Molecular and Biochemical Parasitology, 47 (1991) 63-72
63
© 1991 Elsevier Science Publishers B.V. / 0166-6851/91/$03.50 ADONIS 016668519100069W MOLB IO 01542
A recombinant clone of Wuchereria bancrofti with DNA specificity for human lymphatic filarial parasites Nithyakalyani Raghavan I, Larry A. M c R e y n o l d s 2, Claude V. Maina 2, Stephen M. Feinstone 3, Kunthala J a y a r a m a n 4, Eric A. Ottesen ~and T h o m a s B. N u t m a n 1 ~Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, U.S.A.;2New England Biolabs, Beverly, MA, U.S.A.; 3Laboratory of lnfectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda MD, U.S.A.; and 4Centre for Biotechnology, Anna University, Madras, India (Received 6 September 1990; accepted 10 January 1991 )
In order to understand the immune response to Wuchereria bancrofti and to aid in the diagnosis of W. bancrofti infections, recombinant antigens were identified from a W. bancrofti genomic expression library made in )~gtl 1 using a pool of sera from infected Indian patients. One of the recombinant clones, )~WbN 1, containing a 2.5-kb insert, reacted strongly to a pool of sera from patients with lymphatic filariasis but not to normal human sera. In addition, this clone showed restricted specificity at the genomic level to the major lymphatic filarial parasites W. bancrofti and Brugia malayi but not to the closely related filarial parasite Brugiapahangi or to other filarial and non-filarial species tested. Nucleotide sequence analysis indicated the cloned DNA to have homology to myosinlike myofibrillar proteins. Polymerase chain reaction amplification initiated by specific synthetic oligomers amplified DNA in a species-specific manner from as little as 16 pg of isolated DNA or from one microfilaria. Key words: Lymphatic filariasis; Wuchereria bancrofti; Genomic expression library; Myosin; Polymerase chain reaction
Introduction
Human lymphatic filariasis is caused by the parasites Wuchereria bancrofti, Brugia malayi and Brugia timori. Of the estimated 90 million people infected with these lymphatic-dwelling parasites, more then 90% have bancroftian and less than 10% have brugian filariasis [1,2]. The studies which have been performed to date in the molecular analyCorrespondence address: Nithyakalyani Raghavan, Laboratory of Parasitic Diseases, Bldg. 4, Rm. 126, National Institutes of Health, Betbesda, MD 20892, U.S.A.
Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M38213.
Abbreviations: mf, microfilariae; PBS, phosphate-buffered saline; MBP, maltose-binding protein; SSC, saline sodium citrate buffer; PCR, polymerase chain reaction; ORF, open reading frame.
sis of the filarial genome, however, have focused almost exclusively on brugian species, largely because of the availability of these parasites which can be maintained in experimental animals. Such studies have already identified genes encoding ribosomal RNAs [3], genes expressing antigens of potential use in inducing protective immunity [4, 5] and repetitive DNA sequences [6-8]. Since there is no convenient laboratory model for W. bancrofii infection, all parasites must be collected from human patients with microfilaremia, and thus, there is little information specifically describing the W. bancrofti genome, although a repetitive DNA sequence has recently been described from a W. bancrofti genomic library [9]. The present study, therefore, was designed to construct and characterize a W. bancrofti genomic expression library in )~gtl 1 and to use this library to identify and express genes and gene products important in the study of host-parasite interactions.
64 Materials and Methods
Parasites. Microfilariae of W. bancrofti were obtained from the night-blood of patients with high levels of microfilaremia (> 1000 mf m1-1) living in Madras, India. The blood was diluted 1:10 with phosphate-buffered saline (PBS; 0.01 M KHzPO4/ 0.01 M NazHPO4, pH 7.4, 0.15 M NaC1), layered on a discontinuous, iso-osmotic Percoll (Pharmacia Inc., Piscataway, NJ) gradient (25%-35%) and centrifuged at 400 x g for 30 min at 25°C. The layer containing the microfilariae was collected, diluted with PBS and the microfilariae pelleted at 10 000 x g for 10 min. The pelleted material was resuspended in 2 ml of PBS and filtered through a 5 ~tm Nuclepore membrane (Nuclepore Corporation, Pleasanton, CA). The filter was then placed in a dish containing PBS and gently agitated to allow the microfilariae to migrate out of the filter; these freely moving microfilariae were then pelleted and frozen at-70°C until use. Purification ofDNA. DNA was purified from 2 x 105 microfilariae by freeze-thawing three times on dry ice and then resuspending in 1 ml oflysis buffer (50 mM Tris-Hcl pH 8.0, 50 mM EDTA, 100 mM NaC1, 0.05% SDS, 100 ~tg ml I Proteinase-K, 50 mM 2-mercaptoethanol) [6, 10]. DNA from Brugia malayi, Brugia pahangi, Caenorhabditis elegans, Dirofilaria immitis, Acanthocheilonema viteae, Litomosoides carinii and Onchocerca volvulus was extracted from adult worms in a similar fashion; human placental DNA was provided by F. B. Perler, New England Biolabs. Patient sera and other antisera. The sera used in screening the library consisted of a pool from ten South Indian individuals [11] with high titers of anti-filarial antibody and a spectrum of clinical manifestations of W. bancrofti infection including elephantiasis, microfilaremia and tropical pulmonary eosinophilia. Normal human sera (NHS) were collected from North Americans. The patient sera were frozen in liquid nitrogen within 2-3 h of collection and maintained at -70°C until use. The mouse anti-[3-galactosidase serum (Promega, Madison, WI) was used according to the manufacturer's specifications.
Genomic library construction and immunoscreening. A genomic expression library of W. bancrofti was constructed in ~gtl 1 [12] using a complete EcoRI digest of W. bancrofii genomic DNA. Quantitation of the EcoRI-digested DNA indicated that of the total, approximately 70-75% was in the suitable size range ( 7 kb) could not be packaged by ~gtl 1. The library has certain limitations for use in immunoscreening because of its being generated with EcoRI and thus biasing it toward a less random re-
presentation of the genome. However, this library has been shown to contain and/or express multiple other genes or antigenic gene products including (1) clones with repetitive sequences similar to those described for other filarial species [6-8]; (2) antigens with apparent homology to a B. malayi recombinant expressing a potentially protective antigen [ 11 ]; and (3) a W. bancrofti analogue of Bm22 [27]. In the present study, the recombinant phage XWbN1 was selected from this library because of the strong reactivity of the encoded product with sera from infected patients. It was found to contain a 2.5-kb insert by Southern blot analysis showing species specificity for the DNAs of B. malayi and W. bancrofti, the causative agents of human lymphatic filariasis; the cloned DNA did not hybridize with the DNAs of other filarial species, including the closely related B. pahangi. Such species specificity was unexpected, as the morphology and sequence data from repeated DNAs suggest that B. malayi and B. pahangi are much more closely related to each other than to W. bancrofii [6,7]. It is possible that the sequence has diverged to such an extent in B. pahangi that it is not detected by W. bancrofti probes. The inclusion of a new intron could also alter its ability to be amplified by PCR primers. When induced with IPTG, the protein product expressed by ~,WbN 1 reacted strongly with pooled patient sera and not with normal human sera during immunoscreening. As the malE vector [24,25] offers a convenient method for purifying recombinant antigens, it was used to subclone and express the 2.5-kb ~,WbN1 recombinant fusion product. When induced, a 90-kDa fusion protein (the malE gene codes for a 39 kDa product) was expressed. This finding suggests that the 2.5-kb insert from ~,WbN1 does not express a large protein but only a 51 kDa product, probably because of the presence of the termination signal TAA at the 1380 bp position or by the presence of introns in the genomic DNA. The partial sequence and nucleotide analysis of the cloned DNA showed a high (75%) AT content with a codon bias for an A/T at the third base position; other filarial parasites also have genomes that are very AT-rich [28, 29]. The sequence data obtained indicates that there is a long ORF (1380 bp) starting at the 5' EcoRI site, followed by a termination signal TAA (Fig. 3B). Northern analysis
69 showed the presence of an 8-kb transcript, thereby indicating that the full-length antigen is likely to be much larger than 51 kDa. Since XWBN1 showed restricted specificity to the lymphatic filarial parasites W. bancrofti and B. malayi, the potential of this clone as a diagnostic tool was also considered. However, since it was not a repetitive DNA clone and had homology to a structural molecule such as myosin, the sensitivity and specificity of its detection had to be enhanced before attempting to use it as a diagnostic tool. Therefore, we employed the PCR amplification technique [30,31] using synthetic oligomers enclosing a 161-bp fragment and another set enclosing a 763-bp fragment of the partially sequenced )~WbN 1. Not surprisingly, this technique proved to be sensitive enough to identify the human filarial parasites, with the first set of primers specifically amplifying the 161-bp fragment from 2 ng ofB. malayi and W. bancrofti DNA but not from the related filarial parasites B.pahangi and O. volvulus or from human DNA even under conditions of low stringency. Limiting dilution experiments showed the sensitivity of the technique to be such that it could amplify as little as 16 pg of genomic DNA from B. malayi or the DNA extracted from one microfilaria (estimated to be 500 pg; Fig. 4B, lane 4). In addition, these primers were also able to amplify the different geographical isolates of W. bancrofti. Primers 1 and 3 also amplified a 763-bp product from both B. malayi and W. bancrofti indicating strong homology for )~WbN1 (data not shown). Furthermore, since these PCR products were identified in the present studies using only ethidium bromidestained gels, the sensitivity of the reaction would be even further enhanced if labeled probes were used. The ability of)~WbN 1, showing similarity to a conserved myosin-like molecule, to be used to species specifically amplify DNA using two sets of primers in PCR, can be explained by the degree of identity of this myosin-like clone to other nematode species such as B. malayi (26%), C. elegans (16%) and O. volvulus (15%). The reason for these relatively low homologies could be explained by the possible existence of multiple isoforms of W. bancrofti myosin as has been described for C. elegans [32]. It is also possible that this protein could be a related coiled coil protein, such as paramyosin or tropomyosin. Even though the homology was 26% at
best based on the FASTA sequence comparison, it would still be significant since the shared identities are over the entire sequence [33]. Moreover, the percentage of similarities between ~,WbN1 and each of the other myosin-like sequences is >65% when the conservative substitutions are included (based on the model of evolutionary change in proteins; ref. 34). Nevertheless, these preliminary studies indicate that even structurally conserved molecules can be used to differentiate among closely related, but different, species. The predicted protein sequence of ~WbN1 consists of regions with a series of seven amino acid tandem repeats (a-g) with positions a and d being hydrophobic and the remainder being generally hydrophilic. This arrangement of charge, similar to the other myosins, in conjunction with information from the data bank searches [32, 35-39] and from localization studies of the antigen in the parasite (data not shown) indicates that the cloned product has similarities to myosin. Myosin-like proteins and members of the related paramyosin family have been found to be some of the most highly immunogenic molecules in many helminth and non-helminth parasitic organisms. Indeed, these molecules have, as in the present study, been identified as potent immunogens in generalized immunological screening of expression libraries using either patient sera (26, 38, 40), or immunized animals [41, 42]. That many of these highly immunogenic molecules appear to be conserved among even unrelated organisms suggests that the minor differences in the amino acid sequence alters the protein sufficiently to allow it to become quite antigenic; such a situation has clearly been described for the heat shock proteins of both helminths [43, 44] and protozoa [45, 46]. It has been suggested that massive release of these characteristically intracellular (or strucutural) proteins following the clearance (and presumed death) of the microfilarial form of the parasite is the reason for the immunodominance of these classes of proteins. Thus, understanding further the role played by the myosin-like gene product of ~,WbN1 - - along with other expressed and immunoreactive gene products obtained from this W. bancrofti expression library - - may provide insights not only into the immune mechanisms associated with fil-
70
arial parasite clearance but also into the molecular structure necessary for the induction of an antifilarial immune response.
Acknowledgements The authors would like to thank the WHO for their financial support under which this project was initiated, Dr. D. Comb and Dr. F. B. Perler for their encouragement and support, Dr. B. Slatko and Dr. L. M. Mazzola for their help with the sequencing analysis and the LPD editorial staff for their help in preparing this manuscript.
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individualsin an endemic area. J. Clin. Invest. 83, 14-22. 12 Young, R.A. and Davis, R.W. (1983) Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. USA 80, 1194-1198. 13 Huynh, T.V., Young, R.A. and Davis, R.W. (1985) Constructing and screening cDNA libraries in Xgtl0 and ~,gtl 1. In: DNA Cloning A Practical Approach, Vol.I (D.M. Glover, ed.), pp. 49-78. IRL Press, Oxford. 14 Kafatos, F.C., Jones, C.W. and Efstratiadis, A. (1979) Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucleic Acids Res. 7,1541-1552. 15 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual, pp 382-389. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 16 Auffray, C. and Rougeon, F.(1980) Purification of mouse immunoglobulinheavy-chain mesenger RNAs from total myeloma tumor RNA. Eur. J. Biochem. 107,303-314. 17 Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by guanidium thiocyanate-phenolchloroform extraction. Anal. Biochem. 162, 156-159. 18 Rigby, P.W.J., Dieckmann, M., Rhodes, C. and Berg, P. (1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113,237-251. 19 Yanisch-Perron, C., Vicira, J. and Messing, J.(1985) Improved M 13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33, 103-119 20 Sanger, F., Nicken, S. and Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. 21 Slatko, B.E., Benner, J.S., Jager-Quinton, T., Moran, L.S., Simcox, T.G., Van Cott, E.M. and Wilson, G.G. (1987) Cloning, sequencing and expression of the TaqI restriction and modification system. Nucleic Acids Res. 15, 97819796. 22 Devereux, J., Haeberli, P. and Smithies, O. (1985) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387-395. 23 Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239,487~,90. 24 Guan, C-d., Li, P. and Inouye, H. (1988) Vectors that simplify the expression and purification of foreign peptides in Escherichia coli by fusion to maltose binding protein. Gene 67, 21-30. 25 Maina, C.V., Riggs, P.D., Grandea, A.G., Slatko, B.E., Moran, L.S., Tagliamonte, J.A., McReynolds, L.A. and Guan, C-d. (1988) A Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. Gene 74, 365-373. 26 Donelson, J.E., Duke, B.O.L., Moser, D., Zeng, W., Erondu, N.E., Lucius, R., Renz, A., Karam, M. and Flores, G.Z. (1988) Construction of Onchocerca volvulus cDNA libraries and partial characterization of the cDNA for a major antigen. Mol. Biochem. Parasitol. 31,241-250. 27 Arasu, P., Philipp, M. and Perler, F.B. (1987) Brugia ma-
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layi: recombinant antigens expressed by genomic DNA clones. Exp. Parasitol. 64, 281-291. Maina, C.V., Grandea III, A.G., Tuyen, L.T.K., Asikin, N., Williams, S.A. and McReynolds, L.A. (1987) Dirofilaria immitis: genomic complexity and characterization of a structural gene. Molecular paradigms for eradicating helminthic parasites, pp 193-204, Alan R. Liss, New York. Rothstein, N., Stoller, T.J. and Rajah, T.V. (1988) DNA base composition of filarial nematodes. Parasitol. 97, 7579. Erlich, H.A., Gelfand, D.H. and Saiki, R.K. (1988) Specific DNA amplification. Nature 331,461-462. De Bruijn, M.H.L. (1988) Diagnostic DNA amplification: no respite for the elusive parasite. Parasitol. Today 4, 293295. Miller, D.M., Stockdale, F.E. and Karn, J. (1986) Immunological identification of the genes encoding the four myosin heavy chain isoforms of Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 83, 2305-2309. Pearson, W.R. (1990) Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol. 183, 63-95. Dayhoff, M.D., Schwartz, R.N. and Orcut, B.C. (1978) A model of evolutionary change in proteins. Atlas Protein Sequence Struct. 5,345-352. Kam, J., Brenner, S. and Barnett, L. (1983) Protein structural domains in Caenorhabditis elegans unc-54 myosin heavy chain gene are not separated by introns. Proc. Natl. Acad. Sci. USA 80, 42534257. Jandreski, R., Sole, M. J. and Liew, C.C. (1988) Sequence of a cDNA encoding the Syrian hamster cardiac [3-myosin heavy chain. Nucleic Acids Res. 16, 4737. Warrick, R., De Lozanne, H.M., Leinwand, L.A. and Spudich, J. A. (1986) Conserved protein domains in a myosin heavy chain gene from Dictyostelium discoideum. Proc. Natl. Acad. Sci. USA 83, 9433-9437. Werner, C., Higashi, G.I., Yates, J.A. and Rajan, T.V.
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(1989) Differential recognition of two cloned Brugia malayi antigens by antibody class. Mol. Biochem. Parasitol. 35,209-218. Erondu, N.E. and Donelson J.E. (1990) Characterization of a myosin-likeantigen from Onchocerca volvulus. Mol. Biochem. Parasitol. 40, 213-224. Newport, G.R., Harrison, R.A., McKerrow, J. Tart, P. Kallestad, J. and Agabian, N. (1987) Molecular cloning of Schistosoma mansoni myosin. Mol. Biochem. Parasitol. 26, 29-38. Lanar, D.E., Pearce, E.J., James, S.L. and Sher, A. (1986) Identification of paramyosin as schistosome antigen recognized by intradermally vaccinated mice. Science 234, 593-596. Grandea, A.G. III, Tuyen, L.K., Asikin, N., Davis, T., Philipp, M., Cohen, C. and McReynolds, L.A. (1989) A ~.gtl 1 cDNA recombinant that encodes Dirofilaria immitis paramyosin. Mol. Biochem. Parasitol. 35, 31--41. Selkirk, M.E., Denham, D.A., Partono, F. and Maizels, R.M. (1989) Heat shock cognate 70 is a prominent immunogen in Brugian filariasis. J. Immunol. 143,299-308. Hedstrom, R., Culpepper, J., Schinski, V., Agabian, N. and Newport, G. (1988) Schistosome heat-shock proteins are immunologically distinct host-like antigens. Mol. Biochem. Parasitol. 29, 275-282. Bianco, A.E., Favaloro, J.M., Burkot, T.R., Culvenor, J.G., Crewther, P.E., Brown, G.V., Anders, R.F., Coppel, R.L. and Kemp, D.J. (1986) A repetitive antigen of Plasmodium falciparum that is homologous to heat shock protein 70 of Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 83, 8713-8717. Dragon, E.A., Sias, S.R., Kato, E.A. and Gabe, J.D. (1987) The genome of Trypanosoma cruzi contains a constitutively expressed, tandemly arranged multicopy gene homologous to a major heat shock protein. Mol. Cell Biol. 7, 1271-1275.
63
© 1991 Elsevier Science Publishers B.V. / 0166-6851/91/$03.50 ADONIS 016668519100069W MOLB IO 01542
A recombinant clone of Wuchereria bancrofti with DNA specificity for human lymphatic filarial parasites Nithyakalyani Raghavan I, Larry A. M c R e y n o l d s 2, Claude V. Maina 2, Stephen M. Feinstone 3, Kunthala J a y a r a m a n 4, Eric A. Ottesen ~and T h o m a s B. N u t m a n 1 ~Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, U.S.A.;2New England Biolabs, Beverly, MA, U.S.A.; 3Laboratory of lnfectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda MD, U.S.A.; and 4Centre for Biotechnology, Anna University, Madras, India (Received 6 September 1990; accepted 10 January 1991 )
In order to understand the immune response to Wuchereria bancrofti and to aid in the diagnosis of W. bancrofti infections, recombinant antigens were identified from a W. bancrofti genomic expression library made in )~gtl 1 using a pool of sera from infected Indian patients. One of the recombinant clones, )~WbN 1, containing a 2.5-kb insert, reacted strongly to a pool of sera from patients with lymphatic filariasis but not to normal human sera. In addition, this clone showed restricted specificity at the genomic level to the major lymphatic filarial parasites W. bancrofti and Brugia malayi but not to the closely related filarial parasite Brugiapahangi or to other filarial and non-filarial species tested. Nucleotide sequence analysis indicated the cloned DNA to have homology to myosinlike myofibrillar proteins. Polymerase chain reaction amplification initiated by specific synthetic oligomers amplified DNA in a species-specific manner from as little as 16 pg of isolated DNA or from one microfilaria. Key words: Lymphatic filariasis; Wuchereria bancrofti; Genomic expression library; Myosin; Polymerase chain reaction
Introduction
Human lymphatic filariasis is caused by the parasites Wuchereria bancrofti, Brugia malayi and Brugia timori. Of the estimated 90 million people infected with these lymphatic-dwelling parasites, more then 90% have bancroftian and less than 10% have brugian filariasis [1,2]. The studies which have been performed to date in the molecular analyCorrespondence address: Nithyakalyani Raghavan, Laboratory of Parasitic Diseases, Bldg. 4, Rm. 126, National Institutes of Health, Betbesda, MD 20892, U.S.A.
Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M38213.
Abbreviations: mf, microfilariae; PBS, phosphate-buffered saline; MBP, maltose-binding protein; SSC, saline sodium citrate buffer; PCR, polymerase chain reaction; ORF, open reading frame.
sis of the filarial genome, however, have focused almost exclusively on brugian species, largely because of the availability of these parasites which can be maintained in experimental animals. Such studies have already identified genes encoding ribosomal RNAs [3], genes expressing antigens of potential use in inducing protective immunity [4, 5] and repetitive DNA sequences [6-8]. Since there is no convenient laboratory model for W. bancrofii infection, all parasites must be collected from human patients with microfilaremia, and thus, there is little information specifically describing the W. bancrofti genome, although a repetitive DNA sequence has recently been described from a W. bancrofti genomic library [9]. The present study, therefore, was designed to construct and characterize a W. bancrofti genomic expression library in )~gtl 1 and to use this library to identify and express genes and gene products important in the study of host-parasite interactions.
64 Materials and Methods
Parasites. Microfilariae of W. bancrofti were obtained from the night-blood of patients with high levels of microfilaremia (> 1000 mf m1-1) living in Madras, India. The blood was diluted 1:10 with phosphate-buffered saline (PBS; 0.01 M KHzPO4/ 0.01 M NazHPO4, pH 7.4, 0.15 M NaC1), layered on a discontinuous, iso-osmotic Percoll (Pharmacia Inc., Piscataway, NJ) gradient (25%-35%) and centrifuged at 400 x g for 30 min at 25°C. The layer containing the microfilariae was collected, diluted with PBS and the microfilariae pelleted at 10 000 x g for 10 min. The pelleted material was resuspended in 2 ml of PBS and filtered through a 5 ~tm Nuclepore membrane (Nuclepore Corporation, Pleasanton, CA). The filter was then placed in a dish containing PBS and gently agitated to allow the microfilariae to migrate out of the filter; these freely moving microfilariae were then pelleted and frozen at-70°C until use. Purification ofDNA. DNA was purified from 2 x 105 microfilariae by freeze-thawing three times on dry ice and then resuspending in 1 ml oflysis buffer (50 mM Tris-Hcl pH 8.0, 50 mM EDTA, 100 mM NaC1, 0.05% SDS, 100 ~tg ml I Proteinase-K, 50 mM 2-mercaptoethanol) [6, 10]. DNA from Brugia malayi, Brugia pahangi, Caenorhabditis elegans, Dirofilaria immitis, Acanthocheilonema viteae, Litomosoides carinii and Onchocerca volvulus was extracted from adult worms in a similar fashion; human placental DNA was provided by F. B. Perler, New England Biolabs. Patient sera and other antisera. The sera used in screening the library consisted of a pool from ten South Indian individuals [11] with high titers of anti-filarial antibody and a spectrum of clinical manifestations of W. bancrofti infection including elephantiasis, microfilaremia and tropical pulmonary eosinophilia. Normal human sera (NHS) were collected from North Americans. The patient sera were frozen in liquid nitrogen within 2-3 h of collection and maintained at -70°C until use. The mouse anti-[3-galactosidase serum (Promega, Madison, WI) was used according to the manufacturer's specifications.
Genomic library construction and immunoscreening. A genomic expression library of W. bancrofti was constructed in ~gtl 1 [12] using a complete EcoRI digest of W. bancrofii genomic DNA. Quantitation of the EcoRI-digested DNA indicated that of the total, approximately 70-75% was in the suitable size range ( 7 kb) could not be packaged by ~gtl 1. The library has certain limitations for use in immunoscreening because of its being generated with EcoRI and thus biasing it toward a less random re-
presentation of the genome. However, this library has been shown to contain and/or express multiple other genes or antigenic gene products including (1) clones with repetitive sequences similar to those described for other filarial species [6-8]; (2) antigens with apparent homology to a B. malayi recombinant expressing a potentially protective antigen [ 11 ]; and (3) a W. bancrofti analogue of Bm22 [27]. In the present study, the recombinant phage XWbN1 was selected from this library because of the strong reactivity of the encoded product with sera from infected patients. It was found to contain a 2.5-kb insert by Southern blot analysis showing species specificity for the DNAs of B. malayi and W. bancrofti, the causative agents of human lymphatic filariasis; the cloned DNA did not hybridize with the DNAs of other filarial species, including the closely related B. pahangi. Such species specificity was unexpected, as the morphology and sequence data from repeated DNAs suggest that B. malayi and B. pahangi are much more closely related to each other than to W. bancrofii [6,7]. It is possible that the sequence has diverged to such an extent in B. pahangi that it is not detected by W. bancrofti probes. The inclusion of a new intron could also alter its ability to be amplified by PCR primers. When induced with IPTG, the protein product expressed by ~,WbN 1 reacted strongly with pooled patient sera and not with normal human sera during immunoscreening. As the malE vector [24,25] offers a convenient method for purifying recombinant antigens, it was used to subclone and express the 2.5-kb ~,WbN1 recombinant fusion product. When induced, a 90-kDa fusion protein (the malE gene codes for a 39 kDa product) was expressed. This finding suggests that the 2.5-kb insert from ~,WbN1 does not express a large protein but only a 51 kDa product, probably because of the presence of the termination signal TAA at the 1380 bp position or by the presence of introns in the genomic DNA. The partial sequence and nucleotide analysis of the cloned DNA showed a high (75%) AT content with a codon bias for an A/T at the third base position; other filarial parasites also have genomes that are very AT-rich [28, 29]. The sequence data obtained indicates that there is a long ORF (1380 bp) starting at the 5' EcoRI site, followed by a termination signal TAA (Fig. 3B). Northern analysis
69 showed the presence of an 8-kb transcript, thereby indicating that the full-length antigen is likely to be much larger than 51 kDa. Since XWBN1 showed restricted specificity to the lymphatic filarial parasites W. bancrofti and B. malayi, the potential of this clone as a diagnostic tool was also considered. However, since it was not a repetitive DNA clone and had homology to a structural molecule such as myosin, the sensitivity and specificity of its detection had to be enhanced before attempting to use it as a diagnostic tool. Therefore, we employed the PCR amplification technique [30,31] using synthetic oligomers enclosing a 161-bp fragment and another set enclosing a 763-bp fragment of the partially sequenced )~WbN 1. Not surprisingly, this technique proved to be sensitive enough to identify the human filarial parasites, with the first set of primers specifically amplifying the 161-bp fragment from 2 ng ofB. malayi and W. bancrofti DNA but not from the related filarial parasites B.pahangi and O. volvulus or from human DNA even under conditions of low stringency. Limiting dilution experiments showed the sensitivity of the technique to be such that it could amplify as little as 16 pg of genomic DNA from B. malayi or the DNA extracted from one microfilaria (estimated to be 500 pg; Fig. 4B, lane 4). In addition, these primers were also able to amplify the different geographical isolates of W. bancrofti. Primers 1 and 3 also amplified a 763-bp product from both B. malayi and W. bancrofti indicating strong homology for )~WbN1 (data not shown). Furthermore, since these PCR products were identified in the present studies using only ethidium bromidestained gels, the sensitivity of the reaction would be even further enhanced if labeled probes were used. The ability of)~WbN 1, showing similarity to a conserved myosin-like molecule, to be used to species specifically amplify DNA using two sets of primers in PCR, can be explained by the degree of identity of this myosin-like clone to other nematode species such as B. malayi (26%), C. elegans (16%) and O. volvulus (15%). The reason for these relatively low homologies could be explained by the possible existence of multiple isoforms of W. bancrofti myosin as has been described for C. elegans [32]. It is also possible that this protein could be a related coiled coil protein, such as paramyosin or tropomyosin. Even though the homology was 26% at
best based on the FASTA sequence comparison, it would still be significant since the shared identities are over the entire sequence [33]. Moreover, the percentage of similarities between ~,WbN1 and each of the other myosin-like sequences is >65% when the conservative substitutions are included (based on the model of evolutionary change in proteins; ref. 34). Nevertheless, these preliminary studies indicate that even structurally conserved molecules can be used to differentiate among closely related, but different, species. The predicted protein sequence of ~WbN1 consists of regions with a series of seven amino acid tandem repeats (a-g) with positions a and d being hydrophobic and the remainder being generally hydrophilic. This arrangement of charge, similar to the other myosins, in conjunction with information from the data bank searches [32, 35-39] and from localization studies of the antigen in the parasite (data not shown) indicates that the cloned product has similarities to myosin. Myosin-like proteins and members of the related paramyosin family have been found to be some of the most highly immunogenic molecules in many helminth and non-helminth parasitic organisms. Indeed, these molecules have, as in the present study, been identified as potent immunogens in generalized immunological screening of expression libraries using either patient sera (26, 38, 40), or immunized animals [41, 42]. That many of these highly immunogenic molecules appear to be conserved among even unrelated organisms suggests that the minor differences in the amino acid sequence alters the protein sufficiently to allow it to become quite antigenic; such a situation has clearly been described for the heat shock proteins of both helminths [43, 44] and protozoa [45, 46]. It has been suggested that massive release of these characteristically intracellular (or strucutural) proteins following the clearance (and presumed death) of the microfilarial form of the parasite is the reason for the immunodominance of these classes of proteins. Thus, understanding further the role played by the myosin-like gene product of ~,WbN1 - - along with other expressed and immunoreactive gene products obtained from this W. bancrofti expression library - - may provide insights not only into the immune mechanisms associated with fil-
70
arial parasite clearance but also into the molecular structure necessary for the induction of an antifilarial immune response.
Acknowledgements The authors would like to thank the WHO for their financial support under which this project was initiated, Dr. D. Comb and Dr. F. B. Perler for their encouragement and support, Dr. B. Slatko and Dr. L. M. Mazzola for their help with the sequencing analysis and the LPD editorial staff for their help in preparing this manuscript.
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