ml of.5 M Tris-glycine buffer (ph 8.3). Controls were employed throughout the experiments to assure that the activity observed was rickettsial-specific. Purified, heat-killed C. burnetii was added to normal yolk sacs and repurified. Enyme activity present in these extracts after disruption would be indicative of host enyme contamination. A second control was performed by incubation of whole cells of purified C. burnetii with the reaction mixtures. Enyme activity present in these suspensions was indicative of host enyme absorbed to the exterior of the C. burnetii cells. Cellulose acetate electrophoresis. The electrophoretic migration of aldolase was performed as described by Penhoet et al. (14). The buffer consisted of.6 M sodium barbital containing 1. mm beta-mercaptoethanol adjusted to ph 8.6. A constant current of 1.5 ma per strip was applied for 6 min at 4 C. The strips were stained for activity at 37 C on agar slabs containing.5% Noble agar,.1 M sodium arsenate, 1. mmoles of fructose-1,6-diphosphate,.1 mg of glyceraldehyde- 3-phosphate dehydrogenase per ml,.4 mg of nitroblue tetraolium per ml,.25 mg of phenainemethylsulfate per ml, and.2 mg of NA.D per ml in.1 M glycylglycine buffer (ph 7.5). RESULTS The examination of cell-free extracts of C. burnetii has demonstrated a number of enymes normally associated with the Embden-Meyerhof pathway. As can be seen in Table 1, phosphohexose isomerase, fructose- 1, 6-diphosphatase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase were found to be present in the extracts. Controls employing heatkilled, repurified C. burnetii as well as whole cell assays of viable organisms were routinely incorporated throughout these studies. No enymatic activity was observed with these controls. Considerable difficulty was encountered in our attempts to demonstrate phosphofructokinase activity in C. burnetii extracts. The activity of this enyme was extremely low and difficult to demonstrate consistently. Attempts to increase activity levels by alteration of the ADP/ATP ratio in the reaction mixture as well as varying the ionic environment were unsuccessful. In view of the variability, it is impossible to state at this time whether the enyme is present in the C. burnetii extracts. Initial experiments indicated that glyceraldehyde-3-phosphate dehydrogenase was present although, as with phosphofructokinase, considerable variation occurred with some extracts having negligible activity. The variation in activity of this enyme was eliminated by preincubation of the extracts with reduced glutathione. The activity observed with glutathione-pretreated extracts was approximately three times greater than that observed with untreated extracts (Fig. 1). As expected, linearity of glyceraldehyde-3- phosphate dehydrogenase activity with respect to time was only obtained during the early stages of the assay. Under saturating conditions of substrate and cofactors, ero-order kinetics are not observed in this enyme. Phosphohexose isomerase, fructose- 1, 6-diphosphatase, aldolase, and pyruvate kinase were consistently demonstrable in all rickettsial extracts examined and exhibited little or no variation in activity from one experiment to the next. TABLE 1. Specific activities ofglycolytic enymes in Coxiella burhetiia Enymes Activityb Phosphohexose isomerase Phosphofructokinase Fructose- I, 6-diphosphatase Aldolase Glyceraldehyde-3-phosphate dehydrogenase 5.7 Pyruvate kinase a Each cuvette contained.3 mg of C. burnetii protein in a final volume of 2.5 ml. Reaction conditions for enyme assays are described in the text. b Expressed as nanomoles per minute per milligram of protein.

3 866 McDONALD AND MALLAVIA J. BACTERIOL. c] w w a- a- 4 LAJ a. a- IYS C BURNETII NYS FIG. 1. Effect of reduced glutathione on glyceraldehyde-3-phosphate dehydrogenase activity in C. burnetii. Each cuvette contained.2 mg of protein in a final volume of 2.5 ml. In extracts containing reduced glutathione, 24 Mmoles/ml was added immediately after nucleic acid precipitation. Other reaction conditions are described in the text. Activity is expressed as optical density per milligram ofprotein. It was of interest to examine aldolase in more detail because of its unique role in glycolysis and gluconeogenesis. In addition, the techniques of Richards and Rutter (17) and Rutter (18) allowed the separation of host and rickettsial aldolases by identification of their respective class types. Theoretically, therefore, this would permit an examination of differences (or similarities) between the rickettsial and host enyme. Extracts of normal and infected yolk sacs were also assayed for aldolase activity for comparative purposes. Aldolase was present in all extracts examined (Fig. 2). A 3-min preincubation of the extracts in 8. x 1-3 M ethylenediaminetetraacetic acid (EDTA) completely inhibited C. burnetii aldolase activity which is indicative of class II type aldolase (Fig. 3). In contrast, aldolase from noninfected host tissue showed no change in activity in the presence of EDTA which is characteristic of class I aldolase. The characteristics of the EDTA inhibition of rickettsial aldolase were more closely examined by altering the time at which the inhibitor was added. In this experiment, the reaction was initiated and monitored for a period of time, and at this point EDTA was added. A lag of approximately 1 min occurs after EDTA addition before complete inhibition occurs (Fig. 4). Aldolase activity of both normal and infected host tissues continue unabated after EDTA addition. The effect of K+ on aldolase activity has been suggested by Rutter (18) as a further criterion for classification of aldolases. He reported that class II aldolases are usually stimulated by K+ O FIG. 2. Aldolase activity of C. burnetii, normal, and infected yolk sacs. Each cuvette contained either.4 mg of infected yolk sac (IYS) protein,.6 mg of normal yolk sac (NYS) protein, or.7 mg of C. burnetii protein in a final volume of 2.5 ml. Other reaction conditions are described in the text. Activity is expressed as optical density per milligram ofprotein. addition, whereas class I aldolases remain unaffected. However, when various concentrations of this ion were added to rickettsial and host tissue extracts, no stimulation was observed. Lebher and Rutter (6) have indicated that many aldolases are composed of a series of isoymes which are readily separated by cellulose acetate electrophoresis. It was of interest, therefore, to examine C. burnetii and host extracts with this technique in an attempt to determine the isoymes characteristic of each system. The results obtained are shown in Fig. 5. Both normal and infected yolk sacs exhibited five visible isoyme bands. Only one activity band was observed with the C. burnetii extract, and its migration was approximately 5 mm farther toward the anode than the front isoyme band of host origin. A mixture of C. burnetii and infected yolk sac extract exhibited six activity bands, each corresponding to its migration pattern in the initial separate runs. DISCUSSION The data presented clearly demonstrate that C. burnetii possesses a number of the enymes involved in glycolysis (Embden-Meyerhof pathway). The complete absence of activity in controls designed to reveal host enyme contamination demonstrates that the enyme activity is of rickettsial origin. The inconsistent data obtained during the initial glyceraldehyde-3-phosphate dehydrogenase assays on cell-free extracts of C. burnetii are not

4 VOL EMBDEN-MEYERHOF PATHWAY IN C. BURNE1I86867 IYS U. w.1 a. 4 NYS +[X, IYS C HJRNE,: FIG. 3. Effect of EDTA on aldolase activity. Extracts were incubated in 8 mm EDTA prior to assay. The protein concentration was as in Fig. 2. Activity is expressed as optical density per milligram ofprotein. UA. w 4 a.. o* I, -_ I l O IYS + C. BURNETII - +I) (lfil X ' ti1ioio(j'j C. BURNETII - ORIGIN C BlJRNETII FIG. 5. Cellulose acetate electrophoresis of C. burnetii and yolk sac aldolase. The origin was spotted with either.3 mg of C. burnetii protein,.4 mg of in- NYS fected yolk sac (IYS) protein or.18 mg of normal yolk sac (NYS) protein. The mixture was prepared by mixing nine parts C. burnetii extract with one part I YS extract. Approximately 12,uliters of this mixture was - spotted which represented.21 mg of C. burnetii protein and.15 mg of IYS protein. Electrophoresis was FIG. 4. Effect of EDTA on aldolase activity. The performed as described in the text. reaction was initiated and followed for 3 mi, In, at which time 8 mm EDTA was added. (Arrow indi, [cates point The inability to demonstrate consistently phos- was as phofructokinase in C. burnetii extracts is some- of EDTA addition.) The protein concentration in Fig. 2. Activity is expressed as optical Idensity per what more difficult to understand. It has been milligram of protein. reported that this enyme is an allosteric enyme surprising. Crystalline glyceraldehycde-3-phos- in which the concentration of reactants and their phate dehydrogenase from rabbit m uscle has ratios are critical for maximum activity (1). Alwhen a 1- though several attempts to obtain the proper re- been shown to lose 17% of its activity mg solution is incubated at 3 C for 3 min at action conditions were made, they were by no ph 7.3 (5). The activity loss was repoirted to be means exhaustive. The fact that activity is demuch greater when dilute solutions caf the en- monstrable at times suggests that the enyme is yme were used; however, loss of ac,tivity was present in the organism. It would appear that the prevented by the addition of cysteine or gluta- proper reaction conditions will have to be defined thione. More recently, Bergmeyer (2' reported before increased activity will be observed. that this enyme contains 12 to 16 sulftydryl C. burnetii aldolase was shown to be of the groups which render it extremely susc-eptible to class II type by using criteria established by Riwith these chards and Rutter (17). In contrast, aldolase oxidation and inactivation. Consistent observations are our findings that the a.ddition of present in normal and infected yolk sacs was reduced glutathione to the C. burnetiii extracts shown to be of the class I type. EDTA inhibition resulted in reproducible and increased activity of rickettsial aldolase is characteried by a rapid levels. interference with enyme activity which eventu-

5 868 McDONALD A ID MALLAVIA J. BACTERIOL. ally results in complete inactivation. The chelation of divalent cations required for rickettsial aldolase activity occurs within I min after the addition of EDTA. Since there appears to be no requirement for divalent cations by host (class 1) aldolase, inactivation by EDTA was neither expected nor observed. It has been reported that.1 M K+ may stimulate class II aldolases (18). Although K+ stimulation does not occur with all class II type aldolases, a 34-fold increase in activity has been reported with yeast (18). Addition of this ion to C. burnetii extracts did not alter the aldolase activity. Although it is possible that the enyme is not stimulated in a manner similar to that reported for yeast, a further possibility is that the enyme has been saturated by K+ prior to initial assay. The organisms are purified in a buffer containing.14 M KCI and could take up considerable quantities of K+. Moreover, some is undoubtedly carried over when the cells are resuspended in the experimental buffer. In view of this, it is possible that the activity observed was the stimulated activity and, therefore, no additional K+ stimulation occurred. The data obtained with cellulose acetate electrophoresis reemphasied the difference between rickettsial and host aldolase. The aldolase activity of C. burnetii differed not only in the number of isoyme bands but also in the distance of migration. No difference was observed between the isoyme patterns of normal and infected tissue. Some difference may have been expected since the activity of aldolase from infected tissue is fourfold higher than the activity of normal tissue. This increase in activity was not due to the synthesis of a new isoyme or the alteration of existing isoymes such that they would become distinguishable by electrophoretic pattern changes. Research is currently underway to examine the increases in specific activity of enymes of the glycolytic pathway as a host response to infection. With the demonstration of glucose-6-phosphate dehydrogenase (4) and now several enymes of the glycolytic pathway, a cyclic dissimilation of glucose may be proposed utiliing both the pentose phosphate pathway and the Embden- Meyerhof pathways. It may be that in the in vivo situation glucose uptake by rickettsiae does occur; however, this remains questionable. It has been shown that intact rickettsiae utilie pyruvate (3, 11). Also, rickettsiae have been shown to possess at least a portion of the tricarboxylic acid cycle (13, 16). This leads to the possibility that rickettsiae utilie host materials at the 3- carbon level for energy and synthesis. However, since 5 and 6 carbon sugars are required for cell ~N wall and nucleic acid synthesis, the rickettsiae may further utilie 3-carbon compounds for these purposes. This would require the reversal of the rickettsial glycolytic pathway resulting in the formation of 6-carbon compounds which feed into cell wall or into nucleic acid syntheses via the pentose phosphate pathway. ACKNOWLEDGMENTS This investigation was supported by funds provided for biological and medical research by the State of Washington Initiative Measure no LITERATURE CITED 1. Atkinson, D. E Regulation of enyme function. Annu. Rev. Microbiol. 23: Bergemeyer, H. U Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle, p In H. U. Bergemeyer (ed.), Methods of enyme analysis. Academic Press Inc., New York. 3. Bovernick, M. R., and J. C. Snyder Respiration of typhus rickettsiae. J. Biol. Chem. 154: Consigli, R. A., and D. Paretsky Oxidations of glucose-6-phosphate and isocitrate by Coxiella burnetii. J. Bacteriol. 83: Cori, G. T., M. W. Slein, and C. F. Cori Crystalline D-glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle. J. Biol. Chem. 173: Lebher, H. G., and W. J. 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6 VOL. 17, 1971 EMBDEN-MEYERHOF PATHWAY IN C. BURNETII 869 Methods in enymology, vol. 9. Academic Press Inc., New York. 2. Valentine, W. N., and K. R. Tanaka Pyruvate kinase: clinical aspects, p In S. P. Colowick and N.. Kaplan (ed.), Methods in enymology, vol. 9. Academic Press Inc., New York. 21. Velick, S. F Glyceraldehyde-3-phosphate dehydrogenase from muscle, p In S. P. Colowick and N.. Kaplan (ed.), Methods in enymology, vol. 1. Academic Press Inc., New York.