Dataset Information

Microsatellite in the beta-tubulin gene of Toxoplasma gondii as a new genetic marker for use in direct screening of amniotic fluids.

ABSTRACT: To examine the correlation between Toxoplasma gondii genotype and congenital human toxoplasmosis, the polymorphism of the microsatellite consisting of a dinucleotide (TG) repeat in the intron of the beta-tubulin gene was investigated by PCR. Thirty-four reference strains were studied, including 7 strains virulent in mice and 27 strains avirulent in mice. The seven virulent strains had a (TG)8 microsatellite, and the avirulent strains had a (TG)7 microsatellite. This confirms the dichotomy already observed for virulent and avirulent strains. Additionally, 37 samples of amniotic fluid from infected fetuses were tested. All of them had the (TG)7 microsatellite marker. This result confirms that most of the human cases of congenital toxoplasmosis are due to strains avirulent in mice. Nevertheless, their virulence in human fetuses was obvious, as numerous abnormalities were observed on ultrasonic examination. The new genetic marker is the first one directly used for typing T. gondii isolates without any bias due to cultivation of the parasite. This microsatellite marker is not sufficient to type the strains which are avirulent in mice; however, seeking more polymorphic microsatellites should be worthwhile to obtain new genetic markers for direct screening of biological samples.

SUBMITTER: Costa JM

PROVIDER: S-EPMC230007 | biostudies-other | 1997 Oct

REPOSITORIES: biostudies-other

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Microsatellite in the beta-tubulin gene of Toxoplasma gondii as a new genetic marker for use in direct screening of amniotic fluids.

Costa J M JM Dardé M L ML Assouline B B Vidaud M M Bretagne S S

Journal of clinical microbiology 19971001 10

To examine the correlation between Toxoplasma gondii genotype and congenital human toxoplasmosis, the polymorphism of the microsatellite consisting of a dinucleotide (TG) repeat in the intron of the beta-tubulin gene was investigated by PCR. Thirty-four reference strains were studied, including 7 strains virulent in mice and 27 strains avirulent in mice. The seven virulent strains had a (TG)8 microsatellite, and the avirulent strains had a (TG)7 microsatellite. This confirms the dichotomy alread ...[more]

PMID: 9316904

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Project description:BACKGROUND: Because some Toxoplasma gondii genotypes may be more virulent in pregnant women, discriminating between them appears valuable. Currently, the main genotyping method is based on single copy microsatellite markers, which limit direct genotyping from amniotic fluids (AFs) to samples with a high parasitic load. We investigated whether the multicopy gene B1 could type the parasite with a higher sensitivity. To estimate the amplifiable DNA present in AFs, we first compared three different PCR assays used for Toxoplasma infection diagnosis: the P30-PCR, targeting the single copy gene P30; the B1-PCR, targeting the repeated B1 gene; and RE-PCR, targeting the repeated element. RESULTS: Of the 1792 AFs analyzed between 2008 and 2011, 73 were RE-PCR positive. Of those, 49 (67.1%) were P30-PCR and B1-PCR positive, and 14 (19.2%) additional AFs were B1-PCR positive only.All 63 BI-positive AFs (France n?=?49; overseas n?=?14) could be genotyped based on an analysis of eight nucleotide polymorphisms (SNPs) located within the B1 gene. Following high-resolution melting (HRM) analysis, minisequencing was carried out for each of the eight SNPs. DNA from six reference strains was included in the study, and AFs were assigned to one of the three major lineages (Types I, II, and III). In total, 26 genotypes were observed, and the hierarchical clustering distinguished two clades in lineages II (IIa, n?=?30 and IIb, n?=?4) and III (IIIa n?=?23 and IIIb n?=?6). There was an overrepresentation of overseas isolates in Clade IIb (4/4, 100%) and Clade IIIa (8/22; 36.4%) (p <0.0001), whereas medical interruption and fetal death were overrepresented in Clade IIb (2/4, 50%) and Clade IIIa (4/23, 17.4%) (p?=?0.049). CONCLUSIONS: Although the current genotyping system cannot pretend to replace multilocus typing, we clearly show that targeting the multicopy B1 gene yields a genotyping capacity of AFs around 20% better than when single copy targets are used. The present genotyping method also allows clear identification of genotypes of potential higher virulence.

Project description:Toxoplasma gondii is a widespread protozoan parasite that causes potentially life-threatening opportunistic disease. New inhibitors of parasite replication are urgently needed, as the current antifolate treatment is also toxic to patients. Microtubules are essential cytoskeletal components that have been selectively targeted in microbial pathogens; further study of tubulin in Toxoplasma may reveal novel therapeutic opportunities. It has been noted that ?-tubulin acetylation at lysine 40 (K40) is enriched during daughter parasite formation, but the impact of this modification on Toxoplasma division and the enzyme mediating its delivery have not been identified. We performed mutational analyses to provide evidence that K40 acetylation stabilizes Toxoplasma microtubules and is required for parasite replication. We also show that an unusual Toxoplasma homologue of ?-tubulin acetyltransferase (TgATAT) is expressed in a cell cycle-regulated manner and that its expression peaks during division. Disruption of TgATAT with CRISPR/Cas9 ablates K40 acetylation and induces replication defects; parasites appear to initiate mitosis yet exhibit incomplete or improper nuclear division. Together, these findings establish the importance of tubulin acetylation, exposing a new vulnerability in Toxoplasma that could be pharmacologically targeted. IMPORTANCE Toxoplasma gondii is an opportunistic parasite that infects at least one-third of the world population. New treatments for the disease (toxoplasmosis) are needed since current drugs are toxic to patients. Microtubules are essential cellular structures built from tubulin that show promise as antimicrobial drug targets. Microtubules can be regulated by chemical modification, such as acetylation on lysine 40 (K40). To determine the role of K40 acetylation in Toxoplasma and whether it is a liability to the parasite, we performed mutational analyses of the ?-tubulin gene. Our results indicate that parasites cannot survive without K40 acetylation unless microtubules are stabilized with a secondary mutation. Additionally, we identified the parasite enzyme that acetylates ?-tubulin (TgATAT). Genetic disruption of TgATAT caused severe defects in parasite replication, further highlighting the importance of ?-tubulin K40 acetylation in Toxoplasma and its promise as a potential new drug target.

Project description:Apicomplexa are obligate intracellular parasites that cause important diseases in humans and animals. Manipulating the pathogen genome is the most direct way to understand the functions of specific genes in parasite development and pathogenesis. In Toxoplasma gondii, nonhomologous recombination is typically highly favored over homologous recombination, a process required for precise gene targeting. Several approaches, including the use of targeting vectors that feature large flanks to drive site-specific recombination, have been developed to overcome this problem. We have generated a new large-insert repository of T. gondii genomic DNA that is arrayed and sequenced and covers 95% of all of the parasite's genes. Clones from this fosmid library are maintained at single copy, which provides a high level of stability and enhances our ability to modify the organism dramatically. We establish a robust recombineering pipeline and show that our fosmid clones can be easily converted into gene knockout constructs in a 4-day protocol that does not require plate-based cloning but can be performed in multiwell plates. We validated this approach to understand gene function in T. gondii and produced a conditional null mutant for a nucleolar protein belonging to the NOL1/NOP2/SUN family, and we show that this gene is essential for parasite growth. We also demonstrate a powerful complementation strategy in the context of chemical mutagenesis and whole-genome sequencing. This repository is an important new resource that will accelerate both forward and reverse genetic analysis of this important pathogen.Toxoplasma gondii is an important genetic model to understand intracellular parasitism. We show here that large-insert genomic clones are effective tools that enhance homologous recombination and allow us to engineer conditional mutants to understand gene function. We have generated, arrayed, and sequenced a fosmid library of T. gondii genomic DNA in a copy control vector that provides excellent coverage of the genome. The fosmids are maintained in a single-copy state that dramatically improves their stability and allows modification by means of a simple and highly scalable protocol. We show here that modified and unmodified fosmid clones are powerful tools for forward and reverse genetics.

Project description:The urgent need to develop new antimicrobial therapies has spawned the development of repurposing screens in which well-studied drugs and other types of compounds are tested for potential off-label uses. As a proof-of-principle screen to identify compounds effective against Toxoplasma gondii, we screened a collection of 1,120 compounds for the ability to significantly reduce Toxoplasma replication. A total of 94 compounds blocked parasite replication with 50% inhibitory concentrations of <5 µM. A significant number of these compounds are established inhibitors of dopamine or estrogen signaling. Follow-up experiments with the dopamine receptor inhibitor pimozide revealed that the drug impacted both parasite invasion and replication but did so independently of inhibition of dopamine or other neurotransmitter receptor signaling. Tamoxifen, which is an established inhibitor of the estrogen receptor, also reduced parasite invasion and replication. Even though Toxoplasma can activate the estrogen receptor, tamoxifen inhibits parasite growth independently of this transcription factor. Tamoxifen is also a potent inducer of autophagy, and we find that the drug stimulates recruitment of the autophagy marker light chain 3-green fluorescent protein onto the membrane of the vacuolar compartment in which the parasite resides and replicates. In contrast to other antiparasitic drugs, including pimozide, tamoxifen treatment of infected cells leads to a time-dependent elimination of intracellular parasites. Taken together, these data suggest that tamoxifen restricts Toxoplasma growth by inducing xenophagy or autophagic destruction of this obligate intracellular parasite. IMPORTANCE There is an urgent need to develop new therapies to treat microbial infections, and the repurposing of well-characterized compounds is emerging as one approach to achieving this goal. Using the protozoan parasite Toxoplasma gondii, we screened a library of 1,120 compounds and identified several compounds with significant antiparasitic activities. Among these were pimozide and tamoxifen, which are well-characterized drugs prescribed to treat patients with psychiatric disorders and breast cancer, respectively. The mechanisms by which these compounds target these disorders are known, but we show here that these drugs kill Toxoplasma through novel pathways, highlighting the potential utility of off-target effects in the treatment of infectious diseases.

Dataset Information

Microsatellite in the beta-tubulin gene of Toxoplasma gondii as a new genetic marker for use in direct screening of amniotic fluids.

Publications

Microsatellite in the beta-tubulin gene of Toxoplasma gondii as a new genetic marker for use in direct screening of amniotic fluids.

Similar Datasets

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