TparvaDB: a database to support Theileria parva vaccine development

We describe the development of TparvaDB, a comprehensive resource to facilitate research towards development of an East Coast fever vaccine, by providing an integrated user-friendly database of all genome and related data currently available for Theileria parva. TparvaDB is based on the Generic Model Organism Database (GMOD) platform. It contains a complete reference genome sequence, Expressed Sequence Tags (ESTs), Massively Parallel Signature Sequencing (MPSS) expression tag data and related information from both public and private repositories. The Artemis annotation workbench provides online annotation functionality. TparvaDB represents a resource that will underpin and promote ongoing East Coast fever vaccine development and biological research

Plasmodium bergheisporozoites in nonreplicative vacuole are eliminated by a PI3P-mediated autophagy-independent pathway

The protozoan parasitePlasmodium, causative agent of malaria, invades hepatocytes by invaginating the host cell plasma membrane and forming a parasitophorous vacuole membrane (PVM). Surrounded by this PVM, the parasite undergoes extensive replication. Parasites inside a PVM provoke thePlasmodium-associated autophagy-related (PAAR) response. This is characterised by a long-lasting association of the autophagy marker protein LC3 with the PVM, which is not preceded by phosphatidylinositol 3-phosphate (PI3P)-labelling. Prior to productive invasion, sporozoites transmigrate several cells and here we describe that a proportion of traversing sporozoites become trapped in a transient traversal vacuole, provoking a host cell response that clearly differs from the PAAR response. These trapped sporozoites provoke PI3P-labelling of the surrounding vacuolar membrane immediately after cell entry, followed by transient LC3-labelling and elimination of the parasite by lysosomal acidification. Our data suggest that this PI3P response is not only restricted to sporozoites trapped during transmigration but also affects invaded parasites residing in a compromised vacuole. Thus, host cells can employ a pathway distinct from the previously described PAAR response to efficiently recognise and eliminatePlasmodiumparasites.Host-parasite interactio

Whole-genome sequencing of Theileria parva strains provides insight into parasite migration and diversification in the african continent

The disease caused by the apicomplexan protozoan parasite Theileria parva, known as East Coast fever or Corridor disease, is one of the most serious cattle diseases in Eastern, Central, and Southern Africa. We performed whole-genome sequencing of nine T. parva strains, including one of the vaccine strains (Kiambu 5), field isolates from Zambia, Uganda, Tanzania, or Rwanda, and two buffalo-derived strains. Comparison with the reference Muguga genome sequence revealed 34 814–121 545 single nucleotide polymorphisms (SNPs) that were more abundant in buffalo-derived strains. High-resolution phylogenetic trees were constructed with selected informative SNPs that allowed the investigation of possible complex recombination events among ancestors of the extant strains. We further analysed the dN/dS ratio (non-synonymous substitutions per non-synonymous site divided by synonymous substitutions per synonymous site) for 4011 coding genes to estimate potential selective pressure. Genes under possible positive selection were identified that may, in turn, assist in the identification of immunogenic proteins or vaccine candidates. This study elucidated the phylogeny of T. parva strains based on genome-wide SNPs analysis with prediction of possible past recombination events, providing insight into the migration, diversification, and evolution of this parasite species in the African continent

A bovine lymphosarcoma cell line infected with theileria annulata exhibits an irreversible reconfiguration of host cell gene expression

Theileria annulata, an intracellular parasite of bovine lymphoid cells, induces substantial phenotypic alterations to its host cell including continuous proliferation, cytoskeletal changes and resistance to apoptosis. While parasite induced modulation of host cell signal transduction pathways and NFκB activation are established, there remains considerable speculation on the complexities of the parasite directed control mechanisms that govern these radical changes to the host cell. Our objectives in this study were to provide a comprehensive analysis of the global changes to host cell gene expression with emphasis on those that result from direct intervention by the parasite. By using comparative microarray analysis of an uninfected bovine cell line and its Theileria infected counterpart, in conjunction with use of the specific parasitacidal agent, buparvaquone, we have identified a large number of host cell gene expression changes that result from parasite infection. Our results indicate that the viable parasite can irreversibly modify the transformed phenotype of a bovine cell line. Fifty percent of genes with altered expression failed to show a reversible response to parasite death, a possible contributing factor to initiation of host cell apoptosis. The genes that did show an early predicted response to loss of parasite viability highlighted a sub-group of genes that are likely to be under direct control by parasite infection. Network and pathway analysis demonstrated that this sub-group is significantly enriched for genes involved in regulation of chromatin modification and gene expression. The results provide evidence that the Theileria parasite has the regulatory capacity to generate widespread change to host cell gene expression in a complex and largely irreversible manner

Expression Analysis of the Theileria parva Subtelomere-Encoded Variable Secreted Protein Gene Family

Background The intracellular protozoan parasite Theileria parva transforms bovine lymphocytes inducing uncontrolled proliferation. Proteins released from the parasite are assumed to contribute to phenotypic changes of the host cell and parasite persistence. With 85 members, genes encoding subtelomeric variable secreted proteins (SVSPs) form the largest gene family in T. parva. The majority of SVSPs contain predicted signal peptides, suggesting secretion into the host cell cytoplasm. Methodology/Principal Findings We analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Microarray and quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was largely defined by the parasite genotype and not by host background or cell type, and found to be relatively stable in vitro over a period of two months. Interestingly, immunofluorescence analysis carried out on cell lines established from a cloned parasite showed that expression of a single SVSP encoded by TP03_0882 is limited to only a small percentage of parasites. Epitope-tagged TP03_0882 expressed in mammalian cells was found to translocate into the nucleus, a process that could be attributed to two different nuclear localisation signals. Conclusions Our analysis reveals a complex pattern of Theileria SVSP mRNA expression, which depends on the parasite genotype. Whereas in cell lines established from a cloned parasite transcripts can be found corresponding to a wide range of SVSP genes, only a minority of parasites appear to express a particular SVSP protein. The fact that a number of SVSPs contain functional nuclear localisation signals suggests that proteins released from the parasite could contribute to phenotypic changes of the host cell. This initial characterisation will facilitate future studies on the regulation of SVSP gene expression and the potential biological role of these enigmatic proteins

Stochastic induction of Theileria annulata merogony in vitro by chloramphenicol

Theileria annulata inhabits the cytoplasm of bovine leukocytes where it can be found as a multinucleated schizont. The schizont is the pathogenic stage of the life cycle and by interfering with host signalling pathways, it induces unlimited host cell proliferation and protection against apoptosis. In the infected animal, the schizont differentiates to the merozoite life cycle stage in a process called merogony. This takes place within the host leukocyte, resulting in the production of merozoites that are subsequently released by leukocyte lysis. In established cultures of T. annulata-transformed cells, merogony does not spontaneously occur, but the process can be activated by a shift in temperature. In this study we show that chloramphenicol induces schizont differentiation in proliferating T. annulata-transformed cells. We demonstrate that chloramphenicol-induced merogony is inherently asynchronous and has a quantitative basis. The process is accompanied by the down-regulation of schizont-specific surface proteins, de novo expression of merozoite-specific markers such as Tamr1 and Tams1 and the morphological hallmarks of merogony. Chloramphenicol-induced parasite differentiation was found to be associated with diminished proliferation potential and extensive morphological changes of the host cell, including increased numbers of pseudopodia. Significantly, chloramphenicol treatment can accelerate merogony induced by elevated temperature, supporting postulation that the differentiation event is a stochastic process that can be manipulated to alter the outcome of parasitic infection

Analysis of liver stage development in and merozoite release from hepatocytes

Exoerythrocytic Plasmodium parasites infect hepatocytes and develop to huge multinucleated schizonts inside a parasitophorous vacuole. Finally, thousands of merozoites are formed and released into the host cell cytoplasm by complete disintegration of the parasitophorous vacuole membrane. This, in turn, results in death and detachment of the infected hepatocyte, followed by the formation of merosomes. The fast growth of the parasite and host cell detachment are hallmarks of liver stage development and can easily be monitored. Here, we describe how to translate these observations into assays for characterizing parasite development. Additionally, other recently introduced techniques and tools to analyze and manipulate liver stage parasites are also discussed