Vol. 29, No. 6
JOURNAL OF CLINICAL MICROBIOLOGY, June 1991, p. 1132-1136 0095-1137/91/061132-05$02.00/0 Copyright © 1991, American Society for Microbiology
Typing of Chlamydia trachomatis by Restriction Endonuclease Analysis of the Amplified Major Outer Membrane Protein Gene PATRICIA RODRIGUEZ,' ANTOINE VEKRIS,1 BERTILLE DE BARBEYRAC,l BRIGITTE DUTILH,1 JACQUES BONNET,2 AND CHRISTIANE BEBEAR1* Laboratoire de Bacteriologie, Universite de Bordeaux IH, 146, rue Leo Saignat, 33076 Bordeaux Cedex,' and Institut de Biochimie Cellulaire et Neurobiochimie du Centre National de la Recherche Scientifique, 33077 Bordeaux Cedex,2 France Received 24 October 1990/Accepted 20 February 1991
A procedure was developed for characterization of Chlamydia trachomatis strains by using restriction endonuclease analysis of amplified genes of the major outer membrane protein (MOMP). Reference strains of the 15 serovars (A through K and Li through L3) and clinical isolates were tested. The nucleotide sequences of the MOMP genes of each of the 15 serovars were arbitrarily constructed by using the sequences of the four variable domains known for each serovar and the constant domains of serovar Li. Computer analysis of these sequences indicated that two restriction digestions performed in parallel, one with AluI and the other with HpaII, followed by Hinfl and EcoRI, would allow the theoretical differentiation of 13 serovars. Serovars Ba and Li presented the same theoretical restriction profile. Our typing method consisted of polymerase chain reaction amplification of a fragment of about 1,200 bp of the MOMP gene, followed by restriction endonuclease digestion with the aforementioned enzymes. From the 15 serovars, we obtained 14 different patterns; 13 profiles were serovar specific, while serovars B and Ba presented the same pattern. Application of this typing method to C. trachomatis strains isolated from clinical material gave the same results as the immunotyping method for 14 of 17 strains. Furthermore, restriction endonuclease analysis detected differences within a serovar. This method seems to be promising for epidemiological studies.
Chlamydia trachomatis has been recognized as an important sexually transmitted pathogen and a cause of human ocular disease. The C. trachomatis species is divided into at least 15 serovars, which have been identified by a microimmunofluorescence method by Wang et al. (19) by using antibodies raised against the major outer membrane protein (MOMP) (3). Serovars A, B, Ba, and C are the etiological agents of trachoma; serovars D through K cause primarily oculogenital infections; and serovars Li, L2, and L3 are involved in lymphogranuloma venereum. However, the microimmunofluorescence method has not been widely used in large-scale epidemiological studies, since specific immune sera are generally unavailable. Our goal was to develop a typing method for C. trachomatis based on the analysis of the nucleotide variation of the MOMP gene, which is responsible for the antigenic variation of the protein. The MOMP consists of five constant domains separated by four variable regions and carries antigenic determinants for each serovar. The total MOMP gene sequences for serovars L2, B, and C (16, 17), LI (13), H (6), and E (12) and those of the four variable domains from the 15 serovars (21) have been published. So, we first simulated the 15 nucleotide sequences of the MOMP gene, using the published sequences of variable regions and the constant domains of serovar Li. Then, by computer analysis, we chose restriction endonucleases which would give restriction patterns that differentiated most of the 15 serovars. Finally, the polymorphism of restriction endonuclease digest fragments of the MOMP gene amplified by the polymerase chain reaction (PCR) by using primers localized in the first and the fifth constant region was examined in reference strains of the 15 serovars and in clinical isolates grown on cell culture.
*
Corresponding author.
(This work was presented at the 90th Annual Meeting of the American Society for Microbiology [14].) MATERIALS AND METHODS Bacterial strains. C. trachomatis reference strains of the
following 10 serovars were from the American Type Culture Collection, Rockville, Md.: A (VR 571B), B (VR 573), Ba (VR 347), C (VR 578), D (VR 885), E (VR 348B), F (VR 346), G (VR 878), H (VR 879), and L2 (VR 902B). The strains of five other serovars were from Y. Perol (Hopital Saint-Louis, Paris, France), who received them from the American Type Culture Collection: I (VR 880), J (VR 886), K (VR 887), Li (VR 901B), and L3 (VR 903). Seventeen strains of C. trachomatis, which were isolated from patients, were tested after culture on McCoy cells. Eight randomly selected wild-type strains were obtained from genital specimens in the Laboratoire de Bacteriologie, H6pital Pellegrin, Bordeaux, France. After typing by restriction fragment length polymorphism (RFLP) analysis, the eight strains were sent to K. Persson (Malmo General Hospital, Malmo, Sweden) to be typed by microimmunofluorescence by using type-specific monoclonal antibodies. A panel of 31 different monoclonal antibodies raised against antigens of the MOMP and reactive with one or more of the C. trachomatis serovars was used for serotyping, as described previously (10). Furthermore, nine strains, which were isolated in Malmo from genital specimens and previously serotyped by K. Persson, were selected; one of each of the following serovars was tested by RFLP analysis: B, C, D, F, G, H, I, J, and K. The following clinical isolates were used for specificity testing: Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa, Gardnerella vaginalis, Neisseria gonorrhoeae, Lactobacillus spp., Streptococcus agalactiae, Fusobacterium fusiformis, Clostridium perfringens, and Mycoplasma hom1132
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TYPING OF C. TRACHOMATIS BY RFLP OF THE MOMP GENE
inis. The strains were kept frozen at -70°C until they were tested. C. trachomatis culture and sample preparation for PCR. Reference and clinical strains of C. trachomatis were grown on cycloheximide-treated McCoy cell monolayers. After 72 h of incubation at 35°C, inclusions were detected by a direct fluorescent-monoclonal antibody method (Microtrak; Syva, Palo Alto, Calif.). Serial passages were performed until about 20% of cells were infected. A 100-,ul volume of Chlamydia cell cultures was mixed with 200 RI of lysis buffer (5) containing 10 mM Tris hydrochloride (pH 8), 1 mM EDTA, 0.1% Triton, and 20 ,ug of proteinase K (Boehringer GmbH, Mannheim, Federal Republic of Germany) per ml. Samples were incubated at 55°C for 90 min and then at 95°C for 30 min. DNA extraction from other bacteria. Bacteria were grown in appropriate culture media. Cells were lysed with 50 pug of lysozyme per ml and treated with 1% sodium dodecyl sulfate (wt/vol) containing 0.1 mg of proteinase K per ml. DNA was purified by phenol-chloroform extraction, ethanol precipitation, and RNase treatment, as described previously by Maniatis et al. (8). Oligonucleotides. Two 23-base oligonucleotides (CT1 and CT5) were used as primers for the PCR, and a 21-base internal oligonucleotide (CT3) was used as a probe. CT1 and CT3 were chosen in the sequence of the first conserved domain of the MOMP gene (4), and CT5 was chosen in the sequence of the fifth domain, according to the published sequences of serovars L2, B, and C (16, 17) and Li (13): CT1 = 5'-GCCGClTTTGAGTTCTGCTTCCTC-3'; CT3 = 5'-TC CTTGCAAGCTCTGCCTGTG-3'; CT5 = 5'-ATTrACGTGA GCAGCTCTCTCAT-3'. They were prepared on a DNA synthesizer (model 381A; Applied Biosystems) by the methoxyphosphoramidite method and purified twice by precipitation with 3 volumes of absolute ethanol in the presence of 0.3 M sodium acetate (pH 5). PCR. Amplifications were performed with the thermostable DNA polymerase from Thermus aquaticus (Genofit, Geneva, Switzerland, or New England BioLabs, Inc., Beverly, Mass.). The final reaction mixture contained either 10 pl of extracted nucleic acids from chlamydial reference strains or clinical isolates or 1 ,ug of purified DNA from other microorganisms or human cells; 330 p.M (each) dATP, dCTP, dGTP, and dTTP; 220 ,ug of gelatin per ml; 50 mM KCI; 10 mM Tris hydrochloride (pH 8.4 at 70°C); 1.5 mM MgCl2; and 1 ,uM (each) primers CT1 and CT5 in a total volume of 50 RId. Samples were then heated at 95°C for 10 min and chilled in ice. Condensation droplets were collected by centrifugation at 5,000 x g for 10 s. Each sample received 1.5 U of Taq DNA polymerase and was overlaid with 50 ,u1 of paraffin oil. Thirty cycles of amplification were performed in an automated PCR machine (Tac-Tiq; Genofit). Each cycle consisted of 1 min of denaturation at 95°C, 1 min of annealing at 55°C, and 1 min of extension at 70°C. PCR products were analyzed by electrophoresis of 10 p.l of the amplification mixture on a 1% agarose gel in Tris acetate buffer (8). Purified DNAs from McCoy cells and human leukocytes were used as negative controls. Hybridization. DNA was denatured by 0.2 N NaOH and transferred by capillarity onto a nylon membrane (Compas, Genofit) overnight in a 25 mM NaH2PO4 buffer (pH 6.5). DNA was cross-linked to the membranes by a UV light exposure (312 nm, 2 min). Prehybridization was performed by incubation at 42°C in a solution containing Sx SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.2% sodium dodecyl sulfate, 0.1% N-lauroylsarcosine, and 0.5%
1133
blocking reagent (Boehringer). For the hybridization, 25 ng of [_y-32PIATP 5'-labeled CT3 probe was added. After hybridization at 42°C for 18 h, membranes were washed twice in 0.1% sodium dodecyl sulfate-2x SSC at room temperature for 5 min, and twice in 0.1% sodium dodecyl sulfate0.1 x SSC at 42°C for 15 min. Filters were autoradiographed with Kodak film for 48 h. Computer analysis of MOMP gene sequences. The 15 1.2-kb sequences bracketed by CT1 and CT5 were simulated. They were analyzed by computer with appropriate analysis software (BACHREST and DIGEST programs). The numbers and sizes of the restriction fragments generated by each of 75 different restriction endonucleases were determined in order to find an appropriate restriction treatment of the CT1-CT5 fragment which could give a different electrophoresis profile for each serovar. Restriction treatments of amplified DNA fragments and electrophoretic analysis. Amplified DNA samples were digested in parallel, on one hand, with AluI and, on the other, with the three enzymes HpaII, EcoRI, and Hinfl (Bethesda Research Laboratories). The first digestion was performed with 4 U of AluI on 10 p.l of amplified DNA, using the assay buffer recommended by the manufacturer, for 4 h at 37°C. The second digestion was run on 10 p.l of amplified DNA first with 4 U of HpaII in 10 mM Tris hydrochloride (pH 7.6-10 mM MgCl2 at 37°C for 4 h. HpaII was then denatured by a 10-min incubation at 60°C. Then, 2 p.l of 200 mM Tris hydrochloride (pH 8)-75 mM NaCl was added and samples were incubated overnight at 37°C with 4 U of EcoRI and Hinfl. Analysis of digested DNA was performed by electrophoresis of the total reaction volume on an 8% polyacrylamide gel. RESULTS MOMP gene amplification of the reference strains. Enzymatic amplification with primers CT1 and CT5 yielded products of about 1,200 bp with the 15 reference strains (Fig. 1A). No size variations of amplified products among the 15 serovars were observed on a 1% agarose gel, as might be expected from the small size variations that exist in the variable domains of the MOMP gene (21). A second fragment (about 800 bp) was sometimes detected in some serovars, but this was dependent upon the reaction conditions (Fig. 1A, lanes Ba, H, I, and Li). Different amounts of amplified products were observed because of differences in the starting cell culture lysates. Specificity of the PCR. Amplified fragments were hybridized after Southern transfer with the internal probe CT3 (Fig. 1B). A positive hybridization signal was obtained with the 1,200-bp fragments of each C. trachomatis serovar. We never observed any hybridization signal with the 800-bp fragment that might have been generated by an unspecific amplification. However, in some cases, a smear of highmolecular-weight DNA, which did not appear on ethidium bromide staining, was detected when the CT3 probe was used in the hybridization. This smear could be a network of amplified DNA fragments created during the amplification reaction. Amplifications conducted on DNAs isolated from other microorganisms encountered in the human genital tract or on DNA from human leukocytes or McCoy cells did not yield any band (data not shown). Computer analysis of MOMP gene sequences. According to the results of computer analysis of the MOMP gene sequences, we chose to digest the amplified DNA fragments
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ically similar profiles. Hydrolysis with HpaII-EcoRI-Hinfl differentiated 10 serovars (C, D, E, F, G, H, J, K, L2, and L3), while serovars A-I and B-Ba-Li had similar restriction patterns. Thus, on the basis of the two parallel enzymatic digestions, 13 of 15 serovars of C. trachomatis could theoretically be separated, while serovars Ba and Li presented the same restriction profiles. Analysis of the restriction profiles of the 15 serovars. Because we used serovar Li as a reference for computer analysis, the restriction hydrolysis for this serovar with AluI (Fig. 2A) and HpaII-EcoRI-Hinfl (Fig. 2B) gave the expected electrophoretic patterns (Table 1). However, most of the experimental results obtained with the 14 other serovars differed from the projected values (Table 1). As shown in Table 1 and Fig. 2A, the AluI digestion B A E Ba C D E F G H J KL L2 L a allowed us to separate 10 serovars: A, C, E, F, G, I, J, K, Ov.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Li, and L2, while serovars B-Ba-D and H-L3 exhibited similar patterns and could not been clearly differentiated. As shown in Table 1 and Fig. 2B, the HpaII-EcoRI-Hinfl digestion allowed us to differentiate 11 serovars (D, E, F, G, H, I, J, K, Li, L2, and L3) but not serovars A-C, and B-Ba. So, after the triple hydrolysis, serovar D could be differentiated from serovars B and Ba, and serovar H could be differentiated from serovar L3, whereas AluI hydrolysis gave distinct patterns for serovars A and C. 22{0 ti, _ _~~~~~~~~~~~~~~~~~~~~~~~~~~~~- -0 Thus, AluI and HpaII-EcoRI-Hinfl restriction treatments gave 14 different profiles and permitted complete separation of 13 C. trachomatis serovars. All the reference genital tract FIG. 1. Electrophoretic analysis of PCR products, after amplifiserovars could be identified by this RFLP analysis. Tracation of the MOMP gene from the 15 C. trachomatis serovars by choma serovars B and Ba could not be differentiated after using the CT1 and CT5 primers. (A) Ethidium bromide staining of agarose gel. Lane M contains the DNA molecular weight marker experimental restriction analysis. (lambda DNA digested with EcoRI and HindIll). The letters above Typing of C. trachomatis wild-type strains. The eight ranthe lanes indicate the 15 serovars of C. trachomatis. (B) Southern domly selected isolates obtained from genital tract samples analysis of the same gel by using the internal 5', y-32P-end-labeled were typed by RFLP analysis. Five of the eight strains were CT3 probe. identified as serovar E; there was also one strain each of serovars D, H, and L2 (data not shown). Serovar determination by RFLP analysis was in complete agreement with subsequent immunotyping. separately with AluI and with HpaII-EcoRI-Hinfl in two Because of the limited number of different serovars in the steps: HpaII followed by EcoRI and Hinfl together. For the randomly selected strains, wild-type strains representative two digests, the major differences between each C. trachoof nine other serovars were subjected to analysis. We matis serovar were found among DNA fragments of between identified six serovars (D, F, G, H, J, and K) in this 600 and 100 bp (Table 1). AluI digestion allowed us to collection by RFLP analysis, and these findings were in differentiate five serovars (A, C, J, K, and L2). However, agreement with those of immunotyping (Fig. 3). However, serovars B-D-E; H-I-L3; F-G; and Ba-Li presented theoretTABLE 1. Restriction fragment sizes of the CT1-CT5 sequence of the MOMP gene for the 15 C. trachomatis serovars Restriction fragment size (bp) of fragments of the following serovarsa:
Hydrolysis p
a
p
a
p
a
p
E
D
C
Ba
B
A
a
p
a
p
a
p
a
p
I
H
G
F
a
p
a
p
a
p
Li
K
J a
p
a
p
L3
L2 a
p
a
p
a
AluI 534 530 292 240 237 420 228 225 171 150 225 204 142 142
292 228 204 142
240 490 445 289 240 225 240 420 228 225 204 171 140 225 204 142 142 142
HpaII-EcoRI-
289 240 396 396 396 228 225 228 257 228 225 142 207 210 207 159 159 159 142 107 101
396 506 506 506 450 483 440 534 530 289 240 275 228 506 506 210 240 420 237 410 340 420 171 210 228 228 228 142 240 420 180 171 140 171 140 171 140 171 140 204 210 225 130 171 140 142 142 142 159
Hinfl 481 590 630 630 630 630 868 590 410 410 387 390 225 225 225 225 136 390 225 225 225 170 131 170 216 217 131 120 120 205 a
244 225 706 590 539 510 481 490 481 590 457 490 457 510 627 610 463 460 387 490 225 216 228 230 228 230 387 390 387 390 387 390 411 390 225 220 225 225 350 136 136 136 136 131 167 167 167 136 216 167 156 167 167 167 136 136 225 131 131 156 167 131
p, projected fragment sizes (larger than 100 bp); a, actual fragment sizes (larger than 100 bp) deduced from their migration distances by using the
HaeIlI-digested pBR322 hydrolysate as a reference standard.
TYPING OF C. TRACHOMATIS BY RFLP OF THE MOMP GENE
VOL. 29, 1991
M A B Ba C D E F G H I J K
A
4.14 bWp -
'257 bv-
64
C
-
1.40t)1 >p_
M1 A B Ba C D E F G H I J K Li Lz L3
B 587 bP
-
434 h I,
-
2 5 7t) p
-
1 80 t) p
-
124 bp
-
80 tip
Iqp I
-
I
-
fw
I .il
64
hp-
FIG. 2. Restriction profiles of the 15 C. trachomatis serovars (lanes A to L3) obtained by 8% polyacrylamide gel electrophoresis after restriction hydrolysis of the PCR products. (A) AluI digestion. (B) HpaII-EcoRI-Hinfl digestion. Some sizes of DNA fragments from pBR322, which were digested with HaeIII and used as molecular weight markers (lane M), are given on the left.
RFLP profiles from serovar B and C wild-type strains differed from the profiles of the serovar B and C reference strains and from all the other reference patterns. The total restriction pattern obtained with the wild-type serovar B
L; 2
G
F 2
1
2
1
H 2
1
I 2
1
K
2
1
2
1
2
FIG. 3. Restriction profiles of the nine previously serotyped clinical genital isolates. The letters indicate the types found by using the immunotyping method. Lanes 1 contain the AluI digestions, and lanes 2 contain the HpaII-EcoRI-Hinfl digestions. Some sizes of DNA fragments from pBR322, which were digested with HaeIII and used as molecular weight markers (lane M), are given on the right.
1135
strain clearly differed from that obtained with the serovar B reference strain. The serovar C genital isolate presented a pattern similar to that of the serovar C reference strain after AluI hydrolysis, but not after HpaII-EcoRI-Hinfl hydrolysis. A wild-type serovar I strain by the immunotyping method showed a typical serovar H restriction profile by RFLP analysis (Fig. 3). To reconfirm the results of the immunotyping method, this strain was again examined by K. Persson with different serovar I-specific and serovar H-specific monoclonal antibodies. These new comparisons established the wild-type isolate as being representative of the I immunotype. Furthermore, B and C wild-type immunotypes were confirmed by using different serovar B- and serovar C-specific monoclonal antibodies. DISCUSSION In order to develop epidemiological data and to detect multiple serovar infections (2), accurate and specific typing of C. trachomatis strains is required. Immunotyping, which requires serovar-specific monoclonal antibodies, is the most useful diagnostic method (20). However, this technique has serious limitations, since specific monoclonal antibodies are not commercially available. A common way to type organisms is to use RFLP analysis. Restriction endonuclease analysis of C. trachomatis total DNA has been reported previously (11). Although this method allowed differentiation among biovars, serovars, and strains within a serovar, rather large amounts of purified or labeled DNA and a large number of restriction endonucleases were required. In this report, we described a typing scheme for C. trachomatis strains that combined both PCR and RFLP analyses. Since the MOMP carries the major antigenic determinants which separate the various serovars, analysis of the MOMP gene seemed to be a convenient tool for epidemiological studies. The goal of our study was to develop a typing method based on genotypic differences which could give a classification that is well correlated to that of the 15 known C. trachomatis serovars. This method, although more difficult to employ than immunofluorescence, allows one to type many strains simultaneously and to retain results on preserved gels or photographs. Furthermore, the small number of DNA bands observed by our method enables simple differentiation of strains. In the first step of our typing method, we succeeded in amplifying nearly all of the MOMP gene from reference strains of the 15 serovars and from a selection of clinical isolates. The CT1 and CT5 primer sequences are therefore common to all serovars. As described here, our typing method requires the preliminary growth of C. trachomatis. The PCR could be used on cell culture-grown strains when only about 20% of the McCoy cell line monolayers were infected, while immunotyping requires about 50% of the cells to be infected (10, 18, 20). Using both AluI and HpaII-EcoRI-Hinfl hydrolysis on the amplified MOMP gene, we could experimentally differentiate 13 of 15 reference serovars. The MOMP gene sequences of serovars B and Ba, which have a unique profile, differed only in a small number of bases located in the variable domains (21). Most of the experimental restriction patterns were different from those obtained by computer analysis. Since the five constant domains of the MOMP gene from serovar Li were introduced between the variable regions of the 15 serovars, such differences between theoretical and experimental analyses were expected; indeed, nucleotide sequences of the MOMP gene differed not only in
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J. CLIN. MICROBIOL.
RODRIGUEZ ET AL.
the variable domains but also, to a lesser extent, in the constant domains. The application of this typing method on eight randomly selected chlamydial isolates showed a serovar distribution similar to that noted in previous studies (1, 7, 9, 10, 15, 18). Serovar E occurred frequently (in five of eight strains). We also found one L2 strain, which seems to occur in only about 1% of genital infections (7). The typing of nine other clinical isolates showed restriction patterns corresponding to those obtained for the reference strains in six isolates. For these six isolates, the immunotyping and RFLP identification methods were in agreement. However, some differences were observed for the remaining three clinical isolates (serovars B, C, and I). Thus, genomic differences may exist inside the same serovar. This is to be expected, since small differences in the genome may not affect the protein sequence and modifications in peptide sequence may not affect the epitopes recognized by the monoclonal antibodies used in the immunotyping. The reverse is true. Differences in a given epitope may also not be detected by this particular RFLP analysis. It is interesting that the serovar B and C clinical isolates used in this study came from genital tract specimens, whereas the two serovar B and C reference strains used in this study came from ocular samples. The PCR-RFLP typing method seems promising for epidemiological studies. It provides another marker system which will be useful for obtaining epidemiological data in conjunction with immunotyping.
6. 7. 8.
9. 10.
11. 12. 13.
14.
15.
ACKNOWLEDGMENTS This work was supported by grant 874380228 from the Conseil Regional d'Aquitaine. We thank K. Persson for supplying C. trachomatis isolates and performing immunotyping on all strains.
16.
17. 1. 2.
3.
4.
5.
REFERENCES Barnes, R. C., A. M. Rompalo, and W. E. Stamm. 1987. Comparison of Chlamydia trachomatis serovars causing rectal and cervical infections. J. Infect. Dis. 156:953-958. Barnes, R. C., R. J. Suchland, S.-P. Wang, C.-C. Kuo, and W. E. Stamm. 1985. Detection of multiple serovars of Chlamydia trachomatis in genital infections. J. Infect. Dis. 152:985989. Caldwell, H. D., J. Kromhout, and J. Schachter. 1981. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect. Immun. 31:11611176. Dutilh, B., C. Bebear, P. Rodriguez, A. Vekris, J. Bonnet, and M. Garret. 1989. Specific amplification of a DNA sequence common to all Chlamydia trachomatis serovars using the polymerase chain reaction. Res. Microbiol. 140:7-16. Griffais, R., and M. Thibon. 1989. Detection of Chlamydia
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trachomatis by the polymerase chain reaction. Res. Microbiol. 140:139-141. Hamilton, P. T., and D. P. Malinowski. 1989. Nucleotide sequence of the major outer membrane protein gene from Chlamydia trachomatis serovar I. Nucleic Acids Res. 17:8366. Kuo, C.-C., S.-P. Wang, K. K. Holmes, and J. T. Grayston. 1983. Immunotypes of Chlamydia trachomatis isolates in Seattle, Washington. Infect. Immun. 41:865-868. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Moncan, T., F. Eb, and J. Orfila. 1990. Monoclonal antibodies in serovar determination of 53 Chlamydia trachomatis isolates from Amiens, France. Res. Microbiol. 141:695-701. Persson, K., and S. Osser. 1989. Serovars of Chlamydia trachomatis causing postabortion salpingitis. Eur. J. Clin. Microbiol. Infect. Dis. 8:795-798. Peterson, E. M., and L. M. de la Maza. 1988. Restriction endonuclease analysis of DNA from Chlamydia trachomatis biovars. J. Clin. Microbiol. 26:625-629. Peterson, E. M., B. A. Markoff, and L. M. de la Maza. 1990. The major outer membrane protein nucleotide sequence of Chlamydia trachomatis serovar E. Nucleic Acids Res. 18:3414. Pickett, M. A., M. E. Ward, and I. N. Clarke. 1987. Complete nucleotide sequence of the major outer membrane protein gene from Chlamydia trachomatis serovar Ll. FEMS Microbiol. Lett. 42:185-190. Rodriguez, P., A. Vekris, B. de Barbeyrac, B. Dutilh, J. Bonnet, and C. Bebear. 1990. Typing of Chlamydia trachomatis by restriction endonuclease analysis of amplified MOMP gene. Abstr. 90th Annu. Meet. Am. Soc. Microbiol. 1990, C-333, p. 399. Saikku, P., and S.-P. Wang. 1987. Chlamydia trachomatis immunotypes in Finland. Acta Pathol. Microbiol. Immunol. Scand. Sect. B 95:131-134. Stephens, R. S., R. Mullenbach, R. Sanchez-Pescador, and N. Agabian. 1986. Sequence analysis of the major outer membrane protein gene from Chlamydia trachomatis serovar L2. J. Bacteriol. 168:1277-1282. Stephens, R. S., R. Sanchez-Pescador, E. A. Wagar, C. Inouye, and M. S. Urdea. 1987. Diversity of Chlamydia trachomatis major outer membrane protein genes. J. Bacteriol. 169:38793885. Wagenvoort, J. H. T., R. J. Suchland, and W. E. Stamm. 1988. Serovar distribution of urogenital Chlamydia trachomatis strains in The Netherlands. Genitourin. Med. 64:159-161. Wang, S. P., and J. T. Grayston. 1971. Classification of TRIC and related strains with micro IF, p. 305-321. In R. L. Nichols (ed.), Trachoma and related disorders caused by chlamydial agents. Excerpta Medica, Amsterdam. Wang, S. P., C.-C. Kuo, R. C. Barnes, R. S. Stephens, and J. T. Grayston. 1985. Immunotyping of Chlamydia trachomatis with monoclonal antibodies. J. Infect. Dis. 152:791-800. Yuan, Y., Y.-X. Zhang, N. G. Watkins, and H. D. Caldwell. 1989. Nucleotide and deduced amino acid sequences for the four variable domains of the major outer membrane protein of the 15 Chlamydia trachomatis serovars. Infect. Immun. 57:1040-1049.
JOURNAL OF CLINICAL MICROBIOLOGY, June 1991, p. 1132-1136 0095-1137/91/061132-05$02.00/0 Copyright © 1991, American Society for Microbiology
Typing of Chlamydia trachomatis by Restriction Endonuclease Analysis of the Amplified Major Outer Membrane Protein Gene PATRICIA RODRIGUEZ,' ANTOINE VEKRIS,1 BERTILLE DE BARBEYRAC,l BRIGITTE DUTILH,1 JACQUES BONNET,2 AND CHRISTIANE BEBEAR1* Laboratoire de Bacteriologie, Universite de Bordeaux IH, 146, rue Leo Saignat, 33076 Bordeaux Cedex,' and Institut de Biochimie Cellulaire et Neurobiochimie du Centre National de la Recherche Scientifique, 33077 Bordeaux Cedex,2 France Received 24 October 1990/Accepted 20 February 1991
A procedure was developed for characterization of Chlamydia trachomatis strains by using restriction endonuclease analysis of amplified genes of the major outer membrane protein (MOMP). Reference strains of the 15 serovars (A through K and Li through L3) and clinical isolates were tested. The nucleotide sequences of the MOMP genes of each of the 15 serovars were arbitrarily constructed by using the sequences of the four variable domains known for each serovar and the constant domains of serovar Li. Computer analysis of these sequences indicated that two restriction digestions performed in parallel, one with AluI and the other with HpaII, followed by Hinfl and EcoRI, would allow the theoretical differentiation of 13 serovars. Serovars Ba and Li presented the same theoretical restriction profile. Our typing method consisted of polymerase chain reaction amplification of a fragment of about 1,200 bp of the MOMP gene, followed by restriction endonuclease digestion with the aforementioned enzymes. From the 15 serovars, we obtained 14 different patterns; 13 profiles were serovar specific, while serovars B and Ba presented the same pattern. Application of this typing method to C. trachomatis strains isolated from clinical material gave the same results as the immunotyping method for 14 of 17 strains. Furthermore, restriction endonuclease analysis detected differences within a serovar. This method seems to be promising for epidemiological studies.
Chlamydia trachomatis has been recognized as an important sexually transmitted pathogen and a cause of human ocular disease. The C. trachomatis species is divided into at least 15 serovars, which have been identified by a microimmunofluorescence method by Wang et al. (19) by using antibodies raised against the major outer membrane protein (MOMP) (3). Serovars A, B, Ba, and C are the etiological agents of trachoma; serovars D through K cause primarily oculogenital infections; and serovars Li, L2, and L3 are involved in lymphogranuloma venereum. However, the microimmunofluorescence method has not been widely used in large-scale epidemiological studies, since specific immune sera are generally unavailable. Our goal was to develop a typing method for C. trachomatis based on the analysis of the nucleotide variation of the MOMP gene, which is responsible for the antigenic variation of the protein. The MOMP consists of five constant domains separated by four variable regions and carries antigenic determinants for each serovar. The total MOMP gene sequences for serovars L2, B, and C (16, 17), LI (13), H (6), and E (12) and those of the four variable domains from the 15 serovars (21) have been published. So, we first simulated the 15 nucleotide sequences of the MOMP gene, using the published sequences of variable regions and the constant domains of serovar Li. Then, by computer analysis, we chose restriction endonucleases which would give restriction patterns that differentiated most of the 15 serovars. Finally, the polymorphism of restriction endonuclease digest fragments of the MOMP gene amplified by the polymerase chain reaction (PCR) by using primers localized in the first and the fifth constant region was examined in reference strains of the 15 serovars and in clinical isolates grown on cell culture.
*
Corresponding author.
(This work was presented at the 90th Annual Meeting of the American Society for Microbiology [14].) MATERIALS AND METHODS Bacterial strains. C. trachomatis reference strains of the
following 10 serovars were from the American Type Culture Collection, Rockville, Md.: A (VR 571B), B (VR 573), Ba (VR 347), C (VR 578), D (VR 885), E (VR 348B), F (VR 346), G (VR 878), H (VR 879), and L2 (VR 902B). The strains of five other serovars were from Y. Perol (Hopital Saint-Louis, Paris, France), who received them from the American Type Culture Collection: I (VR 880), J (VR 886), K (VR 887), Li (VR 901B), and L3 (VR 903). Seventeen strains of C. trachomatis, which were isolated from patients, were tested after culture on McCoy cells. Eight randomly selected wild-type strains were obtained from genital specimens in the Laboratoire de Bacteriologie, H6pital Pellegrin, Bordeaux, France. After typing by restriction fragment length polymorphism (RFLP) analysis, the eight strains were sent to K. Persson (Malmo General Hospital, Malmo, Sweden) to be typed by microimmunofluorescence by using type-specific monoclonal antibodies. A panel of 31 different monoclonal antibodies raised against antigens of the MOMP and reactive with one or more of the C. trachomatis serovars was used for serotyping, as described previously (10). Furthermore, nine strains, which were isolated in Malmo from genital specimens and previously serotyped by K. Persson, were selected; one of each of the following serovars was tested by RFLP analysis: B, C, D, F, G, H, I, J, and K. The following clinical isolates were used for specificity testing: Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa, Gardnerella vaginalis, Neisseria gonorrhoeae, Lactobacillus spp., Streptococcus agalactiae, Fusobacterium fusiformis, Clostridium perfringens, and Mycoplasma hom1132
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TYPING OF C. TRACHOMATIS BY RFLP OF THE MOMP GENE
inis. The strains were kept frozen at -70°C until they were tested. C. trachomatis culture and sample preparation for PCR. Reference and clinical strains of C. trachomatis were grown on cycloheximide-treated McCoy cell monolayers. After 72 h of incubation at 35°C, inclusions were detected by a direct fluorescent-monoclonal antibody method (Microtrak; Syva, Palo Alto, Calif.). Serial passages were performed until about 20% of cells were infected. A 100-,ul volume of Chlamydia cell cultures was mixed with 200 RI of lysis buffer (5) containing 10 mM Tris hydrochloride (pH 8), 1 mM EDTA, 0.1% Triton, and 20 ,ug of proteinase K (Boehringer GmbH, Mannheim, Federal Republic of Germany) per ml. Samples were incubated at 55°C for 90 min and then at 95°C for 30 min. DNA extraction from other bacteria. Bacteria were grown in appropriate culture media. Cells were lysed with 50 pug of lysozyme per ml and treated with 1% sodium dodecyl sulfate (wt/vol) containing 0.1 mg of proteinase K per ml. DNA was purified by phenol-chloroform extraction, ethanol precipitation, and RNase treatment, as described previously by Maniatis et al. (8). Oligonucleotides. Two 23-base oligonucleotides (CT1 and CT5) were used as primers for the PCR, and a 21-base internal oligonucleotide (CT3) was used as a probe. CT1 and CT3 were chosen in the sequence of the first conserved domain of the MOMP gene (4), and CT5 was chosen in the sequence of the fifth domain, according to the published sequences of serovars L2, B, and C (16, 17) and Li (13): CT1 = 5'-GCCGClTTTGAGTTCTGCTTCCTC-3'; CT3 = 5'-TC CTTGCAAGCTCTGCCTGTG-3'; CT5 = 5'-ATTrACGTGA GCAGCTCTCTCAT-3'. They were prepared on a DNA synthesizer (model 381A; Applied Biosystems) by the methoxyphosphoramidite method and purified twice by precipitation with 3 volumes of absolute ethanol in the presence of 0.3 M sodium acetate (pH 5). PCR. Amplifications were performed with the thermostable DNA polymerase from Thermus aquaticus (Genofit, Geneva, Switzerland, or New England BioLabs, Inc., Beverly, Mass.). The final reaction mixture contained either 10 pl of extracted nucleic acids from chlamydial reference strains or clinical isolates or 1 ,ug of purified DNA from other microorganisms or human cells; 330 p.M (each) dATP, dCTP, dGTP, and dTTP; 220 ,ug of gelatin per ml; 50 mM KCI; 10 mM Tris hydrochloride (pH 8.4 at 70°C); 1.5 mM MgCl2; and 1 ,uM (each) primers CT1 and CT5 in a total volume of 50 RId. Samples were then heated at 95°C for 10 min and chilled in ice. Condensation droplets were collected by centrifugation at 5,000 x g for 10 s. Each sample received 1.5 U of Taq DNA polymerase and was overlaid with 50 ,u1 of paraffin oil. Thirty cycles of amplification were performed in an automated PCR machine (Tac-Tiq; Genofit). Each cycle consisted of 1 min of denaturation at 95°C, 1 min of annealing at 55°C, and 1 min of extension at 70°C. PCR products were analyzed by electrophoresis of 10 p.l of the amplification mixture on a 1% agarose gel in Tris acetate buffer (8). Purified DNAs from McCoy cells and human leukocytes were used as negative controls. Hybridization. DNA was denatured by 0.2 N NaOH and transferred by capillarity onto a nylon membrane (Compas, Genofit) overnight in a 25 mM NaH2PO4 buffer (pH 6.5). DNA was cross-linked to the membranes by a UV light exposure (312 nm, 2 min). Prehybridization was performed by incubation at 42°C in a solution containing Sx SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.2% sodium dodecyl sulfate, 0.1% N-lauroylsarcosine, and 0.5%
1133
blocking reagent (Boehringer). For the hybridization, 25 ng of [_y-32PIATP 5'-labeled CT3 probe was added. After hybridization at 42°C for 18 h, membranes were washed twice in 0.1% sodium dodecyl sulfate-2x SSC at room temperature for 5 min, and twice in 0.1% sodium dodecyl sulfate0.1 x SSC at 42°C for 15 min. Filters were autoradiographed with Kodak film for 48 h. Computer analysis of MOMP gene sequences. The 15 1.2-kb sequences bracketed by CT1 and CT5 were simulated. They were analyzed by computer with appropriate analysis software (BACHREST and DIGEST programs). The numbers and sizes of the restriction fragments generated by each of 75 different restriction endonucleases were determined in order to find an appropriate restriction treatment of the CT1-CT5 fragment which could give a different electrophoresis profile for each serovar. Restriction treatments of amplified DNA fragments and electrophoretic analysis. Amplified DNA samples were digested in parallel, on one hand, with AluI and, on the other, with the three enzymes HpaII, EcoRI, and Hinfl (Bethesda Research Laboratories). The first digestion was performed with 4 U of AluI on 10 p.l of amplified DNA, using the assay buffer recommended by the manufacturer, for 4 h at 37°C. The second digestion was run on 10 p.l of amplified DNA first with 4 U of HpaII in 10 mM Tris hydrochloride (pH 7.6-10 mM MgCl2 at 37°C for 4 h. HpaII was then denatured by a 10-min incubation at 60°C. Then, 2 p.l of 200 mM Tris hydrochloride (pH 8)-75 mM NaCl was added and samples were incubated overnight at 37°C with 4 U of EcoRI and Hinfl. Analysis of digested DNA was performed by electrophoresis of the total reaction volume on an 8% polyacrylamide gel. RESULTS MOMP gene amplification of the reference strains. Enzymatic amplification with primers CT1 and CT5 yielded products of about 1,200 bp with the 15 reference strains (Fig. 1A). No size variations of amplified products among the 15 serovars were observed on a 1% agarose gel, as might be expected from the small size variations that exist in the variable domains of the MOMP gene (21). A second fragment (about 800 bp) was sometimes detected in some serovars, but this was dependent upon the reaction conditions (Fig. 1A, lanes Ba, H, I, and Li). Different amounts of amplified products were observed because of differences in the starting cell culture lysates. Specificity of the PCR. Amplified fragments were hybridized after Southern transfer with the internal probe CT3 (Fig. 1B). A positive hybridization signal was obtained with the 1,200-bp fragments of each C. trachomatis serovar. We never observed any hybridization signal with the 800-bp fragment that might have been generated by an unspecific amplification. However, in some cases, a smear of highmolecular-weight DNA, which did not appear on ethidium bromide staining, was detected when the CT3 probe was used in the hybridization. This smear could be a network of amplified DNA fragments created during the amplification reaction. Amplifications conducted on DNAs isolated from other microorganisms encountered in the human genital tract or on DNA from human leukocytes or McCoy cells did not yield any band (data not shown). Computer analysis of MOMP gene sequences. According to the results of computer analysis of the MOMP gene sequences, we chose to digest the amplified DNA fragments
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ically similar profiles. Hydrolysis with HpaII-EcoRI-Hinfl differentiated 10 serovars (C, D, E, F, G, H, J, K, L2, and L3), while serovars A-I and B-Ba-Li had similar restriction patterns. Thus, on the basis of the two parallel enzymatic digestions, 13 of 15 serovars of C. trachomatis could theoretically be separated, while serovars Ba and Li presented the same restriction profiles. Analysis of the restriction profiles of the 15 serovars. Because we used serovar Li as a reference for computer analysis, the restriction hydrolysis for this serovar with AluI (Fig. 2A) and HpaII-EcoRI-Hinfl (Fig. 2B) gave the expected electrophoretic patterns (Table 1). However, most of the experimental results obtained with the 14 other serovars differed from the projected values (Table 1). As shown in Table 1 and Fig. 2A, the AluI digestion B A E Ba C D E F G H J KL L2 L a allowed us to separate 10 serovars: A, C, E, F, G, I, J, K, Ov.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Li, and L2, while serovars B-Ba-D and H-L3 exhibited similar patterns and could not been clearly differentiated. As shown in Table 1 and Fig. 2B, the HpaII-EcoRI-Hinfl digestion allowed us to differentiate 11 serovars (D, E, F, G, H, I, J, K, Li, L2, and L3) but not serovars A-C, and B-Ba. So, after the triple hydrolysis, serovar D could be differentiated from serovars B and Ba, and serovar H could be differentiated from serovar L3, whereas AluI hydrolysis gave distinct patterns for serovars A and C. 22{0 ti, _ _~~~~~~~~~~~~~~~~~~~~~~~~~~~~- -0 Thus, AluI and HpaII-EcoRI-Hinfl restriction treatments gave 14 different profiles and permitted complete separation of 13 C. trachomatis serovars. All the reference genital tract FIG. 1. Electrophoretic analysis of PCR products, after amplifiserovars could be identified by this RFLP analysis. Tracation of the MOMP gene from the 15 C. trachomatis serovars by choma serovars B and Ba could not be differentiated after using the CT1 and CT5 primers. (A) Ethidium bromide staining of agarose gel. Lane M contains the DNA molecular weight marker experimental restriction analysis. (lambda DNA digested with EcoRI and HindIll). The letters above Typing of C. trachomatis wild-type strains. The eight ranthe lanes indicate the 15 serovars of C. trachomatis. (B) Southern domly selected isolates obtained from genital tract samples analysis of the same gel by using the internal 5', y-32P-end-labeled were typed by RFLP analysis. Five of the eight strains were CT3 probe. identified as serovar E; there was also one strain each of serovars D, H, and L2 (data not shown). Serovar determination by RFLP analysis was in complete agreement with subsequent immunotyping. separately with AluI and with HpaII-EcoRI-Hinfl in two Because of the limited number of different serovars in the steps: HpaII followed by EcoRI and Hinfl together. For the randomly selected strains, wild-type strains representative two digests, the major differences between each C. trachoof nine other serovars were subjected to analysis. We matis serovar were found among DNA fragments of between identified six serovars (D, F, G, H, J, and K) in this 600 and 100 bp (Table 1). AluI digestion allowed us to collection by RFLP analysis, and these findings were in differentiate five serovars (A, C, J, K, and L2). However, agreement with those of immunotyping (Fig. 3). However, serovars B-D-E; H-I-L3; F-G; and Ba-Li presented theoretTABLE 1. Restriction fragment sizes of the CT1-CT5 sequence of the MOMP gene for the 15 C. trachomatis serovars Restriction fragment size (bp) of fragments of the following serovarsa:
Hydrolysis p
a
p
a
p
a
p
E
D
C
Ba
B
A
a
p
a
p
a
p
a
p
I
H
G
F
a
p
a
p
a
p
Li
K
J a
p
a
p
L3
L2 a
p
a
p
a
AluI 534 530 292 240 237 420 228 225 171 150 225 204 142 142
292 228 204 142
240 490 445 289 240 225 240 420 228 225 204 171 140 225 204 142 142 142
HpaII-EcoRI-
289 240 396 396 396 228 225 228 257 228 225 142 207 210 207 159 159 159 142 107 101
396 506 506 506 450 483 440 534 530 289 240 275 228 506 506 210 240 420 237 410 340 420 171 210 228 228 228 142 240 420 180 171 140 171 140 171 140 171 140 204 210 225 130 171 140 142 142 142 159
Hinfl 481 590 630 630 630 630 868 590 410 410 387 390 225 225 225 225 136 390 225 225 225 170 131 170 216 217 131 120 120 205 a
244 225 706 590 539 510 481 490 481 590 457 490 457 510 627 610 463 460 387 490 225 216 228 230 228 230 387 390 387 390 387 390 411 390 225 220 225 225 350 136 136 136 136 131 167 167 167 136 216 167 156 167 167 167 136 136 225 131 131 156 167 131
p, projected fragment sizes (larger than 100 bp); a, actual fragment sizes (larger than 100 bp) deduced from their migration distances by using the
HaeIlI-digested pBR322 hydrolysate as a reference standard.
TYPING OF C. TRACHOMATIS BY RFLP OF THE MOMP GENE
VOL. 29, 1991
M A B Ba C D E F G H I J K
A
4.14 bWp -
'257 bv-
64
C
-
1.40t)1 >p_
M1 A B Ba C D E F G H I J K Li Lz L3
B 587 bP
-
434 h I,
-
2 5 7t) p
-
1 80 t) p
-
124 bp
-
80 tip
Iqp I
-
I
-
fw
I .il
64
hp-
FIG. 2. Restriction profiles of the 15 C. trachomatis serovars (lanes A to L3) obtained by 8% polyacrylamide gel electrophoresis after restriction hydrolysis of the PCR products. (A) AluI digestion. (B) HpaII-EcoRI-Hinfl digestion. Some sizes of DNA fragments from pBR322, which were digested with HaeIII and used as molecular weight markers (lane M), are given on the left.
RFLP profiles from serovar B and C wild-type strains differed from the profiles of the serovar B and C reference strains and from all the other reference patterns. The total restriction pattern obtained with the wild-type serovar B
L; 2
G
F 2
1
2
1
H 2
1
I 2
1
K
2
1
2
1
2
FIG. 3. Restriction profiles of the nine previously serotyped clinical genital isolates. The letters indicate the types found by using the immunotyping method. Lanes 1 contain the AluI digestions, and lanes 2 contain the HpaII-EcoRI-Hinfl digestions. Some sizes of DNA fragments from pBR322, which were digested with HaeIII and used as molecular weight markers (lane M), are given on the right.
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strain clearly differed from that obtained with the serovar B reference strain. The serovar C genital isolate presented a pattern similar to that of the serovar C reference strain after AluI hydrolysis, but not after HpaII-EcoRI-Hinfl hydrolysis. A wild-type serovar I strain by the immunotyping method showed a typical serovar H restriction profile by RFLP analysis (Fig. 3). To reconfirm the results of the immunotyping method, this strain was again examined by K. Persson with different serovar I-specific and serovar H-specific monoclonal antibodies. These new comparisons established the wild-type isolate as being representative of the I immunotype. Furthermore, B and C wild-type immunotypes were confirmed by using different serovar B- and serovar C-specific monoclonal antibodies. DISCUSSION In order to develop epidemiological data and to detect multiple serovar infections (2), accurate and specific typing of C. trachomatis strains is required. Immunotyping, which requires serovar-specific monoclonal antibodies, is the most useful diagnostic method (20). However, this technique has serious limitations, since specific monoclonal antibodies are not commercially available. A common way to type organisms is to use RFLP analysis. Restriction endonuclease analysis of C. trachomatis total DNA has been reported previously (11). Although this method allowed differentiation among biovars, serovars, and strains within a serovar, rather large amounts of purified or labeled DNA and a large number of restriction endonucleases were required. In this report, we described a typing scheme for C. trachomatis strains that combined both PCR and RFLP analyses. Since the MOMP carries the major antigenic determinants which separate the various serovars, analysis of the MOMP gene seemed to be a convenient tool for epidemiological studies. The goal of our study was to develop a typing method based on genotypic differences which could give a classification that is well correlated to that of the 15 known C. trachomatis serovars. This method, although more difficult to employ than immunofluorescence, allows one to type many strains simultaneously and to retain results on preserved gels or photographs. Furthermore, the small number of DNA bands observed by our method enables simple differentiation of strains. In the first step of our typing method, we succeeded in amplifying nearly all of the MOMP gene from reference strains of the 15 serovars and from a selection of clinical isolates. The CT1 and CT5 primer sequences are therefore common to all serovars. As described here, our typing method requires the preliminary growth of C. trachomatis. The PCR could be used on cell culture-grown strains when only about 20% of the McCoy cell line monolayers were infected, while immunotyping requires about 50% of the cells to be infected (10, 18, 20). Using both AluI and HpaII-EcoRI-Hinfl hydrolysis on the amplified MOMP gene, we could experimentally differentiate 13 of 15 reference serovars. The MOMP gene sequences of serovars B and Ba, which have a unique profile, differed only in a small number of bases located in the variable domains (21). Most of the experimental restriction patterns were different from those obtained by computer analysis. Since the five constant domains of the MOMP gene from serovar Li were introduced between the variable regions of the 15 serovars, such differences between theoretical and experimental analyses were expected; indeed, nucleotide sequences of the MOMP gene differed not only in
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the variable domains but also, to a lesser extent, in the constant domains. The application of this typing method on eight randomly selected chlamydial isolates showed a serovar distribution similar to that noted in previous studies (1, 7, 9, 10, 15, 18). Serovar E occurred frequently (in five of eight strains). We also found one L2 strain, which seems to occur in only about 1% of genital infections (7). The typing of nine other clinical isolates showed restriction patterns corresponding to those obtained for the reference strains in six isolates. For these six isolates, the immunotyping and RFLP identification methods were in agreement. However, some differences were observed for the remaining three clinical isolates (serovars B, C, and I). Thus, genomic differences may exist inside the same serovar. This is to be expected, since small differences in the genome may not affect the protein sequence and modifications in peptide sequence may not affect the epitopes recognized by the monoclonal antibodies used in the immunotyping. The reverse is true. Differences in a given epitope may also not be detected by this particular RFLP analysis. It is interesting that the serovar B and C clinical isolates used in this study came from genital tract specimens, whereas the two serovar B and C reference strains used in this study came from ocular samples. The PCR-RFLP typing method seems promising for epidemiological studies. It provides another marker system which will be useful for obtaining epidemiological data in conjunction with immunotyping.
6. 7. 8.
9. 10.
11. 12. 13.
14.
15.
ACKNOWLEDGMENTS This work was supported by grant 874380228 from the Conseil Regional d'Aquitaine. We thank K. Persson for supplying C. trachomatis isolates and performing immunotyping on all strains.
16.
17. 1. 2.
3.
4.
5.
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