# Molecular Cloning and Characterization ofBabesia orientalis Rhoptry Neck 2BoRON2 Protein

Authors: Ngabu Malobi; Lan He; Long Yu; Pei He; Junwei He; Yali Sun; Yuan Huang; Junlong Zhao
Journal: Journal of Parasitology Research
 (2017)

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## Abstract

Babesiosis caused byBabesia orientalis is one of the most prevalent infections of water buffalo transmitted byRhipicephalus haemaphysaloides causing a parasitic and hemolytic disease. The organelles proteins localized in apical membrane especially rhoptries neck and microneme protein form a complex called moving junction important during invasion process of parasites belonging to apicomplexan group, includingBabesia species. A truncated fragment coding a 936 bps fragment was cloned in pMD-19T and subcloned into pET32 (a)+ expression vector, expressed inE. coli BL21. Purified recombinantBoRON2 was used to produce polyclonal antibody againstBoRON2. Here, we identified the full sequence of gene encoding the rhoptry neck 2 protein that we namedBoRON2 which is 4035 bp in full-length open reading frame without introns, encoding a polypeptide of 1345 amino acids. Western blot of rBoRON2 probed with buffalo positive serum analysis revealed a band of around 150 kDa in parasite lysates, suggesting an active involvement during invasion process. These findings most likely are constructive in perspective of ongoing research focused particularly on water buffalo babesiosis prevention and therapeutics and globally provide new information for genes comparative analysis.


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## Body

## 1. Introduction

Protozoan parasites are a significant cause of mortality in human and animals worldwide. They belong to the phylum Apicomplexa, specifically characterized by the presence of complex specialized organelles at their apical end, and are responsible of important diseases, such as malaria which causes more than 1 million deaths worldwide, toxoplasmosis, cryptosporidiosis, coccidiosis, and babesiosis [1]. Babesiosis caused byBabesia genus causes a huge economic loss to the livestock industry worldwide [2, 3]. The life cycle of these organisms is very complex, involving different stages. InPlasmodium parasites causing malaria, for example, the first stage of infection starts by sporozoites injection into bloodstream by femaleAnopheles (infecting hepatics cells in mammals and salivary glands) followed by entry in liver cells and division named preerythrocytic stage, followed by the release of merozoites into bloodstream which initiate the asexual parasite multiplication stage, and these merozoites’ differentiation leads to male and female gametocytes that invade mosquito midgut cells in which the gametocytes fusion produces a zygote that develops into a motile ookinete that penetrates the midgut wall and forms oocysts [4]. The oocyst enlarged over time and burst leading to sporozoites release which migrate to the mosquito salivary gland where they become susceptible of next infection through blood meal [5].The parasite invasion in host cell is a crucial step in apicomplexan biology infection process, as their extracellular life is limited to a short period of time. This process is achieved in less than 10 seconds and is powered by the glideosome, a macromolecular complex consisting of adhesive proteins and an actomyosin system anchored in the inner membrane complex of the parasite [6]. This invasion involves sequential secretion of the contents of two secretory organelles from the apical complex: the micronemes and rhoptries [7]. Commonly the proteins localized in rhoptries and micronemes mediate interaction between host receptor and parasite during invasion [8]. From the apical complex, the parasite moves through this ring-like structure which is referred to as the moving junction (MJ) and whose function is to generate the parasitophorous membrane from the invaginated host plasma membrane [9]. The importance of rhoptries necks proteins inP. falciparum and inT. gondii as model for apicomplexan parasite [10] has raised concern in identifying the functional characterization of these proteins in other genera, such asNeospora, Theileria, Babesia, Cryptosporidium, and other Plasmodium species. The current model suggests that rhoptry neck protein 2 (RON2) attaches the moving junction to the host cell membrane via its predicted transmembrane domains serving as a receptor for apical membrane antigen 1 (AMA-1), which anchors RONs proteins of junction complex to the parasite surface [10], proving the high importance of rhoptry neck 2 protein function in apicomplexan parasites group.Previous studies onT. gondii identified four rhoptries neck proteins (RON2, RON4, RON5, and RON8) found to be exported from apical rhoptries forming a complex at the host parasite interface [10–12]; however inPlasmodium only the 3 orthologues RON2, RON4, and RON5 found inToxoplasma have been identified [13, 14]. The micronemal protein AMA-1 plays an important role during the tight junction formation with rhoptries proteins and anchors the merozoite to host surface cell leading to the invasion [15]; additionally, although the invasion inhibitory of AMA-1/RON2 interaction has been proved to be crucial forT. gondii andP. falciparum from preventing entry in their respective host cell, the intraspecies interaction is conserved [16].AmongBabesia species, recently, the proteins RON2 ofB. microti andB. divergens were reported to be also homologues to other RON2 proteins of apicomplexan and the purified IgG fromB. divergens RON2 antibodies was shown to be able to inhibit the invasion over 36 hours up to 44% [17]. However, inB. orientalis, AMA-1 protein was identified and has showed to have a similar and relative conservation of domain three (DIII) and the presence of four cysteine motifs amongBabesia species [18]; thus the identification ofB. orientalis RON2 protein and molecular cloning to evaluate the antigenicity of this protein are going to make a new step in order to understand this parasite infection and biochemical regulation mechanism.


## 2. Materials and Method

### 2.1. Merozoite Preparation

B. orientalis collected from water buffalo was cultured in our laboratory as described [19] and kept in nitrogen liquid [20]. The merozoite antigen ofB. orientalis was prepared from blood infected by this parasite according to the modification saponin lysis method [21]. Erythrocytes fromB. orientalis have been washed 3 times by using phosphate buffered saline (PBS) followed by suspension in 9 mL of red blood cells (RBC) lysis buffer (Tiangen, China) and incubation at 37°C for 5 min. After solubilization, the antigen was clarified by centrifugation at 10,000 ×g for 1 h, resuspended in 1 mL PBS, and then stored at −20°C for further use.


### 2.2. Genomic DNA and cDNA Preparation

For DNA and RNA extraction, we collected blood sample from experimentally infected water buffalo with 3% parasitemia into EDTA tubes (Qingdao Pharmacypro Co., Ltd.). Genomic DNA (gDNA) was isolated by using QIAmp DNA Mini Kit™ (Qiagen Hilden, Germany) following the manufacturer protocol. The collected gDNA was stored at −20°C for further use. Total RNA was extracted from 250μL of red blood cells by using TRIzol RNA extraction kit (Invitrogen, USA); RNase inhibitor (RNase OUT recombinant ribonuclease inhibitor Invitrogen, USA) was added; then the cDNA was synthesized by reverse transcription using FastQant RT Kit (Tiangen Biotech (Beijing) Co., Ltd.).


### 2.3. Identification, Amplification, and Sequencing ofBoRON2

The degenerate primer was designed through the consensus sequence found using ClustalW [22] from alignment based upon similarity of related species published RON2 ofB. bovis (XM_001608765.1),B. bigemina (KU696964.1),B. divergens (GU 198499.2), andB. microti (XM_01279409.1) nucleotides sequences from NCBI. The amplified PCR product supposed to represent the partialB. orientalis RON2 was separated in 2% agarose gel ethidium bromide and cleaned using QIAquick gel extraction kit (Qiagen, China) and cloned into pMD-19T cloning vector (Takara Biotechnology, China) for sequencing. The partial amplified sequence was identified in a cDNA library contig in the complete genome sequence ofB. orientalis (unpublished), and then the complete open reading frame was identified by overlapping contigs. By using nBLAST search, the sequence has been confirmed as RON2 regarding the high similarity with its orthologues. The complete sequence was obtained by another PCR amplification using a specific pair of primers (Table 2) and the resulting product was sequenced for confirmation.


### 2.4. Sequence Analysis

The obtainedBoRON2 sequence was analyzed by using bioinformatic tools. SP.4.1 software was used to predict the presence of signal peptide [23]. The transmembrane domain was predicted with TMHMM v.2.0 server [24] and low complexity regions were analyzed by using SMART [25]. Alpha helix and beta sheet were predicted by using Psi Pred server [26]. The presence of repeat was assessed by the sequence tandem repeat using the modeling software XSTREAM [27]. Mega 6 was implemented for phylogenetic tree construction [28]. The tertiary dimensional structure was predicted by using I-Tasser standalone package [29]. For identification of probable anchor location motif scan has been used through Expasy server (http://prosite.expasy.org/). ClustalW was used for alignment and manual correction was applied by using Bioedit [22, 30].


### 2.5. Recombinant Expression and Antibody Production

The gDNA fragment coding a C-terminal region which representsBoRON2914–1226 peptide was amplified to produce a polyclonal antibody by using the expression vector p ET-32(a)+ to generate a recombinant His tag RON2 protein withinSalI andBamhI restrictions sites enzymes underlined with a specific primer (Table 2). After successful sequencing the resulting plasmid was transformed into bacteriaE. coli BL21 strain (Transgen, China). The recombinant protein was expressed after inducing 16 hours at 18°C with IPTG and visualized through SDS-PAGE. The protein expressed as inclusions bodies was solubilized in 8 M urea and eluted at 150 mM via His tag protein pure Ni-NTA resin charged column following the manufacturer’s instruction (Transgen, China). BCA protein assay kit (Beyotime Institute of Biotechnology, China) was used to determine the protein concentration. To generate antirecombinantBoRON2-C serum, 2 rabbits were immunized subcutaneously with 500 μg of rBoRON2-C emulsified in Freund’s complete adjuvant (Sigma, USA) first time at day 0 and then emulsified in Freund’s incomplete adjuvant (FIA) the next 4 times, respectively, at days 20, 35, 45, and 60 followed by antisera collection.


### 2.6. Western Blot Analysis

The reactivity ofBoRON2-C protein for immune response was analyzed by western blot withB. orientalis erythrocytes infected serum and His tag antibody used as positive control and normal serum as negative control. The recombinantBoRON2-C was subjected under reducing condition to 12% SDS-PAGE analysis and transferred to a membrane nitrocellulose (Millipore USA) and then blocking buffer for fixation (1% BSA in TBS with 5% tween) followed by 2 h incubation moderately by shaking at room temperature and probed with secondary antibody Ig rabbit anti-goat. To identify the nativeBoRON2-C in merozoite, rabbit antisera collected from rBoRON2-C immunized rabbit was reacted with parasite lysates subjected to SDS-PAGE 12% followed by transfer electroblotting onto nitrocellulose membrane and then probed with rabbit polyclonal antiserum at 1 : 500 dilutions overnight at 4°C in TBST, after 5 times of washing with TBST, the secondary conjugate rabbit anti-goat antibody was added as final incubation.


## 2.1. Merozoite Preparation

B. orientalis collected from water buffalo was cultured in our laboratory as described [19] and kept in nitrogen liquid [20]. The merozoite antigen ofB. orientalis was prepared from blood infected by this parasite according to the modification saponin lysis method [21]. Erythrocytes fromB. orientalis have been washed 3 times by using phosphate buffered saline (PBS) followed by suspension in 9 mL of red blood cells (RBC) lysis buffer (Tiangen, China) and incubation at 37°C for 5 min. After solubilization, the antigen was clarified by centrifugation at 10,000 ×g for 1 h, resuspended in 1 mL PBS, and then stored at −20°C for further use.


## 2.2. Genomic DNA and cDNA Preparation

For DNA and RNA extraction, we collected blood sample from experimentally infected water buffalo with 3% parasitemia into EDTA tubes (Qingdao Pharmacypro Co., Ltd.). Genomic DNA (gDNA) was isolated by using QIAmp DNA Mini Kit™ (Qiagen Hilden, Germany) following the manufacturer protocol. The collected gDNA was stored at −20°C for further use. Total RNA was extracted from 250μL of red blood cells by using TRIzol RNA extraction kit (Invitrogen, USA); RNase inhibitor (RNase OUT recombinant ribonuclease inhibitor Invitrogen, USA) was added; then the cDNA was synthesized by reverse transcription using FastQant RT Kit (Tiangen Biotech (Beijing) Co., Ltd.).


## 2.3. Identification, Amplification, and Sequencing ofBoRON2

The degenerate primer was designed through the consensus sequence found using ClustalW [22] from alignment based upon similarity of related species published RON2 ofB. bovis (XM_001608765.1),B. bigemina (KU696964.1),B. divergens (GU 198499.2), andB. microti (XM_01279409.1) nucleotides sequences from NCBI. The amplified PCR product supposed to represent the partialB. orientalis RON2 was separated in 2% agarose gel ethidium bromide and cleaned using QIAquick gel extraction kit (Qiagen, China) and cloned into pMD-19T cloning vector (Takara Biotechnology, China) for sequencing. The partial amplified sequence was identified in a cDNA library contig in the complete genome sequence ofB. orientalis (unpublished), and then the complete open reading frame was identified by overlapping contigs. By using nBLAST search, the sequence has been confirmed as RON2 regarding the high similarity with its orthologues. The complete sequence was obtained by another PCR amplification using a specific pair of primers (Table 2) and the resulting product was sequenced for confirmation.


## 2.4. Sequence Analysis

The obtainedBoRON2 sequence was analyzed by using bioinformatic tools. SP.4.1 software was used to predict the presence of signal peptide [23]. The transmembrane domain was predicted with TMHMM v.2.0 server [24] and low complexity regions were analyzed by using SMART [25]. Alpha helix and beta sheet were predicted by using Psi Pred server [26]. The presence of repeat was assessed by the sequence tandem repeat using the modeling software XSTREAM [27]. Mega 6 was implemented for phylogenetic tree construction [28]. The tertiary dimensional structure was predicted by using I-Tasser standalone package [29]. For identification of probable anchor location motif scan has been used through Expasy server (http://prosite.expasy.org/). ClustalW was used for alignment and manual correction was applied by using Bioedit [22, 30].


## 2.5. Recombinant Expression and Antibody Production

The gDNA fragment coding a C-terminal region which representsBoRON2914–1226 peptide was amplified to produce a polyclonal antibody by using the expression vector p ET-32(a)+ to generate a recombinant His tag RON2 protein withinSalI andBamhI restrictions sites enzymes underlined with a specific primer (Table 2). After successful sequencing the resulting plasmid was transformed into bacteriaE. coli BL21 strain (Transgen, China). The recombinant protein was expressed after inducing 16 hours at 18°C with IPTG and visualized through SDS-PAGE. The protein expressed as inclusions bodies was solubilized in 8 M urea and eluted at 150 mM via His tag protein pure Ni-NTA resin charged column following the manufacturer’s instruction (Transgen, China). BCA protein assay kit (Beyotime Institute of Biotechnology, China) was used to determine the protein concentration. To generate antirecombinantBoRON2-C serum, 2 rabbits were immunized subcutaneously with 500 μg of rBoRON2-C emulsified in Freund’s complete adjuvant (Sigma, USA) first time at day 0 and then emulsified in Freund’s incomplete adjuvant (FIA) the next 4 times, respectively, at days 20, 35, 45, and 60 followed by antisera collection.


## 2.6. Western Blot Analysis

The reactivity ofBoRON2-C protein for immune response was analyzed by western blot withB. orientalis erythrocytes infected serum and His tag antibody used as positive control and normal serum as negative control. The recombinantBoRON2-C was subjected under reducing condition to 12% SDS-PAGE analysis and transferred to a membrane nitrocellulose (Millipore USA) and then blocking buffer for fixation (1% BSA in TBS with 5% tween) followed by 2 h incubation moderately by shaking at room temperature and probed with secondary antibody Ig rabbit anti-goat. To identify the nativeBoRON2-C in merozoite, rabbit antisera collected from rBoRON2-C immunized rabbit was reacted with parasite lysates subjected to SDS-PAGE 12% followed by transfer electroblotting onto nitrocellulose membrane and then probed with rabbit polyclonal antiserum at 1 : 500 dilutions overnight at 4°C in TBST, after 5 times of washing with TBST, the secondary conjugate rabbit anti-goat antibody was added as final incubation.


## 3. Result

### 3.1. Molecular Characterization and Bioinformatic Analysis

The obtained gDNA ofBabesia orientalis was amplified and the complete RON2 gene sequence was identified by using specific primer as indicated on Table 2.BoRON2 gene is 4035 bp nucleotides in length, encoding for 1345 amino acid residues with one copy of imperfect repeat sequence one time repeated in positions 824–833 (DDLEK–DDSEK). The amino acid sequence contains 12 cysteine residues as shown in Figure 1 and was predicted 150 kDa by using a computer based molecular weight calculator [31]. The phylogenetic tree shows that the more close species based on alignment ofBoRON2 protein as query isB. bovis (T2Bo strain) RON2 protein belonging into the same clade (Figure 2). They share 72% similarity, followed byB. bigemina with 70% of similarity. The amplification of cDNA has shown an amplicon of similar size with gDNA on gel electrophoresis (Figure 3(a)). Finally, the sequencing of cDNA has confirmed the intronless of this gene. The phylogenetic tree ofBoRON2 protein sequence with homologues was performed to identify the distance between them. The neighbor joining tree shows values of each branch with 0.1 as the length of branch (Figure 2). The protein has 7 conserved cysteine residues among aligned apicomplexan orthologues RON2, and 4 of them are located in C-terminal region alternating with transmembrane domains (Figure 5). In fact, many molecular relevant malaria antigens, actively functional, contain cysteine motifs in their structure forming disulfides bridges and/or occasioning three-dimensional structures fulfilling their biological function [32]. InP. falciparum the protein rhoptry neck 6PfRON6 is conserved among apicomplexans except for rodent malaria orthologue, and rich cysteine region was shown to be involved in parasite survival function [33]. The molecular scanning has revealed a putative phosphotidylinositol specific lipase domain at amino acid positions 1026 to 1086 probably involved in membrane trafficking during parasite invasion and a putative anchoring site during infective process.BoRON2 protein has a putative signal peptide within its 20 first amino acids characterized by the presence of three transmembrane domains all located toward the C-terminal region between residues 1110 and 1251 and three low complexity regions. The relative conservation in C-terminal fragment covering the region of amino acid comprising between 1018 and 1295 (Figure 5) is most likely due to the presence in that region of active site forB. orientalis RON2 interaction and important domains during the invasion process. Neighbor joining phylogenetic tree was built from the protein sequence ofB. orientalis RON2 submitted on GenBank with closely related apicomplexan parasites species as indicated with respective accession number (Table 1) which was obtained by using protein search on NCBI. The tertiary structure sequence is predominantly dominated by the presence of alpha helix (Figure 6) without coiled coils motifs structure in contrary to some reported RON2 orthologues such asP. falciparum andP. vivax [34], which may explain one of the host specificity reasons.Table 1
Apicomplexan species used for alignment and phylogenetic tree.
Organism
Accession number
Babesia bovis
XM_001608815.1
Babesia bigemina
AQU42588.1
Babesia microti
SJK86709.1
Babesia divergens
ADM34975.2
Theileria parva
XP_765541.1
Neospora caninum
XP_003886062.1
Toxoplasma gondii
AAZ38163.1
Eimeria tenella
XP_013231132.1
Plasmodium vivax
ADV19053.1
Plasmodium falciparum
XP_001348669.1
Plasmodium reichenowi
XP_012765563.2
Babesia orientalis
To be submitted
Hammondia hammondi
XP_008888401.1
Plasmodium ovale
SCP05763.1
Plasmodium berghei
CDS50206.1
Plasmodium yoelii
CDU19815.1Table 2
Set of primers used for amplification in this work.
Primer
Sequences (5′-3′)
Remarks
F0
TGGATGMAATGATTTCTGGACCC
Degenerate primer
R0
ACGCACGTTTCCACTTGTTGT
F1
ATGTTTGCGGTTACCCTGGCAACGATCACACTAGTG
ORF
R1
TCAATTAAATACAGTGTATGAGAAGTTGTCGTCAGC
Fa
CGGGATCCATGGCGTTCCAAAGAGCTGCTA
Antibody production
Ra
GCGTCGACTCATTGTACACCTACTGAGAGTGCFigure 1
Schematic representation ofBabesia orientalis RON2 gene with an open reading frame of 4035 bps length, signal peptide is shown is green (1–20), the three transmembrane (TM) domains are shown in blue, in grey are the predicted low complexity (LC) regions, and in red are the twelve cysteine residues contained in that gene. The repeat motif is shown in black between the amino acid regions 824 and 833.Figure 2
Relationship of BoRON2 with other apicomplexan related RON2 proteins. The neighbor joining tree was constructed using protein sequence ofBabesia orientalis RON2 with its homologues found in NCBI, and the respective accessions numbers are indicated. The scale bar shows the length of branch; every 0.1 nucleotides’ difference per 100 is represented.B. orientalis RON2 is in the same clade withB. bovis,B. bigemina, andB. divergens, more close toB. bovis.Figure 3
PCR amplification of rhoptry neck 2 gene of Babesia orientalis from gDNA and cDNA and amplification of fragment used for polyclonal antibody production. (a) M: nucleotide marker (15000 bps), line 1: amplification ofBoRON2 genomic DNA open reading frame showing a specific band of 4035 bp, and line 2: amplification of cDNA showing to have the same size with genomic DNA. (b) Line 1, the amplicon of product used for polyclonal antibody.
(a)
(b)


### 3.2.BoRON2 Immunoblotting Identification

The recombinantBoRON2 antibody from amplification of 936 bp (Figure 3(b)) located between the amino acid regions 914 and 1226 was expressed as His Thioredoxin tag fusion native protein. A strong band of 53 kDa was detected when probing the recombinantBoRON2 with positiveB. orientalis serum of water buffalo (Figure 4(a)). The collected sera from rabbits immunized withBoRON2-C antigen have been used forB. orientalis RON2 protein identification in parasite lysates through immunoblot assay. As indicated on Figure 4(b) a band at ~150 kDa in parasite lysates was observed consistent with the computational predicted size, but no band was observed with the preimmune serum (PI).Figure 4
Western blot of recombinant protein BoRON2-C. (a) A specific band of 53 kDa was detected in reaction of recombinantBoRON2 with antiB. orientalis water buffalo serum on line 1 and line M the prestained protein marker. (b) Immunoblot for detection ofBoRON2 protein in parasite lysates. Line 1, a unique band of around 150 kDa was detected in parasite lysate probed with polyclonalBoRON2 antibody; line 2: no band detected with preimmune serum in parasite lysates.
(a)
(b)Figure 5
Amino acid alignment of Babesia orientalis RON2 and other orthologues. The alignment showing only C-terminal the most conserved region (flanking the region of AA sequence comprising between 1018 and 1295) was generated by using ClustalW. The conserved residues sequences are drawn in black boxes and the four conserved cysteine in C-terminal region in red line among the all proteins RON2 sequences aligned and the three predicted transmembrane regions ofBoRON2 are underlined in blue, the 2 arrows indicating the 5′ and 3′ direction of sequence.Figure 6
Babesia orientalis RON2 tertiary prediction structure. The predicted model with the highest C-score ofB. orientalis RON2 protein.


## 3.1. Molecular Characterization and Bioinformatic Analysis

The obtained gDNA ofBabesia orientalis was amplified and the complete RON2 gene sequence was identified by using specific primer as indicated on Table 2.BoRON2 gene is 4035 bp nucleotides in length, encoding for 1345 amino acid residues with one copy of imperfect repeat sequence one time repeated in positions 824–833 (DDLEK–DDSEK). The amino acid sequence contains 12 cysteine residues as shown in Figure 1 and was predicted 150 kDa by using a computer based molecular weight calculator [31]. The phylogenetic tree shows that the more close species based on alignment ofBoRON2 protein as query isB. bovis (T2Bo strain) RON2 protein belonging into the same clade (Figure 2). They share 72% similarity, followed byB. bigemina with 70% of similarity. The amplification of cDNA has shown an amplicon of similar size with gDNA on gel electrophoresis (Figure 3(a)). Finally, the sequencing of cDNA has confirmed the intronless of this gene. The phylogenetic tree ofBoRON2 protein sequence with homologues was performed to identify the distance between them. The neighbor joining tree shows values of each branch with 0.1 as the length of branch (Figure 2). The protein has 7 conserved cysteine residues among aligned apicomplexan orthologues RON2, and 4 of them are located in C-terminal region alternating with transmembrane domains (Figure 5). In fact, many molecular relevant malaria antigens, actively functional, contain cysteine motifs in their structure forming disulfides bridges and/or occasioning three-dimensional structures fulfilling their biological function [32]. InP. falciparum the protein rhoptry neck 6PfRON6 is conserved among apicomplexans except for rodent malaria orthologue, and rich cysteine region was shown to be involved in parasite survival function [33]. The molecular scanning has revealed a putative phosphotidylinositol specific lipase domain at amino acid positions 1026 to 1086 probably involved in membrane trafficking during parasite invasion and a putative anchoring site during infective process.BoRON2 protein has a putative signal peptide within its 20 first amino acids characterized by the presence of three transmembrane domains all located toward the C-terminal region between residues 1110 and 1251 and three low complexity regions. The relative conservation in C-terminal fragment covering the region of amino acid comprising between 1018 and 1295 (Figure 5) is most likely due to the presence in that region of active site forB. orientalis RON2 interaction and important domains during the invasion process. Neighbor joining phylogenetic tree was built from the protein sequence ofB. orientalis RON2 submitted on GenBank with closely related apicomplexan parasites species as indicated with respective accession number (Table 1) which was obtained by using protein search on NCBI. The tertiary structure sequence is predominantly dominated by the presence of alpha helix (Figure 6) without coiled coils motifs structure in contrary to some reported RON2 orthologues such asP. falciparum andP. vivax [34], which may explain one of the host specificity reasons.Table 1
Apicomplexan species used for alignment and phylogenetic tree.
Organism
Accession number
Babesia bovis
XM_001608815.1
Babesia bigemina
AQU42588.1
Babesia microti
SJK86709.1
Babesia divergens
ADM34975.2
Theileria parva
XP_765541.1
Neospora caninum
XP_003886062.1
Toxoplasma gondii
AAZ38163.1
Eimeria tenella
XP_013231132.1
Plasmodium vivax
ADV19053.1
Plasmodium falciparum
XP_001348669.1
Plasmodium reichenowi
XP_012765563.2
Babesia orientalis
To be submitted
Hammondia hammondi
XP_008888401.1
Plasmodium ovale
SCP05763.1
Plasmodium berghei
CDS50206.1
Plasmodium yoelii
CDU19815.1Table 2
Set of primers used for amplification in this work.
Primer
Sequences (5′-3′)
Remarks
F0
TGGATGMAATGATTTCTGGACCC
Degenerate primer
R0
ACGCACGTTTCCACTTGTTGT
F1
ATGTTTGCGGTTACCCTGGCAACGATCACACTAGTG
ORF
R1
TCAATTAAATACAGTGTATGAGAAGTTGTCGTCAGC
Fa
CGGGATCCATGGCGTTCCAAAGAGCTGCTA
Antibody production
Ra
GCGTCGACTCATTGTACACCTACTGAGAGTGCFigure 1
Schematic representation ofBabesia orientalis RON2 gene with an open reading frame of 4035 bps length, signal peptide is shown is green (1–20), the three transmembrane (TM) domains are shown in blue, in grey are the predicted low complexity (LC) regions, and in red are the twelve cysteine residues contained in that gene. The repeat motif is shown in black between the amino acid regions 824 and 833.Figure 2
Relationship of BoRON2 with other apicomplexan related RON2 proteins. The neighbor joining tree was constructed using protein sequence ofBabesia orientalis RON2 with its homologues found in NCBI, and the respective accessions numbers are indicated. The scale bar shows the length of branch; every 0.1 nucleotides’ difference per 100 is represented.B. orientalis RON2 is in the same clade withB. bovis,B. bigemina, andB. divergens, more close toB. bovis.Figure 3
PCR amplification of rhoptry neck 2 gene of Babesia orientalis from gDNA and cDNA and amplification of fragment used for polyclonal antibody production. (a) M: nucleotide marker (15000 bps), line 1: amplification ofBoRON2 genomic DNA open reading frame showing a specific band of 4035 bp, and line 2: amplification of cDNA showing to have the same size with genomic DNA. (b) Line 1, the amplicon of product used for polyclonal antibody.
(a)
(b)


## 3.2.BoRON2 Immunoblotting Identification

The recombinantBoRON2 antibody from amplification of 936 bp (Figure 3(b)) located between the amino acid regions 914 and 1226 was expressed as His Thioredoxin tag fusion native protein. A strong band of 53 kDa was detected when probing the recombinantBoRON2 with positiveB. orientalis serum of water buffalo (Figure 4(a)). The collected sera from rabbits immunized withBoRON2-C antigen have been used forB. orientalis RON2 protein identification in parasite lysates through immunoblot assay. As indicated on Figure 4(b) a band at ~150 kDa in parasite lysates was observed consistent with the computational predicted size, but no band was observed with the preimmune serum (PI).Figure 4
Western blot of recombinant protein BoRON2-C. (a) A specific band of 53 kDa was detected in reaction of recombinantBoRON2 with antiB. orientalis water buffalo serum on line 1 and line M the prestained protein marker. (b) Immunoblot for detection ofBoRON2 protein in parasite lysates. Line 1, a unique band of around 150 kDa was detected in parasite lysate probed with polyclonalBoRON2 antibody; line 2: no band detected with preimmune serum in parasite lysates.
(a)
(b)Figure 5
Amino acid alignment of Babesia orientalis RON2 and other orthologues. The alignment showing only C-terminal the most conserved region (flanking the region of AA sequence comprising between 1018 and 1295) was generated by using ClustalW. The conserved residues sequences are drawn in black boxes and the four conserved cysteine in C-terminal region in red line among the all proteins RON2 sequences aligned and the three predicted transmembrane regions ofBoRON2 are underlined in blue, the 2 arrows indicating the 5′ and 3′ direction of sequence.Figure 6
Babesia orientalis RON2 tertiary prediction structure. The predicted model with the highest C-score ofB. orientalis RON2 protein.


## 4. Discussion

Apicomplexan phylum contains obligate intracellular parasites characterized by a particular process during host cell invasion involving the secretory organelles essentially rhoptries and micronemes proteins [35]. They form a distinctive structure called moving junction, in the space between host plasma membrane and parasite [33]. Most of these proteins of moving junction identified inT. gondii have been found to be well conserved among apicomplexan parasites, with clear homologues identified in other species such asPlasmodium [36]. The rhoptries neck protein 2 has been shown to be conserved among different reported homologous species [37]. Numerous studies previously have reported both inPlasmodium and inToxoplasma the direct interaction between AMA-1 and RON2 [38], and the occurrence of RON4 in homologuesPlasmodium sp. andBabesia sp. has suggested the conservation of moving junction components across apicomplexan phylum [12].In this work, we reported the RON2 protein ofB. orientalis, which has been shown to be conserved to reported homologues, signifying the importance of this protein in biology of this parasite in infection stages. To our knowledge, this study is the first work to report molecular identification and characterization ofB. orientalis RON2. The recombinant polyclonal antibodyBoRON2 raised was able to detect a unique band at ~150 kDa in parasite lysates, which was consistent with the predicted size. As reported previously, the generated polyclonal antibody RON2 ofB. microti was able to detect a ~170 kDa predominant band consistent to the expected size and a product at ~55 kDa with no clear identity, butB. divergens antibodies identified also a consistent dominant band at ~170 kDa and 2 secondary bands at ~130 kDa and ~60 kDa [17]. Furthermore, in study onP. falciparum RON2 protein, sera in schizont stage have reacted with a band larger than 250 kDa corresponding to the predicted size and an additional band of 80 kDa without specific identity, but probably a Pf RON2 processing product [39]. HoweverP. vivax RON2 polyclonal antibody has detected 2 bands (~220 kDa and ~185 kDa) different to the expected size (240 kDa) suggesting not only a proteolytic process but also a particular behavior during SDS-PAGE running [34]; curiously the same behavior forT. gondii RON2 characterized by an abnormal migration was observed, although the polyclonal antibodyT. gondii RON2 has detected a band less than the predicted size (~150 kDa) cleaved to generate a 120 kDa protein [10].The phylogenetic and BLAST analysis indicate the closeness of taxonomical relationship with other apicomplexan rhoptries proteins RON2, confirming, like for many reportedB. orientalis genes such as AMA-1, Hsp20, Cox1, and Cob genes, thatB. bovis is phylogenetically the closest among all related apicomplexan parasites with 72% sequence similarity; additionally the apicoplast genome is more similar toBabesia bovis apicoplast genome structure [40]. The analysis of cDNA and gDNA has revealed that there are no introns. It has been also reported for AMA-1 nucleotide sequence ofB. orientalis the absence of introns [17], supposing likely that the splicing process involving the apical complex proteins in this genus is a probable inexistent event during protein translation for invasion establishment process. The signal peptide is followed by N-terminal region relatively not conserved harboring 2 low complexity domains needed to be analyzed for functional identification. We noticed the presence of 12 cysteine residues; three between the two hydrophobic regions close to N-terminal region are conserved. The conserved C-terminal region contains 4 conserved cysteine residues, supposing the metabolic importance of that region in this genus; in fact most of apicomplexan parasites RON2, the cysteine-rich globular domain in merozoite surface, or apical proteins are involved in crucial interaction between ligand and surfaces receptors and also during invasion between host and parasite [41–43]. Additionally inP. falciparum merozoite andT. gondii tachyzoite the C-terminal recombinant protein RON2 was able to block the host cell invasion [15]. The relative conservation ofBabesia orientalis C-terminal region presumes most likely the active site for interaction with AMA-1 facilitating this parasite invasion.


## 5. Conclusion

The underlying mechanism of buffalo babesiosis caused byB. orientalisin molecular level passes inevitably through RON2 protein identification. Herein we reportedB. orientalis RON2 which has been identified to be conserved among related species, with a probable C-terminal physiologically and relatively more active. The abundance of cysteine residue is most likely relevant to its function during invasion, which unlikely makes this protein hard for solubilization in active form during experiment for the expression in bacteria. The polyclonal serum has been able to detect the native protein in parasite lysates suggestingBoRON2 as a good potential candidate for vaccine and diagnosis of disease. Therefore, the limitation of doing more experiments for biochemistry and physiology purposes is due to the complication of culturing in vitroB. orientalis parasite.



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*Source: 7259630-2017-07-09.xml*