Biomedical subjects

J C Boothroyd

Publications and source records attributed to J C Boothroyd.

At least 37 records · Page 2Linked to original sources

PubMed2000

Toxoplasma gondii homologue of plasmodium apical membrane antigen 1 is involved in invasion of host cells.

Proteins with constitutive or transient localization on the surface of Apicomplexa parasites are of particular interest for their potential role in the invasion of host cells. We describe the identification and characterization of TgAMA1, the Toxoplasma gondii homolog of the Plasmodium apical membrane antigen 1 (AMA1), which has been shown to elicit a protective immune response against merozoites dependent on the correct pairing of its numerous disulfide bonds. TgAMA1 shows between 19% (Plasmodium berghei) and 26% (Plasmodium yoelii) overall identity to the different Plasmodium AMA1 homologs and has a conserved arrangement of 16 cysteine residues and a putative transmembrane domain, indicating a similar architecture. The single-copy TgAMA1 gene is interrupted by seven introns and is transcribed into an mRNA of approximately 3.3 kb. The TgAMA1 protein is produced during intracellular tachyzoite replication and initially localizes to the micronemes, as determined by immunofluorescence assay and immunoelectron microscopy. Upon release of mature tachyzoites, TgAMA1 is found distributed predominantly on the apical end of the parasite surface. A approximately 54-kDa cleavage product of the large ectodomain is continuously released into the medium by extracellular parasites. Mouse antiserum against recombinant TgAMA1 blocked invasion of new host cells by approximately 40%. This and our inability to produce a viable TgAMA1 knock-out mutant indicate that this phylogenetically conserved protein fulfills a key function in the invasion of host cells by extracellular T. gondii tachyzoites.

Amino Acid Sequence↗

PubMed2000

Ionophore-resistant mutants of Toxoplasma gondii reveal host cell permeabilization as an early event in egress.

Toxoplasma gondii is an obligate intracellular pathogen within the phylum Apicomplexa. Invasion and egress by this protozoan parasite are rapid events that are dependent upon parasite motility and appear to be directed by fluctuations in intracellular [Ca(2+)]. Treatment of infected host cells with the calcium ionophore A23187 causes the parasites to undergo rapid egress in a process termed ionophore-induced egress (IIE). In contrast, when extracellular parasites are exposed to this ionophore, they quickly lose infectivity (termed ionophore-induced death [IID]). From among several Iie(-) mutants described here, two were identified that differ in several attributes, most notably in their resistance to IID. The association between the Iie(-) and Iid(-) phenotypes is supported by the observation that two-thirds of mutants selected as Iid(-) are also Iie(-). Characterization of three distinct classes of IIE and IID mutants revealed that the Iie(-) phenotype is due to a defect in a parasite-dependent activity that normally causes infected host cells to be permeabilized just prior to egress. Iie(-) parasites underwent rapid egress when infected cells were artificially permeabilized by a mild saponin treatment, confirming that this step is deficient in the Iie(-) mutants. A model is proposed that includes host cell permeabilization as a critical part of the signaling pathway leading to parasite egress. The fact that Iie(-) mutants are also defective in early stages of the lytic cycle indicates some commonality between these normal processes and IIE.

Animals↗

PubMed2000

Lytic cycle of Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular pathogen within the phylum Apicomplexa. This protozoan parasite is one of the most widespread, with a broad host range including many birds and mammals and a geographic range that is nearly worldwide. While infection of healthy adults is usually relatively mild, serious disease can result in utero or when the host is immunocompromised. This sophisticated eukaryote has many specialized features that make it well suited to its intracellular lifestyle. In this review, we describe the current knowledge of how the asexual tachyzoite stage of Toxoplasma attaches to, invades, replicates in, and exits the host cell. Since this process is closely analogous to the way in which viruses reproduce, we refer to it as the Toxoplasma "lytic cycle."

Animals↗

PubMed1999

Identification of the pro-mature processing site of Toxoplasma ROP1 by mass spectrometry.

The rhoptries are specialized secretory organelles that function during host cell invasion in the obligate intracellular parasite Toxoplasma gondii. All T. gondii rhoptry proteins studied to date are synthesized as pro-proteins that are then processed to their mature forms. To understand the role of the pro region in rhoptry protein function, we have precisely defined the processing site of the pro-region of the rhoptry protein ROP1. Efforts to determine such processing sites have been prevented by blocked N-termini of mature proteins isolated from T. gondii. To overcome this problem, we have used an engineered form of ROP1 and mass spectrometry to demonstrate that proROP1 is processed to its mature form between the glutamic acid at position 83 and alanine at position 84. These data also show that mature ROP1 lacks substantial post-translational modifications, a result which has important implications for targeting of rhoptry proteins.

Amino Acid Sequence↗

PubMed1999

A Toxoplasma lectin-like activity specific for sulfated polysaccharides is involved in host cell infection.

Toxoplasma gondii is one of the most widespread parasites of humans and animals. The parasite has a remarkable ability to invade a broad range of cells within its mammalian hosts by mechanisms that are poorly understood at the molecular level. This broad host cell specificity suggests that adhesion should involve the recognition of ubiquitous surface-exposed host molecules or, alternatively, the presence of various parasite attachment molecules able to recognize different host cell receptors. We have discovered a sugar-binding activity (lectin) in tachyzoites of T. gondii that plays a role in vitro in erythrocyte agglutination and infection of human fibroblasts and epithelial cells. The ability to agglutinate erythrocytes can be reversed by a variety of soluble glycoconjugates, of which heparin, fucoidan, and dextran sulfate were the most effective. Interestingly, infectivity of tachyzoites for human foreskin fibroblasts, cells that are commonly used to grow T. gondii in vitro, was increased by low concentrations of the sulfated glycoconjugates that inhibited hemagglutination activity (i.e. dextran sulfate and fucoidan) whereas high concentrations inhibited parasite infection. Furthermore, inhibition of glycosaminoglycan biosynthesis and sulfation on the host cells reduced Toxoplasma infectivity. Finally, Toxoplasma tachyzoites showed a reduced ability to infect epithelial cell mutants deficient in the biosynthesis of surface proteoglycans. The probable identity of the hemagglutinin(s) was investigated by 1) direct binding of red blood cells to filter blots of Toxoplasma proteins separated by polyacrylamide gel electrophoresis, and 2) binding of metabolically labeled parasite proteins to fixed mammalian cells. Three parasite bands were thus identified as candidate adhesins. These results suggest that attachment of T. gondii to its target cell is mediated by parasite lectins and that sulfated sugars on the surface of host cells may function as a key parasite receptor.

Animals↗

PubMed1998

Identification of a nuclear protein in Trypanosoma brucei with homology to RNA-binding proteins from cis-splicing systems.

Gene expression in trypanosomes is controlled at the level of pre-mRNA maturation via trans-splicing and polyadenylation and through changes in mRNA stability. To identify the trans- acting factors involved in this regulation, we have used a degenerate PCR approach to clone genes encoding the RNA recognition motif (RRM) consensus. We have identified a single-copy gene encoding a protein (designated RRM1) which contains three consensus RRM motifs, two tandem copies of a retroviral gag-like CCHC 'zinc finger' and an arginine-serine (RS) rich region. Western blotting indicates that RRM1 is expressed in both procyclic and bloodstream-form trypanosomes and has an apparent mobility on SDS-PAGE of ca. 70 Kd. RRM1 is localized in the trypanosome nucleus in substructures which may be functionally analogous to the 'speckles' associated with cis-splicing in higher eukaryotic cells. The structure of RRM1, its pattern of expression and its intracellular location suggest that it may play a role in trans-splicing.

Amino Acid Sequence↗

PubMed1998

Processing of Toxoplasma ROP1 protein in nascent rhoptries.

Secretion in the obligate intracellular parasite, Toxoplasma gondii, occurs through a number of regulated compartments. Among these are the apical organelles known as rhoptries which release their contents as part of the invasion process. We are interested in the processing, targeting and ultimate function of rhoptry proteins (and have focused our analyses on rhoptry protein 1 (ROP1). In this paper, we address the issue of processing: using a number of engineered forms of the ROP1 gene (introduced into a ROP1- background), we show that ROP1 is synthesized as a pre-pro-protein that is subject to proteolytic cleavages to remove the pre-sequence and the 'pro' region, at the N-terminus. Using brefeldin A (BFA) and reduced temperature we show that this processing occurs late in the secretory pathway of the parasite. Immunolocalization studies with epitope-tagged constructs indicate that processing is apparently occurring in the nascent rhoptries of dividing parasites. The results are discussed in the context of the targeting and possible function of the ROP1 protein.

Animals↗

PubMed1998

Development of a stable episomal shuttle vector for Toxoplasma gondii.

The rapid developments in the molecular genetics of Toxoplasma gondii have far reaching implications in treatment and vaccination strategies for this as well as closely related pathogens such as Plasmodium. Although stable transformation of this parasite through homologous and illegitimate genomic integration has provided many of the tools necessary for genetic analysis, subsequent manipulations of the DNA have proven laborious. This report describes the selection and subsequent characterization of a Toxoplasma sequence that permits the episomal maintenance of bacterial plasmids in this parasite. This sequence was isolated from the Toxoplasma genome through selection for episomal stability of a pUC19-based library in the absence of a selectable marker. A 500-base pair fragment was determined to possess the stabilization activity. Transformations of Toxoplasma using vectors possessing this fragment, referred to as EMS (episomal maintenance sequence), demonstrated an elevated stable transformation frequency compared with the vector alone. Mutants deficient in hypoxanthine-xanthine-guanine phosphoribosyltransferase activity were used as a test to see if this gene could be selected from a genomic library using a vector containing the EMS. The success of this test demonstrates the utility of EMS-containing vectors in complementation strategies and the ability of such constructs bearing large fragments of the Toxoplasma genome to be maintained episomally.

Animals↗

PubMed1998

The surface of Toxoplasma: more and less.

As for any intracellular parasite, the surface of the Apicomplexan parasite Toxoplasma gondii must fulfil many functions including a role in attachment, signalling, invasion, transport and interaction with the immune response of the host. In this review, we describe the current state of knowledge on the molecules that are found on the surface of the different developmental stages of this parasite and speculate as to how at least some of these multiple functions are fulfilled. Special emphasis is given to the growing family of surface antigens that are related to the tachyzoite-specific surface antigen 1. We conclude that the surface (of tachyzoites, at least) is both more and less complex than previously thought: there are more proteins present but their sequences suggest that the majority may share a similar overall structure typified by surface antigen 1.

Animals↗

PubMed1998

Molecular Biology's Lessons about Toxoplasma Development: Stage-specific Homologs.

Within intermediate hosts (such as humans), the protozoan parasite Toxoplasma gondii has two life cycle stages: a rapidly replicating form called a tachyzoite and a slowly growing, quiescent form called a bradyzoite. Recently, molecular biology studies have shown that tachyzoites and bradyzoites express a number of homologs (ie. evolutionary related genes)expressed exclusively in one or the other stage. Here, Laura Knoll and John Boothroyd describe examples of how these stage-specific homologs were discovered, and speculate about their regulation and functional significance.

Journal Article↗

PubMed1998

Gene discovery by EST sequencing in Toxoplasma gondii reveals sequences restricted to the Apicomplexa.

To accelerate gene discovery and facilitate genetic mapping in the protozoan parasite Toxoplasma gondii, we have generated >7000 new ESTs from the 5' ends of randomly selected tachyzoite cDNAs. Comparison of the ESTs with the existing gene databases identified possible functions for more than 500 new T. gondii genes by virtue of sequence motifs shared with conserved protein families, including factors involved in transcription, translation, protein secretion, signal transduction, cytoskeleton organization, and metabolism. Despite this success in identifying new genes, more than 50% of the ESTs correspond to genes of unknown function, reflecting the divergent evolutionary status of this parasite. A newly recognized class of genes was identified based on its similarity to sequences known only from other members of the same phylum, therefore identifying sequences that are apparently restricted to the Apicomplexa. Such genes may underlie pathways common to this group of medically important parasites, therefore identifying potential targets for intervention.

Animals↗

PubMed1998

Expressed sequence tag analysis of the bradyzoite stage of Toxoplasma gondii: identification of developmentally regulated genes.

Toxoplasma gondii is a protozoan parasite responsible for widespread infections in humans and animals. Two major asexual forms are produced during the life cycle of this parasite: the rapidly dividing tachyzoite and the more slowly dividing, encysted bradyzoite. To further study the differentiation between these two forms, we have generated a large number of expressed sequence tags (ESTs) from both asexual stages. Previously, we obtained data on approximately 7,400 ESTs from tachyzoites (J. Ajioka et al., Genome Res. 8:18-28, 1998). Here, we report the results from analysis of approximately 2,500 ESTs from bradyzoites purified from the cysts of infected mice. We also report the results from analysis of 760 ESTs from parasites induced to differentiate from tachyzoites to bradyzoites in vitro. Comparison of the data sets from bradyzoites and tachyzoites reveals many previously uncharacterized sequence clusters which are largely or completely specific to one or other developmental stage. This class includes a bradyzoite-specific form of enolase. Combined with the previously identified bradyzoite-specific form of lactate dehydrogenase, this finding suggests significant differences in flux through the lower end of the glycolytic pathway in this stage. Thus, the generation of this data set provides valuable insights into the metabolism and growth of the parasite in the encysted form and represents a substantial body of information for further study of development in Toxoplasma.

Amino Acid Sequence↗

PubMed1998

The surface of Toxoplasma tachyzoites is dominated by a family of glycosylphosphatidylinositol-anchored antigens related to SAG1.

Toxoplasma gondii is an Apicomplexan parasite with a complex life cycle that includes a rapidly dividing asexual stage known as the tachyzoite. The tachyzoite surface has been reported to comprise five major antigens, the most abundant of which is designated SAG1 (for surface antigen 1). At least one of the other four (SAG3) and another recently described minor antigen (SRS1 [for SAG1-related sequence 1]) have previously been shown to be structurally related to SAG1. To determine if further SAG1 homologs exist, we searched a Toxoplasma expressed sequence tag (EST) database and found numerous ESTs corresponding to at least three new genes related to SAG1. Like SAG1, these new SRS genes encode apparently glycosylphosphatidylinositol-anchored proteins that share several motifs and a set of conserved cysteine residues. This family appears to have arisen by divergence from a common ancestor under selection for the conservation of overall topology. The products of two of these new genes (SRS2 and SRS3) are shown to be expressed on the surface of Toxoplasma tachyzoites by immunofluorescence. We also identified strain-specific differences in relative expression levels. A total of 10 members of the SAG1 gene family have now been identified, which apparently include three of the five major surface antigens previously described and one antigen expressed only in bradyzoites. The function of this family may be to provide a redundant system of receptors for interaction with host cells and/or to direct the immune responses that limit acute T. gondii infections.

Amino Acid Sequence↗

PubMed1998

Isolation of developmentally regulated genes from Toxoplasma gondii by a gene trap with the positive and negative selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase.

Within its intermediate host, Toxoplasma gondii switches between two forms: a rapidly replicating tachyzoite and an encysted bradyzoite. Bradyzoites persist within the host throughout its life, hidden from antimicrobial agents and the immune system. The signals that mediate switching are poorly understood. A gene trap was employed to isolate genes whose expression is up-regulated early in the switching of bradyzoites via the negative and positive selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT). T. gondii was transfected with promoterless HXGPRT and negatively selected with 6-thioxanthine to inhibit the growth of tachyzoites expressing HXGPRT. The surviving tachyzoites were then induced for in vitro bradyzoite formation and treated with mycophenolic acid and xanthine to positively select for parasites in which the construct had integrated downstream of a bradyzoite-specific gene. Strains were checked for their ability to differentiate by using Dolichos biflorus agglutinin (a bradyzoite-specific lectin) and a monoclonal antibody against P36 (a bradyzoite-specific surface antigen). After differentiation, all gene-trapped clones had Dolichos immunofluorescence and all but one expressed P36. The sequences flanking the insertion site of this P36-negative strain were homologous to the Toxoplasma family of surface antigens, strongly suggesting that P36 is encoded by the disruptive gene. Genetic mapping and complementation of the P36-negative strain further indicated that the disrupted gene is P36. Reverse transcriptase PCR and S1 nuclease digestion were used to compare mRNA levels during the tachyzoite and bradyzoite stages. The presumptive P36 gene does not appear to regulate its mRNA levels between the two stages, indicating a posttranscriptional mechanism of regulation for early bradyzoite-specific genes.

Amino Acid Sequence↗

PubMed1998

Analysis of Toxoplasma gondii stably transfected with a transmembrane variant of its major surface protein, SAG1.

We have genetically engineered Toxoplasma gondii so that its major surface antigen SAG1 is anchored by a human transmembrane domain (SAG1-TM) instead of its natural GPI anchor (SAG1-GPI) in order to initiate studies to address the function of this protein anchor in parasitic protozoa as well as to get insights into the functional role of SAG1. Our results show that SAG1-TM is correctly folded (at least as judged by the presence of conformationally dependent epitopes) and targeted to the surface of the parasite, indicating that the GPI anchor does not determine its localization nor overall three-dimensional structure. No significant difference was seen in any aspect of the growth of the SAG1-TM mutant. However, compared to the natural SAG1-GPI, SAG1-TM does not form strong associations with itself and/or other molecules in high molecular weight complexes suggesting that allowing such complexes to form may be one role of the GPI anchor. The in vitro half-life of SAG1-TM of extracellular parasites is significantly lower than that of SAG1-GPI suggesting a stabilizing function of the glycolipid anchor against degradation and/or membrane release. Antibodies to SAG1 are shed from SAG1-TM parasites as they invade, just as they are stripped from SAG1-GPI bearing parasites. The stripping, therefore, is unlikely to be driven by the action of lipases.

Amino Acid Sequence↗

PubMed1997

Genetic and biochemical analysis of development in Toxoplasma gondii.

Toxoplasma gondii has recently come under intense study as a model for intracellular parasitism because it has a number of properties that facilitate experimental manipulation. Attention is now being turned towards understanding the developmental biology of this complex parasite. The differentiation between the two asexual stages, the rapidly growing tachyzoites and the more slowly dividing, encysted bradyzoites, is of particular interest. Progression from the former to the latter is influenced by the host's immune response. This paper describes current progress on a number of research fronts, all aimed at understanding the triggers that push the tachyzoite-bradyzoite equilibrium in one or other direction and the changes that occur in gene expression (and ultimately metabolism and function). Chief among the techniques used for these studies are genetics and molecular genetics. Recent progress in these areas is described.

Acetylglucosamine↗

PubMed1997

Genetic analysis in Toxoplasma: gene discovery with expressed sequence tags and rapid mapping of natural polymorphisms.

Genetic analysis of the protozoan parasite Toxoplasma gondii has undergone a rapid expansion in recent years. This is due to effort in a number of laboratories that have worked on the development of molecular genetic techniques. It is also due, however, to the natural biology of this system (including a well-described sexual cycle) that makes possible genetic mapping of the F1 progeny from a cross. In this article, we present a detailed methodology for rapidly mapping natural polymorphisms between the ME49 and CEP strains for which extensive restriction fragment length polymorphism analysis has already been performed. The example we present shows that the failure to detect expression of bradyzoite-specific surface antigens in the CEP strain under conditions that promote differentiation in vitro is not a result of a general failure to express such genes; instead, it is apparently due to antigenic polymorphism in the gene products concerned. This conclusion was reached rapidly and definitively by genetic mapping, whereas molecular approaches would have taken considerably longer. We also show how the recent effort to create an extensive database of expressed sequence tags for this parasite can promote the very rapid discovery of genes that reveal much about the biology of Toxoplasma. The example presented deals with the expression of a family of closely related surface antigens in the tachyzoite stage.

Alleles↗

PubMed1997

Identification and characterization of SRS1, a Toxoplasma gondii surface antigen upstream of and related to SAG1.

Previous investigations of the major surface antigen (SAG1) promoter of Toxoplasma gondii indicated an ability to function bi-directionally in transient transformation assays at least. This suggests there might be another tachyzoite-specific gene being divergently transcribed from the SAG1 promoter in its normal chromosomal location. To investigate this possibility we have characterized the region upstream of SAG1 and report here a co-directional transcription unit coding for a probable GPI-anchored surface protein with homology to SAG1 and SAG3. This antigen, which had not previously been identified in surface iodination experiments is given the acronym SRS1, for SAG1-related sequence 1. Genomic organization and sequence of a full-length cDNA of SRS1 are presented. Antisera against a recombinant SRS1 protein produced in Escherichia coli, recognize a specific band of 46 kDa in parasite lysates which corresponds to the largest of the GPI-anchored proteins by Western blot. The possible role of this previously unidentified surface antigen is discussed.

Amino Acid Sequence↗