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Fractionation of membrane components from tachyzoite forms of Toxoplasma gondii: differential recognition by immunoglobulin M (IgM) and IgG present in sera from patients with acute or chronic toxoplasmosis.

ABSTRACT: Tachyzoite forms of Toxoplasma gondii were subjected to a sequential organic solvent extraction, which allows fractionation of membrane components according to their degrees of hydrophobicity, yielding three fractions named F1 (most hydrophobic) to F3 (least hydrophobic). Fractions F2 (80.85% specificity and 86.95% sensitivity) and F3 (89.36% specificity and 93.61% sensitivity) gave the best results, being preferentially recognized by immunoglobulin M (IgM) and IgG in sera from patients with acute and chronic toxoplasmosis, respectively. Improved scores of specificity (100%) and sensitivity (100%) were achieved when a secondary antibody against human IgG1 instead of total IgG was employed to measure the reactivity of IgG antibodies with the F3 fraction. To purify tachyzoite antigens recognized by human IgM or IgG antibodies, the F2 or F3 fraction was loaded onto an octyl-Sepharose column and eluted with a propan-1-ol gradient. The main antigen(s) recognized by IgM or IgG eluted in a single peak from the octyl-Sepharose resin loaded with either F2 (30 to 50% propan-1-ol) or F3 (15 to 35% propan-1-ol), respectively. These semipurified fractions gave improved scores when used to detect T. gondii-specific IgM (95.7% specificity and 81.8% sensitivity) or IgG (100% specificity and 93. 75% sensitivity) in an enzyme-linked immunosorbent assay. Further biochemical and immunological analyses of antigens partially purified from F2 and F3 indicate that glycoinositolphospholipids are preferentially recognized by IgM, whereas proteins of approximately 30 to 40 kDa are recognized by IgG, elicited during T. gondii infection in humans.

SUBMITTER: Giraldo M

PROVIDER: S-EPMC86463 | biostudies-literature | 2000 Apr

REPOSITORIES: biostudies-literature

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Fractionation of membrane components from tachyzoite forms of Toxoplasma gondii: differential recognition by immunoglobulin M (IgM) and IgG present in sera from patients with acute or chronic toxoplasmosis.

Giraldo M M Cannizzaro H H Ferguson M A MA Almeida I C IC Gazzinelli R T RT

Journal of clinical microbiology 20000401 4

Tachyzoite forms of Toxoplasma gondii were subjected to a sequential organic solvent extraction, which allows fractionation of membrane components according to their degrees of hydrophobicity, yielding three fractions named F1 (most hydrophobic) to F3 (least hydrophobic). Fractions F2 (80.85% specificity and 86.95% sensitivity) and F3 (89.36% specificity and 93.61% sensitivity) gave the best results, being preferentially recognized by immunoglobulin M (IgM) and IgG in sera from patients with acu ...[more]

PMID: 10747125

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Project description:Toxoplasma gondii is a highly successful parasite that infects approximately one-third of the human population and can cause fatal disease in immunocompromised individuals. Systemic parasite dissemination to organs such as the brain and eye is critical to pathogenesis. T. gondii can disseminate via the circulation, and both intracellular and extracellular modes of transport have been proposed. However, the processes by which extracellular tachyzoites adhere to and migrate across vascular endothelium under the conditions of rapidly flowing blood remain unknown. We used microfluidics and time-lapse fluorescence microscopy to examine the interactions between extracellular T. gondii and primary human endothelial cells under conditions of physiologic shear stress. Remarkably, tachyzoites adhered to and glided on human vascular endothelium under shear stress conditions. Compared to static conditions, shear stress enhanced T. gondii helical gliding, resulting in a significantly greater displacement, and increased the percentage of tachyzoites that invaded or migrated across the endothelium. The intensity of the shear forces (from 0.5 to 10 dynes/cm(2)) influenced both initial and sustained adhesion to endothelium. By examining tachyzoites deficient in the T. gondii adhesion protein MIC2, we found that MIC2 contributed to initial adhesion but was not required for adhesion strengthening. These data suggest that under fluidic conditions, T. gondii adhesion to endothelium may be mediated by a multistep cascade of interactions that is governed by unique combinations of adhesion molecules. This work provides novel information about tachyzoite interactions with vascular endothelium and contributes to our understanding of T. gondii dissemination in the infected host. IMPORTANCE Toxoplasma gondii is a global parasite pathogen that can cause fatal disease in immunocompromised individuals. An unresolved question is how the parasites circulate in the body to tissues to cause disease. T. gondii parasites are found in the bloodstream of infected animals and patients, and they have been shown to adhere to and cross the endothelial cells that line blood vessel walls. To investigate these interactions, we devised a microfluidic system to visualize parasites interacting with vascular endothelium under conditions similar to those found in the bloodstream. Interestingly, parasite migration was significantly influenced by the mechanical force of shear flow. Furthermore, we identified a role for the parasite surface protein MIC2 in the initial phase of adhesion. Our study is the first to document T. gondii interactions with endothelium under shear stress conditions and provides a foundation for future studies on the molecules that mediate parasite interaction with the vasculature.

Dataset Information

Fractionation of membrane components from tachyzoite forms of Toxoplasma gondii: differential recognition by immunoglobulin M (IgM) and IgG present in sera from patients with acute or chronic toxoplasmosis.

Publications

Fractionation of membrane components from tachyzoite forms of Toxoplasma gondii: differential recognition by immunoglobulin M (IgM) and IgG present in sera from patients with acute or chronic toxoplasmosis.

Similar Datasets

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