in vitro culture of phytomonas sp. isolated from euphorbia characias. metabolic studies by 1h nmr

J. Euk. Microbrol.. 42(3). 1995. pp 314-320 0 1995 by the Society of Protozoo1op1s1s

In Vitro Culture of Phytomonas Sp. Isolated from Euphorbia characias. Metabolic Studies by 'H NMR

MANUEL ShCHEZ-MORENO,*.' CARMEN FERNANDEZ-BECERRA,* EMILIO ENTRALA,* FRED R. OPPERDOES,** MICHEL DOLLET*** and ANTONIO OSUNA*

*Institute de Biotecnologia, Grupo de Bioquhica y Parasitologia Molecular, Facultad de Ciencias, Universidad de Granada, E-18071 Granada, Spain, and

**Research Unit for Tropical Diseases, International Institute nf Cellular and Molecular Pathology. B- 1200 Brussels, Belgium, and ***IRHO. Labora~orie de Phjdovirologie des Regions Chaudes. CIRAD-ORSTOM, BP5035, 34032 Montpellier cedex 1. France

ABSTRACT. We describe the in vitro culture of Phytomonas species isolated from Euphorbia characias. The best choice between tested media was SDM-79, in which promastigotes, after 6 days of culture, reached cell densities as high as 4 x 10' cells/ml. Cells growing in LIT or MTL medium showed longer division times and lower cell densities. We succeeded in obtaining Phytomonas sp. amastigote and spheromastigote forms in modified GRACE'S medium, yielding transformation rates of up to 70%. Electron microscopy studies were performed in order to characterize the ultrastructural features of these forms obtained in vitro. On the other hand, metabolic studies based on qualitative (nuclear magnetic resonance spectroscopy) and quantitative metabolic methods (enzymatic assays) showed that promastigote forms secreted mainly ethanol, acetate, glycine, glycerol, piruvate and succinate in SDM-79 medium, whereas the major metabolites found after transformation in modified Grace's medium were ethanol, acetate, glycine, piruvate and smaller amounts of glycerol.

Supplementary key words. Carbohydrate metabolism, nuclear magnetic resonance spectroscopy, Phytomonas sp. in vitro culture.

RYPANOSOMATIDS were first described in plants by La- T font, who reported that parasitism in the family Euphor- biaceae more than 80 years ago [22]. Soon afterwards, these organisms were included in the family Trypanosomatidae, being originally described as Leptomonas davidi. In 1909, Donovan proposed [ 161 the creation of a new genus, Phytomonas. in order to differentiate plant from animal trypanosomatids.

Many different plants can be infected by members of the genus Phytomonas; and some species had been associated with sig- nificant economic losses over a wide range of geographical areas [ 14,28,33]. Little is known about the biology ofthese protozoa, mainly due to the lack of an in vitro culture until 1984 [ 13, 231.

These trypanosomatids were found to parasitize the laticif- erous vascular tissue of plants belonging to a variety of families, including Euphorbiaceae, Asclepiadaceae, Compositae, Mora- ceae and Urticaceae [ 131, and in sieve tubes of Palmaceae and Rubiaceae [14, 28, 331, as well as in the edible fruits of Ana- cardiaceae, Punicaceae, Rosaceae and Solanaceae [ 121. The parasite was recently described in some varieties of garden vege- tables and legumes, including beans and soybeans, although nothing is known about the pathogenicity on these crops [ 151.

In contrast to other trypanosomatids, phytomonads were dif- ficult to maintain in culture. At the begining, forms isolated from plants were grown in media used for insect cell culture [ 151. In recent years, some progress in the techniques for in vitro culture have been achieved [20], but the results to date are far from satisfactory. The cell densities reported have been too low, and cultured forms often differ from parasites isolated from plants. The forms found in plants are mainly promastigotes, characterized by a slender, elongated cell body twisted once or more longitudinally, although amastigote forms have occasion- ally been observed.

Recently, we adapted Phytomonas isolated from Euphorbia characias to in vitro culture (301. These cultures showed that the isolated species were strikingly similar to other genera of the family Trypanosomatidae in terms of metabolic behavior, although we noted differences in certain metabolic pathways.

This further approach in the knowledge of the metabolic be- haviour and in vitro culture of Phytornonas sp., would aid the development of effective chemotherapies against plant infec- tion.

' To whom correspondence should be addressed.

MATERIALS AND METHODS Parasite. Species of the genus Phytomonas, isolated from E.

characias in the South of France by Dr. M. Dollet [15], were used as a biological model. The parasites were grown in biphasic blood-agar medium, and were adapted to in vitro culture in the different media used.

In vitro culture. The following culture media were tested: SDM-79 medium [8], LIT medium [9], Grace's medium (Gibco) modified by the addition of Clk and Na+ ions [26, 271, unmodified Grace's and MTL medium [29].

The parasites were inoculated at a density of 1 x lo4 cells/ ml in 25 ml Falcon flasks containing 5 ml of medium, and incubated at 28" C for 12 days. Aliquots of medium and cells were collected for density measurements. Growth was assessed by cell counting in a Neubauer hemocytometer, absorption spec- trophotometry at 550 nm and glucose utilization. Morphological features were analyzed by light microscopy observations of methanol fixed-Giemsa stained cells.

Electron microscopic observation. Parasites obtained from SDM-79 and modified Grace's medium were collected during the exponential growth phase by centrifugation at 1,500 g for 10 min, followed by fixation for 2 h in 0.1 M glutaraldehyde in 2.5% cacodylate buffer (pH 7.2) containing 0.1 M CaCl, and 0.9% NaCl. Fixed cells were washed in the same buffer and postfixed in isotonic saline solution containing 1% osmium te- troxide and 0.08% potassium ferrocyanide. Cells were then dehydrated by acetone washes and embedded in Epon [ 181. Thin sections were stained with uranyl acetate and lead citrate, and examined with an EMCIO Zeiss transmission electron microscope.

For the scanning electron microscope, cells were fixed in glutaraldehyde, adhered to a glass coverslip coated with 0.1% (w/ v) poly-L-lysine, postfixed in OsO, and dehydrated in a series of ethanol washes. Finally, the preparations were dried with COz, sputter-coated with colloidal gold, and examined under a Zeiss EDX DSM 950 scanning electron microscope equipped with a Kevex analysis system.

Sample preparation for 'H NMR. Cell cultures were started by inoculation of 1 ml (approximately 1 x lo6 cells/ml) of a Phytomonas culture at the logarithmic growth phase into 70 ml of the medium tested (SDM-79, modified Grace's or unmodified Grace's) in 250 ml Falcon flasks. On each of the following 14 days, a 5 ml aliquot of culture was drawn for growth assessment. After the cells were counted, they were removed by centrifu-

3 14

SANCHEZ-MORENO ET AL.-IN VITRO CULTURE

gation at 1,500 g for 10 min, and the pH of the medium de- termined. Cell-free media were stored at -20" C until analysis by IH NMR and enzymatic tests.

'H NMR spectroscopy. IH NMR spectra were obtained at 300 MHz on a Bruker AM-300 spectrometer operating in the pulsed Fourier transform mode with quadrature detection [ 191. Probe temperature was maintained at 27" C. Typical acquisition parameters were: 3,287.5 Hz sweep with, 8,192 time domain points, 90" radio frequency pulses, 8 s total recycle time and 160 accumulations. Chemical shifts were expressed as parts per million (ppm) downfield from TSP.

Identification and quantification of metabolites. The chemical shifts used to identify the respective metabolites were in accord with those recorded in the literature [19, 30, 341. By recording spectra with different recycle times, we established that a recycle time of 8 s did not significantly reduce intensity due to partial saturation for the peaks of interest.

Metabolites were quantified enzymatically as described by Bergmeyer [7].

RESULTS AND DISCUSSION One of the aims of the present study was to find an optimal

medium for the in vitro culture of parasites isolated from E. characias. The growth of Phytomonas sp. in the different media tested is shown in Fig 1. The best medium for bulk growing was SDM-79, which after 25 passages, produced approximately 5 x lo7 cells/ml during stationary phase, as previously reported [30]. Growth was exponential during the first 6 days of culture, stationary from day 7 to day 10, and then slightly diminished.

In vitro growth rates obtained by other authors with Phyto- monas species isolated from E. pinea [ 151, E. hyssopifolia [5] and the tomato [3 I ] were slightly lower than the growth rate we obtained with modified SDM-79.

Phytomonas isolated from plants and stained with Giemsa showed promastigote forms typical of flagellate protozoa [ 13, 321. Using the scanning electron microscope, we noted that most organisms growing in vitro were fusiform, with two or three body twists (Fig. 2), while others had no twists (Fig. 3). In addition to these predominant types, we also noted elongated, twisted cells lacking a flagellum, and rounded forms, with or without a flagellum, which were assumed to be intermediate forms [20].

In vitro experiments showed that once the parasite had adapted to the medium, it developed as an elongated promastigote, although after 20 passages it failed to maintain body twists, as was observed in other Phytomonas cultures [5, 151. In contrast with these findings, Jankevicius et al. [20], detected two types of elongated promastigotes when P. serpens was cultured in LIT medium, one longer than the other, and in both cases showing 2 4 body twists. According to these authors, the percentage of cultured forms with body twists depends on the composition of the medium and the duration of culture, with mature cultures producing 20-30% twisted forms.

In cultures with SDM-79 we observed promastigote forms, followed by twisted-body promastigotes from the the sixth day of culture (Fig. 2, 3). The latter forms were similar to the organisms that other authors have isolated from plants, with a mean cell length of 12.7 f 0.3 pm, and a mean flagellum length of 13.6 * 0.57 pm. Promastigotes without body twists and with a mean cell length of 6.5 k 0.25 pm and a mean length of the flagellum of 5.4 -+ 0.6 pm were also observed. These values are similar to those reported by other authors [ 15, 201.

The LIT and MTL media yielded a significantly slower growth, and failed to reach cell densities of 1 x lo6 cells/ml in both cases (Fig. 1). These findings contrast with those of Jankevicius

4ND METABOLISM OF PHYTOMONAS SP. 315

108 t I

: -0- SDM-79

. -0- Mod. GRACE'S

----.------,, .---. +------+------+-.--

I I

0 2 4 6 8 10 12

Time (days)

Fig. 1. Growth curve ofPhyiomonas sp. cultured in different media.

et al. [20], who obtained cell P. serpens densities of about 1.2 x lo7 cells/ml in LIT medium incubated at 28" C.

On the other hand, the effects of osmotic pressure and temperature are known to be important factors in the transformation of trypanosomatids [32]. Osuna et al. [26], reported successful T. cruzi in vitro differentiation, using modified Grace's medium by the addition of NaCl ( 1 25 mEq/l at final concentration). In addition, Na+ ion has been shown to play an important role in the differentiation of T. cmzi forms [27]. In 1978, De Almeida & De Souza [3] suggested that osmotic pressure is one of the factors involved in the differentiation of Herpetomonas sa- rnuelpessoai. However, in T. cruzi, no effect on the differentiation of the protozoa was found when the medium was supple- mented with salts other than NaCI, but in equimolar concentrations as the Cl- salt added, and preserving the osmotic (and viscosity) features of the medium. This suggests a role for Na+ in the differentiation of the parasite [27].

There have been no previous reports of rounded forms, either with or without flagellum, of the genus Phytomonas in vitro. In an attempt to obtain rounded forms in vitro, we used modified Grace's medium [26]. With this medium, cell density reached 1 x lo7 cells/ml, only slightly lower than obtained with SDM- 79 (Fig. 1). The growth curves with the two media were similar, showing an exponential growth phase for 4 days followed by a stationary phase. However, rounded forms with or without flagellum were detected from the fourth day of culture, and the transformation rate reached 70% on day 10 of culture. The different forms are shown in Fig. 4 and 5.

To verify that the transformation of promastigote forms into rounded forms was due to the addition of NaCl to the medium and not to aging of the cultured cells, we prepared in vitro cultures of the parasites in unmodified Grace's medium. After 12 days of culture, no rounded forms had appeared, and cell density was slightly, but not significantly, lower than that obtained with modified Grace's medium (data not shown).

Scanning electron microscopy showed that phytomonads isolated from different plants were markedly different in body and flagellum length. Attias & Souza [5], examining different species of phytomonads isolated from E. hyssopifolia, E. pinea, E. characias and Manihot esculenta, observed that despite these differences in external morphology, all species shared the structures typical of other members of the family Trypanosomatidae.

These findings suggest that modified SDM-79 is an excellent medium for the in vitro culture of Phytomonas isolated from E. characias. Cell densities close to 5 x lo7 cells/ml are ob-

3 16 J. EUK. MICROBIOL., VOL. 42. NO. 3, MAY-JUNE 1995

Fig. 2-5. Different forms of Phvrornonas sp. from Euphorbia characias by scanning electron microscopy. 2 , 3. Promastigote forms with and without body twists. Bar = 2 pm and I prn respectively. 4. Amastigote form. Bar = 1 pm. 5. Spheromastigote form. Bar = I prn.

SANCHEZ-MORENO ET AL.-IN VITRO CULTURE AND METABOLISM OF PHYTOMON-AS SP 317

A

B

4,OO 3 ,OO 2.00 1.00 ppm

Fig. 6. ' H NMR spectra ofPhytornonas sp. medium showing metabolite production and utilization during normal growth in SDM-79 medium. A. Fresh medium. B. After 12 days of culture. Metabolites excreted and chemical shifts were: A, acetate, 1.855 (s); Et, ethanol, I . 185 (t); Glyc, glycerol, 3.565 (s); Gly, glycine, 2.950 (s); P, piruvate, 2.415 (s) and S, succinate, 2.1 15 (s).

tainable, and the packed cell volume per liter of culture is ad- equate for the biochemical studies required for further characterization of the parasite. Modified Grace's medium yielded different forms of Phytomonas sp., including cells similar to the amastigote forms previously isolated only from parasitized tomato plants [20]. We also obtained typical promastigote forms characterized by their elongated, twisted body. Finally, the in vitro organisms we obtained showed no morphological altera- tions in comparison to forms isolated from plants.

Nuclear magnetic resonance spectroscopy is a well-established technique in the study of metabolic pathways in biological systems. To date, most studies have been based on NMR; ' H NMR has only recently been used to investigate parasite metabolism and is expected to become increasingly important in this area, as most biological compounds contain detectable protons [34].

An example of the type of 'H NMR spectrum obtained with SDM-79 is shown in Fig. 6. Fig. 6A illustrates the spectrum obtained with fresh (uninoculated) SDM-79, and Fig. 6B is the spectra given by cell free medium 12 days after inoculation with Phytomonas sp. cells. Additional peaks, corresponding to the major metabolites produced and excreted during growth, were

easily distinguished when compared with fresh medium. Eth- anol, acetate, glycine, glycerol, piruvate and succinate were the metabolites excreted by the parasite.

Glucose consumption by Phytomonas sp. cultured in SDM- 79 was inversely correlated with exponential growth. Glucose consumption was accompanied by a slight fall in the pH of the medium during the first 8 days of culture. This decrease in pH is probably the result of the production of organic acids by the parasite and subsequent excretion into the medium. When the glucose in the medium is depleted, Phytomonas may reutilize these acids as fuels, explaining the increased pH of the medium during the stationary phase (Fig. 7A).

These metabolites were quantified using enzymatic methods (Fig. 7l3-D). The production of some of these compounds is linked to the exponential growth curve, although acetate, piruvate and succinate decreased slightly at the begining of the stationary phase. This decrease may be related to the lack of free glucose in the medium, and the subsequent reuse of these acids by the complete organism, versus the inability of Phyto- monas mitochondria to oxidize these acids [30].

Glycerol became detectable on day 4 of culture, and reached a peak concentration on day 8, decreasing sharply after that.

318 J. EUK. MICROBIOL.. VOL. 42, NO. 3. MAY-JUNE 1995

8

4

A

i I

m v) 0 0 3 - 0

12 I 1 8

I + Glucose I ’ O B 7 8

I a

B

I E

40 a ,

32

28 :-O- Acetate

’ f Plruvare .

20

16

.

0 2 4 6 E 10 12

Time (days) Time (days)

C 1600 I I I 50 T

m c m .- C 0 0 3 v)

+ Succinate I 1280 ’ +Glycerol 1 2 0 .

9 6 0 .

6 4 0 .

0 2 4 6 8 1 0 1 2

5 E Y

D 20 1 5 0

+ Glycine

1 2 0

0 90

0 60

0 30

0 00 4 10 12 0 2 6 8

r E Y

._ 5 i? C

E a

Time (days) Time (days)

Fig. 7. Changes of various metabolite concentrations and pH in SDM-79 medium during growth of Phytomonas sp. (n = 9).

The decrease in glycerol may have been due to its reuse by the parasite as the amount of glucose in the medium decreased.

Von Brand [35] showed that amino acid consumption by T. cruzi epimastigotes during in vitro growth is accompanied by the release of NH,, which is precipitated in the medium as ammonium salts. These findings were confirmed [2, 9, 101, showing that T. cruzi, in contrast to other trypanosomatids, is a strict ammoniotelic organism. Our study has shown that Phy- tomonas sp. isolated from E. characias produces and excretes NH, into the medium during the first four days of culture; however on the fifth day. NH, concentration started to decrease (Fig. 7D), in contrast with T. cmzi [I] .

In modified Grace’s medium, glucose consumption (data not shown) paralleled that recorded in cultures with SDM-79, although the slope of the curve was slightly lower. From the sixth day of culture, glucose consumption increased during the period of transformation from promastigote to amastigote. This in- crease may reflect the greater energy demands of transformation. In contrast with the findings in SDM-79 cultures, the glucose consumption curve in modified Grace’s medium continued to rise until the glucose was completely depleted from the medium. A possible explanation for the shape of the curve is that trans-

formation continued after growth became stationary. As in SDM- 79 cultures, glucose consumption in modified Grace’s medium was accompanied by a slight decrease in the pH of the medium.

The ‘H NMR spectra obtained with modified Grace’s medium (uninoculated) appear in Fig. 8A, and the spectra of medium 12 days after inoculation with Phytomonas sp. cells in the Fig. 8B. Ethanol, acetate, glycine, glycerol and piruvate were again the major metabolites excreted by the parasite, although less acetate was present than in SDM-79. However, although succinate was not detected in Grace’s medium, the signal was clearly detected in SDM-79. The decrease of succinate production may be related to the presence of transformed rounded promastigote forms.

To check this hypothesis, we obtained the ’H NMR spectrum of unmodified Grace’s medium in which phytomonad species had been cultured. The spectra in Fig. 8C show that succinate was clearly detectable in the culture medium, while ethanol production was drastically diminished with respect to those obtained in the spectrum of modified Grace’s medium. These results may confirm that different forms of the parasite could be distinguished not only morphologically, but also in its metabolic behavior.

SANCHEZ-MORENO ET AL.-IN VITRO CULTURE AND METABOLISM OF PHYTOMOMS SP. 319

A

B

C

4.00 3,OO 2.00 1.00 ppm Fig. 8. ‘H NMR spectra of Phytomonas sp. Metabolite production

and utilization during normal growth in Grace’s modified and unmodified medium. A. Fresh medium. B. After 12 days ofculture in modified medium and C. After 12 days of culture in unmodified medium. Me- tabolites excreted and chemical shifts were: A, acetate, 1.855 (s); Et, ethanol, 1.185 (t); Glyc, glycerol, 3.650 (s); Gly, glycine, 3.310 (s); P, piruvate, 2.41 5 (s) and S, succinate, 2.1 15 (s).

Glucose is only partially broken down by trypanosomatids to C 0 2 under either anaerobic or aerobic conditions. A consider- able amount of the carbon skeleton of glucose is excreted into the medium as reduced metabolites [ 1 I].

Our findings show that Phytomonas promastigotes isolated from E. characias grown in vitro under aerobic conditions, ex- crete piruvate together with its derived acetate, ethanol, succinate, glycerol and glycine as end products ofglycolysis. Rounded forms produce more ethanol and less succinate than the promastigote forms. All products are excreted into the culture medium and, under conditions of limited glucose, may be reused by the parasite as energy substrates.

The formation of different metabolites is clearly associated with the forms of the parasite, the stages of growth and the culture media.

It has been known that species of the genera Trypanosoma, Crithidia [6] and Leishmania [24] are not equipped with lactate dehydrogenase, the enzyme responsible in the principal mech- anism for NADH reoxidation during glycolysis. Under anaerobic conditions, this lack is apparently compensated by the a-glycerophosphate conversion to glycerol by a reversion of the glycerol kinase reaction present in glycosomes 1251. This mech- anism appears to be shared by Phytomonas sp. isolated from E. charucias [30]. Thus, the production of glycerol and other fermentation products such as ethanol is probably the result of a partial anaerobic metabolism during the stationary growth phase, or by the inability of the L-a-glycerophosphate-oxidase cycle to cope with all the reduced equivalents produced in glycolysis. The evidence presented above suggests that the catabolic ability of Phytomonas sp. isolated from E. characias is severely limited. Previous studies showed that the mitochondria of this organism cannot oxidize 2-oxoglutarate, succinate or proline. suggesting the absence of a functionally complete Krebs’ cycle [30]. In this way, Phytomonas sp. does not produce succinate by reversing part of the Krebs’ cycle, as has been shown for certain trypanosomatids, such as Trypanosoma brucei procyclic insect stages [ 171 and Crithidia fasciculata [6]. Part of the glyoxylate cycle, comprising the enzymes citrate synthase, aconitase and isocitrate lyase, is most likely responsible for the formation of succinate as an end-product [30]. This pathway also leads to the formation of glyoxylate. The latter compound can be either reutilized in the glyoxylate cycle, or excreted as glycine after transamination. This could be the explanation for the disap- pearance of ammonia from the culture medium during the later stages of growth.

ACKNOWLEDGMENTS We thank Lopez-Martin for technical assistance and Ms Kar-

en Shashok for translating the original manuscript into English.

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Received 1 1 - 7- 92, 9- 19- 94; accepted 12-22- 94

J Euk Microhid . 42(3). 1995. pp 3?&32? 0 1995 by the Soc~ely of Prolozoologisls

Comparison of the 24 kDa Flagellar Calcium-Binding Protein cDNA of Two Strains of Trypanosoma cruzi

LISA M. GODSEL, CHERYL L. OLSON, ZULMIRA G . M. LACAVA’ and DAVID M. ENGMAN’ Departments of Microbiology-Immunology and Pathology and Feinberg Cardiovascular Research Institute,

North western Universiry Medical School, Chicago, Illinois 606 I I

ABSTRACT. DNA sequences encoding the 24 kDa flagellar calcium binding protein (FCaBP) of two strains of Trypanosoma cruzi were found to differ at fourteen positions, six of which result in amino acid differences. Four of the amino acid differences are located within the calcium-binding domains of FCaBP however, none is predicted to affect the calcium-binding ability of the protein. Chro- mosomes harboring the FCaBP gene clusters differ in size among T. cruzi strains.

Supplementary key words. Calcium-binding protein, EF-hand, genetic heterogeneity.

M O N G the various clones routinely isolated from T. cruzi A cDNA expression libraries upon screening with sera from chronically infected humans or experimental animals are those encoding a 24 kDa calcium-binding protein found in the parasite’s flagellum [3]. This protein is variably known as the IF8 protein [S], flagellar calcium binding protein (FCaBP) [3] or

I Current address: Department of Genetics and Morphology, Uni- versity of Brasilia, 709 10 Brasilia, Brasil.

To whom correspondence should be addressed: Northwestern Uni- versity, Department of Pathology, 303 E. Chicago Ave., Chicago, IL 60611.

ALC-I antigen [ I 11, and is encoded by a tandemly arranged gene family ofapproximately 20 members [5]. In this paper, we will refer to the protein as FCaBP, but point out that it is only one o f a number of calcium-binding proteins i n the trypanosome flagellum, the group of which having recently been dubbed the “calflagin” family (L. Ruben, pers. commun.). Although all of the calflagins are localized to the flagellum and possess EF-hand calcium-binding domains, they have different sequences and a wide rangc of molecular masses [7, 141.

To identify antigenic proteins of T. cruzi for use in serodi- agnosis, 80,000 plaque-forming units of a T. cruzi PBOL strain [8] epimastigote c D N A expression library were immunologi-

in vitro culture of phytomonas sp. isolated from euphorbia characias. metabolic studies by 1h nmr

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