TgM2AP participates in Toxoplasma gondii invasion of host cells and is tightly associated with the adhesive protein TgMIC2

Like other members of the medically important phylum Apicomplexa, Toxoplasma gondii is an obligate intracellular parasite that secretes several classes of proteins involved in the active invasion of target host cells. Proteins in apical secretory organelles known as micronemes have been strongly implicated in parasite attachment to host cells. TgMIC2 is a microneme protein with multiple adhesive domains that bind target cells and is mobilized onto the parasite surface during parasite attachment. Here, we describe a novel parasite protein, TgM2AP, which is physically associated with TgMIC2. TgM2AP complexes with TgMIC2 within 15 min of synthesis and remains associated with TgMIC2 in the micronemes, on the parasite surface during invasion and in the culture medium after release from the parasite plasma membrane. TgM2AP is proteolytically processed initially when its propeptide is removed during transit through the golgi and later while it occupies the parasite surface after discharge from the micronemes. We show that TgM2AP is a member of a protein family expressed by coccidian parasites including Neospora caninum and Eimeria tenella. This phylogenic conservation and association with a key adhesive protein suggest that TgM2AP is a fundamental component of the T. gondii invasion machinery.     

The role of sialyl glycan recognition in host tissue tropism of the avian parasite Eimeria tenella

Eimeria spp. are a highly successful group of intracellular protozoan parasites that develop within intestinal epithelial cells of poultry, causing coccidiosis. As a result of resistance against anticoccidial drugs and the expense of manufacturing live vaccines, it is necessary to understand the relationship between Eimeria and its host more deeply, with a view to developing recombinant vaccines. Eimeria possesses a family of microneme lectins (MICs) that contain microneme adhesive repeat regions (MARR). We show that the major MARR protein from Eimeria tenella, EtMIC3, is deployed at the parasite-host interface during the early stages of invasion. EtMIC3 consists of seven tandem MAR1-type domains, which possess a high specificity for sialylated glycans as shown by cell-based assays and carbohydrate microarray analyses. The restricted tissue staining pattern observed for EtMIC3 in the chicken caecal epithelium indicates that EtMIC3 contributes to guiding the parasite to the site of invasion in the chicken gut. The microarray analyses also reveal a lack of recognition of glycan sequences terminating in the N-glycolyl form of sialic acid by EtMIC3. Thus the parasite is well adapted to the avian host which lacks N-glycolyl neuraminic acid. We provide new structural insight into the MAR1 family of domains and reveal the atomic resolution basis for the sialic acid-based carbohydrate recognition. Finally, a preliminary chicken immunization trial provides evidence that recombinant EtMIC3 protein and EtMIC3 DNA are effective vaccine candidates.     

Development of a genetically engineered vaccine against poultry coccidiosis

Coccidiosis is caused by infection with Eimeria spp. The disease is responsible for major economic loss to the poultry industry unless infections are controlled by anticoccidial drugs. John Ellis and Fiona Tomley discuss recent research on the characterization and cloning of antigens from Eimeria spp and advances towards the development of genetically engineered vaccines against poultry coccidiosis.     

Genetic and biological characterisation of three cryptic Eimeria operational taxonomic units that infect chickens (Gallus gallus domesticus)

More than 68 billion chickens were produced globally in 2018, emphasising their major contribution to the production of protein for human consumption and the importance of their pathogens. Protozoan Eimeria spp. are the most economically significant parasites of chickens, incurring global costs of more than UK £10.4 billion per annum. Seven Eimeria spp. have long been recognised to infect chickens, with three additional cryptic operational taxonomic units (OTUs) first described more than 10 years ago. As the world’s farmers attempt to reduce reliance on routine use of antimicrobials in livestock production, replacing drugs that target a wide range of microbes with precise species- and sometimes strain-specific vaccines, the breakthrough of cryptic genetic types can pose serious problems. Consideration of biological characteristics including oocyst morphology, pathology caused during infection and pre-patent periods, combined with gene-coding sequences predicted from draft genome sequence assemblies, suggest that all three of these cryptic Eimeria OTUs possess sufficient genetic and biological diversity to be considered as new and distinct species. The ability of these OTUs to compromise chicken bodyweight gain and escape immunity induced by current commercially available anticoccidial vaccines indicates that they could pose a notable threat to chicken health, welfare, and productivity. We suggest the names Eimeria lata n. sp., Eimeria nagambie n. sp. and Eimeria zaria n. sp. for OTUs x, y and z, respectively, reflecting their appearance (x) or the origins of the first isolates of these novel species (y, z).     

A survey of genes in Eimeria tenella merozoites by EST sequencing

A study of about 500 expressed sequence tags (ESTs), derived from a merozoite cDNA library, was initiated as an approach to generate a larger pool of gene information on Eimeria tenella. Of the ESTs, 47.7% had matches with entries in the databases, including ribosomal proteins, metabolic enzymes and proteins with other functions, of which 14.3% represented previously known E. tenella genes. Thus over 50% of the ESTs had no significant database matches. The E. tenella EST dataset contained a range of highly abundant genes comparable with that found in the EST dataset of T. gondii and may thus reflect the importance of such molecules in the biology of the apicomplexan organisms. However, comparison of the two datasets revealed very few homologies between sequences of apical organelle molecules, and provides evidence for sequence divergence between these closely-related parasites. The data presented underpin the potential value of the EST strategy for the discovery of novel genes and may allow for a more rapid increase in the knowledge and understanding of gene expression in the merozoite life cycle stage of Eimeria spp.     

Proteomic analysis of Plasmodium in the mosquito: progress and pitfalls

Here we discuss proteomic analyses of whole cell preparations of the mosquito stages of malaria parasite development (i.e. gametocytes, microgamete, ookinete, oocyst and sporozoite) of Plasmodium berghei. We also include critiques of the proteomes of two cell fractions from the purified ookinete, namely the micronemes and cell surface. Whereas we summarise key biological interpretations of the data, we also try to identify key methodological constraints we have met, only some of which we were able to resolve. Recognising the need to translate the potential of current genome sequencing into functional understanding, we report our efforts to develop more powerful combinations of methods for the in silico prediction of protein function and location. We have applied this analysis to the proteome of the male gamete, a cell whose very simple structural organisation facilitated interpretation of data. Some of the in silico predictions made have now been supported by ongoing protein tagging and genetic knockout studies. We hope this discussion may assist future studies.     

The microneme proteins EtMIC4 and EtMIC5 of Eimeria tenella form a novel, ultra-high molecular mass protein complex that binds target host cells

Eimeria tenella, in common with other parasitic protozoa of the phylum Apicomplexa, invades host cells using an actinomyosin-powered "glideosome" complex and requires the secretion of adhesive proteins from the microneme organelles onto the parasite surface. Microneme proteins of E. tenella include EtMIC4, a transmembrane protein that has multiple thrombospondin type I domains and calcium-binding epidermal growth factor-like domains in its extracellular domain, and EtMIC5, a soluble protein composed of 11 tandemly repeated domains that belong to the plasminogen-apple-nematode superfamily. We show here that EtMIC4 and EtMIC5 interact to form an oligomeric, ultrahigh molecular mass protein complex. The complex was purified from lysed parasites by non-denaturing techniques, and the stoichiometry was shown to be [EtMIC4](2):[EtMIC5](1), with an octamer of EtMIC4 bound non-covalently to a tetramer of EtMIC5. The complex is formed within the parasite secretory pathway and is maintained after secretion onto the surface of the parasite. The purified complex binds to a number of epithelial cell lines in culture. Identification and characterization of this complex contributes to an overall understanding of the role of multimolecular protein complexes in specific interactions between pathogens and their hosts during infection.