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J C Boothroyd

Publications and source records attributed to J C Boothroyd.

At least 19 recordsLinked to original sources
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

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↗
PubMed1997

Use of Toxoplasma gondii expressing beta-galactosidase for colorimetric assessment of drug activity in vitro.

A microtiter assay for drug evaluation has been developed with a strain of Toxoplasma gondii that expresses bacterial beta-galactosidase. By using chlorophenol red-beta-D-galactopyranoside (CPRG) as the substrate for beta-galactosidase, the efficacy of a drug against the parasite can be determined with a colorimetric readout. Drugs known to have activity against T. gondii (specifically, pyrimethamine, sulfadiazine, atovaquone, and clindamycin) were tested, and efficacies were determined by CPRG cleavage. The 50% inhibitory concentrations determined by the CPRG-based colorimetric assay were similar to those determined by the traditional radiolabelled uracil incorporation assay. Since CPRG is nontoxic to the parasite, viable drug-treated parasites can be obtained at the conclusion of the assay for further evaluation if desired. This assay provides a high-throughput and nonradioactive alternative for the identification of anti-T. gondii compounds.

Animals↗
PubMed1996

Escherichia coli beta-galactosidase as an in vitro and in vivo reporter enzyme and stable transfection marker in the intracellular protozoan parasite Toxoplasma gondii.

We have developed several protocols for the use of beta-galactosidase (betaGal) from Escherichia coli as a reporter enzyme in transfection studies of Toxoplasma gondii (Tg) and as a readily screenable marker for stable transformation. Three Tg expression vectors with different promoters driving lacZ were constructed and shown in transient transfections to differ in their relative expression levels. Using a fluorescent betaGal substrate, it was possible to detect enzymatic activity with as little as 50 ng of transfected lacZ-containing plasmid DNA. When stably transformed intracellular parasites were cultivated in microtiter plates in the presence of the color substrate, chorophenol red-beta-D-galactopyranoside (CPRG), the signal from as few as 400 Tg could be readily detected by eye. Using serial dilutions of transfected parasite cultures in the presence of CPRG, we were able to clone stably expressing betaGal-positive Tg without the need for another selectable marker. Such lacZ transgenics could also be visualized histochemically in the tissue of infected mice. Thus, the application of betaGal to studies on Tg provides not only a much needed second reporter for transient transfection, it also comprises a safe and sensitive marker for the generation and analysis of stably transfected parasites.

Animals↗
PubMed1996

Sequence divergence in a family of variant surface glycoprotein genes from trypanosomes: coding region hypervariability and downstream recombinogenic repeats.

The surface of the parasitic protozoan Trypanosoma brucei spp. is covered with a dense coat consisting of a single type of glycoprotein molecule, the variant surface glycoprotein (VSG). There may be as many as 1,000 genes for VSG within the genome of T. brucei, and the switch of expression from one to another is the phenomenon of antigenic variation. As an approach to understanding the evolution of VSG genes we have determined the genomic DNA sequences of the eight genes encoding the variant surface glycoprotein 117 (VSG) family. From these data we have observed a number of features concerning the relationships between these genes: (1) there is a region of high variability confined to the N-terminus of the coding sequence, and comparison of the sequences with the available X-ray diffraction crystal structures suggests that two of the most variable stretches within the N-terminal domain are present on surface-exposed loops, indicating a role for epitope selection in evolution of these genes; (2) the 29 nucleotides surrounding the splice acceptor site are absolutely conserved in all eight 117 VSG genes; (3) numerous insertion/deletion mutations are located within or immediately downstream of the C-terminal protein-coding sequences: (4) within 500 bp downstream of the insertion/deletion mutations are one or two copies of a repeat motif highly homologous to the recombinogenic 76-bp repeat sequences present upstream of many VSG basic copy genes and the expression-linked copy.

Amino Acid Sequence↗
PubMed1996

Use of chimeric recombinant polypeptides to analyse conformational, surface epitopes on trypanosome variant surface glycoproteins.

Identification of surface-exposed epitopes on the variant surface glycoproteins (VSGs) of African trypanosomes has been complicated by the observation that most such epitopes are highly conformational. As a result, whenever the molecule is broken down for analysis, the epitope is generally lost. We have exploited the existence of closely related gene families to create chimeric molecules in which particular segments of one VSG are placed in the analogous position of a related but antigenically distinct VSG. The process is used in both a positive and negative manner, so that the epitope can be specifically added or destroyed in a given chimera. As an example, we have used this approach to identify the regions involved in reactivity to a monoclonal antibody specific for VSG117 on the surface of live trypanosomes. We show that while deletion of almost any region of VSG117 results in loss of reactivity to this monoclonal antibody, substituting particular regions with the corresponding segment of the structurally related but antigenically distinct VSG FM8.5 restores reactivity in most but not all cases, thereby delimiting the antigenically key regions. Likewise, substituting key regions from VSG117 into FM8.5 confers reactivity on the resulting chimeras. This approach circumvents some of the problems that result from the highly conformational nature of VSG and should allow further elucidation of the biologically relevant antigenic topology of VSGs.

Africa↗
PubMed1995

Toxoplasma gondii: stable complementation of sag1 (p30) mutants using SAG1 transfection and fluorescence-activated cell sorting.

Toxoplasma gondii and the related Apicomplexan protozoan pathogens, Plasmodium, Cryptosporidium, and Eimeria, are obligate intracellular parasites which cause severe disease in their hosts. The recent development of transient transfection of Toxoplasma permits the development of strategies utilizing "reverse genetics" to identify molecules critical to parasite survival within the host. We have utilized transfection of Toxoplasma tachyzoites to stably complement a sag1 (or p30) mutant that does not make detectable SAG1. Transfection of mutants with the wild-type SAG1 gene resulted in transient expression of SAG1 in approximately 15-20% of the transfected population. Stable transformants were enriched by repeated sorting of live parasites using a fluorescein-labeled monoclonal antibody specific for SAG1. Cloned recombinant parasites expressed SAG1 at wild-type levels and maintained expression for over 5 months after transfection (approximately 300 divisions). Cloned transformants (which proved to be siblings) carried both the mutated gene and one copy of the transfected gene which had inserted randomly into the Toxoplasma genome.

Animals↗
PubMed1995

Trypanosoma brucei: molecular cloning of homologues of small GTP-binding proteins involved in vesicle trafficking.

Members of the rab subfamily of GTPases have been implicated as important components in vesicle trafficking in the eukaryotes. Individual rab proteins have a remarkable degree of specific subcellular localization. As a first approach to the study of these proteins in the protozoan parasite Trypanosoma brucei we have undertaken a survey of the rab subfamily using a strategy based on degenerate polymerase chain reaction utilizing the miniexon and the highly conserved WDTAGQE box which comprises part of the GTP-binding site. A number of T. brucei partial cDNAs were obtained from procyclic stage cDNA, and seven of these clones (designated rtb 1, 3-7, 9) were clearly members of the rab family. Northern analysis of poly(A)-selected RNA indicates that rtb1, 3, 4, and 7 are constitutively expressed at low levels in both life-stages of T. brucei. By Southern analysis of trypanosome genomic DNA and specific PCR from an isolated genomic clone, we show that two of the genes, RTB1 and RTB7, are adjacent or at least closely linked in the T. brucei genome, while the other five are dispersed. These data provide important molecular reagents for dissecting the unusual secretory pathway in this organism.

Amino Acid Sequence↗
PubMed1995

Interconnection between organellar functions, development and drug resistance in the protozoan parasite, Toxoplasma gondii.

The protozoan parasite Toxoplasma gondii causes severe disease in animals and humans. In AIDS patients, for example, the encephalitis it produces is a major cause of death. Part of the very successful strategy adopted by the parasite centers on its ability to differentiate from the actively growing tachyzoite form to a chronic, almost latent state called the bradyzoite. The molecular signals and precise triggers involved in this differentiation process are not known. Drugs for treating toxoplasmosis are not capable of clearing the infection apparently because of their inability to eradicate the bradyzoites. Recently, as part of our efforts to understand the mode of action of a promising new drug, atovaquone, we have generated and analysed a mutant that is resistant to this drug. Surprisingly, we found that this mutant is predisposed to spontaneously differentiate from the tachyzoite to bradyzoite form in vitro (Tomavo & Boothroyd, submitted). Given that atovaquone is believed to act on the parasite mitochondria, we were interested to explore the relationship between mitochondrial function and differentiation. We find that atovaquone and a number of other drugs targeted to mitochondria will cause wild type parasites to differentiate from tachyzoites to bradyzoites suggesting some sort of adaptive response to a decrease in mitochondrial activities. The fact that atovaquone-resistant mutants are hypersensitive to clindamycin, a drug believed to work on the putative plastid of these parasites, suggests a model for how the mitochondrion and plastid interact and how they may be tied into the process and state of differentiation. This model is presented and discussed.

AIDS-Related Opportunistic Infections↗