Abstract
The invasive nature of Toxoplasma gondii is closely related to the properties of its cytoskeleton, which is constituted by a group of diverse structural and dynamic components that play key roles during the infection. Even if there have been numerous reports about the composition and function of the Toxoplasma cytoskeleton, the ultrastructural organization of some of these components has not yet been fully characterized. This study used a detergent extraction process and several electron microscopy contrast methods that allowed the successful isolation of the cytoskeleton of Toxoplasma tachyzoites. This process allowed for the conservation of the structures known to date and several new structures that had not been characterized at the ultrastructural level. For the first time, characterization was achieved for a group of nanofibers that allow the association between the polar apical ring and the conoid as well as the ultrastructural characterization of the apical cap of the parasite. The ultrastructure and precise location of the peripheral rings were also found, and the annular components of the basal complex were characterized. Finally, through immunoelectron microscopy, the exact spatial location of the subpellicular network inside the internal membrane system that forms the pellicle was found. The findings regarding these new structures contribute to the knowledge concerning the biology of the Toxoplasma gondii cytoskeleton. They also provide new opportunities in the search for therapeutic strategies aimed at these components with the purpose of inhibiting invasion and thus parasitism.
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Acknowledgments
This research was supported by grant no. 51486 FORDECYT-PRONACES, “CIENCIAS DE FRONTERA 2019 CONACyT” to RMF and by the scholarship from Consejo Nacional de Ciencia y Tecnología (CONACyT, Mexico) to FESR (# 394177). We thank Jorge Fernández-Hernández and Antonieta López-López from the Unit for Experimentation and Production of Laboratory Animals (UPEAL, CINVESTAV-IPN, Mexico) for the supply of the mice. We thank Norma H. Martínez for her help in the grammar correction. Micrographs were obtained at the Electron Microscopy Facility (LaNSE, CINVESTAV-IPN, Mexico).
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Fig. S1
Validation of the polyvalent antibody directed against components of the T. gondii cytoskeleton. PAGE-SDS corresponds to the electrophoretic pattern of whole extract of tachyzoites (WE), cytoskeleton-enriched fraction (Ck), and soluble fraction isolated as supernatant after centrifugation (Snt). Mice polyvalent antibody against the cytoskeleton fraction was tested against the isolated fraction of the T. gondii cytoskeleton by Western blot (Wb). Seven major immunogenic proteins recognized by the polyvalent antibody are indicated by red arrows. IMC1 was tested as positive control for cytoskeleton fraction. The pre-immune serum (C−) did not present any reaction. Antibody against lamin B1 was a negative control for the CK; it recognized only a band in the WE and the Snt but not the CK. Ck, cytoskeleton fraction; Snt, supernatant; WE, whole extract; Ab αCk, polyvalent antibody against Ck (PNG 333 kb)
Fig. S2
IEM of isolated cytoskeletons with pre-immune serum. Mouse pre-immune serum was tested as a negative control for the polyvalent antibody directed against the components of the T. gondii cytoskeleton and then micrographed in the TEM. a Whole cytoskeleton of a tachyzoite. Squares in (a) represent magnifications of different zones of the cytoskeleton such as (b), apical end; (c), subpellicular microtubules; (d), subpellicular network; and (e), posterior end. Scale bars in (a) = 1 μm; and (b)–(e) = 500 nm (PNG 1125 kb)
Fig. S3
IEM of cytoskeletons processed for ultramicrotomy with pre-immune serum and anti-catalase. Isolated cytoskeletons were incubated with pre-immune serum (a, b) and antibody against catalase (c), as negative controls for the polyvalent antibody directed against components of the T. gondii cytoskeleton. Afterwards, samples were processed for ultramicrotomy. Scale bars = 500 nm (PNG 1235 kb)
Supplementary Table S1
Morphometric analysis of the different structures found in Toxoplasma gondii cytoskeleton. The measures of the different structures were performed using Fiji software and the scale bars were obtained directly from the TEM micrographs; values are presented in nm. Statistical analyses were performed using SigmaStat 4.0. The standard deviation (SD) and standard error of the mean (SEM) were obtained from measures done in micrographs of the different structures obtained from at least 60 parasites from about 10 independent processing cycles (DOC 71 kb)
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Díaz-Martin, R.D., Sandoval Rodriguez, F.E., González Pozos, S. et al. A comprehensive ultrastructural analysis of the Toxoplasma gondii cytoskeleton. Parasitol Res 121, 2065–2078 (2022). https://doi.org/10.1007/s00436-022-07534-3
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DOI: https://doi.org/10.1007/s00436-022-07534-3