EXPERIMENTAL

72,236-242 (191)

PARASITOLOGY

Entamoeba

histolytica:

Generation and Characterization Hybrid Clones

FRANCISCO SOL& Departamento

de Gendtica

y Biologi’a A.P.

of

AND ESTHER OROZCO’

Molecular, Centro de Znvestigacih 14-740, Mexico 07000, D.F., Mexico

y de Estudios

Avanzados

Z.P.N.,

SoLfs, F., AND OROZCO, E. 1991. Entamoeba hisrolyrica-Generation and characterization of hybrid clones. Experimental Parasitology 72, 236-242. Transference of DNA to Entamoeba histolyrica was carried out by polyethylene glycol fusion of two amebic clones with different phenotypes. Clone C9, strain HMl:IMSS, was the donor. It is emetine-resistant, highly phagocytic and virulent, and grows in soft agar. Clone L6, strain HMl:IMSS, the recipient, is emetine-sensitive, phagocytic, and virulencedeticient, and it does not grow in soft agar. Clones L6 and C9 have shown high stability in their virulence phenotypes since their isolation more than 5 years ago. Before fusion experiments, clone C9 was incubated in 20 pg/ml bromodeoxyuridine for 24 hr, and then, irradiated with 310 nm light to complete inactivation. Controls ensured that all irradiated trophozoites died after 24 hr of incubation at 37°C. Itradiated C9 trophozoites were fused with L6 trophozoites, and hybrids were selected by their ability to grow in the presence of emetine. All hybrids, independently generated, grew poorly in soft agar and showed both an intermediate emetine-resistance and rate of phagocytosis. Some of them destroyed efficiently cell monolayers, but interestingly, they showed differences in their ability to produce hepatic abscesses in ham&W?..

0 1991 Academic

Press, Inc.

AND ABBREVIATIONS: Enfamoeba hisrolyrica; Genetics; Vlllence; Drua-resistance: Emetine IEmt): Phaaocvtosis (Phag); Cytopathic effect (Cyp); Cytotoxic effect (Cyt); Growth in semisolid ag& (Aga). INDEX

DESCRIF-TOR~

INTRODUCTION

Little is known about the genetic mechanisms underlying the virulence of E~ltamoeba histolytica, the parasite responsible for human amebiasis. Adhesion, phagocytosis, and the ability to destroy cell culture monolayers have been related to the ability of trophozoites, the invasive form of this parasite, to invade tissue hosts (Trissl et al. 1978, Orozco ef al. 1978), but the genetic basis and the relationship among them are unknown. Etner (1971) mixed trophozoites of both NIH-200 and Laredo strains, which are resistant to different drugs, and he found surviving trophozoites resistant to both drugs, suggesting gene transfer by mating. However, the use of noncloned populations and the fact that Laredo is a ’ Present address: Unidad de Microscopia Electnkica, Facultad de Medicina, U.N.A.M. 2 To whom correspondence should be addressed. 236 00144894/91 $3.00 Copyright 0 1991 by Academic Press. Inc. All rights of repmductioa in any form reserved.

histolytica-like strain make these studies dilficult to interpret. Based on an extensive zymodeme analysis of a high number of amebic isolates, Sargeaunt (1985) also proposed that genetic exchange could take place in Entamoeba. These studies suggest that genetic transfer might be a natural process in E. histolytica; but, although intriguing, these studies need to be confirmed. Unfortunately, suitable methodology for gene transfer in this parasite has not been described. We have generated drug-resistant cloned mutants by the selection of surviving trophozoites in lethal doses of emetine, after chemical mutagenesis of trophozoites by ethylmethane sulfonate (Orozco et al. 1985). One group of mutants showed crossresistance to other drugs, and amplification of a multidrug-resistance-like gene (mdr) was detected in them (Sammuelson et al. 1990; Ayala et at., 1990) whereas an-

E. histolytica

HYBRID

other group seemed to be altered at the protein synthesis level (Orozco et al. 1985). We have also generated mutants with a different degree of virulence (Rodriguez and Orozco 1986). All these mutants constitute biological material suitable to carry out genetic studies in E. histolytica. Gene transfer by plasmids or viruses has been carried out only in few protozoan parasite species (Hughes and Simpson 1986; Laban and Wirth 1989). A cell fusion technique has been used to transform eukaryotic cells (Pontecorvo 1971). Hybrid generation by cell fusion has been valuable in the production of monoclonal antibodies (Kohler and Milstein 1975) and it may be an alternative to introduce exogenous DNA into amebic trophozoites. In this paper we report the use of a cell fusion technique to study virulence-related properties in E. histolytica. Hybrid trophozoites were selected using emetine-resistance as a marker and their virulence-related properties were studied. MATERIALSAND

METHODS

Cell cultures. Trophozoites of E. histolytica were cultured in TYI-S-33 medium and harvested as described (Diamond et al. 1978). Growing and colonyforming efficiency (CFE) in semisolid agar were carried out according to Gillin and Diamond (1978). MDCK epithelial cell cultures (Misfeldt et al. 1976) were grown to confluence in 24-well plates (Linbro, VA) in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum (HyClone, CA). Cell fusion. Fusion of E. histolytica trophozoites was carried out by the technique described by Pontecorvo (1971) with several modifications introduced for ameba. Briefly, clone C9 was selected as the donor clone (Orozco et al. 1985) due to its high stability in virulence phenotypes; it is emetine-resistant (EmtR), highly phagocytic @‘hag+), and virulent in vivo (Vir’) and in vitro, the latter defined as the ability of trophozoites to destroy cell culture monolayers (Cyp’). Extracts from C9 trophozoites dislodge cells from culture cell monolayers (Cyt’), and trophozoites form colonies in soft agar (Aga+). Clone L6, another highly stable clone, was selected as the recipient clone (Orozco et al. 1983); it is highly sensitive to emetine (Emt’), nonvirulent (Vir- , Cyp-, Cyt-), phagocytosisdeficient (Phag-), and it does not grow in soft agar t&O.

237

CLONES

Trophozoites (2 x 10’) of clone C9 were grown in TYI-S-33 medium supplemented with 20 &ml of bromodeoxyuridine (BUdR) (Sigma Chemical Co., St. Louis, MO) for 24 hr before fusion experiments. Cells were then washed twice with sterile phosphatebuffered saline (PBS) and distributed in 2-ml aliquots (0.5 x lo5 trophozoites/ml) in 25cm petri dishes (Lux Scientific Co.). Top dishes were removed and bottom dishes containing the trophozoites were placed 20 cm under a 310~nm light source and irradiated for 2 hr with gentle shaking; trophozoites were then transferred to 15ml tubes. Meanwhile, trophozoites from recipient clone L6 were washed twice with sterile PBS, resuspended in 5 ml of sterile PBS, and then mixed with the irradiated C9 trophozoites in a 1: 1 ratio. Cell mixtures were centrifuged at 360g for 5 min, supematants were discarded, and 1 ml of 50% polyethylene glycol (PEG) (MW 4000) (BDH Chemicals LTD, Poole, U.K.), prepared in TYI-S-33 medium without serum was slowly added. After this, 9 ml of PBS was added at a rate of 1 ml/mitt. Cell mixtures were incubated for 5 min at 37°C and centrifuged at 360g for 10 min, and pellets were resuspended in 10 ml of TYI-S-33 medium and distributed in RI-ml culture tubes containing 9 ml of TYI-S-33 medium. Cells were incubated at 37°C for 24 hr and then 3 or 12 kg of emetine/ml was added to each tube. Rates of phagocytosis and virulence. The rates of erythrophagocytosis and the cytopathic and cytotoxic effects were measured as previously described (Trissl et al. 1978; Orozco et al. 1983; Rodriguez and Orozco 1986). Virulence in vivo was measured by inoculation of 2.5 x 10’ trophozoites in anesthetized l-month-old hamsters (Orozco et al. 1983). Livers were extracted 8 days postinoculation to search abscesses. Statistical analysis. All experiments were performed in duplicate and repeated at least three times. The standard deviation was calculated and statistical significance was obtained by Student’s t test. RESULTS

Cell fusion frequency in E. histolytica. Aliquots were taken at different times after the addition of PEG to the mixture of BUdR-inactivated C9 and L6 trophozoites. Under a light microscope, PEG-agglutinated trophozoites were observed in groups of two to three cells in a fashion similar to that presented by PEG-fused mutine cells to produce hybridomas (data not shown). Trophozoites were incubated for 24 hr without emetine to allow for recovery after the treatment. Lethal emetine doses for L6 were then added to the fused cells.

238

SOLfS

AND

After 7 days of incubation in the presence of the drug, no trophozoites were observed in any of the cultures. Three or 4 days later, few trophozoites appeared, which grew to reach a logarithmic growth phase. No trophozoites appeared after a l-month incubation in control cultures containing 12 ug emetine and (a) L6 fused with BUdRtreated L6, and (b) BUdR-treated C9 or clone A (a HMl:IMSS clone identical to C9 except that it is sensitive to emetine) fused with BUdR-treated C9 or clone A (Table I). For each assay, 10culture tubes were inoculated and the average of cultures with surviving amebas was 2.5 per experiment in three different experiments. If we consider that each hybrid population started from one to nine trophozoites, the frequency of hybrid formation, using emetine-resistance as a marker, would be approximately 2.5 x 10e6 (Table I). Hybrid populations C9L6-1, C9L6-2, and C9L6-3 were obtained from three independent experiments. Cloning of hybrid populations. Hybrid populations were cloned when they reached the logarithmic growth phase. Several clones were obtained; two of each hybrid population were studied and results for both were identical (data not shown). Hybrids C9L6-1 and C9L6-3 were unable to

OROZCO

grow in semisolid agar; therefore they were cloned by limited dilution (Orozco et al. 1983), while hybrid C9Ld2 showed a 4% CFE (Table II). Clone L6 does not grow in semisolid agar and clone C9 presents a 87% CFE. (Orozco et al. 1983; Rodriguez and Orozco 1985). Emetine-resistance of hybrid populations. Hybrid selection was carried out by

using different emetine concentrations. First, hybrids C9L6-2 and C9L6-3 were selected in 3 &ml emetine; dose-response curves showed that they died at 5 &ml (Fig. 1). Emetine-resistance of these hybrids, although low, was significantly higher than the one showed by L6 trophozoites, which died at 1 pg/ml, but was lower than C9, which died at 20 &ml (Orozco et al. 1985). Hybrid C9L6-1 was selected using 12 ug/ml emetine and the doseresponse curves showed that it died at 15 kg/ml (Fig. 1). Virulence in vitro of hybrid populations. In order to start the genetic analysis of virulence properties in E. histolytica, the phagocytosis of hybrid populations and their ability to destroy cell culture monolayers were studied. At the end of a lo-min incubation, trophozoites of the three hybrids had ingested a mean of 6 to 7 RBCs

TABLE I Hybrid Generation in E. histolytica Treatment Clone

Number of trophozoites

Irradiation + BUdR

c9 c9 C9lC9 L6 L6/L6 C9lL6 C9/L6

2 x IO’ 2 x 10’ 106/106 2 x 10’ lo%06 106/106 10%06

+ + +I+

C9/L6

106/106

PEG

EMT Wmlf

Cultures wilive cells

+

-T+ +I+I-

+ + +

12 1 1 1 3

O/20 O/20 O/20 O/20 O/20 O/20 4110

+I-

+

12

l/10

Hybrids C9L6-2 C9L6-3 C9L6-4 C9L6-1

Note. Frequency of hybrid formation was 2.5 x lo@, calculated by dividing the number of culture tubes with live cells by the total number of receptor cells. Hybrid C9L6-4 died shortly after its generation.

E. histolytica

239

HYBRID CLONES

TABLE II Cloning of E. histolyrica Hybrids Efficiency Trophozoites c9 L6 C9L6- 1 C9L6-2 C9L6-3

Liquid medium” (%I

Soft agaP m

ND 30 41.4 ND 3.5

81 0 0 4 0

a Number of tubes with growing cells divided by the number of tubes inoculated with a single cell. b Number of colonies in semisolid agar divided by the number of trophozoites inoculated (CFE).

per trophozoite (Fig. 2), while C9 and L6 trophozoites ingested 14.5 and 4 RBCs, respectively. Live trophozoites (1 trophozoite: 1 MDCK cell) and trophozoite lysates (10 trophozoites: 1 MDCK cell) from hybrids C9L6-1 and C9L6-2 destroyed 100% of the MDCK cell monolayers when they were incubated at 37°C for 1 hr, while live trophozoites of hybrid C9L6-3 destroyed only 25% and their extracts had no effect on the cell monolayers integrity (Table III). Clone C9 destroyed the whole cell monolayer under the same conditions and clone L6 destroyed from 0 to 13% of the cell monolayers, as reported (Orozco et al. 1983; Rodriguez and Orozco 1985).

\ i

E

Time

( min)

FIG. 2. Erythrophagocytosis of hybrid clones. Rate of phagocytosis was measured at 37°C after incubation of trophozoites with RBCs (1:lOO) (0) Clone L6. (0) Clone C9. (Cl) Hybrid C9L6-1. (A) Hybrid C9L6-2. (W) Hybrid C9L6-3. Results are the mean of five independent experiments carried out in duplicate. C9L6-1 # L6, P = 0.06; C9L6-2 = L6, P = 0.11; and C9L6-3 # L6, P = 0.08.

Virulence in vivo of hybrid populations. In agreement with the results obtained from the in vitro assays, trophozoites of hybrid C9L6-3 did not produce abscesses in hamsters, but 50% of the animals inoculated with trophozoites of hybrid C9L6-2 developed small abscesses, and 66% of the hamsters inoculated with hybrid C9L6-1 presented large abscesses (Table III). Trophozoites of clone C9 produced large abscesses in 90% of the animals and L6 did not produce abscesses in any animal, as reported (Orozco et al. 1983; Rodriguez and Orozco 1985). DISCUSSION

0

20

5

EtYEETlNF

25

(g/ml)

FIG. 1. Emetine resistance of hybrid clones. Relative growth of hybrid clones was calculated by dividing the rate of growth of the clones in the presence of different emetine concentrations by the rate of growth in the absence of the drug. (A) Clone L6. (0) Clone C9. (0) Hybrid C9L6-1. (A) Hybrid C9L6-2. (A) Hybrid C9L6-3. Experiments were carried out at least five times by duplication.

Genetic analysis of E. histolytica and other protozoa has been difficult because of the lack of a suitable methodology for the transfer of exogenous DNA into these organisms. Cell fusion has been successfully used in different systems to obtain hybrid cells which express a given phenotype (Harris 1985). We showed here that cell fusion in E. histolytica, using previously generated mutants, is a suitable methodology to start genetic studies in this protozoan. In this paper we initiated the analysis of eme-

240

SOLfS

AND OROZCO

TABLE III Virulence in Viva and in Vitro of the E. histolyticu

Hybrids

% Monolayer destruction Clone

CYP”

Cytb

Hamsters with abscesses/Hamsters inoculated

c9 L6 C9L6-1 C9L6-2 C9L6-3

loo 13 (22.43) 100 100 25 (k1.17)

100 0 100 100 0

9/10 (very large abscesses) 0110 7110 (small abscesses) S/10 (small abscesses) o/10

0 Cyp MDCK monolayer destruction in a 1:l (MDCK cellkrophozoite) ratio. b Cyt MDCK monolayer destruction by amebic extracts in a 1:lO (cellkophozoites) ’ Standard deviation.

ratio.

tine-resistance, phagocytosis, cytopathic tance between the parental clones. An inand cytotoxic effects, and the virulence in termediate drug-resistance phenotype of vivo of three hybrid populations. However, hybrid populations has been described for we are aware that a higher number of hy- mammalian hybrid cells (Davidson 1974). brids should be studied in order to accuGrowth in semisolid agar has been assorately define how these properties are ex- ciated with virulence of the strains (Gillin pressed in this parasite. Furthermore, un- and Diamond 1978). Hybrid populations fortunately genetic studies in E. hisfolytica grew deficiently in semisolid agar, as did can only render phenotypic results because the parental clone L6. Hybrid C9L6-1 proeasy to follow molecular markers are not duced large hepatic abscesses in 66% of the available yet. hamsters inoculated, C9L6-2 in SO%, and Inactivation of parental nucleus by a C9L6-3 did not produce any abscess. Our large incorporation of BUdR in the DNA, results show no correlation between the followed by long-time irradiation with 310 CFE of trophozoites and their virulence, in nm light, has been used by Pontecorvo agreement with results obtained with virn(1971) for gene transfer by cell fusion. By lence revertants generated from clone L6 this method, we obtained E. histolytica (Orozco et al. 1983). emetine-resistant hybrid cells with a freThe three hybrids showed a similar rate quency at least lo-fold higher than the gen- of phagocytosis; however, they presented a eration of spontaneous emetine-resistant different ability to destroy cell culture mutants (Orozco et al. 1989) (2.5 X 10V6 vs monolayers. In the multifactorial phenom2.5 x 10W7). Generation of mutants by the enon of virulence, several genes should be treatment of parental cells is improbable, involved. From these results we hypothebecause no surviving C9 trophozoites were sized that there is more than one gene parobtained in experiments using 2 x lo7 tro- ticipating in the phagocytosis and cytophozoites grown in the presence of BUdR pathic effects, some of them may be particand uv irradiated or when L6 trophozoites ipating in both functions, while others may were grown in the presence of 1 p&nil of be specific for each of these virulenceemetine. involved phenomena. Destruction of monoHybrids C9L6-2 and C9L6-3 were ob- layers by hybrid amebic extracts was 100 or tained using 3 pg/ml of emetine, while hy- 0%, as it is in the parental clones, suggestbrid C9L6-1 was obtained using 12 (~g/ml. ing that this property is given by one or a Doseresponse experiments showed that joint group of proteins. The 56kDa major the three hybrids had an intermediate resis- neutral proteinase described by Keene ef

E. histolytica

HYBRID

241

CLONES

TABLE IV Phenotypes of Parental and Hybrid Clones of E. histolytica Clone c9 L6 C9L6- 1 C9L6-2 C9L6-3

Emt bd~) 25” 1 15 5 5

&a

Phag

CYP

CYt

Vir

+ -

+ I I I

+ + + -

+

+ I I -

-

+ + -

Note. Emt, emetine. Aga, colony formation in soft agar. Phag, phagocytosis. Cyp, ability of live trophozoites to destroy cell culture monolayers. Cyt, ability of trophozoite extracts to dislodge cell culture monolayers. Vir, ability of trophozoites to produce liver abscesses in I- to 2-month-old hamsters. I, intermediate numbers between clones C9 and L6. Q Concentration of the drug that kills trophozoites.

al. (1985) could be responsible for the dislodgment of cells from the substrate. In summary, analysis of hybrid populations showed that the three hybrids obtained here are different among them and are also different to the parental clones (Table IV). Hybrid C9L6-1 showed emetine-resistance, rate of phagocytosis, and virulence in vivo intermediate between clones L6 and C9. However, it behaved as clone L6 in its CFE and as clone C9 in its ability to destroy cell culture monolayers. Hybrid C9L6-2 presented rate of phagocytosis and virulence intermediate between the parental clones, but it behaved as clone L6 in CFE and it was closer to C9 in its ability to destroy cell culture monolayers; its emetine resistance was higher than that of clone L6 but was much lower than that of C9, as it was for hybrid C9L6-3. Finally, C9L6-3 showed an intermediate rate of phagocytosis, it behaved as clone L6 in CFE and in its inability to destroy cell culture monolayers and to produce hepatic abscesses in animals. Results suggest that differences may be due to genetic transfer from clone C9 to L6 and give few possibilities for a mutational event as the generating phenomenon for these clones, because it is unlikely that a pleiotropic mutation responsible for the different hybrid phenotypes would occur.

ACKNOWLEDGMENTS This work was partially supported by the MacArthur Foundation (U.S.A.) and CoNaCyT (Mexico). Dr. Orozco was a recipient of a J. S. Guggenheim Award. REFERENCES AYALA, P., SAMUELSON, J., WIRTH, D., AND OROZCO, E. 1991. Entamoeba histolytica: Physiology of multidrug-resistance. Experimental Parasitology, 71, 169-175.

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OROZCO

mosome elimination in somatic cell hybrids. Nature (London) 230, 367-371. RAVDIN, J., CROFT, B., AND GUERRANT, R. 1980. Cytopathogenic mechanisms ofEntamoeba histolyticu. Journal of Experimental Medicine 152, 377-390. RODRIGUEZ, M., AND OROZCO, E. 1986. Isolation and characterization of phagocytosis- and virulencedeficient mutants of Entamoeba histolytica. Journal of Infectious Diseases 154, 27-32. SAMUELSON, J., AYALA, P., OROZCO, E., AND WIRTH, D. F. 1990. Emetine resistant mutants of Entamoeba histolyticn overexpress mRNAs for multidrug resistance. Molecular and Biochemical Parasitology 38, 281-290. SARGEAUNT, P. 1985. Zymodemes expressing possible genetic exchange in Entamoeba histolytica. Transactions of the Royal Society of Tropical Medicine and Hygiene 79, 86-89. SOLIS, F., VARGAS, M. A., AND OROZCO, E. 1986. Transformation genetica en Entamoeba histolytica. Archives de Znvestigacion Medica (Mexico) 17, 3136. TRISSL, D., MARTINEZ-PALOMO, DE LA TORRE, M., AND DE LA

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Received 8 August 1990; accepted 15 October 1990