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A highly sensitive FRET-based approach reveals secretion of the actin-binding protein toxofilin during Toxoplasma gondii infection.

ABSTRACT: We have utilized a highly sensitive approach based on fluorescence resonance energy transfer (FRET) and beta-lactamase (BLA), which we adapted for the detection of Toxoplasma gondii secreted proteins. This assay revealed that the actin-binding protein toxofilin appears to be secreted into host cells during invasion. To determine the function of toxofilin during infection, we engineered a type I (RH strain) parasite with a targeted deletion of the toxofilin gene and compared the phenotypes of control and toxofilin knockout (Deltatxf) parasites in several in vitro assays, including invasion, growth, gliding motility, and egress of the Deltatxf parasites, as well as F-actin staining, phagocytosis and migration of cells infected with Deltatxf parasites or wild-type controls. Despite its apparent secretion into host cells and its ability to bind to and modulate host actin, we observed that toxofilin does not appear to play a role in these processes, under the conditions we examined, and we report these findings here.

SUBMITTER: Lodoen MB

PROVIDER: S-EPMC3411547 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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A highly sensitive FRET-based approach reveals secretion of the actin-binding protein toxofilin during Toxoplasma gondii infection.

Lodoen Melissa B MB Gerke Christiane C Boothroyd John C JC

Cellular microbiology 20090902 1

We have utilized a highly sensitive approach based on fluorescence resonance energy transfer (FRET) and beta-lactamase (BLA), which we adapted for the detection of Toxoplasma gondii secreted proteins. This assay revealed that the actin-binding protein toxofilin appears to be secreted into host cells during invasion. To determine the function of toxofilin during infection, we engineered a type I (RH strain) parasite with a targeted deletion of the toxofilin gene and compared the phenotypes of con ...[more]

PMID: 19732057

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Project description:A widely prevalent disease, toxoplasmosis poses serious health threats to both humans and animals; therefore, development of an ideal DNA vaccine against Toxoplasma gondii is needed eagerly. The purpose of the present study is to assess the protective efficacy of a DNA vaccine encoding the T. gondii toxofilin gene (pEGFP-toxofilin). In addition, toxofilin DNA vaccine combined with the individual adjuvants, alum or monophosphoryl lipid A (MPLA), or a mixture of alum-MPLA adjuvant were screened for their ability to enhance antibody responses.Using bioinformatics, we analyzed the gene and amino acid sequences of the toxofilin protein, recognizing and identifying several potential linear B and T helper (Th)-1 cell epitopes. BALB/c mice were immunized three times with either toxofilin DNA vaccine alone or in combination with the adjuvants such as alum, MPLA or an alum-MPLA mixture. The systemic immune response was evaluated by cytokine, the percentage of CD4 (+) and CD8 (+) T cells and specific antibody measurement. Two weeks after the last immunization, protective efficacy was evaluated by challenging intraperitoneally with 1?×?104 tachyzoites of T. gondii or intragastrically with 20 cysts of T. gondii PRU strain.All experimentally immunized mice developed strong humoral and cellular immune responses compared with the control groups. Moreover, by comparison with the non-adjuvant toxofilin DNA vaccine group, adding alum adjuvant to toxofilin DNA vaccine resulted in an increase in humoral response and a skewed Th2 response. However, the MPLA adjuvant with toxofilin DNA vaccine induced significantly enhanced humoral and Th1-biased immune responses. Importantly, the co-administration of alum-MPLA adjuvant in combination with the toxofilin DNA vaccine shifted the Th2 immune response to a Th1 response compared with the alum-toxofilin group, and elicited the strongest humoral and Th1 responses among all the groups. At the same time, a longer survival time and less cyst amounts against T. gondii infection were also observed in the alum-MPLA-toxofilin group in comparison with single or no adjuvant groups.Toxoplasma gondii toxofilin is a promising vaccine candidate that warrants further development. Co-administration of the alum-MPLA adjuvant mixture with DNA vaccine could effectively enhance immunogenicity and strongly skew the cellular immune response towards a Th1 phenotype.

Dataset Information

A highly sensitive FRET-based approach reveals secretion of the actin-binding protein toxofilin during Toxoplasma gondii infection.

Publications

A highly sensitive FRET-based approach reveals secretion of the actin-binding protein toxofilin during Toxoplasma gondii infection.

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