Microneme Protein Research Articles

Immunomolecular Characterization of MIC-1, a Novel Antigen in Babesia bigemina, Which Contains Conserved and Immunodominant B-Cell Epitopes that Induce Neutralizing Antibodies.

Babesia bigemina is one of the most prevalent species causing bovine babesiosis around the world. Antigens involved in host cell invasion are vaccine targets for this disease but are largely unknown in this species. The invasion process of Babesia spp. into erythrocytes involves membrane proteins from the apical complex. A protein stored in the micronemes, called Micronemal Protein 1 (MIC-1), contains a sialic acid binding domain that participates in the invasion process of host cells and is a vaccine candidate in other apicomplexan parasites. It is not known if there is a homologous gene for mic-1 in B. bigemina. Therefore, the aim of this study was to characterize the mic-1 gene homologue in Babesia bigemina. A gene was found with a microneme adhesive repeat (MAR) domain in the predicted amino acid sequence. Transcription was determined by reverse transcription polymerase chain reaction (RT-PCR). Subsequently, antibodies against peptides containing conserved B-cell epitopes were used to confirm the expression of MIC-1 in intraerythrocytic merozoites. The presence of anti MIC-1 antibodies in cattle naturally infected with B. bigemina was determined and up to 97.4% of the cattle sera (113 out of 116) identified MIC-1 using enzyme-linked immunosorbent assay (ELISA) methods. Finally, antibodies against MIC-1 were able to block 70% merozoite invasion in-vitro.

Modeling and resistant alleles explain the selectivity of antimalarial compound 49c towards apicomplexan aspartyl proteases.

Toxoplasma gondii aspartyl protease 3 (TgASP3) phylogenetically clusters with Plasmodium falciparum Plasmepsins IX and X (PfPMIX, PfPMX). These proteases are essential for parasite survival, acting as key maturases for secreted proteins implicated in invasion and egress. A potent antimalarial peptidomimetic inhibitor (49c) originally developed against Plasmepsin II selectively targets TgASP3, PfPMIX, and PfPMX To unravel the molecular basis for the selectivity of 49c, we constructed homology models of PfPMIX, PfPMX, and TgASP3 that were first validated by identifying the determinants of microneme and rhoptry substrate recognition. The flap and flap-like structures of several reported Plasmepsins are highly flexible and critically modulate the access to the binding cavity. Molecular docking of 49c to TgASP3, PfPMIX, and PfPMX models predicted that the conserved phenylalanine residues in the flap, F344, F291, and F305, respectively, account for the sensitivity toward 49c. Concordantly, phenylalanine mutations in the flap of the three proteases increase twofold to 15-fold the IC50 values of 49c. Compellingly the selection of mutagenized T.gondii resistant strains to 49c reproducibly converted F344 to a cysteine residue.

Apicomplexan C-Mannosyltransferases Modify Thrombospondin Type I-containing Adhesins of the TRAP Family.

In many metazoan species, an unusual type of protein glycosylation, called C-mannosylation, occurs on adhesive thrombospondin type 1 repeats (TSRs) and type I cytokine receptors. This modification has been shown to be catalyzed by the Caenorhabditis elegans DPY-19 protein and orthologues of the encoding gene were found in the genome of apicomplexan parasites. Lately, the micronemal adhesin thrombospondin-related anonymous protein (TRAP) was shown to be C-hexosylated in Plasmodium falciparum sporozoites. Here, we demonstrate that also the micronemal protein MIC2 secreted by Toxoplasma gondii tachyzoites is C-hexosylated. When expressed in a mammalian cell line deficient in C-mannosylation, P. falciparum and T. gondii Dpy19 homologs were able to modify TSR domains of the micronemal adhesins TRAP/MIC2 family involved in parasite motility and invasion. In vitro, the apicomplexan enzymes can transfer mannose to a WXXWXXC peptide but, in contrast to C. elegans or mammalian C-mannosyltransferases, are inactive on a short WXXW peptide. Since TSR domains are commonly found in apicomplexan surface proteins, C-mannosylation may be a common modification in this phylum.

Recent progress in microneme-based vaccines development against Toxoplasma gondii.

Toxoplasmosis is a cosmopolitan zoonotic disease, which infect several warm-blooded mammals. More than one-third of the human population are seropositive worldwide. Due to the high seroprevalence of Toxoplasma gondii infection worldwide, the resulting clinical, mental, and economical complications, as well as incapability of current drugs in the elimination of parasites within tissue cysts, the development of a vaccine against T. gondii would be critical. In the past decades, valuable advances have been achieved in order to identification of vaccine candidates against T. gondii infection. Microneme proteins (MICs) secreted by the micronemes play a critical role in the initial stages of host cell invasion by parasites. In this review, we have summarized the recent progress for MIC-based vaccines development, such as DNA vaccines, recombinant protein vaccines, vaccines based on live-attenuated vectors, and prime-boost strategy in different mouse models. In conclusion, the use of live-attenuated vectors as vehicles to deliver and express the target gene and prime-boost regimens showed excellent outcomes in the development of vaccines against toxoplasmosis, which need more attention in the future studies.

Molecular characterization and protective efficacy of the microneme 2 protein from Eimeria tenella.

Microneme proteins play an important role in the adherence of apicomplexan parasites to host cells during the invasion process. In this study, the microneme 2 protein from the protozoan parasite Eimeria tenella (EtMIC2) was cloned, characterized, and its protective efficacy as a DNA vaccine investigated. The EtMIC2 gene, which codes for a 35.07 kDa protein in E. tenella sporulated oocysts, was cloned and recombinant EtMIC2 protein (rEtMIC2) was produced in an Escherichia coli expression system. Immunostaining with an anti-rEtMIC2 antibody showed that the EtMIC2 protein mainly localized in the anterior region and membrane of sporozoites, in the cytoplasm of first- and second-generation merozoites, and was strongly expressed during first-stage schizogony. In addition, incubation with specific antibodies against EtMIC2 was found to efficiently reduce the ability of E. tenella sporozoites to invade host cells. Furthermore, animal-challenge experiments demonstrated that immunization with pcDNA3.1(+)-EtMIC2 significantly increased average body weight gain, while decreasing the mean lesion score and oocyst output in chickens. Taken together, these results suggest that EtMIC2 plays an important role in parasite cell invasion and may be a viable candidate for the development of new vaccines against E. tenella infection in chickens.

Design of inhibitory peptide targeting Toxoplasma gondii RON4‐human β‐tubulin interactions by implementing structural bioinformatics methods

Toxoplasma gondii an obligate intracellular parasite causes toxoplasmosis in homeothermic animals. Host invasion of this parasite is mediated by the formation of Moving Junction (MJ) complex which encompasses a network of microneme and Rhoptry Neck proteins (RONs) 2/4/5/8. Among these proteins, RON4 is the only cytosolic secretory protein that is considered as a crucial member, as it directly facilitates the motility of MJ complex by interacting with host tubulin. It is also prominently localized at the host-pathogen interface during the invasion, thus projecting it as a potential drug target. The structure of RON4 is yet to be crystallized. Hence, in this study, fold recognition and Free Energy Landscape sampling was performed to predict the plausible 3D structure of RON4. Further, its interacting pattern with the reported crystal structure of human tubulin was analyzed using molecular docking. Subsequently, a β-tubulin based inhibitory peptides were derived based on its interacting interface observed in RON4-β-tubulin docked complex. Following which, a stepwise validation of these peptides for various physico-chemical properties and its homology with antimicrobial peptides were also screened. The peptide (RT_pep) surpassing all these validation filters was modeled and its stability was analysed by Molecular Dynamics simulation. To validate further, the stable conformation of the RT_pep was docked to RON4. Finally, essential molecular dynamics simulation was conducted to determine the stability and atomic motions of native RON4 and also to decipher its association with β-tubulin and RT_pep. All these analyses cumulatively suggest the therapeutic potential of RT_pep in targeting toxoplasmosis.

RON4L1 is a new member of the moving junction complex in Toxoplasma gondii

Apicomplexa parasites, including Toxoplasma and Plasmodium species, possess a unique invasion mechanism that involves a tight apposition between the parasite and the host plasma membranes, called “moving junction” (MJ). The MJ is formed by the assembly of the microneme protein AMA1, exposed at the surface of the parasite, and the parasite rhoptry neck (RON) protein RON2, exposed at the surface of the host cell. In the host cell, RON2 is associated with three additional parasite RON proteins, RON4, RON5 and RON8. Here we describe RON4L1, an additional member of the MJ complex in Toxoplasma. RON4L1 displays some sequence similarity with RON4 and is cleaved at the C-terminal end before reaching the rhoptry neck. Upon secretion during invasion, RON4L1 is associated with MJ and targeted to the cytosolic face of the host membrane. We generated a RON4L1 knock-out cell line and showed that it is not essential for the lytic cycle in vitro, although mutant parasites kill mice less efficiently. Similarly to RON8, RON4L1 is a coccidian-specific protein and its traffic to the MJ is not affected in absence of RON2, RON4 and RON5, suggesting the co-existence of independent MJ complexes in tachyzoite of Toxoplasma.

Integrative transcriptome and proteome analyses define marked differences between Neospora caninum isolates throughout the tachyzoite lytic cycle

Neospora caninum is one of the main causes of transmissible abortion in cattle. Intraspecific variations in virulence have been widely shown among N. caninum isolates. However, the molecular basis governing such variability have not been elucidated to date. In this study label free LC-MS/MS was used to investigate proteome differences between the high virulence isolate Nc-Spain7 and the low virulence isolate Nc-Spain1H throughout the tachyzoite lytic cycle. The results showed greater differences in the abundance of proteins at invasion and egress with 77 and 62 proteins, respectively. During parasite replication, only 19 proteins were differentially abundant between isolates. The microneme protein repertoire involved in parasite invasion and egress was more abundant in the Nc-Spain1H isolate, which displays a lower invasion rate. Rhoptry and dense granule proteins, proteins related to metabolism and stress responses also showed differential abundances between isolates. Comparative RNA-Seq analyses during tachyzoite egress were also performed, revealing an expression profile of genes associated with the bradyzoite stage in the low virulence Nc-Spain1H isolate. The differences in proteome and RNA expression profiles between these two isolates reveal interesting insights into likely mechanisms involved in specific phenotypic traits and virulence in N. caninum. SignificanceThe molecular basis that governs biological variability in N. caninum and the pathogenesis of neosporosis has not been well-established yet. This is the first study in which high throughput technology of LC-MS/MS and RNA-Seq is used to investigate differences in the proteome and transcriptome between two well-characterized isolates. Both isolates displayed different proteomes throughout the lytic cycle and the transcriptomes also showed marked variations but were inconsistent with the proteome results. However, both datasets identified a pre-bradyzoite status of the low virulence isolate Nc-Spain1H. This study reveals interesting insights into likely mechanisms involved in virulence in N. caninum and shed light on a subset of proteins that are potentially involved in the pathogenesis of this parasite.

Characterization of novel microneme adhesive repeats (MAR) in Eimeria tenella

BackgroundThe phylum Apicomplexa comprises a wide variety of parasites of significant medical and economic relevance. These parasites have extremely different host and tissue tropisms; for example Toxoplasma gondii can invade virtually any nucleated cell and infect almost all warm-blooded vertebrates, whereas Eimeria tenella infects only chickens and is restricted in its growth to epithelial cells of the caecum. Proteins released from the microneme secretory organelles (MICs) are critical for apicomplexan invasion of host cells and allow parasites to bind a diverse range of host cell oligosaccharide epitopes. MICs bear modular arrangements of sequences with adhesive proteins and interestingly the sialic-acid binding MAR (microneme adhesive repeat) domain containing proteins (MCPs) are suggested to make significant contributions to the different host and tissue tropisms of T. gondii and E. tenella.ResultsIn this study, we evaluated the binding capacity of Type I MAR domains from novel E. tenella MCPs. Variants of the previously described HxT motif were analysed showing that HxT and VxT variants bind, whereas HxS and YxE variants did not. One of these MCP containing a single MAR (EtMCP2) showed an apical localization when expressed as a fusion with the fluorescent reporter mCherry in transgenic populations and a similar pattern of transcripts per zoite during endogenous development in vitro as the well-characterised microneme protein EtMIC2.ConclusionsVariation in the binding properties of the MAR of different EtMCPs was confirmed and their ability to bind a wider range of sialic acids and terminal linkages should be studied. In addition, transgenesis technology has been used for first time in Eimeria parasites as a rapid tool for the study of endogenous protein localization by fusion with a fluorescent reporter.

A druggable secretory protein maturase of Toxoplasma essential for invasion and egress.

Micronemes and rhoptries are specialized secretory organelles that deploy their contents at the apical tip of apicomplexan parasites in a regulated manner. The secretory proteins participate in motility, invasion, and egress and are subjected to proteolytic maturation prior to organellar storage and discharge. Here we establish that Toxoplasma gondii aspartyl protease 3 (ASP3) resides in the endosomal-like compartment and is crucially associated to rhoptry discharge during invasion and to host cell plasma membrane lysis during egress. A comparison of the N-terminome, by terminal amine isotopic labelling of substrates between wild type and ASP3 depleted parasites identified microneme and rhoptry proteins as repertoire of ASP3 substrates. The role of ASP3 as a maturase for previously described and newly identified secretory proteins is confirmed in vivo and in vitro. An antimalarial compound based on a hydroxyethylamine scaffold interrupts the lytic cycle of T. gondii at submicromolar concentration by targeting ASP3.

Transcriptome modulation of bovine trophoblast cells in vitro by Neospora caninum

Neospora caninum is one of the most efficient transplacentally transmitted pathogens in cattle and is a cause of abortion in this domestic species. The invasion and proliferation of Neospora caninum in the placenta and its dissemination to the foetus are crucial events in the outcome of an infection. In the bovine placenta, the placentomes are formed by maternal caruncles, which are delimited by a maternal epithelium and foetal cotyledons, which are delimited by an epithelial layer named the trophoblast. These epithelia form a physical barrier against foetal infection. Furthermore, trophoblast cells act as an innate immune defence at the foetal-maternal interface. Neospora caninum invades and proliferates in trophoblast cells in vitro, but it is unknown whether host cell modulation events, which affect the immune response and other processes in the trophoblast, occur. In this work, we investigated the transcriptomic modulation by Neospora caninum infection in the bovine trophoblast cell line F3. In addition, two Neospora caninum isolates with marked differences in virulence, Nc-Spain1H and the Nc-Spain7, were used in this study to investigate the influence of these isolates in F3 modulation. The results showed a clear influence on extracellular matrix reorganisation, cholesterol biosynthesis and the transcription factor AP-1 network. Interestingly, although differences in the transcriptome profiles induced by each isolate were observed, specific isolate-modulated processes were not identified, suggesting very similar regulation in both isolates. Differential expression of the N. caninum genes between both isolates was also investigated. Genes involved in host cell attachment and invasion (SAG-related and microneme proteins), glideosome, rhoptries, metabolic processes, cell cycle and stress response were differentially expressed between the isolates, which could explain their variability. This study provides a global view of Neospora caninum interactions with bovine trophoblast cells and of the intra-specific differences between two Neospora caninum isolates with biological differences.

Resistance towards monensin is proposed to be acquired in a Toxoplasma gondii model by reduced invasion and egress activities, in addition to increased intracellular replication.

Monensin (Mon) is an anticoccidial polyether ionophore widely used to control coccidiosis. The extensive use of polyether ionophores on poultry farms resulted in widespread resistance, but the underlying resistance mechanisms are unknown in detail. For analysing the mode of action by which resistance against polyether ionophores is obtained, we induced in vitro Mon resistance in Toxoplasma gondii-RH strain (MonR-RH) and compared it with the sensitive parental strain (Sen-RH). The proteome assessment of MonR-RH and Sen-RH strains was obtained after isotopic labelling using stable isotope labelling by amino acid in cell culture. Relative proteomic quantification between resistant and sensitive strains was performed using liquid chromatography-mass spectrometry/mass spectrometry. Overall, 1024 proteins were quantified and 52 proteins of them were regulated. The bioinformatic analysis revealed regulation of cytoskeletal and transmembrane proteins being involved in transport mechanisms, metal ion-binding and invasion. During invasion, actin and microneme protein 8 (MIC8) are seem to be important for conoid extrusion and forming moving junction with host cells, respectively. Actin was significantly upregulated, while MIC8 was downregulated, which indicate an invasion reduction in the resistant strain. Resistance against Mon is not a simple process but it involves reduced invasion and egress activity of T. gondii tachyzoites while intracellular replication is enhanced.

Recent buzz in malaria research.

In this Commentary, we highlight the latest findings in three active areas of malaria research: Plasmodium biology; host response; and malaria control, prevention and treatment.

Expression of codon-optimized TgMIC16 in three Escherichia coli strains.

In a previous study, we found that rabbit anti-Toxoplasma gondii serum was capable of recognizing truncated T. gondii microneme protein 16 (TgMIC16), indicating that TgMIC16 is an essential antigenic T. gondii protein. However, the broad application of this recombinant protein is limited by its low expression level. In this study, we performed codon optimization of TgMIC16 by changing the codon-adaptation index from 0.22 to 1.0 without altering the amino acid sequence and expressed the optimized gene in three different Escherichia coli strains, followed by comparison of soluble recombinant-protein expression and yield. Our results showed that the recombinant protein rTgMIC16 was expressed as inclusion bodies in all three strains following optimization of induction parameters, and western blot analysis revealed the presence of a ~72-kD recombinant protein as a specific band following purification. A shuffle-expression strain was selected to amplify incubation products and induce expression, resulting in an overall rTgMIC16 yield of ~20mg/L. These findings provide a basis for further investigation of TgMIC16 to elucidate its functions and interaction partners.

Parasites lacking the micronemal protein MIC2 are deficient in surface attachment and host cell egress, but remain virulent in vivo.

Background: Micronemal proteins of the thrombospondin-related anonymous protein (TRAP) family are believed to play essential roles during gliding motility and host cell invasion by apicomplexan parasites, and currently represent major vaccine candidates against Plasmodium falciparum, the causative agent of malaria. However, recent evidence suggests that they play multiple and different roles than previously assumed. Here, we analyse a null mutant for MIC2, the TRAP homolog in Toxoplasma gondii. Methods: We performed a careful analysis of parasite motility in a 3D-environment, attachment under shear stress conditions, host cell invasion and in vivo virulence. Results: We verified the role of MIC2 in efficient surface attachment, but were unable to identify any direct function of MIC2 in sustaining gliding motility or host cell invasion once initiated. Furthermore, we find that deletion of mic2 causes a slightly delayed infection in vivo, leading only to mild attenuation of virulence; like with wildtype parasites, inoculation with even low numbers of mic2 KO parasites causes lethal disease in mice. However, deletion of mic2 causes delayed host cell egress in vitro, possibly via disrupted signal transduction pathways. Conclusions: We confirm a critical role of MIC2 in parasite attachment to the surface, leading to reduced parasite motility and host cell invasion. However, MIC2 appears to not be critical for gliding motility or host cell invasion, since parasite speed during these processes is unaffected. Furthermore, deletion of MIC2 leads only to slight attenuation of the parasite.

Parasites lacking the micronemal protein MIC2 are deficient in surface attachment and host cell egress, but remain virulent in vivo

Background: Micronemal proteins of the thrombospondin-related anonymous protein (TRAP) family are believed to play essential roles during gliding motility and host cell invasion by apicomplexan parasites, and currently represent major vaccine candidates against Plasmodium falciparum, the causative agent of malaria. However, recent evidence suggests that they play multiple and different roles than previously assumed. Here, we analyse a null mutant for MIC2, the TRAP homolog in Toxoplasma gondii. Methods: We performed a careful analysis of parasite motility in a 3D-environment, attachment under shear stress conditions, host cell invasion and in vivo virulence. Results: We verified the role of MIC2 in efficient surface attachment, but were unable to identify any direct function of MIC2 in sustaining gliding motility or host cell invasion once initiated. Furthermore, we find that deletion of mic2 causes a slightly delayed infection in vivo, leading only to mild attenuation of virulence; like with wildtype parasites, inoculation with even low numbers of mic2 KO parasites causes lethal disease in mice. However, deletion of mic2 causes delayed host cell egress in vitro, possibly via disrupted signal transduction pathways. Conclusions: We confirm a critical role of MIC2 in parasite attachment to the surface, leading to reduced parasite motility and host cell invasion. However, MIC2 appears to not be critical for gliding motility or host cell invasion, since parasite speed during these processes is unaffected. Furthermore, deletion of MIC2 leads only to slight attenuation of the parasite.

Eimeria tenella protein trafficking: differential regulation of secretion versus surface tethering during the life cycle

Eimeria spp. are intracellular parasites that have a major impact on poultry. Effective live vaccines are available and the development of reverse genetic technologies has raised the prospect of using Eimeria spp. as recombinant vectors to express additional immunoprotective antigens. To study the ability of Eimeria to secrete foreign antigens or display them on the surface of the sporozoite, transiently transfected populations of E. tenella expressing the fluorescent protein mCherry, linked to endogenous signal peptide (SP) and glycophosphatidylinositol-anchor (GPI) sequences, were examined. The SP from microneme protein EtMIC2 (SP2) allowed efficient trafficking of mCherry to cytoplasmic vesicles and following the C-terminal addition of a GPI-anchor (from surface antigen EtSAG1) mCherry was expressed on the sporozoite surface. In stable transgenic populations, mCherry fused to SP2 was secreted into the sporocyst cavity of the oocysts and after excystation, secretion was detected in culture supernatants but not into the parasitophorous vacuole after invasion. When the GPI was incorporated, mCherry was observed on the sporozites surface and in the supernatant of invading sporozoites. The proven secretion and surface exposure of mCherry suggests that antigen fusions with SP2 and GPI of EtSAG1 may be promising candidates to examine induction of protective immunity against heterologous pathogens.

Parasites lacking the micronemal protein MIC2 are deficient in surface attachment and host cell egress, but remain virulent in vivo

Background: Micronemal proteins of the thrombospondin-related anonymous protein (TRAP) family are believed to play essential roles during gliding motility and host cell invasion by apicomplexan parasites, and currently represent major vaccine candidates against Plasmodium falciparum, the causative agent of malaria. However, recent evidence suggests that they play multiple and different roles than previously assumed. Here, we analyse a null mutant for MIC2, the TRAP homolog in Toxoplasma gondii. Methods: We performed a careful analysis of parasite motility in a 3D-environment, attachment under shear stress conditions, host cell invasion and in vivo virulence. Results: We verified the role of MIC2 in efficient surface attachment, but were unable to identify any direct function of MIC2 in sustaining gliding motility or host cell invasion once initiated. Furthermore, we find that deletion of mic2 causes a slightly delayed infection in vivo, leading only to mild attenuation of virulence; like with wildtype parasites, inoculation with even low numbers of mic2 KO parasites causes lethal disease in mice. However, deletion of mic2 causes delayed host cell egress in vitro, possibly via disrupted signal transduction pathways. Conclusions: We confirm a critical role of MIC2 in parasite attachment to the surface, leading to reduced parasite motility and host cell invasion. However, MIC2 appears to not be critical for gliding motility or host cell invasion, since parasite speed during these processes is unaffected. Furthermore, deletion of MIC2 leads only to slight attenuation of the parasite.

Dual role of the Toxoplasma gondii clathrin adaptor AP1 in the sorting of rhoptry and microneme proteins and in parasite division.

Toxoplasma gondii possesses a highly polarized secretory system, which efficiently assembles de novo micronemes and rhoptries during parasite replication. These apical secretory organelles release their contents into host cells promoting parasite invasion and survival. Using a CreLox-based inducible knock-out strategy and the ddFKBP over-expression system, we unraveled novel functions of the clathrin adaptor complex TgAP1. First, our data indicate that AP1 in T. gondii likely functions as a conserved heterotetrameric complex composed of the four subunits γ, β, μ1, σ1 and interacts with known regulators of clathrin-mediated vesicular budding such as the unique ENTH-domain containing protein, which we named Epsin-like protein (TgEpsL). Disruption of the μ1 subunit resulted in the mis-sorting of microneme proteins at the level of the Trans-Golgi-Network (TGN). Furthermore, we demonstrated that TgAP1 regulates rhoptry biogenesis by activating rhoptry protein exit from the TGN, but also participates in the post-Golgi maturation process of preROP compartments into apically anchored club-shaped mature organelles. For this latter activity, our data indicate a specific functional relationship between TgAP1 and the Rab5A-positive endosome-like compartment. In addition, we unraveled an original role for TgAP1 in the regulation of parasite division. APμ1-depleted parasites undergo normal daughter cell budding and basal complex assembly but fail to segregate at the end of cytokinesis.

Protection induced by virus-like particles containing Toxoplasma gondii microneme protein 8 against highly virulent RH strain of Toxoplasma gondii infection

Toxoplasma gondii (T. gondii) microneme protein 8 (MIC8) represents a novel, functional distinct invasion factor. In this study, we generated virus-like particles (VLPs) targeting Toxoplasma gondii MIC8 for the first time, and investigated the protection against highly virulent RH strain of T. gondii in a mouse model. We found that VLP vaccination induced Toxoplasma gondii-specific IgG and IgG1 antibody responses in the sera. Upon challenge infection with RH strain of T. gondii tachyzoites, vaccinated mice showed a significant increase of both IgG antibodies in sera and IgA antibodies in feces compared to those before challenge, and a rapid expansion of both germinal center B cell (B220+, GL7+) and T cell (CD4+, CD8+) populations. Importantly, intranasally immunized mice showed higher neutralizing antibodies and displayed no proinflammatory cytokine IFN-γ in the spleen. Mice were completely protected from a lethal challenge infection with the highly virulent T. gondii (RH) showing no body weight loss (100% survival). Our study shows the effective protection against T. gondii infection provided by VLPs containing microneme protein 8 of T. gondii, thus indicating a potential T. gondii vaccine candidate.