Molecular and Biochemical Parasitology, 60 (1993) 19-26 © 1993 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/93/$06.00

MOLBIO 01978

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Genetic exchange in Trypanosoma brucei: Selection of hybrid trypanosomes by introduction of genes conferring drug resistance

Wendy Gibson and Hayley Whi t t ing ton Department of Pathology and Microbiology, University of Bristol Veterinary School, Langford, Bristol, UK

(Received 17 December 1992; accepted 5 March 1993)

Genetic exchange in Trypanosoma brucei ssp. can occur when 2 different strains are cotransmitted through the tsetse fly vector. We have introduced heterologous genes for drug resistance (neo or hph) into parental trypanosome lines by electroporation. Drug resistant lines were then crossed in vivo in the fly or in vitro. Hybrids were subsequently selected by double drug resistance. Analysis of trypanosomes from both fly midguts and salivary glands showed the latter to be the probable site of genetic exchange. This is one of the first applications of reverse genetics to a longstanding problem in parasite biology.

Key words: Trypanosome; Trypanosoma brucei; Genetic exchange; Transformation

Introduction

Jenni and colleagues first demonstrated that genetic exchange could take place between Trypanosoma brucei ssp. trypanosomes co- transmitted through tsetse flies (genus Glossi- ha) in the laboratory [1]. Since then several other experimental crosses have been reported [2--4]. Sex is a comparatively rare event and is not an obligatory part of the trypanosome lifecycle, rendering analysis difficult. The de- tails of genetic exchange remain to be elucidated. Although the genotypes of the hybrid progeny are for the most part consis- tent with Mendelian segregation and reassort- ment of parental alleles [1-5], no haploid gametic stage has been substantiated [6,7].

Correspondence address: Wendy Gibson, Department of Pathology and Microbiology, University of Bristol Veterinary School, Langford, Bristol BSI8 7DU, UK.

Abbreviations: SDS, sodium dodecyl sulphate; VSG, variant surface glycoprotein; PFGE, pulsed field gel electrophoresis; neo, neomycin phosphotransferase gene; hph, hygromycin phosphotransferase gene; Hy R, hygromycin B resistant; Gt R, Geneticin (G418) resistant.

Nor is it clear where genetic exchange takes place in the fly and at what lifecycle stage. Two recent reports suggest that hybrid formation occurs at the procyclic stage, either in the tsetse midgut or in vitro [8,9].

Here we have taken advantage of the recent development of methods for the stable trans- formation of trypanosomes by electroporation [10-12] to introduce selectable markers for drug resistance into parental trypanosome lines, so that hybrids can be selected subse- quently by double drug resistance.

Materials and Methods

Trypanosomes. The 2 parental trypanosome clones used were T. b. rhodesiense 058 (MHOM/ZM/74/58 [CLONE B]) [2] and T. b. brucei KP2 (GPAP/CI/82/KP2-I [CLONE 23]) [13]. Procyclic trypanosomes were cultured in Cunningham's medium [14] with 20 mM Hepes and 20 mg ml-1 gentamycin (CM). The plasmid constructs used for transformation were both targetted to integrate into the tubulin locus by homologous recombination.

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The neo construct (pUCTbneo 3) was kindly supplied by Ten Asbroek, Ouellette and Borst [10] and the hph construct (pBnsp-H-T) by Lee and Van der Ploeg [15]. The constructs were linearised and introduced into the procyclics by electroporation (3 pulses of 250 #F at 300 V) as described previously [16]. Transformants were selected using G418 (Geneticin, Gibco) for the neo construct or hygromycin for the hph construct at 50 #g ml-1 final concentration.

These uncloned populations were then cultured in the absence of drug, before equal volumes of culture and horse blood with glucosamine (0.06 M final concentration) [17] were fed via a silicone membrane to groups of 50 teneral male tsetse flies (Glossina morsitans). Flies were maintained thereafter on mem- brane-fed sterile horse blood and dissected 5 weeks later to obtain infected salivary glands, which were then inoculated ip into mice immunosuppressed with cyclophosphamide (200 mg kg -1) to obtain bloodstream form populations. These were then cloned by the hanging drop method to yield parental clones 058H and KP2N. Fig. 1 shows the derivation of these 2 clones in a flow diagram.

In vivo mixing experiment. Fig. 2 is a flow diagram outlining this experiment. 150 teneral male tsetse flies were membrane fed with horse blood supplemented with 0.06 M glucosamine [17] containing approximately equal numbers of bloodstream form 058H and KP2N trypa- nosomes. Flies were maintained thereafter on membrane-fed sterile horse blood. Groups of flies were dissected from 2-9 weeks after the infective feed and the midguts and/or salivary glands examined by microscopy for the presence of trypanosomes.

Midgut procyclics (C populations) were put into CM with 100 mg ml-1 gentamycin, which was reduced to 20 mg m1-1 in subsequent passages. Metacyclics from tsetse salivary glands were amplified in Ross medium (RM) [18,19] for 2-5 days (R populations), before cryopreservation and inoculation into mice as above. Bloodstream form trypanosomes (B populations) were harvested from mouse blood at the first parasitaemic peak by anion

exchange chromatography [20]. At the same time, approximately 10 7 trypanosomes were inoculated into CM to differentiate into procyclics (P populations).

Bloodstream form trypanosomes were cloned by the hanging drop method. Procyclic trypanosomes were cloned by diluting drops containing single trypanosomes, as ascertained by microscopy, with 100 /A of CM and incubating in an atmosphere of 5% CO2 for 7 days (Dukes, P., unpublished observation).

In vitro mixing experiment. Two batches of 30 flies were fed separately with cloned blood- stream form trypanosomes of each transfor- mant, 058H and KP2N, as above. Flies were dissected after 2 weeks and infected midguts cultured on in CM as above. 2.5 x 107 trypanosomes of each parent were mixed in vitro and co-cultured for 28 days, keeping the cell density to approximately 10 7 ml- 1. Half to two-thirds of the culture was removed every 2 days and drug tested as below. An aliquot was also tested for parental drug phenotypes weekly.

Drug tests. Proc)~clics were tested for resis- tance to G418 (Gt '~) and/or hygromycin (Hy R) by adding the drugs at 50 #g ml-1 to 1 ml cultures in a 24-well tissue culture plate. Plates were generally examined every 1 or 2 days for 2 weeks and individual wells passaged as necessary.

Preparation and analysis of trypano- somes. Samples for pulsed field gradient gel electrophoresis (PFGE) were made by lysing and deproteinising trypanosomes in situ in agarose blocks as described [21]. Samples of DNA for restriction analysis were prepared by standard methods [22].

Genomic DNAs were restricted and size- fractionated using 0.7% agarose gels unless otherwise stated in figure legends. PFGE [23] was carried out using an LKB Pulsaphor system with a hexagonal electrode array and 5 phase program [3]. Gels were blotted in the standard way [24], with a preliminary acid wash (0.25 M HC1, 30 min). Nitrocellulose

058 clone B [ J KP2 clone 23 I procyclics [ [proeyellcs [

transform with transform with Hyg construct Neo construct

; 1 select with select with 50ug/ml 50ug/ml hygromycin Gensticin

1 1 transmit through transmit through

tsetse flies tsetse files

; 1 clone bsf clone bsf

Fig. 1. Flow diagram of the derivation of parental clones 058H and KP2N from wild-type procyclics.

filters were hybridised as previously described [25] with the following oligo-labelled DNA probes: (1) neo gene amplified from plasmid pg3neo by PCR; (2) hph gene HindlII-ScaI fragment from plasmid pBABE; (3) fl-tubulin, insert from cDNA clone pTbflT-C2 [26]; (4) VSGll7, 1.5-kb PstI fragment from cDNA

I cotrensmlssion through tsetse flies ]

C- midgut procyclics [ [ R - salivary ,lend metacyclics [

* 1 drag test [ 8 - bloodstream forms ~ - karyotype

l L ~ clones

[ p- procyc.cs ] L-,.-clones

drug test

Fig. 2. Flow diagram of the in vivo cross. Procyclic and salivary gland trypanosomes were isolated from each individual fly. Metacyclics were allowed to differentiate into bloodstream forms and subsequently procyclics for analysis of karyotype and drug resistance phenotype

respectively.

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clone TcV-117.5 [27]; (5) VSGll8, 1.2-kb EcoRI-PstI fragment from clone TcV-clI4 [27]; (6) total kinetoplast DNA from T. b. brucei TSW196 [2]. All post-hybridisational washes were to 0.1 x SSC (standard saline citrate) at 65°C, unless otherwise stated in figure legends.

Results

Transformants. Procyclic forms of the 2 parental trypanosomes were transformed with either the neo or hph genes as shown in the scheme (Fig. 1). Cloned transformants 058H and KP2N were analysed for the presence of the appropriate drug resistance gene in the tubulin locus by hybridisation of genomic digests (not shown) or size-fractionated chro- mosomal DNAs (Fig. 3). Each marker was carried on one of the respective parental tubulin homologues. The hph gene in 058H was on the smaller of the 2 chromosomal bands hybridising with tubulin (compare lanes 1 and 2 in Figs. 3B and D). The neo gene in KP2N was on the larger of the 2 tubulin chromosomal DNAs (compare lanes 7 and 8 in Figs. 3C and D); this chromosomal DNA remains largely in the slot, with some migra- tion of material to the compression area of the gel depending on conditions of the particular PFG run.

In vivo cross. Flies were infected with a mixture of 058H and KP2N bloodstream forms (see Fig. 2) and over the next 9 weeks, groups of flies were dissected at intervals to obtain procyclic (C) populations from the midgut and metacyclic (R) populations from the salivary glands. The overall trypanosome infection rates in this experiment were 101 of 121 (=83%) midguts and 37 of 88 (=42%) salivary glands (Table I).

Most C populations from flies with infected salivary glands were drug tested, plus addi- tional C populations from non-mature infec- tions at 2 and 4 weeks. None of the C populations contained double resistant HyR/ Gt ~ trypanosomes, although both Hy rt and

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TABLE I

Karyotypes and phenotypes of fly-transmitted trypanosome populations

Week No. flies Infections C populations B populations P populations dissected (Drug test) (Karyotype) (Drug test)

Gut SG H N H + N DD O58H KP2N Mix H N H + N DD

2 16 12 ND 0 0 10 0 ND ND 4 17 16 ND 0 0 10 0 ND ND 6 25 21 9 0 0 9 0 8 0 0 3 0 0 0 7 25 19 5 0 0 6 0 5 0 0 5 0 0 0 8 26 21 14 0 0 16 0 6 3 3 6 1 4 3 9 12 12 9 2 0 8 0 5 0 2 5 0 1 2

Totals 121 101 37 2 0 59 0 24 3 5 19 1 5 5

ND, Not done; SG, salivary gland. Drug test results: H, hygromycin-resistant only; N, G418-resistant only; H + N, mixture of hygromycin- and G418-resistant trypanosomes; DD, double drug-resistant trypanosomes.

Gt R trypanosomes, i.e., parents 058H and KP2N, were usually demonstrable by their differential response to the 2 drugs (Table I).

Karyotype analysis of the B populations derived from metacyclics (R populations) showed only 058H in salivary glands from 6 and 7 week flies, but also parent KP2N and mixtures coming through from week 8 on- wards (Table I). The drug resistance phenotype was tested on procyclics (P populations), because of the difficulty of reliably culturing bloodstream forms in vitro. Most bloodstream form samples differentiated rapidly into pro- cyclics, allowing drug tests to be set up after 5- 8 days. In agreement with the karyotype results, drug tests of the P populations showed only Hy R trypanosomes (ie. 058H) in the week 6 and 7 dissected flies, but also mixtures and HyR/Gt R trypanosomes from the week 8 and 9 dissected flies (Table I).

KP2N seems to have lagged behind 058 in invading the salivary glands in this experiment, only appearing at week 8. However, when transmitted alone, nearly 20% of KP2N midguts matured to salivary gland infections after 5 weeks. Table II shows a comparison of karyotype and drug test results for trypano- somes from the week 8 and 9 flies. It can be seen that karyotype is not a good guide to the presence of hybrids. Overall, 10 flies had mixed infections in the salivary glands, but only 5 produced hybrids. Thus, simple co-cultivation of metacyclics or bloodstream forms did not

result in production of hybrids. Trypanosomes were cloned from 2 flies (8/4,

9/6) with double drug resistant trypanosomes, both from selected and unselected populations to obtain a variety of hybrid genotypes. Again, drug resistance phenotype was examined in procyclic forms. Of 12 clones, 7 were HyR/Gt R, while the rest were Hy R only. Hybridisation of PFG gel blots with probes for the hph and neo genes showed both genes to be present in the HyR/Gt R clones and comigrating with the tubulin locus. Fig. 3B and C shows the results obtained for 4 of the hybrid clones. Hybrid clones 5 and 6 were HyR/Gt R and hybridise with both hph and neo probes, while clones 3 and 4 (Hy R) hybridise with hph only. Clones 3- 5 appear to have more than 2 tubulin chromosomes, since there is strong hybridisa- tion to the slot and 2 resolved bands (Fig. 3D).

Some clones were obviously hybrid as judged by karyotype; for example, clones 9/ 6P-1 and 8/4B-2 (lanes 3 and 4 respectively in Fig. 3A), which have a combination of parental chromosomal DNAs in the 1 Mb and 0.5 Mb areas of the gel. However, the 2 HyR/Gt R clones in lanes 5 and 6 have karyotypes resembling that of parent 058H. All clones were judged to be hybrid by analysis of 2 variant surface glycoprotein (VSG) gene families, since the DNA band fingerprints of the clones differed from those of the parents (Fig. 4). The gene rearrangements associated with antigenic variation do not in practice

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1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

Mb

1.1-

1 .0 -

0.88-

0 .25 -

me!

A

1 2 3 4 5 6 7 8 1 2 3

B: Hyg

4 5 6 7 8

C: Neo D: b-Tubulin

Fig. 3. Analysis of transformants 058H and KP2N and hybrid clones. Panel A shows ethidium-stained chromosomal DNAs after separation by FFG gel electrophoresis. Lanes: (1) 058 wild-type; (2) 058H; (3-6) hybrid clones; (7) KP2N; (8) KP2 wild- type. The gel was blotted and each half hybridised with a probe specific for either the hph (B) or neo (C) gene; the blot was subsequently hybridised with a fl-tubulin probe (D - same blot as B), after removal of previous hybridisation by alkali

treatment.

confound this kind of analysis, presumably because most of the genes are silent. Analysis of the inheritance of kinetoplast D N A maxi- circles (equivalent to the mitochondrial D N A of other eukaryotes) by restriction site poly- morphisms (not shown), showed the clones from fly 8/4 to have KP2N type maxi-circles, although these clones mostly resembled 058H by P F G E analysis. The fly 9/6 clones had 058H type maxi-circles.

In vitro mixing experiment. Schweizer et al.

suggested that genetic exchange takes place at the procyclic stage [8,9], because of the appearance of a characteristic heterozygotic isoenzyme band when homozygous procyclics were mixed in vitro, although the hybrids could not be cloned. Following similar meth- odology [9], an in vitro mixing experiment was set up using newly differentiated procyclics. Two procyclic populations were mixed in vitro and co-cultivated for 28 days. Although both parental transfectants could be detected throughout by differential drug resistance, no

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TABLE II

Week/Fly B population P population No. (Karyotype) (Drug test)

8/1 ND H 8/2 O58H H 8/3 KP2N N 8/4 O58H Double resistant 8/5 ND H < N 8/6 Mix Double resistant 8/8 Mix H < N 8/9 KP2N H < < N 8/10 O58H H 8/I 1 O58H H 8/12 Mix Double resistant 8/13 O58H H 8/14 O58H H 8/15 KP2N H < < N

[8,9], we found no evidence of hybrid forma- tion among midgut procyclics either in vivo or in vitro. There are 2 further pointers to the salivary glands as the site of genetic exchange. Firstly, hybrids only appeared in the salivary glands after week 8 when both parents had arrived, although both parents had coexisted in the fly midguts for several weeks. Secondly, as in our previous crosses [2,3], more than one hybrid genotype was present in the salivary glands. It is difficult to believe that more than one hybrid could have made the haphazard and tortuous journey to the salivary glands, unless the midgut contained many. Here, not

Hl/brld c l o n e s

058 058 1 2 3 4 5 6 7 8 9 10 11 12 KP2 9/1 O58H Double resistant 9/2 O58H H 9/3 ND H 9/4 O58H H kb

9/6 Mix Double resistant 9/7 O58H H 3.0. 9/8 O58H H 2.0-

2 . 1 - 9/9 Mix H > N

1 . 7 -

N, G418-resistant; H, hygromycin-resistant; H < N , fewer 1.2. 1 .1 -

hygromycin- than G418-resistant trypanosomes. 0 . 0 .

A: VSG 117 double drug resistant trypanosomes were evident.

To check the ability of low numbers of hybrids to grow through a parental mixture, a tenfold dilution series of procyclics (0.1-1000) of a HyR/Gt R hybrid clone were seeded into a background of 106 058H and 10 6 KP2N trypanosomes in 4 replicate 1-ml wells. 50 #g ml-I of each drug was added and the plate monitored for 2 weeks. All cultures originally containing 10 or more HyR/Gt R trypanosomes grew, as did 1 of 4 cultures seeded with 1 HyR/ Gt R trypanosome; all other cultures died. Less than 10 double drug resistant trypanosomes would therefore have been detectable in our standard drug test.

Discussion

We have successfully used selectable mar- kers to identify trypanosome hybrids after fly transmission. Unlike Schweizer and colleagues

Hybrid c l o n e s

058 058 1 2 3 4 5 6 7 8 9 10 11 12 KP2

1.0- 4

! , i

B: VSG 118

Fig. 4. Comparison of VSG gene families in parents and hybrid clones. HincII (A) or PstI (B) digests were hybridised with probes for VSG genes 117 or 118; post- hybridisational washes were to low stringency (3 x SSC at 65°C). Arrowheads indicate particular DNA fragments which are present in only one parent but some or all hybrid clones. Lanes 1 9 = clones from fly 8/4 B or P populations;

lanes 10-12 =clones from fly 9/6 B or P populations.

even flies with salivary gland hybrids had HyR/ Gt R trypanosomes in their midguts.

Since each drug resistance marker was present on only one parental tubulin homo- logue, Mendelian reassortment of markers would predict the occurrence of hybrids with wild-type or parental drug resistance pheno- types, as well as double drug resistant hybrids. Only the latter could be selected for in this experiment, however. Although several inde- pendent hybrid clones were Hy R only, no Gt R hybrids were found. This may have been due to chance, since relatively few clones were isolated from unselected populations. Alternatively, certain genotypes or phenotypes may have been favoured, either in the fly or during subsequent manipulations. Another complicat- ing factor is that some of the hybrids may be trisomic for the tubulin chromosome, as found previously [28]. Detailed analysis of a larger number of clones will shed light on these issues.

In conclusion, the application of reverse genetics to the problem of mating in trypano- somes has turned a hit or miss experimental system into an eminently manipulable one. The way is clear for definitive analysis of selfing and mating types in trypanosomes.

Acknowledgements

This work was supported by the Medical Research Council. We thank Anneloor ten Asbroek, Marc Ouellette and Piet Borst of the Netherlands Cancer Institute, Mary Lee and Lex Van der Ploeg of Columbia University and John Kelly of the London School of Hygiene and Tropical Medicine for generously provid- ing the DNA constructs and other probes. We are also indebted to Carol Ross of the Centre for Tropical Veterinary Medicine for advice on bsf culture medium.

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