Differences in Genomic DNA Sequences Between Pathogenic

JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1991, p. 2234-2239 Vol. 29, No. 10 0095-1137/91/102234-06$02.00/0 Copyright C) 1991, American Society for Microbiology Differences in Genomic DNA Sequences between Pathogenic and Nonpathogenic Isolates of Entamoeba histolytica Identified by Polymerase Chain Reaction HIROSHI TACHIBANA,1* SEIJI IHARA,2 SEIKI KOBAYASHI,3 YOSHIMASA KANEDA,1 TSUTOMU TAKEUCHI,3 AND YASUSHI WATANABE2 Departments of Parasitology' and Molecular Biology,2 Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-11, and Department of Parasitology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160,3 Japan Received 4 March 1991/Accepted 18 July 1991

A Agtll cDNA library was constructed from the poly(A)+ RNA of trophozoites of Entamoeba histolytica HM-1:IMSS strain. The library was immunologically screened with monoclonal antibody 4G6, which is specific for the 30,000-Mr antigen of pathogenic isolates. A 0.7-kb clone was isolated, and its nucleotide sequence was determined. To examine whether this gene was specific for pathogenic isolates, a polymerase chain reaction was performed by using four sets of primers and the genomic DNA of pathogenic and nonpathogenic isolates as templates. Amplified DNAs were detected not only in pathogenic isolates but also in nonpathogenic isolates. However, when sequences of amplified DNA of these isolates were compared, minor differences were observed. By considering the presence or absence of recognition sites of some endonucleases, it was possible to distinguish between the pathogeniic and nonpathogenic isolates. When various isolates with different zymodemes were examined by polymnerase chain reaction and enzyme digestion, the results of typing were entirely in accord with those of zyknodeme analysis. These results indicate that there is dimorphism in the genomic DNA coding the 30,000-Mr antigen of E. histolytica and that the combined use of the polymerase chain reaction and enzyme digestion is a useful strategy for identification of species and determination of pathogenicity.

Entamoeba histolytica is a protozoan parasite infecting MATERIALS AND METHODS 500 million people worldwide and is associated with asymp- tomatic carriers and symptomatic diseases, such as hemor- Parasites and culture conditions. Trophozoites of patho- rhagic colitis and extraintestinal abscesses (28). Sargeaunt et genic strains of E. histolytica (HM-1:IMSS, HK-9 cll, H303: al. have demonstrated that pathogenic and nonpathogenic NIH, and Rahman) were axenically grown in TYI-S-33 isolates can be distinguished by differences in the electro- medium (5). Pathogenic strain NOT-1 cl2 was monoxenically phoretic patterns of isoenzymes (zymodemes) (20, 22). cultured in TYI-S-33 medium with epimastigotes of Trypano- Recently, monoclonal antibodies (MAbs) which distin- soma cruzi. Trophozoites of pathogenic strains SAW 408, guish between pathogenic and nonpathogenic isolates have SAW 1453, NOT-13, and NOT-25, and nonpathogenic been developed (15, 23). We have also reported that MAb strains SAW 142, NOT-33, and NOT-44 were xenically 4G6, produced against the pathogenic strain HM-1:IMSS of cultured in Robinson's medium (4, 16). Zymodemes of these E. histolytica, reacts only with isolates possessing patho- isolates were determined by the method of Sargeaunt et al. genic zymodemes, regardless of geographic origin and cul- (21). Trophozoites of the E. histolytica-like Laredo strain, ture conditions (24). Western blot (immunoblot) analyses Entamoeba hartmanni, and Entamoeba coli were also xen- have shown that the molecular weight of the component ically cultured in Robinson's medium. Among these para- recognized by the MAb is 30,000. sites, axenic strains of E. histolytica and E. histolytica-like However, zymodeme patterns and reactivities of MAbs Laredo were obtained from L. S. Diamond. SAW strains are phenotypic properties. Indeed, it has been reported that were provided by P. G. Sargeaunt. E. hartmanni, E. coli and zymodeme conversion from nonpathogenic to pathogenic (or the other strains of E. histolytica were isolated in our the reverse) can occur within a cloned culture of some laboratory. Parasites grown in Robinson's medium were strains of E. histolytica during the process of axenization isolated with Percoll as described previously (24). under appropriate growth conditions (1, 11-13). Therefore, Isolation of RNA and DNA. Total RNA of E. histolytica we cannot exclude the possibility that the gene encoding the HM-1:IMSS was recovered by disruption of trophozoites 30,000-Mr antigen may also exist in nonpathogenic isolates. with 5.5 M guanidinium isothiocyanate followed by sedimen- In the present study, we describe the molecular cloning tation in cesium trifluoroacetate (14). The poly(A)+ RNA and sequencing of cDNA coding the 30,000-Mr antigen. In subpopulation was purified on an oligo(dT)-cellulose affinity addition, by using the polymerase chain reaction (PCR) column (Pharmacia LKB, Uppsala, Sweden) (2). Trophozo- technique, we clarified the differences in genomic DNA ite DNA was isolated essentially by the procedure of Huber between pathogenic and nonpathogenic isolates of E. his- et al. (9). Briefly, nuclei were obtained by cell lysis in 1% tolytica. Nonidet P-40 in 10 mM phosphate-buffered saline, (pH 7.4) and then centrifuged at 500 x g at 4°C for 3 min. The pellet was lysed by shaking in a water bath for 2 h at 60°C in lysis buffer (100 mM NaCl, 10 mM Tris-HCI [pH 8.0], 10 mM EDTA, 0.5% sodium N-lauroyl sarcosinate and 0.5 mg of * Corresponding author. proteinase K per ml [20 U/mg]). The DNA was extracted 2234 VOL. 29, 1991 DISTINGUISHING BETWEEN E. HISTOLYTICA ISOLATES BY PCR 2235 twice with phenol-chloroform-isoamyl alcohol (25:24:1, vol/ vol/vol) and then was ethanol precipitated with sodium acetate (18). Construction of cDNA library. A cDNA library was constructed by using cDNA synthesis and Agtll cloning kits kb (Amersham) according to the manufacturer's protocol. The library was immunoscreened with mouse MAb 4G6 (18, 24). Positive clones were detected by using horseradish peroxi- 7.4- dase-labeled anti-mouse immunoglobulin G and Immuno- stain Kit HRP IS-50B (Konica, Tokyo, Japan) as the sub- 5.3- strate. This procedure was repeated until the clones were purified to 100%. Northern (RNA) blot analysis. Northern blot analysis was 2.8- performed by a standard technique (18). Poly(A)+ RNA (5 1.9 ,ug) was electrophoresed in a 1% agarose gel containing 2.2 M formaldehyde and then transferred to a Hybond-N nylon membrane (Amersham), according to the manufacturer's 1.0-g instructions. For a probe, a cDNA insert eluted from a low-melting-point agarose gel was labeled by a multiprime 0.6- DNA labeling system (Amersham), using [a-32P]dCTP (7). 0.4- PCR. Genomic DNA was amplified by PCR (17). The 0-3- reaction mixture contained 10 mM Tris-HCI (pH 8.3), 50 mM KCI, 1.5 mM MgCl2, 0.01% gelatin, 0.2 mM each of the four deoxynucleoside triphosphates, 1 1xM each of the two primers, 2.5 U of Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, Conn.), and genomic DNA as the template in a final volume of 100 RI. The mixture was overlaid with 100 ,ul of light mineral oil. The reactions were amplified for 30 cycles, using an automated bath-type PCR machine TSR-300 FIG. 1. Northern blot analysis of poly(A)+ RNA from E. his- (Iwaki Glass, Tokyo, Japan). Cycling conditions were as tolytica HM-1:IMSS strain with a 32P-labeled cDNA probe. The follows: melting at 94°C for 1 min, annealing at 55°C for 2 numbers to the left correspond to the sizes of the RNA markers. min, and polymerization at 72°C for 2 min. An initial denaturation step of 2 min at 94°C and a final polymerization step of 7 min at 72°C were also included. Portions (10 ,ul) of the amplified products were subjected to electrophoresis in blot analysis of poly(A)+ RNA with this insert revealed a 2% agarose gels, and the presence of specific bands was single band of approximately 0.9 kb (Fig. 1). This size was visualized with UV light after ethidium bromide staining. sufficient for encoding the 30,000-Mr antigen. Sequence Determination of nucleotide sequences. Sequencing was analysis of the insert revealed 714 bp, which included an performed by using the dideoxynucleotide chain termination open reading frame encoding 218 amino acids (Fig. 2). The method (19). A cDNA insert was subcloned into pBluescript translation product deduced from the amino acids had a size II SK+ (Stratagene, La Jolla, Calif.), and unidirectional of 24,500 Mr, which was equivalent to 82% of the 30,000 Mr deletion of the insert was carried out by controlled exonu- identified by Western blotting analysis (24). The nucleotide clease III digestion. Single-stranded DNA was prepared by sequence of this cDNA was almost identical to the sequence using VCSM13 as a helper phage and then sequenced by of a surface antigen of E. histolytica H302:NIH strain using [35S]dCTP and a Sequenase version 2.0 kit (U.S. reported by Torian et al. (27). Three cytosines in the cDNA Biochemicals, Cleveland, Ohio). Double-stranded DNA of the H302:NIH strain were replaced by adenine in the templates amplified by PCR were also sequenced by using HM-1:IMSS strain, as indicated in Fig. 2. However, the [_y-32P]ATP end-labeled primers (pl and p2) and Tth DNA predicted amino acid sequences were identical in both polymerase (Toyobo, Osaka, Japan). strains. Restriction endonuclease digestion. Restriction digests of PCR analysis of genomic DNA differences between patho- amplified DNA were performed according to the manufac- genic and nonpathogenic isolates. To determine whether the turer's recommendations with different endonucleases (To- DNA sequence coding the 30,000-M, antigen of HM-1:IMSS yobo). strain is specific to pathogenic isolates of E. histolytica, Nucleotide sequence accession number. The nucleotide genomic DNA derived from pathogenic and nonpathogenic sequence data reported here have been submitted to DDBJ, isolates was analyzed by PCR, using the four oligonucleotide EMBL, and GenBank Nucleotide Sequence Data Bases and primers shown in Table 1. Incubation of genomic DNA of assigned accession numbers D00871, D00872, and D01079. the HM-1:IMSS strain with four different pairs of primers (pl plus p3, pl plus p4, p2 plus p3, and p2 plus p4) yielded, after 30 PCR cycles, four differently sized products, as RESULTS expected from the cDNA structure (Fig. 3, lane 1). Interest- Cloning and sequencing of cDNA encoding the 30,000-M ingly, comparable amplified products were also detected antigen. The cDNA library containing 106 plaques was when genomic DNA from the SAW 142 strain (nonpatho- immunoscreened with MAb 4G6, and five positive clones genic, Z-I) of E. histolytica was used as the template (Fig. 3, were isolated. One of the clones, containing an EcoRI insert lane 2). Such PCR products were detected when genomic of approximately 0.7 kb, was subcloned into plasmid vector DNA from other strains showing various types of zymo- pBluescript II SK+ and used for further analysis. Northern demes, namely, HK-9 cll (Z-II), H303:NIH (Z-II), Rahman 2236 TACHIBANA ET AL. J. CLIN. MICROBIOL.

1 0 20 30 40 M 1 2 34 56 AA GAG AAA GAA TGT TGT AAA GAA TGT TGT TGT CCA AGA ATA AAA GCA Glu Lys Glu Cys Cys Lys Glu Cys Cys Cys Pro Arg Ilie Lys Ala A 50 60 70 80 90 TTT AAG AAA TTT ATA AAC ACA TTT GAA AAA GCA CAA ATT GGA AAA GAA Phe Lys Lys Phe Ilie Asn Thr Phe Glu Lys Ala Gin Ilie Gly Lys Glu ylOO 110 120 130 140 GCA CCA GAA TTT AAA GCA CCA GCA TAT TGT CCA TGT GGT TCA ATC AAA Ala Pro Giu Phe Lys Ala Pro Ala Tyr Cys Pro Cys Gly Ser Ilie Lys B 150 160 170 180 190 GAG ATT GAT ATT AAT GAA TAT AAA GGA AAA TAT GTT GTA TTG TTG TTT Glu Ilie Asp Ilie Asn Glu Tyr Lys Gly Lys Tyr Val Val Leu Leu Phe 200 210 220 230 TAT CCA TTG GAT TGG ACA TTT GTT TGT CCA ACA GAA ATG ATT GGA TAT Tyr Pro Leu Asp Trp Thr Phe Val Cys Pro Thr Glu Met Ilie Gly Tyr C

NO0 250 260 270 280 3 i54 AGT GAA CTT GCA GGA CAA TTG AAA GAA ATC AAT TGT GAA GTT ATT GGA Ser Giu Leu Ala Gly Gin Leu Lys Giu Ilie Asn Cys Glu Val Ilie Giy 290 300 310 320 330 GTG AGT GTA GAT TCA GTT TAT TGT CAT CAA GCA TGG TGT GAA GCA GAT Val Ser Val Asp Ser Val Tyr Cys His Gin Ala Trp Cys Glu Ala Asp D 340 350 360 370 380 AAA AGT AAA GGA GGA GTA GGA AAG TTG ACA TTC CCA TTA GTA TCA GAT Lys Ser Lys Gly Giy Vai Giy Lys Leu Thr Phe Pro Leu Val Ser Asp 390 400 410 420 430 ATT AAG AGA TGC ATT TCT ATC AAA TAT GGA ATG TTA AAT GTC GAA GCA Ilie Lys Arg Cys Ilie Ser Ilie Lys Tyr Gly Met Leu Asn Vai Giu Ala FIG. 3. Agarose gel separation of PCR products amplified by 440 450 460 470 using different sets of primers. The primer pairs were p1 plus p4 (A), GGA ATT GCA AGA AGA GGA TAT GTC ATC ATT GAT GAT AAA GGA AAA GTA p1 plus p3 (B), p2 plus p4 (C), and p2 plus p3 (D). Different template Giy Ilie Ala Arg Arg Giy Tyr Val Ilie Ilie Asp Asp Lys Gly Lys Val DNAs were used. Lanes: 1, E. histolytica HM-1:IMSS; 2, E. 480 490 500 510 520 SAW 142; 3, E. histolytica-like Laredo; 4, E. hartmanni; AGA TAC ATT CAA ATG AAT GAT GAT GGA ATT GGA AGA TCA ACG GAA GAA histolytica Arg Tyr Ilie Gin Met Asn Asp Asp Gly Ilie Gly Arg Ser Thr Glu Glu 5, E. coli; 6, no template DNA; M, size markers (Hincll-cleaved The and sizes of PCR products are indicated to 530 540 550 560 570 4OX174). positions ACA ATC AGA ATA GTT AAA GCA ATT CAA TTC AGT GAT GAA CAT GGA GCA the right of the gel. Thr Ilie Arg Ilie Vai Lys Ala Ilie Gin Phe Ser Asp Glu His Giy Ala 580 590 600 610 620 GTT TGT CCA CTC AAT TGG AAA CCA GGC AAA GAC ACC ATT GAA CCA ACA Val Cys Pro Leu Asn Trp Lys Pro Gly Lys Asp Thr Ilie Glu Pro Thr guishing between E. histolytica and other Entamoeba spe- 630 640 650 660 670 cies. CCA GAT GGA ATT AAG AAA TAT TTA ACA GCA CAT TAA AACAAACAAGATAATT To evaluate the homology of the nucleotide sequence Pro Asp Gly Ilie Lys Lys Tyr Leu Thr Ala His between pathogenic and nonpathogenic isolates, PCR-ampli- 680 690 700 710 TAATACAAATTATTTTAAAAAAAAAAAAAAAAAAAAAAA fled products of the genomic DNA for HM-1:IMSS and SAW II 142 were sequenced. The results showed the substitution of FIG. 2. Nucleotide sequence of the cloned cDNA and the pre- 22 nucleotides in the 399-bp sequences which were com- dicted amino acid sequence of the coding region. Asterisks indicate pared (5.5%) (Fig. 4A). Of the inferred amino acids, 4.5% (6 the translation stop codon. Arrowheads indicate the nucleotides of 132) were different between these strains (Fig. 4B). The different from the sequence reported by Torian et al. (27). DNA sequence of another nonpathogenic strain, NOT-44 (Z-VIII), was identical with that of the SAW 142 strain. As shown by the arrowhead in Fig. 4A, a single-base discrepancy was observed between the cloned cDNA and the (Z-II), SAW 408 (Z-II), NOT-25 (Z-VII), NOT-13 (Z-XI), PCR-amplified genomic DNA of HM-1:IMSS strain. This SAW 1453 (Z-XIV), NOT-i c12 (Z-XIX), NOT-33 (Z-VIII), resulted in an amino acid change from lysine to arginine. and N-OT-44 (Z-VIII), were used as templates (data not On the basis of DNA differences between pathogenic and shown).' Although incubation of genomic DNA from the E. nonpathogenic isolates, four restriction endonucleases were histolytica-like Laredo strain with primers p1 plus p3 or p2 selected to distinguish between these isolates. As expected, plus p3 p'roduced amplified products, incubation with prim- Hincll, EcoT221, and TaqI digested amplified DNAs from ers p1 plus p4 or p2 plus p4 did not (Fig. 3, lane 3), nor did pathogenic isolates showing different zymodeme patterns the genomic DNA of E. hartmanni and E. coli w'hen incu- but did not cut amplified DNAs from isolates possessing bated 'With the four sets of primers (Fig. 3, lanes 4 and 5). nonpathogenic zymodemes (Fig. SB to D). In contrast, Hinfl These results indicate that incubation of genomic DNA with digested PCR products of both pathogenic and nonpatho- primers p1 plus p4 or p2 plus p4 can be useful for distin- genic isolates, yielding two and three fragments, respec- tively (Fig. 5E).

TABLE 1. Oligonucleotide primers used for PCR DISCUSSION Corresponding The cDNA sequence of the 30,000-Mr antigen reported Primer Sequence Direction ncletDNA here was highly homologous with that of the 29-kDa cys- (bp) teine-rich surface antigen reported by Torian et al. (27). In the overlapping regions, we found only three nucleotide p1 5'TAAAGCACCAGCATATTGTC3' Sense 107-126 substitutions (one in the coding region and two in the p2 5'GTGAAGTTATTGGAGTGAGT3' Sense 274-293 but no differences in the deduced amino p3 5'GATGACATATCCTCTTCTTG3' Antisense 458-439 noncoding region) p4 5'TTAATTCCATCTGGTGTTGG3' Antisense 637-618 acids. In addition, Northern blot analysis demonstrated single bands in each study, although their sizes were slightly VOL. 29, 1991 DISTINGUISHING BETWEEN E. HISTOLYTICA ISOLATES BY PCR 2237 A 10 20 30 v 40 50 60 A TGGTTCAATCAAAGAGATTGATATTAATGAATATAGAGGAAAATATGTTGTATTGTTGTT *************** ******************* *** *********** ** ** ** TGGTTCAATCAAAGAAATTGATATTAATGAATATAAAGGGAAATATGTTGTGTTATTATT 10 20 30 40 50 60 70 80 90 100 110 120 TTATCCATTGGATTGGACATTTGTTTGTCCAACAGAAATGATTGGATATAGTGAACTTGC ******************************************************* **** B TTATCCATTGGATTGGACATTTGTTTGTCCAACAGAAATGATTGGATATAGTGAAGTTGC 70 80 90 100 110 120 1 130 140 150 160 HinfH 170 180 AGGACAATTGAAAGAAATCAATTGTGAAGTTATTGGAGTGAGTGTAGATTCAGTTTATTG ********************************************* ************** AGGACAATTGAAAGAAATCAATTGTGAAGTTATTGGAGTGAGTGTTGATTCAGTTTATTG C 130 140 150 160 HHinf 170 180 190 200 210 220 230Hincl 240 TCATCAAGCATGGTGTGAAGCAGATAAAAGTAAAGGAGGAGTAGGAAAGTTGACATTCCC ************************************************ ** ****** TCATCAAGCATGGTGTGAAGCAGATAAAAGTAAAGGAGGAGTAGGAAAATTAGGATTCCC 190 200 210 220 230 t 240 Hint D 250250 260E260~EcoT221OT221270 280 290 Taqlj 300 ATTAGTATCAGATATTAAGAGATGCATTTCTATCAAATATGGAATGTTAAATGTCGAAGC ****************** ***** ***** ** ******************** *** * ATTAGTATCAGATATTAAAAGATGTATTTCAATTAAATATGGAATGTTAAATGTAGAAAC 250 260 270 280 290 300 310 320 330 340 350 360 AGGAATTGCAAGAAGAGGATATGTCATCATTGATGATAAAGGAAAAGTAAGATACATTCA E **** ** ******************* ******************************** AGGAGTTTCAAGAAGAGGATATGTCATTATTGATGATAAAGGAAAAGTAAGATACATTCA 310 320 330 340 350 360 370 380 390 400 AATGAATGATGATGGAATTGGAAGATCAACGGAAGAAAC ****************************** ******** AATGAATGATGATGGAATTGGAAGATCAACAGAAGAAAC FIG. 5. Agarose gel separation of restriction endonuclease di- 370 380 390 400 gests of PCR products. The PCR products were undigested (A) or were digested with HincIl (B), EcoT22I (C), TaqI (D), or Hinfl (E). B Template DNA from various strains of E. histolytica was amplified 10 20 30 40 50 60 GSIKEIDINEYRGKYVVLLFYPLDWTFVCPTEMIGYSELAGQLKEINCEVIGVSVDSVYC by using primers pl plus p4. Lanes: 1, HM-1:IMSS (Z-II, patho- *********** ************************** ********************* genic, axenic); 2, HK-9 cli (Z-II, pathogenic, axenic); 3, H303:NIH GSIKEIDINEYKGKYVVLLFYPLDWTFVCPTEMIGYSEVAGQLKEINCEVIGVSVDSVYC (Z-II, pathogenic, axenic); 4, Rahman (Z-II, pathogenic, axenic); 5, 10 20 30 40 50 60 SAW 408 (Z-II, pathogenic, xenic); 6, NOT-25 (Z-VII, pathogenic, 70 80 90 100 110 120 xenic); 7, NOT-13 (Z-XI, pathogenic, xenic); 8, SAW 1453 (Z-XIV, HQAWCEADKSKGGVGKLTFPLVSDIKRCISIKYGMLNVEAGIARRGYVIIDDKGKVRYIQ ***************** ********************* * ***************** pathogenic, xenic); 9, NOT-1 cl2 (Z-XIX, pathogenic, monoxenic); HQAWCEADKSKGGVGKLGFPLVSDIKRCISIKYGMLNVETGVSRRGYVIIDDKGKVRYIQ 10, SAW 142 (Z-III [originally, but presently Z-I], nonpathogenic, 70 80 90 100 110 120 xenic]; 11, NOT-33 (Z-VIII, nonpathogenic, xenic); 12, NOT-44 1 30 140 (Z-VIII, nonpathogenic, xenic); M, size markers (HincIl-cleaved MNDDGIGRSTEE 4X174). Arrows indicate the positions of digested fragments. MNDDGIGRSTEE 1 30 1 40 FIG. 4. Comparison of the nucleotide sequence (A) and the amino acid sequence (B) of the genomic DNA from the HM-1:IMSS 4G6 may be composed of a few amino acids specific for (top line) and SAW 142 (bottom line) strains of E. histolytica. pathogenic isolates and, as shown previously, MAb 4G6 is Identity is indicated by asterisks. Arrows indicate the cutting sites of very useful for the identification of pathogenic isolates restriction endonucleases. Arrowheads indicate discrepancies in the regardless of zymodemes, geographic origins, culture condi- nucleotide and deduced amino acid sequences of the genomic DNA tions, or host symptoms (24). of HM-1:IMSS, in comparison with those of the cDNA shown in It is important to determine whether the differences be- Fig. 2. tween pathogenic and nonpathogenic strains are phenotypic or genotypic because zymodeme conversion has been reported in a few isolates (1, 11-13). Recently, DNA probes different. These observations demonstrate that both genes specific for either pathogenic or nonpathogenic strains have encode identical proteins. However, while the 29-kDa anti- been developed (3, 8, 26). More recently, Edman et al. (6) gen was demonstrated to be a surface antigen, we could not demonstrated a significant diversity in nucleotide sequences detect the epitope recognized by MAb 4G6 on the cell between pathogenic and nonpathogenic strains in the 125- surface (24). This discrepancy suggests that the molecule kDa antigen of E. histolytica. The reported variability in recognized by MAb 4G6 either is a precursor of the surface amino acid sequences among different isolates of strain antigen or is a transmembrane protein recognized from HM-1:IMSS was 1%, whereas that between pathogenic and within the cell. nonpathogenic strains was 12 to 13% in the 1.4-kb fragments The amino acid differences in the sequences of the that have been compared. In the present study, although the 30,000-Mr component of the pathogenic and nonpathogenic regions compared were limited to about 400 bp, the variabil- strains that have been compared amounted to only 4.5%. ity in amino acid sequences between pathogenic and non- Indeed, Torian et al. (27) observed that some of the MAbs pathogenic strains was 4.5%, with no diversity observed prepared against the fusion protein with the C terminus of among their respective isolates. This indicates that there is glutathione S-transferase were also reactive with nonpatho- dimorphism in the amino acid sequence of the 30,000-Mr genic isolates. Therefore, the epitope recognized by MAb antigen and the identification of the gene encoding this 2238 TACHIBANA ET AL. J. CLIN. MICROBIOL. antigen is valuable for distinguishing between pathogenic 5. Diamond, L. S., D. R. Harlow, and C. C. Cunnick. 1978. A new and nonpathogenic strains. medium for the axenic cultivation of Entamoeba histolytica and In our observations, direct sequencing of PCR products other Entamoeba. Trans. R. Soc. Trop. Med. Hyg. 72:431-432. 6. Edman, U., M. A. Meraz, S. Rausser, N. Agabian, and I. Meza. from the noncloned genomic DNA of HM-1:IMSS strain 1990. Characterization of an immuno-dominant variable surface showed a single-base discrepancy with a cloned cDNA antigen from pathogenic and nonpathogenic Entamoeba his- sequence. A similar observation has been reported in an- tolytica. J. Exp. Med. 172:879-888. other gene of E. histolytica (10). Such a mismatch may be 7. Feinberg, A. P., and B. Vogelstein. 1983. A technique for due to the misincorporation of nucleotides by Taq or Tth or radiolabeling DNA restriction endonuclease fragments to high both DNA polymerases. However, when the cDNA cloned specific activity. Anal. Biochem. 132:6-13. in Agtll was PCR amplified and then directly sequenced by 8. Garfinkel, L. I., M. Giladi, M. Huber, C. Gitler, D. Mirelman, Tth polymerase, the results were in accord with those in Fig. M. Revel, and S. Rozenblatt. 1989. DNA probes specific for 2. On the other hand, the direct sequencing of PCR-amplified Entamoeba histolytica possessing pathogenic and nonpatho- identical genic zymodemes. Infect. Immun. 57:926-931. products from the cDNA library yielded results 9. Huber, M., L. Garfinkel, C. Gitler, D. Mirelman, M. Revel, and with those from the genomic DNA (data not shown). There- S. Rozenblatt. 1987. Entamoeba histolytica: cloning and char- fore, the possibility that two populations exist in the genomic acterization of actin cDNA. Mol. Biochem. Parasitol. 24:227- DNA cannot be ruled out at present. 235. Edman et al. (6) also demonstrated that PCR amplification 10. Mann, B. J., B. E. Torian, T. S. Vedvick, and W. A. Petri, Jr. of DNA coding the 125-kDa antigen of E. histolytica, fol- 1991. Sequence of a cysteine-rich galactose-specific lectin of lowed by SspI or AccI digestion, showed a distinct differ- Entamoeba histolytica. Proc. Natl. Acad. Sci. USA 88:3248- ence between pathogenic and nonpathogenic isolates. How- 3252. ever, by this procedure, the nonpathogenic E. histolytica- 11. Mirelman, D. 1987. Effect of culture conditions and bacterial like as More associates on the zymodemes of Entamoeba histolytica. Para- Laredo strain is identified pathogenic. recently, sitol. Today 3:37-40. Tannich and Burchard (25) reported that amplification of the 12. Mirelman, D., R. Bracha, A. Chayen, A. Aust-Kettis, and L. S. same gene by using other primers and subsequent digestion Diamond. 1986. Entamoeba histolytica: effect of growth condi- with AccI, TaqI, and XmnI is useful to distinguish between tions and bacterial associates on isoenzyme patterns and viru- isolates of E. histolytica. It has not yet been examined lence. Exp. Parasitol. 62:142-148. whether other human parasitic amoebae may induce errone- 13. Mirelman, D., R. Bracha, A. Wexler, and A. Chayen. 1986. ous amplification products. The present study indicated that Changes in isoenzyme patterns of a cloned culture of nonpath- partial amplification of the genomic DNA coding the ogenic Entamoeba histolytica during axenization. Infect. Im- 30,000-Mr antigen by PCR is also useful for discriminating mun. 54:827-832. between E. and the E. Laredo 14. Okayama, H., M. Kawaichi, M. Brownstein, F. Lee, T. Yokota, histolytica histolytica-like and K. Arai. 1987. High-efficiency cloning of full-length cDNA: strain. Since it is difficult to cultivate nonpathogenic isolates construction and screening of cDNA expression libraries for axenically, we could not avoid contamination by bacterial mammalian cells. Methods Enzymol. 154:3-28. DNA in materials isolated from the parasites grown under 15. Petri, W. A., Jr., T. F. H. G. Jackson, V. Gathiram, K. Kress, xenic conditions. However, PCR technology permits easy L. D. Saffer, T. L. Snodgrass, M. D. Chapman, Z. Keren, and D. amplification of the genomic DNA of E. histolytica for Mirelman. 1990. Pathogenic and nonpathogenic strains of Enta- identification and further analysis. Although it is not clear at moeba histolytica can be differentiated by monoclonal antibod- present whether the 30,000-Mr antigen of pathogenic strains ies to the galactose-specific adherence lectin. Infect. Immun. is responsible for pathogenicity, it should be stressed that 58:1802-1806. differentiating between pathogenic and nonpathogenic 16. Robinson, G. L. 1968. The laboratory diagnosis of human strains on the basis of the endonuclease of PCR- parasitic amoebae. Trans. R. Soc. Trop. Med. Hyg. 62:285-294. digestion 17. Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, amplified DNA is completely in accord with zymodeme G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primer- analysis. directed enzyme amplification of DNA with a thermostable DNA polymerase. Science 239:487-491. ACKNOWLEDGMENTS 18. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor We are grateful to Y. Kawauchi for technical assistance and to W. Laboratory, Cold Spring Harbor, N.Y. Stahl for reviewing the manuscript. 19. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc- This work was supported by Grants-in Aid for Scientific Research ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. from the Ministry of Education, Science and Culture of Japan, USA 74:5463-5467. research aid from the Tokai University School of Medicine, and a 20. Sargeaunt, P. G. 1988. Zymodemes of Entamoeba histolytica, p. grant from the General Organization of Research in Tokai Univer- 370-387. In J. I. Ravdin (ed.), Amebiasis: human infection by sity to H.T. Entamoeba histolytica. John Wiley & Sons, Inc., New York. 21. Sargeaunt, P. G., U. K. Baveja, R. Nanda, and B. S. Anand. REFERENCES 1984. Influence of geographical factors in the distribution of 1. Andrews, B. J., L. Mentzoni, and B. Bjorvatn. 1990. Zymodeme pathogenic zymodemes of Entamoeba histolytica: identification conversion of isolates of Entamoeba histolytica. Trans. R. Soc. of zymodeme XIV in India. Trans. R. Soc. Trop. Med. Hyg. Trop. Med. Hyg. 84:63-65. 78:96-101. 2. Aviv, H., and P. Leder. 1972. Purification of biologically active 22. Sargeaunt, P. G., J. E. Williams, and J. D. Grene. 1978. The globin messenger RNA by chromatography on oligothymidylic differentiation of invasive and non-invasive Entamoeba histolyt- acid-cellulose. Proc. Natl. Acad. Sci. USA 69:1408-1412. ica by isozyme electrophoresis. Trans. R. Soc. Trop. Med. 3. Bracha, R., L. S. Diamond, J. P. Ackers, G. D. Burchard, and D. Hyg. 72:519-521. Mirelman. 1990. Differentiation of clinical isolates of Entamoe- 23. Strachan, W. D., P. L. Chiodini, W. M. Spice, A. H. Moody, and ba histolytica by using specific DNA probes. J. Clin. Microbiol. J. P. Ackers. 1988. Immunological differentiation of pathogenic 28:680-684. and non-pathogenic isolates of Entamoeba histolytica. Lancet 4. Diamond, L. S. 1987. Entamoeba, Giardia and Trichomonas, p. i:561-563. 1-28. In A. E. R. Taylor and J. R. Baker (ed.), In vitro methods 24. Tachibana, H., S. Kobayashi, Y. Kato, K. Nagakura, Y. Kaneda, for parasite cultivation. Academic Press Ltd., London. and T. Takeuchi. 1990. Identification of a pathogenic isolate- VOL. 29, 1991 DISTINGUISHING BETWEEN E. HISTOLYTICA ISOLATES BY PCR 2239

specific 30,000Mr antigen of Entamoeba histolytica by using a 86:5118-5122. monoclonal antibody. Infect. Immun. 58:955-960. 27. Torian, B. E., B. M. Flores, V. L. Stroeher, F. S. Hagen, and 25. Tannich, E., and G. D. Burchard. 1991. Differentiation of W. E. Stamm. 1990. cDNA sequence analysis of a 29-kDa pathogenic from nonpathogenic Entamoeba histolytica by re- cysteine-rich surface antigen of pathogenic Entamoeba histolyt- striction fragment analysis of a single gene amplified in vitro. J. ica. Proc. Natl. Acad. Sci. USA 87:6358-6362. Clin. Microbiol. 29:250-255. 28. Walsh, J. A. 1988. Prevalence of Entamoeba histolytica infec- 26. Tannich, E., R. D. Horstmann, J. Knobloch, and H. H. Arnold. tion, p. 93-105. In J. I. Ravdin (ed.), Amebiasis: human infec- 1989. Genomic DNA differences between pathogenic and non- tion by Entamoeba histolytica. John Wiley & Sons, Inc., New pathogenic Entamoeba histolytica. Proc. Natl. Acad. Sci. USA York.