Leishmania donovani resides in modified early endosomes by upregulating Rab5a expression via the downregulation of miR-494
Fig 2
Leishmania donovani downregulates the expression of miR-494 by degrading c-Jun in macrophages.
A. Leishmania infection modulates the expression of several miR in infected human macrophages as revealed by microarray analysis. Expression fold values are provided in terms of log base 2. Colour scale shown in the right illustrates the relative expression level of miRNAs in Leishmania infected macrophages in comparison to uninfected control. Yellow colour represents an expression level above the mean and Red colour represents the expression level lower than the mean. The whole microarray data have been submitted in Gene Expression Omnibus database (accession number GSE89529). B. TargetScan prediction algorithms showing that 3/-UTR of Rab5a of human and hamster contains an 8-mer target site that precisely matches the seed region of miR-494. C. THP-1 differentiated macrophages were infected with Leishmania (MOI 1:20) and level of expression of miR-494 in infected cells was determined at indicated time by qPCR as described in Materials and Methods. D. Similar experiments were carried out with indicated MOI of infection and level of miR-494 was detected after 12 h. E. THP-1 differentiated human macrophages were infected with Leishmania (MOI 1:20) and level of c-Jun in infected cells was determined at indicated time by Western blot analysis using specific antibody. Actin was used as control. F. Similar experiments were carried out with gp63 secretion deficient transgenic parasites (Ld-Rab1:S22N) and level of c-Jun in infected cells was determined after 6 h of infection by Western blot analysis using specific antibody. All results are represented as mean ± S.D. of three independent experiments and normalized to respective control. Expression of normalized miR-494 or c-Jun in uninfected cells in respective experiment was arbitrarily chosen as one unit. Results of uninfected (*) and infected cells were analyzed by paired t test and levels of significance are indicated by P value.