Veterinary Parasitology 205 (2014) 499–505
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Molecular cloning and characterization of Babesia orientalis rhoptry-associated protein 1 Qian Yu a,b , Lan He a,b,∗ , Wen-Jie Zhang a,b , Jian-Xi Cheng a,b , Jin-Fang Hu a,b , Xiao-Yan Miao a,b , Yuan Huang a,b , Li-Zhe Fan a,b , Muhammad Kasib Khan a,b , Yan-Qin Zhou b , Min Hu a,b , Jun-Long Zhao a,b,∗ a State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China b Key Laboratory of Animal Epidemical Diseases and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China
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Article history: Received 16 June 2014 Received in revised form 13 August 2014 Accepted 14 August 2014 Keywords: Babesia orientalis Rhoptry-associated protein Antigenicity Diagnosis
a b s t r a c t The rhoptry-associated protein 1 (RAP-1) gene of Babesia orientalis was obtained from a cDNA expression library by immunoscreening with B. orientalis-infected water buffalo sera. The nucleotide sequence of the cDNA was 1732 bp with an open reading frame (ORF) of 1434 bp, encoding a polypeptide of 478 amino acid residues with a predicted size of 52.5 kDa. The ORF was cloned into a pGEX-KG plasmid and subsequently expressed as a GST-fusion protein. The recombinant RAP-1 of B. orientalis (rBoRAP-1) was purified and evaluated as an antigen using Western blotting. The native BoRAP-1 was recognized by the antibodies raised in rabbits against rBoRAP-1. Strong immunofluorescence signals were observed in erythrocytes infected with B. orientalis. Phylogentic analysis revealed that B. orientalis fell into a Babesia clade and most closely related to Babesia bovis and Babesia ovis, which was similar to the previous reported trees based on 18S rRNA and HSP70 genes. The present study suggests that the BoRAP-1 might be a potential diagnostic antigen, and the RAP-1 genes can aid in the classification of Babesia and Theileria species. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Tick-transmitted intraerythrocytic babesial parasites cause significant morbidity in humans and domestic animals, manifested predominantly by anemia (Homer et al., 2000; Kjemtrup and Conrad, 2000). It is considered to be
∗ Corresponding authors at: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Shizishan Street, Wuhan 430070, Hubei, China. Tel.: +86 27 87281810. E-mail addresses: [email protected] (L. He), [email protected] (J.-L. Zhao). http://dx.doi.org/10.1016/j.vetpar.2014.08.007 0304-4017/© 2014 Elsevier B.V. All rights reserved.
the second most commonly found parasites in the blood of mammals after trypanosomes (Schnittger et al., 2012). Babesia orientalis is a protozoan parasite transmitted by Rhipicephalus haemaphysaloides, which only infects water buffalo (Liu et al., 1997). Babesiosis, caused by this parasite, is one of the most severe diseases in water buffalo, which results in large economic losses in the central and southern China (Chen, 1984; Liu and Ma, 1987; Zhongling et al., 1986). The clinical symptoms of this disease are fever, anemia, icterus and hemoglobinuria (He et al., 2009; Liu et al., 2005). B. orientalis was distinguished from Babesia bigemina and Babesia bovis in water buffalo and classified as a new species, according to the differences of morphology, transmission and pathogenicity (Liu et al., 1997).
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The Babesia RAP-1 gene was first described in B. bovis (Goff et al., 1988) and sequentially observed in many other Babesia species, including Babesia bigemina, Babesia divergens, Babesia caballi, B. bovis, and Babesia canis (Dalrymple et al., 1993; Ikadai et al., 2000; McElwain et al., 1991; Skuce et al., 1996; Suarez et al., 1991; Zhou et al., 2007). Babesial RAP-1 was tested in several vaccine trials either as an individual candidate or in combined form with other recombinant protein antigens. Experimental evidence suggested that the cattle immunized with RAP-1 elicited strong T-cell and B-cell immune responses (Norimine et al., 2003). As a component of multi-antigen vaccine, three protective antigens, including RAP-1 were identified in B. bovis (Wright et al., 1992). Apicomplexan parasites utilize several rhoptry proteins during invasion into and development within the host cell (Preiser et al., 2000; Reduker et al., 1989). An in vitro erythrocyte-binding assay confirmed that B. bovis RAP-1 was able to bind with the bovine and equine erythrocytes surface, and the binding affinity was weakened by antiRAP-1 MAb (Yokoyama et al., 2002). B. bovis RAP-1 was expressed in sporozoites, and specific RAP-1 antisera could inhibit the sporozoites invasion into host cells (Mosqueda et al., 2002). These studies strongly support that RAP-1 plays a functional role in the biology and development of Babesia parasites. The genetic organization of RAP-1 family is highly complexed in some Babesia species, containing several closely linked genes encoding unique proteins (Dalrymple et al., 1993; Sam-Yellowe, 1996; Skuce et al., 1996; Suarez et al., 1998). Despite of the complexity of the gene loci, all the members of rhoptry-associated protein family have well conserved features, including a N-terminal localized signal peptide, several tandem repeat regions in the C-terminal, four strictly conserved cysteine residues and a 14 aminoacid motif (Dalrymple et al., 1996; Norimine et al., 2003; Sam-Yellowe, 1996; Suarez et al., 1991). The well conserved RAP-1 is also highly immunogenic and has been observed to induce high titer of antibodies in Babesiainfected animals. Thus, the RAP-1 has also been widely used to develop serological diagnostic methods for Babesia parasites (Boonchit et al., 2002; Ikadai et al., 2000; Zhou et al., 2007). In the present study, the gene encoding rhoptryassociated protein 1 (RAP-1) was identified from a B. orientalis cDNA library, which was further confirmed through phylogenetic analysis of gene sequence. The encoding gene was cloned and expressed in Escherichia coli and evaluated its potential use as a diagnostic and vaccine candidate.
Two 1-year-old water buffaloes were purchased from Babesia free area and confirmed to be B. orientalis free by real-time PCR (He et al., 2011). The water buffaloes were splenectomized and subcutaneously injected with 4 ml of B. orientalis-infected blood (Wuhan strain, percentage parasitized erythrocytes (PPE) 1%). Blood samples were collected daily to monitor the parasitemia until it reached up to 3%. All animal experiments described in this article were carried out in compliance with the regulations (no. 5 proclaim of the Standing Committee of Hubei People’s Congress) approved by the Standing Committee of Hubei People’s Congress, P. R. China. The animal protocols were approved by Laboratory Animals Research Centre of Hubei province and the ethics committee of Huazhong Agricultural University (permit number: 4200696657). 2.2. Preparation of genomic DNA The blood from experimentally infected water buffalo was collected in EDTA tubes (BD Vacutainer blood collection tubes, USA). The genomic DNA was extracted from 200 l of B. orientalis-infected blood using a QIAamp DNA mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. DNA samples were stored at −20 ◦ C until further use. 2.3. Cloning and sequencing of the BoRAP-1 gene A cDNA expression library of B. orientalis merozoites, constructed previously (Liu et al., 2009), was screened with the serum of B. orientalis-infected water buffalo. One of the positive clones expected to be RAP-1 were chosen for further analysis. To amplify the B. orientalis RAP-1 gene from gDNA, a pair of primers was designed based on BLAST analysis of B. orientalis genome sequence (unpublished data). The forward primer RAP1-F was 5 -TAC TCA CCT ATA AAA GCC TCT TGC C-3 , and the reverse primer RAP1-R was 5 -CTG TAT GTG TCA AAA AGG GA-3 . The amplified PCR product from both gDNA and cDNA were electrophoresed using 0.8% ethidium bromidestained agarose gel and purified by TIANgel Midi purification Kit (TIANGEN, China). The purified PCR products were ligated into pMD18-T vector (TaKaRa, Japan), and three positive clones from each sample were sequenced using a Dye Terminator Cycle Sequencing reaction in an ABI PRISM 377 DNA sequencer (Sangon, China). The vector primers M13 (−47) and M13 (−48) were used for sequencing. 2.4. Bioinformatics analysis of BoRAP-1
2. Materials and methods 2.1. Parasites and experimental animals B. orientalis strain was previously isolated from water buffalo in Hubei province, China and preserved in liquid nitrogen in State Key laboratory of Agricultural Microbiology, Huazhong Agricultural University, China (Liu et al., 1995).
The isolated gene of B. orientalis RAP-1 was given the name “BoRAP-1”. The deduced amino acid (aa) sequence of BoRAP-1 was subjected to BLASTp analysis in GenBank (Benson et al., 2012). The homologous sequences of related intra-erythrocytic species (Table 1), including BoRAP-1 sequence were aligned using MAFFT version 7 (Katoh and Frith, 2012; Katoh and Standley, 2013) and manually edited using BioEdit version 7.1.11 (Hall, 1999). A
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Table 1 the RAP-1 sequences used in phylogenetic analysis and their amino acid accession numbers. Organism
Accession no.
Organism
Accession no.
B. orientalis B. bovis RAP B. bovis RRA B. caballi RAP B. canis RAP B. bigemina RAP1a B. bigemina RAP1b
AGC60006 AAB84268 XP 001610950 ADF30793 AAA27807 1906304A AAB72094
B. bigemina RAP1c B. divergence RAP B. gibsoni RAP B. equi T. orientalis Shintoku T. annulata RAP T. parva Muguga
AAB82596 CAA89970 ABD52000 XP 004833177 BAM39179 XP 954078 XP 766222
neighbor-joining tree was constructed by MEGA5 (Tamura et al., 2011). The evolutionary divergence was also calculated by MEGA5 using Poisson correction model (Tamura et al., 2011; Zuckerkandl and Pauling, 1965). The deduced protein sequence of BoRAP-1 was subjected to SignalP for analyzing signal peptide (http:// expasy.org/tools/#topology). Tandem repeats regions were determined using Tandem Repeat Finder (http:// tandem.bu.edu/trf/trf.html) and edited manually. Antigen index was analyzed through DNAstar Protean 7.5 (http:// www.dnastar.com/). Motif scanning of the predict protein was performed by PROSITE (http://prosite.expasy.org/).
2.5. Recombinant protein expression and purification of the rBoRAP-1 To amplify the ORF of BoRAP-1 gene, a pair of primers, ExpRAP1-F: 5 -CG GGA TCC ATG AGA GCA ATC AGC AGT-3 , and ExpRAP1-R: 5 -C GAG CTC TTA GTA GTC ATA AAA ATC AAT-3 , containing restriction enzyme sites BamHI and SacI (underlined and italic) were designed. The ORF of BoRAP-1 was cloned into pMD-18T vector, digested and sub-cloned into the expression vector pGEX-KG. The resulting plasmid (pGEX-KG-RAP1) was confirmed by enzyme restriction digestion and sequencing, and subsequently transformed into the E. coli BL21TM (DE3) strain using the standard method. The rBoRAP-1 was expressed as a GST-fusion protein by inducing with 1 mM isopropyl -d-thiogalactoside (IPTG). The recombinant protein was expressed in the soluble-fraction of E. coli and was purified using a GSTrapTM 4B columns (GE Healthcare, USA), according to the manufacturer’s instructions.
2.7. SDS-PAGE and immunoblotting The rBoRAP-1 was separated on 12% SDS-PAGE and transferred onto a nitrocellulose membrane (BioRad, USA). The membrane was blocked with 1% bovine serum albumin (BSA) (Equitech-Bio Inc., USA) and incubated with B. orientalis-infected water buffalo serum (1:200 dilution) for 60 min. After washing three times with PBST, the membrane was incubated with secondary antibodies (1:2000, Bov IgG/HRP, Bioss, China) for 45 min. The membrane was subjected to 3,3 -diaminobenzidine (DAB) (Biomiga, USA) for the visualization of protein bands. Serum from an uninfected water buffalo was used as negative control. 2.8. Identification of BoRAP-1 in merozoites by immunofluorescence The native B. orientalis RAP-1 was identified in merozoites by Indirect Fluorescent Antibody Test (IFAT). Thin blood smears prepared from B. orientalis-infected erythrocytes were dried at 37 ◦ C and fixed with mixed methanol and acetone 1:4 (v/v) at −20 ◦ C for 20 min. The fixed blood smear was probed with the rabbit immune sera against the rBoRAP-1 as primary antibodies (1:100 dilution in PBS; pH 7.4) for 30 min at 37 ◦ C. After washing with PBST three times, the slides were incubated with goat anti-rabbit IgG (H + L) antibodies (Alexa Fluor® 488, Life Technologies) as secondary antibodies (1:200) for 30 min at 37 ◦ C. The slides were washed three times with PBST and mounted by adding 10 l of a 50% glycerol-PBS (v/v) solution and covered with a glass cover slip. The slides were examined using a fluorescence microscope (AxioCam HRc, Zeiss, Germany). 3. Results 3.1. Cloning and characterization of the BoRAP-1
2.6. Preparation of rabbit anti-rBoRAP-1 immune serum Three eight-week-old female Japanese White Rabbits (specific pathogen free, SPF) were used for the preparation of antibodies against rBoRAP-1. The rBoRAP1 (500 g) emulsified in equal amount of Freund’s complete adjuvant (SIGMA) was subcutaneously injected into three rabbits. Two weeks after the immunization, the rabbits were injected with the same amount of antigen emulsified in Freund’s incomplete adjuvant (SIGMA) three times at one week interval. The sera from the immunized rabbits were collected after 7 days of the last immunization and stored at −20 ◦ C until further use.
The cDNA expression library of B. orientalis was screened with B. orientalis-infected buffalo serum, and a positive clone was isolated and sequenced. The full length of this positive clone was 1732 bp, with an ORF of 1434 bp encoding a polypeptide with a molecular weight of 52.5 kDa. The full length of BoRAP-1 gene was also amplified from B. orientalis genomic DNA (Fig. 1). The resulting fragments were ligated into pMD18-T vector and subjected to sequencing. The results showed that the sequence of BoRAP-1 gene obtained from gDNA was exactly same as the sequence from cDNA library. The results indicated that
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Fig. 1. The PCR results of amplifying BoRAP-1 from B. orientalis genome DNA. Lane M: DNA marker; lane 1: BoRAP-1 from gDNA (1732 bp); lane2: negative control.
there is no intron in the BoRAP-1 gene. The nucleotide sequence of BoRAP-1 was submitted to GenBank with accession number JX993940. 3.2. Sequence analysis The nucleotide sequence of BoRAP-1 gene was subjected to BLAST analysis in NCBI. Blastn search indicated that
BoRAP-1 gene had significant similarity with RAP-1 genes of related apicomplexan parasites. It showed highest identity (71%) with B. ovis RAP-1 gene (M91173), followed by B. bovis (68%; FJ588009), B. canis (64%; M91168) and Babesia caballi (63%; EU669865). The BoRAP-1 secondary structure showed several domains conserved in RAP-1 protein family. The BoRAP1 presented a rhoptry-associated protein 1 domain in the N-terminal, which is similar in characterization to other Babesia RAP-1 proteins. Multiple alignment of BoRAP-1 AA sequence and B. bovis (AAB84268), B. ovis (AAA27805) and B. caballi (ADF30793) showed several well defined molecular features of RAP-1 family (Fig. 2), including a serine-rich N-terminal region, a strictly conserved 4 cysteine residues, and a 14 amino-acid motif. However, the seventh proline residue (P) in 14 amino-acid motif of Babesia RAP-1 was replaced by alanine (A) in BoRAP-1. The alignment showed that Babesia RAP-1 AA sequences were highly conserved in the first 300 AA of the N-terminus compared to the sequences of C-terminus. Similar to Babesia RAP-1, a 21 AA signal peptide (1–21 AA residues) with a putative cleavage site was found in the beginning of BoRAP-1 N-terminus. The less conserved C-terminal of BoRAP-1 comprised five tandem repeats regions (TRPs) (Fig. 2). Antigenicity analysis performed by DNAstar showed that the immunogenic peptides were mainly present in the conserved N-terminal region (data not shown). Motif scanning by PROSITE showed several putative post-translation modification sites in the BoRAP-1 AA sequence, including three N-glycosylation sites (AA residues 176 to 179, 206 to 209, and 289 to 292), a cAMPand cGMP-dependent protein kinase phosphorylation site (AA residues 452 to 455), four Protein kinase C phosphorylation sites (AA residues 25 to 27, 52 to 54, 97 to 99, and
Fig. 2. Alignment of the putative amino acid of B. orientalis RAP-1 (BoRAP-1) with the RAP-1 of B. bovis (Bbo), B. ovis (Bov), B. bigemina (Bbi), B. caballi (Bca), B. divergence (Bdi) and B. gibsoni (Bgi). Similar and identical residues are shaded. Black shading indicates that similarity in four or more than four species, gray shading indicates that similarity in three species. The signal peptide cleavage site of BoRAP-1 is indicated by black arrow. Four conserved cysteine residues are indicated by black triangle. The 14 conserved amino acid residues [PLS(L/T/A/V)LPN(E/D)P(A)YQLDAAF] are showing black box. Five repeat motifs [PTKEFFVD(N)T(N)HEKTKD(E)FF(L)E(D)NK(N)VA(G)K] are indicated by underline.
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311 to 313), five Casein kinase II phosphorylation sites (AA residues 52 to 55, 171 to 174, 178 to 181, 184 to 187, and 455 to 458), one tyrosine kinase phosphorylation site (AA residues 112 to 119), and one N-myristoylation site (AA residues 17 to 22). 3.3. Expression and identification of BoRAP-1 The ORF of B. orientalis RAP-1 gene was amplified and ligated into the expression vector pGEX-KG. The resulting plasmid was subsequently subjected to the sequencing and enzyme digestion with BamHI and SacI to confirm the successful insertion of the RAP-1 fragment. The recombinant protein (rBoRAP-1) was expressed as a GST-fused protein in E. coli BL21 strain with a molecular weight of 78 kDa, including an additional 26 kDa peptide of the GST tag, as observed through SDS-PAGE (Fig. 3A). To determine whether antibodies against BoRAP-1 were elicited during B. orientalis infection period, rBoRAP-1 protein was subjected to immunoblotting. The serum of B. orientalis-infected water buffalo recognized a specific band of 78 kDa (Fig. 3B), whereas, the uninfected water buffalo serum did not react with rBoRAP-1 in the Western blot. To identify native RAP-1 in B. orientalis, rabbit antiserum against rBoRAP-1 was prepared and IFAT was performed (Fig. 4). IFAT results showed that the serum from rabbit immunized with rBoRAP-1 interacted with the ring (Fig. 4b) and pear-shaped (Fig. 4a and c) forms of B. orientalis merozoites, displaying the strong immunofluorescence signals. However, no signal was obvious from the uninfected water buffalo erythrocytes, when probed with anti-rBoRAP-1 antibodies. 3.4. Phylogenic analysis A neighbor-joining tree based on Babesia RAP-1 AA sequences has been shown in Fig. 5. B. orientalis fell into a distinct Babesia clade and was most closely related to B. bovis and B. ovis. 4. Discussion Babesia are obligate intracellular protozoan parasites, which invade a wide range of vertebrate hosts. They significantly impair the health of domestic and wild animals,
Fig. 3. SDS-PAGE of expression and purification of the rBoRAP-1 and Western blot analysis. (A) SDS-PAGE expression and purification of the rBoRAP-1. M, molecular weight marker; lane 1, uninduced pGEX-KG; lane 2, induced pGEX-KG; lane 3, supernatant after purification; lane 4, lysates of uninduced cells; lane 5, supernatant of induced lysates. (B) Determination of antibody response of BoRAP-1 in water buffalo antiserum. M, molecular weight marker; lane 1, rBoRAP-1 probed with anti-B. orientalis water buffalo serum; lane 2, rBoRAP-1 probed with uninfected water buffalo serum.
especially the farm and pet animals (Schnittger et al., 2012). The invasion of host erythrocytes by these protozoa is mediated by the molecules located on the merozoite surface and apical complex organelles, including the rhoptry, a unifying structure of the apicomplexan parasites (Perkins, 1992). Accordingly, the rhoptry-associated proteins of Babesia spp. are consistent targets of vaccine research for the past two decades (Brown et al., 1996; Dalrymple et al., 1993; Norimine et al., 2003; Wright et al., 1992). The Babesia rhoptry-associated protein 1 has been identified to be encoded by several closely linked proteins. It was reported that there are two identical genes in B. bovis, four homologous genes in B. bigemina, and five polymorphic linked genes in B. ovis (Sam-Yellowe, 1996; Suarez et al., 1998). The RAP-1 genes may play an
Fig. 4. Identification of native BoRAP-1 in B. orientalis. IFAT analysis of anti-B. orientalis antibody produced in rabbit immunized with rBoRAP-1 and expressed in E. coli. Immunofluroscence signals were observed in B. orientalis (white arrow), and only background signal was observed from erythrocytes.
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B orientalis RAP
69 89
B bovis RAP AAB84268 B ovis RAP AAA27805
94
B caballi RAP ADF30793
52
B bigemina RAP1a 1906304A
77 70
B divergence RAP CAA89970 B canis RAP AAA27807
76 89
B gibsoni RAP ABD52000
24
B bigemina RAP1c AAB82596 61
B bigemina RAP1b AAB72094 B bovis RRA XP001610950 B equi XP 004833177 T orientalis Shintoku BAM39179
97
T annulata RAP XP954078
100 100
T parva Muguga XP 766222 Toxoplasma gondii ROP AAA69859
0.1
Fig. 5. Neighbor-joining phylogenetic tree showing relationship of B. orientalis with related apicomplexa parasites. The trees was constructed based on the amino acid sequences of RAP-1 gene. GenBank accession numbers are indicated.
important function in the erythrocyte binding or invasion, as the RAP-1 may facilitate the parasite adaptation to variant host cells and erythrocyte receptors (Sam-Yellowe, 1996). In this study, a RAP-1 gene was initially identified by immunoscreening of a B. orientalis cDNA library. The complete nucleotide sequence of the BoRAP-1 gene was 1732 bp, with an ORF of 1434 bp encoding a mature protein of 52.5 kDa in size. The BoRAP-1 is intronless, as the sequences obtained from B. orientalis cDNA and gDNA were identical. The RAP-1 has been detected in a majority of Babesia species, which showed high percent identity. This conservation of RAP-1 genes among Babesia parasites may imply a potential key role of RAP-1 in parasite life cycle, especially the invasion process. According to the BLAST analysis, the newly identified BoRAP-1 gene and the putative protein sequence were most similar to B. ovis, followed by B. bovis and B. canis. The phylogenetic tree was constructed based on RAP-1 amino acid sequences, and the topology was consistent with previous analyses based on 18S rRNA genes and HSP70 genes (He et al., 2009; Liu et al., 2005). B. orientalis appeared in a distinct branch of Babesiidae and most closely related to B. bovis and B. ovis. These results suggested that the RAP-1 gene may be evolutionally conserved and can be used in the classification of Babesia species. Multiple alignments of BoRAP-1 AA sequence with the RAP-1 of B. ovis, B. bovis and B. caballi was performed. The results revealed the well-defined features among RAP1 family. A 21 AA signal peptide was identified in the beginning of N-terminus. Four conserved Cys residues were situated in identical positions, implying the conserved tertiary structure of RAP-1. The 14 amino acids motif PLSLPNPYQLDAAF has been observed in all Babesia RAP-1 homologues, though some residue sites have substitutions in certain species. For example, the third serine and sixth
asparagine residues have substitution of other residues in B. ovis and B. caballi. In B. divergens, the tenth leucine and eleventh asparaginase are replaced by valine and glycine, respectively. The seventh proline, which is more stable among the 14 residues, is mutated to alanine in B. orientalis RAP-1. Computer searches based on the 14 AA residues failed to match with any notable region of known proteins, which suggested that the biological functions of this motif remains unknown (Suarez et al., 1991). The motif scanning analysis showed several putative motifs involved in glycosylation and phosphorylation in BoRAP-1. Similarly, these motifs have been identified in several Babesia RAP-1. However, compared to Babesia gibsoni, the erythrocyte binding motifs RGD (aa 156 to 158) and KKNV (aa 313 to 316) were not observed in BoRAP-1 (Terkawi et al., 2009). It has been reported that the Babesia RAP-1 was widely used as diagnostic antigen (Boonchit et al., 2002; Ikadai et al., 2000; Zhou et al., 2007). In this study, the B. orientalisparasitized blood smear was subjected to IFAT analysis, and immunofluorescence signals were observed in different stages of B. orientalis. These indicated that the novel RAP-1 protein exists in B. orientalis. The results were consistent with the previous reports of B. bovis RAP-1 (Yokoyama et al., 2002). According to the results of Western blot, the serum of B. orientalis experimentally infected water buffalo interacted with the rBoRAP-1, indicating that the antibody responses of RAP-1 may be elicited in B. orientalis infection, and rBoRAP-1 could be a potential specific antigen for the detection of B. orientalis antibodies. In addition, the predicted antigenicity sites of BoRAP-1 also showed high antigenicity index (data not shown). There is no standard B. orientalis ELISA assay reported to date. Considering the predicted antigenic potential of BoRAP-1 and the results of Western blot, the protein could be a candidate for developing a stable ELISA method for B. orientalis.
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Overall, in the present study, an intronless rhoptryassociated protein 1 gene of B. orientalis encoding a 52.5 kDa protein was obtained from cDNA library by immunoscreening. This is the first description of RAP1 gene homolog in B. orientalis. Phylogenetic tree based on the RAP-1 amino acid sequences was constructed, the results indicated that B. orientalis appeared in a Babesia clade and is most closely related to B. bovis and B. ovis. The native RAP-1 was identified in B. orientalis merozoites by IFAT. The results suggested that BoRAP-1 could be a potential antigen for the detection of B. orientalis antibodies. Acknowledgements Project support was provided by National Natural Science Foundation of China (31302082), Special Fund for Agro-scientific Research in the Public Interest of China (201003060-01-06), and the National Basic Science Research Program (973 program) of China (2015CB150300). The authors would like to thank Dr. Qing Tao (State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, China) for providing advice on figures and illustrations. References Benson, D.A., Cavanaugh, M., Clark, K., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J., Sayers, E.W., 2012. GenBank. Nucleic Acids Res., gks1195. Boonchit, S., Xuan, X., Yokoyama, N., Goff, W.L., Wagner, G., Igarashi, I., 2002. Evaluation of an enzyme-linked immunosorbent assay with recombinant rhoptry-associated protein 1 antigen against Babesia bovis for the detection of specific antibodies in cattle. J. Clin. Microbiol. 40, 3771–3775. Brown, W.C., McElwain, T.F., Ruef, B.J., Suarez, C.E., Shkap, V., ChitkoMcKown, C.G., Tuo, W., Rice-Ficht, A.C., Palmer, G.H., 1996. Babesia bovis rhoptry-associated protein 1 is immunodominant for T helper cells of immune cattle and contains T-cell epitopes conserved among geographically distant B. bovis strains. Infect. Immun. 64, 3341–3350. Chen, S., 1984. Babesiosis of buffalo caused by a combination of Babesia bigemina and B. bovis. Hubei J. Vet. Sci. Technol. 8, 38–39. Dalrymple, B.P., Casu, R.E., Peters, J.M., Dimmock, C.M., Gale, K.R., Boese, R., Wright, I.G., 1993. Characterisation of a family of multi-copy genes encoding rhoptry protein homologues in Babesia bovis, Babesia ovis and Babesia canis. Mol. Biochem. Parasitol. 57, 181–192. Dalrymple, B.P., Peters, J.M., Böse, R., Wright, I.G., 1996. A polymerase chain reaction method for the identification of genes encoding members of the Bv60/p58 family of rhoptry protein homologues in the genus Babesia. Exp. Parasitol. 84, 96–100. Goff, W., Davis, W., Palmer, G., McElwain, T., Johnson, W., Bailey, J., McGuire, T., 1988. Identification of Babesia bovis merozoite surface antigens by using immune bovine sera and monoclonal antibodies. Infect. Immun. 56, 2363–2368. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser., 95–98. He, L., Feng, H.-H., Zhang, Q.-L., Zhang, W.-J., Khan, M.K., Hu, M., Zhou, Y.-Q., Zhao, J.-L., 2011. Development and evaluation of real-time PCR assay for the detection of Babesia orientalis in water buffalo (Bubalus bubalis, Linnaeus, 1758). J. Parasitol. 97, 1166–1169. He, L., Liu, Q., Quan, M., Zhou, D.-n., Zhou, Y.-q., Zhao, J.-l., 2009. Molecular cloning and phylogenetic analysis of Babesia orientalis heat shock protein 70. Vet. Parasitol. 162, 183–191. Homer, M.J., Aguilar-Delfin, I., Telford, S.R., Krause, P.J., Persing, D.H., 2000. Babesiosis. Clin. Microbiol. Rev. 13, 451–469. Ikadai, H., Osorio, C.R., Xuan, X., Igarashi, I., Kanemaru, T., Nagasawa, H., Fujisaki, K., Suzuki, N., Mikami, T., 2000. Detection of Babesia caballi infection by enzyme-linked immunosorbent assay using recombinant 48-kDa merozoite rhoptry protein. Int. J. Parasitol. 30, 633–635. Katoh, K., Frith, M.C., 2012. Adding unaligned sequences into an existing alignment using MAFFT and LAST. Bioinformatics 28, 3144–3146.
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The test of infected cattle by bite with tick Rhipicephalus haemaphysaloides and injection with parasitized blood of buffalo with babesiosis. Acta Vet. Zootech. Sin. 26, 468–472. Liu, Z., Zhao, J., Ma, L., Yao, B., 1997. Studies on buffalo babesiosis in Hubei Province, China. Trop. Anim. Health Prod. 29, 33S–36S. McElwain, T.F., Perryman, L.E., Musoke, A.J., McGuire, T.C., 1991. Molecular characterization and immunogenicity of neutralization-sensitive Babesia bigemina merozoite surface proteins. Mol. Biochem. Parasitol. 47, 213–222. Mosqueda, J., McElwain, T.F., Stiller, D., Palmer, G.H., 2002. Babesia bovis merozoite surface antigen 1 and rhoptry-associated protein 1 are expressed in sporozoites, and specific antibodies inhibit sporozoite attachment to erythrocytes. Infect. Immun. 70, 1599–1603. Norimine, J., Mosqueda, J., Suarez, C., Palmer, G.H., McElwain, T.F., Mbassa, G., Brown, W.C., 2003. Stimulation of T-helper cell gamma interferon and immunoglobulin G responses specific for Babesia bovis rhoptryassociated protein 1 (RAP-1) or a RAP-1 protein lacking the carboxyterminal repeat region is insufficient to provide protective immunity against virulent B. bovis challenge. Infect. Immun. 71, 5021–5032. Perkins, M., 1992. Rhoptry organelles of apicomplexan parasites. Parasitol. Today 8, 28–32. Preiser, P., Kaviratne, M., Khan, S., Bannister, L., Jarra, W., 2000. The apical organelles of malaria merozoites: host cell selection, invasion, host immunity and immune evasion. Microbes Infect. 2, 1461–1477. Reduker, D.W., Jasmer, D.P., Goff, W.L., Perryman, L.E., Davis, W.C., McGuire, T.C., 1989. A recombinant surface protein of Babesia bovis elicits bovine antibodies that react with live merozoites. Mol. Biochem. Parasitol. 35, 239–247. Sam-Yellowe, T., 1996. Rhoptry organelles of the Apicomplexa: their role in host cell invasion and intracellular survival. Parasitol. 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Molecular cloning and characterization of Babesia orientalis rhoptry-associated protein 1 Qian Yu a,b , Lan He a,b,∗ , Wen-Jie Zhang a,b , Jian-Xi Cheng a,b , Jin-Fang Hu a,b , Xiao-Yan Miao a,b , Yuan Huang a,b , Li-Zhe Fan a,b , Muhammad Kasib Khan a,b , Yan-Qin Zhou b , Min Hu a,b , Jun-Long Zhao a,b,∗ a State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China b Key Laboratory of Animal Epidemical Diseases and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China
a r t i c l e
i n f o
Article history: Received 16 June 2014 Received in revised form 13 August 2014 Accepted 14 August 2014 Keywords: Babesia orientalis Rhoptry-associated protein Antigenicity Diagnosis
a b s t r a c t The rhoptry-associated protein 1 (RAP-1) gene of Babesia orientalis was obtained from a cDNA expression library by immunoscreening with B. orientalis-infected water buffalo sera. The nucleotide sequence of the cDNA was 1732 bp with an open reading frame (ORF) of 1434 bp, encoding a polypeptide of 478 amino acid residues with a predicted size of 52.5 kDa. The ORF was cloned into a pGEX-KG plasmid and subsequently expressed as a GST-fusion protein. The recombinant RAP-1 of B. orientalis (rBoRAP-1) was purified and evaluated as an antigen using Western blotting. The native BoRAP-1 was recognized by the antibodies raised in rabbits against rBoRAP-1. Strong immunofluorescence signals were observed in erythrocytes infected with B. orientalis. Phylogentic analysis revealed that B. orientalis fell into a Babesia clade and most closely related to Babesia bovis and Babesia ovis, which was similar to the previous reported trees based on 18S rRNA and HSP70 genes. The present study suggests that the BoRAP-1 might be a potential diagnostic antigen, and the RAP-1 genes can aid in the classification of Babesia and Theileria species. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Tick-transmitted intraerythrocytic babesial parasites cause significant morbidity in humans and domestic animals, manifested predominantly by anemia (Homer et al., 2000; Kjemtrup and Conrad, 2000). It is considered to be
∗ Corresponding authors at: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Shizishan Street, Wuhan 430070, Hubei, China. Tel.: +86 27 87281810. E-mail addresses: [email protected] (L. He), [email protected] (J.-L. Zhao). http://dx.doi.org/10.1016/j.vetpar.2014.08.007 0304-4017/© 2014 Elsevier B.V. All rights reserved.
the second most commonly found parasites in the blood of mammals after trypanosomes (Schnittger et al., 2012). Babesia orientalis is a protozoan parasite transmitted by Rhipicephalus haemaphysaloides, which only infects water buffalo (Liu et al., 1997). Babesiosis, caused by this parasite, is one of the most severe diseases in water buffalo, which results in large economic losses in the central and southern China (Chen, 1984; Liu and Ma, 1987; Zhongling et al., 1986). The clinical symptoms of this disease are fever, anemia, icterus and hemoglobinuria (He et al., 2009; Liu et al., 2005). B. orientalis was distinguished from Babesia bigemina and Babesia bovis in water buffalo and classified as a new species, according to the differences of morphology, transmission and pathogenicity (Liu et al., 1997).
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The Babesia RAP-1 gene was first described in B. bovis (Goff et al., 1988) and sequentially observed in many other Babesia species, including Babesia bigemina, Babesia divergens, Babesia caballi, B. bovis, and Babesia canis (Dalrymple et al., 1993; Ikadai et al., 2000; McElwain et al., 1991; Skuce et al., 1996; Suarez et al., 1991; Zhou et al., 2007). Babesial RAP-1 was tested in several vaccine trials either as an individual candidate or in combined form with other recombinant protein antigens. Experimental evidence suggested that the cattle immunized with RAP-1 elicited strong T-cell and B-cell immune responses (Norimine et al., 2003). As a component of multi-antigen vaccine, three protective antigens, including RAP-1 were identified in B. bovis (Wright et al., 1992). Apicomplexan parasites utilize several rhoptry proteins during invasion into and development within the host cell (Preiser et al., 2000; Reduker et al., 1989). An in vitro erythrocyte-binding assay confirmed that B. bovis RAP-1 was able to bind with the bovine and equine erythrocytes surface, and the binding affinity was weakened by antiRAP-1 MAb (Yokoyama et al., 2002). B. bovis RAP-1 was expressed in sporozoites, and specific RAP-1 antisera could inhibit the sporozoites invasion into host cells (Mosqueda et al., 2002). These studies strongly support that RAP-1 plays a functional role in the biology and development of Babesia parasites. The genetic organization of RAP-1 family is highly complexed in some Babesia species, containing several closely linked genes encoding unique proteins (Dalrymple et al., 1993; Sam-Yellowe, 1996; Skuce et al., 1996; Suarez et al., 1998). Despite of the complexity of the gene loci, all the members of rhoptry-associated protein family have well conserved features, including a N-terminal localized signal peptide, several tandem repeat regions in the C-terminal, four strictly conserved cysteine residues and a 14 aminoacid motif (Dalrymple et al., 1996; Norimine et al., 2003; Sam-Yellowe, 1996; Suarez et al., 1991). The well conserved RAP-1 is also highly immunogenic and has been observed to induce high titer of antibodies in Babesiainfected animals. Thus, the RAP-1 has also been widely used to develop serological diagnostic methods for Babesia parasites (Boonchit et al., 2002; Ikadai et al., 2000; Zhou et al., 2007). In the present study, the gene encoding rhoptryassociated protein 1 (RAP-1) was identified from a B. orientalis cDNA library, which was further confirmed through phylogenetic analysis of gene sequence. The encoding gene was cloned and expressed in Escherichia coli and evaluated its potential use as a diagnostic and vaccine candidate.
Two 1-year-old water buffaloes were purchased from Babesia free area and confirmed to be B. orientalis free by real-time PCR (He et al., 2011). The water buffaloes were splenectomized and subcutaneously injected with 4 ml of B. orientalis-infected blood (Wuhan strain, percentage parasitized erythrocytes (PPE) 1%). Blood samples were collected daily to monitor the parasitemia until it reached up to 3%. All animal experiments described in this article were carried out in compliance with the regulations (no. 5 proclaim of the Standing Committee of Hubei People’s Congress) approved by the Standing Committee of Hubei People’s Congress, P. R. China. The animal protocols were approved by Laboratory Animals Research Centre of Hubei province and the ethics committee of Huazhong Agricultural University (permit number: 4200696657). 2.2. Preparation of genomic DNA The blood from experimentally infected water buffalo was collected in EDTA tubes (BD Vacutainer blood collection tubes, USA). The genomic DNA was extracted from 200 l of B. orientalis-infected blood using a QIAamp DNA mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. DNA samples were stored at −20 ◦ C until further use. 2.3. Cloning and sequencing of the BoRAP-1 gene A cDNA expression library of B. orientalis merozoites, constructed previously (Liu et al., 2009), was screened with the serum of B. orientalis-infected water buffalo. One of the positive clones expected to be RAP-1 were chosen for further analysis. To amplify the B. orientalis RAP-1 gene from gDNA, a pair of primers was designed based on BLAST analysis of B. orientalis genome sequence (unpublished data). The forward primer RAP1-F was 5 -TAC TCA CCT ATA AAA GCC TCT TGC C-3 , and the reverse primer RAP1-R was 5 -CTG TAT GTG TCA AAA AGG GA-3 . The amplified PCR product from both gDNA and cDNA were electrophoresed using 0.8% ethidium bromidestained agarose gel and purified by TIANgel Midi purification Kit (TIANGEN, China). The purified PCR products were ligated into pMD18-T vector (TaKaRa, Japan), and three positive clones from each sample were sequenced using a Dye Terminator Cycle Sequencing reaction in an ABI PRISM 377 DNA sequencer (Sangon, China). The vector primers M13 (−47) and M13 (−48) were used for sequencing. 2.4. Bioinformatics analysis of BoRAP-1
2. Materials and methods 2.1. Parasites and experimental animals B. orientalis strain was previously isolated from water buffalo in Hubei province, China and preserved in liquid nitrogen in State Key laboratory of Agricultural Microbiology, Huazhong Agricultural University, China (Liu et al., 1995).
The isolated gene of B. orientalis RAP-1 was given the name “BoRAP-1”. The deduced amino acid (aa) sequence of BoRAP-1 was subjected to BLASTp analysis in GenBank (Benson et al., 2012). The homologous sequences of related intra-erythrocytic species (Table 1), including BoRAP-1 sequence were aligned using MAFFT version 7 (Katoh and Frith, 2012; Katoh and Standley, 2013) and manually edited using BioEdit version 7.1.11 (Hall, 1999). A
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Table 1 the RAP-1 sequences used in phylogenetic analysis and their amino acid accession numbers. Organism
Accession no.
Organism
Accession no.
B. orientalis B. bovis RAP B. bovis RRA B. caballi RAP B. canis RAP B. bigemina RAP1a B. bigemina RAP1b
AGC60006 AAB84268 XP 001610950 ADF30793 AAA27807 1906304A AAB72094
B. bigemina RAP1c B. divergence RAP B. gibsoni RAP B. equi T. orientalis Shintoku T. annulata RAP T. parva Muguga
AAB82596 CAA89970 ABD52000 XP 004833177 BAM39179 XP 954078 XP 766222
neighbor-joining tree was constructed by MEGA5 (Tamura et al., 2011). The evolutionary divergence was also calculated by MEGA5 using Poisson correction model (Tamura et al., 2011; Zuckerkandl and Pauling, 1965). The deduced protein sequence of BoRAP-1 was subjected to SignalP for analyzing signal peptide (http:// expasy.org/tools/#topology). Tandem repeats regions were determined using Tandem Repeat Finder (http:// tandem.bu.edu/trf/trf.html) and edited manually. Antigen index was analyzed through DNAstar Protean 7.5 (http:// www.dnastar.com/). Motif scanning of the predict protein was performed by PROSITE (http://prosite.expasy.org/).
2.5. Recombinant protein expression and purification of the rBoRAP-1 To amplify the ORF of BoRAP-1 gene, a pair of primers, ExpRAP1-F: 5 -CG GGA TCC ATG AGA GCA ATC AGC AGT-3 , and ExpRAP1-R: 5 -C GAG CTC TTA GTA GTC ATA AAA ATC AAT-3 , containing restriction enzyme sites BamHI and SacI (underlined and italic) were designed. The ORF of BoRAP-1 was cloned into pMD-18T vector, digested and sub-cloned into the expression vector pGEX-KG. The resulting plasmid (pGEX-KG-RAP1) was confirmed by enzyme restriction digestion and sequencing, and subsequently transformed into the E. coli BL21TM (DE3) strain using the standard method. The rBoRAP-1 was expressed as a GST-fusion protein by inducing with 1 mM isopropyl -d-thiogalactoside (IPTG). The recombinant protein was expressed in the soluble-fraction of E. coli and was purified using a GSTrapTM 4B columns (GE Healthcare, USA), according to the manufacturer’s instructions.
2.7. SDS-PAGE and immunoblotting The rBoRAP-1 was separated on 12% SDS-PAGE and transferred onto a nitrocellulose membrane (BioRad, USA). The membrane was blocked with 1% bovine serum albumin (BSA) (Equitech-Bio Inc., USA) and incubated with B. orientalis-infected water buffalo serum (1:200 dilution) for 60 min. After washing three times with PBST, the membrane was incubated with secondary antibodies (1:2000, Bov IgG/HRP, Bioss, China) for 45 min. The membrane was subjected to 3,3 -diaminobenzidine (DAB) (Biomiga, USA) for the visualization of protein bands. Serum from an uninfected water buffalo was used as negative control. 2.8. Identification of BoRAP-1 in merozoites by immunofluorescence The native B. orientalis RAP-1 was identified in merozoites by Indirect Fluorescent Antibody Test (IFAT). Thin blood smears prepared from B. orientalis-infected erythrocytes were dried at 37 ◦ C and fixed with mixed methanol and acetone 1:4 (v/v) at −20 ◦ C for 20 min. The fixed blood smear was probed with the rabbit immune sera against the rBoRAP-1 as primary antibodies (1:100 dilution in PBS; pH 7.4) for 30 min at 37 ◦ C. After washing with PBST three times, the slides were incubated with goat anti-rabbit IgG (H + L) antibodies (Alexa Fluor® 488, Life Technologies) as secondary antibodies (1:200) for 30 min at 37 ◦ C. The slides were washed three times with PBST and mounted by adding 10 l of a 50% glycerol-PBS (v/v) solution and covered with a glass cover slip. The slides were examined using a fluorescence microscope (AxioCam HRc, Zeiss, Germany). 3. Results 3.1. Cloning and characterization of the BoRAP-1
2.6. Preparation of rabbit anti-rBoRAP-1 immune serum Three eight-week-old female Japanese White Rabbits (specific pathogen free, SPF) were used for the preparation of antibodies against rBoRAP-1. The rBoRAP1 (500 g) emulsified in equal amount of Freund’s complete adjuvant (SIGMA) was subcutaneously injected into three rabbits. Two weeks after the immunization, the rabbits were injected with the same amount of antigen emulsified in Freund’s incomplete adjuvant (SIGMA) three times at one week interval. The sera from the immunized rabbits were collected after 7 days of the last immunization and stored at −20 ◦ C until further use.
The cDNA expression library of B. orientalis was screened with B. orientalis-infected buffalo serum, and a positive clone was isolated and sequenced. The full length of this positive clone was 1732 bp, with an ORF of 1434 bp encoding a polypeptide with a molecular weight of 52.5 kDa. The full length of BoRAP-1 gene was also amplified from B. orientalis genomic DNA (Fig. 1). The resulting fragments were ligated into pMD18-T vector and subjected to sequencing. The results showed that the sequence of BoRAP-1 gene obtained from gDNA was exactly same as the sequence from cDNA library. The results indicated that
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Fig. 1. The PCR results of amplifying BoRAP-1 from B. orientalis genome DNA. Lane M: DNA marker; lane 1: BoRAP-1 from gDNA (1732 bp); lane2: negative control.
there is no intron in the BoRAP-1 gene. The nucleotide sequence of BoRAP-1 was submitted to GenBank with accession number JX993940. 3.2. Sequence analysis The nucleotide sequence of BoRAP-1 gene was subjected to BLAST analysis in NCBI. Blastn search indicated that
BoRAP-1 gene had significant similarity with RAP-1 genes of related apicomplexan parasites. It showed highest identity (71%) with B. ovis RAP-1 gene (M91173), followed by B. bovis (68%; FJ588009), B. canis (64%; M91168) and Babesia caballi (63%; EU669865). The BoRAP-1 secondary structure showed several domains conserved in RAP-1 protein family. The BoRAP1 presented a rhoptry-associated protein 1 domain in the N-terminal, which is similar in characterization to other Babesia RAP-1 proteins. Multiple alignment of BoRAP-1 AA sequence and B. bovis (AAB84268), B. ovis (AAA27805) and B. caballi (ADF30793) showed several well defined molecular features of RAP-1 family (Fig. 2), including a serine-rich N-terminal region, a strictly conserved 4 cysteine residues, and a 14 amino-acid motif. However, the seventh proline residue (P) in 14 amino-acid motif of Babesia RAP-1 was replaced by alanine (A) in BoRAP-1. The alignment showed that Babesia RAP-1 AA sequences were highly conserved in the first 300 AA of the N-terminus compared to the sequences of C-terminus. Similar to Babesia RAP-1, a 21 AA signal peptide (1–21 AA residues) with a putative cleavage site was found in the beginning of BoRAP-1 N-terminus. The less conserved C-terminal of BoRAP-1 comprised five tandem repeats regions (TRPs) (Fig. 2). Antigenicity analysis performed by DNAstar showed that the immunogenic peptides were mainly present in the conserved N-terminal region (data not shown). Motif scanning by PROSITE showed several putative post-translation modification sites in the BoRAP-1 AA sequence, including three N-glycosylation sites (AA residues 176 to 179, 206 to 209, and 289 to 292), a cAMPand cGMP-dependent protein kinase phosphorylation site (AA residues 452 to 455), four Protein kinase C phosphorylation sites (AA residues 25 to 27, 52 to 54, 97 to 99, and
Fig. 2. Alignment of the putative amino acid of B. orientalis RAP-1 (BoRAP-1) with the RAP-1 of B. bovis (Bbo), B. ovis (Bov), B. bigemina (Bbi), B. caballi (Bca), B. divergence (Bdi) and B. gibsoni (Bgi). Similar and identical residues are shaded. Black shading indicates that similarity in four or more than four species, gray shading indicates that similarity in three species. The signal peptide cleavage site of BoRAP-1 is indicated by black arrow. Four conserved cysteine residues are indicated by black triangle. The 14 conserved amino acid residues [PLS(L/T/A/V)LPN(E/D)P(A)YQLDAAF] are showing black box. Five repeat motifs [PTKEFFVD(N)T(N)HEKTKD(E)FF(L)E(D)NK(N)VA(G)K] are indicated by underline.
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311 to 313), five Casein kinase II phosphorylation sites (AA residues 52 to 55, 171 to 174, 178 to 181, 184 to 187, and 455 to 458), one tyrosine kinase phosphorylation site (AA residues 112 to 119), and one N-myristoylation site (AA residues 17 to 22). 3.3. Expression and identification of BoRAP-1 The ORF of B. orientalis RAP-1 gene was amplified and ligated into the expression vector pGEX-KG. The resulting plasmid was subsequently subjected to the sequencing and enzyme digestion with BamHI and SacI to confirm the successful insertion of the RAP-1 fragment. The recombinant protein (rBoRAP-1) was expressed as a GST-fused protein in E. coli BL21 strain with a molecular weight of 78 kDa, including an additional 26 kDa peptide of the GST tag, as observed through SDS-PAGE (Fig. 3A). To determine whether antibodies against BoRAP-1 were elicited during B. orientalis infection period, rBoRAP-1 protein was subjected to immunoblotting. The serum of B. orientalis-infected water buffalo recognized a specific band of 78 kDa (Fig. 3B), whereas, the uninfected water buffalo serum did not react with rBoRAP-1 in the Western blot. To identify native RAP-1 in B. orientalis, rabbit antiserum against rBoRAP-1 was prepared and IFAT was performed (Fig. 4). IFAT results showed that the serum from rabbit immunized with rBoRAP-1 interacted with the ring (Fig. 4b) and pear-shaped (Fig. 4a and c) forms of B. orientalis merozoites, displaying the strong immunofluorescence signals. However, no signal was obvious from the uninfected water buffalo erythrocytes, when probed with anti-rBoRAP-1 antibodies. 3.4. Phylogenic analysis A neighbor-joining tree based on Babesia RAP-1 AA sequences has been shown in Fig. 5. B. orientalis fell into a distinct Babesia clade and was most closely related to B. bovis and B. ovis. 4. Discussion Babesia are obligate intracellular protozoan parasites, which invade a wide range of vertebrate hosts. They significantly impair the health of domestic and wild animals,
Fig. 3. SDS-PAGE of expression and purification of the rBoRAP-1 and Western blot analysis. (A) SDS-PAGE expression and purification of the rBoRAP-1. M, molecular weight marker; lane 1, uninduced pGEX-KG; lane 2, induced pGEX-KG; lane 3, supernatant after purification; lane 4, lysates of uninduced cells; lane 5, supernatant of induced lysates. (B) Determination of antibody response of BoRAP-1 in water buffalo antiserum. M, molecular weight marker; lane 1, rBoRAP-1 probed with anti-B. orientalis water buffalo serum; lane 2, rBoRAP-1 probed with uninfected water buffalo serum.
especially the farm and pet animals (Schnittger et al., 2012). The invasion of host erythrocytes by these protozoa is mediated by the molecules located on the merozoite surface and apical complex organelles, including the rhoptry, a unifying structure of the apicomplexan parasites (Perkins, 1992). Accordingly, the rhoptry-associated proteins of Babesia spp. are consistent targets of vaccine research for the past two decades (Brown et al., 1996; Dalrymple et al., 1993; Norimine et al., 2003; Wright et al., 1992). The Babesia rhoptry-associated protein 1 has been identified to be encoded by several closely linked proteins. It was reported that there are two identical genes in B. bovis, four homologous genes in B. bigemina, and five polymorphic linked genes in B. ovis (Sam-Yellowe, 1996; Suarez et al., 1998). The RAP-1 genes may play an
Fig. 4. Identification of native BoRAP-1 in B. orientalis. IFAT analysis of anti-B. orientalis antibody produced in rabbit immunized with rBoRAP-1 and expressed in E. coli. Immunofluroscence signals were observed in B. orientalis (white arrow), and only background signal was observed from erythrocytes.
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B orientalis RAP
69 89
B bovis RAP AAB84268 B ovis RAP AAA27805
94
B caballi RAP ADF30793
52
B bigemina RAP1a 1906304A
77 70
B divergence RAP CAA89970 B canis RAP AAA27807
76 89
B gibsoni RAP ABD52000
24
B bigemina RAP1c AAB82596 61
B bigemina RAP1b AAB72094 B bovis RRA XP001610950 B equi XP 004833177 T orientalis Shintoku BAM39179
97
T annulata RAP XP954078
100 100
T parva Muguga XP 766222 Toxoplasma gondii ROP AAA69859
0.1
Fig. 5. Neighbor-joining phylogenetic tree showing relationship of B. orientalis with related apicomplexa parasites. The trees was constructed based on the amino acid sequences of RAP-1 gene. GenBank accession numbers are indicated.
important function in the erythrocyte binding or invasion, as the RAP-1 may facilitate the parasite adaptation to variant host cells and erythrocyte receptors (Sam-Yellowe, 1996). In this study, a RAP-1 gene was initially identified by immunoscreening of a B. orientalis cDNA library. The complete nucleotide sequence of the BoRAP-1 gene was 1732 bp, with an ORF of 1434 bp encoding a mature protein of 52.5 kDa in size. The BoRAP-1 is intronless, as the sequences obtained from B. orientalis cDNA and gDNA were identical. The RAP-1 has been detected in a majority of Babesia species, which showed high percent identity. This conservation of RAP-1 genes among Babesia parasites may imply a potential key role of RAP-1 in parasite life cycle, especially the invasion process. According to the BLAST analysis, the newly identified BoRAP-1 gene and the putative protein sequence were most similar to B. ovis, followed by B. bovis and B. canis. The phylogenetic tree was constructed based on RAP-1 amino acid sequences, and the topology was consistent with previous analyses based on 18S rRNA genes and HSP70 genes (He et al., 2009; Liu et al., 2005). B. orientalis appeared in a distinct branch of Babesiidae and most closely related to B. bovis and B. ovis. These results suggested that the RAP-1 gene may be evolutionally conserved and can be used in the classification of Babesia species. Multiple alignments of BoRAP-1 AA sequence with the RAP-1 of B. ovis, B. bovis and B. caballi was performed. The results revealed the well-defined features among RAP1 family. A 21 AA signal peptide was identified in the beginning of N-terminus. Four conserved Cys residues were situated in identical positions, implying the conserved tertiary structure of RAP-1. The 14 amino acids motif PLSLPNPYQLDAAF has been observed in all Babesia RAP-1 homologues, though some residue sites have substitutions in certain species. For example, the third serine and sixth
asparagine residues have substitution of other residues in B. ovis and B. caballi. In B. divergens, the tenth leucine and eleventh asparaginase are replaced by valine and glycine, respectively. The seventh proline, which is more stable among the 14 residues, is mutated to alanine in B. orientalis RAP-1. Computer searches based on the 14 AA residues failed to match with any notable region of known proteins, which suggested that the biological functions of this motif remains unknown (Suarez et al., 1991). The motif scanning analysis showed several putative motifs involved in glycosylation and phosphorylation in BoRAP-1. Similarly, these motifs have been identified in several Babesia RAP-1. However, compared to Babesia gibsoni, the erythrocyte binding motifs RGD (aa 156 to 158) and KKNV (aa 313 to 316) were not observed in BoRAP-1 (Terkawi et al., 2009). It has been reported that the Babesia RAP-1 was widely used as diagnostic antigen (Boonchit et al., 2002; Ikadai et al., 2000; Zhou et al., 2007). In this study, the B. orientalisparasitized blood smear was subjected to IFAT analysis, and immunofluorescence signals were observed in different stages of B. orientalis. These indicated that the novel RAP-1 protein exists in B. orientalis. The results were consistent with the previous reports of B. bovis RAP-1 (Yokoyama et al., 2002). According to the results of Western blot, the serum of B. orientalis experimentally infected water buffalo interacted with the rBoRAP-1, indicating that the antibody responses of RAP-1 may be elicited in B. orientalis infection, and rBoRAP-1 could be a potential specific antigen for the detection of B. orientalis antibodies. In addition, the predicted antigenicity sites of BoRAP-1 also showed high antigenicity index (data not shown). There is no standard B. orientalis ELISA assay reported to date. Considering the predicted antigenic potential of BoRAP-1 and the results of Western blot, the protein could be a candidate for developing a stable ELISA method for B. orientalis.
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Overall, in the present study, an intronless rhoptryassociated protein 1 gene of B. orientalis encoding a 52.5 kDa protein was obtained from cDNA library by immunoscreening. This is the first description of RAP1 gene homolog in B. orientalis. Phylogenetic tree based on the RAP-1 amino acid sequences was constructed, the results indicated that B. orientalis appeared in a Babesia clade and is most closely related to B. bovis and B. ovis. The native RAP-1 was identified in B. orientalis merozoites by IFAT. The results suggested that BoRAP-1 could be a potential antigen for the detection of B. orientalis antibodies. Acknowledgements Project support was provided by National Natural Science Foundation of China (31302082), Special Fund for Agro-scientific Research in the Public Interest of China (201003060-01-06), and the National Basic Science Research Program (973 program) of China (2015CB150300). The authors would like to thank Dr. Qing Tao (State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, China) for providing advice on figures and illustrations. References Benson, D.A., Cavanaugh, M., Clark, K., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J., Sayers, E.W., 2012. GenBank. Nucleic Acids Res., gks1195. Boonchit, S., Xuan, X., Yokoyama, N., Goff, W.L., Wagner, G., Igarashi, I., 2002. Evaluation of an enzyme-linked immunosorbent assay with recombinant rhoptry-associated protein 1 antigen against Babesia bovis for the detection of specific antibodies in cattle. J. Clin. Microbiol. 40, 3771–3775. Brown, W.C., McElwain, T.F., Ruef, B.J., Suarez, C.E., Shkap, V., ChitkoMcKown, C.G., Tuo, W., Rice-Ficht, A.C., Palmer, G.H., 1996. Babesia bovis rhoptry-associated protein 1 is immunodominant for T helper cells of immune cattle and contains T-cell epitopes conserved among geographically distant B. bovis strains. Infect. Immun. 64, 3341–3350. Chen, S., 1984. Babesiosis of buffalo caused by a combination of Babesia bigemina and B. bovis. Hubei J. Vet. Sci. Technol. 8, 38–39. Dalrymple, B.P., Casu, R.E., Peters, J.M., Dimmock, C.M., Gale, K.R., Boese, R., Wright, I.G., 1993. Characterisation of a family of multi-copy genes encoding rhoptry protein homologues in Babesia bovis, Babesia ovis and Babesia canis. Mol. Biochem. Parasitol. 57, 181–192. Dalrymple, B.P., Peters, J.M., Böse, R., Wright, I.G., 1996. A polymerase chain reaction method for the identification of genes encoding members of the Bv60/p58 family of rhoptry protein homologues in the genus Babesia. Exp. Parasitol. 84, 96–100. Goff, W., Davis, W., Palmer, G., McElwain, T., Johnson, W., Bailey, J., McGuire, T., 1988. Identification of Babesia bovis merozoite surface antigens by using immune bovine sera and monoclonal antibodies. Infect. Immun. 56, 2363–2368. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser., 95–98. He, L., Feng, H.-H., Zhang, Q.-L., Zhang, W.-J., Khan, M.K., Hu, M., Zhou, Y.-Q., Zhao, J.-L., 2011. Development and evaluation of real-time PCR assay for the detection of Babesia orientalis in water buffalo (Bubalus bubalis, Linnaeus, 1758). J. Parasitol. 97, 1166–1169. He, L., Liu, Q., Quan, M., Zhou, D.-n., Zhou, Y.-q., Zhao, J.-l., 2009. Molecular cloning and phylogenetic analysis of Babesia orientalis heat shock protein 70. Vet. Parasitol. 162, 183–191. Homer, M.J., Aguilar-Delfin, I., Telford, S.R., Krause, P.J., Persing, D.H., 2000. Babesiosis. Clin. Microbiol. Rev. 13, 451–469. Ikadai, H., Osorio, C.R., Xuan, X., Igarashi, I., Kanemaru, T., Nagasawa, H., Fujisaki, K., Suzuki, N., Mikami, T., 2000. Detection of Babesia caballi infection by enzyme-linked immunosorbent assay using recombinant 48-kDa merozoite rhoptry protein. Int. J. Parasitol. 30, 633–635. Katoh, K., Frith, M.C., 2012. Adding unaligned sequences into an existing alignment using MAFFT and LAST. Bioinformatics 28, 3144–3146.
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