Aerobic and anaerobic glucose metabolism of Phytomonas sp. isolated from Euphorbia characias

E L S E V I E R Molecular and Biochemical Parasitology 67 (1994) 321-331

MOLF_EUI.AR AND BIOCHENICAL P A R A 5 I ~

Aerobic and anaerobic glucose metabolism of Phytomonas sp. isolated from Euphorbia characias

Frang, ois Chaumont a.t, Andr6 N. Schanck b, j. Joseph Blum c, Fred R. Opperdoes a,, a Research Unit.for Tropical D/seases, International Institute of Cellular and Molecular Pathology, ICP-TROP 74.39, Avenue Hippocrate

74, B-1200 Brussels, Belgium b Laboratoire de Chimie-Physique et de Cristallographie, Universit~ Catholique de Louvain, B-1348 Louvain-la-Neuve Belgium

c Division of Physiology, Department of Cell Biology, Duke University Medical Center, Durham, NC, USA

Received 8 March 1994; accepted 12 July 1994

Abstract

Metabolic studies on Phytomonas sp. isolated from the lactiferous tubes of the latex-bearing spurge Euphorbia characias indicate that glucose is the preferred energy and carbon substrate during logarithmic growth. In stationary phase cells glucose consumption was dramatically reduced. Glucose consumption and end-product formation were measured on logarithmically growing cells, both under aerobic (air and 95% 02/5% CO 2) and anaerobic (95% N2/5% CO 2 and 100% N 2) conditions. The rate of glucose consumption slightly increased under anaerobic conditions indicating that Phytomonas lacks a 'reverse Pasteur' effect contrary to the situation encountered in Leishmania major. Major end-products of glucose catabolism under aerobic conditions, detected by enzymatic and NMR measurements, were acetate, ethanol and carbon dioxide and under anaerobic conditions ethanol, glycerol and carbon dioxide. Smaller amounts of pyruvate, succinate, L-malate, L-lactate, phosphoenolpyruvate, alanine and aspartate were also detected.

Keywords: Phytomonas; Glucose metabolism; Anaerobiosis; Carbon dioxide; Carbon balance

1. Introduction

Trypanosomatids of the genus Phytomonas are etiological agents of diseases affecting economically important crops [1], but they also parasitize many

Abbreviations: SHAM, salicylhydroxamic acid; PEPCK, phosphocnolpyruvate carboxykinase

* Corresponding author. Tel: 32-2-764.74.39; Fax: 32-2- 762.68.53. E-mail: [email protected]

1 Present address: Unit6 de Biochimie Physioiogique, Univer- sit6 Catholique de Louvain, Place Croix du Sud 2-20, B-1348 Louvain-la-Neuve, Belgium

plants and fruits without any apparent pathogenicity. The phytomonads that infect plants have the appear- ance of promastigotes and have structures typical of all members of the Trypanosomatidae: a single unit membrane, forming a flagellar pocket, a kinetoplast- containing single tubular mitochondrion and glyco- somes [2,3]. Characterization of Phytomonas' metabolism has lagged behind because of the diffi- culties of growing the parasite in vitro. Recently, we succeeded in the axenic culture of these parasites isolated from the lactiferous tubes of the latex-bearing spurge Euphorbia characias, and some aspects of its

0166-6851/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 6 - 6 8 5 1 ( 9 4 ) 0 0 1 4 1 - 3

322 F. Chaumont et al. / Molecular and Biochemical Parasitology 67 (1994) 321-331

carbohydrate metabolism have been described [3,4]. A detailed investigation of the metabolism of this trypanosomatid is of interest not only because of its pathogenicity to plants, but also because of its evolu- tionary relationship to the other trypanosomatid flag- ellates.

None of the trypanosomatids studied are capable of fully degrading glucose to carbon dioxide under aerobic conditions, and a great proportion of glucose carbon is excreted into the medium as fermentative metabolites which may differ according to the species (for review, see [5-7]). Phytomonas excretes cellu- lose-, starch- and pectin-degrading enzymes into the external medium and utilizes glucose, fructose and mannose as the major energy substrates [3]. Ill- Nuclear magnetic resonance spectroscopy was used to study the consumption of glucose and to identify as end-products of carbohydrate metabolism acetate, ethanol, succinate, glycine, pyruvate and glycerol. No quantitative data were provided. The glycolytic enzymes from hexokinase to phosphoglycerate ki- nase as well as glycerol kinase, glycerol-3-phosphate dehydrogenase, phosphoenol pyruvate carboxykinase (PEPCK) and malate dehydrogenase have all been detected in homogenates of Phytomonas sp. and have been localized to glycosomes. Moreover, pyru- vate kinase, alcohol dehydrogenase and malic en- zyme have been detected in these cells [3,4].

In this paper, we describe a quantitative analysis of the aerobic and anaerobic glucose metabolism of Phytomonas sp. isolated from E. characias. Acetate, ethanol and CO 2 are the major end-products under aerobic conditions and ethanol, CO 2 and glycerol are mainly produced under anaerobiosis.

2. Materials and methods

2.1. Growth and collection of organisms

Phytomonas sp. isolated from Euphorbia chara- cias [8] were grown at 28°C in SDM-79 medium [3,9] in Falcon flasks. Cells were collected from culture medium by centrifugation at 1500 X g for 5 min at room temperature. The pellet of cells was washed twice and resuspended in ice-cold Hanks' balanced salt solution, pH 7.1 (HBSS). Samples of cell suspensions were taken for protein measurement.

2.2. Metabolite measurements at various partial pressures of 0 2 and CO 2

Cells at a final concentration of about 1 mg protein ml-1 and glucose at a final concentration of 4 mM were added to 50-ml Falcon tubes containing HBSS buffer in the presence of 5% CO 2 and 10 mM NaHCO 3. The tubes were incubated at 26°C in a shaker bath (approx. 100 rpm). Four different gas mixtures were used: air, O2/CO 2 (95%/5%), N 2 and N2/CO 2 (95%/5%). Prior to and during the incubations, aliquots were removed from the incuba- tion mixture at constant time intervals and immersed in boiling water for 90 s, placed on ice, centrifuged (1500 X g, 15 min, 4°C) and the supernatants ana- lyzed for metabolites using enzymatic assays as de- scribed by Bergrneyer [10] or using appropriate kits from Boehringer Mannheim. For amino acid analy- sis, the supernatant was flushed through a C18 Sep- Pak cartridge (Waters Associates, Milford, MA) and analyzed using high performance liquid chromatog- raphy and fluorescence techniques [11]. The protein concentration was determined by the fluorescamine method [12] using bovine serum albumin as standard.

2.3. Measurement of 14C0 2 release and ;4C incorpo- ration into macromolecules

The method previously described by Blum [13] was adopted. A washed cell suspension (0.7 ml) was added to 50-ml Erlenmeyer flasks containing 1.3 ml HBSS buffer supplemented with 6 mM glucose and 0.2 /xCi [1-14C]glucose, [6-14 C]glucose or [U- 14C]glucose resulting in a final glucose concentration of 3.9 mM and a protein concentration of 0.5-0.8 (mg protein) m1-1 The flasks were capped with rubber serum stoppers and incubated in a shaker bath for 1 h at 26°C. The reaction was stopped by inject- ing 0.3 ml of 13% (v /v ) perchloric acid and 14C02 was collected for 1 h in plastic center wells to which 0.2 ml hyamine hydroxide was added. The center wells were then placed in a vial containing 5 ml of scintillation fluid and the radioactivity determined in a liquid scintillation counter. The flasks were run in triplicate and the average of the three measurements was used as a datapoint. In order to determine glu- cose consumption, a control flask was incubated in

F. Chaumont et aL / Molecular and Biochemical Parasitology 67 (1994) 321-331 323

the same conditions except that the reaction was stopped by immersion in a boiling water bath for 90 s and the supernatant assayed for glucose.

The pellet of the perchloric acid-treated cells was washed 3 times in 5% trichloroacetic acid and then solubilized in hyamine hydroxide and radioactivity counted in a liquid scintillation counter.

2.4. NMR measurements

Phytomonas cells were collected, washed and re- suspended in HBSS buffer except that 10 mM Na2HPO4/10 mM KH2PO 4 were substituted for Hepes. Cells at a final concentration of about 3 mg protein ml - 1 and [1-t3C]glucose at a final concentra- tion of 9 mM were incubated at 26°C in the presence of 10 mM NaHCO 3 with O2/CO 2 (95%/5%) or N2/CO 2 (95%/5%). After 1 h incubation, the mix- ture were treated as above. The supernatant contain- ing the extracellular and intracellular metabolites was filtered through a 22-/xm Millipore filter and frozen until NMR measurements.

L~C-NMR spectra were acquired at 125.7 MHz on an AM 500 Bruker spectrometer. Spectra were ob- tained under the inverse-gated decoupling mode for suppression of the nuclear Overhauser effect in order to obtain quantitative data [14]. Typical Fourier

0 • • i •

0 2O

2" ( n

! • • ! - • i • •

4 0 6 0 8 0 1 Time (h)

6O

"7 ,50 ~.

°i • 30

2 0

l o

0

00

Fig. 1. Growth curve and glucose consumption by Phytomonas. Culture medium was inoculated with 5x10 s cells m1-1. At different times of growth, cells concentrations were determined and aliquots were collected, washed and incubated in HBSS medium containing 4 mM glucose. Glucose consumption was measured as described in Materials and methods. ([] cell density; • glucose consumption)

Transform parameters were: spectral width, 15 kHz; 32000 data points; 60 ° pulse angle (10 IXS); recycle time, 6 s; 5000-10000 transients were accumulated, a line broadening of 1.5 Hz was applied to the free induction decays before Fourier transformation. NMR tubes with a diameter of 10 mm were used and 20% 2H20 was added to the sample for field frequency stabilization on the 2H-NMR signal. Dioxane was used as reference signal at 67.6 ppm. Chemical shifts are accurate at 0.1 ppm.

2.5. Materials

[1-14 C]Glucose, [6-t4C]glucose and [U-14C]glu - cose were from NEN Research Products• [1-13C]Glu- cose (98.95% 13C enriched) and hyamine hydroxide were from Sigma• All other chemicals were of reagent grade.

3 . R e s u l t s

3.1. Glucose consumption during growth

Glucose consumption of washed Phytomonas cells was dependent on the phase of growth (Fig. 1). Glucose utilization was very high (50 nmol min-1 (mg protein) -1) in early log phase, but decreased dramatically as the cultures approached stationary phase. Therefore, unless stated otherwise, in further experiments designed to quantify glucose consump- tion and metabolite production, cells were taken during the logarithmic phase of growth when they had reached a density of approximately 1 X 10 7 cells ml - t .

3.2. Glucose consumption and metabolite production under aerobic and anaerobic conditions

Rates of glucose consumption and end-product formation under aerobic as well as under anaerobic conditions, both in the presence and absence of CO 2, were determined to obtain information about the parasite's capacity of aerobic and anaerobic glycoly- sis and to monitor its capabilities of carbon dioxide fixation. The rates of glucose consumption and metabolite production were constant with respect to time for the duration of the measurements (60 min)

324 F. Chaumont et al. / Molecular and Biochemical Parasitology 67 (1994) 321-331

2000

"7 • glucose .=: • acetate

7 • ethanol ~1 glycerol

o~ 1000

0, Air O2/CO2 N2 N2/CO2

200.

~-~ 100. o E

• pyrtwate • L-rnalate • L-lactate [ ] succinate

0

Air O2/CO2 N2 N2/CO2

Fig. 2. Rates of glucose consumption and product formation by Phytomonas incubated under aerobic and anaerobic conditions in the presence and absence of CO 2. Cells were incubated in HBSS buffer containing 4 mM glucose and metabolite concentrations were measured as described in Materials and Methods. Results are the mean + SD of 3 or 5 experiments.

under all conditions used (data not shown). No major variations in glucose consumption by cells incubated under the four conditions were observed (Fig. 2). Anaerobiosis led to a slight increase ( P < 0.01, Stu- dent's t-test for paired experiments) in glucose con- sumption (Pasteur effect) as observed in most other cells, contrary to the situation encountered in Leish- mania major where a reverse Pasteur effect was observed [15].

The nature and the quantities of the metabolic end-products excreted by Phytomonas depended on the gas mixtures used (Fig. 2). Under aerobic condi- tions, acetate was the major end-product of glucose metabolism, whereas ethanol was the main product formed under anaerobic conditions. Ethanol was also produced under aerobic incubations to an extent dependent on the experiment. These variations prob- ably reflect a partial anaerobiosis in the test tube during incubation. In addition to ethanol, significant amounts of glycerol were produced under anaerobio-

sis. The cells also produced L-lactate; no D-lactate was detected. The amount of L-lactate formed in- creased markedly under anaerobic conditions in the absence, but not in the presence of CO 2. Pyruvate and L-malate were minor products of glucose metabolism when the cells were incubated under air, 95% 0 2 / 5 % CO2, or N 2. The amount of these two products increased over two-fold, however, under 95% N2/5% CO 2. This increase in pyruvate plus malate was about equal to the decrease in L-lactate caused by the presence of CO 2 under anaerobic conditions (Fig. 2), suggesting CO 2 fixation via PEPCK. No fumarate or glycerol-3-phosphate were detected under any of the conditions studied. Very small amounts of succinate were produced in the presence of CO 2, but not in its absence.

An amino acid analysis of a complete Phy- tomonas cell suspension in HBSS medium supple- mented with 4 mM glucose prior to incubation under aerobic conditions using an amino-acid analyser, re- vealed as the major amino acids proline (200 nmol mg- l ) , alanine (96 nmol mg -1) and glycine (97 nmol mg -1). After 60-min incubation, we observed a decrease in the concentration of proline ( - 7 6 nmol mg -1 ) and an increase of alanine ( + 65 nmoi mg- l ) . A minor change in the amounts of glycine present in the medium was also observed ( + 9 nmol mg- l ) .

3.3. Incorporation of label from [U- 14C], [1-14C] and [6-14C]glucose into 14602 and macromolecules

Under aerobic conditions less than 50% of the glucose carbon utilized was recovered in the form of the above identified end products. To obtain informa- tion about the amount of glucose carbon excreted as volatile CO 2 or incorporated into macromolecules, Phytomonas ceils were incubated in the presence of [1-14C]-, [6-14C] - o r [U-14C]glucose under an atmo- sphere of air (Table 1). About 22% of the [U- 14C]glucose carbon was converted to CO 2. The rate

14, 14 of CO 2 from [1- C]glucose exceeded that from [6-14C] glucose by over 4-fold. This observation is consistent with the existence of an appreciable flux of the glucose carbon through the pentose-phosphate pathway. An approximate estimation [16] revealed that about 4% of the total amount of glucose con- sumed was metabolized via the pentose-phosphate

F. Chaumont et al. / Molecular and Biochemical Parasitology 67 (1994) 321-331 325

Table 1 Rates of production of ]4COz and incorporation of 14C into macromolecules by Phytomonas cells incubated in the presence of air with either [1-14C] -, [6J4C]- or [UJ4C]glucose

nmol (mg protein ) - ] h - 1

Glucose 14CO2 TCA pellet

[1-14 C]glucose 1134:1:206 141+:29(12.4%) 29+3(2.6%) [6J4C]glucose 1134+:206 30+: 4(2.7%) 38+:4(3.4%) [UJ4C]glucose 999+:255 215 +45 (21.5%) 20+:3 (2.0%)

Results are the mean+ SD of 4 experiments and are also ex- pressed as a percentage of glucose molecules utilized.

pathway. The rate of 14CO2 production from [6- ]4C]glucose was 13% of that from [UJ4C]glucose. This is consistent with the production of acetate as the major end-product under aerobic conditions, since acetate formation results in a greater CO 2 production from C3 and C4 of glucose. A small amount of the labeled glucose was incorporated into trichloroacetic acid (TCA) precipitable products (Table 1). The amount of radioactivity incorporated was smaller from [U-14C]glucose than from [6-14C]glucose, con-

s i s ten t with the fact that a significant part of the glucose C1 ends up in CO 2 and that the remaining C5-moiety is preferentially incorporated into nucleic acids. In the same way, given the higher CO: pro- duction from [1-14 C]glucose than [6-14 C]glucose, less incorporation in the TCA pellet was observed from

[1-]4C]glucose ( P < 0.01, Student's t-test for paired experiments).

3.4. Carbon balance under aerobic and anaerobic conditions

Assuming that the acetate and ethanol produced under anaerobic conditions resulted from a decarbox- ylation of pyruvate by pyruvate decarboxylase and that the CO 2 thus released was not reincorporated to any significant extent, 80% of the glucose carbon utilized could be accounted for by the metabolites produced by the Phytomonas cells under anaerobic conditions. This assumption, however, implies that under anaerobiosis CO 2 is also a significant end product of Phytomonas metabolism. Because CO 2 measurements were not possible under a constant flow of N 2, anaerobic conditions were mimicked using salicylhydroxamic acid (SHAM), an inhibitor of mitochondrial glycerophosphate oxidase, the ter- minal oxidase of Phytomonas sp [3]. Various SHAM concentrations were used in order to mimic the anaerobic situation. As shown in Fig. 3, a concentra- tion of 10 /zM SHAM led to an ethanol production similar to that observed under an N 2 atmosphere whereas a concentration of above 100 /.LM SHAM was required to obtain a similar amount of glycerol. This is consistent with a previous observation that

i t -

Q.

g o E c "

2000

1000

• Air • SHAM 10 uM • SHAM 50 uM [ ] SHAM 100 uM [ ] SHAM 200 uM • N2

(*)

* : i n d e p e n d a n t e x p e r i m e n t

glucose acetate e thano l glycerol

Fig. 3. Rates of glucose consumption and product formation by Phytomonas incubated in the presence of SHAM. Cells were incubated in HBSS buffer containing 4 mM glucose with increasing concentration of SHAM. As control, ceils were incubated in the presence of air or N 2. Glucose, acetate, ethanol and glycerol concentrations were measured as described in Materials and Methods. Incubations were conducted simultaneously with the same cell preparation except those in the presence of 200 /xM SHAM whose results are mean of 3 independent experiments.

326 F. Chaumont et al. / Molecular and Biochemical Parasitology 67 (1994) 321-331

Table 2 Rates of production of 14CO 2 and incorporation of ~4C into macromolecules by Phytomonas cells incubated in presence of 200 ~M SHAM with [U-14C]glucose

[U- 14 Clglucose

nmol (mg protein)- 1 h - 1

Glucose 14CO 2 TCA pellet

1411:1=194 318:t:20(22.5%) 18:1:1(1.3%)

Results are the mean :t: SD of 4 experiments and are also ex- pressed as a percentage of glucose molecules utilized.

70% of the total (Table 3). Thus 30% of the glucose carbon under anaerobic and 25% under aerobic con- ditions was not accounted for. However, when simi- lar experiments were carried out using Phytomonas cells harvested at the early stationary phase of growth, carbon recovery increased to about 95% (Table 3). This raised the suspicion that during the logarithmic phase of growth one or more as yet unidentified end-product(s) were being produced.

100 /xM S H A M inhibited 0 2 uptake by try- panosomes over 90% [17].

The production of t4CO2 from [O-14C]glucose by cells incubated in the presence of 200 /xM SHAM was similar (22.5%) to what was observed in the presence of air (Table 2). This amount is slightly lower than the theoretical amount that should have been released through the formation of acetate and ethanol. Under the same conditions only 1% of the glucose consumed was incorporated into TCA-in- soluble macromolecules, against 2% under aerobic conditions, suggesting that anaerobic cells perform less biosynthetic activities.

Under aerobic conditions, the sum of the glucose carbon reaching the end-products detected by enzy- matic assays, plus 14CO2 and the carbon incorpo- rated into macromolecules, accounted for only about

3.5. NMR identification o f products

In order to detect any thus far unidentified end products of glucose metabolism, we conducted NMR experiments. Phytomonas cells were incubated with [l-13C]glucose in the presence of O 2 / C O 2 and N 2 / C O 2 and the supernatants coming from boiled cell-incubation mixtures (intracellular + extracellular content) were analyzed by NMR. This technique allows the detection of all major products except for CO 2. To obtain quantitative information on the metabolites produced, the integrated value of each signal was calibrated to the amount of glucose con- sumed as determined enzymatically, assuming a CO 2 production of 12.4% as shown in Table 1.

In general, the NMR spectra confirmed the results obtained with the enzymatic detection of the metabo-

Table 3 Rates of glucose consumption and product formation by Phytomonas incubated under aerobic and anaerobic conditions

Metabolite Logarithmic phase nmol (mg protein)- 1 h - 1 Stationary phase nmol (mg protein)- 1 h- 1

O2/CO2 N2/CO 2 O2/CO 2 N2/CO 2

Glucose 1277 1674 837 1330 Acetate 1077 (28.1%) 167 (3.3%) 1183 (47.1%) 160 (4.0%) Ethanol 472 (12.3%) 1354 (27.0%) 150 (6.0%) 1891 (47.4%) Glycerol 23 (0.9%) 366 (10.9%) 79 (4.7%) 281 (10.6%) Pyruvate 50 (2.0%) 85 (2.5%) 93 (5.5%) 87 (3.3%) Succinate 28 (1.5%) 17 (0.7%) 35 (2.8%) 20 (1.0%) L-lactate 59 (2.3%) 94 (2.8%) 24 (1.4%) 84 (3.2%) L-malate 17 (0.9%) 82 (3.3%) 13 (1.0%) 41 (2.1%) Percent glucose carbon into products 48.0% 50.5 % 68.5% 71.6 % CO 2 21.5% 22.5% 22.2% ND TCA pellet 2.0% 1.3% 2.8% ND Total 71.5% 74.3% 92.0%

Phytomonas cells were harvested at logarithmic phase (mean of 5 experiments) or at early stationary phase of growth (I experiment). Samples of cell suspensions were incubated in HBSS buffer containing 4 mM glucose and metabolite concentrations were measured as described in Materials and methods. The percentage of glucose carbons released as products was calculated from the total number of carbons in each compound. The rate of 14CO2 and TCA pellet were measured from [U-t4Clglucose in the presence of air or 200 /~M SHAM (logarithmic phase, mean of 4 experiments; stationary phase, 1 experiment). ND, not determined.

F. Chaumont et al. /Molecular and Biochemical Parasitology 67 (1994) 321-33l 327

lites. Acetate and ethanol were mainly produced under aerobiosis (Fig. 4A) and ethanol and glycerol under anaerobiosis (Fig. 4B). Among the minor sig- nals observed, glucose labeled at position C-2, C-3, C-4, C-5 and C-6 was present but in a ratio similar to a control of buffer containing 9 mM [1-t3C]glucose, indicating a contamination of the [1-~3 Clglucose with

glucose labeled in the other positions. An ethanol C-1 signal was also detected as previously reported for Crithidia fasciculata by De los Santos et al. [18], who attributed this to the natural abundance of 13C in ethanol. However, this peak seemed too large to be explained by such a natural abundance. The nature of the ethanol C-1 peak remains to be elucidated.

A

PIEP C-3

~o

GkJ C-1

Acet I;tOH C-2 C'2

Glu Et~'l Pvr Olu c-e C-! C-2~-3~C-4K:-6 Qv c-3 . su¢ c-3

" ' ' " " | r

PPN

i

2O

Ala C-3

~ tx61

L _ _ _

B G~ C-I

PEP C-3

.1.

A e e t

C-2 Lgc

cl CO/ EROS1 Pyr { Ci3 C-1/C-3 C-1

C-3

EtOH C-2

C-3

L _

(x6 )

• • ' - - " = , ' ' ! ' ' . r | , - .

l~o 9'o a'o 7o 6'0 ~'0 ,'0 3'o 2o ;o PPN

Fig. 4. Proton de, coupled ~3C-NMR spectra of Phytomonas incubated with [1J3C]glucose at 26°C for 1 h in the presence of O2/CO2 (A) or N2/CO 2 (B). Final glucose concentrations, as determined enzymatic.ally, were 5.7 mM and 4.8 mM, respectively.

328 F. Chaumont et al. / Molecular and Biochemical Parasitology 67 (1994) 321-331

G-6..P-<-~

Pentose Phosphate Pathway

Glucose /

Glycosome Glucose

ATP ,~ ADP

G-6-P

F-6-P ~ ATP

~-~ ADP FDP

NAD+

NADH OAA

-->- DHAP < > GAP

~ D +

.->- G-3-P 1,3-DPGA

ATP ADP CO 2 IGlycerol] 3-PGA

It~lveerol I 3-PGA NADP i ~ - , r - j

NADPH ~

2-PGA

¢

NAD + NAOH ~ ~ NADH NAD +

I, ruv.,or I

~ . _ _ L . _ . ~ - - - I . . - - - ~ - . . . . F u m a r a t e ~

Ltm" Fig. 5. Scheme of the major pathways of glucose metabolism in Phytomonas. Major products of glucose metabolism are in grey boxes. Abreviations used : G-6-P, glucose 6-phosphate; F-6-P, fructose 6-phosphate; FDP, fructose 1,6-bisphosphate; GAP, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; G-3-P, glycerol 3-phosphate; 1,3-DPGA, 1,3-bisphosphoglycerate; 2-PGA, 2-phosphog- lycerate; 3-PGA, 3, pbosphoglycerate; PEP, pbosphoenolpyruvate; OAA, oxaloacetate. ? indicates that the enzyme fumarase is not detected [3].

F. Chaumont et al. / Molecular and Biochemical Parasitology 67 (1994) 321-331 329

In addition to the metabolites detected enzymati- cally, alanine C-3 and aspartate C-3 were present and represented about 2% of the glucose consumption in both conditions. Interestingly, alanine and aspartate formation increased when the cells were in late-log phase (data not shown). These amino acids are prob- ably synthesized from pyruvate and oxaloacetate through transamination from glutamate. Their pro- duction might be linked to the reoxidation of gly- colytic NADH via glutamate dehydrogenase, which is present in Phytomonas (activity of 24 mU (mg protein)-1). PEP C-3 was also present in the cell lysate (about 3% of the glucose consumption) con- firming the potential role of this metabolite as accep- tor of CO z.

Despite the fact that we detected by NMR the production of some alanine and aspartate (less than 5% of the total, see above) that were not identified enzymatically, the most important quantitative differ- ences between the two methods came from the major metabolites. With NMR about 20% more glucose carbon was retrieved in ethanol and acetate under aerobic conditions and in ethanol and glycerol under anaerobic conditions, suggesting an underestimation of these metabolites by our enzymatic methods.

4. Discussion

The Phytomonas sp. isolated from Euphorbia characias is an organism highly specialized for gly- colysis. We have previously shown that it excretes hydrolytic enzymes involved in the degradation of the polysaccharide constituents of the plant cell wall and of disaccharides, and that it consumes the result- ing monosaccharides at an extremely high rate [3]. This high rate of glycolysis is forced upon the organism by the absence of oxidative phosphoryla- tion through the complete repression of mitochon- drial activity, as reflected by the absence of de- tectable cytochromes [3].

During logarithmic growth, mainly glucose is consumed as energy and carbon source. As the cells approach stationary phase, glucose consumption is almost arrested. The study of glucose metabolism of Trypanosoma cruzi (epimastigotes) and Crithidia spp. [19] has shown that these two organisms con- sume glucose preferentially to amino acids for

growth, whereas the reverse is true for L. mexicana promastigotes. The first two organisms use glucose during the entire logarithmic phase of growth, whereas L. mexicana uses glucose only at the end of the logarithmic phase and at the beginning of the stationary phase [19].

The pathways of glucose metabolism in Phy- tomonas sp. are illustrated in Fig. 5. The major end-products of aerobic glucose catabolism are ac- etate and CO 2. Under anaerobic conditions, mainly ethanol, CO 2 and glycerol are produced. Ethanol production has previously been reported in Crithidia [19,20], and should be related to glycolytic NADH reoxidation mediated by pyruvate decarboxylase and a NAD-linked alcohol dehydrogenase. It is interest- ing that in the presence of a N2/CO 2 atmosphere, other fermentation products are produced in addition to ethanol and glycerol: i.e. lactate, malate and succi- nate. This indicates that not all the reducing equiva- lents produced by the glycolytic pathway can be taken care of by the enzyme alcohol dehydrogenase. Alternative routes for the reoxidation of glycolyti- cally produced NADH must be invoked and these are catalyzed by the cytosolic lactate dehydrogenase and the glycosomal glycerol-3-phosphate dehydroge- nase and malate dehydrogenase. Each of these en- zymes has previously been demonstrated in Phy- tomonas sp. [3]. The complex pattern of fermentation products cannot be explained by the presence of various phenotypes in our Phytomonas stock, since identical results were obtained after a recloning of the Phytomonas strain.

Studies of the carbohydrate metabolism of C. fasciculata [18] and T. cruzi [21] using NMR have shown that phosphoenolpyruvate serves as the accep- tor of the primary CO2-f'Lxation reaction. This results in the formation of oxaloacetate and malate and leads in these Trypanosomatidae to the excretion of succinate. The central role of PEPCK in energy metabolism in insect-stage trypanosomatids has been illustrated in the case of T. cruzi epimastigotes, using 3-mercaptopicolinic acid, a powerful inhibitor of this enzyme [22]. Inhibition of PEPCK led to a 2.2-fold reduction in the anaerobic production of succinate and a similar decrease in glucose consump- tion, while the production of alanine, via the transamination of pyrnvate, increased by 3-fold.

In the presence of carbon dioxide, Phytomonas

330 F. Chaumont et al. / Molecular and Biochemical Parasitology 67 (1994) 321-331

cells produce higher amounts of malate and pyru- vate, also suggesting CO 2 fixation by PEPCK, but a very low amount of succinate was produced. This result is in agreement with the fact that no fumarase activity and only small amounts of fumarate reduc- tase activity were detected in this organism [3] and this contrasts with the situation encountered in the other Trypanosomatidae: i.e., T. cruzi, C. fasciculata and L. mexicana, which produce significant amounts of succinate [19]. Rather than being converted to fumarate, malate is decarboxylated to pyruvate by malic enzyme, present in Phytomonas cells [3], pro- ducing NADPH for cytosolic metabolic processes. These carboxylation and decarboxylation reactions are probably regulated by subceUular compartmenta- tion to prevent futile cycling which might interfere with NADH reoxidation.

All the lactate produced was L-lactate. No D- lactate could be detected, indicating that the methyl- glyoxal bypass is not operational in this organism. We have specifically investigated the possibility of D-lactate production by Phytomonas sp., since Dar- ling et at. [23] reported that D-lactate is produced by at least four species of Leishmania, while T. lewisi produces both isomers and T. brucei procyclics only L-lactate.

Phytomonas sp. is remarkably well adapted to its stay in the sugar-rich latex fluid of the Euphorbia plant. Hydrolases required for the conversion of disaccharides into glucose and fructose are excreted by Phytomonas. The hexoses so produced provide it with an unlimited source of carbon and energy through either aerobic or anaerobic glycolysis. Re- markable is the complete absence in this organism of mitochondrial metabolism. A similar situation exists in the bloodstream form of the African trypanosome T. brucei which, however, exclusively produces pyruvate under aerobic conditions and equimolar amounts of glycerol and pyruvate under anaerobic conditions [24]. A complete suppression of mito- chondrial activity and a total absence of cy- tochromes, has been reported also in the case of another trypanosomatid Herpetomonas muscarum ingenoplastis, but details on its metabolic end-prod- ucts are not available [25].

The separation of the African trypanosome from the main line of trypanosomatid evolution took place several hundreds of millions of years ago, well be-

fore the other representatives of the family Try- panosomatidae diverged [26]. The utilization of the glycolytic pathway as the sole carbon and energy source, together with a complete suppression of mi- tochondrial activity, have now been found in at least three distantly related representatives within the try- panosomatid family. Moreover, a recent report that in the culture-adapted trypanosomatid Leishmania tarentolae most of the guide RNAs required for the editing of mitochondrial transcripts have been lost [27], suggests that also this organism is able to survive in the absence of mitochondrial activity. Together this indicates that such a metabolic flexibil- ity must have represented an ancestral property, rather than a recent adaptation to life in a specific host. Probably it is this metabolic flexibility that has al- lowed descendants of the ancestral trypanosomatid to adapt to the great variety of metazoan hosts, ranging from plants via insects, fishes, amphibians and rep- tiles to mammals, that are all parasitized today by trypanosomatids.

Acknowledgments

The authors wish to thank Mrs. Dominique Cot- tem for the cultivation of Phytomonas. This work was financially supported by a grant from the Com- mission of the EEC, Science and Technology for Development Programme, TS3 CT 92-0077.

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