Sue McKellar

Characterization of a polymorphic Theileria annulata surface protein (TaSP) closely related to PIM of Theileria parva: implications for use in diagnostic tests and subunit vaccines.

Theileria annulata is a tick-transmitted protozoan that causes tropical theileriosis, an often fatal leukoproliferative disorder of cattle. To characterize and identify parasite proteins suitable as diagnostic antigens and/or vaccine candidates, a cDNA clone encoding a macroschizont stage protein was isolated and characterized (here designated TaSP). The gene, present as a single copy within the parasite genome, is transcribed in the sporozoite and schizont stage and codes for a protein of about 315 amino acids, having a predicted molecular weight of 36 kDa. Allelic variants were found within single parasite isolates and between isolates originating from different geographical regions. The N-terminal part contains a predicted signal peptide and the C-terminal section encodes membrane-spanning regions. Comparison of a number of cDNA clones showed that both these sequence regions are conserved while the central region shows both size and amino acid sequence polymorphism. High identity of the N- and C-terminal regions with the polymorphic immunodominant molecule (PIM) of Theileria parva (identity of 93%), the existence of a central polymorphic region and two short introns within genomic clones suggest that the presented gene/protein may be the T. annulata homologue of PIM. However, the central region of TaSP has no significant identity with PIM, contains no repetitive peptide motifs and is shorter, resulting in a lower molecular weight. The existence of the predicted secretion signal peptide and membrane spanning regions suggest that TaSP is located at the parasite membrane.

Selection of diversity at putative glycosylation sites in the immunodominant merozoite/piroplasm surface antigen of Theileria parasites☆

The immunodominant merozoite/piroplasm surface antigen of Theileria parasites has potential as a diagnostic reagent and as a component of a sub-unit vaccine. This molecule is known to be antigenically diverse, and it is important to determine the nature and extent of this heterogeneity. In the present study nucleotide sequences, representing alleles of the gene (Tams1) encoding this molecule in Theileria annulata were compared to each other and to sequences of homologous genes in Theileria sergenti, Theileria buffeli and Theileria parva. This analysis revealed that a region of the polypeptide which contains putative N-linked glycosylation sites is particularly diverse and, in analogy to retroviral systems, may indicate selection of variable glycosylation sites or amino acid epitopes to evade the bovine immune response. This conclusion was also made from the results of a phylogenetic analysis which compared the variable region of the genes with a second region, which appeared to show no bias for diversity or functional constraint. The results indicated that the variable sequence encoding putative glycosylation sites has diverged, both within and between Theileria species, at a much faster rate than the rest of the molecule. Southern blot analysis of T. annulata populations from within a single geographical region detected six possible variant Tams1 alleles. However, a correlation between restriction-fragment-length polymorphism (RFLP) patterns detected by the Tams1-1 gene probe and geographical location could not be made. In addition, although a high prevalence of one particular RFLP was found, this is unlikely to be the result of a clonal population structure, as we present evidence for significant parasite genotypic variability within a single endemic region.

Phylogenetic analysis of Theileria and Babesia equi in relation to the establishment of parasite populations within novel host species and the development of diagnostic tests

The divergence of parasites is important for maintenance within an established host and spread to novel host species. In this paper we have carried out phylogenetic analyses of Theileria parasites isolated from different host species. This was performed with small subunit ribosomal RNA sequences available in the data bases and a novel sequence amplified from Theileria lestoquardi DNA. Similar phylogenetic studies were carried out with sequences representing the major merozoite/piroplasm surface antigen (mMPSA) from the data base, and novel sequences representing 2 mMPSA alleles from T. lestoquardi, a full length sequence of a Theileria taurotragi mMPSA gene and partial sequences of two new allelic variants of the Babesia equi mMPSA gene homologue. The analysis indicated that the pathogenic sheep parasite T. lestoquardi has most probably evolved from a common ancestor of T. annulata. Interestingly, the level of mMPSA sequence diversity found for T. lestoquardi was surprisingly low, while diversity between the B. equi sequences was higher than that found within any of the classical Theileria species. The possible implications of these results for the establishment of Theileria parasites within novel species are discussed. Extensive cross-reactivity of a range of antisera was found when tested against recombinant mMPSA polypeptides from different Theileria (including B. equi) species. The cross-reactivity between mMPSA polypeptides and sequence diversity are relevant for the development of species specific diagnostic tests.

A Theileria annulata DNA Binding Protein Localized to the Host Cell Nucleus Alters the Phenotype of a Bovine Macrophage Cell Line

ABSTRACT The apicomplexan parasite Theileria annulata is the only intracellular eukaryote that is known to induce the proliferation of mammalian cells. However, as the parasite undergoes stage differentiation, host cell proliferation is inhibited, and the leukocyte is eventually destroyed. We have isolated a parasite gene (SuAT1) encoding an AT hook DNA binding polypeptide that has a predicted signal peptide, PEST motifs, nuclear localization signals, and domains which indicate interaction with regulatory components of the higher eukaryotic cell cycle. The polypeptide is localized to the nuclei of macroschizont-infected cells and was detected at significant levels in cells that were undergoing parasite stage differentiation. Transfection of an uninfected transformed bovine macrophage cell line, BoMac, demonstrated that SuAT1 can modulate cellular morphology and alter the expression pattern of a cytoskeletal polypeptide in a manner similar to that found during the infection of leukocytes by the parasite. Our findings indicate that Theileria parasite molecules that are transported to the leukocyte nucleus have the potential to modulate the phenotype of infected cells.

Selection for Antigenic Diversity of Tams1, the Major Merozoite Antigen of Theileria annulataa

ABSTRACT: Tams1, the major merozoite/piroplasm surface antigen of Theileria annulata has the potential to be a component of a diagnostic ELISA test and be included in a recombinant subunit vaccine. However, the observation that this antigen displays diversity could constrain these applications. In this paper we have extensively characterized Tams1 diversity at the DNA level, using a PCR/sequencing strategy. Up to 44 alleles have been cloned and sequenced. The comparison of these alleles has identified regions of sequence conservation, variability and hyper‐variability. Computer analysis of these alleles has indicated that positive selection may operate on certain regions of Tams1. Expression and Western blot analysis of selected alleles has indicated that sequence diversity is reflected in altered antigenicity and a continuum of relatedness and antibody cross recognition may exist. The possible function of the sequence conservation and polymorphism within Tams1 is discussed in relation to protein structure, host cell invasion and immune evasion.

Infection of bovine cells by the protozoan parasite Theileria annulata modulates expression of the ISGylation system.

The apicomplexan parasite, Theileria annulata, dedifferentiates and induces continuous division of infected bovine myeloid cells. Re‐expression of differentiation markers and a loss of proliferation occur upon treatment with buparvaquone, implying that parasite factors actively maintain the altered status of the infected cell. The factors that induce this unique transformation event have not been identified. However, parasite polypeptides (TashAT family) that are located in the infected leucocyte nucleus have been postulated to function as modulators of host cell phenotype. In this study differential RNA display and proteomic analysis were used to identify altered mRNA and polypeptide expression profiles in a bovine macrophage cell line (BoMac) transfected with TashAT2. One of the genes identified by differential display was found to encode an ubiquitin‐like protease (bUBP43) belonging to the UBP43 family. The bUBP43 gene and the gene encoding its ubiquitin‐like substrate, bISG15, were expressed at a low level in T. annulata‐infected cells. However, infected cells were refractory to induction of elevated bISG15 expression by lipopolysaccharide or type 1 interferons while TashAT2‐transfected cells showed no induction when treated with camptothecin. Modulation of the ISGylation system may be of relevance to the establishment of the transformed infected host cell, as ISGylation is associated with resistance to intracellular infection by pathogens, stimulation of the immune response and terminal differentiation of leukaemic cells.

TashHN, a Theileria annulata encoded protein transported to the host nucleus displays an association with attenuation of parasite differentiation

The intracellular apicomplexan parasite, Theileria annulata, manipulates its bovine host cell by over‐riding the cells natural apoptotic response and inducing proliferation of the infected leukocyte. We have recently identified a T. annulata encoded family of polypeptides (TashATs) with characteristics that indicate that they are involved in control of host cell gene expression. Here we present data on another member of this family, TashHN, showing that it is located to the parasite and host cell nucleus. Immunoblot analysis demonstrated that, unlike TashAT2 and 3, TashHN displays three forms, the largest of which is enriched in the host nuclear fraction and appears to be phosphorylated. Northern and 5 prime race analyses identified multiple TashHN RNA species in infected cells that have retained the ability to differentiate. These transcripts showed subtly different kinetics, but all decreased during differentiation to the merozite, and two showed reduced levels prior to down‐regulation of the other TashATs. In addition, analyses of multiple cell lines that have become severely attenuated in their potential to differentiate, indicated a substantial increase in TashHN expression, with host nuclear reactivity particularly enhanced.

Directing differentiation in Theileria annulata: old methods and new possibilities for control of apicomplexan parasites

Apicomplexan parasites are major pathogens of humans and domesticated animals. The ability of these organisms to evade the host immune response and the emergence of drug-resistant parasites indicates a need for the identification of novel control strategies. Ideally, selected targets should be shared by a range of apicomplexans and fundamental to parasite biology. One process of apicomplexan biology which may provide this type of target is the molecular regulation of stage differentiation. This paper has reviewed studies carried out on differentiation of Theileria annulata and has highlighted general similarities with other apicomplexan differentiation steps. Similarities include asynchrony of differentiation, the loss (attenuation) of differentiation potential and an association between reduced proliferation and differentiation. In addition, novel data are presented assessing a possible role for a signal transduction mechanism or a direct involvement of classical heat-shock polypeptides in regulating differentiation of T. annulata in vitro. These studies, and previously published data, have led to the postulation that progression to the next stage of the life-cycle can be predetermined and involves the attainment of a quantitative threshold by regulators of gene expression. A modification of this model takes into account that for certain in-vitro systems, or differentiation steps in vivo, the process has to be initiated by alteration of the extracellular environment. Work which has shown that the time taken to achieve differentiation can be increased or decreased is also outlined. The ability to change the timing of differentiation suggests that the associated regulatory mechanism could be manipulated directly to significantly influence the outcome of an apicomplexan infection. The observation that a number of existing drugs and control strategies may exert their protective effect by altering differentiation potential supports this possibility.

Modulation of NF-kappaB activation in Theileria annulata-infected cloned cell lines is associated with detection of parasite-dependent IKK signalosomes and disruption of the actin cytoskeleton.

Apicomplexan parasites within the genus Theileria have the ability to induce continuous proliferation and prevent apoptosis of the infected bovine leukocyte. Protection against apoptosis involves constitutive activation of the bovine transcription factor NF‐κB in a parasite‐dependent manner. Activation of NF‐κB is thought to involve recruitment of IKK signalosomes at the surface of the macroschizont stage of the parasite, and it has been postulated that additional host proteins with adaptor or scaffolding function may be involved in signalosome formation. In this study two clonal cell lines were identified that show marked differences in the level of activated NF‐κB. Further characterization of these lines demonstrated that elevated levels of activated NF‐κB correlated with increased resistance to cell death and detection of parasite‐associated IKK signalosomes, supporting results of our previous studies. Evidence was also provided for the existence of host‐ and parasite‐dependent NF‐κB activation pathways that are influenced by the architecture of the actin cytoskeleton. Despite this influence, it appears that the primary event required for formation of the parasite‐dependent IKK signalosome is likely to be an interaction between a signalosome component and a parasite‐encoded surface ligand.

A role for tertiary structure in the generation of antigenic diversity and molecular association of the Tams1 polypeptide in Theileria annulata

The major merozoite-piroplasm surface antigen (mMPSA) of Theileria annulata, Tams1, is known to be antigenically diverse. The possession of variable N-linked glycosylation sites and removal of monoclonal antibody 5E1 reactivity by mild periodate treatment suggested, previously, that divergent epitopes may be conferred by secondary modification. This study has shown that monoclonal antibody 5E1 and polyspecific antisera raised against the native protein react against divergent amino acid epitopes that are dependent on a molecular conformation that is sensitive to periodate. Therefore, no experimental evidence exists to confirm the sequence prediction that Tams1 undergoes N-linked glycosylation. Data is also presented indicating that the conformation of the antigen results in presentation of divergent regions on the external surface of the molecule, while conserved regions are more likely to be internal and hidden. In addition, non-reducing SDS-PAGE analysis demonstrated that Tams1 can undergo molecular association to form homo-dimers, trimers and multimers. The potential influence of tertiary structure and inter-molecular association on Tams1 diversity and function is discussed.