Abstract
Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Western blot analysis showed that this antibody reacted with a polypeptide of approximately 42 kDa in the whole-cell lysates of parasites targeting mammals (T. cruzi, Trypanosoma brucei and Leishmania major), insects (Angomonas deanei, Crithidia fasciculata, Herpetomonas samuelpessoai and Strigomonas culicis) and plants (Phytomonas serpens). A single polypeptide of approximately 42 kDa was detected in the whole-cell lysates of T. cruzi cultured epimastigotes, metacyclic trypomastigotes and amastigotes at similar protein expression levels. Confocal microscopy showed that actin was expressed throughout the cytoplasm of all the tested trypanosomatids. These data demonstrate that actin expression is widespread in trypanosomatids.
Highlights
Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein
When used in western blotting to probe for the presence of actin in trypanosomatids, this antibody reacted with a peptide of approximately 42 kDa in the whole-cell lysates of multiple species (Fig. 1A), including parasites targeting mammals (T. cruzi epimastigotes, T. brucei procyclic forms and L. major promastigotes), insects (A. deanei, C. fasciculata and S. culicis) and plants (P. serpens)
A mouse T. cruzi actin gene (TcActin) polyclonal antibody was cross-reacted with different trypanosomatids, including some monoxenic species found in insects (A. deanei, C. fasciculata, H. samuelpessoai and S. culicis) as well as a plant species (P. serpens)
Summary
Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Actin filaments have been only poorly visualised in trypanosomatids through transmission electron microscopy (Sahasrabuddhe et al 2004) Their presence has been demonstrated by immunofluorescence in the cytoplasm of the pathogenic species Trypanosoma brucei (García-Salcedo et al 2004), Trypanosoma cruzi (de Souza et al 1983, Mortara 1989, Melo et al 2008, Cevallos et al 2010) and Leishmania major (Sahasrabuddhe et al 2004). The subcellular localisation of T. cruzi actin was evaluated using a homologous antibody and an immunofluorescence analysis of the different developmental forms of this parasite (epimastigotes, bloodstream trypomastigotes and amastigotes) This assay revealed many rounded and punctuated structures, resembling patches, distributed throughout the cytoplasm, which had no association with subpellicular microtubules or with the nuclear or kinetoplast DNA (Melo et al 2008). We have analysed the expression of this protein in different developmental forms of T. cruzi (cultured epimastigotes, in vitro-derived trypomastigotes and cell culture-derived amastigotes) and studied its subcellular localisation by confocal microscopy
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