Effect of EhCoactosin over-expression on growth of trophozoites.

<p>The overexpression and underexpression of EhCoactosin in trophozoites is regulated by a tetracycline inducible promoter. (A) Immunoblot analysis of amebic cells with recombinant constructs. Cells carrying vector alone in presence and absence of tetracycline showed similar levels of EhCoactosin (lane 1 and 2 from left). Cells carrying EhCoactosin in the sense orientation showed over-expression (sense) of protein upon tetracycline induction (lane 4–6 from left) in comparison to cells with vector alone (lane 1and 2). The antisense construct (lane 7) and the vector alone (TOC) (lane 1 and 2) in the absence of tetracycline did not show significant differences in EhCoactosin levels. Induction of EhCoactosin antisense RNA in the presence of tetracycline (lanes 8–10 from left) reduced the expression of EhCoactosin in comparison to only vector containing cells in the presence of tetracycline (lane 2). The equal loading of total lysate has been shown by probing for EhCaBP1, which is not affected by inducing agent. (B) Erythrophagocytosis by trophozoites overexpressing and underexpressing EhCoactosin for fixed time of 15 min. (C) and (D) Cells with indicated constructs with and without tetracycline were incubated with human erythrocytes for indicated time points and then were fixed and stained with TRITC-Phalloidin. Initiation of phagocytosis was marked by accumulation of actin at the phagocytic cups. Fifty cells were randomly selected for each experiment and the number of phagocytic cups present in all the cells was then counted. The experiment was carried out independently thrice and statistical significance was tested by ‘t test’. The * marks the p values in figure. (E) Growth curve of <i>E. histolytica</i> trophozoites overexpressing and underexpressing EhCoactosin over 72 h. The growth is monitored in the absence and presence of tetracycline.