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ABSTRACT |
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vector ZAP express. Sequences of 145 clones established that the libraries predominantly contain sequences from S. scabiei, enabling a major sequencing program to begin. Among those sequenced to date, cDNAs encoding S. scabiei homologues of 3 house dust mite allergens—the M-177 apolipoprotein, glutathione S-transferase, and paramyosin—were identified. The availability of cDNA libraries from S. scabiei var. hominis and S. scabiei var. vulpes and the emerging public sequence databases from both opens up new possibilities in scabies research. |
INTRODUCTION |
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Molecular studies on scabies have been limited to date because of the difficulty of obtaining sufficient mites. A system of growing dog-derived mites on the ears of New Zealand white rabbits, which provides access to S. scabiei var. canis and allows the testing of potential vaccines, has been developed.4,5 A library of cDNA clones from S. scabiei var. vulpes has been constructed from mites obtained from red foxes, and the sequence of a cDNA clone encoding paramyosin has been characterized.6 In the accompanying article by Harumal et al., we describe the construction of a cDNA expression library cloned in the vector pGEX4T-2 from S. scabiei var. hominis, the isolation of a homologue of the house dust mite allergen Mag/M-1777,8 by immunoscreening and a vaccination/ challenge trial with recombinant fused polypeptide in the rabbit/S. scabiei var. canis system. We report here the construction of cDNA libraries from S. scabiei var. hominis cloned in a bacteriophage vector (ZAP express, Stratagene, La Jolla, CA). This has the advantage of using the packaging system to generate a large number of clones from small amounts of cDNA. Then they can be converted en masse into plasmids suitable for sequencing after library amplification.
S. scabiei var. hominis mites were collected, and mRNA was prepared from approximately 1,000 mites as described (see Harumal et al.). Approximately 65% of the purified mRNA was used to prepare oligo-dT primed, double-stranded cDNA, which was directionally cloned into the ZAP express vector using Stratagene’s ZAP express cDNA synthesis kit (catalogue No. 200403). cDNA was size fractionated on a Sepharose CL-2B column to yield 3 fractions. The fractions were ligated independently into the vector and packaged using Stratagene’s Gigapack III gold packaging extracts (catalogue No. 200451). A total of approximately 3 x 105 independent clones were generated in the 3 primary libraries Yv4 (with the largest cDNA), Yv5, and Yv6. The primary libraries were amplified yielding between 3 x 109 and 2 x 1010 plaque forming units/ml. Clones were excised to phagemids as described by the manufacturer and used to infect Escherichia coli strain XLOLR before sequencing. Insert sizes ranged from 300 bp to at least 2.5 kb.
Twenty phagemid clones from each of the 3 libraries were sequenced by the dideoxy chain termination procedure from the T3 primer using ABI PRISM BigDye terminator (Applied Biosystems, Foster City, CA, part No. 4303153) and an ABI PRISM 377 DNA sequencer. These sequences were manually trimmed of vector and low-quality sequence. An additional 96 clones from the Yv4 library were sequenced in 96-well plate format from the T3 primer using BigDye terminator and an ABI PRISM 3700 capillary DNA analyzer, under contract by the Australian Genome Research Facility. The program phred9 was used for automatic base calling and trimming of vector and low-quality sequences (phred score <20).
All 145 successful sequences were compared with the Gen-Bank nonredundant (nr) databases using the basic local alignment search tool (BLAST).10 Sequences also were compared with the GenBank expressed sequence tag (EST) database using BLASTn, to check for identity with ribosomal RNAs or any of the available S. scabiei var. vulpes anonymous ESTs. We had found previously (see Harumal et al.) that the cDNA was contaminated with human DNA, presumably from skin cells in the gut of the mites; it was important to ascertain the level of contamination in the ZAP express library before starting a major EST project. Sequences giving a best BLASTn result against a human DNA sequence with a score of 
200 (chosen to minimize the number falsely assigned) against the GenBank nr database were considered to be human DNA contamination (n = 3), whereas those with a best BLAST result of E > 0.01 were not considered to have a statistically significant match (n = 39). For the remaining sequences (n = 103), the E value, accession number, and description of the most significant BLAST result (BLASTn or BLASTx) are shown in Table 1
. These sequences have been deposited at GenBank, and their accession numbers are listed in Table 1. The 39 sequences that did not give a significant match have been deposited in GenBank EST database as probable Sarcoptes scabiei var. hominis sequences under accession numbers BM276682 to BM276719 (except for YV4B03; see later). The closest matches to 56 of the sequences are nonvertebrate metazoan sequences, principally Drosophila melanogaster (n = 23) and Caenorhabditis elegans (n = 9), for which most such data have been published. These are exemplified by BLASTn matches to ribosomal sequences (Yv4-1, Yv4-13, Yv5-16, Yv6-16, Yv4E02, and Yv4E05), which have strong homology to lower metazoa.
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). An additional 3 sequences (Yv5-6, Yv5-15, and Yv4H03) that had no significant BLAST matches against the GenBank nr databases had significant matches with 1 of the var. vulpes ESTs (data not shown). This initial characterization confirms that the library described here consists predominantly of clones derived from S. scabiei sequences with limited contamination by human sequences and is suitable for the start of an EST sequence library.
Clones Yv4D01 and Yv4E09 had a most significant match to the house dust mite Dermatophagoides farinae Mag allergen,7 whereas clone Yv6-6 matched with the Euroglyphus maynei homologue, the high-molecular-weight allergen precursor M-1778 (Table 1). These sequences, corresponding to an apolipoprotein in hemolymph, are some of the most frequent cDNA clones in our library (Table 1 and unpublished data). We previously have identified a cloned sequence designated S. scabiei antigen 1 (Ssag1—EST database accession No. BM176880) corresponding to M-177, by screening with antibodies from scabies-infested rabbits (see Harumal et al.). The combined contiguous sequence of Yv4D01, Yv4E09, and Yv6-6 was submitted to GenBank as S. scabiei antigen 1 (Ssag1) with the accession number AF462196.
A BLASTx search using Yv4A08 revealed a highly significant match with the D. pteronyssinus allergen glutathione S-transferase (GST)11 (E = 1e-82; accession No. AAB32224). The complete coding sequence of the scabies GST was submitted to GenBank with the accession number AF462789. The S. scabiei GST homologue is a potential candidate for a scabies allergen.
Clone Yv4B03 is included in Table 1 despite being classified as a technical failure (phred score <20), as a BLASTn search using the low-quality data revealed homology with the S. scabiei var. vulpes paramyosin clone6 (E = 1e-18; accession No. AF317670), the only scabies protein coding sequence previously published. Further sequencing of Yv4B03 (accession No. AF462195) allowed the comparison of the fox and human mite sequences over a 1,948-bp region, which showed that the fox mite sequence differed at 1 nucleotide position and had an insertion of 3 nucleotides.
Clone Yv4-12 displayed significant homology at the amino acid level with cysteine proteases, in particular the Boophilus microplus cathepsin L–like enzyme12 (E = 2e-45; accession No. AAF61565), a cysteine protease with optimal enzyme activity occurring at an acidic pH. We previously have discussed the hypothesis that hydrolytic enzymes of the scabies gut may be potential vaccine molecules (see Harumal et al.). It would be relevant to investigate whether this molecule is an enzyme secreted into the gut of the mite.
Clone Yv4E01 was homologous to vitellogenin from Pimpla nipponica (E = 1e-06), and further sequencing has revealed much more extensive homology (unpublished data). Egg antigens are the targets of delayed-type hypersensitivity reactions in schistosomiasis and have been used successfully as vaccines that suppressed granuloma formation and reduced mortality in Schistosoma mansoni infected mice.13 Although nothing is known at the moment, it is possible that egg antigens play some role in the immunology of scabies. A new avenue of investigation will be opened by the identification of such clones.
Two clones (Yv5-9 and Yv6-12) encoded a protein with a substantial number of internal repeats. Because this is an abundant molecule (unpublished results), it would be relevant to investigate whether it is the target of antibody responses in humans or animals or both. If so, it could be a candidate immunodiagnostic molecule.
Many molecules identified here offer the potential to extend previous studies on the relationships of scabies mites from various mammalian hosts, including the clones Yv6-17 encoding cytochrome oxidase subunit I, Yv5-8 encoding cytochrome oxidase subunit II, and Yv4D09 encoding the cytochrome Bc1 complex. Although microsatellite studies led us to conclude that mites clustered by host rather than geographic origin,14 studies on a fragment of the mitochondrial 12S RNA gene agreed with the concept that all mites are the same species.15
Limitations on availability of experimental material from which to generate S. scabiei var. hominis cDNA libraries have been overcome here by using mites obtained from skin fragments in the bedding of crusted scabies patients and by taking advantage of the highly efficient ZAP express/packaging system. The 3 S. scabiei var. hominis cDNA libraries described here have allowed the start of a program to generate 50,000 scabies sequences. Although the current libraries will require normalization to remove abundant sequences16 when the rate of new sequence acquisition in this program declines, they are sufficiently large (3 x 105 clones) to allow this. The beginning of EST databases derived from mites from humans and foxes and our demonstration that it is now technically feasible to carry out vaccination/challenge studies with cloned scabies antigens (see Harumal et al.) should facilitate greatly research on scabies.
Received January 7, 2002. Accepted for publication June 27, 2002.
Acknowledgments: The authors thank Danny Wilson for assistance with sequencing at QIMR, and Rob Slade and Peter Wilson for assistance with sequencing at the Australian Genome Research Facility.
Financial support: This work was supported by the Australian National Health and Medical Research Council (grant 137206 and Program in Medical Genomics grant 219175).
Reprint requests: Bart J. Currie, Menzies School of Health Research, P.O. Box 41096 Casuarina, NT 0811 Australia, E-mail: bart{at}menzies.edu.au
Authors’ addresses: Katja Fischer, Deborah C. Holt, and David J. Kemp, The Queensland Institute of Medical Research, The Australian Centre for International and Tropical Health and Nutrition, and The University of Queensland, Brisbane 4029, Australia, Telephone: 61-7-33620402, Fax: 61-7-33620104. Pearly Harumal, Shelley F. Walton, and Bart J. Currie, The Menzies School of Health Research, Darwin 0811, Australia, Telephone: 61-8-89228196, Fax: 61-8-89275187.
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REFERENCES |
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