Molecular Characterization and Immunological Evaluation of Truncated Babesia microti Rhoptry Neck Protein 2 as a Vaccine Candidate

Babesia microtiis 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 inB. microtiparasites.In vitroexperiments were carried out to investigate the role of BmRON2 proteins during theB. microtiinvasion process andin vivoexperiments to investigate immunoprotection. Homologous sequence alignment and molecular phylogenetic analysis indicated that BmRON2 showed similarities with RON2 proteins of otherBabesiaspecies. 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 withB. microti.Immunofluorescence analysis indicated that the BmRON2 protein was located at the apical end of merozoites, at the opposite end of the nucleus.In vitrored blood cell invasion inhibition studies withB. microtirBmRON2 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 andB. microtiinfection 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.

Detection of the Rhoptry Neck Protein Complex in Plasmodium Sporozoites and Its Contribution to Sporozoite Invasion of Salivary Glands

ABSTRACT In the Plasmodium life cycle, two infectious stages of parasites, merozoites and sporozoites, share rhoptry and microneme apical structures. A crucial step during merozoite invasion of erythrocytes is the discharge to the host cell membrane of some rhoptry neck proteins as a complex, followed by the formation of a moving junction involving the parasite-secreted protein AMA1 on the parasite membrane. Components of the merozoite rhoptry neck protein complex are also expressed in sporozoites, namely, RON2, RON4, and RON5, suggesting that invasion mechanism elements might be conserved between these infective stages. Recently, we demonstrated that RON2 is required for sporozoite invasion of mosquito salivary gland cells and mammalian hepatocytes, using a sporozoite stage-specific gene knockdown strategy in the rodent malaria parasite model, Plasmodium berghei. Here, we use a coimmunoprecipitation assay and oocyst-derived sporozoite extracts to demonstrate that RON2, RON4, and RON5 also form a complex in sporozoites. The sporozoite stage-specific gene knockdown strategy revealed that both RON4 and RON5 have crucial roles during sporozoite invasion of salivary glands, including a significantly reduced attachment ability required for the onset of gliding. Further analyses indicated that RON2 and RON4 reciprocally affect trafficking to rhoptries in developing sporozoites, while RON5 is independently transported. These findings indicate that the interaction between RON2 and RON4 contributes to their stability and trafficking to rhoptries, in addition to involvement in sporozoite attachment. IMPORTANCE Sporozoites are the motile infectious stage that mediates malaria parasite transmission from mosquitoes to the mammalian host. This study addresses the question whether the rhoptry neck protein complex forms and functions in sporozoites, in addition to its role in merozoites. By applying coimmunoprecipitation and sporozoite stage-specific gene knockdown assays, it was demonstrated that RON2, RON4, and RON5 form a complex and are involved in sporozoite invasion of salivary glands via their attachment ability. These findings shed light on the conserved invasion mechanisms among apicomplexan infective stages. In addition, the sporozoite stage-specific gene knockdown system has revealed for the first time in Plasmodium that the RON2 and RON4 interaction reciprocally affects their stability and trafficking to rhoptries. Our study raises the possibility that the RON complex functions during sporozoite maturation as well as migration toward and invasion of target cells.