Rhoptry proteins affect the placental barrier in the context of Toxoplasma gondii infection: Signaling pathways and functions.

Toxoplasma gondii is an opportunistic and pathogenic obligate intracellular parasitic protozoan that is widespread worldwide and can infect most warm-blooded animals, seriously endangering human health and affecting livestock production. Toxoplasmosis caused by T. gondii infection has different clinical manifestations, which are mainly determined by the virulence of T. gondii and host differences. Among the manifestations of this condition, abortion, stillbirth, and fetal malformation can occur if a woman is infected with T. gondii in early pregnancy. Here, we discuss how the T. gondii rhoptry protein affects host pregnancy outcomes and speculate on the related signaling pathways involved. The effects of rhoptry proteins of T. gondii on the placental barrier are complex. Rhoptry proteins not only regulate interferon-regulated genes (IRGs) to ensure the survival of parasites in activated cells but also promote the spread of worms in tissues and the invasive ability of the parasites. The functions of these rhoptry proteins and the associated signaling pathways highlight relevant mechanisms by which Toxoplasma crosses the placental barrier and influences fetal development and will guide future studies to uncover the complexity of the host-pathogen interactions.

A conserved motif in the immune-subdominant RAP-1 related antigen of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle.

Introduction: Babesia bovis, a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily in B. bovis, as strong candidates for the development of subunit vaccines. Both RAP-1 and RRA share conservation of a group of 4 cysteines and amino acids motifs at the amino terminal end (NT) of these proteins. Methods and results: Sequence comparisons among the RRA sequences of several B. bovis strains and other Babesia spp parasites indicate a high level of conservation of a 15-amino acid (15-mer) motif located at the NT of the protein. BlastP searches indicate that the 15-mer motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. AlphaFold2 structure predictions suggest partial exposure of the 15-mer on the surface of RRA of three distinct Babesia species. Antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in iELISA, and rabbit antibodies against the 15-mer react with the surface of free merozoites in immunofluorescence. Discussion and conclusion: The presence of the 15-mer-like regions in dynein and ATP-binding proteins provides a rationale for investigating possible functional roles for RRA. The demonstrated presence of a surface exposed B-cell epitope in the 15-mer motif of the B. bovis RRA, which is recognized by sera from protected bovines, supports its inclusion in future subunit epitope-based vaccines against B. bovis.

Protists: Eukaryotic single-celled organisms and the functioning of their organelles.

Organelles are membrane bound structures that compartmentalize biochemical and molecular functions. With improved molecular, biochemical and microscopy tools the diversity and function of protistan organelles has increased in recent years, providing a complex panoply of structure/function relationships. This is particularly noticeable with the description of hydrogenosomes, and the diverse array of structures that followed, having hybrid hydrogenosome/mitochondria attributes. These diverse organelles have lost the major, at one time, definitive components of the mitochondrion (tricarboxylic cycle enzymes and cytochromes), however they all contain the machinery for the assembly of Fe-S clusters, which is the single unifying feature they share. The plasticity of organelles, like the mitochondrion, is therefore evident from its ability to lose its identity as an aerobic energy generating powerhouse while retaining key ancestral functions common to both aerobes and anaerobes. It is interesting to note that the apicoplast, a non-photosynthetic plastid that is present in all apicomplexan protozoa, apart from Cryptosporidium and possibly the gregarines, is also the site of Fe-S cluster assembly proteins. It turns out that in Cryptosporidium proteins involved in Fe-S cluster biosynthesis are localized in the mitochondrial remnant organelle termed the mitosome. Hence, different organisms have solved the same problem of packaging a life-requiring set of reactions in different ways, using different ancestral organelles, discarding what is not needed and keeping what is essential. Don't judge an organelle by its cover, more by the things it does, and always be prepared for surprises.

Cryogenic electron tomography reveals novel structures in the apical complex of Plasmodium falciparum.

Intracellular infectious agents, like the malaria parasite, Plasmodium falciparum, face the daunting challenge of how to invade a host cell. This problem may be even harder when the host cell in question is the enucleated red blood cell, which lacks the host machinery co-opted by many pathogens for internalization. Evolution has provided P. falciparum and related single-celled parasites within the phylum Apicomplexa with a collection of organelles at their apical end that mediate invasion. This apical complex includes at least two sets of secretory organelles, micronemes and rhoptries, and several structural features like apical rings and a putative pore through which proteins may be introduced into the host cell during invasion. We perform cryogenic electron tomography (cryo-ET) equipped with Volta Phase Plate on isolated and vitrified merozoites to visualize the apical machinery. Through tomographic reconstruction of cellular compartments, we see new details of known structures like the rhoptry tip interacting directly with a rosette resembling the recently described rhoptry secretory apparatus (RSA), or with an apical vesicle docked beneath the RSA. Subtomogram averaging reveals that the apical rings have a fixed number of repeating units, each of which is similar in overall size and shape to the units in the apical rings of tachyzoites of Toxoplasma gondii. Comparison of these polar rings in Plasmodium and Toxoplasma parasites also reveals them to have a structurally conserved assembly pattern. These results provide new insight into the essential and structurally conserved features of this remarkable machinery used by apicomplexan parasites to invade their respective host cells. IMPORTANCE: Malaria is an infectious disease caused by parasites of the genus Plasmodium and is a leading cause of morbidity and mortality globally. Upon infection, Plasmodium parasites invade and replicate in red blood cells, where they are largely protected from the immune system. To enter host cells, the parasites employ a specialized apparatus at their anterior end. In this study, advanced imaging techniques like cryogenic electron tomography (cryo-ET) and Volta Phase Plate enable unprecedented visualization of whole Plasmodium falciparum merozoites, revealing previously unknown structural details of their invasion machinery. Key findings include new insights into the structural conservation of apical rings shared between Plasmodium and its apicomplexan cousin, Toxoplasma. These discoveries shed light on the essential and conserved elements of the invasion machinery used by these pathogens. Moreover, the research provides a foundation for understanding the molecular mechanisms underlying parasite-host interactions, potentially informing strategies for combating diseases caused by apicomplexan parasites.

Evaluation of the immunoprotective effect of the recombinant Eimeria intestinalis rhoptry protein 25 and rhoptry protein 30 on New Zealand rabbits.

BACKGROUND: Rabbit coccidiosis is a parasitism caused by either one or multiple co-infections of Eimeria species. Among them, Eimeria intestinalis is the primary pathogen responsible for diarrhea, growth retardation, and potential mortality in rabbits. Concerns regarding drug resistance and drug residues have led to the development of recombinant subunit vaccines targeting Eimeria species as a promising preventive measure. The aim of this study was to assess the immunoprotective efficacy of recombinant subunit vaccines comprising EiROP25 and EiROP30 (rhoptry proteins (ROPs)) against E. intestinalis infection in rabbits. METHODS: Cloning, prokaryotic expression, and protein purification were performed to obtain EiROP25 and EiROP30. Five groups of fifty 35-day-old Eimeria-free rabbits were created (unchallenged control group, challenged control group, vector protein control group, rEiROP25 group, and rEiROP30 group), with 10 rabbits in each group. Rabbits in the rEiROP25 and rEiROP30 groups were immunized with the recombinant proteins (100 µg per rabbit) for primary and booster immunization (100 µg per rabbit) at a two-week intervals, and challenged with 7 × 104 oocysts per rabbit after an additional two-week interval. Two weeks after the challenge, the rabbits were euthanized for analysis. Weekly collections of rabbit sera were made to measure changes in specific IgG and cytokine level. Clinical symptoms and pathological changes after challenge were observed and recorded. At the conclusion of the animal experiment, lesion scores, the relative weight increase ratio, the oocyst reduction rate, and the anticoccidial index were computed. RESULTS: Rabbits immunized with rEiROP25 and rEiROP30 exhibited relative weight gain ratios of 56.57% and 72.36%, respectively. Oocysts decreased by 78.14% and 84.06% for the rEiROP25 and rEiROP30 groups, respectively. The anticoccidial indexes were 140 and 155. Furthermore, there was a noticeable drop in intestinal lesions. After the primary immunization with rEiROP25 and rEiROP30, a week later, there was a notable rise in specific IgG levels, which remained elevated for two weeks following challenge (P < 0.05). Interleukin (IL)-2 levels increased markedly in the rEiROP25 group, whereas IL-2, interferon gamma (IFN-γ), and IL-4 levels increased substantially in the rEiROP30 group (P < 0.05). CONCLUSION: Immunization of rabbits indicated that both rEiROP25 and rEiROP30 are capable of inducing an increase in specific antibody levels. rEiROP25 triggered a Th1-type immune protection response, while rEiROP30 elicited a Th1/Th2 mixed response. EiROP25 and EiROP30 can generate a moderate level of immune protection, with better efficacy observed for EiROP30. This study provides valuable insights for the promotion of recombinant subunit vaccines targeting rabbit E. intestinalis infection.

A candidate virulence factor of Eimeria tenella (EtROP30) predicted by virulence enhancement of transgenic Toxoplasma gondii.

Difficulties of in vitro culture and genetic manipulation of Eimeria tenella have hindered the screening of virulence factors in this parasite. In this study, the E. tenella rhoptry protein 30 (EtROP30) was expressed in Toxoplasma gondii (RH∆Ku80-EtROP30), and its effect on the proliferation and virulence of parasites was investigated. The results revealed that the expression of EtROP30 had no impact on the invasion and egress processes. However, the RH∆Ku80-EtROP30 strain formed larger plaques compared to the RH∆Ku80, indicating that the EtROP30 expression promotes T. gondii proliferation. Furthermore, the RH∆Ku80-EtROP30 strain exhibited greater pathogenicity, resulting in earlier mortality and shorter overall survival time compared to RH∆Ku80. These results imply that EtROP30 expression facilitates parasite intracellular proliferation and virulence in mice, suggesting that EtROP30 might be a candidate virulence factor of E. tenella.

A rhoptry protein, localizing in the bulb region of rhoptries, could induce protective immunity against Eimeria tenella infection.

Background: Rhoptry organelle proteins (ROPs) secreted by apicomplexan parasites play important roles during parasites invasion and survival in host cells, and are potential vaccine candidates against apicomplexan diseases. Eimeria tenella (E. tenella) is one of the most noteworthy apicomplexan species, which causes hemorrhagic pathologies. Although dozens of putative E. tenella ROP sequences are annotated, most ROP proteins are not well studied. Methods: In this study, an E. tenella ROP21 gene was identified and the recombinant EtROP21 protein (rEtROP21) was expressed in Escherichia coli. The developmental expression levels, localization, and protective efficacy against E. tenella infection in chickens were studied. Results: An EtROP21 gene fragment with an open reading frame (ORF) of 981 bp was obtained from the Beijing strain of E. tenella. The rEtROP21 has a molecular weight of approximately 50 kDa and was recognized by rEtROP21-immunized mouse serum. Two specific protein bands, about 43 KDa and 95 KDa in size, were detected in the whole sporozoite proteins using the rEtROP21-immunized chicken serum. RT-qPCR analysis of the E. tenella ROP21 gene (EtROP21) revealed that its mRNA levels were higher in merozoites and sporozoites than in sporulated and unsporulated oocysts. Immunofluorescence and immunoelectron analyses showed that the EtROP21 protein predominantly localizes in the bulb region of rhoptries distributed at anterior, posterior, and perinuclear regions of E. tenella sporozoites. Immunization and challenge experiments revealed that immunizing chickens with rEtROP21 significantly increased their average body weight gain while decreasing mean lesion score and oocyst output (P <0.05). When compared with the challenged control group, the rEtROP21-immunized group was associated with a significantly higher relative weight gain (90.2%) and a greater reduction in oocyst output (67%) (P <0.05). The anticoccidial index of the rEtROP21-immunized group was 163.2. Chicken serum ELISA revealed that the levels of the specific anti- rEtROP21 antibody, IFN-γ, and IL-4 were significantly higher in the rEtROP21-immunized group than in the challenged control group (P <0.05). Conclusion: These results indicate that rEtROP21 can induce a high level of specific immune response and it is a potential candidate for the development of vaccines against E. tenella infection in chickens.

Dense granule protein 3 of Toxoplasma gondii plays a crucial role in the capability of the tissue cysts of the parasite to persist in the presence of anti-cyst CD8+ T cells during the chronic stage of infection.

Toxoplasma gondii establishes chronic infection by forming tissue cysts, and this chronic infection is one of the most common parasitic infections in humans. Our recent studies revealed that whereas CD8+ T cells of genetically resistant BALB/c mice have the capability to remove the tissue cysts of the parasite through their perforin-mediated activities, small portions of the cysts are capable of persisting in the presence of the anti-cyst CD8+ T cells. It is currently unknown how those small portions of the cysts resist or escape the T-cell immunity and persist in the hosts. In the present study, we discovered that the cysts, which persisted in the presence of the perforin-mediated CD8+ T-cell immunity, have significantly greater mRNA levels for four dense granule proteins, GRA1, GRA2, GRA3, and GRA7, and one rhoptry protein, ROP35, than the total population of the cysts present in the absence of the T cells. In addition, increased levels of mRNA for GRA1, GRA3, and ROP35 in the cysts significantly correlated with their successful persistence through the condition in which greater degrees of reduction of the cyst burden occurred through anti-cyst CD8+ T cells. In addition, GRA3-deficient T. gondii displayed significantly enhanced elimination of the cysts by anti-cyst CD8+ T cells when compared to the wild-type parasite. These results indicate that GRA3 is a key molecule that mediates in the capability of T. gondii cysts to persist by resisting or evading the anti-cyst activity of CD8+ T cells during the later stage of infection.

A conserved complex of microneme proteins mediates rhoptry discharge in Toxoplasma.

Apicomplexan parasites discharge specialized organelles called rhoptries upon host cell contact to mediate invasion. The events that drive rhoptry discharge are poorly understood, yet essential to sustain the apicomplexan parasitic life cycle. Rhoptry discharge appears to depend on proteins secreted from another set of organelles called micronemes, which vary in function from allowing host cell binding to facilitation of gliding motility. Here we examine the function of the microneme protein CLAMP, which we previously found to be necessary for Toxoplasma gondii host cell invasion, and demonstrate its essential role in rhoptry discharge. CLAMP forms a distinct complex with two other microneme proteins, the invasion-associated SPATR, and a previously uncharacterized protein we name CLAMP-linked invasion protein (CLIP). CLAMP deficiency does not impact parasite adhesion or microneme protein secretion; however, knockdown of any member of the CLAMP complex affects rhoptry discharge. Phylogenetic analysis suggests orthologs of the essential complex components, CLAMP and CLIP, are ubiquitous across apicomplexans. SPATR appears to act as an accessory factor in Toxoplasma, but despite incomplete conservation is also essential for invasion during Plasmodium falciparum blood stages. Together, our results reveal a new protein complex that mediates rhoptry discharge following host-cell contact.

Functional screening reveals Toxoplasma prenylated proteins required for endocytic trafficking and rhoptry protein sorting.

In the apicomplexans, endocytosed cargos (e.g., hemoglobin) are trafficked to a specialized organelle for digestion. This follows a unique endocytotic process at the micropore/cytostome in these parasites. However, the mechanism underlying endocytic trafficking remains elusive, due to the repurposing of classical endocytic proteins for the biogenesis of apical organelles. To resolve this issue, we have exploited the genetic tractability of the model apicomplexan Toxoplasma gondii, which ingests host cytosolic materials (e.g., green fluorescent protein[GFP]). We determined an association between protein prenylation and endocytic trafficking, and using an alkyne-labeled click chemistry approach, the prenylated proteome was characterized. Genome editing, using clustered regularly interspaced short palindromic repaet/CRISPR-associated nuclease 9 (CRISPR/Cas9), was efficiently utilized to generate genetically modified lines for the functional screening of 23 prenylated candidates. This identified four of these proteins that regulate the trafficking of endocytosed GFP vesicles. Among these proteins, Rab1B and YKT6.1 are highly conserved but are non-classical endocytic proteins in eukaryotes. Confocal imaging analysis showed that Rab1B and Ras are substantially localized to both the trans-Golgi network and the endosome-like compartments in the parasite. Conditional knockdown of Rab1B caused a rapid defect in secretory trafficking to the rhoptry bulb, suggesting a trafficking intersection role for the key regulator Rab1B. Further experiments confirmed a critical role for protein prenylation in regulating the stability/activity of these proteins (i.e., Rab1B and YKT6.1) in the parasite. Our findings define the molecular basis of endocytic trafficking and reveal a potential intersection function of Rab1B on membrane trafficking in T. gondii. This might extend to other related protists, including the malarial parasites. IMPORTANCE The protozoan Toxoplasma gondii establishes a permissive niche, in host cells, that allows parasites to acquire large molecules such as proteins. Numerous studies have demonstrated that the parasite repurposes the classical endocytic components for secretory sorting to the apical organelles, leaving the question of endocytic transport to the lysosome-like compartment unclear. Recent studies indicated that endocytic trafficking is likely to associate with protein prenylation in malarial parasites. This information promoted us to examine this association in the model apicomplexan T. gondii and to identify the key components of the prenylated proteome that are involved. By exploiting the genetic tractability of T. gondii and a host GFP acquisition assay, we reveal four non-classical endocytic proteins that regulate the transport of endocytosed cargos (e.g., GFP) in T. gondii. Thus, we extend the principle that protein prenylation regulates endocytic trafficking and elucidate the process of non-classical endocytosis in T. gondii and potentially in other related protists.

New Allele-Specific Oligonucleotide (ASO) amplifications for Toxoplasma gondii rop18 allele typing: Analysis of 86 human congenital infections in Brazil.

This study aimed to detect and differentiate Toxoplasma gondii by the allele typing of its polymorphic rop18 gene. For this purpose, a novel genotyping system using allele-specific oligonucleotides (ASOs) was designed, consisting of three ASO pairs. The first and third pairs specifically amplify rop18 allele I and allele III, while the second pair amplify both allele I and II. Genomic DNA from 86 congenital infections was analyzed by ASO-PCRs, successfully typing 82 (95.35%) samples. The remaining 4 samples (4.65%) required sequencing and single nucleotide polymorphism (SNP) analysis of the amplification products. The distribution of samples according to rop18 alleles was: 39.5% of allele III, 38.4% of allele II, 19.8% of mixed rop18 alleles (I/III or II/III), and 2.3% of allele I. The six severely compromised infants exhibited the highest parasite load levels and were infected during the first and early second trimesters of pregnancy. Among these cases, two were associated with rop18 allele I parasites, two with mixed rop18 alleles (I/III), one with allele II, and one with allele III parasites. In conclusion, all severe cases of congenital toxoplasmosis were infected during early pregnancy, but they were not exclusively associated with rop18 allele I parasites, as observed in murine toxoplasmosis. Furthermore, nearly one-fifth of parasites were non-archetypal, exhibiting more than one rop18 allele, indicating a higher genetic diversity of Toxoplasma gondii in this South American sample. Overall, a robust T. gondii rop18 allele typing was developed and suggested that congenital toxoplasmosis in humans involves complex mechanisms beyond the parasite genotype.

Bioinformatics-based prediction and screening of immunogenic epitopes of Toxoplasma gondii rhoptry proteins 7, 21 and 22 as candidate vaccine target.

Introduction: Toxoplasmosis is a well-known zoonotic disease caused by Toxoplasma gondii. The main causes of the disease range from eating undercooked or contaminated meat and shellfish to cleaning litter trays into which cats that excreted toxoplasma via faeces. This pathogen can live for a very long time, possibly a lifetime, within the bodies of humans and other animals. Aims and objectives: This study aimed to predict and analyse candidate immunogenic epitopes for vaccine development by evaluating the physio-chemical properties, multiple sequence alignment, secondary and tertiary structures, phosphorylation sites, transmembrane domains, and signal peptides, of T. gondii rhoptry proteins ROP7, ROP21, and ROP22 using bioinformatics tools. Methods: To find immunogenic epitopes of rhoptry proteins, numerous bioinformatics web servers were used containing multiple sequence alignment, physiochemical properties, antigenicity and allergenicity, post-translational modification sites (PTMs), signal peptides, transmembrane domains, secondary and tertiary structures, and screening of predicted epitopes. We evaluated immunogenic linear B-cell epitopes as candidate proteins for vaccine development. Results: Nine epitopes were identified for each protein, and analysis of immunogenicity, revealed three candidate epitopes for ROP7, one for ROP21, and four for ROP22. Among all candidate epitopes, ROP22 contained the most immunogenic epitopes with immunogenicity score of 0.50575. Conclusion: We acquired detailed information on predicted immunogenic epitopes using in-silico methods. The results provide a foundation for further experimental analysis of toxoplasmosis, and potential vaccine development.

Screening of apical membrane antigen-1 (AMA1), dense granule protein-7 (GRA7) and rhoptry protein-16 (ROP16) antigens for a potential vaccine candidate against Toxoplasma gondii for chickens.

Toxoplasmosis is a zoonotic disease caused by the protozoan parasite, Toxoplasma gondii known to infect almost all animals, including birds and humans globally. This disease has impacted the livestock industry and public health, where infection of domestic animals increases the zoonotic risk of transmission of infection to humans, threatening public health. Hence the need to discover novel and safe vaccines to fight against toxoplasmosis. In the current study, a novel multiepitope vaccine was designed using immunoinformatics techniques targeting T. gondii AMA1, GRA7 and ROP16 antigens, consisting of antigenic, immunogenic, non-allergenic and cytokine inducing T-cell (9 CD8+ and 15 CD4+) epitopes and four (4) B-cell epitopes fused together using AAY, KK and GPGPG linkers. The tertiary model of the proposed vaccine was predicted and validated to confirm the structural quality of the vaccine. The designed vaccine was highly antigenic (antigenicity = 0.6645), immunogenic (score = 2.89998), with molecular weight of 73.35 kDa, instability and aliphatic index of 28.70 and 64.10, respectively; and GRAVY of -0.363. The binding interaction, stability and flexibility were assessed with molecular docking and dynamics simulation, which revealed the proposed vaccine to have good structural interaction (binding affinity = -106.882 kcal/mol) and stability when docked with Toll like receptor-4 (TLR4). The results revealed that the Profilin-adjuvanted vaccine is promising, as it predicted induction of enhanced immune responses through the production of cytokines and antibodies critical in blocking host invasion.

Roles of the RON3 C-terminal fragment in erythrocyte invasion and blood-stage parasite proliferation in Plasmodium falciparum.

Plasmodium species cause malaria, and in the instance of Plasmodium falciparum is responsible for a societal burden of over 600,000 deaths annually. The symptoms and pathology of malaria are due to intraerythocytic parasites. Erythrocyte invasion is mediated by the parasite merozoite stage, and is accompanied by the formation of a parasitophorous vacuolar membrane (PVM), within which the parasite develops. The merozoite apical rhoptry organelle contains various proteins that contribute to erythrocyte attachment and invasion. RON3, a rhoptry bulb membrane protein, undergoes protein processing and is discharged into the PVM during invasion. RON3-deficient parasites fail to develop beyond the intraerythrocytic ring stage, and protein export into erythrocytes by the Plasmodium translocon of exported proteins (PTEX) apparatus is abrogated, as well as glucose uptake into parasites. It is known that truncated N- and C-terminal RON3 fragments are present in rhoptries, but it is unclear which RON3 fragments contribute to protein export by PTEX and glucose uptake through the PVM. To investigate and distinguish the roles of the RON3 C-terminal fragment at distinct developmental stages, we used a C-terminus tag for conditional and post-translational control. We demonstrated that RON3 is essential for blood-stage parasite survival, and knockdown of RON3 C-terminal fragment expression from the early schizont stage induces a defect in erythrocyte invasion and the subsequent development of ring stage parasites. Protein processing of full-length RON3 was partially inhibited in the schizont stage, and the RON3 C-terminal fragment was abolished in subsequent ring-stage parasites compared to the RON3 N-terminal fragment. Protein export and glucose uptake were abrogated specifically in the late ring stage. Plasmodial surface anion channel (PSAC) activity was partially retained, facilitating small molecule traffic across the erythrocyte membrane. The knockdown of the RON3 C-terminal fragment after erythrocyte invasion did not alter parasite growth. These data suggest that the RON3 C-terminal fragment participates in erythrocyte invasion and serves an essential role in the progression of ring-stage parasite growth by the establishment of the nutrient-permeable channel in the PVM, accompanying the transport of ring-stage parasite protein from the plasma membrane to the PVM.

Rhoptry neck protein 4 plays important roles during Plasmodium sporozoite infection of the mammalian liver.

During invasion, Plasmodium parasites secrete proteins from rhoptry and microneme apical end organelles, which have crucial roles in attaching to and invading target cells. A sporozoite stage-specific gene silencing system revealed that rhoptry neck protein 2 (RON2), RON4, and RON5 are important for sporozoite invasion of mosquito salivary glands. Here, we further investigated the roles of RON4 during sporozoite infection of the liver in vivo. Following intravenous inoculation of RON4-knockdown sporozoites into mice, we demonstrated that sporozoite RON4 has multiple functions during sporozoite traversal of sinusoidal cells and infection of hepatocytes. In vitro infection experiments using a hepatoma cell line revealed that secreted RON4 is involved in sporozoite adhesion to hepatocytes and has an important role in the early steps of hepatocyte infection. In addition, in vitro motility assays indicated that RON4 is required for sporozoite attachment to the substrate and the onset of migration. These findings indicate that RON4 is crucial for sporozoite migration toward and invasion of hepatocytes via attachment ability and motility.IMPORTANCEMalarial parasite transmission to mammals is established when sporozoites are inoculated by mosquitoes and migrate through the bloodstream to infect hepatocytes. Many aspects of the molecular mechanisms underpinning migration and cellular invasion remain largely unelucidated. By applying a sporozoite stage-specific gene silencing system in the rodent malarial parasite, Plasmodium berghei, we demonstrated that rhoptry neck protein 4 (RON4) is crucial for sporozoite infection of the liver in vivo. Combined with in vitro investigations, it was revealed that RON4 functions during a crossing of the sinusoidal cell layer and invading hepatocytes, at an early stage of liver infection, by mediating the sporozoite capacity for adhesion and the onset of motility. Since RON4 is also expressed in Plasmodium merozoites and Toxoplasma tachyzoites, our findings contribute to understanding the conserved invasion mechanisms of Apicomplexa parasites.

Overexpression of Eimeria tenella Rhoptry Kinase 2 Induces Early Production of Schizonts.

Eimeria tenella is an obligate intracellular parasite responsible for avian coccidiosis. Like other apicomplexan parasites, such as Toxoplasma gondii, cell invasion and intracellular development rely on apical organelle content discharge, named micronemes and rhoptries. Some rhoptry (ROP) kinases (ROPK) are key virulence factors in T. gondii. To date, among the 28 ropk genes carried by E. tenella, only two to four were confirmed by proteomic analysis or immunostaining to be expressed at the sporozoite stage. We have previously shown that EtROP1 is implicated in the inhibition of host cell apoptosis by interacting with the cellular p53. This work functionally described the second ROP kinase expressed at the sporozoite stage in E. tenella. EtROP2 is an active kinase that phosphorylates cell substrates of approximately 50 kDa. Its overexpression leads to the shortening of the prepatent period and to the early development of first-generation schizonts. Conduction of RNA sequencing analysis and reverse transcriptase quantitative PCR (RT-qPCR) on the host cell allowed us to identify the mitogen-activated protein kinase (MAPK) pathway and the transcription factor cFos to be upregulated by EtROP2. We also showed by immunofluorescence assay that the active kinase EtROP2 is implicated in the p38 MAPK pathway activation. We established here that EtROP2 activates the p38 MAPK pathway through a direct or indirect phosphorylation, leading to the overexpression of the master transcription factor cFos known to be implicated in E. tenella development. IMPORTANCE Rhoptries are specialized secretory organelles found in zoite stages of apicomplexan parasites. In addition to well-conserved rhoptry neck proteins, their protein consists mostly of kinase proteins, highly divergent from eukaryotic kinases. Some of those kinases are described as major virulence factors in Toxoplasma gondii, secreted into the host cell to hijack signaling pathways. Most of those kinases remain to be characterized in Eimeria tenella. Deciphering their cellular function is a prerequisite to supporting their relevance as a druggable target in development of new means of Eimeria tenella control. Secreted divergent kinases that interact with host cell partners to modulate pathways are good candidates, as they coevolve with their host targets to ensure their function within the host and are less prone to mutations that would lead to drug resistance. The absence of any orthologous kinase in host cells makes these parasite kinases a promising drug target candidate.

Plasmodium falciparum rhoptry neck protein 4 has conserved regions mediating interactions with receptors on human erythrocytes and hepatocyte membrane.

Plasmodium falciparum-related malaria represents a serious worldwide public health problem due to its high mortality rates. P. falciparum expresses rhoptry neck protein 4 (PfRON4) in merozoite and sporozoite rhoptries, it participates in tight junction-TJ formation via the AMA-1/RON complex and is refractory to complete genetic deletion. Despite this, which PfRON4 key regions interact with host cells remain unknown; such information would be useful for combating falciparum malaria. Thirty-two RON4 conserved region-derived peptides were chemically synthesised for determining and characterising PfRON4 regions having high host cell binding affinity (high activity binding peptides or HABPs). Receptor-ligand interaction/binding assays determined their specific binding capability, the nature of their receptors and their ability to inhibit in vitro parasite invasion. Peptides 42477, 42479, 42480, 42505 and 42513 had greater than 2% erythrocyte binding activity, whilst peptides 42477 and 42480 specifically bound to HepG2 membrane, both of them having micromolar and submicromolar range dissociation constants (Kd). Cell-peptide interaction was sensitive to treating erythrocytes with trypsin and/or chymotrypsin and HepG2 with heparinase I and chondroitinase ABC, suggesting protein-type (erythrocyte) and heparin and/or chondroitin sulphate proteoglycan receptors (HepG2) for PfRON4. Erythrocyte invasion inhibition assays confirmed HABPs' importance during merozoite invasion. PfRON4 800-819 (42477) and 860-879 (42480) regions specifically interacted with host cells, thereby supporting their inclusion in a subunit-based, multi-antigen, multistage anti-malarial vaccine.

Cryptosporidium uses multiple distinct secretory organelles to interact with and modify its host cell.

Cryptosporidium is a leading cause of diarrheal disease in children and an important contributor to early childhood mortality. The parasite invades and extensively remodels intestinal epithelial cells, building an elaborate interface structure. How this occurs at the molecular level and the contributing parasite factors are largely unknown. Here, we generated a whole-cell spatial proteome of the Cryptosporidium sporozoite and used genetic and cell biological experimentation to discover the Cryptosporidium-secreted effector proteome. These findings reveal multiple organelles, including an original secretory organelle, and generate numerous compartment markers by tagging native gene loci. We show that secreted proteins are delivered to the parasite-host interface, where they assemble into different structures including a ring that anchors the parasite into its unique epicellular niche. Cryptosporidium thus uses a complex set of secretion systems during and following invasion that act in concert to subjugate its host cell.

The strategies of NLRP3 inflammasome to combat Toxoplasma gondii.

Infection with the protozoan parasite Toxoplasma gondii (T. gondii) results in the activation of nucleotide-binding domain leucine-rich repeat containing receptors (NLRs), which in turn leads to inflammasome assembly and the subsequent activation of caspase-1, secretion of proinflammatory cytokines, and pyroptotic cell death. Several recent studies have addressed the role of the NLRP3 inflammasome in T. gondii infection without reaching a consensus on its roles. Moreover, the mechanisms of NLRP3 inflammasome activation in different cell types remain unknown. Here we review current research on the activation and specific role of the NLRP3 inflammasome in T. gondii infection.

An apical membrane complex for triggering rhoptry exocytosis and invasion in Toxoplasma.

Apicomplexan parasites possess secretory organelles called rhoptries that undergo regulated exocytosis upon contact with the host. This process is essential for the parasitic lifestyle of these pathogens and relies on an exocytic machinery sharing structural features and molecular components with free-living ciliates. However, how the parasites coordinate exocytosis with host interaction is unknown. Here, we performed a Tetrahymena-based transcriptomic screen to uncover novel exocytic factors in Ciliata and conserved in Apicomplexa. We identified membrane-bound proteins, named CRMPs, forming part of a large complex essential for rhoptry secretion and invasion in Toxoplasma. Using cutting-edge imaging tools, including expansion microscopy and cryo-electron tomography, we show that, unlike previously described rhoptry exocytic factors, TgCRMPs are not required for the assembly of the rhoptry secretion machinery and only transiently associate with the exocytic site-prior to the invasion. CRMPs and their partners contain putative host cell-binding domains, and CRMPa shares similarities with GPCR proteins. Collectively our data imply that the CRMP complex acts as a host-molecular sensor to ensure that rhoptry exocytosis occurs when the parasite contacts the host cell.