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.
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► Guidelines are provided for analyzing new genotypes of Toxoplasma gondii and categorizing those using population genetic tools. ► Analysis of strains of T. gondii previously ...designated as “atypical” reveals a new clonal lineage in North America referred to as group 12. ► Group 12 has a strongly clonal population structure. ► The abundant type 2 lineage has been the parental strain for at least three examples of natural recombinations that led to clonal expansion.
Toxoplasma gondii is a widespread parasite of animals that causes zoonotic infections in humans. Previous studies have revealed a strongly clonal population structure in North America and Europe, while strains from South America are genetically separate and more diverse. However, the composition within North America has been questioned by recent descriptions of genetically more variable strains from this region. Here, we examined an expanded set of isolates using sequenced-based phylogenetic and population analyses to re-evaluate the population structure of T. gondii in North America. Our findings reveal that isolates previously defined by atypical restriction fragment length polymorphism patterns fall into two discrete groups. In one case, these new isolates represent variants of an existing lineage, from which they differ only by minor mutational drift. However, in the second case, it is evident that these isolates define a completely new lineage that is common in North America. Support for this new lineage was based on phylogeny, principle components analysis, STRUCTURE analyses, and statistical analysis of gene flow between groups. This new group, referred to as haplogroup 12, contains divergent genotypes previously referred to as A and X, isolated from sea otters. Consistent with this, group 12 was found primarily in wild animals, as well as occasionally in humans. This new lineage also has a highly clonal population structure. Analysis of the inheritance of multilocus genotypes revealed that different strains within group 12 are the products of a single recombination event between type 2 and a unique parental lineage. Collectively, the archetypal type 2 has been associated with clonal expansion of a small number of lineages in the North, as a consequence of separate but infrequent genetic crosses with several different parental lines.
Worldwide, approximately two billion people are chronically infected with Toxoplasma gondii with largely unknown consequences.
To better understand long-term effects and pathogenesis of this common, ...persistent brain infection, mice were infected at a time in human years equivalent to early to mid adulthood and studied 5-12 months later. Appearance, behavior, neurologic function and brain MRIs were studied. Additional analyses of pathogenesis included: correlation of brain weight and neurologic findings; histopathology focusing on brain regions; full genome microarrays; immunohistochemistry characterizing inflammatory cells; determination of presence of tachyzoites and bradyzoites; electron microscopy; and study of markers of inflammation in serum. Histopathology in genetically resistant mice and cytokine and NRAMP knockout mice, effects of inoculation of isolated parasites, and treatment with sulfadiazine or alphaPD1 ligand were studied.
Twelve months after infection, a time equivalent to middle to early elderly ages, mice had behavioral and neurological deficits, and brain MRIs showed mild to moderate ventricular dilatation. Lower brain weight correlated with greater magnitude of neurologic abnormalities and inflammation. Full genome microarrays of brains reflected inflammation causing neuronal damage (Gfap), effects on host cell protein processing (ubiquitin ligase), synapse remodeling (Complement 1q), and also increased expression of PD-1L (a ligand that allows persistent LCMV brain infection) and CD 36 (a fatty acid translocase and oxidized LDL receptor that mediates innate immune response to beta amyloid which is associated with pro-inflammation in Alzheimer's disease). Immunostaining detected no inflammation around intra-neuronal cysts, practically no free tachyzoites, and only rare bradyzoites. Nonetheless, there were perivascular, leptomeningeal inflammatory cells, particularly contiguous to the aqueduct of Sylvius and hippocampus, CD4+ and CD8+ T cells, and activated microglia in perivascular areas and brain parenchyma. Genetically resistant, chronically infected mice had substantially less inflammation.
In outbred mice, chronic, adult acquired T. gondii infection causes neurologic and behavioral abnormalities secondary to inflammation and loss of brain parenchyma. Perivascular inflammation is prominent particularly contiguous to the aqueduct of Sylvius and hippocampus. Even resistant mice have perivascular inflammation. This mouse model of chronic T. gondii infection raises questions of whether persistence of this parasite in brain can cause inflammation or neurodegeneration in genetically susceptible hosts.
Partial cDNA sequences or Expressed Sequence Tags (ESTs) have proven to be an economical way to gain information about expressed genes in a variety of organisms. Further, ESTs can be generated for ...strain or developmental stage comparisons. Currently there are over 10 000 ESTs for
Toxoplasma gondii derived from RH tachyzoite, ME49 tachyzoite and ME49 bradyzoite cDNA libraries. A set of Web pages and tools has been developed to provide easy access and rapid analysis of these data. Top Hits lists,
T. gondii-specific databases\search tools and cluster analyses can be browsed or used to rapidly gain insight into the structure and potential function of genes\proteins held within the database. The previously characterised
Eimeria protein Etp100 has been used to demonstrate how it is possible to use these tools to extract and assemble information about the putative
T. gondii homologue.
Like other members of the medically important phylum Apicomplexa, Toxoplasma gondii is an obligate intracellular parasite that secretes several classes of proteins involved in the active invasion of ...target host cells. Proteins in apical secretory organelles known as micronemes have been strongly implicated in parasite attachment to host cells. TgMIC2 is a microneme protein with multiple adhesive domains that bind target cells and is mobilized onto the parasite surface during parasite attachment. Here, we describe a novel parasite protein, TgM2AP, which is physically associated with TgMIC2. TgM2AP complexes with TgMIC2 within 15 min of synthesis and remains associated with TgMIC2 in the micronemes, on the parasite surface during invasion and in the culture medium after release from the parasite plasma membrane. TgM2AP is proteolytically processed initially when its propeptide is removed during transit through the golgi and later while it occupies the parasite surface after discharge from the micronemes. We show that TgM2AP is a member of a protein family expressed by coccidian parasites including Neospora caninum and Eimeria tenella. This phylogenic conservation and association with a key adhesive protein suggest that TgM2AP is a fundamental component of the T. gondii invasion machinery.
A common feature of microarray experiments is the occurrence of missing gene expression data. These missing values occur for a variety of reasons, in particular, because of the filtering of poor ...quality spots and the removal of undefined values when a logarithmic transformation is applied to negative background-corrected intensities. The efficiency and power of an analysis performed can be substantially reduced by having an incomplete matrix of gene intensities. Additionally, most statistical methods require a complete intensity matrix. Furthermore, biases may be introduced into analyses through missing information on some genes. Thus methods for appropriately replacing (imputing) missing data and/or weighting poor quality spots are required.
We present a likelihood-based method for imputing missing data or weighting poor quality spots that requires a number of biological or technical replicates. This likelihood-based approach assumes that the data for a given spot arising from each channel of a two-dye (two-channel) cDNA microarray comparison experiment independently come from a three-component mixture distribution--the parameters of which are estimated through use of a constrained E-M algorithm. Posterior probabilities of belonging to each component of the mixture distributions are calculated and used to decide whether imputation is required. These posterior probabilities may also be used to construct quality weights that can down-weight poor quality spots in any analysis performed afterwards. The approach is illustrated using data obtained from an experiment to observe gene expression changes with 24 hr paclitaxel (Taxol) treatment on a human cervical cancer derived cell line (HeLa).
As the quality of microarray experiments affect downstream processes, it is important to have a reliable and automatic method of identifying poor quality spots and arrays. We propose a method of identifying poor quality spots, and suggest a method of repairing the arrays by either imputation or assigning quality weights to the spots. This repaired data set would be less biased and can be analysed using any of the appropriate statistical methods found in the microarray literature.
The ability to clone large fragments of DNA in yeast artificial chromosomes (YAC's) has created the possibility of obtaining global physical maps of complex genomes. For this application to be ...feasible, most sequences in complex genomes must be able to be cloned in YAC's, and most clones must be genetically stable and colinear with the genomic sequences from which they originated (that is, not liable to undergo rearrangement). These requirements have been met with a YAC library containing DNA fragments from Drosophila melanogaster ranging in size up to several hundred kilobase pairs. Preliminary characterization of the Drosophila YAC library was carried out by in situ hybridization of random clones and analysis of clones containing known sequences. The results suggest that most euchromatic sequences can be cloned. The library also contains clones in which the inserted DNA is derived from the centrometric heterochromatin. The locations of 58 clones collectively representing about 8 percent of the euchromatic genome are presented.
The pathogenesis of diabetes associated with hemochromatosis is not known. We therefore examined glucose homeostasis and β-cell function in mouse models of hemochromatosis. Mice with targeted ...deletion of the hemochromatosis gene (Hfe−/−) on the 129/Sv genetic background exhibited a 72% increase in iron content in the islets of Langerhans compared with wild-type controls. Insulin content was decreased in Hfe−/− mice by 35%/pancreas and 25%/islet. Comparable decreases were seen in the mRNA levels of β-cell-specific markers, ins1, ins2, and glucose transporter 2. By 6–8 months, islets from Hfe−/− mice were 45% smaller, associated with increased staining for activated caspase 3 and terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling. Islets from Hfe−/− mice were also desensitized to glucose, with half-maximal stimulation of insulin secretion seen at 16.7 ± 0.9 mm glucose in perifused islets from Hfe−/− mice compared with 13.1 ± 0.6 mm glucose in wild-type animals. Carbonyl protein modification, a marker for oxidative stress, was increased by 58% in Hfe−/− islets. Despite decreased islet size, Hfe−/− mice exhibited enhanced glucose tolerance. Fasting serum insulin levels were comparable between Hfe−/− and Hfe+/+ mice, but were 48% lower in the Hfe−/− mice 30 min after challenge. Similar results were seen in mice carrying an Hfe mutation analogous to the common human mutation (C282Y) and in mice fed excess dietary iron. Hfe−/−mice on the C57BL6 background exhibited decreased glucose tolerance at 10–12 months due to an inability to increase insulin levels as they aged. We conclude that iron excess results in β-cell oxidant stress and decreased insulin secretory capacity secondary to β-cell apoptosis and desensitization of glucose-induced insulin secretion. This abnormality alone, however, is insufficient to cause diabetes.
Analysis of DNA sequences from the 5′ end of 239 directionally cloned
Toxoplasma gondii RH strain tachyzoitederived cDNAs revealed significant similarity to several classes of genes/proteins ...including 24 ribosomal proteins, five metabolic enzymes, four cell-cycle regulators and 15 previously cloned
T. gondii genes. The remaining sequences with no significant match include several which were recovered more than once. The variety and redundancy of expressed sequence tags (ESTs GenBankTM accession numbers T62239–T62475) in this sample suggest that the tachyzoite cDNA library reflects tachyzoite gene expression. A large scale EST effort should uncover many new genes and provide a wealth of information about genes involved with the growth and proliferation of tachyzoites.
Toxoplasma gondii, an obligate intracellular protozoan of the phylum Apicomplexa, is estimated to infect over a billion people worldwide as well as a great many other mammalian and avian hosts. ...Despite this ubiquity, the vast majority of human infections in Europe and North America are thought to be due to only three genotypes. Using a genome-wide analysis of single-nucleotide polymorphisms, we have constructed a genealogy for these three lines. The data indicate that types I and III are second- and first-generation offspring, respectively, of a cross between a type II strain and one of two ancestral strains. An extant T. gondii strain (P89) appears to be the modern descendant of the non-type II parent of type III, making the full genealogy of the type III clonotype known. The simplicity of this family tree demonstrates that even a single cross can lead to the emergence and dominance of a new clonal genotype that completely alters the population biology of a sexual pathogen.
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