Current and Emerging Approaches to Studying Invasion in Apicomplexan Parasites

Mital, Jeffrey; Ward, Gary E.

doi:10.1007/978-0-387-78267-6_1

Cited by 12 publications

(9 citation statements)

References 252 publications

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“…S1 and Text S1) that inhibited parasite invasion and motility and reproducibly induced the TgMLC1 mobility shift. It was difficult to establish structure-activity relationships among these analogs due to the semi-quantitative nature of the in vivo assays [27] and problems with compound stability and solubility. However, we did not identify any analogs that induced the modification of TgMLC1 without also inhibiting motility and invasion, consistent with the hypothesis that the observed block in these processes results from the modification of TgMLC1.…”

Section: Resultsmentioning

confidence: 99%

A Small-Molecule Inhibitor of T. gondii Motility Induces the Posttranslational Modification of Myosin Light Chain-1 and Inhibits Myosin Motor Activity

et al. 2010

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Toxoplasma gondii is an obligate intracellular parasite that enters cells by a process of active penetration. Host cell penetration and parasite motility are driven by a myosin motor complex consisting of four known proteins: TgMyoA, an unconventional Class XIV myosin; TgMLC1, a myosin light chain; and two membrane-associated proteins, TgGAP45 and TgGAP50. Little is known about how the activity of the myosin motor complex is regulated. Here, we show that treatment of parasites with a recently identified small-molecule inhibitor of invasion and motility results in a rapid and irreversible change in the electrophoretic mobility of TgMLC1. While the precise nature of the TgMLC1 modification has not yet been established, it was mapped to the peptide Val46-Arg59. To determine if the TgMLC1 modification is responsible for the motility defect observed in parasites after compound treatment, the activity of myosin motor complexes from control and compound-treated parasites was compared in an in vitro motility assay. TgMyoA motor complexes containing the modified TgMLC1 showed significantly decreased motor activity compared to control complexes. This change in motor activity likely accounts for the motility defects seen in the parasites after compound treatment and provides the first evidence, in any species, that the mechanical activity of Class XIV myosins can be modulated by posttranslational modifications to their associated light chains.

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Section: Resultsmentioning

confidence: 99%

A Small-Molecule Inhibitor of T. gondii Motility Induces the Posttranslational Modification of Myosin Light Chain-1 and Inhibits Myosin Motor Activity

et al. 2010

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“…Because of the haploid nature of the Toxoplasma genome, classic forward genetic screens have proven difficult (reviewed in ref. 3). In addition, gene knockouts can result in upregulation of related proteins or signaling pathways that can make it difficult to interpret the true function of gene products using this technology.…”

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confidence: 99%

Chemical genetic screen identifies Toxoplasma DJ-1 as a regulator of parasite secretion, attachment, and invasion

Hall¹,

Reese²,

Weerapana

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Toxoplasma gondii is a member of the phylum Apicomplexa that includes several important human pathogens, such as Cryptosporidium and Plasmodium falciparum, the causative agent of human malaria. It is an obligate intracellular parasite that can cause severe disease in congenitally infected neonates and immunocompromised individuals. Despite the importance of attachment and invasion to the success of the parasite, little is known about the underlying mechanisms that drive these processes. Here we describe a screen to identify small molecules that block the process of host cell invasion by the T. gondii parasite. We identified a small molecule that specifically and irreversibly blocks parasite attachment and subsequent invasion of host cells. Using tandem orthogonal proteolysis-activity-based protein profiling, we determined that this compound covalently modifies a single cysteine residue in a poorly characterized protein homologous to the human protein DJ-1. Mutation of this key cysteine residue in the native gene sequence resulted in parasites that were resistant to inhibition of host cell attachment and invasion by the compound. Further analysis of the invasion phenotype confirmed that modification of Cys127 on TgDJ-1 resulted in a block of microneme secretion and motility, even in the presence of direct stimulators of calcium release. Together, our results suggest that TgDJ-1 plays an important role that is likely downstream of the calcium flux required for microneme secretion, parasite motility, and subsequent invasion of host cells.

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“…Host cell invasion is a complex process and is relatively conserved among the members of this taxon [ 59 ]. Genomic DNA extracted from parasites after cultivation for 24 h can be used as a template for quantitatively assessing invasion [ 60 ]. Sporozoites pre-incubated with 2.5% anti-rCSUI_005805 antibodies prior to infection of IPEC-J1 cells slightly reduced the number of intracellular parasites whereas inhibition of host-cell invasion by porcine anti- C. suis serum antibodies in the same dilution was two-fold higher, indicating that CSUI_005805 does not play a leading role in the invasion of the host-cell, at least not by itself.…”

Section: Discussionmentioning

confidence: 99%

Cloning, expression and molecular characterization of a Cystoisospora suis specific uncharacterized merozoite protein

et al. 2017

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BackgroundThe genome of the apicomplexan parasite Cystoisospora suis (syn. Isospora suis) has recently been sequenced and annotated, opening the possibility for the identification of novel therapeutic targets against cystoisosporosis. It was previously proposed that a 42 kDa uncharacterized merozoite protein, encoded by gene CSUI_005805, might be a relevant vaccine candidate due to its high immunogenic score, high expression level and species-specificity as determined in silico.MethodsThe 1170 bp coding sequence of the CSUI_005805 gene was PCR amplified and cloned into the bacterial expression vector pQE-31. The specificity of the expressed recombinant protein was evaluated in an immunoblot, and relative levels of expression in different developmental stages and subcellular localization were determined by quantitative real-time PCR and indirect immunofluorescence assay, respectively.ResultsThe CSUI_005805 gene encoded for a 389 amino acid protein containing a histidine-rich region. Quantitative RT-PCR showed that CSUI_005805 was differentially expressed during the early development of C. suis in vitro, with higher transcript levels in merozoites compared to sporozoites. The recombinant protein was specifically recognized by sera from chicken immunized with recombinant CSUI_005805 protein and sera from piglets experimentally infected with C. suis, all of which suggested that despite prokaryotic expression, the recombinant CSUI_005805 protein maintained antigenic determinants and could elicit an immune response in the host. Immunofluorescence labelling and confocal microscopy revealed localization primarily at the surface of the parasite.ConclusionsThe results suggest that CSUI_005805 is highly expressed in merozoites and might thus be critical for their survival and establishment inside host cells. Owing to its specificity, localization and expression pattern, CSUI_005805 could be exploited as an attractive candidate for alternative control strategies against C. suis such as vaccines.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-017-2003-1) contains supplementary material, which is available to authorized users.

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Current and Emerging Approaches to Studying Invasion in Apicomplexan Parasites

Cited by 12 publications

References 252 publications

A Small-Molecule Inhibitor of T. gondii Motility Induces the Posttranslational Modification of Myosin Light Chain-1 and Inhibits Myosin Motor Activity

A Small-Molecule Inhibitor of T. gondii Motility Induces the Posttranslational Modification of Myosin Light Chain-1 and Inhibits Myosin Motor Activity

Chemical genetic screen identifies Toxoplasma DJ-1 as a regulator of parasite secretion, attachment, and invasion

Cloning, expression and molecular characterization of a Cystoisospora suis specific uncharacterized merozoite protein

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