This article appeared in a journal published by Elsevier The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited In most cases authors are permitted to post their version of the article (e.g in Word or Tex form) to their personal website or institutional repository Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Molecular & Biochemical Parasitology 178 (2011) 40–45 Contents lists available at ScienceDirect Molecular & Biochemical Parasitology Short communication Secretion of a new spherical body protein of Babesia bovis into the cytoplasm of infected erythrocytes Mohamad Alaa Terkawi a , Faasoa Junior Seuseu a , Putut Eko-Wibowo a , Nguyen Xuan Huyen a , Yuka Minoda a, Mahmoud AbouLaila a, Satoru Kawai b, Naoaki Yokoyama a, Xuenan Xuan a, Ikuo Igarashi a,∗ a b National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-13, Inada-cho, Obihiro, Hokkaido 080-8555, Japan Department of Tropical Medicine and Parasitology, Dokkyo Medical University, Mibu, Tochigi 321-0293, Japan a r t i c l e i n f o Article history: Received 10 September 2010 Received in revised form February 2011 Accepted 14 February 2011 Available online 13 March 2011 Keywords: Babesia bovis Spherical body protein Secretion Erythrocytes a b s t r a c t A cDNA encoding a new Babesia bovis spherical body protein (BbSBP-4) was reported to have no significant homology to other apicomplexan proteins or previously reported B bovis spherical body proteins In the present study, we further examined the molecular characteristics of BbSBP-4 including the expression and cellular localization of the BbSBP-4 An anti-rBbSBP-4 mouse serum specifically reacted to a 41-kDa native protein B bovis in Western blot analysis The immunoelectron microscopic examination confirmed the localization of BbSBP-4 in spherical bodies, but not in the nucleus, rhoptries, and micronemes Interestingly, the protein was found to be localized not only in the spherical body of B bovis but also in the cytoplasm of infected erythrocytes (iRBC) at the later stage of parasite development The confocal laser microscopic examination and Western blot analysis demonstrated the increased accumulation of BbSBP-4 in the cytoplasm of iRBC and in the supernatant of cultivated B bovis during the late developmental stage of the parasite These results suggest that BbSBP-4 was secreted from spherical body into cytoplasm of iRBC during the late developmental stage of the parasite before the rupture of infected RBC Taken together, BbSBP-4 might play an important role as a secreted protein in the intracellular development and/or survival of B bovis © 2011 Elsevier B.V All rights reserved Babesia parasites are obligate intraerythrocytic protozoa, taxonomically classified in the phylum Apicomplexa, class Piroplasmida, order Piroplasmida, which are capable of invading a broad range of vertebrate hosts, subsequently leading to serious economic problems within the livestock industry throughout tropical and subtropical regions of the world [1] The life cycle is relatively similar among the order, and it requires a vertebrate host for the asexual stage as well as invertebrate hosts for the sexual stage Babesia parasites initiate infection to mammalian hosts by sporozoites, which are transmitted through the bites of infected ticks; subsequently, the merozoites invade and replicate within the infected erythrocytes (iRBC), eventually leading to acute babesiosis Babesia bovis is the most economically important species because of its impact on the cattle industry; it has ability to modify the iRBC sequestered in microcapillaries of the kidneys, lungs, and brain, resulting in organ failure and systemic shock, leading to death [1–3] Cattle that have recovered from acute infection become asymptomatic carriers, where the parasites persist in blood ∗ Corresponding author Tel.: +81 155 49 5641; fax: +81 155 49 5643 E-mail address: igarcpmi@obihiro.ac.jp (I Igarashi) 0166-6851/$ – see front matter © 2011 Elsevier B.V All rights reserved doi:10.1016/j.molbiopara.2011.02.006 for years and recrudescence of parasitemia can occur at irregular intervals [3] Although bovine babesiosis can be controlled with vaccination or treatment with antiparasitic drugs, the available vaccines are live and have safety concerns, and many drugs have been withdrawn from the market due to safety or resistance problems [3] Therefore, the development of new strategies, including vaccination and chemotherapy, is highly required for controlling the disease and reduce the hazards of infection Apicomplexan parasites are defined by their characteristic fine structures, including the apical complex, which consists of rhoptries, micronemes, and dense granules Spherical bodies are known as unique organelles that present only in the babesial apical complex and are analogous to dense granules in other members of the genera [4] Proteins derived from those organelles are believed to have critical functions, including host cell interaction and modification for parasite invasion, growth, and egression [5,6] Indeed, some of these proteins are released and incorporated into their host cell environment during the invasion or post-invasion step, leading to favorable conditions for the survival of the parasite [6] While rhoptry and microneme proteins participate in the process of parasite attachment and invasion, spherical body proteins seem to play a role in the post-invasion step, aiding in parasite growth [5,6] Author's personal copy M.A Terkawi et al / Molecular & Biochemical Parasitology 178 (2011) 40–45 Three proteins localized in the B bovis spherical body organelles have been previously identified and characterized A gene encoding the 77–80 kDa protein (BbSBP-1) was isolated from cDNA expression library screened by naturally infected bovine sera On the other hand, BbSBP-2 (225-kDa) and BbSBP-3 (135-kDa) were isolated using monoclonal antibodies raised against spherical bodies [7–9] All of these proteins were found to localize to the cytoplasmic face of the infected RBC membrane in a similar manner to the dense granule proteins identified in Plasmodium spp and Toxoplasma gondii Indeed, the dense granule proteins are released post-invasion either into the cytoplasm of host cells or within a parasitophorous vacuole, aiding in stabilizing the parasite environment and facilitating the interaction between the parasites and the erythrocyte membrane [5,6,10] However, understanding the biological functions of the proteins derived from these organelles, particularly those involved in B bovis invasion, growth, and egression, is undoubtedly necessary for the development of effective chemotherapeutic agents or a protective vaccine Recently, a new cDNA encoding the fourth spherical body protein (BbSBP-4) was deposited in the GenBank [11] However, detailed molecular and biological characterizations of this protein have not been studied Therefore, a study describing the molecular features, structure and cellular localization of BbSBP-4 was made for better understanding of the biology of the parasites The BbSBP4 of B bovis Texas strain [12] contains an open reading frame (ORF) of 1119 bp that was predicted to result in a 41-kDa mature protein consisting of 372 amino acids with an isoelectric point (IP) of 4.22 A putative signal peptide was found in the N-terminal hydrophobic region with a predicted cleavage site between amino acid residues 22 and 23 (http://www.cbs.dtu.dk/services/SignalP/) The computer analysis of potential subsequence motifs in the BbSBP-4 amino acid sequence (http://myhits.isb-sib.ch/cgi-bin/motif scan) revealed the presence of numerous phosphorylation sites distributed across the entire length of the protein, a domain (FAINT) extending over amino acid residues 207–285 and a glutamic acidrich region extending over amino acid residues 301–373 (Fig 1A) Likewise, BbSBP-3 seemed to possess a FAINT domain that is present in a large number of secreted proteins of Theileria spp [13] However, it was earlier suggested that both the Theileria and Babesia spp., encode an array of proteins with FAINT domains that are likely to be secreted into the host cytoplasm and act as mediators of physiological changes associated with intracellular parasitism by piroplasms [14] BbSBP-4 had no significant identity with proteins from other member of Apicomplexan parasites (http://blast.ncbi.nlm.nih.gov), although a little possible homology found in B bigemina (www.sanger.ac.uk/Projects/B bigemina/), but it is highly dissimilar In addition, the comparison of the amino acid sequence of BbSBP-4 with previously identified BbSBPs did not show any significant identity Next, the presence or absence of an intervening sequence in the coding region of the genomic B bovis DNA was examined The obtained genomic sequence was colinear with the cDNA clone in length and content, indicating the absence of introns in the BbSBP-4 gene (data not shown) Genomic DNA analysis (www.ncbi.nlm.nih.gov//genomes/geblast) has revealed that the BbSBP-4 exists as a single copy gene in chromosome extending between 356,249 and 357,756 bp of the genome In support of this finding, Southern blot analysis of the genomic DNA probed with the BbSBP-4 gene demonstrated a single hybridizing band after digestion with appropriate enzymes that can cut outside of the gene (data not shown) Thereafter, the geographic conservation of the BbSBP-4 gene was evaluated in the genomic DNA samples of field isolates from Texas, Brazil, Thailand, Mongolia, Ghana, and South Africa As calculated using DNASTAR software (DNASTAR software; NetWell Corporation, Tokyo, Japan) of BbSBP-4 sequences aligned by CLUSTAL W method, the gene isolated from cultured Texas strain (AB594813) demonstrated high identities with other isolates; 100% 41 Fig Genetic characterizations of the Babesia bovis spherical body protein (BbSBP-4) (A) Graphic depiction of BbSBP-4 The B bovis cDNA BbSBP-4 (accession number: AF486506) contains a complete ORF encoding the predicted protein shown in the bar below, with characteristic structures: a signal peptide (1–22 aa) at the N-terminal, a faint domain and a glutamic acid-rich region (Glu-RP) at the C-terminal The Texas strain of B bovis was maintained in bovine RBCs, and total RNA was extracted from the B bovis-infected bovine RBCs using Trizol (Sigma, St Louis, MO, USA) and used as a template for the subsequent one-step RT-RNA reaction (Takara, Tokyo, Japan) with the gene-specific primer The DNA fragment was subcloned into a pCR® 4-TOPO® plasmid (Invitrogen, CA, USA) and then sequenced using an automated sequencer (ABI PRISM 3100 Genetic Analyzer, USA) (B) SDS–PAGE and Western blot analysis of recombinant and native BbSBP-4 12% SDS–PAGE stained with Coomassie blue: recombinant BbSBP-4 (lane 1) and GST (lane 2) Western blot of recombinant protein: rBbSBP-4 (lane 3) and GST (lane 4) probed with B bovisinfected serum Western blotting of native BbSBP-4: B bovis-infected RBC lysate (lane 5); B bigemina-infected RBC lysate (lane 6); and normal bovine RBC (lane 7) probed with anti-rBbSBP-4 serum with MEX isolate (AF486506), 99.2% with field isolates from Brazil (AB569300), 99.1% from Mongolia (AB569302), 99% from Thailand (AB571871), 96.7% from Ghana (AB569301), and 96.4% from South Africa (AB569303) To study molecular characterizations of BbSBP-4, the entire DNA fragment encoding the BbSBP-4 gene (lacking the signal peptide sequence) was subcloned into a pGEX-4T-1 vector and expressed as a glutathione S-transferase fusion protein [12] with a molecular mass that was estimated to be 67-kDa in SDS–PAGE, including a 26kDa GST tag (Fig 1B, lane 1) The recombinant BbSBP-4 (rBbSBP-4) specifically reacted with the serum collected from experimentally infected cattle with B bovis (Fig 1B, lane 3) but not with that from cattle experimentally infected with B bigemina in Western blot analyses (data not shown), suggesting the specific antigenicity of rBbSBP-4 Thereafter, the anti-rBbSBP-4-specific immune serum [12] was used to identify the native protein in the B bovis lysate by Western blot analysis A single band corresponding to a 41-kDa native BbSBP-4 was detected in the B bovis lysate but not in the B bigemina lysate or normal bovine RBC (Fig 1A, lanes 5, 6, and 7, respectively) The detected size was consistent with the expected molecular weight of mature BbSBP-4 Since a localization study often helps interpret the functional role of a particular protein, the indirect immunofluorescent antibody test [15] and immunoelectron microscopy [16] were undertaken to determine the intracellular localization of the BbSBP-4 protein in B bovis merozoites using the anti-rBbSBP-4-specific immune serum Confocal Author's personal copy 42 M.A Terkawi et al / Molecular & Biochemical Parasitology 178 (2011) 40–45 Fig Cellular localization of BbSBP-4 (A) Confocal laser microscopic observation of BbSBP-4 in thin blood smears of B bovis-infected RBCs fixed with solution contained 95% methanol and 5% acetone and stained with specific antibodies (panels a–e) Anti-rBbSBP-4 serum (green) and nuclear staining of propidium iodide (red) Reactivity of anti-BbSBP-4 serum with free merozoite (a), ring stage (b), sequentially dividing forms (c), late-dividing forms (d), and rupturing form of infected erythrocytes (e) Panel (1) overlaid image of BbSBP-4 (green) and nuclear staining (red) and (2) overlaid image of BbSBP-4 (green) and nuclear staining (red) on phase-contrast images of the parasites (B) Immunoelectron microscopy of intraerythrocytic merozoites stained with anti-BbSBP-4 serum Gold particles were associated mainly with a spherical body located in the anterior half of the mature merozoite AC: apical complex; M: micronemes; R: rhoptries; S: spherical body; N: nucleus Scale bar = 0.1 m, Original magnification = ×120,000 laser microscopy of both extracellular and intracellular parasites demonstrated the cytoplasmic localization of BbSBP-4 through various developmental stages of the parasite (Fig 2A) By immunoelectron microscopic examination, gold particles were observed mainly in the spherical bodies of B bovis merozoites but not in the nucleus, rhoptries, and micronemes (Fig 2B) Infected erythrocytes incubated with control mouse IgG did not have any particles bound to them (data not shown) Moreover, the fluorescence appeared to be restricted within the parasite body during the early phase of single merozoites (Fig 2A, panel a), the ring-form stage (Fig 2A, panel b), and the early cell-division stage of parasites (Fig 2A, panel c) Interestingly, BbSBP-4 was looked to be discharged into the cytoplasm of infected RBC in the late stages of the parasites, as shown by the presence of fluorescent staining within the cytoplasm of infected RBC (Fig 2A, panels d and e) The secretion appeared to occur only during the late stages of parasite development and to increase gradually until the rupture of infected RBC These results indicated that BbSBP-4 localizes to spherical bodies of B bovis merozoites and discharges into the cytoplasm of infected RBC in a stage-dependent manner To further define the secretion of BbSBP4, co-localization studies were carried out with rabbit immune sera specific to rBbRAP-1 and rBbSBP-1 [12], known as secreted proteins within the cytoplasm of infected RBC While the secreted form of BbRAP-1 was shed into the infected RBC during or shortly after invasion (Fig 3A, panels a and b), BbSBP-4 appeared in the later stage of the dividing form (Fig 3A, panels d and e) At the early stage of the ring form, BbSBP-1 was detected within the cytoplasm of the parasite in a pattern overlapped by BbSBP-4 (Fig 3A, panel f), while, at a later stage, BbSBP-1 appeared to be uniformly distributed throughout the cytoplasm with high intensity near the membrane of infected RBC, persisting throughout the developmental stages of parasites (Fig 3A, panels g–j) Moreover, the overall percentage of fluorescence of the secreted form of each protein was determined in the blood smears The shed form of BbSBP-1 was detected in 93.9% of all infected RBC, while the percentages were 19.95% for BbSBP-4 and 9.81% for BbRAP-1 (Fig 3B) The presence of BbSBP-4 within the cytoplasm of infected RBC was puzzling as to whether this protein can be released into the supernatant of the B bovis culture Therefore, Western blotting of the supernatant of the culture with the anti-rBbSBP-4 serum was performed, and an apparent band of the 41-kDa protein corresponding to the native BbSBP-4 was observed Notably, the quantity of the detected protein in the supernatant of the culture harvested on the third day of culture seemed to be higher than that detected in the supernatant of the culture harvested on days and In contrast, BbRAP-1, known as a soluble culture-derived exoantigen [17], was detected in almost the same amount in the supernatant of the culture from days and (Fig 3C) These results were consistent with the observations made in the course of co-localization studies Furthermore, the effects of purified anti-rBbSBP-4 rabbit IgG on the growth of B bovis were examined in vitro [18] Test cultures were maintained with medium contained 0.5, 1, and mg/ml of anti-rBbSBP-4 IgG, while control Author's personal copy M.A Terkawi et al / Molecular & Biochemical Parasitology 178 (2011) 40–45 43 Fig Secretion nature of BbSBP-4 (A) Confocal laser microscopic observation of a co-immunostaining study of BbSBP-4 in thin blood smears of B bovis-infected RBC Co-immunostaining study of anti-rBbSBP-4 serum (green) with anti-rBbRAP-1 serum (red) (panels (a-e)) and with anti-BbSBP-1 serum (red) (panels (f-j)) during the developmental stages of parasites Reactivity of the anti-rBbSBP-4 serum and anti-rBbRAP-1CT serum with free merozoites during invasion (a), after invasion (b), in the ring stage (d), with sequentially dividing forms (d), and with the rupturing form (e) Reactivity of the anti-rBbSBP-4 serum and anti-rBbSBP-1 serum with the ring form (f and g), with the sequentially dividing form (h and i), and with the rupturing form of erythrocytes (j) Panel (1) overlaid image of BbSBP4 (green) and BbRAP-1 or BbSBP-1 (red) and (2) overlaid image of BbSBP-4 (green) and BbRAP-1 or BbSBP-1 (red) on phase-contrast images of the parasites Scale bar m (B) Percentage of the shed form of BbSBP-1, BbSBP-4, and RAP-1 in infected RBC Thin blood smears harvested from three independent experiments were fixed, stained with each antiserum, and then examined by confocal laser microscopy The shed form of each protein was calculated from the total infected RBC (C) Detection of BbRAP-1 and BbSBP-4 in the supernatant of the B bovis culture The supernatants were harvested every day after culturing, centrifuged at 40,000 × g for h at ◦ C, concentrated by acetone precipitation, and then subjected to SDS–PAGE and Western blotting The blots were probed with a specific antiserum to detect the secreted protein cultures were either with mg/ml anti-GST IgG or without IgG [15] All cultures demonstrated rapid growth of parasites, and there was no significant difference in the growth of parasites between controls and test cultures, although parasitemias in the culture contained mg/ml anti-rBbSBP-4 IgG showed tendency to be lower than these in controls In addition, no morphological changes on the parasites were observed over three days of culture (data not shown) Apicomplexan parasites, including Plasmodium spp and T gondii, release granule contents into the parasitophorous vacuole (PV) shortly after invasion, stabilizing the parasite environment and producing changes in the vacuole membrane which are postulated to affect nutrient acquisition [5,10] Babesia spp., unlike other Api- complexa, rapidly escapes from its vacuole after their invasion and proliferates freely in the cytoplasm of RBC, which allows it more direct access to host nutrients This adaptation requires an insensitive network and vesicle-like structures within the host cells that facilitate protein trafficking, nutrient uptake, and delivery between the parasites and RBC membrane compartment [19] However, the presence of spherical body proteins in the cytosol of infected RBCs during the life cycle of the merozoite suggests their crucial functions in the survival, growth, and development of parasites The increase in babesial proteins exporting into the cytosol and membrane is associated with alteration in the characteristics of infected RBC, including membrane permeability, rigidity, and deformability [20,21] It is most probable that the persistence of these structures Author's personal copy 44 M.A Terkawi et al / Molecular & Biochemical Parasitology 178 (2011) 40–45 within the cytoskeleton of infected RBC maintains the mechanical integrity and stability of the host cells during their intracellular replication, preventing RBC lysis and consequent parasite release The dramatic changes in the infected RBCs caused by the parasite-derived proteins are not only in their biochemical properties but also in the shape and morphology, as seen, for example, in the membrane protrusions (ridges) of B bovis-infected RBC [19] These structure alterations contribute to the sequestration of infected RBC in organ capillaries, where the variant erythrocyte surface antigen (VESA1) on the surface of the ridge structure serves as an RBCs ligand for cytoadhesion This phenomenon is important for the survival of the parasites, allowing the infected RBC to adhere to host endothelial cells of microcapillaries and subsequently escape from destruction in the spleen [19,22] Therefore, the potential function of BbSBPs might include the stabilization of the parasite environment, alteration of the membrane permeability required for nutrient acquisition, and formation of the ridge structure Differently from the previously reported BbSBPs, which were detected within the cytoplasmic face of the infected RBC membrane shortly after parasite invasion [7–9] BbSBP-4 appeared to be discharged into the cytoplasm of RBC at a later stage and to increase gradually until the rupture of RBCs This characteristic, coupled with the high conservation of BbSBP-4 among different isolates and the absence of homology in B bigemina, suggests a potential role in parasite virulence; probably, BbSBP-4 contributes to the formation of the ridge structure Ridge structures are completely absent from the surface of B bigemina-infected RBCs, a species closely related to B bovis that causes a much less severe disease with lower mortality [23] Elucidating the precise function of BbSBPs as a modulator of host RBCs is crucial for a better understanding of the virulence and pathogenesis of parasites and beneficial for developing new therapeutic strategies to combat this important disease Further study including investigation of the possibility of BbSBP-4 to interact with the vesicles of RBCs and other member of SBPs might be very necessary as step toward understanding of the function of this molecule The failure of anti-BbSBP-4 IgG to inhibit the growth of the parasites suggests that BbSBP-4 is not involved in invasion of B bovis into RBCs The inability of purified IgG to neutralize the free merozoite in vitro is most probably due to the inaccessibility of the antibody to BbSBP-4 found within the cytoplasm of free merozoites at early stage of invasion In contrast, antiserum to dense granule proteins of a variety of Apicomplexan parasites inhibits the growth of the parasites in vitro [10,24] These inhibitory effects appear to reflect interference of the antibodies with events occurring during or shortly after invasion Even in B bovis, several studies have shown the inhibitory effects of antiserum raised against molecules involved in the invasion process; such molecules are usually derived from cell membrane, micronemes or rhoptries [6] After the entry of the parasites inside the RBCs, the antibodies are less effective and the degeneration of parasites inside the erythrocyte is due to the interaction with the soluble mediators of cell-mediated immunity [25] However, the secretion nature of BbSBP-4 at the late stage of infection and its consequent release from RBCs might make this molecule more accessible to a host immune cells, this attribute is very important for subunit vaccine candidate and diagnostic antigen [26] A recent study has shown that BbSBP-4 is highly antigenic during the infection and has the best diagnostic performance in detection antibody to B bovis infection in cattle as compared to other antigens derived from cell membrane, micronemes or rhoptries [12] In summary, we have identified and characterized a unique 41kDa protein that has no significant identity to other Apicomplexan parasites and is highly conserved among field isolates from differently geographic regions The protein was localized to the spherical body of B bovis and was discharged into the cytoplasm of infected RBC at a later stage of parasite development These findings are expected to provide a basis for future work to exploit the function of spherical body protein in the development and survival of these parasites Acknowledgments This work was supported by a grant from the Global COE Program (J02) and a Grant-in-Aid for Scientific Research (A-22248035), both from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and a program for the Promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN), and a cooperative research grant (22-23 joint-6) from the National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine The first author was supported by a research grant fellowship from the Japanese Society for the Promotion of Science (JSPS) for young scientists The second, third and fourth authors were supported by Japan International Cooperation Agency (JICA) References [1] Kuttler KL Worldwide impact of babesiosis In: Ristic M, editor Babesiosis of domestic animals Boca Raton, FL, USA: CRC Press, Inc.; 1988 p 1–22 [2] McCosker PJ The global importance of babesiosis In: Ristic M, Kreier JP, editors Babesiosis New York: Academic Press; 1981 p 1–24 [3] Bock R, Jackson L, De Vos A, Jorgensen W Babesiosis of cattle Parasitology 2004;129:247–69 [4] Potgieter FT, Els HJ The fine structure of intra-erythrocytic stages of Babesia bovis merozoite surface-exposed epitopes Infect Immun 1977;59:3340–2 [5] Preiser P, Kaviratne M, Khan S, Bannister L, Jarra 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Identification and characterization of putative secreted antigens from Babesia microti J Clin Microbiol 2003;41(2):723–9 ... of a new spherical body protein of Babesia bovis into the cytoplasm of infected erythrocytes Mohamad Alaa Terkawi a , Faasoa Junior Seuseu a , Putut Eko-Wibowo a , Nguyen Xuan Huyen a , Yuka Minoda... Minoda a, Mahmoud AbouLaila a, Satoru Kawai b, Naoaki Yokoyama a, Xuenan Xuan a, Ikuo Igarashi a, ∗ a b National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary... structures: a signal peptide (1–22 aa) at the N-terminal, a faint domain and a glutamic acid-rich region (Glu-RP) at the C-terminal The Texas strain of B bovis was maintained in bovine RBCs, and total