Molecular and Biochemical Parasitology', 54 (1992) 185-200 185 © 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00

MOLBIO01791

Characterization of carbohydrate metabolism and demonstration of glycosomes in a Phytomonas sp. isolated from Euphorbia characias

a* • a** M a n u e l S a n c h e z - M o r e n o , D e m e t e r Lasz t l ty , Isabel le C o p p e n s a'b

and F r e d R. O p p e r d o e s a

aResearch Unit for Tropical Diseases and bCell Biology Unit, International Institute of Cellular and Molecular Pathology and University of Louvain Medical School, Brussels, Belgium

(Received 2 January 1992; accepted 29 April 1992)

Phytomonas sp. isolated from Euphorbia characias was adapted to SDM-79 medium. Cells isolated in the early stationary phase of growth were analyzed for their capacity to utilize plant carbohydrates for their energy requirements. The cellulose- degrading enzymes amylase, amylomaltase, invertase, carboxymethylcellulase, and the pectin-degrading enzymes polygalacturonase and oligo-D-galactosiduronate lyase were present in Phytomonas sp. and were all, except for amylomaltase, excreted into the external medium. Glucose, fructose and mannose served as the major energy substrates. Catabolism of carbohydrates occurred mainly via aerobic glycolysis according to the Embden-Meyerhof pathway, of which all the enzymes were detected. Likewise, the end-products of glycolysis, acetate and pyruvate, glycerol, succinate and ethanol were detected in the culture medium, as were the enzymes responsible for their production. Mitochondria were incapable of oxidizing succinate, 2-oxoglutarate, pyruvate, malate and proline, but had a high capacity to oxidize glycerol 3-phosphate. This oxidation was completely inhibited by salicylhydroxamic acid. No cytochromes could be detected either in intact mitochondria or in sub-mitochondrial particles. Mitochondrial respiration was not inhibited by antimycin, azide or cyanide. The glycolytic enzymes, from hexokinase to phosphoglycerate kinase, and the enzymes glycerol kinase, glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, malate dehydrogenase and adenylate kinase, were all associated with glycosomes that had a buoyant density of about 1.24 g cm- I in sucrose. Cytochemical staining revealed the presence of catalase in these organelles. The cytosolic enzyme pyruvate kinase was activated by fructose 2,6-bisphosphate, typical of all other pyruvate kinases from Kinetoplastida. The energy metabolism of the plant parasite Phytomonas sp. isolated from E. characias resembled that of the bloodstream form of the mammalian parasite Trypanosoma brucei.

Key words: Phytomonas; Carbohydrate metabolism; Glycolysis; Glycosome; Hydrolase; Respiration

Correspondence address: F.R. Opperdoes, ICP-TROP 74.39, Avenue Hippocrate 74, B-1200 Brussels, Belgium. Tel.: 32-2- 764 74 39; Fax: 32-2-762 68 53. E-mail: Opperdoes@trop.ucl.- ac.be.

Present addresses: *Grupo de Bioquimica y Parasitologia Molecular, Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias, Universidad de Granada, Granada, Spain. **Department of Plant Physiology, Faculty of Natural Sciences, Eotv6s University, Budapest, Hungary.

Abbreviations." NMR, nuclear magnetic resonance; GAPDH, glyceraldehyde phosphate dehydrogenase; PG1, phosphoglu- cose isomerase; PGM, phosphoglucomutase; ALAT, alanine aminotransferase; HK, hexokinase; ALDO, aldolase; TIM, triosephosphate isomerase; PEPCK, phosphoenol pyruvate carboxykinase; PFK, phosphofructokinase; GDH, glycerol-3-

Introduction

Flagellated trypanosomatids of the genus Phytomonas are parasites of plants, responsible for economically important diseases [1]. They are the etiological agents in devastating crop epiphytotics, but may also parasitize many plants without apparent pathogenicity. The parasites are thought to be transmitted to plants through the bite of phytophagous

phosphate dehydrogenase; Malic, malic enzyme; PK, pyruvate kinase; ADK, adenylate kinase; GK, glycerol kinase; PGK, phoshoglycerate kinase; MDH, malate dehydrogenase.

186

insects harboring the flagellate in their gut and salivary glands [1,2]. In the plant they para- sitize the lactiferous tubes that contain the milky substance known as latex and the phloem and xylem of palm trees and other plants where they may exert a pathogenic effect.

The lack of isolates and cultures of Phytomonas spp. has precluded adequate studies of the morphological and biochemical characteristics of this group of flagellates. Only recently has it become possible to obtain axenic cultures of the parasites isolated from various plants [3-5] which allowed compara- tive electron microscopic studies to be done in a number of Phytomonas species [6], but little to nothing is known about nutritional needs and metabolism in this genus.

Phytomonas spp. all have structures typical of the members of the family of the Trypano- somatidae [6-8]. They have a kinetoplast, tubular mitochondria with few cristae and glycosome-like organelles. Glycosomes con- tain the first part of the glycolytic pathway and are presumed to belong to the peroxisome family of organelles. They have been described for the other major representatives of the trypanosomatids, i.e., Trypanosoma spp. [9,10], Leishmania spp. [11], and Crithidia spp. [12], but direct evidence for the presence of glycosomes in Phytomonas spp. has been lacking so far.

Ultrastructural studies on the isolate from the plant Euphorbia characias has revealed the presence of many glycosome-like structures that were unevenly distributed within the cell. They often look like stacks of flattened disks, which occasionally are associated with bundles of filamentous structure [6]. In addition the Phytomonas species isolated from E. characias contained more (W. De Souza, personal communication) of these organelles then any other trypanosomatid analyzed to date.

In this paper we show that a Phytomonas sp. isolated from E. characias is highly specialized in the utilization of carbohydrates from its plant host. We present a characterization of the major energy-producing pathways in this organism and biochemically characterize its

peroxisome-like organelles and show that these microbodies are true glycosomes. Part of this work has already been published elsewhere in a preliminary form [13].

Materials and Methods

Organism. A Phytomonas sp. was originally isolated from E. characias by Dollet et al. [3] and grown at 28°C in a biphasic medium made of 1.5% blood agar-base and 9% defibrinated rat blood with an overlay of Roitman's medium [14]. Upon arrival of the organism in Brussels it was immediately transferred to SDM-79 medium [15]. After 3 passages of the organism in this monophasic medium it was able to grow with a doubling time of 12 h to a density >5 x 107 ml -~. Cells were harvested at the late log(phase of growth (cell density 5 x 107 m l - ) and thoroughly washed in

isotonic homogenization buffer containing 0.25 M sucrose/25 mM Tris-HCl/1 mM EDTA, pH 7.8, by centrifugation at 3000 rev./min (1000 x g) for 5 rain in the SS-34 rotor of a Sorvall RC-5 centrifuge at 4°C.

Cell fractionation. Homogenates of Phytom- onas sp. were prepared by grinding with silicon carbide abrasive grain as described previously [16]. Differential centrifugation was performed in a Sorvall RC-5 Superspeed refrigerated centrifuge equipped with a SS-34 rotor. The final step at 40000 rpm was carried out in a Beckman L5-50 preparative ultracentrifuge with a 50-Ti rotor. The sequential centrifuga- tion scheme was as follows: unbroken cells, nuclei and debris were sedimented at 3500 rev./ min (1500 x g) for 10 min (nuclear fraction). From the resulting cytoplasmic extract (post- nuclear supernatant) a granular fraction was separated at 11 000 rev./min (14500 x g) for 10 min and a microsomal fraction was separated by centrifugation at 40 000 rev./min (139000 x g) for 1 h from the final super- natant. The nuclear, granular and microsomal pellets were washed once with homogenization buffer after resuspension in a Potter tissue homogenizer with loosely fitting pestle.

For density equilibration a post-nuclear supernatant (14500 × g for 10 min) was layered on top of a linear sucrose gradient containing 25 mM Tris-HC1, pH 7.4 and 1 mM EDTA and centrifugation was carried out in a Beckman VTi-50 rotor at 48000 rev./min (190000 × g) for 150 min as previously described [17]. Gradient fractions were collec- ted from the bot tom of the gradient by suction. Protein content and density of the fraction were measured immediately prior to storage of the fractions at - 8 0 ° C for later analysis. The presentation of the distribution patterns is as described by Beaufay and Amar-Costesec [18].

Preparation of mitochondria and submitochon- drial particles. Mitochondria were prepared as described by Toner and Weber [19]. Submitochondrial particles were prepared as described by Kusel and Storey [20]. Difference spectra were recorded on a Aminco-Chance DW-2 double beam dual wavelength spectro- photometer at room temperature over the wavelength range of 400-650 nm as described previously [17].

Respiration studies. Polarographic experi- ments were carried out with a Clark-type oxygen electrode, calibrated against oxygen- saturated water and a sodium dithionite solution. The oxygen electrode vessel had a volume of 1.5 ml and contained 350 mM sucrose, 20 mM morpholinopropane sulfonate buffer, pH 7.0.

Lateno' studies. For the determination of the degree of latency of a number of particle- bound enzymes a washed preparation of a granular fraction was used. Free activity was measured in an isotonic enzyme assay mixture to which 0.25 M sucrose was added. Total activity was measured under identical condi- tions but after the addition of 0.2 % Triton X- 100.

Enzyme determinations. The glycolytic en- zymes were assayed as described by Misset and Opperdoes [21]. Pyruvate kinase activity was measured after the addition of fructose

187

2,6-bisphosphate as described by Van Schaf- tingen et al. [22]. Malate dehydrogenase, adenylate kinase and phosphoenolpyruvate carboxykinase were assayed according to Opperdoes et al. [17]. Fumarase, succinate dehydrogenase and fumarate reductase were measured according to Klein et al. [23]. Alcohol dehydrogenase and lactate dehydro- genase were measured according to Bergmeyer [24]. Catalase activity was determined by the UV method [25]. Acid phosphatase was assayed as described previously [17]. Amylase [26]', amylomaltase [27], invertase [27], carboxy- methylcellulase [28], pectin-degrading enzymes [29], proteinase [30] and ribonuclease [31] were all measured according to published proce- dures.

Measurement of excreted enzymes. To de- monstrate the excretion of hydrolases from Phytomonas sp. intact cells were incubated in isotonic phosphate buffer for 2 h at 25°C. After incubation the cells were removed by centrifugation and the remaining activity in the supernatant was measured.

Preparation of samples and : H-NMR spectro- scopy. Cell cultures were started by inocula- tion of 200 ml of SDM-79 medium in 300 ml flasks with 2 ml of a mid-log phase culture of Phytomonas sp. (approximately 5 × 106 cells ml-1). Samples (5 ml) were removed from the culture flasks periodically (at 0, 6, 12, 24, 30, 48, 60 h) and the cell numbers were determined from the turbidity at 550 nm. After removal of the cells by centrifugation the pH of each sample was determined. The cell-free medium was stored frozen at - 2 0 ° C until further use.

1H-NMR spectra were obtained at 300 MHz on a Brucker CXP-300 spectrometer operating in the pulsed Fourier transform mode with quadrature detection. Probe temperature was maintained at 27°C. Typical acquisition para- meters were: 3287.5 Hz sweep with, 8192 time domain addresses, 90 ° radio frequency pulses, 8 s total recycle time and 160 accumulations. Where difference spectra were required, the time domain data were zero filled to 16384 addresses before Fourrier transformation.

188

Chemical shifts are expressed as parts per million (ppm) downfield from TSP. The resonances of a number of metabolites were determined by the addition of standards and their chemical shifts measured.

Electron microscopy. Phytomonas were re- suspended in 1.2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 and fixed for 60 min. Cells (10 s ml - I ) were filtered [32] through a nitrocellulose filter (0.1 ~m diameter pores, Millipore Co., Bedford, MA). Postfixa- tion was performed for 1 h at 4°C in a solution containing 1% osmium tetroxide and 2% potassium ferrocyanide. Samples were then dehydrated and embedded in an epoxy-resin mixture [33]. Sections were stained first with 3% uranyl acetate and then with lead citrate [34] and examined with a Philips EM 301 microscope at 60 kV.

Cytochemistry. For detection of catalase activity, glutaraldehyde-fixed cells were wa- shed twice in 0.1 M cacodylate buffer, then incubated for 60 min at 37°C in a medium containing 3,3-diaminobenzidine (Sigma Chem- ical Company, USA) at a concentration of 0.5 mg ml 1 in 0.05 M Tris-HC1 buffer, pH 9.0, and 0.02% H202 [35]. Controls were subjected to 10 mM 3-amino-l,2,4-triazole as inhibitor of catalase activity, in the incubation medium. The sections were stained only with lead citrate.

Results

Utilization of nutrients by Phytomonas sp. We have investigated whether Phytomonas sp. was capable of converting starch and cellulose into monosaccharides that could serve as nutrients for the parasite. Table I shows that Phytomonas contained high activities of amy- lase, amylomaltase and invertase, all of which are involved in the conversion of starch into glucose and fructose (see pathway in Fig. 1). In addition carboxymethyl cellulase, an enzyme of cellulose degradation, was present in high activity. Pectin, another major component of

the plant cell wall, is degraded by the combined action of polygalacturonase and oligo-D- galactosiduronate lyase. These 2 enzymes were also present in Phytomonas homoge- nates. Interestingly, all the enzymes involved in the degradation of plant polysaccharides, except amylomaltase, were excreted into the medium. Their activities were not only easily detectable in supernatants of cultures but also in the supernatant after short-term incubations of live cells in isotonic phosphate buffer (Table I). This was not the result of release of enzymes due to cell lysis, since hexokinase and malate dehydrogenase could not be detected in such supernatants. Acid phosphatase and acid ribonuclease were also excreted under these conditions, while both acidic and neutral proteinase were not. After differential centri- fugation of a Phytomonas homogenate amylo- maltase and proteinase activities were predominantly found in the high speed super- natant. Amylase, invertase, carboxymethyl cellulase, polygalacturonase and oligo-o-galac- tosiduronate lyase were mainly associated with particulate material (not shown).

The monosaccharides glucose (10 raM), fructose (10 mM), and mannose (10 raM) all supported respiration of intact washed cells. With glucose as the substrate the rate of oxygen consumption was 130 nmol 02 min (mg protein) i. The disaccharide sucrose (10 raM) did not support respiration. Succinate, glycerol and glycerol 3-phosphate, all at a

TABLE l

Specific activities of hydrolytic enzymes in a Phytomonas homogenate and in a supernatant after 2 h of incubation at 25°C

Enzyme Specific activity (mU m g i)

Homogenate Supernatant

Acid phosphatase 143 22 Amylase 307 46 Amylomaltase 43 nd ~ Invertase 249 38 Carboxymethylcellulase 139 21 Potygalacturonase 267 40 Oligo-D-galactosiduronate lyase + + b Proteinase 13 nd Acid ribonuclease 331 159

aNot detectable. bpresent, but specific activity not determined.

189

C e l l u l o s ~

1 2 Starch ) Maltose . ~ G l u c o s e ..

Sucrose ) Fructose 3

Glycolysis

/ 2-Keto-3-deoxy-6-phosphogluconic acid ~ " ~ ' ~ " ~

Trlose-phosphate

" ~ " Pyruvate 10 9

2- Keto-3-deoxy-6-gluco nic acid

D-Tagaturonic acid

D-AItronic acid

D-Galacturcnic acid Oligogalacturonic acid

~ 5a,b

Pectin

Enzvmes

1 Amylase 2 Amylomaltase 3 Invertase 4 Carboxymethyl cellulase 5a Polygalacturonase 5b Olid-D-galactosiduronate lyase 6 Uronic acid isomerase 7 D-Altronic acid dehydrogenase 8 D-Altronic acid dehydrase 9 2-keto-3-deoxygluconate kinase 10 2-keto-3-deoxygluconate aldolase

Fig. 1. Pathways of polysaccharide degradation.

concentration of 15 mM could all be oxidized by intact cells. Potassium cyanide (10 mM) and antimycin (2 mM) had no effect on the respiration of intact cells. 50 mM sodium azide had a slight inhibitory effect (15%), while 2 mM SHAM inhibited respiration by more than 50%.

End products of carbohydrate catabolism. To identify the major metabolites that were excreted into the culture medium by Phytomonas, the composition of the medium was analyzed by the technique of 1H-NMR spectroscopy. Fig. 2 shows a typical result. The upper trace (i) represents the fresh medium before inoculation of cells, while the middle

trace (ii) is the spectrum of the medium after Phytomonas had grown to stationary phase under aerobic conditions. The difference spectrum (trace iii) shows that the major carbohydrate utilized was glucose (3.780 ppm) while the major products excreted were acetate (1.855 ppm) and ethanol (1.185 and 3.657 ppm). Glycerol (3.565 ppm), succinate (2.380 ppm) and pyruvate (2.415 ppm) were also present but as minor products. Fig. 3 shows how the pH and the various metabolites in the culture medium changed with growth. During logarithmic growth glucose was con- tinuously consumed but after all glucose was exhausted from the medium the cells went into stationary phase. During growth the medium

190

I

I I I

] ? c e t a t e Ethanol

! 1,1 J Ethanol , 1 Pyruvate

S u c c i n a t e

Glucose [ I I I

4 3 2 1 p p m

Fig. 2. Proton-NMR spectra of Phytomonas sp. cultures. Trace (i), fresh culture medium before inoculation of cells. Trace (ii), culture medium after Phytomonas had grown to stationary phase. Trace (iii), difference spectrum of trace (ii) minus trace (i). Chemical shifts are expressed as parts per million (ppm): glucose, 3.780 (d); acetate, 1.855 (s); ethanol, 1.185 (t) and 3.657 (d); glycerol, 3.565 (t); succinate, 2.380 (s) and pyruvate, 2.415 (s). (s), singlet;

(d), doublet; (t), triplet.

slowly acidified due to the production of carboxylic acids, such as acetic, pyruvic and succinic acid.

Screening ./or mitochondrial activities. Both isolated mitochondria and sub-mitochondrial particles were screened for the presence of cytochromes by making reduced-minus oxi- dized difference spectra at room temperature. No absorption typical of cytochromes could be detected, while under the same conditions and at the same protein concentration a control sample of rat-liver mitochondria revealed clear peaks for the cytochromes a, b and c.

When mitochondria were mixed with glycer- ol 3-phosphate in an oxygen electrode, a several fold increase in mitochondrial oxygen consumption was observed (Fig. 4). This enhanced respiration was stimulated by the presence of 1 mM ADP, but could not be inhibited by the classical inhibitors of the respiratory chain such as KCN (10 mM), antlmycin (2 mM) or sodium azide (50 raM).

TABLE 11

Specific activities of enzymes in homogenates of Phytomonas sp.

Enzyme Specific activity (mUmg 1)

Hexokinase 78 Glucosephosphate isomerase 266 Phosphofructokinase 143 Aldolase 147 Triosephosphate isomerase 106 Glyceraldehyde phosphate dehydrogenase 134 Phosphoglycerate kinase 213 Enolase 45 Pyruvate kinase 49 Alcohol dehydrogenase 7 Phosphoglucomutase 25 Glucose 6-phosphate dehydrogenase 4 Glycerol kinase 56 Glycerol 3-phosphate dehydrogenase 146 Phosphoenolpyruvate carboxykinase 17 Malate dehydrogenase 357 Malic enzyme 153 Fumarate reductase 8 Succinate dehydrogenase 124 Fumarase nd Malate synthase 7 Isocitrate lyase 6 Adenylate kinase 18 Alanine aminotransferase 21 Catalase 31

0.8-

"~ 0.7'

o. 0.6' 0

0.5 0

D e n s i t y

i

20 40 60 Time (hrs)

Growth of Phytomonas

80

12

10'

6 -

8 -= 4

2

0 0

glucose

20 40 60 Time (hrs)

7 . 6

" 7 .4

7 . 2

" 7 . 0

6 . 8

6 . 6

8O

- r

191

Excretion of metabolites

200

160-

c

= 120"

~ 80"

40'

Ethanol

0 - • i • • i • • i • •

20 40 60 Time (hrs)

20"

cn 15" m c

>" 10" m

Pyruvale 8 Succinate

5 " / ,

0 ~ . . 80 0 20 40 60

Time (hrs)

Fig. 3. Evolution of pH and various metabolite concentrations in the culture medium during growth of Phytomonas sp. Glucose was measured enzymatically. Acetate, pyruvate, succinate and glycerol concentrations were estimated from the peak

intensities in the ~H-NMR spectra.

Salicylhydroxamic acid (SHAM, 1 mM), an inhibitor of cyanide-insensitive respiration, both in plants and in Trypanosoma brucei, completely inhibited the respiration by Phytomonas mitochondria.

Screening for enzymes. Total homogenates of Phytomonas sp. were screened for the presence of enzymes that have been described for several representatives of the Trypanosomatidae and that could serve to identify the various subcellular constituents of Phytomonas and the associated metabolic pathways. Table II shows that in Phytomonas homogenates the enzymes of the Embden-Meyerhof pathway of glycolysis and glycerol-3-phosphate dehydro- genase were present. In addition phosphoenol-

pyruvate carboxykinase, malate dehydrogen- ase, fumarate reductase and succinate dehy- drogenase, were present. Fumarase activity could not be detected. In other trypanosoma- tids, these enzymes are involved in the reductive pathway of succinate production. In addition glycerol kinase and alcohol dehydro- genase were present at relatively low specific activities, in line with the observed formation of ethanol and glycerol as minor end-products of Phytomonas' carbohydrate catabolism. Lactate dehydrogenase was not detected. Malic enzyme was also present in Phytomonas. Catalase a marker for peroxi- somes and associated with the glycosomes ot certain Kinetoplastida (i.e., Crithidia sp. and Trypanoplasma sp.; refs. 12 and 36) was

192

250

E "~" 200 U~

o E c c 0

¢" 100 O C O O C ~) 50

X o

/Glycerol 3-phosphate

p " - k ~ n t imyci n

Azide

I I I I

2 4 6 8 10

T i m e (ra in ) Fig. 4. Oxygen consumption by Phytomonas mitochondria. For conditions, see Materials and Methods. Additions: glycerol 3-P (15 mM), ADP (I mM), KCN (10 mM), antimycin (2 #M), sodium azide (50 mM), salicylhydrox-

amic acid (SHAM, 1 raM).

present, but at very low activity. The glyox- ylate cycle enzymes malate synthase and isocitrate lyase were also detected.

Latency of glycolytic enzymes. As a first indication for an association of glycolytic enzymes with glycosomes, we have searched for latency of enzyme activity in freshly prepared particle fractions. Various degrees of latency were measured for a number of glycolytic enzymes and glycerol-3-phosphate dehydrogenase, glycerol kinase, malate dehy- drogenase and phosphoenolpyruvate carboxy- kinase. Glucose-6-phosphate dehydrogenase, a cytosolic enzyme in trypanosomatids [37], did not show any latency.

Cell fractionation. Fig. 5 shows the distribu- tion profiles of a number of selected enzymes after fractionation of a Phytomonas homoge- nate by differential centrifugation. The glyco- somal enzymes hexokinase, glucosephosphate isomerase, phosphofructokinase, aldolase, triosephosphate isomerase, glyceraldehyde- phosphate dehydrogenase, glycerol-3-phos- phate dehydrogenase, glycerol kinase and

4 ~- GADPH PGI PGM ALAT

o

4

~0 >

o 101%

_~ 0 I

0 , ,

ALDO TIM PEPCK

r 1 ,r- GDH MALIC

123% 78%

7.75

PK ADK 83% 78%

8 0 %

J / 100 0 100 0 100 0

PROTEIN (%)

MDH 114%

! "-7

0 r - - 0 100

Fig. 5. Distribution profiles of a number of selected enzymes in fractions obtained by differential centrifuga- tion of a Phytomonas sp. homogenate. The fractions are plotted in the order of their isolation (from left to right): nuclear, large granular, small granular, microsomal and final supernatant. Percentage of recovery for each enzyme

is indicated.

phosphoglycerate kinase, were all predomi- nantly associated with the small granular fraction. Also, the enzymes malate dehydro- genase and PEP-carboxykinase had distribu- tions similar to that of the glycosomal markers. Glucose-6-phosphate dehydrogenase, phos- phoglucose mutase, enolase, pyruvate kinase, and alcohol dehydrogenase behaved as soluble enzymes. Approximately half of the malic enzyme and alanine aminotransferase was associated with the large granule fraction.

Fig. 6 shows the fractionation of a post- nuclear extract by isopycnic centrifugation in a sucrose gradient. The glycolytic enzymes together with those of glycerol metabolism and malate dehydrogenase and phosphoenol- pyruvate carboxykinase, all banded at a

193

>.- (O Z u.I : ) O uJ

15 PROT (R=111)

10

o "-7_,

15 TIM 1R=1081

10

0

15 G A P - D H (R=80)

10

0 ~ 15 PGK (R=246)

lO F; 0

15 GDH 1R=711

5

0 , , 1 ,

1.05 1.15 1.25

HK (R=104) - -

PGI (R=154)

J - PK (R=93)

MDH 1R~106)

/ PFK (R=93) 19'°7.E~ ENOLASE

ALDO (R=74)

GK (R=103)

PGM (R=164)

/ . MALIC (R=IOO)

ALAT ,{R=356)

' F

7 G6PDH ~ ) (R=90)

1.05 1.15 1.25 I.;5 1.~5 i.,15 i.;5 135 i.)5 DENSITY ( g . c m 3 )

PEP-CK (R=155)

Fig. 6. Distr ibution profiles of a post-nuclear extract o f a homogena te of Phytomonas after isopycnic centr ifugation on a linear sucrose gradient. Recoveries for protein and each of the enzymes is indicated in parentheses.

density of 1.24 g cm -3, clearly distinct from the bulk of the protein. Pyruvate kinase, enolase and glucose-6-phosphate dehydrogen- ase and phosphoglucose mutase behaved as soluble enzymes. Malic enzyme and alanine aminotransferase had both a bimodal distribu- tion with a soluble and a particle-bound component that banded at a density of 1.17 g cm -3, in agreement with a mitochondrial localization. Adenylate kinase was mainly soluble with a minor contribution at glycoso- mal density.

Properties of pyruvate kinase and phosphofruc- tokinase. We have reported previously that T. brucei and other representatives of the Trypanosomatidae contain a unique type of pyruvate kinase which is allosterically regu- lated by fructose 2,6-bisphosphate [22]. Such a type of regulation has furthermore only been found in one other eukaryotic organism, Trypanoplasma borelli [36], also a member of the order Kinetoplastida. We have, therefore, studied the effect of phosphoenolpyruvate on the activity of the Phytomonas enzyme in the presence of various concentrations of fructose

194

2,6-bisphosphate. The saturation curve of pyruvate kinase in the absence of fructose 2,6-bisphosphate was sigmoidal (Hill coeffi- cient n = 1.4) with a low affinity (S0.5 = 1.0 mM) for phosphoenolpyruvate (not shown). Increasing concentrations of fructose 2,6-bis- phosphate rendered the saturation curve more hyperbolic. At 1/~m fructose 2,6-bisphosphate,

the Phytomonas pyruvate kinase had a S0.5 for phosphoenolpyruvate of 0.09 mM with a Hill coefficient of 1 (n = 1.02). Inorganic phos- phate in the millimolar range counteracted the stimulatory effect of fructose 2,6-bisphosphate as was previously described for the other Trypanosomatidae [22]. Similar to the phos- phofructokinases of other Trypanosomatidae

\

O

" • ~ , ~ ~ u : 7 , ~ : ~ • ....

Fig. 7. (A) Electron micrographs of cells of Phytomonas sp. F, flagellum; FP, flagellar pocket; K, kinetoplast; M, mitochondrion; G, glycosome; N, nucleus; C, costa; L, lipid droplet; M-ER, mitochondrion-endoplasmic reticulum complexes. (Magnification × 18000.) (B) Phytomonas sp. showing abundant glycosome-like structures. L, lipid droplet

(Magnification × 35000.)

195

the enzyme from Phytomonas did not respond to fructose 2,6-bisphosphate (not shown).

Morphological demonstration of glycosome-like organelles. Figs. 7 and 8 show electron micrographs of in vitro cultured Phytomonas sp. The cells are characterized by a flagellum, a kinetoplast and a costa. The mitochondrion was tubular with barely any christae (Fig. 7A). Microbody-like organelles with an electron- dense matrix surrounded by a single membrane were abundantly present in the cytoplasm of

the cell. These microbodies were rather homo- geneous in appearance with a diameter not exceeding 0.3/~m (Fig. 7B). They were mostly ellipsoid or globular in shape. Fig. 8A shows that these microbodies, after incubation with hydrogen peroxide and diaminobenzidine at pH 8.5, gave a positive reaction for catalase which was absent from control sections that were incubated with either diaminobenzidine alone, or with aminotriazole, an inhibitor of catalase (Fig. 8B).

J f-

B

196

Fig. 8. Phytomon positively stained -,- . . . . . . . . . . . . ,~,t,. o~ ceJJS with diaminobenzidine and hydrogen peroxide, the microbodies wer~

(A); this staining was absent in controls incubated in the presence of aminotriazole indicated by arrows. (Magnification × 46000.) (B). The microbodies arc

Discussion

The main interest of this study was to demonstrate that the peroxisome-like organ- elles that were reported to be abundantly present in a Phytomonas sp. isolated from E. characias [3,4] were in fact glycosomes and that in the genus Phytomonas the glycolytic path- way is compartmentalized in a way similar to that in the other Trypanosomatidae.

All the enzymes of the Embden-Meyerhof pathway of glycolysis and alcohol dehydro- genase were present in homogenates of Phytomonas sp., in agreement with its capacity to convert glucose into pyruvate, acetate and ethanol with high efficiency. In addition the enzymes phosphoenolpyruvate carboxykinase, malate dehydrogenase and fumarate reductase, all involved in the reductive pathway of succinate production, were present. Interest- ingly, fumarase was not detectable in fresh Phytomonas lysates, while 2 enzymes of the glyoxylate cycle (malate synthase and isocitrate lyase) were present. This may suggest that the formation of succinate does not involve fumarate as an intermediate, but is the result of a cleavage of the glyoxylate cycle inter- mediate isocitrate into glyoxylate and succi- nate. Glyoxylate may then be reutilized in the glyoxylate cycle or transaminated to glycine. In this respect it is of interest to note that one of us (Sanchez-Moreno, unpublished) has recent- ly detected the metabolic end-product glycine in Phytomonas sp. cultures.

The absence of detectable cytochromes and the fact that respiration is not inhibited by the classical inhibitors of the mitochondrial re- spiratory chain, such as cyanide and antimy- cin, indicates that in this cultured form of Phytomonas the glycolytic pathway serves as the major, if not the only, source of ATP within the cell.

We conclude that the first 7 enzymes of the glycolytic pathway, two enzymes of glycerol metabolism, phosphoenol pyruvate carboxyki- nase and part of the malate dehydrogenase and adenylate kinase activities in Phytomonas sp. are compartmentalized within glycosomes. This conclusion is based on several lines of

197

evidence: firstly, the behavior of these enzymes in cell fractionation experiments was similar to that found for a number of other glycosome- containing trypanosomatids. Secondly, various degrees of latency, indicative of their localiza- tion within a membrane-bounded particle was observed. Thirdly, this particle had an equilib- rium density in sucrose of about 1.24 g cm -3, similar to the value reported for the glyco- somes of the various other Trypanosomatidae. Finally, pyruvate kinase was regulated in the same unique manner as has been described for the other glycosome-containing trypanosoma- tid species Trypanosoma and Leishmania [22].

The peroxisomal marker enzyme catalase had an activity that was too low to allow any reliable localization by biochemical techniques. Fortunately cytochemical staining for catalase activity allowed the localization of this enzyme inside Phytomonas microbodies. There is no direct evidence that these catalase-positive microbodies are identical to the biochemically characterized glycosomes. However, from the fact that in 2 other Kinetoplastid organisms (i.e., Crithidia sp. [12] and Trypanoplasma sp. [36] catalase and glycolytic enzymes have been co-localized in the same organelle, we infer that glycolytic enzymes and catalase are also present in Phytomonas sp. in one and the same organelle: a glycosome.

Phytomonas is now, after the demonstration of glycosomes in Trypanosoma brucei [9], T. cruzi [10], Le&hmania spp. [11], and Crithidia sp. [12], the fourth genus within the Trypano- somatidae that have biochemically been shown to contain glycosomes. In addition, Trypano- plasma borelli, belonging to the Kinetoplastid suborder Bodonina, has also been shown to contain glycosomes [36].

Phytomonas sp. has a very high glycolytic capacity which easily surpasses that of the bloodstream form of T. brucei [38]. Its mitochondria are not capable of oxidizing 2- oxoglutarate, succinate and proline, suggesting the absence of a functional Krebs' cycle. Similarly to T. brucei where lactate dehydro- genase is also lacking, some of the reducing equivalents generated in the glycolytic pathway are most likely reoxidized by a dihydroxyace-

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tone phosphate: glycerol 3-phosphate cycle, involving a glycosomal glycerol-3-phosphate dehydrogenase and a mitochondrial salicylhy- droxamic acid-sensitive glycerol-3-phosphate oxidase, present in the Phytomonas mitochon- drion. The fact that Phytomonas also produces fermentation products, such as glycerol, etha- nol and succinate, could be explained either by a partial anaerobiosis of the cell cultures during early stationary growth or by an insufficient capacity of the glycerol-3-phos- phate oxidase to cope with all the reducing equivalents produced in glycolysis.

The demonstration that Phytomonas active- ly excretes enzymes involved in the degrada- tion of starch to di- and monosaccharides, together with cellulose- and pectine-degrading enzymes, indicates that it is well adapted to the conditions prevalent in the plant and that it lives at the expense of its host. Both the high rate of consumption of plant nutrients and the production of fermentive end-products, together with the active excretion of acid phosphatase, ribonuclease, cellulose-degrad- ing and pectine-degrading enzymes, are prob- ably all directly related to the pathology that may be observed in infected plants.

Acknowledgements

The authors would like to thank Drs. W. De Souza and I. Roitman for providing the Phytomonas strain isolated by Dr. Dollet from Euphorbia characias and Dr. Anna Kiss for preparing some of the electron micrographs.

This work was supported by the Belgian State Prime Minister's Office--Science Policy Programming Grant No. 88/93-122.

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