Genotypic polymorphisms in experimental metastatic dermal leishmaniasis

MOLECULAR

ELSEVIER

i&EMICfiL PARASITOLOGY

Molecular and Biochemical Parasitology 69 (1995) 197-209

Genotypic polymorphisms in experimental metastatic dermal leishmaniasis Raquel S. Pacheco a,*, Julia Elvira Martinez b, Liliana Valderrama Hooman Momen a, Nancy G. Saravia b a Department of Biochemistry and Molecular Biology, Institute Oswald0 Crux, FIOCRUZ, Au. Brasil4365, b Centro International

b,

Manguinhos

21045. Rio de

Janeiro, Brazil de Inuestigaciones Medicas, CIDEIM, Cali, Colombia

Received 6 July 1994; revised 14 November 1994; accepted 22 November 1994

Abstract Molecular karyotype and kDNA restriction analyses were utilized to examine the genetic heterogeneity and plasticity of the Leishmaniu (Viunniu) guyanensis strain WHI/BR/78/M5313, composed of metastatic and non-metastatic populations. Cloning revealed that the strain was constituted by multiple closely related populations that were distinguishable by restriction fragment polymorphisms in kDNA. Size polymorphisms in molecular karyotype were not detected. Passage of clones in hamsters and recovery of parasites from cutaneous metastatic lesions yielded evidence of further genetic heterogeneity among some of the progeny populations. Overall, six kDNA minicircle restriction patterns or schizodemes were observed among clones, subclones and progeny. Although the possibility that population heterogeneity was not resolved by cloning cannot be ruled out, subcloning and kDNA restriction analysis to determine whether the putative clones consisted of homogeneous populations showed the schizodeme of subclones of 3 out of 4 clones to be identical to the clone of origin, while a subclone of the fourth had a co-efficient of similarity of 0.95. Metastasis did not segregate with a particular schizodeme: all six restriction profiles were represented among populations isolated from metastatic lesions and some clones with the same restriction profile did not produce metastatic lesions. The strain from which the clones, subclones and progeny were derived had a kDNA restriction pattern identical to the most prevalent schizodeme (38%) among these subpopulations. This finding together with the reappearance of the repertoire of schizodemes found among clones in the populations recovered from metastatic lesions in hamsters inoculated with a single clone, suggest that sequence polymorphisms in kDNA can emerge during infection. Keywords:

Minicircle; Kinetoplast DNA; Schizodeme; Karyotype; Metastasis; Leishmania

1. Introduction

Abbreviations: kDNA, kinetoplast DNA; TAPE, transverse alternating field electrophoresis; RFLP, restriction fragment length polymorphism * Corresponding author.

Secondary

manifestations

tion are among

the most

disease

presentations

to manage,

epidemiologically [l-3]. and recurrent cutaneous

0166-6851/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166-6851(94)00212-6

of

destructive

Leishmania

infec-

and challenging both clinically

and

In the Americas, metastasis and mucosal leishmaniasis

198

R.S. Pacheco et al. /Molecular

and Biochemical Parasitology

are associated with Leishmania of the braziliensis complex, also known as the Viannia subgenus. Whether Leishmania involved in the development of Table 1 Results of schizodeme analyses of metastatic and non-metastatic progeny, isolated from metastatatic lesions in hamsters Clone code

Generation

a

_

Clone 1 Clone 5

First Clone 8 First Clone 9 First -

Clone 11

secondary disease are intrinsically distinguishable from other parasites of the same taxon is unknown. However, individual strains of L (v). panamensis

clones of L. guyanensis

Progeny analized/ no. hamsters _ 2/2 _ l/l 3/3 _ 412

First

positive positive

positive

136 6 6 2

positive

First -

g/3

2,326 1

positive

First Third

912 2/l 2/l _ 6/3 l/l 2/2 l/l _ l/l _

1,6 1 1 6 6 1 1 1 1 6 1

8/3 _

1,6,3 1 1 1 1 1 1 1.1 1 1 1 1 1 1

Clone 23 Clone 20

First _ First Subclone 20A Subclone 20D Subclone 20E

_

Clone 10 _ -

Clone 19 Clone 17

a Generation refers to a hamster passage. b Number of hamsters from which progeny ’ Production of cutaneous lesions.

positive

2/l _ 2/2 _

_

17A 17B 17C 17D 17E

positive

First

First Second Third Fourth _

Subclone Subclone Subclone Subclone Subclone

1 1 4 4 4 5 1 1

positive

Clone 22

19E

b

192 1

Clone 21

Subclone

Metastatic phenotype

6

First

10F

Schizodeme

and corresponding

312

Clone 16

Subclone

strain WHI/BR/78/M5313

First Clone 15

Clone 13

69 (199.5) 197-209

_ -

analyzed

derived.

positive

positive

positive positive

negative negative negative

’

R.S. Pacheco et al. /Molecular

and Biochemical Parasitology

and L (v). guyanensis differ in their propensity to disseminate and to produce metastatic disease in the hamster model [4]. In endemic areas where multiple Leishmania species and variants are concurrently transmitted, recurrent disease can be the outcome of either reinfection or relapse of a prior infection [5]. Distinction between relapse and reinfection can sometimes be achieved by the phenotypic and genotypic characterization of parasites isolated from primary and recurrent lesions [5]. Nevertheless, the polyclonality of some isolates [6], possibly as a result of a heterogeneous infecting inoculum, and/or the accumulation of infections, is a potential confounder in the interpretation of genotypic disparity between parasites isolated from primary and secondary lesions. Likewise, the endogenous generation of new polymorphisms, particularly in kinetoplast DNA minicircles, which are considered to evolve more rapidly than nuclear DNA [7,8], or the emergence of minor parasite subpopulations as a consequence of selection in vivo or propagation in vitro are difficult to ascertain in natural infections. We have exploited an experimental model of metastatic disease to determine whether genetically distinct populations emerge during infection and the development of secondary lesions. In this study we examined the genetic variability of a strain of L. (V) guyanensis that is highly metastatic in the hamster [4]. Molecular karyotype and restriction fragment length polymorphisms of kDNA were utilized to dissect the population structure of the strain. Clonal populations derived from this strain were characterized with respect to genetic polymorphisms during infection and the development of metastatic lesions in hamsters through multiple generations of passage in vivo. Both intuitive and numerical taxonomic methods were utilized to analyze genetic polymorphisms and to infer relationships among the clonal populations and related strains of the Viannia subgenus.

2. Materials

and methods

2.1. Isolates Fifteen clones, 10 subclones and 58 progeny populations from metastatic lesions produced by the

69 (1995) 197-209

highly metastatic L. 04 guyanensis WHI/BR/78/M5313 were analyzed. Tables 2 summarize, respectively, the characteristics different clones and progeny, and the isolates (v) guyanensis, L. (V) panamensis and WHO ence strains included in the analyses.

199

strain 1 and of the of L. refer-

2.2. Cloning Promastigotes of L. (V) guyanensis strain WHl/BR/78/M5313 were grown in Senekjie’s culture medium [9]. Parasites were harvested in stationary phase after eight days in culture [lo] and serially diluted to 1000 promastigotes ml-‘. One hundred microliter aliquots were spread on Senekjie’s blood agar medium in sterile plastic Petri dishes (100 X 15 mm). These were incubated at 27°C for seven days, by which time colonies of cells appeared in some of the dishes. The smallest colonies were aspirated using a Pasteur pipette and inoculated into tubes of Senekjie’s medium to which had been added 0.1 ml PBS, containing 1% (v/v) penicillin-streptomycin. Clones were subsequently propagated in diphasic culture and on the 6th day of growth the parasites were inoculated into Schneider’s Drosophila medium supplemented with heat-inactivated 10% fetal bovine serum, 1000 units ml-’ penicillin, 1000 units ml-’ streptomycin (Gibco, Grand Island, NY) and 1% glutamine (Gibco). Stationary phase promastigotes were inoculated into hamsters to evaluate metastatic behavior and to obtain progeny populations from metastatic lesions. The same culture utilized to prepare the inoculum for hamsters was cryopreserved in liquid nitrogen in order to conserve the clones at early passage following their derivation. 2.3. Infection of hamsters to assess metastatic behavior

A subline of hamsters from the Chester Beatty Golden strain (Charles River Laboratories) was used for these studies. Groups of 2-4 male hamsters per clone were inoculated subcutaneously in the snout at 5-6 weeks of age using 5 X lo6 stationary phase parasites in 0.1 ml PBS pH 7.2. Clinical examination of hamsters to detect cutaneous metastases was carried out beginning at two weeks post-inoculation. This evaluation consisted of examining the ears, fore

R.S. Pacheco et al. /Molecular

200

Table 2 List of Leishmania isolates and WHO reference

and Biochemical Parasitology

69 (1995) 197-209

strains included in this study

Code

Host

Place of isolation

Lesion

Identification

MS313 *

Lutzomyia whitmani Human

Brazil

-

Brazil

CL

Human

Panama

CL

Human

Brazil

CL

2025R

Human

Colombia

CL

2387R

Human

Colombia

CL

2238R

Human

Colombia

CL

2406R

Human

Colombia

CL

2509R

Human

Colombia

CL

2249

Human

Colombia

CL

2249R

Human

Colombia

CL

2249 cl.3

Human

Colombia

CL

2170

Human

Colombia

CL

2170R

Human

Colombia

CL

2485F

Human

Colombia

CL

2485 c1.F

Human

Colombia

CL

2485N

Human

Colombia

CL

2485R

Human

Colombia

CL

IM74

Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Human

Brazil

CL

L. guyanensis WHI/BR/78/M5313 L. guyanensis HOM/BR/75/M4147 L. panamensis HOM/PA/71/LS94 L. braziliensis HOM/BR/75/M2903 L. panamensis HOM/COL/84/2025R L. panamensis HOM/COL/84/2387R L. panamensis HOM/COL/84/2238R L. panamensis HOM/COL/84/2406R L. panamensis HOM/COL/84/2509R L. panamensis HOM/COL/84/2249 L. panamensis HOM/COL/84/2249R L. panamensis HOM/COL/84/2249~1.3 L. panamensis HOM/COL/84/2170 L. panamensis HOM/COL/84/2170R L. panamensis HOM/COL/84/2485F L. panamensis HOM/COL/84/2485cl.F L. panamensis HOM/COL/84/2485N L. panamensis HOM/COL/84/2485R L. guyanensis DID/BR/8O/IM74 L. guyanensis DID/BR/80/IM77 L. guyanensis DID/BR/80/IM242 L. guyanensis HOM/BR/80/IM276 L. guyanensis DID/BR/80/IM277 L. guyanensis HOM/BR/80/IM288 L. guyanensis CHO/BR/80/IM235 L. guyanensis CHO/BR/SO/IM371

M4147 Ls94 M2903

IM77 IM242 IM276 IM277 IM288 IM32.5 IM371

* * *

Brazil Brazil Brazil

CL

Didelphis marsupialis Human

Brazil

CL

Brazil

CL

Choloepus didacvlus Choloepus didactylus

Brazil Brazil

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Parasitology

201

69 (199.5) 197-209

Table 2 (continued) Code

Host

Place of isolation

Lesion

Identification

AHMS

Human

Brazil

CL

RC

Human

Brazil

CL

HJ9

Human

Brazil

CL

L. guyanensis HOM/BR/80/AHMS L. braziliensis HOM/BR/81/RC L. braziliensis HOM/BR/81/HJ9

* WHO reference

strain; R, recurrent

lesion; N, lesion on the neck; F, lesion on the foot; cl, clone; CL, cutaneous

and hind paws, ence of lesions inoculation site. tally confirmed

tail, skin and genitalia for the pressuspected to be metastases from the Only lesions that were parasitologiwere considered metastases.

2.4. Derivation

of progeny populations

Parasites isolated from selected cutaneous metastatic lesions were propagated for genotypic analyses and re-inoculation into two hamsters each. These progeny populations of individual clones were re-inoculated into hamsters and re-isolated from metastatic lesions for up to four sequential passages (generations l-4). 2.5. kDNA extraction enzymes

and digestion

with restriction

The technique of extraction and analysis of kDNA restriction profiles (schizodeme analysis) has been previously described [11,12]. The term schizodeme (schizo = division, deme = population) is used to define populations having similar kDNA minicircle restriction profiles [13]. Two micrograms of purified kDNA were digested with the restriction enzymes Rsal (GT/AC), HinfI (G/AnTC) and Mb01 (/GATC) in the appropriate buffers according to manufacturer’s instructions. Maxi and minicircle kDNA fragments were separated in 5-10% linear polyacrylamide gradient gels then silver stained. 2.6. Numerical

analysis

The profiles of kDNA minicircles obtained with the restriction enzyme RsaI, were compared using the Jaccard’s coefficient of similarity, to determine

leishmaniasis.

the proportion of mismatched bands between pairs of isolates. Each minicircle band (fragment) was coded with a number, starting with 1 for the largest fragment. The Similarity Matrix produced by comparison of all possible combinations between pairs of isolates was transformed into a dendrogram using the UPGMA algorithm [14]. The numerical analysis was performed using the NTSYS-pc software program (version 1.70, Exeter Software, 100 North Country Rd, Setauket, NY). 2.7. Sample preparation

for karyotype

analysis

Late logarithmic phase promastigote cultures were chilled and harvested by centrifugation at 4°C. The promastigotes were resuspended in fresh Schneider’s Drosophila medium with 10% fetal bovine serum (FCS) to 5 X lo8 ml-‘. The cells were then embedded in 1% low melting point agarose (Gibco, Grand Island, NY) in PBS equilibrated at 42°C. The cell/agarose mixture was immediately distributed into a chilled block former (at 4°C). After solidifying, the blocks were placed into 10 ml of lysis mixture (0.5 m EDTA pH 9.0; 1% N-lauryl sarcosine; 0.5 mg ml - ’ proteinase K), incubated at 51°C with gentle shaking for 48 h, then stored at 4°C until required. 2.8. Transverse alternating trophoresis (TAFE)

pulsed-field

elec-

Leishmania chromosomes were separated by transverse alternating field electrophoresis (TAFE) on a Gene Line II system (Beckman Instruments, Fullerton, CA). Lambda DNA concatemers and whole chromosomes from the yeast Saccharomyces

H.S. Pacheco et al. /Molecular

202

and Biochemical Parasitology

cereuisiae were used to estimate the size of chromosomes (Beckman Instruments). Gel preparation and electrophoresis were performed according to the protocols in the GeneLine II system Operating Instructions (Beckman Instruments). Two different conditions were utilized to obtain optimal separation of the maximum number of chromosomes. To separate smaller chromosomes, a TAFE electrophoresis program was run at 15°C and 350 milliamps for 12 h with a 30-s pulse time for the first stage, and for 24 h with a 60-s pulse time for the second stage. To resolve larger chromosomes, separation was performed’at 15°C and 350 mA for 36 h with a 75-s pulse time. Gels were stained in ethidium bromide (2.5 pg ml-‘), destained in distilled water and photographed under ultraviolet transillumination.

3. Results 3.1. Genotypic polymorphisms among clones Molecular chromosomal

karyotype analyses revealed identical banding patterns for all clones, which

69 (1995) 197-209

were also identical to that of the strain of origin (Fig. 1). In addition the molecular karyotypes of progeny populations of clones 13 and 22 were found to be indistinguishable. TAFE conditions that optimized the separation of large and smaller chromosomes, allowed the identification of sixteen chromosomes bands ranging in size from 300 to 1800 kilobases. The intensity of 3 bands suggested multiple chromosomes that were not separable under the conditions employed. Restriction analysis of kDNA using RsaI, HinfI and Mb01 yielded evidence for clonal heterogeneity based on polymorphisms within minicircles and maxicircles. Four different RsaI restriction profiles (schizodemes 1, 4, 6 and 2) were observed among the 15 clones analyzed, and two additional profiles (schizodemes 3 and 5) were detected among progeny isolated from metastatic lesions. The 6 patterns generated by RsaI were intuitively grouped as a spectrum ranging from least to most complex (Fig. 2). The original strain and the majority of clones (S/15) shared an intermediate profile, designated schizodeme 1. RsaI yielded more restriction fragments and polymorphisms than Hi&I or MboI. The

Size

Kb

460 370 290 245

Fig. 1. TAFE analyses of L. tv) guyanensis strain WHI/BR/78/M5313

and 12 clones showing identity of molecular

karyotypes.

R.S. Pacheco et al. /Molecular

and Biochemical Parasitology

restriction profiles obtained with HinfI and h4boI distinguished four of the six schizodemes revealed by RsuI restriction (data not shown). 3.2. Restriction

(schizodeme)

analysis of subclones

Four clones were subcloned in order to determine whether the cloning procedure had effectively yielded populations deriving from a single organism. Nine subclones obtained from 3 clones demonstrated RsaI restriction profiles of minicircles that were indistinguishable from the clone of origin. However, one of these, subclone 20D, presented a single additional restriction fragment in the maxicircles. A fourth clone, yielded a subclone 19E with an RsaI restric-

203

69 (1995) 197-209

tion pattern having an additional fragment in the minicircle profile, and a coefficient of similarity of 0.95 with the clone of origin based on numerical taxonomic methods (Fig. 3 and Table 1). This difference constituted a profile intermediate between schizodeme 1, 3 and 6.

3.3. Relation between expression and schizodeme

of metastatic

trait

Though all clones did not produce metastatic lesions, all schizodemes were represented among the 12 metastatic clones and their progeny (Table 1). Schizodemes 1 (5/12), 2 (l/12), 4 (2/12) and 6

Fig. 2. Electrophoresis in polyacrylamide gradient gel (5-10%) showing RsaI schizodeme analysis of clones and progeny of L. (VJ guyanensis WHI/BR/78/M5313 (silver staining). (A) Schizodemes 1, 2, 4 and 6 were found among original clones, schizodemes 3 and 5 were detected in metastatic lesions. (B) Enlarged photograph of kDNA minicircle regions showing different degrees of heterogeneity. Arrows indicate regions of polymorphism.

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69 (1995) 197-209

Fig. 3. RsaI schixodeme analysis of original clones and their subclones showing predominance of a genotypic pattern (schixodeme 1) and overall identity between subclones and clone of origin. M5313, uncloned parental strain. Subclone 19 E presents an additional restriction fragment in the minicircle (arrow) and subclone 20D an additional fragment in the maxicircle profile (arrow).

(4/12) were represented among the original metastatic clones analyzed in this study. Schizodeme 3 was detected among the progeny of clones 20 and 21, whereas schizodeme 5 was only detected in the progeny of clone 8. Moreover, progeny of clones 13 and 22 evaluated through four generations of passage in hamsters yielded schizodeme(s) distinct from that of the respective originating clone, yet metastatic behavior was stable.

vealed the emergence of schizodemes distinct from that of the original inoculum at different generations. Clone 13 (schizodeme 1) yielded 1st generation progeny of schizodeme 1 and 6 (Fig. 4A); progeny analyzed from subsequent generations all pertained to schizodeme 1. Clone 22 (schizodeme 6) consistently yielded progeny of the initial schizodeme 6 in the first generation supporting the clonal origin of the inoculum (Fig. 4B) yet schizodeme 1 emerged in the second through fourth generations.

3.4. Genotypic analyses of progeny of selected clones Progeny of eleven clones were analyzed by RsaI restriction. Progeny of seven clones presented schizodemes different from the originating clone (Table 1). Results obtained with progeny populations of clones 13 and 22 isolated from metastatic lesions over four generations of passage in hamsters re-

3.5. Comparison of genotypic polymorphisms at the intraspecific and interspecific levels with that of clones Results obtained in prior studies utilizing both schizodeme analysis [12] and molecular karyotype

R.S. Pacheco et al./Molecular

and Biochemical Parasitology

[5,15] have revealed polymorphisms among isolates of L. (I4 guyanensis and between these and other species of the Viannia subgenus. Application of numerical analysis to the data obtained by restriction analysis of kDNA indicated a higher degree of similarity among the clones of L. (v) guyanensis strain M5313 than among isolates of the same species or among species of the same subgenus (Fig. 5). However, strains of each species did not necessarily cluster together based upon RsaI restriction polymorphisms. The identical molecular karyotype of clones of the metastatic strain under study also contrasted with the karyotype heterogeneity observed among strains of L. 04. panamensis [15j.

69 (1995) 197-209

4. Discussion In this study we have demonstrated genotypic heterogeneity in the mitochondrial DNA from clones and progeny of a strain of L. (V) guyanensis (WHI/BR/78/M5313), which has been shown to be composed of metastatic and non-metastatic populations (Martinez et al., data not shown). Although chromosome size polymorphisms were not detected in the molecular karyotype of clones or progeny, genomic changes not discernable as gross modifications in the molecular karyotype, may be revealed by hybridization [15] and/or restriction analyses of individual chromosomes. Restriction enzyme analyses

12

123456

205

1234

34

667 B

Fig. 4. kDNA restriction profiles observed after RsaI digestion. (A) Metastatic clone 13 and first generation progeny showing the presence of two genotypic profiles. Lanes 1-3, schizodeme 1; lanes 4-6, schizodeme 6. (B) First generation progeny of metastatic clone 22 showing the same schizodeme (schizodeme 6) as the progenitor clone (lane 1). (C) Second (lane 11, third (lanes 2 and 3) and fourth (lane 4) generation progenies of metastatic clone 22 showing the emergence of schizodeme 1.

R.S. Pacheco et al. /Molecular

206

and Biochemical Parasitology 69 (1995) 197-209

of kDNA minicircles yielded six different profiles (schizodemes) among the cloned, subcloned and progeny populations originating from this strain. Restriction polymorphisms were found predominantly in the minicircles of the LDNA, and to a lesser extent in the maxicircles. Whether the observed genotypic polymorphisms reflect genetic plasticity or indicate that some of the putative clones originated from more than one promastigote cannot be unequivocally ascertained from our data. Nevertheless, the clonal origin of the populations was supported by the results of kDNA restriction analysis of subclones. Likewise, the fact that the restriction profile of the uncloned parental strain was less complex than 3 of the 5 other profiles evident among clones and progeny, indicates that the strain profile could not have been generated by a compos-

L-. 0.2

0.4

0.6

ite of the constituent clones. The reproducibility of these six profiles allows us to affirm that the variations were not due to the amount of kDNA loaded on the gel, partial digestion or artifacts. The endogenous generation of polymorphisms in kDNA sequences should also be considered as a possible basis of the observed genotypic variations. Progeny of 7/11 clones passaged in hamster displayed kDNA restriction patterns that diverged from that of the clone of origin (Table 1). Pertinent in this regard is the finding that multiple subclones of clone 20 (schizodeme 1) all yielded minicircle restriction profiles identical to that of the clone, yet two other schizodemes, 3 and 6, besides schizodeme 1, were found among the first generation progeny of this clone (Table 1). The restriction polymorphism generating an additional band in the maxicircles of sub-

0.8

I.0 51 s2 s3 56 $5 s4

A -

202% 2387R 2406G 2238R 2509R 2170R

1

~

~

::z, B 2485N 2485R 248% 2249 2249~1 2249R Itl242 111276 i;::; c 11177 Ill277 AHtlS

Ii-l288 -

RC HJ9 +

2170 M2903 M4147 11174

Fig. 5. Phenetic analysis of RsaI restriction data from clones of L. (ti guyanemis M5313 and different isolates of Leishmania of the Viannia subgenus. The similarity matrix transformed into a dendogram shows groups separated by different levels of Jaccard’s coefficient of similarity (Sj). (A) Clones of L. (V) guyanensis M5313 (schizodemes l-6). (B) Different isolates of L. (v) panamensis from recurrent human lesions. (C) Different isolates of L. (V) guyanensis from human and reservoir hosts.

R.S. Pacheco et al./Molecular

and Biochemical Parasitology 69 (1995) 197-209

clone 20D was not found in schizodemes 3 or 6 (or any other schizodeme) and would not therefore account for the appearance of these schizodemes in progeny populations of clone 20. Further supporting the possibility of the endogenous generation of kDNA polymorphisms, re-cloning of clone 22 by flow cytometry showed 10 randomly selected subclones to have a kDNA restriction pattern identical to the source clone, schizodeme 6. Sequence divergence in minicircles does not seem to be random [16-181. In this study, most minicircle classes within each population shared the same restriction sites for RsaI, and Hinfl and M601, i.e., a common basic restriction profile was evident. Comparison of the least complex restriction profile (schizodeme 4) consisting of 19 minicircle fragments, with increasingly complex restriction profiles revealed that each ‘new’ fragment was also present in the next most complex profile. Interestingly, the subclone 19E of clone 19 (schizodeme 1) displayed one additional restriction fragment also present in schizodemes 3 and 6, yet lacked a fragment found in each of these two schizodemes. This subclone may therefore represent a genotype in transition to the more complex schizodemes. The finding that the strain from which the clones were obtained, as well as most (8/15) of the clones and progeny analyzed, pertain to schizodeme 1 whose profile is qualitatively and quantitatively intermediate between the least and most complex schizodemes, suggest that either this is the dominant population and/or this schizodeme may represent the most stable conformation of minicircle classes. Whether selection influenced the frequency distribution of particular schizodemes among the clones obtained in culture, or the emergence of populations with different restriction profiles during sequential passage in hamsters, cannot be determined from our observations. Although progeny were isolated and reinoculated into naive hamsters based upon the development of metastatic lesions, metastatic behavior did not segregate with any particular schizodeme. Furthermore, schizodemes 1 and 6 were most frequently represented among clones obtained by plating on blood agar as well as by re-isolation from metastatic lesions during in vivo passage. Therefore the presence of a particular schizodeme among the clonal and progeny populations may merely reflect

207

growth characteristics or numerical predominance within a heterogeneous population. Sequence rearrangements or recombinatorial events such as those described by Rogers and Wirth [19,20] represent another plausible mechanism for the generation of genotypic heterogeneity observed in these studies. However, the finding that schizodemes of the progeny of individual clones were sometimes distinct from the clone of origin but indistinguishable from the schizodeme of other clones from the same strain, implies a mechanism that reproducibly generates a particular repertoire of heterogeneity. Evidence of sequence differences in minicircles was not detectable using minicircle sequences as probes (not shown). The extensive and uniform pattern of hybridization with kDNA from these discrete populations contrasts with the phenomenon of transkinetoplastidy [21]. The sequence changes observed under the presumably non-selective conditions of the current study and those resulting from the highly selective pressure of drugs such as arsenite and tunicamycin [21,22] could reflect the same phenomenon, which is driven to extreme expression by selection. The common basic restriction profile, and the repetitive appearance of a limited number of schizodemes are consistent with expansion and contraction of discrete minicircle populations as a possible explanation for the apparent fluctuations in population structure observed during the passage of metastatic L. (v) guyanensis clones in vivo. The notion that expansion and contraction of minicircle populations might account for the observed polymorphisms, presumes that all of the polymorphic minicircle sequences are present in the original strain, either within subpopulations of parasites or subpopulations of minicircles present at variable frequency, in each of the constituent organisms. Since schizodeme analysis can detect parasite subpopulations that constitute 1% or more of the total population 1231, polymorphisms not evident in the schizodeme of the progenitor strain but observed in the restriction profiles of clones and/or progeny should either have been present in less than 1% of the parasite population or represent a very minor subpopulation of minicircles. Restriction analysis of kDNA by itself, does not always discriminate between strains of the same and

208

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and Biochemical

different species of the Viannia subgenus. This fact and the finding of polymorphisms among the progeny of clones passaged in hamsters suggest that the detection of distinct kDNA restriction profiles in Leishmania isolated from primary and recurrent lesions in the same individual, in the absence of other genotypic or phenotypic evidence, may not distinguish reactivation from reinfection. On the other hand, identical kDNA restriction profiles in isolates from primary and recurrent lesions provides virtual proof of relapse. Molecular karyotype was found to be highly stable in the same cloned populations passaged in hamsters. These observations confirm the utility of molecular karyotype in distinguishing relapse and reinfection, and schizodeme analysis in establishing relapse as a mechanism of recurrent leishmaniasis. The biological significance of genotypic polymorphisms in minicircle kDNA remains an enigma. However, the maintenance of minicircle heterogeneity may offer a selective advantage [24], and has been suggested as a source of genetic information for RNA editing [2.5-271. Minicircles of L. tarentolae and Trypanosoma brucei encode guide RNAs for RNA editing [28,29]. RNA editing is extensive in T. brucei [30] which is known to exhibit great minicircle heterogeneity. Hence it is conceivable that the observed microheterogeneity in clones and progeny of L. guyanensis strain 5313 is related to genetic information involved in or resulting from RNA editing. The results of these studies provide evidence that polymorphisms detectable by restriction analyses of kDNA, especially in minicircle sequences, can emerge during infection with putative clonal populations. The mechanisms that generate these polymorphisms, the participation of selection in the transition from one restriction profile to another and whether the transition is linked to RNA editing or to a change in virulence will require further investigation.

Acknowledgements This work received financial support from United States National Institute of Health (NIH grant No. I P50 AI30603) and UNDP/World Bank/WHO Special Program for Research and Training in Tropical

Parasitology

69 (1995) 197-209

Diseases. The study was conducted while R.S.P. was in receipt of a WHO Research Training Grant (ID 910767). We also wish to thank Dr. Bart Vanderborght from Innogenetics, Belgium, Dr. Barbara Herwaldt of the U.S. Centers for Disease Prevention and Control and Dr. Sylvie LeBlancq, Columbia University for the critical review of the manuscript and Marize Q. Pires for technical assistance. DNA probes were provided by Drs. Wim Degrave and Octavia Femandes, Instituto Oswald0 Cruz, FIOCRUZ. Subcloning of clone 22 by flow cytometry was performed by Dr. David Leiby, Transmissible Diseases Department, American Red Cross.

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