,-lnn. 1st. Super. Sanità Vol. 21, N . 3 (1985). pp. 219-306

RECOMBINANT DNA TECHNIQUES AS POTENTIAL DIAGNOSTIC MEANS

P. Wilairat, N. Tirawanchai, C. Intapruk, S. Tungpradabkul, R. Sertsrivanich, S. Panyim & Y. Yuthavong

Department of Biochemistry and Center for Molecular Genetics and Genetic Engineering, Faculty of Science, Mahidol University, Bangkok, Thailand

Summary. - Recombinant DNA techniques are providing new important tools in malaria research. Molecular cloning of Plasmodial repetitive DNA sequences have generated probes that are capable pf distinguishing between different parasite isolates as well as identifying malaria infected blood and mosquito samples.

Riassunto (Tecniche di DNA ricombinante come potenziali mezzi diagnostici). - Le tecniche del DNA ricombinante forniscono nuovi e importanti mezzi di ricerca nel campo della malaria. I1 clonaggio molecolare di sequenze di DNA ripetitive ha permesso di ottenere sonde molecolari capaci di discriminare tra diversi isolati e di identificare campioni infetti di sangue e di zanzare.

The current persistance and spread of malaria in tropical regions o£ the world constitute a major health problem, not only to people living in endemic areas but also to travelers visiting such regions (1). It has been estimated that over 200 million people annually are affected by the disease and the mortality among children in Africa alone is close to one million. Contro1 of malaria is hampered by the rising cost of insecticides and the concern for their possible detrimental impact on the environment, and by the rapid appearance of resistance in the parasites, in particular Plasmodium falciparum, to anti- malarials.

This global problem has stimulated research into the basic biology of the parasite and its relationship with the human host and insect vector, in the hope that such new information will provide additional armory with which to combat the disease. Many fundamental questions remain to be answered. How genetically diverse is P. falciparum in a given geographical region? By what mechanism does resistance to antimalarials occur and how quickly does it spread throiigh the more drug-sensitive parasite population? Which are the important insect vectors?

One of the simplest method employed to demonstrate genetic differences in malaria parasites is enzyme electrophoresis (2). Carter and McGregor (3) vere the first to show that single isolates of P. falciparum from individuals in Gambia often exhibited different electrophoretic forms of one or more enzymes. More recent analysis of P. falciparum isolates from Gambia, Tanzania, Thailand, and Brazil have indicated that P. falciparum exists as a single, worldwide species containing potentially interbreeding organisms (4). The simplicity of the electrophoretic method is offset by the paucity of suitable enzyme markers. For P. falciparum only four enzymes, glucose phosphate isomerase, lactate dehydrogenase, adenosine deaminase and peptidase, have so far proven useful, although other enzymes will undoubtedly be found. Thus enzyme studies alone will not reveal the fu11 potential of the genetic variations present in natura1 populations of P. falciparum.

High resolution two-dimensional gel electrophoresis of proteins can resolve

over 1000 different molecular species (5). Taking advantage of the lack of endogenous protein synthesis of red cells, Tait (6) was able to compare metabolically radiolabelled parasite proteins extracted from P. falciparum isolates maintained under in vitro culture conditions. Al1 isolates f rom South East Asia can be distinguished from the Gambian isolates, and £or isolates from a single geographical region in Thailand, variations in the protein pattern are discernable. Two main problems are encountered using two-dimensional gel elec- trophoresis: that o£ obtaining absolutely identica1 gel patterns for comparison purposes and the inability o£ identifying the protein spots. The latter problem could be overcome using an "immunoblot" technique (7) provided that appropriate antibodies are available.

The recent development of hybridomas to produce monoclonal antibodies has provided an extremely powerful t001 with which to characterize parasite antigens. McBride and co-workers (8) have prepared monoclonal antibodies in mice against two P. falciparum isolates from Thailand and have used this battery of antibodies to distinguish between isolates from Asia, Africa and Australasia. No geographically specific antigenic types could be identified. However Kidson and co-workers (9) produced monoclonal antibodies against Papua New Guinea (PNG) isolates which recognized schizonts of al1 seven PNG isolates but not the isolates from Thailand, Nigeria, Ghana and The Netherlands.

Al1 the above methods are based on phenotypic properties, and genotype analysis at the DNA leve1 would greatly expand the existing range of methods for strain classification. This could be done simply by examination of the DNA patterns produced following digestion with restriction endonucleases. Since the complexity of P. berghei DNA has been shown by Dore and co-workers (10) to be comparable to Trypanosoma brucei, which had been strain characterized based on size analysis of endonuclease restricted nuclear DNA (111, a similar approach should be possible to differentiate malaria parasites. DNA from P. falciparum, K1 isolate, maintained in culture over a period of some three years, and from cloned strains of rodent parasites were obtained (12). Contaminating mouse white blooa cells were removed by passage of infected blood over a column of cellulose CF11. The DNA preparations were deproteinized with Pronase CB, digested with RNase and extracted with phenol. DNA samples were digested to completion with the desired restriction endonuclease at 37°C for 2h. Size separation was conducted by electrophoresis in 0.7% agarose and the DNA pattern visualized with ethidium bromide staining. Molecular size markers were ADNA fragments generated by digestion with HindlII.

Fig. 1 shows the patterns o£ DNA from the various species of parasites following digestion with the restriction endonucleases PstI and EcoRI. The undigested DNA materia1 appeared as a high molecular weight band of about 50 kb which upon treatment with the enzyme was reduced in size, appearing as 2 fluorescent background along the length of the gel. However distinct bands o£ parasite DNA fragments were readily and reproducibly observed. These banding patterns are characteristic for a particular species of parasite.

The complexity o£ the banding patterns is dependent upon the type o£ restriction endonuclease employed in the digest'ion. Fig. 1 shows that EcoRI digestion gave greater pattern variations than digestion with PstI. Digestion with BamHI failed to clearly differentiate between P. berghei, P.chabaudi, L yoelii and P. falciparum (12). Plasmodial DNA has a high AT content (13,141 and thus restriction sites composed mainly of GC bases would be fewer in number, and the number o£ fragments generated would be accordingly reduced.

It wns not possible to distinguish between the two cloned P. yoelii YM and 33X strains, between drug sensitive P. chabaudi and drug resistant clones derived from it (12), nor between P. falciparum K1 isolate from Thailand and 6112 isolate from Gambia (data not shown). To extend the sensitivity of this approach it would be necessary to use specific DNA probes to hybridize with the parasite DNA fragments in order to determine the distribution of the sequences of the probes in the parasite genome.

Fig. 1. - Aga digested with with PstI (e) (h) and EcoRI

.rose gel profiles of DNA from P. falciparum, undigested (a) and PstI (b) and EcoRI (C); P. chabaudi. undigested (d) and digested I

and EcoRI (f); P. yoelii, undigested (g) and digested with PstI ! (i); and Aphage digested with Hind 111 ( s ) .

Fig. 2. - Cloning strategy of P. falciparum, K1 isolate, in E. coli.

With this goal in mind, we extracted DNA from P. falciparum, K1 isolate, digested it with PstI and EcoRI and cloned these DNA fragments in E. coli using as vector the. plasmid pBR322, at its PstI/Eco~1 cloning sites (see Fig.2). Transformants that were ampicillin-sensitive and tetracycline-resistant were selected. Out of some 72 transformants generated, a clone containing the plasmid designated pBRK1-14 was chosen as a source for obtaining the DNA probe.

The recombinant plasmid pBRK1-14 hybridized with intermediate intensity compared with recombinant plasmids from the other transformants when tested in "dot-blot" hybridization assay using 3ZP-labelled parasite genomic DNA as the radiolabelled probe. pBRK1-14 contains a parasite DNA insert of about 760 nucleotides in length (Fig. 3) which has one RsaI restriction site close to the PstI cloning site.

Fig. 3. - Map of the recombinant plasmid, pRRK1-14.

The sequence of the 760 nucleotide insert has been parti.ally determined using the Maxam-Gilbert sequencing method (15). The insert has an AT content of 82% and contains tandem repeating sets of the tetranucleotides TTTA, AATT, TTAA and AAAT.

DNA from four P. falciparum isolates were digested either with EcoRI or doubly digested with EcoRI and PstI, size fractionated on 0.7% agarose gel and Southern blot transferred onto a nitrocellulose sheet (16). Fig.4 shows that autoradiographic patterns obtained following hybridization with 32P-labelled pBRK1-14 probe. The differences in the size distribution of hybridizable bands observed between the K1 (Thai) and G112 (Gambian) isolates allow for facile discrimination between isolates collected from two different geographical locations.

A distinction could be made between an isolate kept in liquid nitrogen followirig collection and that (marked with an asterisk) maintained for a number of years under continuous in vitro culture, based on minor quantitative differences in the hybridization bands of both K1 and G112 isolates. Other restriction enzymes used in these comparative studies included AluI, HindIII and Sau961; al1 provided Southern blot patterns that could differentiate between K1 and G112 isolates.

Two other potentially useful applications of cloned parasite DNA probes are in the detection of infected human blood and of infected mosquitoes.

Fig. 4. - Southern blot analysis o£ P. falciparum DNA from K1 and G112 isolates. Lane(a) contains pBFX1-14 and (b) PstI-digested LDNA

i HYBRIDIZED' with

ig. 5. - Dot blot hybridization of DNA from salivary gland (2 G1) and gut (2 1 U) pooled from two Anopheles dirus nosquitoes infected with P. falciparum; I ninfected Anopheles dirus (An); P. falciparum (Kl); P. vivax ( P V ) ; P. chabaudi ' ?C); and human cells (Hu).

Franzen and co-workers (17) have produced a P.falciparum DNA probe, pRepHind, capable of detecting parasitaemia levels of 0.001% in 50 p1 blood using a spot hybridization assay. The probe did not cross-react with parasite DNA of P. vivax, P. chabaudi or P. yoelii. or with human DNA. Out of eighteen P.falciparum-infected blood samples analyzed, one false negative result was obtained. This type of diagnostic test would be beneficial in a laboratory situated in a developed country where little skill in parasitology is available. The requirement for radioattive isotopes would make the method less practical for routine application in a developing country where visual microscopic examination of blood smears still remains the method of choice.

Detection of malaria infection in mosquito vector has traditionally depended upon manual dissection of individual mosquitoes and microscopic examination of the gut for parasite oocysts (indicating an infected mosquito) and of the salivary gland for parasite sporozoites (indicating an infective mosquito). The species of malaria parasite in the insect cannot be identified by morphological examination. To alleviate this tedious and labour-intensive method, Nussenzweig and co-workers (18) have introduced an immunoradiometric assay using monoclonal antibodies to the circumsporozoite protein antigens to detect P. falciparum in an individual or pools of mosquitoes.

Southern blot hybridization of the recombinant pBRK1-14 probe with DNA extracted from laboratory-infected Anopheles dirus mosquitoes also detected P. falciparum both in che gut and salivary gland regions (Fig. 5).

The pBRK1-14 probe did not cross hybridize with 100 ng DNA from P. vivax, P. chabaudi. Anopheles dirus and human white cells. A minimum of 0.5 ng of parasite DNA could be detected in the assay. The suitability of using recombinant DNA probes in epidemiological surveys will depend on answers concerning minimum number of oocysts or sporozoites capable of being detected in an infected mosquito, procedure for collecting and trasporting mosquitoes to the laboratory for DNA extraction, and development of nonradioactive detection techniques. A P. vivax-specific DNA probe should also be produced.

A great dea1 of effort in applying recombinant DNA techniques to malaria research has been directed towards identifying specific parasite genes coding for antigens that may constitute a malaria vaccine (19). Nevertheless, we have indicated that recombinant DNA techniques could also produce reagents that are potentially useful in providing basic information regarding the genetic diversity of malaria parasites in a given geographical location and their transmission in both man and insect. Such knowledge will be of primary impor- tante in determining the success of any malaria vaccination program.

Acknowledgements

Research in the authors' laboratory was supported by grants from the Rockefeller Foundation Great Neglected Diseases of Mankind Network and Mahidol University. The secretarial assifitance of Ms. Thitika Vajarodaya is greatly appreciated.

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