<i>Leishmania donovani</i> resides in modified early endosomes by upregulating Rab5a expression via the downregulation of miR-494

<div><p>Several intracellular pathogens arrest the phagosome maturation in the host cells to avoid transport to lysosomes. In contrast, the <i>Leishmania</i> containing parasitophorous vacuole (PV) is shown to recruit lysosomal markers and thus <i>Leishmania</i> is postulated to be residing in the phagolysosomes in macrophages. Here, we report that <i>Leishmania donovani</i> specifically upregulates the expression of Rab5a by degrading c-Jun via their metalloprotease gp63 to downregulate the expression of miR-494 in THP-1 differentiated human macrophages. Our results also show that miR-494 negatively regulates the expression of Rab5a in cells. Subsequently, <i>L</i>. <i>donovani</i> recruits and retains Rab5a and EEA1 on PV to reside in early endosomes and inhibits transport to lysosomes in human macrophages. Similarly, we have also observed that <i>Leishmania</i> PV also recruits Rab5a by upregulating its expression in human PBMC differentiated macrophages. However, the parasite modulates the endosome by recruiting Lamp1 and inactive pro-CathepsinD on PV via the overexpression of Rab5a in infected cells. Furthermore, siRNA knockdown of Rab5a or overexpression of miR-494 in human macrophages significantly inhibits the survival of the parasites. These results provide the first mechanistic insights of parasite-mediated remodeling of endo-lysosomal trafficking to reside in a specialized early endocytic compartment.</p></div

<i>Leishmania donovani</i> infection specifically upregulate the expression of Rab5a in macrophages.

<p>A. <i>Leishmania</i> infected THP-1 differentiated human macrophages were lysed at different time points after infection and levels of indicated Rabs were determined by Western blot analysis using specific antibodies. Actin was used as loading control. Lower panel indicates the quantitation of the respective data. Lower band of Rab11 corresponding to uninfected control was only used for quantitation of Rab11 in infected cells. B. Levels of different Rabs in infected and uninfected human macrophages at indicated time points were determined by qPCR as described in Materials and Methods. C. Levels of different isoforms of Rab5 in uninfected and infected human macrophages were determined by Western blot analysis using specific antibodies. All results are represented as mean ± S.D. of three independent experiments and normalized to respective control. Expression of normalized indicated Rab in uninfected cells was arbitrarily chosen as one unit. Results of the indicated groups were analyzed by paired <i>t</i> test and levels of significance are indicated by <i>P</i> value.</p

Schematic representation of mechanism of survival of <i>Leishmania donovani</i> in early endocytic compartment in macrophages.

<p><i>Leishmania</i> after entering into human macrophages secretes gp63 which degrades c-Jun to downregulate the expression of miR-494 and thereby overexpress Rab5a in the infected cells. Subsequently, <i>Leishmania</i> recruits and retains Rab5a, EEA1 on PV and inhibits transport to lysosome. Interestingly, <i>Leishmania</i> also recruits inactive CathepsinD and Lamp1 on PV to reside in a modified early endocytic compartment in human macrophages.</p

<i>Leishmania donovani</i> containing PV specifically recruits Rab5a and EEA1 in human macrophages.

<p>THP-1 differentiated human macrophages were infected with <i>Leishmania</i> and recruitment of Rab5a (A), EEA1 (B), Rab7 (C) were determined after 24 h of infection by immuno-staining with specific antibody as described in Materials and Methods. Uninfected cells were used as control. <i>Leishmania</i> and macrophage nucleus were stained with propidium iodide (Red). All results are representative of three independent experiments. D. Results are represented as mean ± S.D. of three independent experiments and expressed as percentage of <i>Leishmania</i>-PV positive for indicated markers after counting 100 cells.</p

miR-494 negatively regulates the expression of Rab5a.

<p>A. To determine miR-494 mediated regulation of hereterologous expression of Rab5a chimeric construct, pmir-GLO chimeric construct containing Rab5a 3^/-UTR or its mutant were cotransfected with 40 nM miR-494 or control mimic miR into semiconfluent HeLa cells. After 48 h, cells were lysed and luciferase activity was measured as described in Materials and Methods. Results are represented as mean ± S.D. of three independent experiments and expressed as relative luciferase activity compared to miR-494 untreated control cells arbitrarily chosen as one unit. To determine miR-494 mediated regulation of endogenous expression of Rab5 isoforms, HeLa (B) or THP-1 differentiated human macrophages (C) were transfected with 40 nM miR-494 and levels of different isoforms of Rab5 were determined as described in Materials and Methods. Results are represented as mean ± S.D. of three independent experiments and demonstrated as relative expression of Rab5 isoforms compared to untreated control arbitrarily chosen as one unit. Inset (C) shows the level of expression of miR-494 in THP-1 transfected cells. D. THP-1 differentiated human macrophages were transfected with indicated concentrations of miR-494 and levels of Rab5a protein were determined by Western blot analysis as described in Materials and Methods. Actin was used as control. Results are represented as mean ± S.D. of three independent experiments and normalized to respective control. Expression of normalized Rab5a in uninfected cells was arbitrarily chosen as one unit. Results of control (*) and miR-494 overexpressed cells were analyzed by paired <i>t</i> test and levels of significance are indicated by <i>P</i> value.</p

<i>Leishmania donovani</i> downregulates the expression of miR-494 by degrading c-Jun in macrophages.

<p>A. <i>Leishmania</i> 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 <i>Leishmania</i> 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 <i>Leishmania</i> (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 <i>Leishmania</i> (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 <i>t</i> test and levels of significance are indicated by <i>P</i> value.</p

<i>Leishmania donovani</i> containing PV specifically recruits Rab5a human PBMC differentiated macrophages.

<p>A. Levels of Rab5a in infected and uninfected PBMC differentiated macrophages after 24 h of infection were determined by qPCR as described in Materials and Methods. B. Human PBMC differentiated macrophages were infected with <i>Leishmania</i> (MOI 1:20) and level of expression of miR-494 in uninfected and infected cells was determined at indicated time by qPCR as described in Materials and Methods. All results are represented as mean ± S.D. of three independent experiments and normalized to respective control. Expression of normalized respective data in uninfected cells was arbitrarily chosen as one unit. C. Human PBMC differentiated macrophages were infected with <i>Leishmania</i> and recruitment of Rab5a was determined after 24 h of infection by immuno-staining with specific antibody as described in Materials and Methods. Uninfected cells were used as control. Results are representative of three independent experiments.</p

<i>Leishmania donovani</i> inhibits its transport to lysosomes in macrophages.

<p>A. Lysosomes of THP-1 differentiated human macrophages were prelabeled with internalization of Dextran Texas Red for 24 h and subsequently, cells were infected with Fluoresbrite-YG-latex beads (Green) as described in Materials and Methods (upper panel). Similarly, lysosomes of THP-1 differentiated human macrophages were labeled with Green fluorescent labeled latex beads and cells were immune-stained with anti-CathepsinD (middle panel) or anti-Lamp1 (middle panel) antibody as described in Materials and Methods. THP-1 differentiated human macrophages were coinfected with <i>Leishmania</i> and Green fluorescent labeled latex beads and chased for 24 h at 37°C and their localization was determined by confocal microscopy (lower panel). <i>Leishmania</i> and macrophage nucleus were stained with propidium iodide (Red). All results are representative of three independent experiments. B. Results are represented as mean ± S.D. of three independent experiments and expressed as percentage of latex-bead containing phagosomes positive for indicated markers after counting 100 cells. C. THP-1 differentiated human macrophages were infected with <i>Leishmania</i> or Green fluorescent labeled latex beads. Cells were incubated for 24 h at 37°C and lysosomes were labeled with DQ-BSA Red as described in Materials and Methods. <i>Leishmania</i> and macrophage nucleus were stained with Hoechst (Blue). Cells were viewed in an LSM 510 Meta confocal microscope using an oil immersion objective. Results are expressed as mean percentage of total fluorescence per cell ± S.D. of 100 independent indicated cells.</p

Rab5a function is required for the survival of parasites in macrophages.

<p>A. THP-1 differentiated human macrophages were transfected with 50 nM Rab5a specific siRNA or control RNA and levels of different isoforms of Rab5 were determined after 48 h by Western blots analysis. B. THP-1 differentiated human macrophages were transfected with 50 nM miR-494 or Rab5a specific siRNA and subsequently cells were infected with <i>Leishmania</i> promastigotes as described in Materials and Methods. Parasites load in the infected macrophages were microscopically estimated at indicated time. Results are expressed as numbers of parasites present in 100 macrophages ± S.D. from three independent experiments. Results of control (*) and Rab5a siRNA/miR-494 overexpressed cells were analyzed by paired <i>t</i> test and levels of significance are indicated by <i>P</i> value. C. Confocal images of the representative experiment showing the parasite load in macrophages. <i>Leishmania</i> and macrophage nucleus were stained with propidium iodide (Red). D. THP-1 differentiated human macrophages were transfected with 50 nM miR-494 and infected with <i>Leishmania</i> for 24 h as described in Materials and Methods. Recruitment of Rab5a on <i>Leishmania</i>-PV in miR-494 overexpressed human macrophages was detected using specific antibody by confocal microscopy. Results are represented of three independent experiments.</p

Recruitment of Lamp1 and CathepsinD on <i>Leishmania donovani</i> containing PV.

<p>A. THP-1 differentiated human macrophages were infected with <i>Leishmania</i> and recruitment of Lamp1 and CathepsinD were determined after 24 h of infection by immuno-staining with specific antibody as described in Materials and Methods. Uninfected cells were used as control. <i>Leishmania</i> and macrophage nucleus were stained with propidium iodide (Red). B. Rab5a or its mutants were overexpresses in HeLa cells as GFP fusion protein and cells were immuno-stained with anti-CathepsinD or anti-Lamp1 antibody (C). Cells were viewed under confocal microscope to determine the localization of indicated protein. All results are representative of three independent experiments. D. THP-1 differentiated human macrophages were infected with live or dead <i>Leishmania</i> and chased for 24 h. Finally, cells were lysed and size of the CathepsinD was determined by Western blot analysis using specific antibody. Results are represented as mean ± S.D. of three independent experiments and expressed a ratio of mature CathepsinD and pro-CathepsinD compared to ratio in dead infected cells arbitrarily chosen as one unit. Results of dead and live parasite infected cells were analyzed by paired <i>t</i> test and levels of significance are indicated by <i>P</i> value. E. Similarly, size of the CathepsinD was determined in Rab5a:WT, Rab5a:Q79L or Rab5a:S34N overexpressed HeLa cells using specific antibody. Results are represented as mean ± S.D. of three independent experiments and expressed as a ratio of mature CathepsinD and pro-CathepsinD compared to ratio in control cells normalized to actin was arbitrarily chosen as one unit.</p