Molecular and Biochernical Parasitology, 49 (1991) 297 302 ~ 1991 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/91/$03.50 ADONIS 016668519100452Q

297

MOLB10 01633

Ribosomal RNA genes of 'pathogenic' and 'nonpathogenic' Entamoeba histolytica are distinct C. G r a h a m C l a r k a n d L o u i s S. D i a m o n d Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, U.S.A. (Received 29 May 1991; accepted 24 July 1991)

Most infections with Entamoeba histolytica are asymptomatic. Two forms of the organism can be distinguished biochemically, and this finding has been explained by two distinct hypotheses: (1) there are two morphologically indistinguishable species, one of which causes disease; (2) there is one species which exists in two interconvertible forms, one of which causes disease. Knowledge of which hypothesis is correct has major implications for evaluation and treatment of carriers. We have studied the ribosomal RNA genes of the two forms hypothesising that, if E. histolytica is one species, there should be no differences between them, We have found that the ribosomal RNA genes of the two forms are quite distinct, which supports the hypothesis that E. histolytica is two species. Key words: Entamoeba histolytica; Pathogenicity; Polymerase chain reaction; Riboprinting; Ribosomal RNA gene

Introduction

Entamoeba histolytica parasitises perhaps 10% of the world population, causing an estimated 40 million cases of dysentery annually, and over 40000 deaths, many from extraintestinal lesions [1]. However, 90% of infections are asymptomatic, which raises the question of whether it is necessary to treat all carriers. Addressing the dichotomy in the course of infection, Brumpt, in 1925 [2], proposed the existence of two morphologically indistinguishable species, one a potential pathogen, the other a harmless commensal. His theory gained few supporters until it was shown that amoebae from cases of active disease ('pathogenic' strains) could be distinguished from amoebae in most asymptomatic Correspondence address." C. Graham Clark, Laboratory of Parasitic Diseases, NIA1D, Building 4/126, NIH, Bethesda MD 20892, U.S.A. Abbreviations." rRNA, ribosomal RNA; PCR, polymerase chain reaction; 'P', 'pathogenic' E. histolytica; 'NP', 'nonpathogenic' E. histolytiea.

carriers ('nonpathogenic' strains) by isoenzyme patterns [3]. While these and subsequent [4-10] data lend credence to the two-species hypothesis, conversion of isoenzyme patterns of 'nonpathogenic' strains to those of 'pathogenic' strains has been reported [11-13], supporting the existence of distinct but interconvertible forms of a single species. If E. histolytica is two species, we reasoned that the ribosomal R N A gene sequences of 'pathogenic' and 'nonpathogenic' strains might be distinguishable, whereas, if it is one species, the genes should be identical. We report here that the genes are quite distinct.

Materials and Methods

Cultures. The strains examined (Table I) are all in the senior author's cryopreserved collection, except for two monkey isolates which were cultured from the faeces of one rhesus macaque and one baboon in the N I H animal facility.

298

Riboprinting. Cells were grown, harvested and D N A purified as previously described [14]. Small subunit ribosomal R N A genes were amplified using PCR, based on the method of Medlin et al. [15], passed over spin columns to remove residual primers, and digested with A

restriction enzymes. T h e presence o f bacterial D N A in p r e p a r a t i o n s f r o m xenic E. histolytica cultures does not interfere with the P C R reactions. T h e p r i m e r s used were: R N A - l i k e 5' .. C C A G C T G C A G A T C T G G T T G A T C C T G C C A G T . . 3 ' ; a n d anti-sense - 5 ' . . C C G C T -

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Fig. 1. (A) Comparison of riboprints from 'pathogenic' and "nonpathogenic" strains of E. histolytica. The ethidium bromide stained gel shown here illustrates PCR amplified small subunit r R N A genes digested with several of the restriction enzymes with which 'NP' and 'P' strains can be distinguished. In addition to these, the enzymes BglII, Sau3AI and Asul also discriminate between the two groups. The 'P' strain used was HI-1372:AIIMS. The 'NP' strain was SAW760RR cl A. The marker (M) is a mixture of bacteriophage 2 DNA digested with EcoRI + Hindlll and plasmid pBR322 DNA digested with Mspl. (B) Restriction map comparison of 'P' and 'NP' strains. The restriction map of the 'P' strain small subunit rRNA gene was derived from an unpublished E. histolytica sequence kindly provided by M.L. Sogin. The riboprints of all 'P" strains exactly match the fragment sizes predicted from the sequence. The 'NP' map was derived by incorporating restriction site differences and confirmed by double digestion. The variable sites illustrated in Fig. 1A are marked with an asterisk. A, HaelII;

B, Xbal; C, EcoRl; D, Dral; E, DdeI; F, HinfI; H, HhaI; L, Alul; M, Mspl; R, Rsal; S, Sau96I; T, Taql; X, Xhol.

299 TABLE 1 Isolates o f E. histolytica used in this s t u d y Strain designation

P/NP

Geographic origin a

Source

HM-I:IMSS cl 6 ~ 200:NIH cl 2 ~ HK-9 cl 2 a HU-1 :CDC a HU- 1:M USC a HU-2:MUSC a R a h m a n cl 2 ~ Jawa 103 HI-1372:AIIMS HX-I:UCLA NIH:0283:l SAW408RR cl A SAW755CR cl B SAW891R cl B SAWI734R cl A R CDC:0784:4 TI-ET-80 UoB: lc SAW1734R cl A R S A W I 4 2 R R cl A SAW760RR cl A SAW937R cl A Rustom CDC:0784:4 WI:1285:1 NIH:0581 : 1 NIH:I083:2 NIH:0187:I NIH:0890:l NIH:0191:2

P P P P P P P P P P P P P P P pb pb P NP NP NP NP NP NP NP NP NP NP NP NP c NP c

Mexico Uncertain Korea U.S.A. U.S.A. U.S.A. Uncertain Uncertain Uncertain India Uncertain Burkina Faso Mexico India Mexico Ethiopia Guatemala Ethiopia Norway Ethiopia India Uncertain Mexico Uncertain Guatemala Uncertain Uncertain Uncertain Uncertain Uncertain Uncertain

M. De la Torre This laboratory G.W. Healy G.W. Healy G.W. Healy W.B. Lushbaugh R.A. Neal R.A. Nea[ R.A. Neal V.K. Vinayak M. Voge This laboratory P.G. Sargeaunt P.G. Sargeaunt P.G. Sargeaunt D. Mirelman D. Mirelman D. Mirelman D. Mirelman D. Mirelman P.G. Sargeaunt P.G. Sargeaunt P.G. Sargeaunt R.A. Neal D.M. Moss This laboratory This laboratory This laboratory This laboratory This laboratory This laboratory

~These strains or the p a r e n t strains are available from the A m e r i c a n T y p e C u l t u r e Collection. b ' I s o e n z y m e - c o n v e r t e d ' strains (refs. I I a n d 12). ~Strains from m o n k e y s r h e s u s m a c a q u e ( N I H : 0 8 9 0 : l ) a n d b a b o o n (NIH:0191:2). ~ ' U n c e r t a i n ' m e a n s either that the patient h a d travelled to m o r e t h a n one e n d e m i c area, or that d o c u m e n t a t i o n is inadequate.

GCAGGATCCTTCCGCAGGTTCACCT..3' (Synthecell Corp., Rockville, MD). P C R

reactions were as described [14] with 30 cycles of 94°C/1 min, 55°C/1.5 rain, 72°C/2 rain in a PTC-100 thermal cycler (MJ Research, Inc.). The D N A fragments were separated on 2.6% agarose gels (3:1; NuSieve: Seakem ME agarose (FMC)) in Tris-borate buffer.

Sequencing. The PCR amplification products from 2 'P' and 2 'NP' strains were cloned using the TA cloning kit (Invitrogen Corp.) and pooled recombinants for each strain were partially sequenced using a Sequenase kit (US Biochemical Corp.), the amplification primers,

and rRNA sequencing primers (BoehringerMannheim). In addition, genomic rDNA clones from one 'P' and one 'NP' strain were examined (HM-I:IMSS cl 6 [16] and SAW1734R cl AR, respectively, kindly provided by D. Mirelman; not shown). Based on the derived sequences, primers that were predicted to amplify 'P' rDNA specifically were designed. The sequences of the 'P'-specific primers (Operon Technologies, Inc., Alameda CA) are: R N A - l i k e - 5 ' . . G G C C A A T T C A T T C A A T G A A T T G A G . . 3 ' ; anti-sense - 5'..CTCA G A T C T A G A A A C A A T G C T T C T C . . 3'. Am-

plification was as above.

300

Results and Discussion

Of the 780 bases of ~P' and 'NP' r R N A sequence which were compared 17 positions (2.2%) differed between the two forms while being conserved within each group (Fig. 3). Of these sequence differences 12 represented transitions, 4 transversions and 1 an insertion/deletion event; all are located in the recognized 'variable' regions of small subunit r R N A sequences [17], and most are point mutations. Our ability to discriminate among the small subunit r R N A genes of 'P' and 'NP' strains supports the two-species hypothesis. However, when we examined strains in which the isoenzyme profile had been reportedly converted (CDC:0784:4 and SAW1734R cl AR: refs. 11 and 12), we observed a concomitant alteration in the riboprints from ~NP' to 'P' patterns. Because the r R N A genes are a multicopy family [16,18], it is possible that there was amplification of a minor r D N A component during isoenzyme conversion [19]. To search for a low copy number or 'master-P' r R N A gene in a ~NP' strain, we designed primers based on the regions sequenced above to amplify 'P' r D N A specifically. These primers are 24 and 25 bases long, cover regions with 5 and 6 mismatches, respectively, between "P' and 'NP' strains, and flank an 876-bp fragment

We recently described a technique that allows us to discriminate among closely related species of Entamoeba by examination of restriction enzyme site polymorphisms in PCR-amplified small subunit ribosomal R N A g e n e s - 'riboprinting' [14]. While the ribosomal D N A fragment patterns of "pathogenic' CP') and "nonpathogenic' ('NP') strains are in most cases identical, a few restriction enzymes reveal differences between the two forms (Fig. I A). Mapping these variable restriction sites revealed that they are widely dispersed (Fig. 1B): therefore, more than one region of the gene is different. To date, we have examined 18 'P' and 13 'NP' strains from across the world (Table I). All strains have been examined with 12 restriction enzymes and many with several additional enzymes. The riboprint pattern of all 'P' strains is identical and distinct from the pattern seen in all 'NP' strains (including two from monkeys). To examine the underlying nature of these restriction site differences, we have sequenced regions of cloned, small subunit r R N A genes from 3 'P' and 3 'NP' strains, including the RsaI, DdeI, XbaI and Sau96I polymorphic sites. The first of these is illustrated in Fig. 2.

P 1

NP 2

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3

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A A

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Fig. 2. rDNA sequence in the region of a restriction site polymorphism. Cloned, PCR-generated, small subunit rRNA genes of two 'P" and two 'NP' strains were partially sequenced. The rRNA region illustrated here was obtained using the small subunit rRNA 5' amplification primer and covers the polymorphic RsaI site present only in 'NP' strains. The sequences of the ~P' strains exactly match the unpublished E. histolytica sequence of M.L. Sogin, except for a single base transition detected in SAW891R cl B (Fig. 3). Strains: 1, HI-1372:AIIMS; 2, SAW891R cl B; 3, SAW937R cl A; 4, SAW760RR cl A.

301

of the gene. Under the P C R conditions used, the primers amplify r D N A from 'P' strains but not from 'NP' strains (Fig. 4A). To estimate the sensitivity of this detection method, variable numbers of 'P' cells were mixed with a fixed number of 'NP' cells before extracting the D N A and amplifying with the 'P'-specific primers. We can easily detect as few as 1 'P' in 104 'NP' cells (Fig. 4B). Occasionally, small amounts of product are seen in the control 'NP' amplification (lane 4), but this D N A is completely digested with Sau96I (lane 8), a 'NP'-specific restriction enzyme (Fig. 1B), indicating that it results from nonspecific priming in the 'NP' gene. Thus, if there is a 'P' gene in a 'NP' isolate, it must be present at a ratio of less than 1 in 104. Since the estimated number of r R N A genes per cell is 200-400 [16,18], we can infer that there is less than one 'P' gene in each 'NP' cell and, therefore, since some of these strains are cloned lines, that 'P' ribosomal R N A genes are not found in the 'NP' genome. This contrasts with a report of 'P'-specific repetitive D N A in a 'NP' strain

[19]. T h e s e p r i m e r s m a y a l s o p r o v e t o b e u s e f u l for the rapid diagnosis of 'P' infections. In the absence of reports of isoenzyme conversion and the accompanying DNA and antigenic changes, the proposed existence of two species would be widely accepted. Indeed,

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Fig. 3. Sequence differences between +pathogenic' and 'nonpathogenic' ribosomal RNA genes. (A-D) The DNA sequence surrounding the detected base differences is illustrated. |n each case the RNA-like 'NP' sequence is given in full while only those bases differing in the 'P' sequence are shown. Restriction sites affected by the variation are marked. Numbering is derived from the unpublished E. histolytica sequence of M.L. Sogin. The asterisk marks the site of intra-'P' heterogeneity detected in SAW891R cl B (T-*C).

Fig. 4. (A) Specific amplification of 'P' rDNA. A selection of strains is illustrated here, each amplified with the 'P'specific primers (Materials and Methods). As a control, the complete small subunit gene was amplified from the same DNA preparations, and portions of each amplification were mixed before electrophoresis on a 1.2% agarose gel. Lanes 1 and 2 contain DNAs amplified from cloned small subunit rRNA genes of strains SAW891R cl B ('P') and SAW760RR cl A ('NP'). In lanes 3-8, amplifications of genomic D N A from strains H I - 1 3 7 2 : A I I M S ('P'), SAW760RR cl A ('NP'), N1H:0283:l ('P'), U o B : l c ('NP'), H U - I : C D C ('P'), and SAW937R cl A ('NP'), respectively, are shown. The marker (M) is bacteriophage )+ DNA digested with EcoRI + HindIII. (B) Sensitivity of 'W-specific primers. Strains SAW760RR cl A ('NP') and 200:NIH cl 2 ('P') were grown separately and equal number of SAW760 were mixed with variable numbers of 200:NIH cells in ratios of 100:1 to 10000:1 before extracting the DNA. Equal amounts of DNA from the mixtures and from the SAW760 control (lanes 4 and 8) were then amplified with the 'P'-specific primers. The amplification products were ethanol precipitated and half of each was digested with the restriction enzyme Sau96I (lanes 5 8) before loading on a 1.2% agarose gel. The mix ratios shown are 100:1 (lanes 1 and 5), 1000:l (lanes 2 and 6), and 10000:1 (lanes 3 and 7). The estimated total number of cell equivalents of D N A in the 10 000:1 amplification (lanes 3 and 7) were 18 750 'NP' and < 2 'P', assuming quantitative DNA recovery during isolation. The marker (M) is as in (A).

302

the estimated genetic distance between the small subunit rRNAs of 'P' and 'NP' strains of E. histolytica (2.2%) is comparable to that between human and mouse rRNAs, lsoenzyme conversion has been reported from two laboratories [11-13]. We have attempted to replicate those results ourselves using the same strains and following the published methodologies, but, despite considerable effort, we have not met with success (unpublished results). Much of the previous D N A evidence for the distinctiveness of 'P' and 'NP' E. histolytica was derived from genes encoding molecules potentially involved in pathogenesis [4, 8 10]. Such sequence differences could be interpreted as being directly linked to the variability in virulence between the two forms. The ribosomal R N A genes are immune to such concerns. All the available data, including those presented above, support the notion that E. histolytica, as currently defined, is, in fact, two distinct species. Acknowledgements We thank Dr. David Mirelman for providing the genomic r D N A clones and his converted strains, and Dr. Mitchell Sogin for communicating his unpublished E. histolytica rRNA sequence. References 1 Walsh, J.A. (1986) Problems in recognition and diagnosis of amebiasis: estimation of the global magnitude of morbidity and mortality. Rev. Infect. Dis. 8, 228 238. 2 Brumpt, E. (1925) Etude sommaire de l"Entamoeba dispar' n.sp., amibe fi kystes quadrinucl~es, parasite de l'homme. Bull. Acad. Med. (Paris) 94, 942 952. 3 Sargeaunt, P.G., Williams, J.E. and Grene, J.D. (1978) The differentiation of invasive and non-invasive Entamoeba histolytica by isozyme electrophoresis. Trans. R. Soc. Trop. Med. Hyg. 72, 519 521. 4 Strachan, W.D., Spice, W.M., Chiodini, P.L., Moody, A.H. and Ackers, J.P. (1988) Immunological differentiation of pathogenic and non-pathogenic isolates of Entarnoeba histolytica. Lancet i, 561 562. 5 Garfinkel, L.I., Giladi, M., Huber, M., Gitler, C., Mirelman, D., Revel, M. and Rozenblatt, S. (1989) DNA probes specific for Entamoeba histolytica possessing pathogenic and nonpathogenic zymodemes. Infect. Immun. 57, 926 931. 6 Tannich, E., Horstmann, R.D., Knobloch, J. and

Arnold, H.H. (1989) Genomic D N A differences between pathogenic and nonpathogenic Entamoeha histolytica. Proc. Natl. Acad. Sci, USA 86, 5118 5122. 7 Tachibana, H., Kobayashi, S., Kato, Y., Nagakura, K., Konada, Y. and Takeuchi, T. (1990) Identification of a pathogenic isolate-specific 30000 Mr antigen of Entamoeba histo@tica using a monoclonal antibody. Infect. lmmun. 58, 955 960. 8 Edman, U., Meraz, M.A., Rausser, S., Agabian, N. and Meza, 1. (1990) Characterization of an immunodominant variable surface antigen from pathogenic and nonpathogenic Entamoeba histolytica. J. Exp. Med. 172, 879 888. 9 B[akely, P., Sargeaunt, P.G. and Reed, S.L. (1990) An immunogenic 30-kDa surface antigen of pathogenic clinical isolates of Entamoeba histoh, tica. J. Infect. I)is. 162, 949 954. 10 Tannich, E., Scholze, H., Nickel, R. and Horstmann, R.D. (1991) Homologous cysteine proteinases of" pathogenic and nonpathogenic Entamoeha histolytica. J. Biol. Chem. 266, 4798 4803. 11 Mirelman, D., Bracha, R., Chayen, A.. Aust-Kettis, A. and Diamond, L.S. (1986) Entarnoeha histoh,tica: effects of growth conditions and bacterial associates on isoenzyme patterns and virulence. Exp. Parasitol. 62, 142-148. 12 Mirelman, D., Bracha, R., Wexler, A. and Chaycn, A. (1986) Changes in isoenzyme patterns of a cloned culture of nonpathogenic Entarnoeba histoO,tica during axenization. Infect. lmmun. 54, 827 832. 13 Andrews, B.J., Mentzoni, L. and Bjorvatn, B. (19911) Zymodeme conversion of isolates of Entar~2oeha histoh, tica. Trans. R. Soc. Trop. Med. Hyg. 84, 63 65. 14 Clark, C.G. and Diamond, L.S. (1991) The Laredo strain and other Entamoeba histolytica-like amoebae are Entamoeba moshkovskii. Mol. Biochem Parasitol. 46. 11 18. 15 Medlin, L., Elwood, H.J., Stickel, S. and Sogin, M.L. (1988) The characterization of enzymatically amplified eukaryotic 16S-like rRNA coding regions. Gene 71, 491 499. 16 Huber, M., Koller, B., Gitler, C., Mirelman, D., Revel, M., Rozenblatt, S. and Garfinkel, L. (1989) Entamoeha histolytica ribosomal RNA genes are carried on palindromic circular DNA molecules. Mol. giochem. Parasitol. 32, 285 296. 17 Raue, H.A., Klootwijk, J. and Musters, W. (1988) Evolutionary conservation of structure and function of high molecular weight ribosomal RNA. Prog. Biophys. Mol. Biol. 51, 77 129. 18 Bhattacharya, S., Bhattacharya, A. and Diamond, L.S. (1988) Comparison of repeated DNA from strains of Entamoeba histolytica and other Entamoeba. Mol. Biochem. Parasitol. 27, 257 262. 19 Mirelman, D., Bracha, R., Rozenblatt, S. and Gartin~ kel, L.I. (1990) Repetitive DNA elements characteristic of pathogenic Entamoeba histolytica strains can also be detected after po[ymerase chain reaction in a cloned nonpathogenic strain. Infect. lmmun, 58, 1660 1663.