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.

Dynamically expressed genes provide candidate viability biomarkers in a model coccidian

Eimeria parasites cause enteric disease in livestock and the closely related Cyclospora cayetanensis causes human disease. Oocysts of these coccidian parasites undergo maturation (sporulation) before becoming infectious. Here, we assessed transcription in maturing oocysts of Eimeria acervulina, a widespread chicken parasite, predicted gene functions, and determined which of these genes also occur in C. cayetanensis. RNA-Sequencing yielded ~2 billion paired-end reads, 92% of which mapped to the E. acervulina genome. The ~6,900 annotated genes underwent temporally-coordinated patterns of gene expression. Fifty-three genes each contributed >1,000 transcripts per million (TPM) throughout the study interval, including cation-transporting ATPases, an oocyst wall protein, a palmitoyltransferase, membrane proteins, and hypothetical proteins. These genes were enriched for 285 gene ontology (GO) terms and 13 genes were ascribed to 17 KEGG pathways, defining housekeeping processes and functions important throughout sporulation. Expression differed in mature and immature oocysts for 40% (2,928) of all genes; of these, nearly two-thirds (1,843) increased their expression over time. Eight genes expressed most in immature oocysts, encoding proteins promoting oocyst maturation and development, were assigned to 37 GO terms and 5 KEGG pathways. Fifty-six genes underwent significant upregulation in mature oocysts, each contributing at least 1,000 TPM. Of these, 40 were annotated by 215 GO assignments and 9 were associated with 18 KEGG pathways, encoding products involved in respiration, carbon fixation, energy utilization, invasion, motility, and stress and detoxification responses. Sporulation orchestrates coordinated changes in the expression of many genes, most especially those governing metabolic activity. Establishing the long-term fate of these transcripts in sporulated oocysts and in senescent and deceased oocysts will further elucidate the biology of coccidian development, and may provide tools to assay infectiousness of parasite cohorts. Moreover, because many of these genes have homologues in C. cayetanensis, they may prove useful as biomarkers for risk.

Role of Plasmodium berghei ookinete surface and oocyst capsule protein, a novel oocyst capsule-associated protein, in ookinete motility

Background Plasmodium sp., which causes malaria, must first develop in mosquitoes before being transmitted. Upon ingesting infected blood, gametes form in the mosquito lumen, followed by fertilization and differentiation of the resulting zygotes into motile ookinetes. Within 24 h of blood ingestion, these ookinetes traverse mosquito epithelial cells and lodge below the midgut basal lamina, where they differentiate into sessile oocysts that are protected by a capsule. Methods We identified an ookinete surface and oocyst capsule protein (OSCP) that is involved in ookinete motility as well as oocyst capsule formation. Results We found that knockout of OSCP in parasite decreases ookinete gliding motility and gradually reduces the number of oocysts. On day 15 after blood ingestion, the oocyst wall was significantly thinner. Moreover, adding anti-OSCP antibodies decreased the gliding speed of wild-type ookinetes in vitro. Adding anti-OSCP antibodies to an infected blood meal also resulted in decreased oocyst formation. Conclusion These findings may be useful for the development of a transmission-blocking tool for malaria. Graphical abstract

Molecular identification and biological characterization of Cryptosporidium muris from camels (Camelus bactrianus) in China

Background Cryptosporidium is an opportunistic pathogen that infects a wide variety of vertebrates. The aim of the present study was to characterize Cryptosporidium spp. isolates from Bactrian camels and to foster further understanding of the biological characteristics of the pathogen. Methods Fecal specimens were collected from two 4-year-old Bactrian camels resident at the Kaifeng City Zoo in China and examined for Cryptosporidium. Fecal specimens were screened using the floatation method, and then genomic DNA was extracted from the oocysts and identified by nested-PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene, the actin gene and the Cryptosporidium oocyst wall-protein (COWP) gene. Subtype analysis was performed based on four minisatellite (MS) loci (MS1, MS2, MS3 and MS16) that were aligned and phylogenetically analyzed to determine the species and subtype of Cryptosporidium. We then established a BALB/c mice infection model and further verified the results through clinical status, pattern of oocyst excretion and histological examination. Results Cryptosporidium oocyst isolates from the two Bactrian camels had an average (± standard deviation) size of 7.49 ± 0.13 × 5.70 ± 0.10 μm (n = 50). The sequencing and phylogenetic analysis confirmed the species as C. muris. Multilocus sequence typing analysis indicated that the subtypes were M13, M4, M1 and M5. Following the inoculation of BALB/c mice, we found that the prepatent period and number of oocysts per gram increased with increasing infective dose. Oocysts were first detected in the feces of BALB/c mice at 7–8 days post-infection (dpi), with levels peaking twice thereafter, at 15–16 dpi and 19–20 dpi. Histology and scanning electron microscopy studies showed that the stomach contained gastric pits filled with Cryptosporidium that adhered to the surface of gastric mucosa gland epithelial cells, causing the latter to deform, swell and become disordered. Conclusions The findings of this study indicated that oocysts isolated from Bactrian camels were from C. muris. This is the first report of C. muris isolated from camels in China. More epidemiological data are needed to understand the prevalence and transmission of C. muris in camels in different geographic areas. Graphical abstract

Use of Oxidative Stress Responses to Determine the Efficacy of Inactivation Treatments on Cryptosporidium Oocysts

Cryptosporidium oocysts are known for being very robust, and their prolonged survival in the environment has resulted in outbreaks of cryptosporidiosis associated with the consumption of contaminated water or food. Although inactivation methods used for drinking water treatment, such as UV irradiation, can inactivate Cryptosporidium oocysts, they are not necessarily suitable for use with other environmental matrices, such as food. In order to identify alternative ways to inactivate Cryptosporidium oocysts, improved methods for viability assessment are needed. Here we describe a proof of concept for a novel approach for determining how effective inactivation treatments are at killing pathogens, such as the parasite Cryptosporidium. RNA sequencing was used to identify potential up-regulated target genes induced by oxidative stress, and a reverse transcription quantitative PCR (RT-qPCR) protocol was developed to assess their up-regulation following exposure to different induction treatments. Accordingly, RT-qPCR protocols targeting thioredoxin and Cryptosporidium oocyst wall protein 7 (COWP7) genes were evaluated on mixtures of viable and inactivated oocysts, and on oocysts subjected to various potential inactivation treatments such as freezing and chlorination. The results from the present proof-of-concept experiments indicate that this could be a useful tool in efforts towards assessing potential technologies for inactivating Cryptosporidium in different environmental matrices. Furthermore, this approach could also be used for similar investigations with other pathogens.

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

ABSTRACT 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.

Role of Plasmodium berghei ookinete surface and oocyst capsule protein, a novel oocyst capsule-associated protein, in ookinete motility

Background: Plasmodium sp., which causes malaria, must first develop in mosquitoes before being transmitted. Upon ingesting infected blood, gametes form in the mosquito lumen, followed by fertilization and differentiation of the resulting zygotes into motile ookinetes. Within 24 hours of blood ingestion, these ookinetes traverse mosquito epithelial cells and lodge below the midgut basal lamina, where they differentiate into sessile oocysts that are protected by a capsule. Methods: We identified an ookinete surface and oocyst capsule protein (OSCP) that is involved in ookinete motility as well as oocyst capsule formation. Results: We found that knockout of OSCP in parasite decreases ookinete gliding motility and gradually reduces the number of oocysts. On day 15 after blood ingestion, the oocyst wall was significantly thinner. Moreover, adding anti-OSCP antibodies decreased the gliding speed of wild type ookinetes in vitro. Adding anti-OSCP antibodies to an infected blood meal also resulted in decreased oocyst formation. Conclusion: These findings may be useful for the development of transmission-blocking tools for malaria.

Insulinase-like Protease 1 Contributes to Macrogamont Formation in Cryptosporidium parvum

ABSTRACT The apicomplexan parasite Cryptosporidium parvum contains an expanded family of 22 insulinase-like proteases (INS), a feature that contrasts with their otherwise streamlined genome. Here, we examined the function of INS1, which is most similar to the human insulinase protease that cleaves a variety of small peptide substrates. INS1 is an M16A clan member and contains a signal peptide, an N-terminal domain with the HXXEH active site, followed by three inactive domains. Unlike previously studied C. parvum INS proteins that are expressed in sporozoites and during merogony, INS1 was expressed exclusively in macrogamonts, where it was localized in small cytoplasmic vesicles. Although INS1 did not colocalize with the oocyst wall protein recognized by the antibody OW50, immune-electron microscopy indicated that INS1 resides in small vesicles in the secretory system. Notably, these small INS1-positive vesicles were often in close proximity to large OW50-positive vacuoles resembling wall-forming bodies, which contain precursors for oocyst wall formation. Genetic deletion of INS1, or replacement with an active-site mutant, resulted in lower formation of macrogamonts in vitro and reduced oocyst shedding in vivo. Our findings reveal that INS1 functions in the formation or maturation of macrogamonts and that its loss results in attenuated virulence in immunocompromised mice. IMPORTANCE Cryptosporidiosis is a debilitating diarrheal disease in young children in developing countries. The absence of effective treatments or vaccines makes this infection very difficult to manage in susceptible populations. Although the oral dose of oocysts needed to cause infection is low, infected individuals shed very high numbers of oocysts, readily contaminating the environment. Our studies demonstrate that the protease INS1 is important for formation of female sexual stages and that in its absence, parasites produce fewer oocysts and are attenuated in immunocompromised mice. These findings suggest that mutants lacking INS1, or related proteases, are useful for further characterizing the pathway that leads to macrogamont maturation and oocyst wall formation.

Molecular Methods for the Detection of Toxoplasma gondii Oocysts in Fresh Produce: An Extensive Review

Human infection with the important zoonotic foodborne pathogen Toxoplasma gondii has been associated with unwashed raw fresh produce consumption. The lack of a standardised detection method limits the estimation of fresh produce as an infection source. To support method development and standardisation, an extensive literature review and a multi-attribute assessment were performed to analyse the key aspects of published methods for the detection of T. gondii oocyst contamination in fresh produce. Seventy-seven published studies were included, with 14 focusing on fresh produce. Information gathered from expert laboratories via an online questionnaire were also included. Our findings show that procedures for oocyst recovery from fresh produce mostly involved sample washing and pelleting of the washing eluate by centrifugation, although washing procedures and buffers varied. DNA extraction procedures including mechanical or thermal shocks were identified as necessary steps to break the robust oocyst wall. The most suitable DNA detection protocols rely on qPCR, mostly targeting the B1 gene or the 529 bp repetitive element. When reported, validation data for the different detection methods were not comparable and none of the methods were supported by an interlaboratory comparative study. The results of this review will pave the way for an ongoing development of a widely applicable standard operating procedure.

Effect of household bleach on the structure of the sporocyst wall of Toxoplasma gondii

Toxoplasma gondii oocysts are responsible for food- and water-borne infections in humans worldwide. They are resistant to common chemical disinfectants, including chlorinated products, presumably due to the structure and molecular nature of the oocyst wall but also the sporocyst wall. In this study, we used fluorescence microscopy and transmission electron microscopy to characterise the structure of both the oocyst and sporocyst walls, exposed to household bleach. Bleach removed the outer layer of the oocyst wall and the outer layer of the wall of sporocysts exposed due to rupture of the oocyst wall. The loss of the outer sporocyst wall layer was associated with a decrease in its autofluorescence, which can be linked to the degradation of dityrosine cross-link proteins, and loss of Maclura pomifera lectin-reactive glycoproteins. This study suggests that the inner layers of the oocyst and sporocyst walls are the main structures responsible for the resistance of the parasite to household bleach.