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F Tomley

Publications and source records attributed to F Tomley.

13 recordsLinked to original sources
PubMed2006

Enzymes of type II fatty acid synthesis and apicoplast differentiation and division in Eimeria tenella.

Apicomplexan parasites, Eimeria tenella, Plasmodium spp. and Toxoplasma gondii, possess a homologous plastid-like organelle termed the apicoplast, derived from the endosymbiotic enslavement of a photosynthetic alga. However, currently no eimerian nuclear encoded apicoplast targeted proteins have been identified, unlike in Plasmodium spp. and T. gondii. In this study, we demonstrate that nuclear encoded enoyl reductase of E. tenella (EtENR) has a predicted N-terminal bipartite transit sequence, typical of apicoplast-targeted proteins. Using a combination of immunocytochemistry and EM we demonstrate that this fatty acid biosynthesis protein is located in the apicoplast of E. tenella. Using the EtENR as a tool to mark apicoplast development during the Eimeria lifecycle, we demonstrate that nuclear and apicoplast division appear to be independent events, both organelles dividing prior to daughter cell formation, with each daughter cell possessing one to four apicoplasts. We believe this is the first report of multiple apicoplasts present in the infectious stage of an apicomplexan parasite. Furthermore, the microgametes lacked an identifiable apicoplast consistent with maternal inheritance via the macrogamete. It was found that the size of the organelle and the abundance of EtENR varied with developmental stage of the E. tenella lifecycle. The high levels of EtENR protein observed during asexual development and macrogametogony is potentially associated with the increased synthesis of fatty acids required for the rapid formation of numerous merozoites and for the extracellular development and survival of the oocyst. Taken together the data demonstrate that the E. tenella apicoplast participates in type II fatty acid biosynthesis with increased expression of ENR during parasite growth. Apicoplast division results in the simultaneous formation of multiple fragments. The division mechanism is unknown, but is independent of nuclear division and occurs prior to daughter formation.

Amino Acid Sequence↗
PubMed2003

High-pressure freezing in the study of animal pathogens.

High-pressure freezing is applicable to both morphological and immunocytochemical studies. We are investigating the morphogenesis of foot-and-mouth disease virus and African swine fever virus by the use of high-pressure freezing of infected cells. Foot-and-mouth disease virus particles are not detected in sections of conventionally immersion-fixed infected cells, but when the cells are prepared by high-pressure freezing, newly formed virions are readily seen throughout the cell. We report two methods for high-pressure freezing of virally infected cells: first, two sapphire discs frozen 'face to face' with a narrow spacer to prevent cell damage and, second, a fibrous filter substrate that can be easily cut into discs to fit into the freezing planchettes. Cells readily adhere to the fibres in vitro, and the complete disc can be rapidly transferred to the planchettes for freezing. Immunolabelling studies of the microneme proteins of the parasite Eimeria tenella indicate that high-pressure freezing followed by freeze-substitution in acetone with uranyl acetate allows high-sensitivity immunolabelling for these proteins.

African Swine Fever Virus↗
PubMed2001

Genomic organisation and developmentally regulated expression of an apicomplexan aspartyl proteinase.

The cDNA for an aspartyl proteinase, termed eimepsin, was isolated from an Eimeria tenella sporulated oocyst library and the deduced amino acid sequence found to be almost identical to a previously described aspartyl proteinase from E. acervulina (97.4% amino acid identity). An E. tenella cosmid clone covering the entire eimepsin gene was cloned and characterised. Sequencing revealed that the eimepsin gene spans 2.9 kb and consists of 18 exons and 17 introns. The 5' flanking region sequence of the gene contains a putative transcriptional promoter sequence (TATAAA box) and three potential transcription initiator sites (Inr sites). Expression of eimepsin at the mRNA and protein level is developmentally regulated during oocyst sporulation. The eimepsin transcript was detected in unsporulated oocysts and increased in abundance during the early part of sporulation when the oocyst undergoes nuclear division and blast formation. Thereafter, the level of the eimepsin transcript decreases and in the excysted sporozoite, no eimepsin-specific RNA was detected. Expression of eimepsin lags behind transcript expression by some hours, and the protein accumulates in the oocyst during sporocyst and sporozoite formation.

Amino Acid Sequence↗
PubMed2001

Aspartyl proteinase genes from apicomplexan parasites: evidence for evolution of the gene structure.

Aspartyl proteinases are a widely distributed family of enzymes. All vertebrate aspartyl proteinases share a conserved nine-exon gene structure, but in other organisms the structure of aspartyl proteinase genes varies considerably. The exon-intron patterns generally reflect phylogeny based on amino acid sequences. However, close comparison of these gene structures reveals some striking features, such as the conservation of intron positions and intron phases between aspartyl proteinases from nematodes and apicomplexans. Here, we discuss the implications of gene structure for the possible evolution of the aspartyl proteinase family, with particular reference to the plasmepsins of Plasmodium falciparum and eimepsin from Eimeria tenella.

Amino Acid Sequence↗
PubMed2000

Differential localisation of an Eimeria tenella aspartyl proteinase during the infection process.

Aspartyl proteinases are essential for the survival of many pathogens. A single copy gene in species of Eimeria encodes an aspartyl proteinase, which we propose should be called eimepsin to conform to the commonly used names of this family of proteinases. An epitope map, constructed using BIAcore technology, confirmed the specificity of 14 mAbs for eimepsin and defined four antigenic domains, which were conserved between native and recombinant forms of eimepsin. In resting sporozoites, mAb defining antigenic domains I and II stained the refractile body organelles, whereas those defining antigenic domains III and IV stained cytoplasmic granules. During host cell invasion, the staining patterns of mAb defining antigenic domains I, III and IV changed dramatically with the apical tips of invading sporozoites becoming strongly stained. In contrast, mAb defining antigenic domain II continued to stain only the refractile bodies. During early schizogony, mAb to all four domains stained the single fused refractile body, but when schizonts matured, mAb to antigenic domains I, III and IV stained the apical tip of merozoites whereas those to antigenic domain II continued to follow the developmental redistribution of the refractile body. Irrespective of localisation, mAb to three antigenic domains recognised a polypeptide of 49 kDa, which from N-terminal sequencing corresponds to a mature form of eimepsin. Staining with fluorescent pepstatin localised a mature, active form of eimepsin to the refractile bodies of the sporozoite, schizont and first generation merozoite. It remains to be determined whether eimepsin has a catalytic function within the refractile body or whether the activated enzyme is stored in the refractile body so that it can be rapidly redistributed to the apical tip during parasite invasion.

Animals↗
PubMed2000

Mapping and expression of microneme genes in Eimeria tenella.

Microneme organelles are located at the apical tip of invading stages of all apicomplexan parasites and they contain proteins that are critical for parasite adhesion to host cells. In this paper, we have utilised the process of oocyst sporulation in Eimeria tenella to investigate the timing of expression of components of the microneme organelle, at both mRNA and protein levels. Two time-course studies showed that there is a high level of synchrony in the sporulation process, especially during the time period when sporozoites are formed. Western blotting showed that the expression of five microneme proteins (EtMIC1-5) is differentially regulated and highly co-ordinated during sporulation with the proteins being detected only towards the end of the process, as the sporozoites matured within the sporocysts. In contrast, mRNA for all five of these microneme proteins was detected some 10-12 h earlier in sporulation than when the corresponding proteins were seen. Overall these data suggest that the expression of proteins destined for the microneme is regulated both at the transcriptional and translational level. The single copy genes encoding EtMIC1-5 are not clustered on the genome, but are found on four different chromosomes.

Animals↗
PubMed2000

Induction of secretion and surface capping of microneme proteins in Eimeria tenella.

Micronemes are secretory organelles of the invasive stages of apicomplexan parasites and contain proteins that are important for parasite motility and host cell invasion. We have examined the induction of microneme secretion in the coccidian Eimeria tenella. When sporozoites were added to MDBK cells in culture, microneme proteins were secreted, capped backwards over the parasite surface and deposited onto underlying host cells from the posterior end of gliding parasites. Induction of secretion was also achieved by the addition of foetal calf serum, or purified albumin, to extracellular sporozoites. Microneme secretion per se was not dependent on parasites being able to move or to invade host cells. However, in the presence of cytochalasin D, which disrupts actin polymerisation and prevents parasite movement, microneme proteins were secreted from the apical tip but were not capped backwards over the sporozoite surface. These observations support the hypothesis that microneme proteins function as ligands which, when secreted out onto the parasite surface, form a link, either directly or indirectly, between the sub-pellicular actin myosin cytoskeletal motor of the parasite and the surface of target host cells.

Animals↗
PubMed1997

Techniques for isolation and characterization of apical organelles from Eimeria tenella sporozoites.

Apical organelles contain molecules that are of critical importance for the interaction of all apicomplexan parasites with their target host cells. Thus, there is considerable interest in characterizing and understanding the function of molecules that reside in these organelles. Large numbers of surface-sterilized oocysts of Eimeria tenella, an apicomplexan coccidian of the chicken, can be routinely obtained from the animal host, and invasive sporozoites, which contain abundant apical organelles, can be rapidly prepared from these oocysts in the laboratory. Thus, E. tenella is proving to be an amenable parasite for subcellular fractionation techniques that allow the direct isolation and characterization of apical organelles. In this paper, a series of protocols is described for the large-scale culture of E. tenella parasites, the preparation of invasive sporozoites, the isolation of apical organelles, and the use of in vitro culture for localization and functional studies.

Animals↗
PubMed1994

Antigenic diversity of the asexual developmental stages of Eimeria tenella.

Total polypeptides from unsporulated and sporulated oocysts, sporozoites and the first two generations of merozoites of Eimeria tenella were fractionated by electrophoresis through polyacrylamide gels. The parasites are complex and the arrays of polypeptides differ for each of the developmental stages indicating that there is stage-specific control of gene expression. In particular, first generation merozoites display a markedly different polypeptide profile to that of either sporozoites or second generation merozoites. Changes in antigenicity during sporulation and the antigenic relationships between the three asexual zoite stages were examined by probing electroblotted polypeptides with a panel of antisera raised in rabbits to purified preparations of each stage. Antigenic cross-reactivity is well maintained throughout sporulation even though the sizes of antibody-reactive polypeptides change. In contrast there is a marked lack of cross-reactive epitopes between sporozoites, first and second generation merozoites.

Animals↗
PubMed1991

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.

Journal Article↗
PubMed1991

Recombinant vaccines for poultry.

The poultry industry relies on intensive farming to supply meat and eggs at relatively low cost. Many thousands of individuals are reared in enclosed houses which are left vacant for only short times between successive crops. Disease control is essential for the successful raising of poultry and this includes a need for cheap, effective vaccines against the major diseases.

Animals↗
PubMed1988

Sequence analysis of an 11.2 kilobase, near-terminal, BamHI fragment of fowlpox virus.

The nucleotide sequence of an 11.2 kilobase fragment of the fowlpox virus genome is presented. The fragment comes from near one end of the genome and contains part of the terminal inverted repeat. Twenty open reading frames (ORFs) are predicted from the sequence and are classified into 13 major and seven minor ORFs. The 100 base pairs immediately upstream of each ORF are up to 83% AT-rich, with some motifs similar to those seen in vaccinia virus early gene promoters. The TTTTTNT element which has been identified as a termination signal for vaccinia virus early genes is also found downstream of several ORFs. Three ORFs are predicted to specify polypeptides with significant homology to proteins coded by genes near termini of orthopoxvirus genomes: the vaccinia virus 42K early gene and 32.5K host range gene, and the cowpox virus 38K red pock gene. In addition, there are two families of ORFs within the fragment which potentially encode related polypeptides. One of these, family B, contains three ORFs which are related to those of chicken and rat hepatic lectins.

Amino Acid Sequence↗