A Time Point Proteomic Analysis Reveals Protein Dynamics of Plasmodium Oocysts.

The oocyst is a sporogonic stage of Plasmodium development that takes place in the mosquito midgut in about 2 weeks. The cyst is protected by a capsule of unknown composition, and little is known about oocyst biology. We carried out a proteomic analysis of oocyst samples isolated at early, mid, and late time points of development. Four biological replicates for each time point were analyzed, and almost 600 oocyst-specific candidates were identified. The analysis revealed that, in young oocysts, there is a strong activity of protein and DNA synthesis, whereas in mature oocysts, proteins involved in oocyst and sporozoite development, gliding motility, and invasion are mostly abundant. Among the proteins identified at early stages, 17 candidates are specific to young oocysts. Thirty-four candidates are common to oocyst and the merosome stages (sporozoite proteins excluded), sharing common features as replication and egress. Western blot and immunofluorescence analyses of selected candidates confirm the expression profile obtained by proteomic analysis.

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.

Plasmodium sporozoite excystation involves local breakdown of the oocyst capsule.

Plasmodium oocysts develop on the abluminal side of the mosquito midgut in relatively small numbers. Oocysts possess an extracellular cell wall-the capsule-to protect them from the insect's haemolymph environment. To further maximise transmission, each oocyst generates hundreds of sporozoites through an asexual multiplication step called sporogony. Completion of transmission requires sporozoite egress from the capsule (excystation), but this process remains poorly understood. In this study, we fused the parasite-encoded capsule protein Cap380 with green fluorescent protein in a transgenic P. berghei line, allowing live fluorescence imaging of capsules throughout sporogony and sporozoite excystation. The results show that capsules progressively weaken during sporulation ultimately resulting in sporozoite exit through small holes. Prior to formation of the holes, local thinning of the capsule was observed. Our findings support an excystation model based on local, rather than global, weakening of the capsule likely facilitated by local re-orientation of sporozoites and apical secretion.

Plasmodium berghei oocysts possess fatty acid synthesis and scavenging routes.

Malaria parasites carry out fatty acid synthesis (FAS) in their apicoplast organelle via a bacterially related (type II) enzymatic pathway. In the vertebrate host, exoerythrocytic Plasmodium stages rely on FAS, whereas intraerythrocytic stages depend on scavenging FA from their environment. In the mosquito, P. falciparum oocysts express and rely on FAS enzymes for sporozoite formation, but P. yoelii oocysts do not express, nor depend on, FAS enzymes and thus rely on FA scavenging to support sporogony. In P. berghei, FAS enzymes are similarly expendable for sporogony, indicating it conforms to the P. yoelii scenario. We show here that P. berghei, unexpectedly, expresses FAS enzymes throughout oocyst development. These findings indicate that P. berghei can employ FAS alongside FA scavenging to maximise sporogony and transmission, and is more similar to P. falciparum than previously assumed with respect to FA acquisition by the oocyst. The ability of oocysts to switch between FAS and scavenging could be an important factor in the non-competitive relationship of resource exploitation between Plasmodium parasites and their mosquito vectors, which shapes parasite virulence both in the insect and vertebrate.

Label-free quantitative detection and comparative analysis of lysine acetylation during the different life stages of Eimeria tenella.

Proteome-wide lysine acetylation has been documented in apicomplexan parasite Toxoplasma gondii and Plasmodium falciparum. Here, we conducted the first lysine acetylome in unsporulated oocysts (USO), sporulated 7 h oocysts (SO 7h), sporulated oocysts (SO), sporozoites (S), and the second generation merozoites (SMG) of Eimeria tenella through a 4D label-free quantitative technique. Altogether, 8532 lysine acetylation sites on 2325 proteins were identified in E. tenella, among which 5445 sites on 1493 proteins were quantified. In addition, 557, 339, 478, 248, 241, and 424 differentially expressed proteins were identified in the comparisons SO7h vs USO, SO vs SO7h, SO vs USO, S vs SO, SMG vs S, and USO vs SMG, respectively. The bioinformatics analysis of the acetylome showed that the lysine acetylation is widespread on proteins of diverse functions. Moreover, the dynamic changes of lysine acetylome among E. tenella different life stages revealed significant regulation during the whole process of E. tenella growth and stage conversion. This study provides a beginning for the investigation of the regulate role of lysine acetylation in E. tenella and may provide new strategies for anticoccidiosis drug and vaccine development. Raw data are publicly available at iProX with the data set identifier PXD040368.

Molecular characterization and protective efficacy of vacuolar protein sorting 29 from Eimeria tenella.

Introduction: Vacuolar protein sorting 29 (VPS29) is a core component of the retromer-retriever complex and is essential for recycling numerous cell-surface cargoes from endosomes. However, there are no reports yet on VPS29 of Eimeria spp. Methods: Here, we cloned and prokaryotically expressed a partial sequence of Eimeria tenella VPS29 (EtVPS29) with RT-PCR and engineered strain of Escherichia coli respectively. The localization of the VPS29 protein in E. tenella sporozoites was investigated with immunofluorescence (IFA) and overexpression assays. And its protective efficacy against E. tenella infection was investigated in chickens with the animal protection test. Results: An EtVPS29 gene fragment with an ORF reading frame of 549 bp was cloned. The band size of the expressed recombinant protein, rEtVPS29, was approximately 39 kDa and was recognized by the chicken anti-E. tenella positive serum. EtVPS29 protein was observed widely distributing in the cytoplasm of E. tenella sporozoites in the IFA and overexpression assays. rEtVPS29 significantly increased average body weight gain and decreased mean lesion score and oocyst output in chickens. The relative weight gain rate in the rEtVPS29-immunized group was 62.9%, which was significantly higher than that in the unimmunized and challenged group (P < 0.05). The percentage of reduced oocyst output in the rEtVPS29 immunized group was 32.2%. The anticoccidial index of the rEtVPS29-immunized group was 144.2. Serum ELISA also showed that rEtVPS29 immunization induced high levels of specific antibodies in chickens. Discussion: These results suggest that rEtVPS29 can induce a specific immune response and is a potential candidate for the development of novel vaccines against E. tenella infections in chickens.

Characterization of CpCaM, a protein potentially involved in the growth of Cryptosporidium parvum.

Cryptosporidium parvum is an important apicomplexan parasite causing severe diarrhea in both humans and animals. Calmodulin (CaM), a multifunctional and universal calcium-binding protein, contributes to the growth and development of apicomplexan parasites, but the role of CaM in C. parvum remains unknown. In this study, the CaM of C. parvum encoded by the cgd2_810 gene was expressed in Escherichia coli, and the biological functions of CpCaM were preliminarily investigated. The transcriptional level of the cgd2_810 gene peaked at 36 h post infection (pi), and the CpCaM protein was mainly located around the nucleus of the whole oocysts, in the middle of sporozoites and around the nucleus of merozoites. Anti-CpCaM antibody reduced the invasion of C. parvum sporozoites by 30.69%. The present study indicates that CpCaM is potentially involved in the growth of C. parvum. Results of the study expand our knowledge on the interaction between host and Cryptosporidium.

Late Embryogenesis Abundant Proteins Contribute to the Resistance of Toxoplasma gondii Oocysts against Environmental Stresses.

Toxoplasma gondii oocysts, which are shed in large quantities in the feces from infected felines, are very stable in the environment, resistant to most inactivation procedures, and highly infectious. The oocyst wall provides an important physical barrier for sporozoites contained inside oocysts, protecting them from many chemical and physical stressors, including most inactivation procedures. Furthermore, sporozoites can withstand large temperature changes, even freeze-thawing, as well as desiccation, high salinity, and other environmental insults; however, the genetic basis for this environmental resistance is unknown. Here, we show that a cluster of four genes encoding Late Embryogenesis Abundant (LEA)-related proteins are required to provide Toxoplasma sporozoites resistance to environmental stresses. Toxoplasma LEA-like genes (TgLEAs) exhibit the characteristic features of intrinsically disordered proteins, explaining some of their properties. Our in vitro biochemical experiments using recombinant TgLEA proteins show that they have cryoprotective effects on the oocyst-resident lactate dehydrogenase enzyme and that induced expression in E. coli of two of them leads to better survival after cold stress. Oocysts from a strain in which the four LEA genes were knocked out en bloc were significantly more susceptible to high salinity, freezing, and desiccation compared to wild-type oocysts. We discuss the evolutionary acquisition of LEA-like genes in Toxoplasma and other oocyst-producing apicomplexan parasites of the Sarcocystidae family and discuss how this has likely contributed to the ability of sporozoites within oocysts to survive outside the host for extended periods. Collectively, our data provide a first molecular detailed view on a mechanism that contributes to the remarkable resilience of oocysts against environmental stresses. IMPORTANCE Toxoplasma gondii oocysts are highly infectious and may survive in the environment for years. Their resistance against disinfectants and irradiation has been attributed to the oocyst and sporocyst walls by acting as physical and permeability barriers. However, the genetic basis for their resistance against stressors like changes in temperature, salinity, or humidity, is unknown. We show that a cluster of four genes encoding Toxoplasma Late Embryogenesis Abundant (TgLEA)-related proteins are important for this resistance to environmental stresses. TgLEAs have features of intrinsically disordered proteins, explaining some of their properties. Recombinant TgLEA proteins show cryoprotective effects on the parasite's lactate dehydrogenase, an abundant enzyme in oocysts, and expression in E. coli of two TgLEAs has a beneficial effect on growth after cold stress. Moreover, oocysts from a strain lacking all four TgLEA genes were more susceptible to high salinity, freezing, and desiccation compared to wild-type oocysts, highlighting the importance of the four TgLEAs for oocyst resilience.

Targetome Analysis of Malaria Sporozoite Transcription Factor AP2-Sp Reveals Its Role as a Master Regulator.

Malaria transmission to humans begins with sporozoite infection of the liver. The elucidation of gene regulation during the sporozoite stage will promote the investigation of mechanisms of liver infection by this parasite and contribute to the development of strategies for preventing malaria transmission. AP2-Sp is a transcription factor (TF) essential for the formation of sporozoites or sporogony, which takes place in oocysts in the midguts of infected mosquitoes. To understand the role of this TF in the transcriptional regulatory system of this stage, we performed chromatin immunoprecipitation sequencing (ChIP-seq) analyses using whole mosquito midguts containing late oocysts as starting material and explored its genome-wide target genes. We identified 697 target genes, comprising those involved in distinct processes parasites experience during this stage, from sporogony to development into the liver stage and representing the majority of genes highly expressed in the sporozoite stage. These results suggest that AP2-Sp determines basal patterns of gene expression by targeting a broad range of genes directly. The ChIP-seq analyses also showed that AP2-Sp maintains its own expression by a transcriptional autoactivation mechanism (positive-feedback loop) and induces all TFs reported to be transcribed at this stage, including AP2-Sp2, AP2-Sp3, and SLARP. The results showed that AP2-Sp exists at the top of the transcriptional cascade of this stage and triggers the formation of this stage as a master regulator. IMPORTANCE The sporozoite stage plays a central role in malaria transmission from a mosquito to vertebrate host and is an important target for antimalarial strategies. AP2-Sp is a candidate master transcription factor for the sporozoite stage. However, study of its role in gene regulation has been hampered because of difficulties in performing genome-wide studies of gene regulation in this stage. Here, we conquered this problem and revealed that AP2-Sp has the following prominent features as a master transcription factor. First, it determines the repertory of gene expression during this stage. Second, it maintains its own expression through a transcriptional positive-feedback loop and induces all other transcription factors specifically expressed in this stage. This study represents a major breakthrough in fully understanding gene regulation in this important malarial stage.

Genetic disruption of nucleoside transporter 4 reveals its critical roles in malaria parasite sporozoite functions.

All protozoan parasites are lacking the pathway to synthesize purines de novo and therefore they depend on their host cells to provide purines. A number of highly conserved nucleoside transporter (NT) proteins are encoded in malaria parasite genomes, of which NT1 is characterized in Plasmodium falciparum and P. yoelii as a plasma membrane protein that is responsible for salvage of purines from the host, and NT2 is an endoplasmic membrane NT protein. Whereas NT3 is only present in primate malaria parasites, little is known about NT4, which is conserved in all malaria parasite species. Herein, we targeted NT4 gene for deletion in P. berghei. NT4 knockout parasites developed normally as blood stages, ookinetes and formed oocysts with sporozoites compared with wild-type (WT) P. berghei ANKA parasites. However, nt4(-) sporozoites showed significantly decreased egress from oocysts to hemolymph, significant reduction of colonization of the salivary glands, and complete abolishment of infection of the mammalian host by salivary gland and hemolymph sporozoites. Therefore, we identify NT4 as a NT that is important, not for replication and growth, but for sporozoite infectivity functions.

Plasmodium berghei leucine-rich repeat protein 1 downregulates protein phosphatase 1 activity and is required for efficient oocyst development.

Protein phosphatase 1 (PP1) is a key enzyme for Plasmodium development. However, the detailed mechanisms underlying its regulation remain to be deciphered. Here, we report the functional characterization of the Plasmodium berghei leucine-rich repeat protein 1 (PbLRR1), an orthologue of SDS22, one of the most ancient and conserved PP1 interactors. Our study shows that PbLRR1 is expressed during intra-erythrocytic development of the parasite, and up to the zygote stage in mosquitoes. PbLRR1 can be found in complex with PbPP1 in both asexual and sexual stages and inhibits its phosphatase activity. Genetic analysis demonstrates that PbLRR1 depletion adversely affects the development of oocysts. PbLRR1 interactome analysis associated with phospho-proteomics studies identifies several novel putative PbLRR1/PbPP1 partners. Some of these partners have previously been characterized as essential for the parasite sexual development. Interestingly, and for the first time, Inhibitor 3 (I3), a well-known and direct interactant of Plasmodium PP1, was found to be drastically hypophosphorylated in PbLRR1-depleted parasites. These data, along with the detection of I3 with PP1 in the LRR1 interactome, strongly suggest that the phosphorylation status of PbI3 is under the control of the PP1-LRR1 complex and could contribute (in)directly to oocyst development. This study provides new insights into previously unrecognized PbPP1 fine regulation of Plasmodium oocyst development through its interaction with PbLRR1.

The transcriptome from asexual to sexual in vitro development of Cystoisospora suis (Apicomplexa: Coccidia).

The apicomplexan parasite Cystoisospora suis is an enteropathogen of suckling piglets with woldwide distribution. As with all coccidian parasites, its lifecycle is characterized by asexual multiplication followed by sexual development with two morphologically distinct cell types that presumably fuse to form a zygote from which the oocyst arises. However, knowledge of the sexual development of C. suis is still limited. To complement previous in vitro studies, we analysed transcriptional profiles at three different time points of development (corresponding to asexual, immature and mature sexual stages) in vitro via RNASeq. Overall, transcription of genes encoding proteins with important roles in gametes biology, oocyst wall biosynthesis, DNA replication and axonema formation as well as proteins with important roles in merozoite biology was identified. A homologue of an oocyst wall tyrosine rich protein of Toxoplasma gondii was expressed in macrogametes and oocysts of C. suis. We evaluated inhibition of sexual development in a host-free culture for C. suis by antiserum specific to this protein to evaluate whether it could be exploited as a candidate for control strategies against C. suis. Based on these data, targets can be defined for future strategies to interrupt parasite transmission during sexual development.

Characterization of the Eimeria tenella rhoptry protein with a nuclear localization sequence (EtROP30).

Rhoptry proteins (ROPs), secreted by specific rhoptry organelles of apicomplexan parasites, are determinants of parasite pathogenesis and sources of vaccine candidates. Twenty-eight ROPs of Eimeria tenella have been predicted by genomic approaches, and in the present study, E. tenella rhoptry protein 30 (EtROP30) was characterized. Subcellular localizations of EtROP30 in sporozoites and merozoites were in the apical complex and rhoptry-like bulb, suggesting that EtROP30 is a member of ROPs in E. tenella. Sequence analysis showed that EtROP30 contained an N-terminal secretory signal, a protein kinase domain with eight E. tenella-specific rhoptry kinase 1 subfamily (ROPK-Eten1) motifs, and a C-terminal nuclear localization sequence (NLS), making EtROP30 the only ROP that contains both a secretory signal and an NLS in E. tenella. Subsequent experiments showed that EtROP30 was a secreted protein in the sporozoite stage, relying on NLS for migration to the host nucleus. In addition, EtROP30 showed significantly higher expression levels in the parasite merozoite stage, indicating that EtROP30 plays a critical role during parasite reinvasion and development and may be a viable option as a vaccine candidate for anti-parasitic infection. The immunization protection efficacies of EtROP30 were evaluated. Significant improvements in mean body weight gain, reduction of cecum lesion score, and number of oocysts excreted were observed, indicating that EtROP30 has good immunogenicity against E. tenella. In the present study, a ROP of E. tenella with secretory and nuclear localization characteristics has been identified, and proved to be an effective vaccine candidate against this parasite.

Comparative Phosphoproteomic Analysis of Sporulated Oocysts and Tachyzoites of Toxoplasma gondii Reveals Stage-Specific Patterns.

Toxoplasma gondii is an obligate intracellular protozoan of severe threat to humans and livestock, whose life history harbors both gamic and apogamic stages. Chinese 1 (ToxoDB#9) was a preponderant genotype epidemic in food-derived animals and humans in China, with a different pathogenesis from the strains from the other nations of the world. Posttranslational modifications (PTMs) of proteins were critical mediators of the biology, developmental transforms, and pathogenesis of protozoan parasites. The phosphoprotein profiling and the difference between the developmental phases of T. gondii, contributing to development and infectivity, remain unknown. A quantitative phosphoproteomic approach using IBT integrated with TiO2 affinity chromatography was applied to identify and analyze the difference in the phosphoproteomes between the sporulated oocysts and the tachyzoites of the virulent ToxoDB#9 (PYS) strain of T. gondii. A total of 4058 differential phosphopeptides, consisting of 2597 upregulated and 1461 downregulated phosphopeptides, were characterized between sporulated the oocysts and tachyzoites. Twenty-one motifs extracted from the upregulated phosphopeptides contained 19 serine motifs and 2 threonine motifs (GxxTP and TP), whereas 16 motifs identified from downregulated phosphopeptides included 13 serine motifs and 3 threonine motifs (KxxT, RxxT, and TP). Beyond the traditional kinases, some infrequent classes of kinases, including Ab1, EGFR, INSR, Jak, Src and Syk, were found to be corresponding to motifs from the upregulated and downregulated phosphopeptides. Remarkable functional properties of the differentially expressed phosphoproteins were discovered by GO analysis, KEGG pathway analysis, and STRING analysis. S8GFS8 (DNMT1-RFD domain-containing protein) and S8F5G5 (Histone kinase SNF1) were the two most connected peptides in the kinase-associated network. Out of these, phosphorylated modifications in histone kinase SNF1 have functioned in mitosis and interphase of T. gondii, as well as in the regulation of gene expression relevant to differentiation. Our study discovered a remarkable difference in the abundance of phosphopeptides between the sporulated oocysts and tachyzoites of the virulent ToxoDB#9 (PYS) strain of T. gondii, which may provide a new resource for understanding stage-specific differences in PTMs and may enhance the illustration of the regulatory mechanisms contributing to the development and infectivity of T. gondii.

Eimeria proteins: order amidst disorder.

Apicomplexans are important pathogens that cause severe infections in humans and animals. The biology and pathogeneses of these parasites have shown that proteins are intrinsically modulated during developmental transitions, physiological processes and disease progression. Also, proteins are integral components of parasite structural elements and organelles. Among apicomplexan parasites, Eimeria species are an important disease aetiology for economically important animals wherein identification and characterisation of proteins have been long-winded. Nonetheless, this review seeks to give a comprehensive overview of constitutively expressed Eimeria proteins. These molecules are discussed across developmental stages, organelles and sub-cellular components vis-à-vis their biological functions. In addition, hindsight and suggestions are offered with intention to summarise the existing trend of eimerian protein characterisation and to provide a baseline for future studies.

Molecular characterization and immune protection by cystathionine ß-synthase from Eimeria tenella.

Eimeria tenella is an obligate intracellular apicomplexan parasite that causes avian coccidiosis and leads to severe economic losses in the global poultry industry. Cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CGL) act together to generate H2S in the reverse transsulfuration pathway. In this study, E. tenella Cystathionine ß-synthase (EtCBS) was cloned using rapid amplification of cDNA 5'-ends (5'RACE) and characterized, and its immunoprotective effects were evaluated. The recombinant EtCBS protein (rEtCBS) was expressed and successfully recognized by anti-sporozoites (Spz) protein rabbit serum. EtCBS mRNA levels were highest in Spz by qPCR, and the protein expression levels were higher in unsporulated oocysts (UO) than in other stages by Western blot. Indirect immunofluorescence showed that EtCBS protein was found on the surface of Spz and second-generation merozoites (Mrz). The invasion inhibition assays showed that rabbit anti-rEtCBS polyclonal antibodies effectively inhibited parasite invasion host cells. Chickens immunized with rEtCBS protein showed prominently increased weight gains and decreased oocyst output compared to nonimmunized and infected control group. The results suggest that EtCBS could be a potential vaccine candidate against E. tenella.

Toxoplasma gondii Tyrosine-Rich Oocyst Wall Protein: A Closer Look through an In Silico Prism.

Toxoplasmosis is a global threat with significant zoonotic concern. The present in silico study was aimed at determination of bioinformatics features and immunogenic epitopes of a tyrosine-rich oocyst wall protein (TrOWP) of Toxoplasma gondii. After retrieving the amino acid sequence from UniProt database, several parameters were predicted including antigenicity, allergenicity, solubility and physico-chemical features, signal peptide, transmembrane domain, and posttranslational modifications. Following secondary and tertiary structure prediction, the 3D model was refined, and immunogenic epitopes were forecasted. It was a 25.57 kDa hydrophilic molecule with 236 residues, a signal peptide, and significant antigenicity scores. Moreover, several linear and conformational B-cell epitopes were present. Also, potential mouse and human cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes were predicted in the sequence. The findings of the present in silico study are promising as they render beneficial characteristics of TrOWP to be included in future vaccination experiments.

Acute infection with Toxoplasma gondii oocysts preferentially activates non-neuronal cells expressing serotonin in the jejunum of rats.

The interaction of Toxoplasma gondii with the gastrointestinal tract of its host is highly regulated. Once ingested, the parasite crosses the epithelium without altering the permeability of the intestinal barrier. Nevertheless, many studies report alterations ranging from structural to functional damage in cells and tissues that make up the wall of the small and large intestine. Although the immune response to the parasite has been extensively studied, the role of serotonin (5-HT) in toxoplasmosis is poorly understood. Here we investigate the distribution of cells expressing 5-HT and its effects on cells and tissues of the jejunal wall of rats after 2, 3, or 7 days of T. gondii infection. KEY RESULTS: Our results show that transposition of the jejunal epithelium by T. gondii leads to ruptures in the basement membrane and activation of the immune system, as confirmed by the decrease in laminin immunostaining and the increase in the number of mast cells, respectively. CONCLUSIONS AND INFERENCES: We showed an increase in the number of enterochromaffin cells and mast cells expressing 5-HT in the jejunal wall. We also observed that the percentage of serotonergic mast cells increased in the total population. Thus, we can suggest that oral infection by T. gondii oocysts preferentially activates non-neuronal cells expressing 5-HT. Together, these results may explain both the changes in the extracellular matrix and the morphology of the enteric ganglia.

Global transcriptome landscape of the rabbit protozoan parasite Eimeria stiedae.

BACKGROUND: Coccidiosis caused by Eimeria stiedae is a widespread and economically significant disease of rabbits. The lack of studies on the life-cycle development and host interactions of E. stiedae at the molecular level has hampered our understanding of its pathogenesis. METHODS: In this study, we present a comprehensive transcriptome landscape of E. stiedae to illustrate its dynamic development from unsporulated oocysts to sporulated oocysts, merozoites, and gametocytes, and to identify genes related to parasite-host interactions during parasitism using combined PacBio single-molecule real-time and Illumina RNA sequencing followed by bioinformatics analysis and qRT-PCR validation. RESULTS: In total, 12,582 non-redundant full-length transcripts were generated with an average length of 1808 bp from the life-cycle stages of E. stiedae. Pairwise comparisons between stages revealed 8775 differentially expressed genes (DEGs) showing highly significant description changes, which compiled a snapshot of the mechanisms underlining asexual and sexual biology of E. stiedae including oocyst sporulation between unsporulated and sporulated oocysts; merozoite replication between sporulated oocysts and merozoites; and gametophyte development and gamete generation between merozoites and gametocytes. Further, 248 DEGs were grouped into nine series clusters and five groups by expression patterns, and showed that parasite-host interaction-related genes predominated in merozoites and gametocytes and were mostly involved in steroid biosynthesis and lipid metabolism and carboxylic acid. Additionally, co-expression analyses identified genes associated with development and host invasion in unsporulated and sporulated oocysts and immune interactions during gametocyte parasitism. CONCLUSIONS: This is the first study, to our knowledge, to use the global transcriptome profiles to decipher molecular changes across the E. stiedae life cycle, and these results not only provide important information for the molecular characterization of E. stiedae, but also offer valuable resources to study other apicomplexan parasites with veterinary and public significance.

Expanding the Known Repertoire of C-Type Lectin Receptors Binding to Toxoplasma gondii Oocysts Using a Modified High-Resolution Immunofluorescence Assay.

The environmental stage of the apicomplexan Toxoplasma gondii oocyst is vital to its life cycle but largely understudied. Because oocysts are excreted only by infected felids, their availability for research is limited. We report the adaptation of an agarose-based method to immobilize minute amounts of oocysts to perform immunofluorescence assays. Agarose embedding allows high-resolution confocal microscopy imaging of antibodies binding to the oocyst surface as well as unprecedented imaging of intracellular sporocyst structures with Maclura pomifera agglutinin after on-slide permeabilization of the immobilized oocysts. To identify new possible molecules binding to the oocyst surface, we used this method to screen a library of C-type lectin receptor (CLR)-human IgG constant region fusion proteins from the group of related CLRs called the Dectin-1 cluster against oocysts. In addition to CLEC7A that was previously reported to decorate T. gondii oocysts, we present experimental evidence for specific binding of three additional CLRs to the surface of this stage. We discuss how these CLRs, known to be expressed on neutrophils, dendritic cells, or macrophages, could be involved in the early immune response by the host, such as oocyst antigen uptake in the intestine. In conclusion, we present a modified immunofluorescence assay technique that allows material-saving immunofluorescence microscopy with T. gondii oocysts in a higher resolution than previously published, which allowed us to describe three additional CLRs binding specifically to the oocyst surface.IMPORTANCE Knowledge of oocyst biology of Toxoplasma gondii is limited, not the least due to its limited availability. We describe a method that permits us to process minute amounts of oocysts for immunofluorescence microscopy without compromising their structural properties. This method allowed us to visualize internal structures of sporocysts by confocal microscopy in unprecedented quality. Moreover, the method can be used as a low- to medium-throughput method to screen for molecules interacting with oocysts, such as antibodies, or compounds causing structural damage to oocysts (i.e., disinfectants). Using this method, we screened a small library of C-type lectin receptors (CLRs) present on certain immune cells and found three CLRs able to decorate the oocyst wall of T. gondii and which were not known before to bind to oocysts. These tools will allow further study into oocyst wall composition and could also provoke experiments regarding immunological recognition of oocysts.