Molecular and Biochemical Parasitology, 16 (1985) 173-183 Elsevier

MBP 00567

173

PROTEIN VARIATION IN C L O N E S OF P L A S M O D I U M F A L C I P A R U M

D E T E C T E D BY TWO D I M E N S I O N A L E L E C T R O P H O R E S I S

BRIAN FENTON. ALISON WALKER* and [)AVID WAI.LIKt!R

Department of Genetics'. University of Edinburgh. West Mains Road, l:'dingburgh Elf9 3.IN. S~otland. U.K.

(Received 19 February 1985; accepted 15 April 1985)

Two dimensional clectrophorcsis has been used toexaminc protein variation in clones of two Plasmodium falciparurn isolates. Variant forms of 12 proteins were detected. Five genetically distinct parasite types were identified in one isolate, and two in the second isolate. Examination of uncloncd parasites using this technique showed that the frequency of each genotype altered during six months of culture.

Key words: Two dimensional electrophoresis: Plasmodium fah'iparum; Malaria; Protein variation

INTRODUCTION

It is now well established that isolates of the human malaria parasite Plasmodium

falciparum lYequently conta in mixtures of genetically diverse organisms. This was first

demonst ra ted in studies on the enzyme characteristics of cloned parasite lines; isolates

charactcrised by two electrophoretic forms of the enzyme glucose phosphate isomcr-

ase wcre shown by cloning to contain two types of parasite, each possessing single

enzyme forms [ 1-4]. Such cloning studies have also demonst ra tcd the occurrence of

parasites of differing drug-scnsitivities [2-51, antigens [4] and gametocyte-producing

ability [5] within single infections.

The technique of two d imens ional polyacrylamide gel elcctrophoresis (2D-PAGE)

is especially useful in studies on strain characterisat ion as it enables variat ions in many

proteins to be studied at the same time. In P.falciparum the method has been used to

demonst ra te variat ions in fourtcen proteins among isolates of different countries [6].

In Thai land , each of thirteen isolates could be dist inguished by their posscssion of at

least one characteristic protein form detected by this method (A. Walker and A. Tait,

in preparat ion) .

* Present address: Department of Medical Protozoologv, l.ondon School of I'l?'gicnc and Tropical Me- dicine. Keppel Street, l.ondon WCI.F 7H-I'. L'.K.

0166-6851/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

174

The aim of the present work was to use the 2D-PAGE technique to examine the

protein composition of two isolates of P. falciparum, and of cloned parasite lines derived from them. We show that certain proteins possess more than one torm in the

uncloned material, while only single forms arc prcsent in the clones. In one isolate, five genetically distinct types of parasite have been identified, while in the second two types have been found.

MATIRIALS AND ME'FIIOI)S

Parasites. Two P. falciparum isolates from Thailand were studied. Isolate T9 was obtained from a patient at Mac Sod, near Tak, and PB- 1 from a patient at Phraphutta- bat. The parasites were established in continuous culture in Bangkok and brought to

Edinburgh by Dr. Sodsri Thaithong for cloning. The two isolates were shown to be parasite mixtures from a study of their enzymes (glucose phosphate isomerase and adenosine deaminase) by starch gel electrophoresis [7]. Cloning was carried out on two

occasions on T9 and once on PB-I by Drs. Sodsri Thai thong and V.E. Rosario, using a dilution technique [1]. The present work was carried out on fifteen such diluted

cultures. Fourteen were assumed, from their enzymes, to be clones, while the remain- ing culture (T9-105) possessed two forms of glucose phosphate isomerase, and was

thus derived from more than one parasite.

Radio-labelling of parasites. Parasites were grown by the Trager and Jensen petri- dish method [8], using RPMI 1640 medium (Gibco)supplemented with HEPES buffer

(Sigma Chemical Co.), human type 'O ' erythrocytes and human serum (Edinburgh and South East Scotland Blood Transfusion Service). Each petri dish contained 1.5 ml culture, at a haematocrit of 5%. When the cultures had achieved a parasitaemia of 4-5%, as much as possible of the medium was removed and replaced with EMEM medium (Eagle's minimal medium modified with Earle's salts - Gibco) lacking

methionine, para-amino-benzoic acid (PABA) and glutamine, and supplemented with HEPES (5.94 g l-t), PABA (1 mg l-t), hypoxanthine (50 mg l-t), reduced glutathione

(0.6 g l -t) and glucose (2 g l-t). 50 laCi of [35Slmethionine (Amersham International) was added to each culture. Parasites were incubated in this medium for 12 h under

normal candle-jar conditions. All cultures were asynchronous and contained ring- forms, trophozoites and schizonts at the time of radio-labelling.

Preparation of parasites for electrophoresis. After incubation, parasites were released from the host cell using saponin [7], the process being carried out at 4°C instead of 37°C. Lysis of free parasites was then carried out using O'Farrel l ' s buffer [9], 80 tal of" buffer being sufficient for each 1.5 ml culture. Any remaining intact parasites were ruptured by freeze-thawing. After centrifugation, incorporation of radio-labelled amino acids was determined by liquid scintillation counting of trichloroacetic acid

(TCA) precipitable material [6]. Typical counts were 2-6 × 106 cpm 10 -6 organisms. Samples containing approximately 2 × 105 cpm were applied to each gel.

175

2D-PAGE. Labelled parasite preparations were subjected to 2D-PAGE using the Tait [6] modification of O'Farrell 's technique [9]. An iso-electric focussing gel of an

extended pH 5-7 range was loaded with the parasite preparation and focussed lbr 12 h. The second dimension gel was 10% SDS-polyacrylamide, and electrophoresis in this gel was at 120 mA h. The gel was then fixed and fluorographed [ I0], then exposed to pre-flashed Kodak X-omat film. Development of the film took place over 2-3 weeks.

Standardisation of iso-electric focussing was carried out by measuring the pH of l- mm segments of gel equilibrated in 1 ml 25 mM KCI and plotting the gradient. SDS-PAGE standardisation was carried out by co-electrophoresing standard molecu- lar weight markers (Sigma SDS-6H).

Two dimensional gels of each parasite line were comparcd for variation of individ- ual proteins in either iso-electric point or molecular weight. In somc instances mixcd preparations of two parasite lines were subjected to elcctrophoresis on a single gel, in order to determine whether certain proteins werc identical or different in the two lines.

Terminology of protein variants. In this survey, a protein which is found to exhibit variation in position between different parasite lines is designated by a capital letter (A, B, C, etc.) and the individual variant forms of each protein by numbers (e.g.A. 1, A.2, A.3, etc.). The numbering of each variant has no significance, but reflects their ordcr of discovery.

RESULTS

Autoradiography of 2D gels of all parasite lines revealed more than I00 labelled parasite proteins. The majority of these appeared to be invariant in position in all lines. Fourteen, however, exhibited variation detectable by charge or relative molecular mass differences. Fig. la is a composite map illustrating all forms of these proteins detected among P. falciparum isolates cxamined to datc (including some isolatcs not described in this paper).

The principal finding of this work was that the uncloned isolates T9 and PBI possessed more thao one form of variable proteins, while the dilutcd cultures (except T9-105) possessed only single forms. Full results are given in Table I. Fig. 1 shows autoradiographs of uncloned T9 (b) and PB-I (c); in the T9 gel, 2 forms of proteins A, F, G and K are arrowed, and in the PB-I gel different lorms of protein D are arrowed. Fig. 2 illustratcs four of the diluted cultures cxhibiting single forms of proteins of A, D, F, G and K.

Fig. 3 illustrates examples of gels containing artificial mixtures of ccrtain diluted cultures. These illustrate clearly the differences in position of some of the variant protein forms. For example, the two forms of protein D in a mixture of cultures T9-96 and T9-100 can bc readily distinguished. Since these are artificial mixtures, the variants are prcsent in equal intensitics. This contrasts with some of the uncloned

176

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a :--,-+;.. B ~ . . . . :"" + , 0 # . . . . . " .-. . . . . . . .

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..~ I ,=,~.. . . . . . q P , D

i l l : ..... :

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m o7 "5 "6 '7 '8

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P O I N T . S O E L E C T R I C

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4 b o

~ , . . ~ ... :. ,.,.:.~l~,~ -

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Fig. 1. (a) Compos i t e map of all pro te ins in P. falciparum detected by 2 D - P A G E . Protein spots exh ib i t ing

var ia t ion are indicated by let ters and are tmshadcd. The origin of the first d imens ion gel is marked "O'. The

Y-axis denotes the relat ive mobi l i ty ( M r) o f a series of s t andard proteins: their sizes arc given in k i loda l tons

(kDa) . (b,c) A u t o r a d i o g r a p h s of u n d o n c d isolates T9 and PB-I. The protein pos i t ions that i l lustrate tv.o

var iant forms arc A, F. ( ; and K i n ' l 9 and D i n P B - I . T h e rest o f t h c c l e a r l y visible var iable proteins are also

indicated.

177

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Fig. 2. A u l o r a d i o g r a p h s o f representati,,'es from lhe diluted cultures that yielded pure genotypes. As in Fig. ]

the variant protein spots of interest are i n d i c a t e d .

material in which some variants appear less intense than others, representing unequal proportions of different genotypes. This can be seen in Fig. 1 where, in uncloned 1"9, protein form F.3 is less intense than F.2.

Uncloned T9 was examined by 2 D - P A G E on five occasions during a period of 7 months' continuous culture. The protein composit ion was found to change during this

179

g 4 /

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O

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l ' ig. 3. Au to r ad iog raphs of art if icial mixtt, res o f l a b d l c d el<rues made as dc:-,c ribed (Mater ia ls and Methods) .

The types of mixtures are indica ted at the top left hand corner.

time (Table II). Some protein forms, e.g.A.2, were present in early cultures, but absent later. In other instances, e.g.E.3 and K.3, forms detectable at the start disappeared during subsequent cultures, but reappeared later. Finally, some protein forms absent at the start appeared transiently, and then apparently disappeared (e.g.G. 1, K.5). The changing patterns imply changes in the frequencies of particular genotypes during the period of cuht, re.

180

TABI,E II

The variant proteins of culture 19

Variant T9 Variation versus time in culture ('o,.ecks in culture) polypcptide spot 8 16 211 28 31)

A 2,4 4.2" 4 4 4

C 1.2 2 2,3 2 2

D _h 4 4.5 _t, 4.5

E 1,3 _t, I 2,3" 2

F 2. I" 1,3 1,3.2" 3. I".2" 3.2"

G 2 1.2 1.2 2 2 1 _b 1.2 1.2 1.2 _b . j h ") -~ .~ I,

K 3,6;' 5,6' 5.6" 6. Y' 6 I. _b I I _h _~

M _h I I I h

N -~' I I I -~'

As in Table I each variable protein is represented A-N. Samples were taken during 30 weeks of culture and

the composition of each is presented.

;' Minority.

b Not easily distinguished in autoradiograph.

DISCUSSION

This work illustrates clearly the genetic heterogeneity that exists in uncloned isolates

of P. falciparum. We have shown that the isolates T9 and PB- 1 possess more than one

form of certain proteins detectable by 2D-PAGE, while the majori ty of diluted

cultures derived from these isolatcs possess only single forms. The var iant forms of

each protein are most probably due to allelic variation of the genes de termining them.

Since the blood forms of malaria parasites are haploid [ 11], clones possess only one

allele of each gene, which wot, ld be cxpected to determine only a single protein form.

Parasite cultures exhibit ing two or more forms can be presumed to comprise mixtures

of more than one haploid genotype.

Therc have been two previous studies using the 2 D - P A G E technique to study I'. falciparum, by Tait [6] and Brown et al. [12]. The protein patterns in these tw.o studies

are very similar to those obta ined in the present work. Some of the variable proteins in

the three studies can be cross-identified readily, and these are indicated in Table I. The

nature of most of the proteins remains to be established. We know, however, that the

protein designated 'A ' here is an antigen associated with schizonts and merozoites,

probably corresponding to Pf 220 of Brown ct al. [12], P 190 of flail et al. [ 13],and to

an antigen of M.W. 195 kDa of Holder and Freeman [ 14], which exhibits considerable

181

antigenic diversity among different isolates [ 15]. Forms of protein 'B' in the present

study probably correspond to S-antigens as studied by Brown et al. [12].

One isolate (S 118) studied by Tail [6] was considered to be a mixture of genetically

distinct clones, as it possessed two forms of each of five variable proteins. Brown et al. [12] also considered one of their cultures (NF7) to be a mixture as its protein

composition changed tb[lowing 4 months of culture. In the present work, we have shown that both T9 and PB-I are mixtures. By diluting an early culture ofT9 we have

been able to distinguish five types of parasite, each bearing its own set of distinct

polypeptides (Table I). Four of these types were obtained as pure clones, while the fifth

(1"9-105) was present only as a mixture, this culture containing two forms of A, C and

F; further dilution of this culture should produce a pure fifth genotype. The findings

reported here confirm the preliminary report by Thaithong et al. [4] concerning

2D-PAGE characters of these clones, with the following exceptions. Protein E of most

cultures has now proved to be type 3 apart from cultures 94 (type 2)and 105 (type 1).

Proteins A, C and D of clone 98 have now been shown to be respectively, types 2, 1 and

4. Thus, the number of parasite types identified by 2D-PAGE in isolate T9 is five.

Using other markers, e.g. drug resistance, Thaithong et al. [4] were able to find an

additional two clonal types in this isolate. A limitation of the 2D-PAGE technique as used in this work is that proteins of

parasite types forming only a small proportion of each preparation may not be

detected. Absence of a particular variant in uncloned parasite material may not,

therefore, be due to a complete absence of a given genotype, but rather to its presence

in numbers too low to be detectable. Some of the proteins may also exhibit a

stage-specificity, e.g. they may be expressed only in schizonts. In the present work,

however, the parasite cultures were asynchronous, containing all stages of asexual

erythrocytic parasites at the time of examination; absence of a given protein, therefore,

was unlikely to have been due to absence of a specific parasite stage in the preparation. During passage of uncloned T9. we observed changes in protein composition,

implying that certain clones became selectively favoured (Table II). This observation agrees with that of Rosario [ I], using enzyme markers to follow changes in genotype

frequencies. The reasons for these changes are not understood. ]hey may be due to variations in culture components during the period of observation, e.g. addition of

different sera or red cells, providing more favourablc growth conditions for some genotypes than for others. At the final stage of examination, it appears that parasites

with the characters of clone 94 were predominant.

The 2D-PAGI'," protein characters of clones have been found to be stable following

at least 6 months of culture. The technique is thus a good method of strain characteris- ation which can be used in conjunction with other characters such as enzymes,

drug-sensitivity and antigens. The complete characterisation of the T9 clones has been reported elsewhere [4]. With regard to PB-1, two types of parasite could be identified by 2D-PAGE, both of which were similar to two of the T9 clonal types (Table I); these

182

two PB-1 types have also been shown to differ from one another by enzymes and drug-sensitivity [4].

The 2D-PAGE technique has thus been shown to be extremely valuable for P. falciparum strain characterisation, and for examining the genetic composition of individual isolates. Although more complicatcd than cnzymc clectrophoresis it has

advantages: many gene products are studied on a single gcl, the cxisting markers give a very large number of combinations, cnough tk)r over a million diffcrent two dimension- al polypeptide maps. In current work we have bcen ablc to dctcct the variablc

proteins by Coomassie bluc and by silvcr staining, thus simplifying the procedure. Wc

also hope to investigate the nature ofthc variable proteins, in addition to those already identified as antigens. It can bc expected that the method will be of value in, for example, identifying alterations in proteins concerned in drug-resistance, and such an approach could lead to the dcvelopment of diagnostic probes for resistant parasites.

ACKNOWI.EI-)GEMENTS

We thank the Wellcome Trust, the Medical Research Council and the World Health Organization (TDR) for support. We also thank Richard Fawcett for expert technical assistance, Sodsri Thaithong and Virgilio Rosario for use of their clones, and F. Johnston and A.M. McEwan for photographic help. We are grateful to l)r. Andrew

Tait and Professor G.H. Beale for discussion and advice, and thank the staff of the Edinburgh and South East Scotland Blood Tranfusion Service.

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