Evidence of genetic recombination in Leishmania

253

Molecular and Biochemical Parasitology, 46 (1991) 253-264 © 1991 Elsevier Science Publishers B.V. / 0166-6851/91/$03.50 ADONIS 016668519100160G MOLBIO 01530

Evidence of genetic recombination in Leishmania 1 *

John M. Kelly ~, Janette M. Law ~, Caroline J. C h a p m a n ' , Gulllaume J.J.M. Van Eys 2 and David A. Evans ~ ~Department of Medical Parasitology, London School of ttygiene and Tropical Medicine, London, U.K.: and 2Department ~[ Tropical Hygiene, Royal Tropical Institute, Amsterdam, The Netherlands (Received 7 November 1990; accepted 17 December 1990)

In the genus Leishmania there has been no convincing demonstration of genetic exchange, and it has been proposed that reproduction is clonal. However, preliminary characterisation of two strains of Leishmania isolated from wild animals in a zoonotic focus of cutaneous leisbmaniasis in the Eastern Province of Saudi Arabia, has suggested that they may represent hybrids of Leishmania major and Leishmania arabica. Evidence presented here strongly supports this hypothesis. Isoenzyme analysis and molecular karyotyping of cloned organisms indicated that the putative hybrids are distinct from other species of Leishmania, and possess characteristics of both L. major and L. arabica. Experiments using highly specific probes demonstrated that kinetoplast minicircle DNA from the putative hybrid contained L. major-specific, but not L. arabica-specific sequences. DNA fingerprinting data obtained using 6 genomic DNA probes were consistent in all cases with a L. majopJL, arabica recombinant genotype, and implied both diploidy and allelic segregation. These observations suggest that sexual reproduction may generate genetic diversity within natural Leishmania populations. Key words: Leishmania; Genetic exchange; DNA fingerprinting; PIoidy

Introduction Kinetoplastid protozoan parasites of the genus Leishmania are the causative agents of the various

human and animal leishmaniases [1]. Classification of organisms within this genus has traditionCorrespondence address: D.A. Evans, Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC I E 7HT, U.K. *Present address: Cell and Molecular Biology Department, Cancer Research Institute, Chester Beatty Laboratories, Fulham Road, London SW3, U.K. Abbreviations: kDNA, kinetoplast DNA; cDNA, complementary DNA; RFLP, restriction fragment length polymorphism; hsp70, the gene encoding the 70-kDa heat shock protein: CHEFE, clamped homogenous electric field electrophoresis; ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase: ES, esterase; GPI, glucose phosphate isomerase; MDH, malate dehydrogenase; MPI, mannose phosphate isomerase; NH, nucleoside hydrolase; PEP-D, peptidase-D; PGM, phospboglucomutase; 6PGD, 6-phosphogluconate dehydrogenase; SOD, superoxide dismutase.

ally been based on geographical and clinico-biological criteria. More precise taxonomic characterisation has resulted from the application of isoenzyme analysis [2-4], chromosome separation techniques [5-7], and the use of species-specific DNA probes [8-11] and monoclonal antibodies [ 12,13]. Despite these and other advances in Leishmania molecular genetics, genetic exchange has not been demonstrated in this genus. In addition the ploidy of Leishmania is uncertain, although it has been reported that some housekeeping genes are diploid [14,15]. Population genetics analysis has, on the other hand, suggested that in natural populations of Leishmania, reproduction is predominantly or entirely an asexual process [16]. Resolution of this question would represent a fundamental advance in our understanding of the biology of this medically important genus. Genetic exchange resulting in the formation of distinct hybrid organisms has been demonstrated in the laboratory for a related kinetoplastid flagellate Trypanosoma brucei [17-21]. Hybrid T. brucei

254 clones were isolated after mixed cyclical transmission of two parental clones through tsetse flies. An increased DNA content was noted in some but not all hybrids. The mechanism of hybrid formation has not been determined, but most data are consistent with a classical Mendelian mechanism, although it has been suggested that mating could involve fusion of parental nuclei followed by loss of DNA and return towards a diploid state [18]. The possibility that at least some hybrids arise from a mating process involving haploid gametes cannot be excluded [ 19,22]. The inheritance of kDNA minicircles appears to be from both parents [23] whereas that of maxicircle kDNA appears uniparental [20,211. Recently evidence consistent with genetic recombination within the genus Leishmania has come from the characterisation of two new strains isolated from a fat-tailed desert rat (Psammomys obesus) and a feral dog (Canisfamiliaris) captured in the A1-Ahsa oasis of the Eastern Province of Saudi Arabia [24[. In this region Leishmania major and Leishmania arabica exist sympatrically, with L. major causing human and animal cutaneous leishmaniasis and L. arabica infecting dogs and desert rats [25,26], A single sandfly species, Phlehotomus papatasi, has been implicated as the vector of both parasites [24]. Isoenzyme analysis of cloned organisms from the large number of isolates made in the Al-Ahsa oasis revealed two with banding patterns intermediate between L. major and L. arabica [24]. Consistent with this, analysis of chromosomal DNA separaled by orthogonal field alternation gel electrophoresis (OFAGE) revealed an intermediate pattern of hyhridisation to an undefined L. donovani genomic DNA fragment [24]. One interpretation of these data is that the new strains represent a hybrid forna between L. major and L. arabit'd. In order to extend these observations, genomic DNA from L. major, L. arabica and the putative hybrids has been analysed by molecular karyotyping and DNA fingerprinting, and the kDNA profiles examined using species-specific probes. The results of these and other experiments reported here provide further evidence of genetic exchange in Leishmania.

TABLE I L e i s h m a n i a strains

Organism

Code MRHO/SU/59/P L. mcuor MHOM/SU/73/5ASKH L. m a j o r MHOM/SN/00/DK1 L. m a l o r MHOM]IR/00/NIH173 L. m a j o r MPSM/TN/87/RON101 L. major MHOM/IL/83/LRC-L351 L. m~qor MPSM/IL/83/PSAM398 L. mcuor MHOM/SA/84/JISH118 L. mcuor MPSM/SA/84/JISH252 L. major MMER/IN/73/GTBM L. malor ISAL/IN/73/SDTBM L. r/Ill o r MHOM/SA/84/KFUH7352 L. nla or MHOM/IL/67/JER1CHO-II L. g e r b i l l i MRHO/CN/60/GERBILL| L. a r a b i c a MPSM/SA/83/JISH220 L. arabica MPSM/SA/83/JISH224 L. arabica MPSM/SA/84/JISH231 L. arabica MPSM/SA/84/JISH238 L. arabica MCAN/SA/84/MD94 'Hybrid' MCAN/SA/83/MD26 'Hybrid' MRHO/SA/84[JISH249 ~Clonedorganisms. L . 117(i o 1

Zymodeme LON 1" LON l LON 1 LON1 LON 1 LON2 LON3 LON4~ LON4* LON5 LON6 LON65 LON70 LON25 LON64* LON64* LON64 LON64 LON64 LON62~ LON62~

Materials and Methods Growth o f parasites. All the strains of Leishmania used in this study were isolated and cultured

as described previously [24-26]. A detailed list of these strains is given in Table I. Leishmanial promastigotes from culture were cloned by the hanging drop and capillary tube cultivation method [27], and except where stated otherwise, all experiments were carried out using cloned organisms. Theorganismsused in this study were characterised on the basis of isoenzyme patterns obtained with 1 1 enzymes following thin-layer starch gel electrophoresis [28], and dendrograms of cluster analyses of isoenzyme data were constructed using Jaccard's coefficient [3].

lsoenzyme characterisation.

Probes pBAT and pBT4 correspond to single copy genes located on a 1200kb chromosome of L. donovani [29]. The pBT4 gene encodes an antigen which is recognised by sera from the majority of visceral leishmaniasis patients. Probe pCTI is a complementary DNA (eDNA) clone which corresponds to the 3'-end of

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Fig. 1. Diagrammatic representation of the isoenzyme variation observed in 11 enzymes after thin-layer starch gel electrophoresis. Zymodemes LONI-6, LON65 and LON70 all L. major. LON62 the 2 'hybrid' strains, LON64 5 strains ofL. arabica and LON25 L. gerbilli.

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Fig. 2. Cluster analysis of Leishmania isoenzyme data (thinlayer starch gel electrophoresis): group average.

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the L. donovani 70-kDa heat shock protein gene (hsp70). In L. donovani, these genes are arranged as a series of direct tandem repeats on a 1000-kb chromosome [30]. Probe L-met9 corresponds to the Y-untranslated region of a L. donovani gene which is a member of a complex multigene family. The genes are restricted to a 700-kb chromosome, and this probe is specific to Old World species of Leishmania (M.K. Howard, personal communication). Probes pDK 10 and pDK20 correspond to fragments of the genome of L. major strain MHOM/SN/00/DK 1. The fragments were selected from a genomic DNA library by differential hybridisation. They hybridise to all 'Old World' species of Leishmania, but provide different patterns for the taxa after cleavage with most restriction enzymes. The clones have been described in more detail elsewhere [31 ].

256

kDNA probes. The kDNA probes were produced by a differential screening procedure (C.J.C., MS. in preparation). The L. major specific probe (CMSP-3) contains a 500 bp sequence derived from L. major (JISH252) minicircle kDNA. The L. arabica-specific probe (CASP-5) contains a 280-bp sequence ofL. arabica (JISH220) minicircle kDNA. Both probes represent multicopy sequences. Analysis of genomic DNA.

Leishmania promastigotes (1-5 × 109) were harvested, washed in phosphate-buffered saline (pH 7.2) and pelleted by centrifugation. The pellet was resuspended in 1-5 ml DNA extraction buffer (50 mM EDTA/1% sodium lauryl sarcosinate/50 mM Tris-HCl, pH 8.0); proteinase K (Sigma) was added to a final concentration of 100 ~tg ml ~ and the mixture incubated at 37°C for 3-24 h. After phenol/chloroform extraction (×3) the DNA was precipitated by the addition of two volumes of ethanol. Standard protocols were used for Southern analysis [32]. DNA was radiolabelled with [~-32p]dCTP using a random priming procedure (Amersham International). Blots were washed at 0.2 × SSC (0.3 M NaC1/0.03 M sodium

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The preparation of promastigotes for the fractionation of chromosome-sized parasite DNA was as described [6]. Clamped, homogenous electric field electrophoresis (CHEFE) [33] was performed using a BioRad CHEF DRII system. Electrophoresis was performed as described in the figure legends.

Isolation ofkDNA.

The method used was essentially that of Kennedy [34] with the following modifications. The cell pellet (10 ~° parasites) was washed twice in NET 100 (100 mM NaC1/100 mM EDTA/10 mM Tris-HC1, pH 8.0) and resuspended in a solution of 3% sodium sarcosine and 1 mg ml -~ proteinase K in NET at a cell concentration of 10 9 ml -~. This was passed through a 23G hypodermic needle and incubated at 37°C overnight. The DNA was then centrifuged and purified as described [34]. Results

The putative hybrid strains are different from other species of Leishmania. The isoenzyme patterns

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Fig. 3. Comparison of chromosome separation patterns obtained with organisms cloned from the 2 putative hybrid strains. Chromosome samples were obtained from clone MD26 ( 1) and JISH249 (2) and separatedon 1.2%agarosegels by CHEFE, using the followingswitching times: (A) 1 s:30 min; 70 s:18 h; 150 s:24 h and (B) 1 s:30 min; 40 s:42 h. Molecular sizes given in Mb.

obtained for 11 enzymes (ES, GPI, 6PGD, PGM, SOD, PEP-D, NH, ALAT, ASAT, MPI and MDH) with 8 zymodemes of L. major (LON1-6, 65 and 70), with 5 strains ofL. arabica (LON64), 2 strains of the putative hybrid (LON62) and 2 strains of Leishmania gerbilli (LON25) are shown in Fig. 1. Of the 8 known zymodemes of L. major, zymodeme LON4 predominates in Saudi Arabia. Over the past 8 years we have examined more than 300 isolates of Saudi Arabian L. major, and all but one (LON65) have isoenzyme typed as zymodeme LON4 [25], suggesting that reproduction is predominantly clonal. Similarly the 5 strains of L. arabica (LON64) were isoenzymically distinct from all other known Leishmania taxa, and indistinguishable from each other, as were the 2 putative hybrid strains and the 2 strains ofL. gerbilli (Fig. 1). With 4 enzymes (GPI, MPI, 6-PGD and ES) the putative 'hybrids' gave classical heterozygous isoenzyme banding patterns in contrast to the generally homozygous patterns given by L. major (LON3 heterozygous in 6PGD) and L. arabica (Fig. 1). The heterozygous isoenzyme patterns displayed by the

257 'hybrid' organisms for GPI and 6PGD were those expected with two codominant alleles and a dimeric enzyme, and in MPI and ES with two codominant alleles and a monomeric enzyme. The isoenzyme data were subjected to cluster analysis (Jaccard's coefficient) [3] and a similarity dendrogram plotted. The dendrogram (Fig. 2) shows the 8 zymodemes of L. major as closely similar and clearly distinct from L. arabica and L. gerbilli, with the putative hybrid organisms lying between the similarity indices of L. major and L. arabica, but slightly closer to the former. Although the two 'hybrid' clones were found to be phenotypically identical by isoenzyme analysis, this did not extend to their karyotype. Whereas the chromosomes greater than 500 kb appeared very similar or identical on CHEFE gels (Fig. 3), there was heterogeneity in both the size and number of the smaller chromosomes (300-450 kb). DNA fingerprinting data are consistent with a recombinant genotype. In the DNA fingerprinting experiments genomic DNA from L. major, L. arabica and the putative hybrid was analysed using 6 different DNA probes. The most striking feature of the results was the observation that bands present in the tracks containing DNA from the 'hybrid' were always present in either the L. major or L. arabica tracks or both. A hybrid organism and its parental strains would present a profile such as this. ProbepCT1. This probe corresponds to the hsp70 genes which in Leishmania species occur as a multigene family arranged largely as direct tandem repeats [30,35]. After digestion with BamHI (Fig. 4A), two predominant bands were identified in both the L. arabica (3.7 kb, 7.0 kb) and L. major (3.7 kb, 9.0 kb) lanes. These can be related to the three predominant bands observed in the MD26 ('hybrid') lane (3.7 kb, 7.0 kb and 9.0 kb). Other restriction enzymes gave similar patterns for L. major, L. arabica and the 'hybrid' strains (Fig. 4A). Given this heterozygous pattern and the observation that hsp70 genes are confined to a single locus [30], these data are consistent with this chromosome being diploid at least in the case of the putative hybrid. Probes pBT4 and pBAT. Similar interpretations can be drawn from the data obtained with these pro-

bes. For example, with pBT4, 2 predominant bands can be identified in the SacII lane of MD26 ('hybrid') DNA (5.4 kb, 7.0 kb) (Fig. 4B). These can be related to the bands present in the L. major (5.4 kb) and L. arabica (7.0 kb) lanes. With probe pBAT, 2 bands can be identified in the lane containing HindIII-digested MD26 DNA (6.0 kb, 10 kb) (Fig. 4C). This compares with single bands present in the L. major (6.0 kb) and L. arabica (10 kb) lanes. The pBT4 and pBAT genes are single copy and are genetically linked on a 1200-kb chromosome [29]. Heterozygosity at a single locus as observed here is evidence for both diploidy and a recombinant genotype. Probe L-met9. Using this probe a similar type of Southern hybridisation pattern was obtained to those described above, i.e., all bands present in the track containing DNA from cloned MD26 ('hybrid') can be related to bands in either L. major or L. arabica tracks (e.g., SacII digest, Fig. 5A). When the probe was hybridised with blots containing chromosome-sized molecules, predominant single bands were detected in the L. major (950 kb) and L. arabica (1100 kb) tracks (Fig. 5C) which corresponded with 2 bands present in the MD26 lane. This suggests that the 950-kb L. major and 1100- kb L. arabica chromosomes are homologues and that the MD26 'hybrid' organism has at least one copy of each (see also ref. 24). Similar observations with T. brucei laboratory-generated hybrids have been interpreted as implying diploidy [36]. Probes pDKIO and pDK20. The relationship of the 'hybrid' to other reference strains was investigated using probes pDK10 and pDK20. Southern analysis of the genomic DNA from the hybrid and reference strains using several restriction enzymes showed bands associated with L. major as well as L. arabica in the hybridisation pattern of strain MD26 (Fig. 6). Hybridisation patterns of PvuII digests with pDK10 showed 8.6 kb, 3.0 kb, 2.4 kb and a double band around 1.9 kb forL. major and a double band around 2.5 kb for L. arabica. The putative hybrid has all these bands. Hybridisation of HindIII digests with pDK20 gave 2 bands ( 12 kb and 5.0 kb) forL. major and 3 bands (12 kb, 6.6 kb and 4.5 k b ) for L. arabica. The 'hybrid' (MD26) displays a combination of the L. major and L. arabica patterns

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Fig. 6. Genetic fingerprint analysis. Genomic DNA from L. major strains RON101 (lane 1) and 5ASKH (lane 2), L. arabica strains JISH224 (lane 3) and JISH 238 (lane 4) and the putative hybrid MD26 (lane 5) were digested with Pvull (A) and HindIII (B) and probed with pDK10 (A) and pDK20 (B). Molecular sizes are given in kb. with bands at 12 kb, 6.6 kb, 5.0 kb and 4.5 kb in size. Taking into account that MD26 is a cloned strain, these observations are further evidence both of genetic exchange and the diploidy of Leishmania. Minicircle kDNA from ihe putative hybrid contains L. major-specific sequences. Minicircle kDNA species-specific probes were used as an additional approach to clarify the relationship of the putative hybrid to L. major and L. arabica. The L. arabica specific probe (CASP-5) was hybridised with a Southern blot containing restriction digested total DNA from the three organisms (Fig. 7A). Under the conditions used (see Fig. 7), there was no indication of hybridisation with kDNA from L. major or cloned MD26, even after long exposure. This experiment confirms the species specificity of CASP5 and demonstrates the non-relatedness of MD26 ('hybrid') and L. major kDNA to L. arabica kDNA, at least as judged by sequences present in this probe. kDNA isolated from L. arabica, MD26 and two strains of L. major (JISH252, NIH173) was analysed by Southern hybridisation using probe CMSP-5. This probe hybridises with kDNA from allL. major strains against which it has been tested,

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Fig. 7. Hybridisation with species-specifickDNA probes. (A) Southern blot of restriction-digestedtotal DNA from ( 1) L. arabica JISH224; (2) clone MD26; (3) L. majorJISH252 hybridised with L. arabica-specific kDNA probe CASP-5 (0.1 x SSC, 65°(7). H, HindIII; P, PstI; B, BamHI. (B, C, D) Southern blots of restriction-digested kDNA (B, Taql; C, HaeIII; D, HhaI) hybridised with L major-specific probe CMSP-5 (Methods). kDNA was isolated from (1) L. arabica MD94; (2) clone MD26; (3) L. major JISH252; (4) L. major NIH 173. Molecular sizes are given in kb. with the exception of 2 strains from the Soviet Union (P-strain and 5ASKH) (Fig. 7; C.J.C. unpublished data). A considerable variation in both the extent and pattern of hybridisation was observed amongst L. major strains, thus reflecting kDNA heterogeneity. The probe also hybridises with MD26 kDNA and to a much lesser extent with L. arabica kDNA (Fig. 7). It does not cross-react with kDNA from any other species of Leishmania. The pattern of hybridisation obtained is illustrated in Fig. 7. With TaqI (Fig. 7B)-digested kDNA, the probe hybridised to the same predominant band in MD26 and the 2 L. major strains. Several bands were detected with HaeIII-cut kDNA (Fig. 7C), some of which were common between the L. major strains and MD26, some of which were

261 species-specific. With HhaI cut kDNA (Fig. 7D), a predominant L. major-specific band (500 bp) was observed. In addition, an 800-bp band which was present in the MD26 lane (Fig. 7) was also detected in the L. major lanes. These data indicate that the MD26 'hybrid' clone contains minicircle kDNA sequences which are L. major-specific, but that the profile of hybridisation is not identical to that of kDNA from the L. major JISH252 or NIH 173 strains. Discussion The genus Leishmania has been considered to be predominantly asexual [16], but several pieces of evidence presented here, strongly support the hypothesis that a newly isolated Leishmania strain is a naturally occurring hybrid of L. major and L. arabica. Evidence of cross-species genetic exchange within natural populations would have medical and taxonomic implications, such as the potential for the transfer of drug resistance, changes in pathogenicity, and may indeed change our ideas of the' species concept' in the genus Leishmania. Several criteria can be applied to determine whether natural populations undergo genetic exchange. The new Leishmania strain conforms to these criteria.

Opportunity for interaction of parental strains. Close association is an obvious prerequisite for genetic exchange. The 'hybrid' strains described here were isolated in a small geographical area from animals which are hosts of both L. major and L. arabica [26]. A single sandfly species, P. papatasi is thought to act as the vector for both species. Evidence for a recombinant genotype. In most cases sexual reproduction will result in the inheritance of genetic characteristics by simple Mendelian rules to produce a recombinant genotype. The relationship between progeny and their putative parental strains can be tested by DNA fingerprinting. Using six DNA probes we have demonstrated that each RFLP identified in the 'hybrid' strains can be traced to either L. major or L. arabica. These data indicate that the 'hybrid' has a genotype expected of a L. major/L, arabica recombinant.

Independent transmission of mitochondrial and nuclear DNA sequences. In eukaryotes inheritance of mitochondrial DNA sequences is usually uniparental and independent of nuclear DNA. Analysis of kDNA minicircles from the 'hybrid' demonstrated its relatedness to L. major kDNA and non-relatedness to L. arabica kDNA, in contrast to other genotypic (RFLPs) and phenotypic (isoenzyme) markers which were intermediate between the two species. The extensive heterogeneity in the hybridisation pattern observed between L. major strains with probe CMSP-5 (C.J.C., unpublished) makes it difficult to identify a potential 'parental' strain. Conformity to these criteria is not proof that sexual reproduction occurs, since all evidence obtained from natural populations is by nature circumstantial. However, the data are difficult to correlate with an organism in which reproduction is completely asexual. On this basis we propose that although reproduction within Leishmania would appear to be predominantly clonal, sexual reproduction occurs at a low, but as yet undefined level. Other possible explanations for our observations could be that the MD26 and JISH249 strains represent a hitherto undescribed species which originated from a common ancestor of L. major and L. arabica and has retained some characteristics of both. The consistency of the DNA fingerprint experiments in which bands present in the MD26 track were always present in the L. major or L. arabica lanes (Figs. 4 ~ ) , suggests that the 'hybrid' hypothesis is the more likely. The data provide no insight into the sites or mechanisms that may be involved in genetic exchange, although it can be inferred that unlike the situation in T. brucei [23] inheritance of minicircle kDNA is uniparental. The isoenzymic identity of the two 'hybrid' strains suggests that they were derived from a single mating event which has been followed by clonal reproduction. Heterogeneity in the size of smaller chromosomes (Fig. 3) does not conflict with this hypothesis. Such polymorphisms have been observed to occur within clonal populations [37] and to occur spontaneously. It has been suggested from analysis of available isoenzymic data that genetic exchange in Leishmania does not occur or is a very infrequent event [16], although other potential instances may come

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to light given the increasing availability of genetic markers. This rarity may reflect that progeny from occasional sexual reproduction within clonally reproducing populations only become detectable when the hybrid phenotype confers a selective advantage. Although cell fusion between cultured promastigotes of Leishmania infantum and of Leishmania tropica has recently been observed [38], attempts to demonstrate genetic exchange in Leishmania under laboratory conditions have so far been unsuccessful. Application of genetic techniques so that hybrids can be isolated on the basis of their ability to express sets of acquired selectable drug markers [39,40] may be one approach to achieving this goal.

Acknowledgements. This work received financial support in the form of a grant from the United Kingdom Medical Research Council. Caroline Chapman was a Medical Research Council student. We thank Keith Howard for the use of the L-met9 probe, and Michael Miles and Martin Taylor for critical reading of the manuscript.

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