Abstract
Cryptosporidium parasites are known to be highly divergent from other apicomplexan species at evolutionary and biological levels. Here we provide evidence showing that the zoonotic Cryptosporidium parvum also differs from other apicomplexans, such as Toxoplasma gondii, by possessing only two tubulin-based filamentous structures, rather than an array of subpellicular microtubules. Using an affinity-purified polyclonal antibody against C. parvum β-tubulin (CpTubB), we observed a long and a short microtubule that are rigid and stable in the sporozoites and restructured during the intracellular parasite development. In asexual development (merogony), the two restructuring microtubules are present in pairs (one pair per nucleus or merozoites). In sexual developmental stages, tubulin-based structures are detectable only in microgametes, but undetectable in macrogametes. These observations indicate that C. parvum parasites use unique microtubule structures that differ from other apicomplexans as part of their cytoskeletal elements.
Highlights
These observations indicate that C. parvum parasites use unique microtubule structures that differ from other apicomplexans as part of their cytoskeletal elements
Apicomplexan parasites are unicellular organisms named after their unique apical complex that is composed of specific cytoskeletal elements and secretory organelles
In the case of Toxoplasma gondii tachyzoites for which cytoskeletons were more thoroughly studied, tubulin-based structures include conoid and a pair of intra-conoid microtubules as part of the apical complex, and 22 evenly distributed subpellicular microtubules in a left-handed spiral that emerge from the apical polar ring (APR) and extend down to two-thirds the length of the tachyzoites [4,5]
Summary
Anti-CpTubB antibody: Tubulin-based structures are composed of α- and β-tubulin heterodimers that can be labeled by an antibody specific to either α- or β-tubulin. Peptide was synthesized by ChinaPeptides Company (Shanghai, China), linked to keyhole limpet hemocyanin (KLH) via maleimidobenzoyl-N-hydroxysuccinimide ester in-house [16], and used to immunize two specific pathogen-free rabbits using a standard antibody production protocol [17]. Specific antibody was affinity-purified by a nitrocellulose membrane-based protocol with slight modification [18]. 4 mL of 1:20 diluted antisera for h at room temperature and overnight at 4 ◦ C, five washes with TBST and elution with 1 mL glycine elution buffer (GBST; 0.2 M glycine, 0.15 M NaCl, 0.05% Tween-20, pH 2.7), and immediate neutralization of eluted antibody with 50 μL of.
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