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Identification and Characterization of the Rhoptry Neck Protein 2 in Babesia divergens and B. microti.

ABSTRACT: Apicomplexan parasites include those of the genera Plasmodium, Cryptosporidium, and Toxoplasma and those of the relatively understudied zoonotic genus Babesia In humans, babesiosis, particularly transfusion-transmitted babesiosis, has been emerging as a major threat to public health. Like malaria, the disease pathology is a consequence of the parasitemia which develops through cyclical replication of Babesia parasites in host erythrocytes. However, there are no exoerythrocytic stages in Babesia, so targeting of the blood stage and associated proteins to directly prevent parasite invasion is the most desirable option for effective disease control. Especially promising among such molecules are the rhoptry neck proteins (RONs), whose homologs have been identified in many apicomplexan parasites. RONs are involved in the formation of the moving junction, along with AMA1, but no RON has been identified and characterized in any Babesia spp. Here we identify the RON2 proteins of Babesia divergens (BdRON2) and B. microti (BmRON2) and show that they are localized apically and that anti-BdRON2 antibodies are significant inhibitors of parasite invasion in vitro Neither protein is immunodominant, as both proteins react only marginally with sera from infected animals. Further characterization of the direct role of both BdRON2 and BmRON2 in parasite invasion is required, but knowledge of the level of conformity of RON2 proteins within the apicomplexan phylum, particularly that of the AMA1-RON2 complex at the moving junction, along with the availability of an animal model for B. microti studies, provides a key to target this complex with a goal of preventing the erythrocytic invasion of these parasites and to further our understanding of the role of these conserved ligands in invasion.

SUBMITTER: Ord RL

PROVIDER: S-EPMC4862700 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Identification and Characterization of the Rhoptry Neck Protein 2 in Babesia divergens and B. microti.

Ord Rosalynn L RL Rodriguez Marilis M Cursino-Santos Jeny R JR Hong Hyunryung H Singh Manpreet M Gray Jeremy J Lobo Cheryl A CA

Infection and immunity 20160422 5

Apicomplexan parasites include those of the genera Plasmodium, Cryptosporidium, and Toxoplasma and those of the relatively understudied zoonotic genus Babesia In humans, babesiosis, particularly transfusion-transmitted babesiosis, has been emerging as a major threat to public health. Like malaria, the disease pathology is a consequence of the parasitemia which develops through cyclical replication of Babesia parasites in host erythrocytes. However, there are no exoerythrocytic stages in Babesia, ...[more]

PMID: 26953328

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Project description:Babesia microti is a protozoan that infects red blood cells. Babesiosis is becoming a new global threat impacting human health. Rhoptry neck proteins (RONs) are proteins located at the neck of the rhoptry and studies indicate that these proteins play an important role in the process of red blood cell invasion. In the present study, we report on the bioinformatic analysis, cloning, and recombinant gene expression of two truncated rhoptry neck proteins 2 (BmRON2), as well as their potential for incorporation in a candidate vaccine for babesiosis. Western blot and immunofluorescence antibody (IFA) assays were performed to detect the presence of specific antibodies against BmRON2 in infected mice and the localization of N-BmRON2 in B. microti parasites. In vitro experiments were carried out to investigate the role of BmRON2 proteins during the B. microti invasion process and in vivo experiments to investigate immunoprotection. Homologous sequence alignment and molecular phylogenetic analysis indicated that BmRON2 showed similarities with RON2 proteins of other Babesia species. We expressed the truncated N-terminal (33-336 aa, designated rN-BmRON2) and C-terminal (915-1171 aa, designated rC-BmRON2) fragments of the BmRON2 protein, with molecular weights of 70 and 29 kDa, respectively. Western blot assays showed that the native BmRON2 protein is approximately 170 kDa, and that rN-BmRON2 was recognized by serum of mice experimentally infected with B. microti. Immunofluorescence analysis indicated that the BmRON2 protein was located at the apical end of merozoites, at the opposite end of the nucleus. In vitro red blood cell invasion inhibition studies with B. microti rBmRON2 proteins showed that relative invasion rate of rN-BmRON2 and rC-BmRON2 group is 45 and 56%, respectively. Analysis of the host immune response after immunization and B. microti infection showed that both rN-BmRON2 and rC-BmRON2 enhanced the immune response, but that rN-BmRON2 conferred better protection than rC-BmRON2. In conclusion, our results indicate that truncated rhoptry neck protein 2, especially its N-terminal fragment (rN-BmRON2), plays an important role in the invasion of host red blood cells, confers immune protection, and shows good potential as a candidate vaccine against babesiosis.

Project description:Babesiosis is a zoonosis and an important blood-borne human parasitic infection that has gained attention because of its growing infection rate in humans by transfer from animal reservoirs. Babesia represents a potential threat to the blood supply because asymptomatic infections in man are common, and blood from such donors can cause severe disease in certain recipients. Extracellular vesicles (EVs) are vesicles released by cells that contain a complex mixture of proteins, lipids, glycans, and genetic information that have been shown to play important roles in disease pathogenesis and susceptibility, as well as cell-cell communication and immune responses. In this article, we report on the identification and characterization of EVs released from red blood cells (RBCs) infected by two major human Babesia species-Babesia divergens from in vitro culture and those from an in vivo B. microti mouse infection. Using nanoparticle tracking analysis, we show that there is a range of vesicle sizes from 30 to 1,000 nm, emanating from the Babesia-infected RBC. The study of these EVs in the context of hemoparasite infection is complicated by the fact that both the parasite and the host RBC make and release vesicles into the extracellular environment. However, the EV frequency is 2- to 10-fold higher in Babesia-infected RBCs than uninfected RBCs, depending on levels of parasitemia. Using parasite-specific markers, we were able to show that ~50%-60% of all EVs contained parasite-specific markers on their surface and thus may represent the specific proportion of EVs released by infected RBCs within the EV population. Western blot analysis on purified EVs from both in vivo and in vitro infections revealed several parasite proteins that were targets of the host immune response. In addition, microRNA analysis showed that infected RBC EVs have different microRNA signature from uninfected RBC EVs, indicating a potential role as disease biomarkers. Finally, EVs were internalized by other RBCs in culture, implicating a potential role for these vesicles in cellular communication. Overall, our study points to the multiple functional implications of EVs in Babesia-host interactions and support the potential that EVs have as agents in disease pathogenesis.

Project description:BACKGROUND: Plasmodium vivax malaria remains a major health problem in tropical and sub-tropical regions worldwide. Several rhoptry proteins which are important for interaction with and/or invasion of red blood cells, such as PfRONs, Pf92, Pf38, Pf12 and Pf34, have been described during the last few years and are being considered as potential anti-malarial vaccine candidates. This study describes the identification and characterization of the P. vivax rhoptry neck protein 1 (PvRON1) and examine its antigenicity in natural P. vivax infections. METHODS: The PvRON1 encoding gene, which is homologous to that encoding the P. falciparum apical sushi protein (ASP) according to the plasmoDB database, was selected as our study target. The pvron1 gene transcription was evaluated by RT-PCR using RNA obtained from the P. vivax VCG-1 strain. Two peptides derived from the deduced P. vivax Sal-I PvRON1 sequence were synthesized and inoculated in rabbits for obtaining anti-PvRON1 antibodies which were used to confirm the protein expression in VCG-1 strain schizonts along with its association with detergent-resistant microdomains (DRMs) by Western blot, and its localization by immunofluorescence assays. The antigenicity of the PvRON1 protein was assessed using human sera from individuals previously exposed to P. vivax malaria by ELISA. RESULTS: In the P. vivax VCG-1 strain, RON1 is a 764 amino acid-long protein. In silico analysis has revealed that PvRON1 shares essential characteristics with different antigens involved in invasion, such as the presence of a secretory signal, a GPI-anchor sequence and a putative sushi domain. The PvRON1 protein is expressed in parasite's schizont stage, localized in rhoptry necks and it is associated with DRMs. Recombinant protein recognition by human sera indicates that this antigen can trigger an immune response during a natural infection with P. vivax. CONCLUSIONS: This study shows the identification and characterization of the P. vivax rhoptry neck protein 1 in the VCG-1 strain. Taking into account that PvRON1 shares several important characteristics with other Plasmodium antigens that play a functional role during RBC invasion and, as shown here, it is antigenic, it could be considered as a good vaccine candidate. Further studies aimed at assessing its immunogenicity and protection-inducing ability in the Aotus monkey model are thus recommended.

Dataset Information

Identification and Characterization of the Rhoptry Neck Protein 2 in Babesia divergens and B. microti.

Publications

Identification and Characterization of the Rhoptry Neck Protein 2 in Babesia divergens and B. microti.

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