Single copy Babesia microti hsp70

MOLECULAR

i2LEMICAL PARAsIToLoGy ELSEVIER

Molecular

and Biochemical

Parasitology

83 (1996) 241-246

Short communication

Single copy Babesia wticroti hsp70’ Hyun-Bae

Jie”, Craig W. Bailey”, Bimal K. Ray”, D. Mark Estes”, Nirbhay C. Andrew Carson”,*

Kumarb,

“Departmem of’ b’eterinary Pathohiology. College of Veterinary Medicine, University qf’ Missouri. Columbia. MO 65211. USA bDepartment of Molecular Microbiology and Immunology, School of Hygiene and Public Health, Johns Hopkins Unirersit~. Baltimore. MD, USA

Received

Keywords:

Heat

13 September

1996; revised 2 October

shock protein; Bahesia microti;

Human

1996; accepted

babesiosis;

hsp70;

3 October

1996

Babesiosis

Babes& microti is a malaria-like protozoan which is an obligate parasite of red blood cells (RX). Transmission to humans by Ix-odes dummini [I] or Ixodes scupuluris [2] causes hemolytic anemia and can result in severe illness. In the US there have been about 250 reported cases of human babesiosis caused by B. microti [3]. Due to similarity in agents and diseases, B. microti has been used as a model for the study of human malaria and bovine babesiosis, both associated with diseases of worldwide importance [4]. Heat shock proteins have been found in all species of organisms ranging from bacteria and plants to humans. Hsp70 is perhaps the most highly conserved and abundant of this family of stress * Corresponding author: Tel.: + I 573 8847640: fax: + 1 573 8845050. ’ Note: Nucleotide sequence data reported in this paper are available in the GenbankTM database under the accession number U53448. 0166-6851!96:$15.00

Copyright

fIISO166-6851196)02767-3

CI 1996 Elsevier

Science ?? All rights

proteins [5]. The amino acid sequence of human hsp70 has a 50% sequence identity with Escherichia coli hsp70 and 73% with Drosophila hsp70. The E. coli homologue is encoded by a single gene, Dnak, but eukaryotic cells typically contain multiple copies of hsp70 genes. Plasmodium _fulciparum contains at least five hsp70-like genes, located on four different chromosomes [6]. Similarly. there are four copies of the hsp70 genes in Schistosomu species [7] and eight copies of hsp70 in Leishmuniu amuzonensis [B]. The expression patterns of the hsp70 family of molecules are divided into three groups: those that are constitutively expressed (heat shock cognate protein or hsc70) and others that are heat inducible (hsp70) or glucose-regulated (grp78). We here report on a putative single copy hsp70 homologue in B. microti. The gene was detected using the polymerase chain reaction (PCR), cloned by generation of a B. microti genomic library and sequenced. reserved

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Babesia microti was obtained from the American type culture collection (ATCC # 30222) and maintained by passage in female BALB/c mice. Mice were infected intraperitoneally or subcutaneously with B. microti-parasitized RBC. Giemsastained blood films were monitored for percent parasitized erythrocytes (PPE) and blood collected near peak PPE. Inocula were prepared from a single lot of frozen stock (stabilate), preserved in 20% polyvinylpyrolidone and stored in liquid nitrogen. B. microti-infected whole blood ( z 70-80 PPE) was collected from mice and passed through a cellulose (Whatman, Chicago, IL) column to remove white blood cells (WBC). The eluate was diluted in 1 x VYMS solution [9] and centrifuged at 800 x g for 15 min. Pelleted RBC were resuspended in an equal volume of 1 x VYMS, overlaid on 1.5 vol of density gradient solution (Ficoll-Paque, Pharmacia, Uppsala, Sweden) and centrifuged at 400 x g for 40 min. RBC were lysed by suspending in an equal volume of lysis buffer (10 mM NaCl, 10 mM EDTA, 10 mM Tris-HCl pH7.5, 1% SDS) and incubated at 37°C for 30 min. Parasite DNA was isolated by a previously described method [lo]. DNA from P. jtilciparzmr-infected human RBC (provided by Dr J.B. Jensen, Brigham Young University) and normal murine white blood cells (WBC) were used for comparison. The P. falciparum and murine DNA were prepared as described for B. microti DNA [lo]. B. nricroti-infected RBC also served as a source of parasite RNA. Total RNA was extracted using the guanidine isothiocynate/phenolchloroform method [l 11. Three sets of oligonucleotide primers, representing an hsp70 amino acid sequence [8,12-161 conserved in 16 species, was synthesized by the DNA core facility, University of Missouri Columbia. Forward (5’ CARGCNACNAARGAYGCNGG 3’) and reverse (5’ GCNACNGCYTCRTCNGGRTT 3’) primers (where N = A. C, G or T; Y = C or T; and R = A or G) represented QATKDAG and NPDEAVA respectively [ 151. The third codon position was kept degenerate to allow for different codon usage. All primers had a degeneracy of 512. Templates for amplification included B. microti and P. falciparum genomic DNA and P. ,faZciparum grp78 cDNA [14]. PCR

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was performed in 50 pl volume of 10 mM TrisHCl (pH 8.3) 50 mM potassium chloride, 1.5 mM magnesium chloride with 0.55 1 pg template DNA, 200 /tM each of dNTPs, 2.5-5.0 pM combinations of reverse and forward oligonucleotide primers and 2.5-5.0 units of Tth thermostable DNA polymerase (Epicentre Technologies, Madison, WI). The PCR sequence was composed of 60 s denaturation at 94°C 60 s annealing at 55°C and 90 s chain extension at 72°C for 30 cycles. Following completion of amplification, 10 pl of the reaction mixture was fractionated by electrophoresis in a 0.8”% agarose gel, stained with ethidium bromide and photographed under UV light. Major amplified DNA products were eluted and purified using a GENECLEAN II Kit (Bio 101, La Jolla, CA) prior to sequencing. The B. Microti PCR product, the sequence of which most resembled a consensus hsp70 gene sequence in the amplified region, was used as a probe to screen a B. microti genomic DNA library. The PCR amplification product was also used as a probe for hybridization to a Southern blot of EcoRI, HirrdIII and PstI digests of B. microti genomic DNA and a Northern blot of total RNA [17]. Blots were washed under high stringency conditions (65°C 0.1 x SSC - 0.1% SDS) to avoid nonspecific binding. The probe was labeled using a random priming kit (Stratagene, La Jolla, CA). B. microti genomic DNA (10 pg) was digested with EcoRI (based on Southern blot analysis), purified with GENECLEAN II kit and ligated into the pUC18 vector also digested with EcoRI and dephosphorylated (Pharmacia Biotech., Uppsala, Sweden). After incubation overnight at 16”C, the recombinant vector was used to transform E. coli MV1304. Selected recombinant clones were screened to detect the presence of insert containing the B. microti hsp70 sequence by colony blot hybridization [18]. DNA sequencing was performed by Taq Cycle sequencing reactions, using dideoxy terminators, at the DNA core facility of the University of Missouri. The DNA sequence of B. microti hsp70 (Bm/hsp70) was used to deduce the amino acid sequence, and compared with that of P. ,fidciparum hsp70, grp78, BALB/c mouse and human hsp70 by computer program (Hibio Mac DNASIS Pro.. Hitachi. San Bruno, CA).

H.-B.

Jie et 01. / Molecular

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PCR performed with one of the three sets of degenerate primers combined with B. microti DNA template produced a 0.65 kb fragment, which was of expected size. The most effective primers were: forward 5’ CAA GCA ACC AAG GAT GCT GG 3’ and reverse 5’ GC CAC AGC CTC ATC TGG GTT 3’. The P. falciparum grp78 cDNA and P. falcipurum genomic DNA, used as positive controls, also produced 0.65 kb bands. Sequences of the PCR fragments from P. falciparum genomic DNA and grp78 cDNA perfectly matched the known P. jillcipurum grp78 sequence [14]. The deduced amino acid sequence of 0.65 kb PCR product from B. microti showed a high degree of sequence identity to the hsp70 family. Single major bands of about 4-6 kb were observed when B. microti genomic DNA digests were blotted and probed with the 0.65 kb PCR product to detect the hsp70 gene, suggesting that B. microti 11~~70 is a single copy gene. By contrast there was no signal from murine white blood cell genomic DNA, digested with the same restriction enzymes, indicating that the probe is specific for Bmihsp70 and does not cross react with the murine homologue. One positive clone (Bm195) was isolated by screening the EcoRI genomic library. Bm195 was digested with EcoRI and hybridized with 0.65-kb probe. The clone contained a 6 kb insert in accord with the Southern blot analysis. The Bm/hsp70 position in the 6 kb insert of clone 195 was determined by restriction enzyme mapping, PCR analysis and DNA sequencing. About 3 kb of DNA existed upstream from the 5’ end of Bm/ hsp70 and roughly 1 kb downstream from the 3’ end. RNA was isolated from parasitized RBCs and assayed for expression of hsp70 by Northern blot analysis. Signal from the 0.65 kb probe was detected at about 2.3 kb (Fig. 1). DNA sequencing revealed that the Bm195 clone contained the full length Bmihsp70 gene with no intergenic region. The intronless state may be suggestive of heat inducibihty since inducible forms of hsp70 molecules have been reported to lack introns [21]. Functionally, intronless hsp70 genes may serve as more rapid responders to stress conditions than intron-containing hsp70 genes because RNA

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splicing is unnecessary. Bm/hsp70 consisted of a single open reading frame of 1935 nucleotides (645 amino acids represented) encoding a calculated protein of 70 349 Da. The 0.65 kb PCR probe sequence matched perfectly with nucleotides 469- 1119. The codon usage in Bm/hsp70 showed preference for A or T in the third position (61.2%). These observations are in accord with A + T content (56.8%) of the Bm/hsp70 gene. The extent of sequence conservation of the coding region is similar at the amino acid and nucleotide levels. This implies that there is a constraint on third position diversity of codons [22]. It has also been noted that codon usage in hsp70 genes is

Fig. 1. Northern blot of B. microti RNA was electrophoretitally fractionated after denaturation with glyoxal and dimethyl sulfoxide and probed with 0.65 kb PCR product.

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H.-B. Jie et al. / Molecular and Biochemical Parasitology 83 (1996) 241-246

Fig. 2. Alignment of B. microti hsp70 with the related protein from other species. Identical residues are denoted by asterisks (*), addition(s) or gap(s) in the sequences are indicated by dash (-_). Rows: (t) B. microti hsp70; (2) P. falc@arum hsp70; (3) P. fblciparurn grp78: (4) mouse hsp70: (5) human hsp70.

biased [23]. Clostridium perfringens hsp70 [ 121 and P. fa~ciparum grp78 [14] also reportedly show strong preference for A and T in the third position, whereas Halobacterium marismortui reflected a strong bias toward codons with either G or C in third position [16]. These observations are in accord with nucleotide content of each species. The

amino acid composition of Bm/hsp70 included 61% hydrophilic amino acids, indicating that this protein is hydrophilic with several hydrophobic regions. Sequence similarity between B. microti and other species is higher in the N-terminal domain than in the C-terminal part and less in the final 10 kDa region of the hsp70 molecules. This

H.-B. Jie et al. 1 Molecular and Biochemical Parasitology 83 (1996) Nl62446

constant feature is one of the typical characteristics of the hsp70 proteins. The only notable structural feature is the presence of two perfect repeats of the sequence Gly-Gly-Phe-Pro-Gly at the C-terminal end. This structure is different from the typical Gly-Gly-Met-Pro repeat sequence that may represent an evolutionary feature characteristic of some parasites (P. fulciparum, T. cruzi). However, a comparison of the sequences related to the various species in Fig. 2 indicates that the hsp70 C-terminal ends are characteristically Gly-rich. Alignment of deduced amino acid sequence of Bm/hsp70 with the known P. falciparum homologue and murine and human (B. microti host) counterparts (Fig. 2) shows extensive homology through its entire length. Pairwise alignment of the hsp70 amino acid sequence using the MAXIMUM MATCHING program of the DNAsis package (Hibio Mac DNAsis pro Hitachi, San Bruno, CA) revealed that Bm/hsp70 showed 71% similarity with P. falcipurum hsp70 and 55% similarity with P. fulciparum grp78, indicative of its identity as an hsp70 homologue. Comparison of the sequence of Bm/hsp70 with murine and human (B. microti hosts) homologues both showed 68% similarity. The observed high degree of sequence similarity in hsp70 sequence over their entire length indicates that hsp70 constitutes one of the most conserved proteins known. Heat shock proteins retain more than 50% of amino acid sequence identity between pathogen and host counterparts, often with long identical stretches of amino acids. Nevertheless, hsp-reactive T cells and antibodies have been found to be immunodominant during and after multiple pathogenic bacterial and parasitic infections [24]. However, due to their high degree of sequence conservation, antigenic mimicry between the host protein and that of the pathogen has also been found to be associated with autoimmune diseases such as systemic lupus erythematosus in which high levels of antibodies against the self-hsp70 and hsp90 are found in patient sera [25]. Hsp65 also seems to be involved in protection against arthritis [26] or exacerbation (adjuvant induced arthritis) in the rat. Hsp70 molecules in other species are commonly products of a multi-gene

245

family, making the B. microtilhsp70 unusual. Only P. cynomolgi, P. vivax and P. brasilianum have been previously reported to yield a hybridization pattern indicative of a single copy hsp70 gene [19]. Since hsp70 proteins perform functions which may be critical [20] for parasite survival, the existence of a single copy of this gene in B. microti presents an opportunity for further studies on the effect of gene ablation and use of hsp70 as a protective immunogen.

Acknowledgements

This study was partially supported by NIH grant AI3 1589 and the University of Missouri College of Veterinary Medicine. The authors thank Mrs Ellen Swanson for expert assistance in manuscript preparation.

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