Vol. 29, No. 10
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1991, p. 2234-2239
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 500 million people worldwide and is associated with asymptomatic carriers and symptomatic diseases, such as hemorrhagic colitis and extraintestinal abscesses (28). Sargeaunt et al. have demonstrated that pathogenic and nonpathogenic isolates can be distinguished by differences in the electrophoretic patterns of isoenzymes (zymodemes) (20, 22). Recently, monoclonal antibodies (MAbs) which distinguish between pathogenic and nonpathogenic isolates have been developed (15, 23). We have also reported that MAb 4G6, produced against the pathogenic strain HM-1:IMSS of E. histolytica, reacts only with isolates possessing pathogenic zymodemes, regardless of geographic origin and culture conditions (24). Western blot (immunoblot) analyses have shown that the molecular weight of the component recognized by the MAb is 30,000. However, zymodeme patterns and reactivities of MAbs are phenotypic properties. Indeed, it has been reported that zymodeme conversion from nonpathogenic to pathogenic (or the reverse) can occur within a cloned culture of some strains of E. histolytica during the process of axenization under appropriate growth conditions (1, 11-13). Therefore, we cannot exclude the possibility that the gene encoding the 30,000-Mr antigen may also exist in nonpathogenic isolates. In the present study, we describe the molecular cloning and sequencing of cDNA coding the 30,000-Mr antigen. In addition, by using the polymerase chain reaction (PCR) technique, we clarified the differences in genomic DNA between pathogenic and nonpathogenic isolates of E. histolytica.
*
Corresponding author. 2234
MATERIALS AND METHODS Parasites and culture conditions. Trophozoites of pathogenic strains of E. histolytica (HM-1:IMSS, HK-9 cll, H303: NIH, and Rahman) were axenically grown in TYI-S-33 medium (5). Pathogenic strain NOT-1 cl2 was monoxenically cultured in TYI-S-33 medium with epimastigotes of Trypanosoma cruzi. Trophozoites of pathogenic strains SAW 408, SAW 1453, NOT-13, and NOT-25, and nonpathogenic strains SAW 142, NOT-33, and NOT-44 were xenically cultured in Robinson's medium (4, 16). Zymodemes of these isolates were determined by the method of Sargeaunt et al. (21). Trophozoites of the E. histolytica-like Laredo strain, Entamoeba hartmanni, and Entamoeba coli were also xenically cultured in Robinson's medium. Among these parasites, axenic strains of E. histolytica and E. histolytica-like Laredo were obtained from L. S. Diamond. SAW strains were provided by P. G. Sargeaunt. E. hartmanni, E. coli and the other strains of E. histolytica were isolated in our laboratory. Parasites grown in Robinson's medium were isolated with Percoll as described previously (24). Isolation of RNA and DNA. Total RNA of E. histolytica HM-1:IMSS was recovered by disruption of trophozoites with 5.5 M guanidinium isothiocyanate followed by sedimentation in cesium trifluoroacetate (14). The poly(A)+ RNA subpopulation was purified on an oligo(dT)-cellulose affinity column (Pharmacia LKB, Uppsala, Sweden) (2). Trophozoite DNA was isolated essentially by the procedure of Huber et al. (9). Briefly, nuclei were obtained by cell lysis in 1% 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 proteinase K per ml [20 U/mg]). The DNA was extracted
VOL. 29, 1991
DISTINGUISHING BETWEEN E. HISTOLYTICA ISOLATES BY PCR
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 (Amersham) according to the manufacturer's protocol. The library was immunoscreened with mouse MAb 4G6 (18, 24). Positive clones were detected by using horseradish peroxidase-labeled anti-mouse immunoglobulin G and Immunostain Kit HRP IS-50B (Konica, Tokyo, Japan) as the substrate. This procedure was repeated until the clones were purified to 100%. Northern (RNA) blot analysis. Northern blot analysis was performed by a standard technique (18). Poly(A)+ RNA (5 ,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 instructions. For a probe, a cDNA insert eluted from a low-melting-point agarose gel was labeled by a multiprime DNA labeling system (Amersham), using [a-32P]dCTP (7). PCR. Genomic DNA was amplified by PCR (17). The 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 (Iwaki Glass, Tokyo, Japan). Cycling conditions were as follows: melting at 94°C for 1 min, annealing at 55°C for 2 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 2% agarose gels, and the presence of specific bands was visualized with UV light after ethidium bromide staining. Determination of nucleotide sequences. Sequencing was performed by using the dideoxynucleotide chain termination method (19). A cDNA insert was subcloned into pBluescript II SK+ (Stratagene, La Jolla, Calif.), and unidirectional deletion of the insert was carried out by controlled exonuclease III digestion. Single-stranded DNA was prepared by using VCSM13 as a helper phage and then sequenced by using [35S]dCTP and a Sequenase version 2.0 kit (U.S. Biochemicals, Cleveland, Ohio). Double-stranded DNA templates amplified by PCR were also sequenced by using [_y-32P]ATP end-labeled primers (pl and p2) and Tth DNA polymerase (Toyobo, Osaka, Japan). Restriction endonuclease digestion. Restriction digests of amplified DNA were performed according to the manufacturer's recommendations with different endonucleases (To-
yobo).
Nucleotide sequence accession number. The nucleotide sequence data reported here have been submitted to DDBJ, EMBL, and GenBank Nucleotide Sequence Data Bases and assigned accession numbers D00871, D00872, and D01079.
RESULTS Cloning and sequencing of cDNA encoding the 30,000-M antigen. The cDNA library containing 106 plaques was immunoscreened with MAb 4G6, and five positive clones were isolated. One of the clones, containing an EcoRI insert of approximately 0.7 kb, was subcloned into plasmid vector pBluescript II SK+ and used for further analysis. Northern
2235
kb 7.4-
5.3-
2.81.9
1.0-g
0.60.40-3-
FIG. 1. Northern blot analysis of poly(A)+ RNA from E. histolytica HM-1:IMSS strain with a 32P-labeled cDNA probe. The numbers to the left correspond to the sizes of the RNA markers.
blot analysis of poly(A)+ RNA with this insert revealed a single band of approximately 0.9 kb (Fig. 1). This size was sufficient for encoding the 30,000-Mr antigen. Sequence analysis of the insert revealed 714 bp, which included an open reading frame encoding 218 amino acids (Fig. 2). The translation product deduced from the amino acids had a size of 24,500 Mr, which was equivalent to 82% of the 30,000 Mr identified by Western blotting analysis (24). The nucleotide sequence of this cDNA was almost identical to the sequence of a surface antigen of E. histolytica H302:NIH strain reported by Torian et al. (27). Three cytosines in the cDNA of the H302:NIH strain were replaced by adenine in the HM-1:IMSS strain, as indicated in Fig. 2. However, the predicted amino acid sequences were identical in both strains. PCR analysis of genomic DNA differences between pathogenic and nonpathogenic isolates. To determine whether the DNA sequence coding the 30,000-M, antigen of HM-1:IMSS strain is specific to pathogenic isolates of E. histolytica, genomic DNA derived from pathogenic and nonpathogenic isolates was analyzed by PCR, using the four oligonucleotide primers shown in Table 1. Incubation of genomic DNA of 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 expected from the cDNA structure (Fig. 3, lane 1). Interestingly, comparable amplified products were also detected when genomic DNA from the SAW 142 strain (nonpathogenic, Z-I) of E. histolytica was used as the template (Fig. 3, lane 2). Such PCR products were detected when genomic DNA from other strains showing various types of zymodemes, namely, HK-9 cll (Z-II), H303:NIH (Z-II), Rahman
2236
J. CLIN. MICROBIOL.
TACHIBANA ET AL.
40 30 20 10 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
M 1 2 34 56
A
90 80 70 60 50 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 140 130 120 110 ylOO 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
190 180 170 160 150 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
230 220 210 200 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
280 270 260 250 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
NO0
330 320 310 300 290 GTG AGT GTA GAT TCA GTT TAT TGT CAT CAA GCA TGG TGT GAA GCA GAT Ala Asp Glu Gin Ala Val Ser Val Asp Ser Val Tyr Cys His Trp Cys
3 i54
D
380 370 360 350 340 AAA AGT AAA GGA GGA GTA GGA AAG TTG ACA TTC CCA TTA GTA TCA GAT Phe Leu Val Ser Asp Thr Pro Lys Ser Lys Gly Giy Vai Giy Lys Leu
430 420 410 400 390 ATT AAG AGA TGC ATT TCT ATC AAA TAT GGA ATG TTA AAT GTC GAA GCA Vai Ala Met Leu Asn Giu Ser Ilie Ilie Ilie Lys Arg Cys Lys Tyr Gly 470 460 440 450 GGA ATT GCA AGA AGA GGA TAT GTC ATC ATT GAT GAT AAA GGA AAA GTA Giy Ilie Ala Arg Arg Giy Tyr Val Ilie Ilie Asp Asp Lys Gly Lys Val
510 520 500 490 480 AGA TAC ATT CAA ATG AAT GAT GAT GGA ATT GGA AGA TCA ACG GAA GAA Arg Tyr Ilie Gin Met Asn Asp Asp Gly Ilie Gly Arg Ser Thr Glu Glu 560 570 550 540 530 ACA ATC AGA ATA GTT AAA GCA ATT CAA TTC AGT GAT GAA CAT GGA GCA Thr Ilie Arg Ilie Vai Lys Ala Ilie Gin Phe Ser Asp Glu His Giy Ala
620 600 610 590 580 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 670 660 640 650 630 CCA GAT GGA ATT AAG AAA TAT TTA ACA GCA CAT TAA AACAAACAAGATAATT Pro Asp Gly Ilie Lys Lys Tyr Leu Thr Ala His
710 680 690 700 TAATACAAATTATTTTAAAAAAAAAAAAAAAAAAAAAAA
II
FIG. 2. Nucleotide sequence of the cloned cDNA and the predicted amino acid sequence of the coding region. Asterisks indicate the translation stop codon. Arrowheads indicate the nucleotides different from the sequence reported by Torian et al. (27).
(Z-II), SAW 408 (Z-II), NOT-25 (Z-VII), NOT-13 (Z-XI), SAW 1453 (Z-XIV), NOT-i c12 (Z-XIX), NOT-33 (Z-VIII), and N-OT-44 (Z-VIII), were used as templates (data not shown).' Although incubation of genomic DNA from the E. histolytica-like Laredo strain with primers p1 plus p3 or p2 plus p3 p'roduced amplified products, incubation with primers p1 plus p4 or p2 plus p4 did not (Fig. 3, lane 3), nor did the genomic DNA of E. hartmanni and E. coli w'hen incubated 'With the four sets of primers (Fig. 3, lanes 4 and 5). These results indicate that incubation of genomic DNA with primers p1 plus p4 or p2 plus p4 can be useful for distin-
FIG. 3. Agarose gel separation of PCR products amplified by using different sets of primers. The primer pairs were p1 plus p4 (A), p1 plus p3 (B), p2 plus p4 (C), and p2 plus p3 (D). Different template DNAs were used. Lanes: 1, E. histolytica HM-1:IMSS; 2, E. histolytica SAW 142; 3, E. histolytica-like Laredo; 4, E. hartmanni; 5, E. coli; 6, no template DNA; M, size markers (Hincll-cleaved The positions and sizes of PCR products are indicated to 4OX174). the right of the gel.
guishing between E. histolytica and other Entamoeba species. To evaluate the homology of the nucleotide sequence between pathogenic and nonpathogenic isolates, PCR-amplifled products of the genomic DNA for HM-1:IMSS and SAW 142 were sequenced. The results showed the substitution of 22 nucleotides in the 399-bp sequences which were compared (5.5%) (Fig. 4A). Of the inferred amino acids, 4.5% (6 of 132) were different between these strains (Fig. 4B). The 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 PCR-amplified genomic DNA of HM-1:IMSS strain. This resulted in an amino acid change from lysine to arginine. On the basis of DNA differences between pathogenic and nonpathogenic isolates, four restriction endonucleases were selected to distinguish between these isolates. As expected, Hincll, EcoT221, and TaqI digested amplified DNAs from pathogenic isolates showing different zymodeme patterns but did not cut amplified DNAs from isolates possessing nonpathogenic zymodemes (Fig. SB to D). In contrast, Hinfl digested PCR products of both pathogenic and nonpathogenic isolates, yielding two and three fragments, respec-
tively (Fig. 5E). TABLE 1. Oligonucleotide primers used for PCR Corresponding Primer
p1 p2 p3 p4
Sequence 5'TAAAGCACCAGCATATTGTC 3' 5'GTGAAGTTATTGGAGTGAGT 3' 5'GATGACATATCCTCTTCTTG 3' 5'TTAATTCCATCTGGTGTTGG 3'
Direction
Sense Sense Antisense Antisense
ncletDNA (bp) 107-126 274-293 458-439 637-618
DISCUSSION The cDNA sequence of the 30,000-Mr antigen reported here was highly homologous with that of the 29-kDa cysteine-rich surface antigen reported by Torian et al. (27). In the overlapping regions, we found only three nucleotide substitutions (one in the coding region and two in the noncoding region) but no differences in the deduced amino acids. In addition, Northern blot analysis demonstrated single bands in each study, although their sizes were slightly
DISTINGUISHING BETWEEN E. HISTOLYTICA ISOLATES BY PCR
VOL. 29, 1991
A 60 v 40 50 30 20 10 TGGTTCAATCAAAGAGATTGATATTAATGAATATAGAGGAAAATATGTTGTATTGTTGTT ***************
*******************
***********
***
2237
A
** **
**
TGGTTCAATCAAAGAAATTGATATTAATGAATATAAAGGGAAATATGTTGTGTTATTATT 10
30
20
60
50
40
120 110 100 90 80 70 TTATCCATTGGATTGGACATTTGTTTGTCCAACAGAAATGATTGGATATAGTGAACTTGC *******************************************************
****
B
TTATCCATTGGATTGGACATTTGTTTGTCCAACAGAAATGATTGGATATAGTGAAGTTGC 70
80
90
100
110
120
130
140
150
160
Hinf H 1 170
180
AGGACAATTGAAAGAAATCAATTGTGAAGTTATTGGAGTGAGTGTAGATTCAGTTTATTG *********************************************
**************
AGGACAATTGAAAGAAATCAATTGTGAAGTTATTGGAGTGAGTGTTGATTCAGTTTATTG 130
140
150
160
190
200
210
220
H
Hinf
170
180
230Hincl
C
240
TCATCAAGCATGGTGTGAAGCAGATAAAAGTAAAGGAGGAGTAGGAAAGTTGACATTCCC ************************************************
******
**
TCATCAAGCATGGTGTGAAGCAGATAAAAGTAAAGGAGGAGTAGGAAAATTAGGATTCCC 230 t 240 220 210 200 190
Hint
Taql 260~EcoT221 280 290 300 j 260E OT221270 250250 ATTAGTATCAGATATTAAGAGATGCATTTCTATCAAATATGGAATGTTAAATGTCGAAGC ******************
*****
*****
**
********************
***
D
*
ATTAGTATCAGATATTAAAAGATGTATTTCAATTAAATATGGAATGTTAAATGTAGAAAC 300 280 290 270 250 260 360 350 340 330 320 310 AGGAATTGCAAGAAGAGGATATGTCATCATTGATGATAAAGGAAAAGTAAGATACATTCA ****
**
*******************
E
********************************
AGGAGTTTCAAGAAGAGGATATGTCATTATTGATGATAAAGGAAAAGTAAGATACATTCA 320
310
330
340
350
360
400 370 380 390 AATGAATGATGATGGAATTGGAAGATCAACGGAAGAAAC ******************************
********
AATGAATGATGATGGAATTGGAAGATCAACAGAAGAAAC 400 370 390 380
B 20
10
40
30
50
60
GSIKEIDINEYRGKYVVLLFYPLDWTFVCPTEMIGYSELAGQLKEINCEVIGVSVDSVYC ***********
**************************
*********************
GSIKEIDINEYKGKYVVLLFYPLDWTFVCPTEMIGYSEVAGQLKEINCEVIGVSVDSVYC 60 50 40 10 20 30 70
80
90
110
100
120
HQAWCEADKSKGGVGKLTFPLVSDIKRCISIKYGMLNVEAGIARRGYVIIDDKGKVRYIQ *****************
*********************
*
*****************
HQAWCEADKSKGGVGKLGFPLVSDIKRCISIKYGMLNVETGVSRRGYVIIDDKGKVRYIQ 70
80
1 30
140
90
100
110
120
MNDDGIGRSTEE
MNDDGIGRSTEE 1 30
FIG. 5. Agarose gel separation of restriction endonuclease digests of PCR products. The PCR products were undigested (A) or were digested with HincIl (B), EcoT22I (C), TaqI (D), or Hinfl (E). Template DNA from various strains of E. histolytica was amplified by using primers pl plus p4. Lanes: 1, HM-1:IMSS (Z-II, pathogenic, axenic); 2, HK-9 cli (Z-II, pathogenic, axenic); 3, H303:NIH (Z-II, pathogenic, axenic); 4, Rahman (Z-II, pathogenic, axenic); 5, SAW 408 (Z-II, pathogenic, xenic); 6, NOT-25 (Z-VII, pathogenic, xenic); 7, NOT-13 (Z-XI, pathogenic, xenic); 8, SAW 1453 (Z-XIV, pathogenic, xenic); 9, NOT-1 cl2 (Z-XIX, pathogenic, monoxenic); 10, SAW 142 (Z-III [originally, but presently Z-I], nonpathogenic, xenic]; 11, NOT-33 (Z-VIII, nonpathogenic, xenic); 12, NOT-44 (Z-VIII, nonpathogenic, xenic); M, size markers (HincIl-cleaved 4X174). Arrows indicate the positions of digested fragments.
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 (top line) and SAW 142 (bottom line) strains of E. histolytica. Identity is indicated by asterisks. Arrows indicate the cutting sites of restriction endonucleases. Arrowheads indicate discrepancies in the nucleotide and deduced amino acid sequences of the genomic DNA of HM-1:IMSS, in comparison with those of the cDNA shown in Fig. 2.
different. These observations demonstrate that both genes encode identical proteins. However, while the 29-kDa antigen was demonstrated to be a surface antigen, we could not detect the epitope recognized by MAb 4G6 on the cell surface (24). This discrepancy suggests that the molecule recognized by MAb 4G6 either is a precursor of the surface antigen or is a transmembrane protein recognized from within the cell. The amino acid differences in the sequences of the 30,000-Mr component of the pathogenic and nonpathogenic strains that have been compared amounted to only 4.5%. Indeed, Torian et al. (27) observed that some of the MAbs prepared against the fusion protein with the C terminus of glutathione S-transferase were also reactive with nonpathogenic isolates. Therefore, the epitope recognized by MAb
4G6 may be composed of a few amino acids specific for pathogenic isolates and, as shown previously, MAb 4G6 is very useful for the identification of pathogenic isolates regardless of zymodemes, geographic origins, culture conditions, or host symptoms (24). It is important to determine whether the differences between pathogenic and nonpathogenic strains are phenotypic or genotypic because zymodeme conversion has been reported in a few isolates (1, 11-13). Recently, DNA probes specific for either pathogenic or nonpathogenic strains have been developed (3, 8, 26). More recently, Edman et al. (6) demonstrated a significant diversity in nucleotide sequences between pathogenic and nonpathogenic strains in the 125kDa antigen of E. histolytica. The reported variability in amino acid sequences among different isolates of strain HM-1:IMSS was 1%, whereas that between pathogenic and nonpathogenic strains was 12 to 13% in the 1.4-kb fragments that have been compared. In the present study, although the regions compared were limited to about 400 bp, the variability in amino acid sequences between pathogenic and nonpathogenic strains was 4.5%, with no diversity observed among their respective isolates. This indicates that there is dimorphism in the amino acid sequence of the 30,000-Mr antigen and the identification of the gene encoding this
2238
TACHIBANA ET AL.
antigen is valuable for distinguishing between pathogenic and nonpathogenic strains. In our observations, direct sequencing of PCR products from the noncloned genomic DNA of HM-1:IMSS strain showed a single-base discrepancy with a cloned cDNA sequence. A similar observation has been reported in another gene of E. histolytica (10). Such a mismatch may be due to the misincorporation of nucleotides by Taq or Tth or both DNA polymerases. However, when the cDNA cloned in Agtll was PCR amplified and then directly sequenced by Tth polymerase, the results were in accord with those in Fig. 2. On the other hand, the direct sequencing of PCR-amplified products from the cDNA library yielded results identical with those from the genomic DNA (data not shown). Therefore, the possibility that two populations exist in the genomic DNA cannot be ruled out at present. Edman et al. (6) also demonstrated that PCR amplification of DNA coding the 125-kDa antigen of E. histolytica, followed by SspI or AccI digestion, showed a distinct difference between pathogenic and nonpathogenic isolates. However, by this procedure, the nonpathogenic E. histolyticalike Laredo strain is identified as pathogenic. More recently, Tannich and Burchard (25) reported that amplification of the same gene by using other primers and subsequent digestion with AccI, TaqI, and XmnI is useful to distinguish between isolates of E. histolytica. It has not yet been examined whether other human parasitic amoebae may induce erroneous amplification products. The present study indicated that partial amplification of the genomic DNA coding the 30,000-Mr antigen by PCR is also useful for discriminating between E. histolytica and the E. histolytica-like Laredo strain. Since it is difficult to cultivate nonpathogenic isolates axenically, we could not avoid contamination by bacterial DNA in materials isolated from the parasites grown under xenic conditions. However, PCR technology permits easy amplification of the genomic DNA of E. histolytica for identification and further analysis. Although it is not clear at present whether the 30,000-Mr antigen of pathogenic strains is responsible for pathogenicity, it should be stressed that differentiating between pathogenic and nonpathogenic strains on the basis of the endonuclease digestion of PCRamplified DNA is completely in accord with zymodeme
analysis.
ACKNOWLEDGMENTS We are grateful to Y. Kawauchi for technical assistance and to W. Stahl for reviewing the manuscript. This work was supported by Grants-in Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, research aid from the Tokai University School of Medicine, and a grant from the General Organization of Research in Tokai University to H.T. REFERENCES 1. Andrews, B. J., L. Mentzoni, and B. Bjorvatn. 1990. Zymodeme conversion of isolates of Entamoeba histolytica. Trans. R. Soc. Trop. Med. Hyg. 84:63-65. 2. Aviv, H., and P. Leder. 1972. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA 69:1408-1412. 3. Bracha, R., L. S. Diamond, J. P. Ackers, G. D. Burchard, and D. Mirelman. 1990. Differentiation of clinical isolates of Entamoeba histolytica by using specific DNA probes. J. Clin. Microbiol.
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4.
Diamond, L. S. 1987. Entamoeba, Giardia and Trichomonas, p.
1-28. In A. E. R. Taylor and J. R. Baker (ed.), In vitro methods for parasite cultivation. Academic Press Ltd., London.
J. CLIN. MICROBIOL.
5. Diamond, L. S., D. R. Harlow, and C. C. Cunnick. 1978. A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans. R. Soc. Trop. Med. Hyg. 72:431-432. 6. Edman, U., M. A. Meraz, S. Rausser, N. Agabian, and I. Meza. 1990. Characterization of an immuno-dominant variable surface antigen from pathogenic and nonpathogenic Entamoeba histolytica. J. Exp. Med. 172:879-888. 7. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13. 8. Garfinkel, L. I., M. Giladi, M. Huber, C. Gitler, D. Mirelman, M. Revel, and S. Rozenblatt. 1989. DNA probes specific for Entamoeba histolytica possessing pathogenic and nonpathogenic zymodemes. Infect. Immun. 57:926-931. 9. Huber, M., L. Garfinkel, C. Gitler, D. Mirelman, M. Revel, and S. Rozenblatt. 1987. Entamoeba histolytica: cloning and characterization of actin cDNA. Mol. Biochem. Parasitol. 24:227235. 10. Mann, B. J., B. E. Torian, T. S. Vedvick, and W. A. Petri, Jr. 1991. Sequence of a cysteine-rich galactose-specific lectin of Entamoeba histolytica. Proc. Natl. Acad. Sci. USA 88:32483252. 11. Mirelman, D. 1987. Effect of culture conditions and bacterial associates on the zymodemes of Entamoeba histolytica. Parasitol. Today 3:37-40. 12. Mirelman, D., R. Bracha, A. Chayen, A. Aust-Kettis, and L. S. Diamond. 1986. Entamoeba histolytica: effect of growth conditions and bacterial associates on isoenzyme patterns and virulence. Exp. Parasitol. 62:142-148. 13. Mirelman, D., R. Bracha, A. Wexler, and A. Chayen. 1986. Changes in isoenzyme patterns of a cloned culture of nonpathogenic Entamoeba histolytica during axenization. Infect. Immun. 54:827-832. 14. Okayama, H., M. Kawaichi, M. Brownstein, F. Lee, T. Yokota, and K. Arai. 1987. High-efficiency cloning of full-length cDNA: construction and screening of cDNA expression libraries for mammalian cells. Methods Enzymol. 154:3-28. 15. Petri, W. A., Jr., T. F. H. G. Jackson, V. Gathiram, K. Kress, L. D. Saffer, T. L. Snodgrass, M. D. Chapman, Z. Keren, and D. Mirelman. 1990. Pathogenic and nonpathogenic strains of Entamoeba histolytica can be differentiated by monoclonal antibodies to the galactose-specific adherence lectin. Infect. Immun. 58:1802-1806. 16. Robinson, G. L. 1968. The laboratory diagnosis of human parasitic amoebae. Trans. R. Soc. Trop. Med. Hyg. 62:285-294. 17. Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primerdirected enzyme amplification of DNA with a thermostable DNA polymerase. Science 239:487-491. 18. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 19. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 20. Sargeaunt, P. G. 1988. Zymodemes of Entamoeba histolytica, p. 370-387. In J. I. Ravdin (ed.), Amebiasis: human infection by Entamoeba histolytica. John Wiley & Sons, Inc., New York. 21. Sargeaunt, P. G., U. K. Baveja, R. Nanda, and B. S. Anand. 1984. Influence of geographical factors in the distribution of pathogenic zymodemes of Entamoeba histolytica: identification of zymodeme XIV in India. Trans. R. Soc. Trop. Med. Hyg. 78:96-101. 22. Sargeaunt, P. G., J. E. Williams, and J. D. Grene. 1978. The differentiation of invasive and non-invasive Entamoeba histolytica by isozyme electrophoresis. Trans. R. Soc. Trop. Med. Hyg. 72:519-521. 23. Strachan, W. D., P. L. Chiodini, W. M. Spice, A. H. Moody, and J. P. Ackers. 1988. Immunological differentiation of pathogenic and non-pathogenic isolates of Entamoeba histolytica. Lancet i:561-563. 24. Tachibana, H., S. Kobayashi, Y. Kato, K. Nagakura, Y. Kaneda, and T. Takeuchi. 1990. Identification of a pathogenic isolate-
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specific 30,000Mr antigen of Entamoeba histolytica by using a monoclonal antibody. Infect. Immun. 58:955-960. 25. Tannich, E., and G. D. Burchard. 1991. Differentiation of pathogenic from nonpathogenic Entamoeba histolytica by restriction fragment analysis of a single gene amplified in vitro. J. Clin. Microbiol. 29:250-255. 26. Tannich, E., R. D. Horstmann, J. Knobloch, and H. H. Arnold. 1989. Genomic DNA differences between pathogenic and nonpathogenic Entamoeba histolytica. Proc. Natl. Acad. Sci. USA
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86:5118-5122. 27. Torian, B. E., B. M. Flores, V. L. Stroeher, F. S. Hagen, and W. E. Stamm. 1990. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica. Proc. Natl. Acad. Sci. USA 87:6358-6362. 28. Walsh, J. A. 1988. Prevalence of Entamoeba histolytica infection, p. 93-105. In J. I. Ravdin (ed.), Amebiasis: human infection by Entamoeba histolytica. John Wiley & Sons, Inc., New York.
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1991, p. 2234-2239
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 500 million people worldwide and is associated with asymptomatic carriers and symptomatic diseases, such as hemorrhagic colitis and extraintestinal abscesses (28). Sargeaunt et al. have demonstrated that pathogenic and nonpathogenic isolates can be distinguished by differences in the electrophoretic patterns of isoenzymes (zymodemes) (20, 22). Recently, monoclonal antibodies (MAbs) which distinguish between pathogenic and nonpathogenic isolates have been developed (15, 23). We have also reported that MAb 4G6, produced against the pathogenic strain HM-1:IMSS of E. histolytica, reacts only with isolates possessing pathogenic zymodemes, regardless of geographic origin and culture conditions (24). Western blot (immunoblot) analyses have shown that the molecular weight of the component recognized by the MAb is 30,000. However, zymodeme patterns and reactivities of MAbs are phenotypic properties. Indeed, it has been reported that zymodeme conversion from nonpathogenic to pathogenic (or the reverse) can occur within a cloned culture of some strains of E. histolytica during the process of axenization under appropriate growth conditions (1, 11-13). Therefore, we cannot exclude the possibility that the gene encoding the 30,000-Mr antigen may also exist in nonpathogenic isolates. In the present study, we describe the molecular cloning and sequencing of cDNA coding the 30,000-Mr antigen. In addition, by using the polymerase chain reaction (PCR) technique, we clarified the differences in genomic DNA between pathogenic and nonpathogenic isolates of E. histolytica.
*
Corresponding author. 2234
MATERIALS AND METHODS Parasites and culture conditions. Trophozoites of pathogenic strains of E. histolytica (HM-1:IMSS, HK-9 cll, H303: NIH, and Rahman) were axenically grown in TYI-S-33 medium (5). Pathogenic strain NOT-1 cl2 was monoxenically cultured in TYI-S-33 medium with epimastigotes of Trypanosoma cruzi. Trophozoites of pathogenic strains SAW 408, SAW 1453, NOT-13, and NOT-25, and nonpathogenic strains SAW 142, NOT-33, and NOT-44 were xenically cultured in Robinson's medium (4, 16). Zymodemes of these isolates were determined by the method of Sargeaunt et al. (21). Trophozoites of the E. histolytica-like Laredo strain, Entamoeba hartmanni, and Entamoeba coli were also xenically cultured in Robinson's medium. Among these parasites, axenic strains of E. histolytica and E. histolytica-like Laredo were obtained from L. S. Diamond. SAW strains were provided by P. G. Sargeaunt. E. hartmanni, E. coli and the other strains of E. histolytica were isolated in our laboratory. Parasites grown in Robinson's medium were isolated with Percoll as described previously (24). Isolation of RNA and DNA. Total RNA of E. histolytica HM-1:IMSS was recovered by disruption of trophozoites with 5.5 M guanidinium isothiocyanate followed by sedimentation in cesium trifluoroacetate (14). The poly(A)+ RNA subpopulation was purified on an oligo(dT)-cellulose affinity column (Pharmacia LKB, Uppsala, Sweden) (2). Trophozoite DNA was isolated essentially by the procedure of Huber et al. (9). Briefly, nuclei were obtained by cell lysis in 1% 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 proteinase K per ml [20 U/mg]). The DNA was extracted
VOL. 29, 1991
DISTINGUISHING BETWEEN E. HISTOLYTICA ISOLATES BY PCR
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 (Amersham) according to the manufacturer's protocol. The library was immunoscreened with mouse MAb 4G6 (18, 24). Positive clones were detected by using horseradish peroxidase-labeled anti-mouse immunoglobulin G and Immunostain Kit HRP IS-50B (Konica, Tokyo, Japan) as the substrate. This procedure was repeated until the clones were purified to 100%. Northern (RNA) blot analysis. Northern blot analysis was performed by a standard technique (18). Poly(A)+ RNA (5 ,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 instructions. For a probe, a cDNA insert eluted from a low-melting-point agarose gel was labeled by a multiprime DNA labeling system (Amersham), using [a-32P]dCTP (7). PCR. Genomic DNA was amplified by PCR (17). The 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 (Iwaki Glass, Tokyo, Japan). Cycling conditions were as follows: melting at 94°C for 1 min, annealing at 55°C for 2 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 2% agarose gels, and the presence of specific bands was visualized with UV light after ethidium bromide staining. Determination of nucleotide sequences. Sequencing was performed by using the dideoxynucleotide chain termination method (19). A cDNA insert was subcloned into pBluescript II SK+ (Stratagene, La Jolla, Calif.), and unidirectional deletion of the insert was carried out by controlled exonuclease III digestion. Single-stranded DNA was prepared by using VCSM13 as a helper phage and then sequenced by using [35S]dCTP and a Sequenase version 2.0 kit (U.S. Biochemicals, Cleveland, Ohio). Double-stranded DNA templates amplified by PCR were also sequenced by using [_y-32P]ATP end-labeled primers (pl and p2) and Tth DNA polymerase (Toyobo, Osaka, Japan). Restriction endonuclease digestion. Restriction digests of amplified DNA were performed according to the manufacturer's recommendations with different endonucleases (To-
yobo).
Nucleotide sequence accession number. The nucleotide sequence data reported here have been submitted to DDBJ, EMBL, and GenBank Nucleotide Sequence Data Bases and assigned accession numbers D00871, D00872, and D01079.
RESULTS Cloning and sequencing of cDNA encoding the 30,000-M antigen. The cDNA library containing 106 plaques was immunoscreened with MAb 4G6, and five positive clones were isolated. One of the clones, containing an EcoRI insert of approximately 0.7 kb, was subcloned into plasmid vector pBluescript II SK+ and used for further analysis. Northern
2235
kb 7.4-
5.3-
2.81.9
1.0-g
0.60.40-3-
FIG. 1. Northern blot analysis of poly(A)+ RNA from E. histolytica HM-1:IMSS strain with a 32P-labeled cDNA probe. The numbers to the left correspond to the sizes of the RNA markers.
blot analysis of poly(A)+ RNA with this insert revealed a single band of approximately 0.9 kb (Fig. 1). This size was sufficient for encoding the 30,000-Mr antigen. Sequence analysis of the insert revealed 714 bp, which included an open reading frame encoding 218 amino acids (Fig. 2). The translation product deduced from the amino acids had a size of 24,500 Mr, which was equivalent to 82% of the 30,000 Mr identified by Western blotting analysis (24). The nucleotide sequence of this cDNA was almost identical to the sequence of a surface antigen of E. histolytica H302:NIH strain reported by Torian et al. (27). Three cytosines in the cDNA of the H302:NIH strain were replaced by adenine in the HM-1:IMSS strain, as indicated in Fig. 2. However, the predicted amino acid sequences were identical in both strains. PCR analysis of genomic DNA differences between pathogenic and nonpathogenic isolates. To determine whether the DNA sequence coding the 30,000-M, antigen of HM-1:IMSS strain is specific to pathogenic isolates of E. histolytica, genomic DNA derived from pathogenic and nonpathogenic isolates was analyzed by PCR, using the four oligonucleotide primers shown in Table 1. Incubation of genomic DNA of 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 expected from the cDNA structure (Fig. 3, lane 1). Interestingly, comparable amplified products were also detected when genomic DNA from the SAW 142 strain (nonpathogenic, Z-I) of E. histolytica was used as the template (Fig. 3, lane 2). Such PCR products were detected when genomic DNA from other strains showing various types of zymodemes, namely, HK-9 cll (Z-II), H303:NIH (Z-II), Rahman
2236
J. CLIN. MICROBIOL.
TACHIBANA ET AL.
40 30 20 10 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
M 1 2 34 56
A
90 80 70 60 50 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 140 130 120 110 ylOO 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
190 180 170 160 150 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
230 220 210 200 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
280 270 260 250 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
NO0
330 320 310 300 290 GTG AGT GTA GAT TCA GTT TAT TGT CAT CAA GCA TGG TGT GAA GCA GAT Ala Asp Glu Gin Ala Val Ser Val Asp Ser Val Tyr Cys His Trp Cys
3 i54
D
380 370 360 350 340 AAA AGT AAA GGA GGA GTA GGA AAG TTG ACA TTC CCA TTA GTA TCA GAT Phe Leu Val Ser Asp Thr Pro Lys Ser Lys Gly Giy Vai Giy Lys Leu
430 420 410 400 390 ATT AAG AGA TGC ATT TCT ATC AAA TAT GGA ATG TTA AAT GTC GAA GCA Vai Ala Met Leu Asn Giu Ser Ilie Ilie Ilie Lys Arg Cys Lys Tyr Gly 470 460 440 450 GGA ATT GCA AGA AGA GGA TAT GTC ATC ATT GAT GAT AAA GGA AAA GTA Giy Ilie Ala Arg Arg Giy Tyr Val Ilie Ilie Asp Asp Lys Gly Lys Val
510 520 500 490 480 AGA TAC ATT CAA ATG AAT GAT GAT GGA ATT GGA AGA TCA ACG GAA GAA Arg Tyr Ilie Gin Met Asn Asp Asp Gly Ilie Gly Arg Ser Thr Glu Glu 560 570 550 540 530 ACA ATC AGA ATA GTT AAA GCA ATT CAA TTC AGT GAT GAA CAT GGA GCA Thr Ilie Arg Ilie Vai Lys Ala Ilie Gin Phe Ser Asp Glu His Giy Ala
620 600 610 590 580 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 670 660 640 650 630 CCA GAT GGA ATT AAG AAA TAT TTA ACA GCA CAT TAA AACAAACAAGATAATT Pro Asp Gly Ilie Lys Lys Tyr Leu Thr Ala His
710 680 690 700 TAATACAAATTATTTTAAAAAAAAAAAAAAAAAAAAAAA
II
FIG. 2. Nucleotide sequence of the cloned cDNA and the predicted amino acid sequence of the coding region. Asterisks indicate the translation stop codon. Arrowheads indicate the nucleotides different from the sequence reported by Torian et al. (27).
(Z-II), SAW 408 (Z-II), NOT-25 (Z-VII), NOT-13 (Z-XI), SAW 1453 (Z-XIV), NOT-i c12 (Z-XIX), NOT-33 (Z-VIII), and N-OT-44 (Z-VIII), were used as templates (data not shown).' Although incubation of genomic DNA from the E. histolytica-like Laredo strain with primers p1 plus p3 or p2 plus p3 p'roduced amplified products, incubation with primers p1 plus p4 or p2 plus p4 did not (Fig. 3, lane 3), nor did the genomic DNA of E. hartmanni and E. coli w'hen incubated 'With the four sets of primers (Fig. 3, lanes 4 and 5). These results indicate that incubation of genomic DNA with primers p1 plus p4 or p2 plus p4 can be useful for distin-
FIG. 3. Agarose gel separation of PCR products amplified by using different sets of primers. The primer pairs were p1 plus p4 (A), p1 plus p3 (B), p2 plus p4 (C), and p2 plus p3 (D). Different template DNAs were used. Lanes: 1, E. histolytica HM-1:IMSS; 2, E. histolytica SAW 142; 3, E. histolytica-like Laredo; 4, E. hartmanni; 5, E. coli; 6, no template DNA; M, size markers (Hincll-cleaved The positions and sizes of PCR products are indicated to 4OX174). the right of the gel.
guishing between E. histolytica and other Entamoeba species. To evaluate the homology of the nucleotide sequence between pathogenic and nonpathogenic isolates, PCR-amplifled products of the genomic DNA for HM-1:IMSS and SAW 142 were sequenced. The results showed the substitution of 22 nucleotides in the 399-bp sequences which were compared (5.5%) (Fig. 4A). Of the inferred amino acids, 4.5% (6 of 132) were different between these strains (Fig. 4B). The 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 PCR-amplified genomic DNA of HM-1:IMSS strain. This resulted in an amino acid change from lysine to arginine. On the basis of DNA differences between pathogenic and nonpathogenic isolates, four restriction endonucleases were selected to distinguish between these isolates. As expected, Hincll, EcoT221, and TaqI digested amplified DNAs from pathogenic isolates showing different zymodeme patterns but did not cut amplified DNAs from isolates possessing nonpathogenic zymodemes (Fig. SB to D). In contrast, Hinfl digested PCR products of both pathogenic and nonpathogenic isolates, yielding two and three fragments, respec-
tively (Fig. 5E). TABLE 1. Oligonucleotide primers used for PCR Corresponding Primer
p1 p2 p3 p4
Sequence 5'TAAAGCACCAGCATATTGTC 3' 5'GTGAAGTTATTGGAGTGAGT 3' 5'GATGACATATCCTCTTCTTG 3' 5'TTAATTCCATCTGGTGTTGG 3'
Direction
Sense Sense Antisense Antisense
ncletDNA (bp) 107-126 274-293 458-439 637-618
DISCUSSION The cDNA sequence of the 30,000-Mr antigen reported here was highly homologous with that of the 29-kDa cysteine-rich surface antigen reported by Torian et al. (27). In the overlapping regions, we found only three nucleotide substitutions (one in the coding region and two in the noncoding region) but no differences in the deduced amino acids. In addition, Northern blot analysis demonstrated single bands in each study, although their sizes were slightly
DISTINGUISHING BETWEEN E. HISTOLYTICA ISOLATES BY PCR
VOL. 29, 1991
A 60 v 40 50 30 20 10 TGGTTCAATCAAAGAGATTGATATTAATGAATATAGAGGAAAATATGTTGTATTGTTGTT ***************
*******************
***********
***
2237
A
** **
**
TGGTTCAATCAAAGAAATTGATATTAATGAATATAAAGGGAAATATGTTGTGTTATTATT 10
30
20
60
50
40
120 110 100 90 80 70 TTATCCATTGGATTGGACATTTGTTTGTCCAACAGAAATGATTGGATATAGTGAACTTGC *******************************************************
****
B
TTATCCATTGGATTGGACATTTGTTTGTCCAACAGAAATGATTGGATATAGTGAAGTTGC 70
80
90
100
110
120
130
140
150
160
Hinf H 1 170
180
AGGACAATTGAAAGAAATCAATTGTGAAGTTATTGGAGTGAGTGTAGATTCAGTTTATTG *********************************************
**************
AGGACAATTGAAAGAAATCAATTGTGAAGTTATTGGAGTGAGTGTTGATTCAGTTTATTG 130
140
150
160
190
200
210
220
H
Hinf
170
180
230Hincl
C
240
TCATCAAGCATGGTGTGAAGCAGATAAAAGTAAAGGAGGAGTAGGAAAGTTGACATTCCC ************************************************
******
**
TCATCAAGCATGGTGTGAAGCAGATAAAAGTAAAGGAGGAGTAGGAAAATTAGGATTCCC 230 t 240 220 210 200 190
Hint
Taql 260~EcoT221 280 290 300 j 260E OT221270 250250 ATTAGTATCAGATATTAAGAGATGCATTTCTATCAAATATGGAATGTTAAATGTCGAAGC ******************
*****
*****
**
********************
***
D
*
ATTAGTATCAGATATTAAAAGATGTATTTCAATTAAATATGGAATGTTAAATGTAGAAAC 300 280 290 270 250 260 360 350 340 330 320 310 AGGAATTGCAAGAAGAGGATATGTCATCATTGATGATAAAGGAAAAGTAAGATACATTCA ****
**
*******************
E
********************************
AGGAGTTTCAAGAAGAGGATATGTCATTATTGATGATAAAGGAAAAGTAAGATACATTCA 320
310
330
340
350
360
400 370 380 390 AATGAATGATGATGGAATTGGAAGATCAACGGAAGAAAC ******************************
********
AATGAATGATGATGGAATTGGAAGATCAACAGAAGAAAC 400 370 390 380
B 20
10
40
30
50
60
GSIKEIDINEYRGKYVVLLFYPLDWTFVCPTEMIGYSELAGQLKEINCEVIGVSVDSVYC ***********
**************************
*********************
GSIKEIDINEYKGKYVVLLFYPLDWTFVCPTEMIGYSEVAGQLKEINCEVIGVSVDSVYC 60 50 40 10 20 30 70
80
90
110
100
120
HQAWCEADKSKGGVGKLTFPLVSDIKRCISIKYGMLNVEAGIARRGYVIIDDKGKVRYIQ *****************
*********************
*
*****************
HQAWCEADKSKGGVGKLGFPLVSDIKRCISIKYGMLNVETGVSRRGYVIIDDKGKVRYIQ 70
80
1 30
140
90
100
110
120
MNDDGIGRSTEE
MNDDGIGRSTEE 1 30
FIG. 5. Agarose gel separation of restriction endonuclease digests of PCR products. The PCR products were undigested (A) or were digested with HincIl (B), EcoT22I (C), TaqI (D), or Hinfl (E). Template DNA from various strains of E. histolytica was amplified by using primers pl plus p4. Lanes: 1, HM-1:IMSS (Z-II, pathogenic, axenic); 2, HK-9 cli (Z-II, pathogenic, axenic); 3, H303:NIH (Z-II, pathogenic, axenic); 4, Rahman (Z-II, pathogenic, axenic); 5, SAW 408 (Z-II, pathogenic, xenic); 6, NOT-25 (Z-VII, pathogenic, xenic); 7, NOT-13 (Z-XI, pathogenic, xenic); 8, SAW 1453 (Z-XIV, pathogenic, xenic); 9, NOT-1 cl2 (Z-XIX, pathogenic, monoxenic); 10, SAW 142 (Z-III [originally, but presently Z-I], nonpathogenic, xenic]; 11, NOT-33 (Z-VIII, nonpathogenic, xenic); 12, NOT-44 (Z-VIII, nonpathogenic, xenic); M, size markers (HincIl-cleaved 4X174). Arrows indicate the positions of digested fragments.
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 (top line) and SAW 142 (bottom line) strains of E. histolytica. Identity is indicated by asterisks. Arrows indicate the cutting sites of restriction endonucleases. Arrowheads indicate discrepancies in the nucleotide and deduced amino acid sequences of the genomic DNA of HM-1:IMSS, in comparison with those of the cDNA shown in Fig. 2.
different. These observations demonstrate that both genes encode identical proteins. However, while the 29-kDa antigen was demonstrated to be a surface antigen, we could not detect the epitope recognized by MAb 4G6 on the cell surface (24). This discrepancy suggests that the molecule recognized by MAb 4G6 either is a precursor of the surface antigen or is a transmembrane protein recognized from within the cell. The amino acid differences in the sequences of the 30,000-Mr component of the pathogenic and nonpathogenic strains that have been compared amounted to only 4.5%. Indeed, Torian et al. (27) observed that some of the MAbs prepared against the fusion protein with the C terminus of glutathione S-transferase were also reactive with nonpathogenic isolates. Therefore, the epitope recognized by MAb
4G6 may be composed of a few amino acids specific for pathogenic isolates and, as shown previously, MAb 4G6 is very useful for the identification of pathogenic isolates regardless of zymodemes, geographic origins, culture conditions, or host symptoms (24). It is important to determine whether the differences between pathogenic and nonpathogenic strains are phenotypic or genotypic because zymodeme conversion has been reported in a few isolates (1, 11-13). Recently, DNA probes specific for either pathogenic or nonpathogenic strains have been developed (3, 8, 26). More recently, Edman et al. (6) demonstrated a significant diversity in nucleotide sequences between pathogenic and nonpathogenic strains in the 125kDa antigen of E. histolytica. The reported variability in amino acid sequences among different isolates of strain HM-1:IMSS was 1%, whereas that between pathogenic and nonpathogenic strains was 12 to 13% in the 1.4-kb fragments that have been compared. In the present study, although the regions compared were limited to about 400 bp, the variability in amino acid sequences between pathogenic and nonpathogenic strains was 4.5%, with no diversity observed among their respective isolates. This indicates that there is dimorphism in the amino acid sequence of the 30,000-Mr antigen and the identification of the gene encoding this
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antigen is valuable for distinguishing between pathogenic and nonpathogenic strains. In our observations, direct sequencing of PCR products from the noncloned genomic DNA of HM-1:IMSS strain showed a single-base discrepancy with a cloned cDNA sequence. A similar observation has been reported in another gene of E. histolytica (10). Such a mismatch may be due to the misincorporation of nucleotides by Taq or Tth or both DNA polymerases. However, when the cDNA cloned in Agtll was PCR amplified and then directly sequenced by Tth polymerase, the results were in accord with those in Fig. 2. On the other hand, the direct sequencing of PCR-amplified products from the cDNA library yielded results identical with those from the genomic DNA (data not shown). Therefore, the possibility that two populations exist in the genomic DNA cannot be ruled out at present. Edman et al. (6) also demonstrated that PCR amplification of DNA coding the 125-kDa antigen of E. histolytica, followed by SspI or AccI digestion, showed a distinct difference between pathogenic and nonpathogenic isolates. However, by this procedure, the nonpathogenic E. histolyticalike Laredo strain is identified as pathogenic. More recently, Tannich and Burchard (25) reported that amplification of the same gene by using other primers and subsequent digestion with AccI, TaqI, and XmnI is useful to distinguish between isolates of E. histolytica. It has not yet been examined whether other human parasitic amoebae may induce erroneous amplification products. The present study indicated that partial amplification of the genomic DNA coding the 30,000-Mr antigen by PCR is also useful for discriminating between E. histolytica and the E. histolytica-like Laredo strain. Since it is difficult to cultivate nonpathogenic isolates axenically, we could not avoid contamination by bacterial DNA in materials isolated from the parasites grown under xenic conditions. However, PCR technology permits easy amplification of the genomic DNA of E. histolytica for identification and further analysis. Although it is not clear at present whether the 30,000-Mr antigen of pathogenic strains is responsible for pathogenicity, it should be stressed that differentiating between pathogenic and nonpathogenic strains on the basis of the endonuclease digestion of PCRamplified DNA is completely in accord with zymodeme
analysis.
ACKNOWLEDGMENTS We are grateful to Y. Kawauchi for technical assistance and to W. Stahl for reviewing the manuscript. This work was supported by Grants-in Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, research aid from the Tokai University School of Medicine, and a grant from the General Organization of Research in Tokai University to H.T. REFERENCES 1. Andrews, B. J., L. Mentzoni, and B. Bjorvatn. 1990. Zymodeme conversion of isolates of Entamoeba histolytica. Trans. R. Soc. Trop. Med. Hyg. 84:63-65. 2. Aviv, H., and P. Leder. 1972. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA 69:1408-1412. 3. Bracha, R., L. S. Diamond, J. P. Ackers, G. D. Burchard, and D. Mirelman. 1990. Differentiation of clinical isolates of Entamoeba histolytica by using specific DNA probes. J. Clin. Microbiol.
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