SOME ASPECTS OF THE CARBCHYDRATE METABOLISM OF CERTATN TRICHOMONADS Thesis for tin Degree of Pb, D. MICHIGAN STATE UNIVERSITY Gordon Paul Lindblom 1957 Cl This is to certify that the thesis entitled Some Aspects of the Carbohydrate Metabolism of Certain Trichomonads presented by / Gordon Paul Lindblom has been accepted towards fulfillment of the requirements for M.— degree in _Miczobialogy /’f _ - a , r ‘A .- ///;LZ//(2 1m L/X 3, ”41 2/4111 // Major professor; Date _Ma.y 9: 195'] LIBRARY Michigan Stave University SOME ASPECTS OF THE CARBOHYDRATE METABOLISM OF CERTAIN TRICHOHDNADS BY Gordon Paul Lindblom A THESIS Subndtted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirement: for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1957 ACKNOWLEDGEMENTS The author wishes to express his sincere thanks to his major adviser, Dr. w. D. Lindquist, for his constant encouragement and helpful advice; and for making avail- able certain research funds for equipment and supplies, without which this work could not have been done. He also wishes to thank Dr. W. N. Mack, who permitted use of the Raytheon Sonic Oscillator; Dr. R. N. Costilow, for the use of the Beckman DU spectrophotometer and other instruments; and Dr. C. L. San Clemente for loaning a micro-Kjeldahl digestion apparatus. Special thanks are also due Dr. D. T. Clark, Dr. J. J. Stockton, Mr. I. L. Dahljelm.and his staff, Dr. H. L. Sadoff, Dr. R. U. Byerrum, Dr. Hans Lillevik, and Dr. Sam Rosen, who contributed certain essential materials and who offered useful suggestions during the course of this work . Finally, to all those whose suggestions, help, or encouragement contributed in any way to the completion of this project, the author expresses his sincere appreciation. TABLE OF CONTENTS INTRODUCTIONOOOOOOOOOCOOOOOOOOOO00.000.000.0001 REVIEW OF Tm LITERATURE. O C O O O O O O O O O O O O O O O O O 0 C“ A. The par‘Sit.' O O O O O O C O O O O O O O O I O O O O O O O 0 O O 0% B. Protozoan physiolegy and metabolism..... 1. G.n°r.1000000000.0.0.000000000000006 2. Flagell.t°3e O O. O O O O O. O. O O O O O 0 O O O O. .8 3. TP‘. Chomon‘a O O O O l O O I O O O O O O O O O O O O O O O .9 TIE PEQOBI‘EMOOOOOOOOOOOOO0.000.000.0000000000022 MATEIIIALS AND IMTHODSOIOOOOOOCOI. ....... 0.00.23 orgtn18MS.................................23 Culturll mothOdBooco......................23 M1nomotry................................o25 COIl'frO. OXtr‘Ctseeeeeeeeeeeeeeeeeeeeeeeezs Chbmic‘l mothods..........................25 Enzyme determinations.....................26 EXPERIMENTAL RESULTS.........................28 Growth studies............................28 Changes in metabolic activity during culture........................38 Substrate utilization and inhibitors......h5 Hydrogen production by these trichomonads.51 Enzymes of intermediary metabolism........59 DISCUSSIONeoeeeeeeeeeeeeeeeeeeeeee00.000.000.70 SUMMARYeeeeeeeeeeeeeeeeeeeeeeoeeooeee0000000081 REFERENCESeeeeeeeeeeeeeeeeeeeoeeeeeeeeeeeeeeeah ii INTRODUCTION Studies of the chemical activities of micro-organisms have been largely confined to the bacteria. The primary reason for this has been the relative ease with which large quantities of bacteria are cultured, compared to the difficulties encountered with other organisms, es- pecially the protozoa. With the development of more satisfactory methods for growth of protozoa information on their metabolic activities has been gradually accum- ulating. This information, however, is still very sketchy and incomplete. Only a few species have been studied and for these there is but little quantitative information. It has been found that protozoa do not always follow the well-travelled metabolic pathways found in the bacteria, but carry on their cellular chemistry with many modifica- tions and innovations - the details of which are still obscure. Much of the work in this area of research to date has logically been concerned with accumulating basic information regarding the type of metabolism possible, the substrates utilized and those which are necessary, the optimum conditions for the organism's physiological activities, and the effects of various external agents on growth.and metabolism. A few reports on characterization of intracellular enzymes and cell contents have appeared but these are the exception. It is obvious that the study of the biochemistry of protozoa lags far behind that of the bacteria. This is not only a consequence of the problems inherent in working with these organisms but is also due indirectly to the fact that these organisms have been most often studied by biologists who were not specially trained in chemistry. These workers concentrated on elucidating the often complex life cycles of these organisms, and describing their cellular morphology, but did not until recently consider their biochemistry. These barriers are being rapidly removed by the entrance into basic biological research of men trained in chemistry as well as biology. Still, a mass of basic information remains to be detailed. Many more protozoa must be grown in pure culture and investigated for possibly hitherto unknown growth factors, must be examined for their activities in protein, carbohydrate, and lipid metabolism, and infor- mation obtained regarding their enzyme content. The enzymes must be characterized and then compared with those found in other protozoa and other micro-organisms whose chemistry is better understood in order to make the greatest contribution to comparative biochemistry. The flagellate, Trichomonas vaginalis, because of its importance as a parasite of humans, is one of the organisms which has received some attention in this regard. Methods for growing the organism are available and.some of its requirements are known. Other workers have produced brief reports on similar work with.T£i- trichomonas foetus, a pathogen of cattle. However, to date there has been no regular report of research on the metabolic activities of other species of Trichomonas or related forms with an attempt to dis- cover differences, if any, and to relate these to para- sitic habitat. It was the purpose of this work to investigate the application of methods, which had been found to be of value in the study of other microbes, to the identifica- tion of some of the biochemical processes in the carbo- hydrate metabolism of four species of trichomonads. REVIEU OF THE LITLRATURE 5;,Thegparasites The organisms known variously as Trichomonas, Tritrichomonas, Pentatrichomonas, Trichomastix, etc., are flagellates of the order Trichomonadida Kirby, l9u7. Those with which the present work is concerned are placed by Kirby (19k?) in the family Trichomonadidae Wenyon, 1926. At least 90 species of these organisms have been described (Morgan, l9hu), and until Kirby proposed a reclassification the taxonomy was rather confused. Even now, all workers evidently do not accept Kirby's termin- ology. For example, LaPage (1956), Ryley (1955a, 1955b), and Menalosino and Hartman (195h) prefer to place these organisms in the one genus Trichomonas.‘ Kirby's class- ification is used in this work. These protozoa are somewhat pyriform in shape, the posterior end more pointed than the anterior, with 3 to S anteriorly-attached flagella. One of these is directed backwards and united with the body by a well-developed undulating membrane. A stiff fibril, called the £232; , is seen to run along the base of the undulating membrane. In some species a row of deeply staining chromatin granules is present near the goats. A supporting struc- ture, giving stiffness to the body, and often projecting from the posterior end, is the axostyle. Granules are sometimes seen within this structure also. There is a single, oval nucleus near the anterior end of the‘gxg- £21l2° A slit-like cytestome is located anteriorly on the side opposite the undulating membrane. The organisms divide by longitudinal fission and, although there are some reports to the contrary, are not thought to have a cystic stage. The organisms in the present study were Tritrichomonas foetus (Riedmflller, 1928) Henrich and Emmerson, 1933. a parasite of the reproductive tract of cattle (a cause of abortion); Pentatrichomonas gallinarum.(Martin and Robert- son, 1912) Mesnil, l9lu, a parasite of gallinaceous birds; and Trichomonas £31; Gruby and Delefond, lth, a commensal from the intestinal tract of the pig (S23 scrofa). Of the latter, two separate isolates were studied. One was from the normal intestinal habitat. The other was from.the nasal passages (turbinates) of a pig with atrophic rhin- itis. Although attempts have been made to incriminate this organism as the cause of the condition (Spindler, 22'2l., 1953; Switzer, 1951), others (Levine, gt'gl., 19Sh; Lindquist and Sanborn, 1953) have not been able to produce the infection under experimental conditions. It is not yet known what relation, if any, the two organisms have to each other. Buttrey (1956) has reported that the so-called "fecal form” of T:_gui£ is actually a mixture of two types of organisms, a large form and a small form, both of which are distinctly different from that found in the nasal cavity. He believes there is morphologic similarity between the "nasal form" and'z; foetus, and calls it a Tritrichomonas. Sanborn (1955) and Rothenbacher (1956) found that serological differences existed when they produced anti- bodies to these organisms in rabbits. For purposes of this report they will be referred to as the nasal and fecal forms of 2; suis. g; Protozoanphysiology and metabolism 1: General - The biochemistry of protozoa was a rather neglected field of research until about 1951. Al- though some reports had appeared, they were generally unrelated. They concerned the basic nutritional needs of various parasitic flagellates, amoebae, and ciliates; with only a small amount of work on metabolism. A collection of the pertinent data to 1951 was published by Lwoff (1951). An elaboration of some of the metabolic work (primarily on flagellates), and suggestions for future research, was presented by von Brand (1952). The most recent general review (Hutner and Lwoff, 1935) gives a resume of investigations beyond those in the 1951 volume. Discussions are included on comparative flagellate biochemistry (mostly free-living organisms), ciliate nutrition (especially the interesting steroid requirements of Paramecium) and metabolism, slime mold development, mutualistic protozoa, and the physiological basis of chemotherapy of parasitic disease. Despite these excellent recent advances, the amount of information regarding protozoan life processes seems very small when compared to other organisms. Many proto- zoa have not been grown in pure culture, let alone studied chemically. The basic metabolic patterns, so long familiar in other cells, are often extremely modified in protozoa; and details about these aberrant pathways are lacking. Also, growth factors necessary for some protozoa turn out to be related to metazoan vitamins and hormones. In general, a large mass of detail must first be accumulated before generalizations logically can be made. It would seem that two statements by Hutner (1955) are pertinent here. He says, when speaking of the vast amount of work on cultures which yet remains: "...it may demand courage - even the taking of vows of academic poverty - to pursue the lonely, risky enterprise of taming new organisms, yet the growth of protozoology owes more to these pioneers than to those who in biochemical language belabor the point that protozoal protoplasm.resembles other protoplasm." Again, in reference to the use of fundamental research rather than direct, more immediately useful approaches, he says: "(There is) danger (in) the temptation to attack difficult but important problems uneconomically - by frontal assault. The meager results of this strategy when applied to the . . . exacting parasitic protozoa (are obvious). Perhaps the lasting value of these volumes may be their hints on how such scientific outposts may be outflanked when they cannot be stormed." ‘2; Flaggllates - The flagellates which have received the most attention from researchers are the hemo-parasitic Trypanosomidae, which have primitive relatives in the green algae, the chrysomonads (from brown algal stock), and Trichomonas (also from green stock, but farther re- moved than the trypanosomes). Study of the carotenoid pigments of these primitive organisms indicates a sexual function for them (Hutner and Provasoli, 1955). The pathogenic trypanosomes have been studied by several workers (e.g. von Brand, 1952; Harvey, l9h9; Ryley, 1956). Most of them have been found to possess a conventional Meyerhof-Embden glycolytic pathway at least as far as pyruvate, and an oxidative system insensitive to cyanide. Phagotropy has been studied in Paranema, a protozoan related to the green Euglena but without chlorophyll and therefore more'lnimal'in nature. These experiments, designed to analyze, if possible, the biochemical memn- ing of "planm vs. animal", have shown a more specific lipid requirement for Paranema. Several flagellates have been shown to require vitamin 812 - either intact or in a folic acid form (Hutner, 1955). A group of organisms (e.g. Chilomonas) have been found to utilize acetate as a sole source ef carbon. That these "acetate flagellates" have an extremely different internadiary metabolism is becoming apparent. Recent work has shown that sugar phosphates are somehow involved in their metabolism (Hutner and Provasoli, 1955). A few erganisms (e.g. Euglena, Chilomonas, Chlamydo- .92222) give evidence of a conventional tricarboxylic acid (Krebs) cycle (Hutner and Provasoli, 1955). Intracellular polysaccharides have been isolated from several protozoa. One of these, paramylum (frem.Euglena), is a 1-3' glucan, a form unknown in plants, metazoa, er bacteria (Hutner and Provasoli, 1955). An intracellular starch has also been purified from‘gglytomella‘ggggg (Barker and Bourne, 1955). 3;_Trichomonas - Since the present work concerns trichomonad metabolism, the reports of previous research will be considered in greater detail. Witte (1933) showed that‘T; foetus could tolerate a pH range of 5.5 to 8.5. Cailleau (1937a) and Ried- mflller (1936) reported that pH 6.5-7.6 was the optimum range for T; foetus in culture. Morista (1939) found the optimum pH to be 6.6-7.8 with the limits 5.6 and 6.h compatible with survival. Johnson (l9h0)and Trussell 10 and Johnson (l9hl) found‘gs viginalis to reach maximum population densities in culture at a pH value between 5.5 and 6.0 The extreme ranges, beyond which.multiplica- tion ceased, were 5.0 and 7.55. Morgan (l9h2) also studied the pH of cultures of 2;.foetus and reported the final value (when all organisms were dead) was usually 5.3 to 5.5. He also found (Morgan and Whitehair, 19h3) that this organism lives in the reproductive tract of cows at a pH of about 7.1-7.2. The only report at vari- ance with these is that of Tanabe (1925) who reported the optimum pH for culture of trichomonads from.man, rat, and owl to be 8.0-8.2 in a complex medium. In 19h1 Lyford published results she obtained by varying the environment of bacteria-free cultures of Tiffoetus. Some of her more pertinent results follow: (a) the organism.was found to require complex nutrients such as egg yolk, blood, or serum - the latter allowing best growth when used in “0% concentration; (b) the maximum.popu1ation depended on the number of organisms inoculated; (c) growth of'2& foetus in a medium containing dextrose resulted in a decrease in pH of the medium to about h.8; (d) addition of as little as 1% of old culture fil- trates to new culture mediumiresulted in decreased max- imum population density. The inhibiting substance 11 (presumably a metabolite) was not identified; (o) the glycogen content of the cells (as determined by direct inspection of iodine-stained organisms) became progressively less as a culture aged. There have appeared several articles dealing with the nutritional requirements of T: vaginalis (Sprince and.Kupferberg,l9h7a, l9h7b; Sprinco, 19h8; Kupferberg, 33 31., l9h8; Sprince, 33 31., l9h9). In general these have dealt with the unknown factor(s) in blood serum necessary for growth of the organism in bacteria-free culture. A requirement for pantothenic acid and phos- phate has been establisod, but a completely defined medium has not been developed. Specific metabolicireactions of the various tricho- monads have received little attention until relatively recently. von Brand (1950) said "...it is singular that the easily available trichomonads have so for re- ceived but little attention...". Earlier work (Cailleau, 1937a) indicated that T; foetus utilized glucose, galac- tose, lactose, fructose, maltose, saccharose, raffinoso, dextrin, and.solublo starch.with a fall in culture pH to 5.7 - 6.0, but glycerol was not fermented. Another species,'T; columbao (I 2;,gallinae), used glucose, galactose, maltose, saccharose, dextrin, and soluble starch, but only weakly fermented fructose, lactose, and inulin. This same worker (Cailleau, 1937b) also 12 reported in some detail on utilization of storols by trichomonads. Later, Trussell and Johnson (19hl) found that T; vaginalis fermented glucose, maltose, starch, dextrin, and glycogen in aerobic culture. Fructose and galactose were only slightly utilized, and lactose was not attacked. Willens, Massart, and Posters (19h2) were the first to apply manometric techniques to the study of the metab- olism.of a trichomonad. Using 2; hepatica (= T; ggllinae) they found that glucose, fructose, and mannoso were util- ized with an average respiratory quotient of 0.87 in pH 7.h phosphate buffer. (It is interesting to note that they used only 106 organisms in each rospiremeter flask which gave extremely small oxygen uptakes - of the order of 6 microliters per hour.) Lactate, pyruvate, succinate, hexose diphosphate, and glucose-l-phosphate were not oxidized. The oxygen uptake was unaffected by cyanide, azide, fluoride, or 2,h-dinitrophenol. Suzuoki and Suzuoki (1951a) were the first to study carbohydrate metabolism.in‘2; foetus to any great extent. They found that oxygen uptake with glucose as substrate was maximal at a sodium.chloride concentration of 0.19- 0.26 M and a pH of 7.0-7.6. Of 27 substrates tested, only glucose, fructose, mannose, gala ctoso, sucrose, and maltose stimulated respiration appreciably; while Cu 13 dicarboxylic acids were neither utilized nor activating. Pyruvato and lactate were not oxidized aerobically. Studies of inhibitors showed that those character- istically antagonistic to enzymes of the Meyerhof-Embden glycolytic pathway (iodoacetate and fluoride) were most effective in decreasing oxygen uptake when glucose was used as substrate. Iodoacetate inhibited respiration 50% at a concentration of about 2.5 x 10-.5 M, and fluoride was equally as effective at about 0.001 M. Hydroxylamino and cyanide were ineffective at concentrations less than 0.01 M, and azide only inhibited respiration about 12% at a concentration of 0.1 M. 2,h-dinitrophonol produced about 75% inhibition at a concentration of approximately 0.01 M. Evidence was presented that for every molecule of oxygen consumed during respiration, one acid equivalent was formed per molecule of glucose used. The major acid . produced was found to be succinic acid. Small amounts of lactic and pyruvic acids were also identified. These workers (Suzuoki and Suzuoki, 1951a, 1951b) also presented evidence that hydrogen was produced from glucose by 2;_feetus under anaerobic conditions, a fact earlier reported by Andrews and von Brand (1938). Pyru- vato and formato stimulated the endogenous hydrogen production only slightly. Formic dehydrogenaso was identified in the cells, but no evidence could be found for either formic hydrogonlyase er hydrogenase. The presence of catalase and cytochrome b, but not cytochromes a or c, was demonstrated. In a later paper from Japan (Ninemiya and Suzuoki, 1952) the metabolism of‘2;'vaginalis was discussed, and comparisons made with‘E; foetus. Optimum conditions for respiration in the presence of glucose were given as 0.1-0.2 M NaCl, pH n.5-6.o, and 5% or loss oxygen. In- crease in oxygen tension resulted in a decreased rate of respiration for 2; vaginalis (also shown by Johnson, 19u2) but an increased rate for 2;,foetus. It was shown that .E vaginalis possessed no catalaso activity as did L foetus so it was postulated that accumulation of hydrogen peroxide accounted for the inhibitory effect of oxygen. or 13 substrates tested with‘g; vaginalis, maltose and glucose were oxidized rapidly, lactate and pyruvate were oxidized at a rate about half that for glucose, and the others (including formate and acetate) were not util- ized at all. The oxygen uptake was inhibited by iodo- acetate, arsenite, fluoride, and p-mercuribenzoato. Cy- anide, malonate, and azide had no effect. Tho inhibition by sulfhydryl-binding agents was considered by Baernstein (1955) as presumptive evidence for the presence of triesophesphato dehydrogenase. It was found that glucose increased hydrogen pro- duction to about four times the endogenous level, while 15' pyruvate increased it only about twice the endogenous. Hydrogen evolution with glucose as substrate was maximal in phosphate buffer at about pH 6.2.. Kupferberg, =£,gl., (l953) followed cultures of T; vaginalis in simplified trypticase serum (STS) medium Kupferberg, gt‘al., l9u8). They noted a change in pH of the medium from 6.0 to 5.75 in AB hours with an in- crease in population from about 10,000 per ml to 1,800,000 per ml. Lactic acid was found to be the major acid pro- duced by this organism in this medium. Its production paralleled the pH change. Their studies on oxygen uptake in the presence of glucose showed that respiration was of the order of 162 micreliters per 108 cells per hour, that it was propor- tional to the number of cells employed, and that the respiratory quotient decreased with time of respiration. A CO -fixation product was isolated using radio- 2 active carbon dioxide, but its nature was not determined. In conflict with the reports of Ninemiya and Suzuoki (1952) and Read (1953) no gas other than 002 was identi- fied by these workers as a product of 2; vaginalis metab- olism. Read and Rothman (1955) reported later that this organism produces hydrogen only under anaerobic conditions. By far the most detailed work on trichomonad metab- olism was reported by Ryley (1955a). In his work on T&_foetus attention was focused mainly on the glycogen 16 reserves stored by the flagellate. An earlier paper (Stewart, 1938) had reported that as growth progresses (and as carbohydrate is consumed) in culture, the number and per cent of forms containing glycogen inclusions (as identified with iodine stain) increases to the time of maximum population and decreases thereafter..Using uB-hour cultures Ryley obtained an average endogenous qO liters OZ/mg N/hr) of 176. 2 (micro- Of 22 substrates tested glucose, fructose, mannese, galactose, and lactose stimulated this endogenous respir- ation by 50% or more. Maltese gave a stimulation between 10% and 25%. Formate, acetate, lactate, citrate, and succinate were reported to stimulate respiratory activity less than 10%. Pyruvato was without effect. Under anaerobic conditions CO was released from 2 a bicarbonate medium.with a qu of 325. This was in- 02 creased by addition of exogenous substrate. 0f inhibitors studied, the following gave the in- dicated per cent inhibition of endogenous aerobic and anaerobic activity- % inhibiti on Inhibitor Conc. aerobic anaerobic Sodium.?luorido 0305 M 8 1h Iodoacetate 0.01 M 80 -- " 0.001 M 61 -- " 0.0003 M ." 88 Hydrexylamino 0.01 M 10 -- 2,h-DNP 0.001 M 12 -- 8-0H-quinolino 0.001 M -- 10 Sodium azide 0.02 M. -- 26 Arsenito 0.02 M -- 22 KCN 0.01 M. -- 25 17 Arsenite, malonate, 8-hydroxy-quineline, and azide stimp ulated aerobic metabolism.rather than inhibiting it. Studies on glycogen utilization and formation showed that under aerobic conditions the organism increased its glycogen reserves when incubated with glucose, galactose, or lactose. With maltose a not decrease of glycogen re- sulted. Anaerobically the sumo was observed, but in the presence of lactose and galactose the organism stored very little intracellular polysaccharide. This material was presented by Ryloy (loc. cit.) to show that data on oxygen uptake or 002 production in the presence of exogenous carbohydrate cannot be considered as representative of "utilization" of those sugars, if by that term is meant complete immediate breakdown to CO2 and H 0 (or to acid 2 end products) to gain cell energy. Andrews and von Brand (1938), in studies on is foetus, had found that the rate of glucose consumption was not uniform throughout growth, being high during periods of active multiplication and low during periods of decreasing population. Analysis of the medium after anaerobic glycolysis by T; foetus (Ryley, 1955a) revealed succinic acid and acetic acid as the major products. 002 was assimilated (fixed) in the process, and considerable hydrogen was also produced. With cell-free preparations (made in a Mickie disintegrater) evidence was obtained for the presence of l8 amylase, maltase, phosphorylase, hexokinase, phospho- glucomutase, ketoisomerase, a metal-activated aldolase, and a system.oxidizing triesophesphato which was DPN dependent. Wirtschafter (l95h) presented evidence for the exis- tence of hexokinaso and aldolase in.2&_vgginalis, and in- dicated that two strains of the organism differed in their content of these enzymes. Baernstein (1955) characterized the aldolase of E;_ vaginalis in sonic homogenates of the organism and found it to be activated by cobaltous and ferrous ions, but inhibited by the trivalent forms of these metals. A short abstract appeared (Read, 1955) indicating that work had been done on the comparison of the metabolic activities of T_._ vaginalis and T: gallinae and, although quantitative differences were implied, no data were given in the abstract. It was stated that E;_gallinao was found to oxidize all Krebs cycle intermediates, the first report of this activity for these flagellates, and in contrast to the statement by Willems, gt_gl; (l9u2) that succinic acid is not utilized by this organism. Recently, Wirtschafter and Jahn (1956) and wirtschaf- tor, Saltman, and Jahn (1956) reported on the aerobic and anaerobic pathways of carbohydrate degradation in.2; vaginalis. Phosphoglucomutase, alpha-glycenphosphate dehydrogenase, lactic dehydrogenase, and malic dehydrogen- 19 see were demonstrated in homogenates of this organism. Tricarboxylic acid cycle intermediates other than malate were not utilized. Glucose-l-phosphate, fructose-6-phosphate, g1ucose-6-phosphate, fructose-1,6-diphosphate, 2-phospho- glyceric acid, 3—phosphoglyceric acid, and phOSpho-enol pyruvate were identified by chromatography of reaction mixtures. A radioactive product which incorporates a cm label from pyruvate was found but not identified. All the foregoing authors have failed to give specific details about the age of the culture used, and therefore, comparison of work was difficult unless it was assumed that metabolic activity was relatively constant over a period of about 2k-96 hours. That this assumption is not valid has recently been indicated by Doran (1956a). He found that the qOZ of 2; foetus decreased from a high of 277 at 20 hours to 190 at k8 hours with much variation between. A further decrease to 65 was observed with organisms 120 hours old. Acid formation from.glucose also decreased with time. Variations in metabolic activity in Th_vaginalis were noted by Read and Rothman (1955) who attributed the differences to the quality of the media, and the age and strain of the inoculum. Using u8-hour cultures Doran (1956b) studied aerobic carbohydrate metabolism 0f.$s foetus and the three forms of ‘.1_‘_._ gu_i_s_ (small cecal form, large cecal form, and nasal form). His results regarding substrate oxidation.were as 20 follows: Th. small large Substrate foetus cecal cecal nasal Glucose *x - x x Galactose *x e x x Mannose *x - x x Fructose *x o x x Maltese x x x x Lactose o o x x Raffinose o o x x Trehalose x o x x TCA inter-me d. o e e e x-utilized o-not utilized *-utilized at rate 3 times faster than others. In further studies iodoacetate and arsenite were shown to inhibit oxygen uptake by all forms. 2‘.foetus and the nasal form of 2&‘3233 were also inhibited by fluoride and 8-hydrexy-quinoline. Cyanide, azide, arsen- ate, 2,u-dinitrophenol, and malonate showed no antagonistic action. Read (1953) reported that .anaerobio gas production by 2&rgaginalis at pH 5.8 was inhibited 70% by 10"3 M potassium cyanide, but was not affected under aerobic conditions. He also noted that carbon monoxide inhibited anaerobic gas production completely in the dark, but illum— ination of the cellsieversed the effect. The conclusion reached was that an iron porphyrin was involved in anaerobic metabolism - the first time this has been reported for animals. Suzuoki and Suzuoki (1951a) havezreported evi- ' dence for the presence of cytochrome b in'gs foetus, 21 while Ryley (1955a) was unable to find any cytochromes. Riedmflller (1936) also failed to detect any cytochrome pigments. Purification and physico-chemical characterization of the intracellular glycogens of 2;_foetus and 2a.&£il: 1233 have been performed by Manners and Ryley (1955). Feinberg and Morgan (1953) have also described the isolation of a pelyglucese from‘2;_foetus. These workers also found another polysaccharide with an attached amino acid moiety. This substance contained rhamnese, fucese, xylose, galac- tose, and glucosamine plus ten amino acids. This latter substance was shown to be non-antigenic in rabbits. It is evident from.the foregoing review that much basic research is yet necessary to elucidate the complex biochemical activities of the trichomonads. Additional and confirmative data on standard conditions, substrate utilization, inhibitors, and enzyme content is urgently needed, especially from species other than 2; foetus and.2; vaginalis. 22 THE PRO BUSM The present researchivas undertaken to investigate some aspects of carbohydrate metabolism in four trichomonads. After much.preliminary work, the probleniresolved itself into the following component parts: (a) (b) (e) (d) (e) (1‘) study of growth of the organisms in bacteria- free culture under usual laboratory conditions; determination of changes in the metabolic activity of the organisms during growth in artificial media,and standardization of conditions for manometric study; investigation of substrate utilization and effects of various inhibitors; investigation of the production of hydrogen by these organisms; analysis of cell-free sonic homogenates for some enzymes likely involved in intermediary metabolism; comparison of the organisms on the basis of any differences which might be found to exist, in such items as respiratory activity, enzyme content, effect of inhibitors, and accumulation of end products. 23 MATERIALS AND METHODS Organisms - The four organisms used in this study were obtained in bacteria-free culture from earlier work (Sanborn, 1955) in this department. They had been isolated originally from their appropriate animal hosts in the veterinary clinic of Michigan State University, and had been carried in culture in a modified CPLMFmedium since. As listed earlier, the organisms used were Tritricho- .EEEEE foetus, Pentatrichomonas gallinarum, and nasal and fecal forms of Trichomonas suis. For the various experiments appropriate dilutions of the cultures were made and the organisms counted in a hemacytometer chamber as for white blood cells. The usual dilution was 1:10. Then, if the four corner squares in the chamber are used, multiplication of the count obtained by 25,000 gives the number of organisms per m1. Cultural methods - Several media were tried in an effort to find one suitable for the present work. The complexity of the CPLM medium rendered it difficult to make in large quantities, and its agar content made it useless for harvesting large quantities of organisms by centrifugation. Several commercially-available dehydrated culture media were found to support good growth of these protozoa when supplemented with serum. The serum used was dehy- drated Beef Blood Serum (Difco) which was made in a 20% i-eystoine-peptene-liver-malteso 2h (of normal) concentration, filtered through gauze and paper, sterilized by gravity passage through a Seitz filter, and added to all cultures so as to give a final concen- tration of 1-2%. Adequate growth was obtained at this concentration and the organisms did not show the high rate of endogenous respiration reported by others. Three types of cultures were used routinely in this study. Stock cultures were maintained in 20 ml amounts of Difco Brewer Thioglycollate Medium.in 25 x 150 mm test tubes. Transfers were made weeklyeind the cultures in- cubated at 370 C for about 36 hours. They were then placed at room temperature where the cultures remained viable for about 3 weeks. Special preparatory cultures were planted prior to inoculation of larger amounts of media for bulk growth of the organisms. These were made by inoculating 20 m1 of NIH Thioglycollate Broth (Case or Difco) in 25 x 150 mm test tubes with.about 1.5 ml of stock culture and incubating about 36 hours at 37° C. The contents of one tube was then inoculated into a 250 md Erlenmeyer flask containing 100 m1 of the same (NIH) medium. This culture was incubated the required length of time (12-72 hours) and harvested by centrifugation in 50 ml amounts at 1500 RPM (approx. 500 RCF) in an International No. 2 horizontal centrifuge. The sedimented organisms were washed twice with 0.9% NaCl, and finally taken up with an appropriate amount of 0.9% NaCl or (for sonic 25 extract preparations) 0.9% KCl. In most cases where whole cells were used the dilution was sudi that each ml contained approximately 50 million (5 x 107) flagellates. Manometgy,- The experiments on respiration and fer- mentationlaere conducted in accord with standard manometric practice (Umbreit, 1989) at 370 C in a Precision Warburg 20-unit respirometer with a shaking rate of 120 per min. Gas atmospheres were altered as desired by gassing the flasks while in the bath through.a lO-place gassing man- ifold. Gases used were nitrogen, 5% 002 - 95% N2, and hydro gen. Cell-free extracts - These were prepared by harvest- ing cells as described above (after 36 hours of incubation), diluting to approximately 108 cells per ml with ice-cold 0.9% KCl, and subjecting the suspension to vibrations in the chilled (20 C) cup of a Raytheen 10 kc Sonic Oscille ator for 7 minutes. This treatment time was sufficient to rupture practically all the cells, the cloudy, almost opaque suspension being changed to a faintly epalescent solution. The cellular debris was removed by centrifuging lightly and the extract was immediately frozen in 5 m1 amounts at -20° C. ' Chemical methods — Succinic acid was assayed manomet- rically as described by Umbreit (l9u9) using a succinexidase preparation from fresh pig heart. Pyruvato was measured by the procedure of Lu (1939) as described in modified form 26 by Umbreit (19u9), and lactic acid by the method of Barker and Summerson (l9u1). Reducing sugar was estimated by the ferricyanide method of Folin and Malmros (1929), and fructose by the procedure given by Roe (l93h). Inorganic phosphate was determined by the procedure of Fiske and SubbaRow (1925). Estimation of nitrogen was by direct nesslerization after digestion in 5 N sulfuric acid and 30% 3202 and neutralization with 5.5 N KOH. The general procedure as given by Umbreit (l9u9). including the mod- ified Nessler's solution, was used. Digestion was on a micro-Kjeldahl digestion.rack for about half the deter- minations; the others were digested in the even as described. Colorimetric determinations were made with the aid of a Bausch and Lomb Spectronic-20 spectrophotometer. 'Eggymo determinations - Catalase activity was assayed manometrically by measurement of oxygen released from 10 micremoles of H202. Hydrogenase was assayed by measuring hydrogen uptake manometrically in an atmosphere of hydrogen and in the presence of 25 micromoles of methylene blue with.KOH in the center well of the flask. Fermic hydro- genlyase was determined by following CO2 and H2 formation from 25 micromoles of pyruvate under an atmosphere of nitrogen. Formic dehydrogenase activity was measured by estimating C0 production from.50 micromoles of formats 2 in the presence of 25 micromoles of methylene blue under a nitrogen atmOSphere. 27 Hexokinase activity was assayed by the manometric method of Colowick and Kalckar (l9h1)e Phosphoglucomutase by the method given by Najjar (1955), phosphohexoisomerase by measuring fructose-6-phosphate formation from glucose-6- phosphate as described by Slein (1955), and aldolase and phosphofructekinase by the "hydrazone chromogen" procedure of Sibley and Lehninger (1989). Phosphoglyceromutase and enelase activities were indicated by measuring pyruvate formation from phosphoglyceric acid in reaction mixtures as described by Vandemark .nd Wood (1956). The "phosphoro- clastic” reaction was studied with the method of Koepsell (1955). Dehydrogenase activities (other than formic dehydro- genase) were identified by following the change in absorb- ance at 3&0 millimicrens in the presence of specific sub- strate and diphesphopyridine nucleotide or triphosphopyr- idine nucleotide in a Beckman DU spectrophotometer, and in two cases, by the Thunberg methylene blue decolorization method. Details regarding these procedures, and modifications thereof, are given in appropriate context. All general chemicals were of highest purity. Metabolic intermediates were purchased from Nutritional Biochemical Corporation and fromiGeneral Biochemicals, Inc. Certain special organic reagents were products of Eastman Organic Chemical Division of Eastman Kodak Company. 28 EXPERIMENTAL RESULTS Growth studies - The nature of the research made it desirable that as simple a medium as possible be employed, so that it could be made in large quantities with a min— imum of work. The CPLM medium of Johnson and Trussell (19h3), and the STS broth of Kupferberg‘gg'21; (l9h8) were both too complex for routine use, and both contained a small amount of agar which was undesirable because of the difficulty in harvesting the cells in its presence. Therefore a search was made for commercially-available products containing tryptone or phytone, yeast extract, carbohydrate, and a reducing agent such as thioglycollate or cysteine, but no agar. Several commercial dehydrated products*with the above requirements were found to support growth of the flagellates, but not all to the same extent. Population counts were consistently about 1.5-2.0 times higher in NIH Thioglycollate Broth (Case or Difco) than in any of the others. Counts between 3-h million organisms per ml in 36-h8 hours were obtained routinely when a sufficiently large inoculum.was employed. This medium was therefore adopted and used as described earlier. In an effort to obtain general information regarding * - Eugonbroth (BBL) Tryptone Soy Broth (Case) Brucella Broth (Albimi) Cystine Trypticase Agar (BBL) Trypticase Soy Broth (BBL) (tried for maintenance cultures by stab inoc.) 29 carbohydrate breakdown during growth in this medium, replicate cultures were inoculated with each of the various organisms and sampled periodically for 3 days. At each time, determinations were made of population, pH, titratable acidity (using 0.01 M NaOH to titrate 5 ml of culture with brom—thymol blue as the indicator), and reducing sugar. In certain samples lactic, pyruvic, and succinic acids were assayed. Figures l-u show the changes occurring in the cultures of the four organisms over the three day period. The figures are not directly comparable since the original inoculum differed slightly in each case, varying from 110 to 160 thousand cells per ml of culture. The pH was between 6.0 and 6.3 at the time of the population peak for each species. Figure 5, which repre- sents average values from several cultures of these organ- isms, indicates that when the pH of the culture reached 5.0-5.2 only very few living organisms remained. These few disappeared in a few hours at this pH. (It is perhaps well to mention at this point that if the cultures are removed from the incubator at about the time of the pop- ulation maximum, the organisms remain viable for longer periods of time due to their decreased reproduction rate at room temperature. Without agar, however, the popu- lation declines much more rapidly than in its presence - as in the stock cultures.) millions of organisms Nun-F" pH mM 1 N NaOH per 100 ml culture 2000‘ micro- grams 1‘ p31 1000 Figure l; 30 I POPULATION l zu us hours Changes in a Bacteria-free Culture of Tri- trichomonas foetus During Three Days Growth n NIH oegcoIIate Broth at 37 C. 31 millions 0" 3 .. POPULATION organisms 2 l 7.0‘ pH 6 e S 6.0 l l l l J ’ 2.0 , 21+ hours “8 7«2 TEN 'rrrluneu: NaOH - aclnrrv per 100 ml .Or- culture 1 l . ! 2h MB hours 2000 aeoucme m1 cro_ 800”! grams per mi 1000 . l 1 I L 1 4g_ 21; 1+8 72 hours Figure §;, Changes in a Bacteria-free Culture of Penta- tridhomonas gallinarum.During Three Days Growth in NI og ycollate Broth at 37 C. 32 1, L million: 3. of organisms 2. 1 POPULATION pH mm 1 N NaOH per 100 ml culture 200 micro- grams per ml lOOO 1 I I I I 2h 1&8 7% hours Figgre 2; Changes in a Bacteria-free Culture of Tricho- monas suis (fecal strain) During Three Bays Growth In NIH Thioglycollate Broth at 37 C. 33 millions 3 _ POPULATION MM l N NaOH per 100 m1 culture 2000 REDUCING micro- SUGAR grams per "‘1 1000 a l A I I L fi— ZLL h8 72 hours Figgge g=_ Changes in a Bacteria-free Culture of Trighg- monas suis (nasal strain) During Three Days GroiEh.In NIH Thioglycollate Broth at 37 C. 3h Figure fig. Average Relationship of pH to Population in Cultures of Four Trichomonads in NIH Thioglycollate Broth.at 37 C. i b E e E b b D b C b . 0 : O . Q " ..... 5‘ . . b ’l’ .‘i p I . .‘ . p ’I . \. . 2.0 l- I b ’. . “ O p I. . \ O ' \ a D t ‘ . ,’ \ _ I e‘ I ‘ ' n . l, “ . b I o I . ’I O. b O Q E 1.0 - ,o ‘. O 0) ’ '1’ O. *4 a g » . '\ to .{I .‘O I ‘4 b ' . O I O. a. I l o ’ I ‘ p I a I, “ ‘30.; >0 ‘ O 0 I . | H v H i H . '. E ’ ‘ \ e 0 \ | ‘ \ Q \ \ g \ O \ b ‘. O \ \ ‘\ ‘0 O I ‘ ‘s \ \s P \ \\ a \\ A a j e a a a \ 7.0 6.6 602 §08 g1; 5:0 35 That this pH drop is parallel.d by an increase in titratable acidity and a decrease in the reducing sugar is apparent from Figs. l-h. Analysis of the culture medium at various times during incubation showed that except for the fecal strain of 2s.22l21 and to a lesser extent for 2; foetus, the acidity could largely be accainted for by an increase in lactic, succinic, and py- ruvic acids. This is shown (for 70-75 hrs.) in Table 1. As can be seen, succinic acid was the major acid produced by all but the fecal strain of 2&,ggég, Of the acids shown, lactic was produced in greatest quantity by this latter organism. However, over 50% of the total acidity is unaccounted for in these analyses for 2;,ggig (fecal). The lactic/pyruvic ratio was about 2.5 for the two swine forms, while 2; gallinarum and I; foetus produced approx- imately equal amounts of each acid. From these data it appears that the nasal form of T; £B§3_produces much more acid in these cultures than the others. In an effort roughly to quantitate these differences Table 2 was prepared from the figures for total acidity and organism count at the time of maximum population. Again, from this table, it is obvious that the metabolic activities of the nasal organism.under these cultural conditions result in the production of a greater amount of acidic end products than the others. 36 eaSpHSo HE ooa and momz 2 SE n as enduHSQ as 00H sea chow oamwnocoe ms oapapwapau hpaefioa SE a * madness mansuaauss asses do a ADV oaspHSo as ooa sea :8 Adv 2... 0&3 0.3 0.0m 41R 2: firmwuot oo0.a m:a.m mom. mmw. mdm. any Aammmcv masm .9 --- m.~: e.m o.mw o.ma Ans x.n: opp som.H Hoe. owe. mmm. zoa. lav Assumes was» .a nun 0.00 N.oa ~.ma o.©o Any ».anlobk me.H mmH.H was. Hos. mom. Ame sssscsflasm,.m 2... TS. T: «.3 ad‘m 3v EC. mom.” mom.H and. 00H. mm . Adv espoom .8 as asseso< * uses eso< ewes suscamao manageauae 85m oH>5hhm capoaa oacfioosm Snavoz epsaaoohamoane mHz a“ opsuaso moan uaaaouomm 2a newcoEoonae asom hp coaposeoam vao< H mqm<9 37 o.a~m oo:.a mo.m assmscv mesm_qm alum 21H Hum :89: 22.. um o.osm cam. as.m essaessamm_qm 0.0ma om». m0.m mssooe_qw AME OOH pod AME nod mEmucwmaO moaz z EEO asap ecofiaaaev Smacwmao m OH pom vao< was» as had coaumasdom mmewhocoz :8 uvHo< oHnmpwapHa EdEaxmz asses: ossaaooaHmOflne m Goauosooam an< one Hz Ca moaSuHsO undoeonowna Ca hufincea coHumHSQOA no noauwaeaaoo N mamom m.mmm m.ommlev :11: I: H.mem e.c¢e e.mmm m.:am 3.5:mAev Aasmmev mass .9 mm.o Hm.o emno mm.o oo.H on m.mmm :.ecm «.mos m.mmm o.omoanv .Iu m.som m.mmm m.emm :.som m.ommlsv assumes mass .9 HHH No.0 NH.H m0.o em.o ass 1.0 m c.0mm m.omm o.~ea s.emaaev .1. m.oim 0.5mm m.mmm m.eafl e.momflsv esascssasm .m -.o No.0 as. No.0 01.0 on ~.scs e._mm :.esm e.mea s.:melpv In - 0.5:m. m.mce m.c:m mumsm 3.H00hsv masses .e on o: on on om ma m x a o m z a a o a amusemeo emooSHO meHoanoaz om mo mosemeam emu CH hHHmoHAueECsz UQLSmwoz me easuaso eeamnmahepoem CH Luzoaw no maso: Om mamas: mOwcoEonoueB psoz mo hue>apo< haoumpfimmem : memes UHOd a any cospoaeoee edge 0» eessnssspe moons Ass Ham mam wee How has I: OOH m.@m 0.0m ~.OmAwV Amemcv mHSm . com omm es: mm: Nam Ans n: cos H.eo o.e¢ m.ms m.m:aav Assumes swam . mmm can ism. moo any .I: I: 00H o.me 0.0: H.maxsv asewesHHmmv.a mam Hon mm: mm: ems any I: OOH :.m~ m.mm 0.~m m.mmhsv menace . m: 0: on ON NH smHCswaO : o m 2 H m o 4 emoosz mOHoanon om mo eocemoam exp CH m>.O mm as nemusm seasobamon CH meadoSonoHaa neon hp GQHposnonm OHo< m memes T; suis (fecal) at 12 hours. ) It dropped during the actively growing phase and then increased again until by A8 hours practically 100% of the CO produced was due to 2 acid formation. The q , on the other hand, declined acid with time in these experiments. Substrate utilization and inhibitors - Twenty-one compounds were tested for their ability to stimulate the respiration of the organisms in this study. They can be divided into three groups: (a) intermediates of the Meyerhof-nmbden glycolytic pathway, (b) tricarboxylic acid cycle intermediates, and (c) mono- and di-saccharide sugars. Table 6 shows the results obtained when the substrate (usually 50 micromoles) was aided to the organ- isms from the sidearm of a standard Warburg vessel. The figures given are ratios of the oxygen uptake in the presence of substrate to the endogenous oxygen uptake. It can be seen that most of the compounds tested had no stimulatory effect; in fact, many of them were decided- ly inhibitory. Only the sugars and, in a few cases, for- mats, pyruvate, lactate, and malate stimulated oxygen up- take. Of the sugars, those which stimulated respiration he most were generally glucose, mannose, and fructose. Galactose was included in this group for all except 2; gallinarum, which also did not oxidize fructose very rapidly. The uisaccharides were less effective in M6 .eocemnw muH CH omega: nemmxo on» on openness» on» go codename 0:» CH 0x090: cowhwo mo OHpea on» one coaam neasmHm 00.0 30.0 00.0 m0.0 HoaeohH0 o.H 00.0 00.0 so.H euepmoe 4.0 H0.0 m0.o me.o opeepse m0.o 00.0 N0.0 mm.o oudGuocfim 00.0 00.0 m.o mm.o openness no.0 mm.o o..H 00.0 opeHez 00.0 0m.o 00.0 00.0 opeaupsamopoauyu 00.0 00.0 mm.0 No.0 opepeoeHeHO 00.0 00.0 ma.H no.0 essence 00.0 Om.H 00.0 00.0 suspend mm.0 00.0 mm.H H0.0 oue>shhm mm.0 H0.0 H0.0 nn eueaeohHmondmozm 0m.H 00.H 0O.H nu openamondHu omoxem 30.0 m0.0 nu nu epsndmonduHuemoosHO m0.H mO.H 00.0 mm.H emoaosw m0.H 0H.H 00.0 mO.H enOpomH 0m.H 00.0 NH.H :H.H owoprz 0m.m OO.H 0H.H 0:.H omouowHeo m:.m m0.H 0m.H Nw.H emouosaa mm.m mw.H Om.H m~.H amends: oo.N HO.N oo.H om.H emooflHc A20 mH3m .8 Amy mHSm 4H. EsaecHHHOw dM mspeom 4H epaaumnsm mOwsosonoHnB seem 0o COHumaHdmem on» no nepeapmnsm mSOHpm> no vacuum 0 mam<9 inc1easing the rate of oxygen uptake, undoubtedly due to the necessity for hydrolysis before they could be utilized. Lactose and sucrose failed to stimulate the respiration of a; ggllinarum. All of the sugars were utilized at greater rates by the nasal Zé‘ggig. Ryley (1955a) indicated that a net synthesis of intracellular glycogen took place during utilization of exogenous<:arbohydrate. In an attempt to repeat parts of this work, the glycogen content of the cells used to measure aerobic sugar utilization was determined before and after each experiment. The results (shown in'Table 7) indicated that for most sugars used under aerobic conditions by 1; gallinarum and the two forms of‘gg‘ggig there was a synthesis of intracellular polysaccharide. However, it was not possible to demonstrate this with our strain of T; foetus, except for maltose. The results for that organism.are in direct opposition to those of Ryley, who reported that maltose was the only carbohydrate which did 223 cause intracellular synthesis. The endogenous cmtrols for these experiments for T; foetus were unsatisfactory because of accidental contamination with sugar, so therefore the results obtained for this organism are in all probability in error. The low respiratory rates given for formate, pyruvate, and lactate for all but 3; gillinarum.may be due to the production of hydrogen which would obscure the respiratory uB H.H\ O.m\ H.m\ N.On emoaoSm O.H\ H.m\ N..O\ HmHI 0009064 sq N0. 00. sex :33. 0.0- :.m\ _.H\ ~.Hu emosoeaae NeM‘ OeNl meOl OeHI OQOUOEE ..N\ . :.O\ 0.H\ 0.0n amends: 0.0x m.m\ m.m\ T0- 3830 :0 3% 4.0 at 3% am seamstress .0 301.60de AsewoapHc HHeo EsamHHHHE oueaumpsm Led pceHe>sto emoous neHoanoHE may a 0 z 4 m 0 z m 0 O O M H 0 a m z maemsm msOHae> no moHoanon 0m 59H: anon H ueuwDSocH Con: ocecoeonoHaB adom CH owedno dowooth AstHHeoeaacH 0 Wanda #9 picture. This is discussed in a later section. Ten inhibitors of various enzymatic reactions were tested for their effect on aerobic metabolism of the four organisms. The results are given in Table 8. The most potent antagonist was iodoacetate, a pow- erful inhibitor of glycolysis. Fluoride, another glycoly- sis inhibitor, was only slightly effective in high concen- trations. Other agents, such as 8-hydroxy-quinoline and 2,h-dinitrophenol with E; allinarum, and hydroxylamine with g; gallinarum and the fecal T; 3213, gave slight inhibitions. Arsenate was appreciably stimulatory with the fecal T; 53$; and T;_foetus, and 2,h-dinitrophmnol also stimulated these two as well as the nasal pig foam. Arsenite showed very slight inhibition only with.§:’ggll: inarum and T; foetus. Malonate did not affect respiration- another indication that the Krebs tricarboxylic acid cycle is not functional in these organisms. All four organisms were found to possess catalase activity. Incubation of cell suspensions containing 108 organisms with 10 micromoles H202 resulted in a rapid evolution of oxygen. The reaction was complete within 10 minutes. With 2.5 x 107 (i as many) organisms the reaction was slower, requiring 30 minutes for completion 'with.the nasal T;_ggig and §;_ggllinarum.and h5 minutes with the fecal E. suis and _T__._ foetus. TABLE Effect of Various Inhibitors on the Respiration of Four Trichomonads in the Presence of 250 hicromoles Glucose Inhibitor Conc.(M) T; P. 2;. ‘2; foetus gallinarum suis(F) suis(N) Arsenate .01 1.56 0.87 1.52 1.18 .001 1.16 1.01 1.19 1.01 m .0001 0.99 0.95 T‘ Arsenite .01 0.90 0.91 0.99 1.10 ' .001 0.87 0.88 0.95 Cyanide .1 0.62 0.99 .01 1.08 0.98 1.09 0.92 Fluoride e1 0078 0.90 1. .01 1.00 0.85 1.05 1.08 h“ Hydroxylamine .l 0.81 , 0.69 .01 1.06 0.87 0.8h 1.03 Iodoacetate .01 0.10 0.20 0.3M 0.13 .001 O. 1 0.2h 0.39 0.30 .0001 0.96 0.80 0.83 Malonate .01 1.02 1.11 0.99 1.02 2,11'DNP .001 1 e26 0 07? low; 1.20 .0001 1.21 0.98 1.05 1.00 8'0H‘Q e01 Oe88 .001 1.18 1.11 1.01 A21d6 e1 0096 .01 1.01 1.00 0.82 .001 0.89 ¥ Figures given are the ratio of oxygen uptake in the Presence of inhibitor to the oxygen uptake in its absence. Inhibitors were made up to concentration shown in 10% glucose and 0.5 m1 of this solution added to cells from flask sidearm after equilibration at 37° C. 51 Cnrbon dioxide production from bicarbonate under '55; N2 - 5% 00‘2 was strongly inhibited in all organisms 3y iodoucetate, but not by arsenite or fluoride. This is shown in Table 9. TABLE 9 Effect of Three Inhibitors on the Anaerobic Met- abolism of Four Trichomonads in the Presence of 50 Micromoles of Glucose INHIBITORS Organism Arsenite Iodoacetate Fluoride .007 H .01 M .02 M y '1'; foetus . 1.21 0.22 1&3 g: gallinarum 1.07 0.214. 1.27 3L. _s_1_i_i_£ (feca1)1.09 0.33 1.01 3; 932.3 (nasal)1.18 0.22 1.12 Figures given are the ratio of C0 production in the presence of inhibitor to the 802 production in its absence. gydrogen production by these trichomonads - As pre- viously mentioned, several authors have reported that an alkali-insoluble gas is produced by cultures of _‘1_‘_._ foetus and 1. vaginslis. The gas has been rather conclusively identified as hydrogen. The four organisms employed in this study were all observed to produce large amounts of gas when grown in culture media containing small amounts (0.05%) of agar, or in media without agar if the fluid was more than about one inch in depth. This gas production was evidenced by evolution of many gas bubbles when the agar-containing Eu...; 1 52 alturo was agitated with a pipette, or by the accumulation f n. froth on the surface of the broth medium. Shaking of Ather type of culture resulted in the release of more gas. l'hoso characteristics became less pronounced with increasing ago of the culture, disappearing almost entirely by about '72 hours. The figures given in Table 10 represent a series of oxperiments wherein the evolution of this gas (calculated as 32) from a glucose substrate by suspensions of organ- isms of different cultural ages was determined manometrical- 1y under a nitrogen atmosphere. It can be seen that pro- duction of this gas was greatest by organisms from the 12-hour culture with a general decrease from then on. A slight, unexplained, increase occurred with 36-hour organ- isms. TABLE 10 Evolution of an Alkali-insoluble Gas (Hydrogen) From a Glucose Substrate by Trichomonad Suspen- sions Under Anaerobic Conditions Organism Age of Culture in Hours 12 2h 36 g; foetus 100 3M; 1.7.3 17.3 P. gallinarum 100 30.9 60.3 50.5 T.suis (fecal) 100 27.1 20.8 110.9 '1' ans (nasal) 100 36.2 69.8 59.2 Figures for gas evolution are expressed as per cent of the twelve-hour level. 53 It: has been postulated (Umbreit, 1952) that tricho- ad hydrogen evolution is due to the action of a formie rogonlynse enzyme system, although this has not been won. Also, Ryley (1955a) thought it was "...1ike1y at 3: foetus contains a phosphoroclastic system similar that of Clostridium butEicum", wherein hydrogen, urbon dioxide, and acetyl phosphate are formed from yruvate without formate intervention. Thirty-hour organisms showed a slight increase in nydrogen production when supplied with 50 micromoles of lactate, pyruvate, or formate under anaerobic conditions (N2), as shown in Table 11. TABLE 11 Stimulation of Hydrogen Production of Triehomonad Suspensions by Three Substrates Organism Lactate Aruvate Formats T. foetus (a) 6 0 67 — (b) Sea " 62o? 2; gallinarum (a) 59 10 30 (b) 314.1; 5.9 17.11 T. suis(fecal) (a) 0 7.11 0 — —- (b) ' 20.1 e- T. suis(nasal)(a) 0 19 0 — — (b) - 19.9 - (a) per cent stimulation of endogenous rate (0) nicroliters 112 per mg cell nitrogen (qHZ) The actual amounts of hydrogen produced were rather snail as can be seen from the table. In general, it appeared that more 002 was produced than Hz,but determin- Sh .one of this gas in combination with hydrogen under see conditions have been shown to be erroneous (Ryley, SSe) since CO2 is fixed by the organisms. Some of the experiments also showed a net assimilation of 002, 'ement :ien standard methods were used for calculating the exchange 05,! L 5.. .;‘41‘.£"s'h. f two gases.- Formic hydrogenlyase is reported to be an adaptive '1. ).-_~n“~‘i_ - . ' s enzyme in such organisms as Salmonella (Stokes, 1956, and others). It is best formed by organisms grown under re- ducing conditions (deep broth) in the presence of glucose and a mixture of amino acids or other nitrogen source. Cells which do not form this enzyme during growth can often be adapted in the presence of glucose, formate, and amino acids, and the adaptation followed by manometric m0t110d8e Figure 8 shows the results of an experiment in which cells were incubated with glucose, formate, and casein hydrolysate (Casamino Acids, Bacto) under a nitrogen atmo- sphere. KOH was present in the center well of the flasks to absorb 002. T_._ foetus and _l_’_._ gallinarum showed a typical adaptation curve after a 15-minute lag. 1'; suis (fecal) showed no lag period and the nasal form failed to show any activity. The curves for 1: foetus and _I_’_._ gall- inarum decreased after 30 minutes, long before the theo- retical amount of 82 had been liberated from the formats. Fornic hydrogenlyase has often been identified as SS 2:. £952.22?- , 50" is a ho b llinaz'um rum—l} ., . r 30. \. 0 micro- 0! liters / \9 Q- hydrogen 90% 20 1 1e __._ suis (nasal) fl 1 30 60 Time in mi nut es Figure 8;, Response of Four Trichomonad Suspen- sions to Formats, Glucose, and Casein Hydrolysate Under Nitrogen Atmosphere (Hydrogenlyase Adaptation-?) Warburg flask contained: 7 1.m1 organisms (5 x 10 ) OeS m1 TM buffer pH 6e14, 0.5 ml 0.1 M Na formats 0.5 m1 3% Casamino Acids (Difco)(sidearm) 0.3 ml 10% glucose (sidearm) o 2 ml 20% KOH (center well) 56 mixture of two enzymes - formic dehydrogenase, which tmlyzoe the breakdown of formic acid to 211/ / 2e', and 'drogenmee, which converts these products to gaseous ydrogen. (Organisms have ,however, been found which pperently either produce hydrogen without having one of shame enzymes, or which have both but yet do not produce g‘.. ‘§La.; we? the. Consequently the status of these enzymes is still not completely clear). Attempts were made to demonstrate the actions of these enzymes in suspensions of the four trichomonads. ‘-'--—-—— Observation of gas evolution from formate under nitrogen in the presence of methylene blue as a hydrogen acceptor gave the results shown in Figure 9, indicating a rather weak but definite formic dehydrogenase activity. Figures 10 and 11 show the results obtained when the cells were incubated with no exogenous substrate in the presence of methylene blue or sodium fumarate in an atmosphere of hydrogen. 1: gallinarug took up the most hydrogen with the nasal E 52}; showing the greatest activity of the other three. For these three fumarate was an even poorer acceptor for hydrogen, while with L. gallinarum, it acted slightly better than methy- lene blue. In order to investigate Ryley's suggestion that a 'phosphoroclastic" split of pyruvate perhaps occurred in these organisms, the assay procedure of Koepsell (1955) 120 100 80 microliters CO or 2 P ndlligram cell nitrogen 60 no 20 57 WNH 8 '9 e m suis (nasal) al inarum s eca1) 3 J 8 £1 enous endOS . A 60 minutes Figure 2; Formic Dehydrogenase Activity of Harburg flask contained: 1 m1 organisms (5 x 107) 1 ml TM buffer pH 6.h Suspensions of Trichomonad Cells 0.5 ml 0.05 M methylene blue 0.5 ml 0.1 M sodium formate (sidearm) 58 Warburg flask contained: 100 _ 1 ml organisms (5 x 107) .3 ml TM buffer pH 6.h 80 c 0.5 m1 M/500 Methylene blue 0.2 ml 20% KOH (center well) 60 . micro- a P. gallinarum liters -— H2 no s;T;.suis (nasal) 1 ‘ a 20 —e_'I_'_._ foetus ‘ NE; suis (fecal) ‘: 13' 30 60 ‘ Time in minutes Figw 10, Hydrogenase Activity of Cell Suspensions of Four Trichomonads with Methylene Blue as Hydrogen Acceptor Warburg flask contained: Same as listed for Fig. 9 except 0.5 m1 100 0.1 M Na fumarate used ( instead of methylene blue. Egggallinarum T. suis (nasal) - 3 T: suis (fecal) - ‘1’ ——1‘T7 s 15 30 60 Time in minutes Figure 11. Hydrogenase Activity of Cell Suspensions of Four Trichomonads with Fumarate as Hydrogen Acceptor S9 was employed with cell-free sonic homogenates of the organisms. No increase in gas production in the presence of pyruvate over the endogenous rats was observed for any of the homogenates. Enzymes of intermediary metabolism - Certain glyco- lytic and oxidative enzymes have been identified in cell- free preparations of Z; foetus and.2;_vaginalis. Exper- iments were therefore designed to investigate the presence of these and other enzymes of carbohydrate metabolism.in sonic homogenates of the four trichomonads in this study. Evidence for strong hexokinase activity was demon- strated in all the homogenates by following CO evolution 2 from a bicarbonate buffer in the presence of adenesine triphosphate (ATP) and.Mg/y. Figure 12 indicates the results of this experiment. This enzyme catalyzes the phosphorylation of glucose by ATP (Reaction 1). (1) Glucose ,1 ATP—————9 Glucose-G-phosphate / ADP This results in the liberation of one acid equivalent Per mole of glucose phosphorylated, and this acid liber- ates 002 from the buffer. It can be seen that the nasal fon- is most active of the four. (2) Glucose-l-phosphatc -—————9'Glucose-6-ph03phatc Evidence for phosphoglucomutase, the enzyme respon- sible for reaction (2), and therefore important in poly- aaccharide breakdown, was measured by incubating the homogenates with two micromoles glucose-l-phosphate moo f 1200 " 1000. 800 - 600 > (A 200 microliters C02 per milligram homogenate N Figure 12 e 60 T foe/us Harburg flask contained: Main compartment- 1.5 m1 homogenate 0.5 ml 0.12 M NaHCO 0.5 ml 0.12 M glucoge Sidearm- 0.3 ml 0.0145 M ATP with 0.21 micromoles MgCl2 0.2 m1 0.0a M NaHCO3 1 21? do ed Time in minutes Hexokinase Activity of Trichomonad Sonic Homogenates. 61 (acid-labile phosphate) and measuring the decrease in acid-labile phosphate (glucose-6-phosphats is more stable to acid hydrolysis under the conditions of this experi- ment). Table 12 shows the results of these determinations. TABLE 12 Phosphoglucomutase Activity of Trichomonad Sonic Homogenates Increase in Acid- Stable Phosphate Organism. Homogenate (micromoles per (ml.) mg cell nitrogen) T4’foetus 0.1 0.9 0.3 1.8 P; ggllinarum 0.1 0.h 0.3 0.1 2‘_suis (fecal) 0.1 0.6 0.3 1.3 T;_ggig_(nasal) 0.1 0.2 0.3 0.0 I The reaction mixture contained 0.1 m1 .006 M MgSO , 0.1 m1 glucose-l-PO (.02 M), 0.1 ml 0.1 cysteine HCl, plus e yme. Reaction stop- ped with 1 m1 5 N H280“. (pH during reaction 3 7.5) _T; foetus and the fecal strain of _T_._ _s__u_i_s_ showed definite activity but _P_. gallinarum and the nasal _T_._ £133 were in- active. (3) glucose-G-phoephate-———afructose-6-phosphate Phosphohexoisomerase activity (Reaction 3) was ex- tremely rapid in these homogenates, but a definite in- crease in fructose ester, as measured by the HCl-resor- cinol method of Roe (193k), was seen in all the incubatss. Figure 13 gives the data for these experiments. 62 0 .85 Homogenates* fl. 0.6 lat *-all organisms gave 5&0 essentially the my 0.11 same curve. 0 .2 EndogenouL 9- a______a. 3 h S Time of incubation in minutes Figure 13. Phosphohexoisomerase Activity in Trichomonad Sonic Homogenates 63 The phosphorylation of fructose-6-phosphate by ATP, the next step in glycolysis (Reaction 2;), is catalyzed by the enzyme phosphofructokinase. (4) fructose-6-phosphate I ATP—efiuctose-l,6-dtP04 / ADP Evidence was obtained for presence of this enzyme in the homogenates of all the organisms using the same method as that for aldolase (Reaction 5) except that fructose-6-phosphate and ATP were used instead of hexose C11 pho Sph‘t . e dihydrozycoetene phosphate (5) fructose-1,6-dtPO4—9 / 3-phosphoglyceraldehyde The data are shown in Figures 114-17. Several attempts to demonstrate triosephosphate isomerase activity (Reaction 6) by omitting hydrazine from the digestion mixture for aldolase, on the assumption that isomerase would cause an increase in dihydrexyacetone phosphate thereby increasing the "chromogen", were unsuccessful. (6) 3-phosphcglycercldehyde a—) dihydroryscetone phosphate In every case, a decrease in color resulted on omission 0f hydrazine. Baernstein and Rees (1952) and Baernstsin (1953) were able to show isomerase activity in L M by this method, and Vandemark and Wood (1956) used it to 8how evidence of the enzyme in Microbacterium lacticum. It is interesting to note however, that Baernstsin (1955) ““108 no mention of this in connection with later work on 1’1 Ohomonas vaginal i s . Measurement of pyruvic acid formed on incubation of 0.5“ o.u) formation (5h0 mp) chromogen formation (514.0 1751) chromogsn Figures 1g-11. 0 e O o O O 15 Time in minutes Figure 1h.-2¢ foetus 1’ >11 ) <3 <3 <3 0 0 e v: F? V T chromogen (5u0 c: h‘ 15 Time in minutes Figure 16.-2; suis (F) 61+ 15 Time in minutes Figure 15.-§; gallinarum 15 Time in minutes Figure ll;-T; suis (N) 30 Aliolase and Phosphofructokinase Activity of Trichomonad Sonic Homogenates. 65 homogenates with phosphoglyceric acid is an indication of the presence of phosphoglyceromutase and enolass (Reactions 7 and 8). (99 3-phesphoglycertc acid-—92-phosphoglyoerio acid (B) 2-pheephoglycertc actd-—.(en01)phosphopyruvtc acid fun - The data given in Table 13 show that pyruvic acid in- creased in the tubes containing T; foetus and §;‘ggl_- inarum homogenates, but not in the others. TABLE 13 $- Phosphoglyceromutase and Enolase Activity in Sonic Homogenatss of Four Trichomonads Micrograme pyru- Organism Incubation vate formed per time (min) ng homogenate N g;_ro.tus 1o S-h """'"" 20 13 .2 2; gallinarum. 10 1.6 20 5.6 T; suis (fecal) 10 0.0 20 0.0 T; suis (nasal) 10 0.0 20 0.0 The reaction mixture contained 2 ml 0.25 M glycylglycine buffer pH 7.h, 1 ml 0.05 M phospho- 1ycsric acid, 2 ml homogenate, and water to 8.5 m1. 1 ml aliquots removed at 10 and 20 minutes and added to 0.2 m1 h0% TCA. Additional studies were done to determine the existence in sonic homogenates of the trichomonads of malic, lactic, glycerol, glucose, and glucose-6-phosphate dehydrogenases, and fumarase. No evidence was obtained for the presence of glycerol 66 dehydrogenase, glucose dehydrogenase, or fumarase, enzymes which are active in reactions (9). (10), and (11), respec- t 1761’s (9) glycerol / DPI-edihydrenacetone / DPNH (10) glucose / DPN—tglucenolactene / DPIIH -e gluconate (11) fumarate / £30 -—ena1ate An active malic dehydrogenase (Reaction 12) was found in homogenates of P; gallinarum and the nasal T;_gg$g as shown in Figure 18, by following the increase in den- sity at 3&0 millimicrons of a reaction mixture containing diphosphopyridine nucleotide (DPN). (12) salate / UPI—eoralacetate / DPNH In Thunberg experiments the same homogenates exhibited a reducing effect on methylene blue. A weak, but definite, liberation of gas (002) occurred when E; gallinarum.homogenate (but not the others) was incubated with potassium malats and DPN (Figure 19). This corresponds to a "malic enzyme" activity (Reaction 13) seen in Lactobacillus arabinosus (Blanchard, g£_gl., 1950). (13) malate———->Iaotate { 002 No work has been published on the possible importance of the hexose monophosphate shunt in trichomonad metabolism, although Sprince, at 51. (1953) have indicated that ribo- nucleic acids are important for growth of these organisms. The first enzyme in this pathway, glucose-6-phosphate 6? Cuvette contained: 'fg9 0.1 1 m1 glycine .06 M(pH 10) .909 0.5 ml 0.1 M malate .0 002 m1 .009 M DPN Q. ’ 0.08» Oel “11 110171086th / Chang in 0.D. at 0.06b . 380 mu 0 ___. _.——-" ’ 0002' ondo. or no DPN l 2 3 . Time in minutes Figure 18; Melic Dehydrogenase Activity of Trichomonad Sonic Homegenates Harburg flask contained: (fir’fifi ‘VTffii‘fifi‘n‘! 0.25 ml P0 buffer (pH 6.h) 0.25 ml 0.62 M MnClZ 30 0.25 ml 0.009 M DPN t 0.25 ml malate (0.6 M) unarum 1.3 m1 “tor Y0 M 0.7 m1 homogenate ” 20 in sidearm micro- liters C02 endo. 4 10 15 30 66 Time in minutes Figure 19. "Malic enzyme" activity in I; gallinarum Sonic Homogenate 68 dehydrogenase, which catalyzes reaction (1h), has been reported from various micro-organisms. (14) glucose-6-P04 ,1 TPNdG-phosphogluconate / TPNH In certain organisms it has been shown to be highly specific for one or the other of the pyridine nucleotides, while in others the enzyme appears to be active with either DPN or TPN. Very active glucose-6-phosphate dehydrogenase activity, which was specific for TPN, was observed in three tricho- monad homogenates. The nasal strain of‘g;‘ggig showed weak activity. Figure 20 shows the results obtained when increase in density at 3&0 millimicrons was followed in a DU spectrophotometer. 0.2 Change 0.D. at 3&0 0.1 69 L Cuvette contained: . 1 0.1 ml .0015 M TPN * 0.25 ml glycylglycine(pH7.5) 2 - 0.2 m1 homogenate I . (Q l-P. gallinarum 2-1": foetus 3- . suis (fecal) b _ V suis (nasal) e, . W ___g__, ends. or DPN for TPN 1 2 3 Time in minutes Figure 20. Glucose-6-phosphate Dehydrogenase Activity in Trichomonad Sonic Homogenates 70 DISCUSSION The trichomonad flagellates appear to possess a rather strange type of carbohydrate metabolism. It con- sists, at least in part, of many of the reactions of the well-known Meyerhof-Embden pathway; but the organisms also have the ability to perform other reactions, not obviously connected with glycolysis. The tricarboxylic acid cycle does not appear to be present, although detailed studies with cell-free homogenates have not yet been reported. In general, the organisms studied to date have be- haved as facultative anaerobes. They gave evidence of preferring an environment of low oxygen tension, but still showed considerable activity under aerobic conditions. They metabolized carbohydrate substrates to a mixture of organic acids, notably succinic, lactic, acetic, and pyru- vic, and also produced carbon dioxide and hydrogen. Evi- dence is also accumulating for the aerobic utilization of other substrates, such as pyruvate and malate, with- out evidence for the Krebs cycle. In general outline, the organisms in the present study followed the pattern outlined above. However, certain differences were found to exist between the various species and, in some cases, differences from other work were noted. The respiration of these organisms was affected by the age of the culture and the type and pH of the buffer 71 solution used in the experiments. Therefore, comparison of respiratory rates of certain organisms (e.g., Kupfer- berg, at 51., 1953) without other experimental data is not strictly valid. The organisms in this study showed the greatest activity when less than 2h hours old, with a gradual decrease from 2h-72 hours. This, and a fall in R.Q., has been indicated for other protozoa by Hut- chens (19u1)(Chilomonas), Baker and Baumberger (l9ul) 9 (Tetrahymena), and Ryley (1955b)(Strigomonas). When 5 the trichomonads were supplied with exogenous carbohy- drate however there was no appreciable change in the R.Q. of organisms up to A8 hours old, showing that the flagel- lates were capable of actively using an external energy source even if their resting metabolic rate is slowed. It is still unknown, however, whether this respiration provides energy for growth. The data on acid production showed that these flag- ellates produced more acid when harvested during the early and late periods of their growth, with a lag in between. Since acid production is usually evidence of an anaerobic metabolism, this result would indicate that in young cultures, and also as the organism ages, the glycolytic pathway is preferred. The nasal form of T; £E£§ was exceptional because of its constantly high rate of acid production. This was also reflected in the fact that a greater amount of 72 acid end products accumulated in cultures of this organ- ism. In fact, this rather striking difference often made it difficult to work with cultures of the nasal strain since toxic pH levels were reached before satis- factory populations were attained. Additional evidence for increased glycolysis in the nasal form was offered by the observation of a more active hexokinase and the fact that utilization of all the sugars tested was great- er than in the other three organisms tested. Sugars utilized by E;_foetus and the swine forms in the present study were essentially the same as reported by others. However, it is important to note that Ryley's (1955a) comment that strain differences apparently do exist in these protozoa can be confirmed by this work. The MSU strain of T; foetus resembled that used by Doran (1956b) more closely than either that of Ryley (loc. cit.) or Suzuoki and Suzuoki (1951a). All utilized glucose, fructose, mannose, and galactose, but Ryley reported that lactose was also oxidized by his strain, and the Japan- ese workers mentioned sucrose and maltose as stimulatory. Doran (loc. cit.) indicated that maltose was only slightly used, while the MSU organism used sucrose, but attacked maltose only slightly. The use of all sugars by the nasal g; gals, as was shown here, was also reported by Doran. The production of hydrogen by these protozoa is of great interest from the standpoint of comparative bio- ills-.9811". 1 (VI!!! I'iol'nqll (ii 1.31)]. .1 (h 73 chemistry. Suzuoki and Suzuoki (1955a) reported an ac- tive formic dehydrogenase in 2; foetus, but no evidence for hydrogenase or formic hydrogenlyase. Ryley (1955s) was unable to detect any dehydrogenase activity. The organisms in this study all showed a strong formic dehy- drogenase, but only a weak indication of hydrogenase. Formic hydrogenlyase was indicated only slightly by the adaptation experiment reported here. This evidence, however, together with the negative results of the "phosphoroclastic split” reaction suggests that here hydrogen is produced by a method more similar to Escher- ighig and Salmonella than Clostridium (i.e., like the facultative organisms rather than like the anaerobes). Nevertheless, the two forms of 2:,gglg appeared to re- lease hydrogsn from pyruvate and not from formats. The phosphoroclastic enzyme is known to be very labile and, although every precaution was taken, it is possible that it became inactivated during preparation or storage of the sonic homogenates. This could explain the apparent discrepancy and more detailed work on this enzyme is indicated. These organisms differ from other parasitic flagel- lates studied to date (e.g., Trypanosomidae) in being able to degrade carbohydrate past the pyruvate stage. Evidence has been available for many of the enzymes in the Meyerhof-Embden scheme for T; vaginalis and.2; foetus. 711. In this work the presence of certain key enzymes in this scheme has been indicated for all the organisms. This confirms earlier work on 2; foetus and provides similar information.for the other organisms studied. The inability to demonstrate triosephosphate isomer- ase activity by the methods involving omission of hydra- zine from the digestion mixtures is interesting. This result might probably be explained by assuming that an active alpha-glycerophosphats dehydrogenase is present in all these organisms as has been reported for 2;.ggglg- ‘3113. This enzyme would form.alpha-glycerophosphate from the excess dihydroxyacstone phosphate obtained, and thus cause a reduction in color. This dehydrogenase acyivity was not tested in these organisms since a puri- fied substrate was not available. However, since glycerol is not used by these organisms, the importance of this reaction is not clear. The marked inhibitory effect of iodoacetate indicates that glycolysis normally proceeds past the triosephosphate stages: but it is also important to note that fluoride (an enolass inhibitor) was antagonistic (aerobically) to only'T;.foetus and T; gallinarum, the organisms which showed pyruvate formation from phosphoglyceric acid. This suggests that at least these latter two organisms produce pyruvate (and perhaps lactate) by conventional means under anaerobic conditions. The 7S stimulation of acid production by fluoride under anaer- obic conditions could perhaps result from the inhibition of enolass and a coupled oxidation-reduction of two moles of triosephosphate to give alpha-glycerophosphate and 3-phosphoglyceric acid, as has been postulated by Ryley (1955b) for Strigomonas oncopslti. Arsenate stimulation is compatible with the presence of a triosephosphate oxidizing system since addition of this ion increases the available esterifying material, and oxidation of the arseno-phesphoglyceraldehyds may not require arsenate acceptors, thus not limiting the rate of the reaction (Werkman and Schlenk, 1951). This has been observed by Speck and Evans (l9h5) in their studies on glycolysis in malaria parasites. The pathway of formation of the organic acids formed as end products of trichomonad metabolism presents an interesting problem. In the absence of detailed infor- mation, speculation as to the possible schemes involved is the best that can be done. The presence of enzyme systems active with malate is especially interesting. Malate can obviously arise from pyruvate or lactate by carbon diexide fixatien (the Wood- flerkman reaction). It could then be degraded through fumaric acid te succinic acid. (Failure in this work to demonstrate fumarase might be due to unrecognized experimental error; hewever, fumarate was shown to act 76 as a hydrogen acceptor, especially with g; gallinarum.) The efficiency of the organisms in fixing C0 would deter- 2 mdns the small amount of pyruvic and lactic acids found as end products at any one time. It is important to note that Baernstsin and Rees (1952) indicated a malate system in.2:.ggggi. They suggested that a linkage of this sys- tem to glutamic and aspartic acids through transaminase enzymes might prove to be more important than the glucose system in explaining respiratory activity. Under anaerobic conditions pyruvate could form.1ac- tic and acetic acids, as well as 002, by the well-known Krebs dismutation (Reaction 15). (15) 2 pyruvate—alum” / acetate ,1 6'02 Pyruvate could also, as indicated before, partici- pate in a ”phosphereclastic" reaction giving rise to for- mate and acetyl phosphate ('active' 02) and eventually te hydrogen and 002. The possibility of a 2-02 condensation to succinate or malate appears here. Another interesting possibility is the probable presence of a hexose monophosphate shunt mechanism.in these organisms, suggested by the demonstration of glucose-6-phosphate dehydrogenase. By this pathway triosephosphate and an ”active” C2 fragment are formed. A 2-C condensation ts succinate or malate as referred 2 to earlier might be possible here. Also, Ochoa, g£_;l. (1950) have shown that fixation of CO2 by pyruvate in 77 the presence of reduced TPN (frem the glucose-6-phes- phate dehydrogenase reaction) can give rise to malate. Another hypothesis would involve the formation of ribulose-S-phosphate via the hexose nonophosphate shunt followed by its splitting into triosephosphate plus a 02 fragment which could condense with ribose forming sedoheptulose. This C7 sugar could possibly split to another triosephosphate and a C compound which might then proceed to succinate. u The identity of the tsnminal oxidizing systems of these organisms remains an unanswered question. Insen- sitivity of respiration to cyanide and azide, and the lack of evidence fer cytochrome absorption bands in spectroscopic examinations, speak against this type of system. In all probability the flavin nucleotides are important in the trichomonads although much more work remains before details will be apparent. The scheme shown in Figure 21 is an outline of the above discussion, showing the possibilities which exist in the breakdown of carbohydrates by trichomonads. It appears that detailed investigation of the reactions involving pyruvate and malate is indicated. Besides attempting to investigate certain aspects of carbohydrate metabolism in these trichomonads, it was also a purpose of this work to discover, if possible, biochemical differences between the organisms employed, 78 _polysaccharides glucose _ngPOu—e glucos e-6-P0u—e 6-phos phoglucona te fructose-6-P0u i HMSc hexose diphosphate ribulos'e-S-POu--—-fi I dihydroxyace one P0“ """ '3-phosphog ceraldehydee-u.c3 / .02 l alpha-glycerophosphate 1,3 diphosphoglycerate C I . 2 1’ C5 I, 3- -phosphoglycerate “- : I I 2:phosphoglycerate 7 I \ l phospho-enal-pyruvate \ 12 \ lactic_/ 99093135 ......... pyruvicacid\ \ i can 69.9.9121 , ‘ \ ,' \\\ / \ (/C02) \ I ‘\j 90 lactic acid \ \I \\/ | , C \ 3c; tic / formic! :(/COZ)QY§§ ‘\ {’4 scid 8°19 . $ \ l *mlatet—goxalacetate I ’ 3 w H l acetyl P0 {-312 ,l c I/ . \ H I ( \\ " I’ l ‘gaspartic acid' 'x2 ," : glutamic acid: | \ L::-? ----- -'/ \\ ( : IN-I‘ \ifumarate I \~‘\‘ I “‘~Lsuccinate ? ' 51;. ............... l fi-HMS= hexose monophosphate shunt Reactions which have been observed in trichomonads are indicated by solid lines. Those which are postulated are indicated by broken lines. The products which have been identified in any trichomonad are underlined in red. Figure 21. Possible Metabolic Inter-relationships in the Carbohydrate Metabolism of Trichomonads 79 especially the two swine forms. The apparently greater acid-producing capacity of the nasal I:_guig has already been mentioned. Also, this organism.appears to differ from its fecal counterpart by the fact that ever 50% of its acid production can be accounted for as succinic acid. The fecal strain forms succinic as only 15% of its total acid, and also evidently produces a great deal of volatile acids (unaccounted for in the present analyses). With regard to this acid for- mation the nasa1.l;’ggi£ more closely resembled T; foetus than'g;‘ggig (fecal). This resemblance has been pointed out, on morphological grounds, by Buttrey (1956). The respiration of both swine forms was more stimu- lated by exogenous carbohydrate than that of either L foetus or P. gglIinarum. Respiratory stimulation by disaccharides also differed, lactose and maltose being used only by the nasal form, and sucrose only by T; foetus. Intracellular glycogen storage was considerably greater in _T_._ _sliig (fecal) than in T_._ _s_1_1_i_s_ (nasal), suggesting that the nasal organism, more than the fecal form, required or preferred supplied, rather than formed, carbohydrate for energy. Both swine organisms also differed from _T_._ foetus and g; gallinarum by not utilizing lactate or formate for increased hydrogen production. This, and their weak response in the hydrogenlyase adaptation experiment, 80 would seem to indicate the presence of a "phosphore- elastic" system in these organisms. On the other hand, evidence for phesphoglycerie acid dissimilation to pyru- vate was not demonstrated in homogenates of the swine organisms, but was found in the other two. 2; gallinarum.showed considerably more malate activ- ity than any of the others, a possible indication that this intermediate is more important in its metabolism. 81 SUMMARY (1) T; foetus, g; gallinarum, and the focal and nasal (2) (3) forms of 2:’£213 were found to grow well in.NIH Thioglycollate Broth (Case or Difco) with the addi- tion of l% sterile beef serum (Difco Beef Blood Serum, Dehydrated). Populations sufficient for mmometric experiments were obtained in 30-150 hours if the original inoculum.was reasonably large. Special preliminary cultures were used to insure this. During growth.large amounts of acid were produced from glucose by all the organisms. The nasal 23.5213 hewever, was the most active in this regard. The pH at the time of maximum population was between 6.0 and 6.3 for all species and living ergmnisms disappeared almost entirely at pH 5.2. These re- sults are similar to data previously reported for other trichomonads. Succinic acid was the major acid produced by T"feetus, f;_gallinarum, and the nasal 2; suis, accounting for ever 50% of the total acid formed in each case. 2; guig (fecal) produced mmre lactic than succinic ‘Cid. but much of its acid is unaccounted for in these experiments. It is postulated that this might be acetic acid. 82 (h) The endogenous metabolic activity of the organisms (6) (7) was shown to vary with age at the time of harvest from culture, being greatest in young forms, and decreasing with advancing age. The composition of buffers was also shown to be important in the results obtained. With an external supply of carbohydrate the R.Q. of the organisms was constant for cultural ages of lZ-hb hours. Glucose, mannose, fructose, and galactose were most active in stimulating respiratory activity. 2; gallinarum, however, used fructose and galactose more slowly than the others. Dissccharides did not stimulate the respiration as well as did the monosaccharidos. Maltose and lactose were used by the nasal form only, and sucrose by 1; foetus-alone. Intracellular glycogen synthesis was indicated with most of the sugars. Iodoacetate was the most potent inhibitor of the aerobic or anaerobic metabolism of all the species studied. Arsenate was stimulatory to all but 3; ggllinarum, and malonate failed to affect the respiration of any of the organisms. Hydrogen peroxide was not inhibitory since all the flagellates possessed strong catalase activity. Hydrogen production by these organisms decreased with cultural ago; and it was stimulated by formate (8) (9) 83 in.1& foetus and 2; gallinarum, and by pyruvate in the swine forms. Presumptive evidence for a hydrogenlyase system.(at least in g; foetus and '2; gallinarum) was obtained. Formdc dehydrogenase activity was marked in all the species. Hexokinase, phosphohexoisomerase, phosphofructo- kinase. aldolase, and glucose-é-phosphate dehydrogen- ase were demonstrated in sonic homogenates of each of the four organisms. Phosphoglucomutase activity was seen in preparations of 2; foetus and the fecal I; suis. Production of pyruvic acideron phospho- glyceric acid, an indication of phosphpglyceromutase and enclose activities, was seen only in 1; foetus and g; gallinarum. the only two which exhibited susceptibility to fluoride inhibition. _T_=_ 3113.3 (focal) and 2; gallinarum were found to possess malic dehydrogenase, and E; g;llinarun.also showed "malic enzyme" activity. Possible biochemical inter-relationships, based on available information for trichomonad metabolism, are discussed. It is felt that investigation of reactions involving malate and pyruvate will provide a key to understanding the complex life processes of these flagellates. 81+ RmFEHbNCES Andrews, J.M., and von Brand, T., 1938, Quantitative studies of glucose consumption by Trichomonas Mn 521-. 2.215.; £53. (1). 136-1117:- Baernstein, H.D., 1953, The enzyme systems of the culture form of Tgypanosoma cruzi., Ann. N.Y. Acada SCio 26-) p 9 -9 e Baernstsin, H.D., 1955. Aldolase in Trichomonas vaginalis., Exptl. Parasitol.y§ (h). 323-33H. Baernstein, H.D., and Ross, c.w., 1952, Aldolase in the culture form of T anosoma cruzi. Exptl. Parasitol. L (3). - . ' Baker, 3.6.8., and Baumberger, J.P., 19ul, The respiratory rate and the cytochrome content of a ciliate protozoan (Tetrahémena geleii). J. Cell. Comp. PhiSiOle E p "’ e . _- Barker, S.A., and Bourne, E.J., 1955. Composition and synthesis of the starch of Pol tomella coeca., pp. u5-56, in S.B. Hutner and i. Ewoff, eds. 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Lindblom ABSTRACT It was the purpose of this work to investigate the metabolic activities toward carbohydrates of four species of trichomonads, to relate the information gained to other flag- ellates which had been studied, and to discover, if possible, differences between the four species. It was found that all species studied (2; foetus, 2; gallinarum, and 1; 32;! nasal and fecal strains) grew well in NIH Thioglycollate broth with the addition of 1% sterile beef serum. During growth in this medium the organisms pro- duced various acid products from the glucose it contained. Succinic acid was the major acid produced by E; foetus, g; gallinarum, and the nasal 2; suis, accounting for more than 50% of the total acid in each case. The fecal 2;.ggig pro- duced more lactic acid than succinic acid, but about h5¢ of the total acid was unaccounted for (probably volatile acid such as acetic). Pyruvic acid was found in small amounts in all cultures. The nasal form of 1;_ggig produced much.more total acid than the others. During growth.the pH of the medium fell to about 5.2 at which point all organisms were dead. The pH at the time of maximwm population was between 6.0 and 6.3. These findings were consistent with other work on 1; foetus and 2; vaginalis. The metabolic activities of the organisms (oxygen uptake, 002 production, hydrogen evolution, anaerobic acid formation) varied with the age of the organisms. nespiratory activity was highest at about 12 hours, but varied considerably thereafter. Hydrogen was produced in greatest amount when the organisms Gordon P. Lindblom were about 12 hours old, showing a gradual decline thereafter. Acid production was high at 12 hours, fell between 20-30 hours, and rose again by hB hours. With an external carbohydrate source the R.Q. of the organisms was consistent throughout growth. Glucose, mannose, fructose, and galactose were most active in stimulating respiration. Disaccharides were slowly utilized. Intracellular glycogen synthesis occurred on incubation with most of the sugars. Iodoacetate and fluoride were the most ac- tive inhibitors of sugar utilization. Malonate was not effective. Cyanide and azidewere ineffective also, as was H202 since all the organisms showed strong catalase activity. Evidence was obtained for the presence in these organisms of hexokinase (greatest activity in the nasal:form), phospho- hexoisomerase, phosphofructokinase, aldolase, and glucose-6- phosphate dehydrogenase. Phosphoglucomutase was demonstrated in 2; foetus and the fecal 2; suis. 2; foetus and Z; gallinarum showed evidence of phosphoglyceromutase and enolass. §;_suis (fecal) and 2; gallinarum were found to Possess malic dehydro- genase,and g; gallinarum gave evidence of "malic" enzyme activity. Formic dehydrogenase activity was marked in all forms, and pre- sumptive evidence for a formic hydrogenlyase system was obtained (at least for 2; foetus and 3; gallinarum). ‘ A discussion is presented indicating that investigations of those reactions which could involve pyruvate and malate (with a possible linkage with a hexose mononhosphate shunt) xnight provide a key to a more complete understanding of trichomonad metabolism of carbohydrates. V4 . -. MN?" L ~33»- we. WY 1153““ IE5 \ H "7|!ififlfilfilflfijflufififlflflfiflififlufiflfiflfifi