Several intracellular pathogens arrest the phagosome maturation in the host cells to avoid transport to lysosomes. In contrast, the Leishmania containing parasitophorous vacuole (PV) is shown to recruit lysosomal markers and thus Leishmania is postulated to be residing in the phagolysosomes in macrophages. Here, we report that Leishmania donovani 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, L. donovani 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 Leishmania 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.
Leishmania donovani (Ld) is an obligate intracellular parasite which causes visceral leishmaniasis in the mammalian hosts that affects annually about 12 million people worldwide [1]. This parasite is postulated to reside and replicate in a phagolysosomal compartment in mouse macrophages as the parasites acquire lysosomal markers such as Lysosome Associated Membrane Protein 1 (Lamp1), Lamp2 and CathepsinD on PV [2,3]. However, not much is known about how Leishmania is surviving in such detrimental compartment. Interestingly, it has been shown that Leishmania excludes Vesicular Proton-ATPase on PV by inhibiting the recruitment of Synaptotagmin V to prevent the acidification in mouse macrophages [4]. Consequently, some recent studies have shown that Leishmania also modulates the recruitment of Rab7 [5] and ER markers [6] on PV in mouse macrophages. Thus, the Leishmania possibly resides in a hybrid compartment, but the nature of the compartment is not well characterized [7]. Since Rab GTPases are the central regulators of membrane trafficking pathways [8,9, 10], targeting the function of Rab proteins is one of the commonly used mechanisms exploited by intracellular pathogens to subvert their lysosomal targeting [11,12,13,14,15,16]. Moreover, successful intracellular pathogens are also shown to modulate the expression of host cytokines to establish a safe niche inside the host cells [17,18,19]. Consequently, we have shown that cytokines can specifically modulate the expression of different Rabs [20]. Thus, it is possible that intracellular pathogens might also modulate the expression of Rab GTPases in the host cells. In addition, recent studies have shown that several intracellular pathogens such as S. typhimurium, M. tuberculosis and L. monocytogenes target the host microRNAs (miRNA) to modulate the expression of host proteins for their successful infection and survival [21,22,23]. Thus, it is tempting to speculate that Leishmania might target the host miRNA (s) to modulate the expression of endocytic Rab GTPases in infected macrophages. Here, we have shown that L. donovani upregulates the expression of Rab5a in human macrophages by inhibiting the expression of miR-494 and retains Rab5a and EEA1 on PV to survive in an early endocytic (EE) compartment. Thus, in contrast to previous perception, our results demonstrate that L. donovani modulates endo-lysosomal pathway to reside in a modified early endocytic compartment and inhibits lysosomal transport in macrophages.
To determine whether Leishmania infection modulates the expression of different Rab GTPases, human THP-1 monocytic cell line were differentiated into macrophages (human macrophages) and were infected with L. donovani promastigotes (MOI 1: 20). Subsequently, the cellular contents of various Rabs were determined by Western blot analysis using specific antibodies at indicated time points of infection. About 4-fold and 2-fold increase in the cellular content of Rab5 and Rab11, respectively, were observed after 24 h of infection (Fig 1A) in comparison to uninfected control cells. No significant change in the levels of expression of Rab4 and Rab7 were observed in infected and uninfected cells. To determine whether higher level of Rab5 expression was due to transcriptional activation, we checked transcript levels of different endocytic Rab GTPases by Real Time PCR (qPCR) in infected human macrophages. Similar to protein expression, our results showed about 3-fold and 2-fold induction in the levels of Rab5a and Rab11 mRNA expression, respectively, after 24 h of L. donovani infection in comparison to uninfected control cells (Fig 1B). Previous studies demonstrated that Rab5 has three isoforms namely, Rab5a, Rab5b and Rab5c in mammalian cells [24]. Interestingly, we observed that L. donovani infection does not significantly alter the expression of Rab5b and Rab5c (Fig 1B). These results were further confirmed by Western blot analysis using isoform-specific antibodies (Fig 1C) and limited dilution semi-quantitative RT-PCR (S1 Fig). To understand the mechanism of upregulation of Rab5a expression in Leishmania infected human macrophages, we compared the miRNA profiles of uninfected and infected macrophages. Our results showed that expressions of 29 miRNAs are upregulated by more than 2-fold in L. donovani infected macrophages whereas expressions of 19 miRNAs are found to be downregulated by about 2-fold in infected cells in comparison to uninfected cells (Fig 2A). As miRNA negatively regulates the expression of their target gene, we screened those miRNAs which were downregulated in Leishmania infected human macrophages to identify their putative target sites in 3/-regulatory region of Rab5a using TargetScan prediction algorithms. This analysis predicted that 3/-UTR of Rab5a mRNA of human cells contains an 8-mer target site (5/-AUGUUUCA-3/) located between 191–198 nucleotides that precisely matches the seed region (positions 1–8) of miR-494 (Fig 2B). Interestingly, we observed that 8-mer target site of miR-494 is well conserved in the 3/-UTR of Rab5a in hamster whereas it is completely absent in the 3/-UTR of Rab5a in mice (Fig 2B). Our analysis also predicted (S2 Fig) that miR-494 has highest affinity to its complimentary target site for Rab5a (mirSVR score: -1. 2494) compared to Rab5b (mirSVR score: -0. 0086) and Rab5c (mirSVR score: -0. 1579) in human cells. Consequently, we determined the level of expression of miR-494 in L. donovani infected human macrophage (MOI 1: 20) at different time periods of infection. Our results showed about 60% inhibition of the expression of miR-494 in L. donovani infected macrophages after 24 h of infection in comparison to uninfected cells (Fig 2C). We also found that infection with MOI (MOI 1: 40) led to higher suppression of miR-494 than with MOI (MOI 1: 20) after 12 h of infection (Fig 2D). These results indicated that inhibition of miR-494 expression in infected macrophages is also dependent on the extent of infection. To determine how L. donovani infection downregulates the expression of miR-494 in macrophages, we checked for the level of c-Jun subunit of AP-1 transcription factor in infected cells as it was previously reported that miR-494 expression is regulated by AP-1 transcription factor [25]. Previous studies demonstrated that AP-1 is composed of c-Jun, c-Fos family of proteins. Interestingly, we found that c-Jun subunit level is significantly lower (~50%) in human macrophages after 6 h of infection with L. donovani in comparison to uninfected cells (Fig 2E). It was reported earlier that Leishmania gp63 is a metalloprotease which is secreted into host cells and degrades several host proteins including some transcription factors like NF-kB, STAT1 and AP-1 to alter gene expression [26,27]. Therefore, to determine how Leishmania infection lowers the c-Jun level in infected macrophages, we infected the human macrophages with L. donovani overexpressing LdRab1: S22N, a gp63 secretion deficient parasite [28]. Interestingly, our results showed (Fig 2F) that degradation of c-Jun is significantly lower in LdRab1: S22N infected cells in comparison to infection with Ld: WT cells. These results were substantiated by the fact that overexpression of gp63 in Raw 264. 7 mouse macrophages significantly degrades c-Jun (S3 Fig). Thus, Leishmania degrades c-Jun in infected human macrophages via their gp63 to inhibit the expression of miR-494. In order to validate the regulation of Rab5a expression by miR-494, we prepared chimeric construct by ligating 3/-UTR of human Rab5a with luciferase as heterologous reporter. First, the human 3/-UTR of Rab5a was identified from human genome database and amplified (1350 bp) from cDNA prepared from THP-1 macrophage using appropriate primers. Subsequently, amplified product (1350 bp) was digested and cloned in pmir-GLO dual luciferase vector at SacI/XhoI restriction sites. To determine the specificity, we also made another chimeric construct of 3/-UTR of Rab5a containing mutation in miR-494 recognition element (5/-CGACACGG-3/). The chimeric construct containing 3/-UTR region of Rab5a or its mutant was co-transfected with miR-494 mimic into HeLa cells and firefly luciferase reporter activity was measured after 48 h of transfection. Co-transfection of 3/-UTR region of human Rab5a and a nonspecific miR mimic into HeLa cells was used as a control. The result presented in the Fig 3A showed that transfection with the miR-494 (40 nM) reduces about 50% luciferase activity of Rab5a 3/-UTR reporter, whereas about 20% inhibition of mutant Rab5a 3/-UTR reporter was detected. These results indicated that miR-494 binds with miR-494 recognition element present in the 3/-UTR of human Rab5a to repress the expression of Rab5a. To demonstrate that miR-494 specifically regulates the expression of Rab5a, HeLa (Fig 3B) and THP-1 (Fig 3C) cells were transfected with miR-494 mimic (40 nM) or control mimic and levels of Rab5 isoforms were determined by qPCR using specific TaqMan probes. We observed that miR-494 specifically inhibits about 50% expression of Rab5a mRNA compared to the control mimic in both cells types. No significant changes were observed in Rab5b and Rab5c levels in miR-494 transfected cells. Finally, we checked the expression of Rab5a protein after 48 h of transfection of indicated concentration of miR-494 in THP-1 macrophages by Western blot analysis using specific antibody. Our results demonstrated that overexpression of miR-494 inhibits Rab5a expression in a concentration dependent manner. More than 80% inhibition of Rab5a protein expression was observed in 100 nM miR-494 transfected human macrophages in comparison to the control miR transfected cells (Fig 3D). Similar results were also observed in HeLa cells overexpressing miR-494 (S4 Fig). These results demonstrated that miR-494 regulates the expression of Rab5a in human cell. Subsequently, we determined whether L. donovani recruits Rab5a on Leishmania containing parasitophorous vacuole (Leishmania-PV) in macrophages. Indeed, we found that Rab5a is specifically recruited on Leishmania-PV (Fig 4A) after 24 h of infection in human macrophages. In contrast, cells infected with dead parasites were unable to recruit Rab5a and it was found to be localized on discrete small punctate vesicular structures as observed in uninfected cells. Among the different isoforms of Rab5, interestingly our results showed that Rab5a is predominantly recruited (S5A Fig) and retained on Leishmania-PV for at least 48 h (S5B Fig). Moreover, we also found that Leishmania-PV also recruits (Fig 4B) and retains (S5C Fig) Early Endosome Associated Antigen (EEA1), a Rab5 effector. Moreover, we also observed higher recruitment of Rab5a and EEA1 on PV at 24 h in comparison to 6 h. In addition, results presented in the Fig 4C showed that Leishmania-PV does not recruit Rab7 possibly to block transport to lysosomes. We also used anti-Rab8 and anti-Rab9 antibodies as control and found that Leishmania-PV does not recruit Rab9 and Rab8 indicating that Rab5a recruitment on PV is specific (S5D Fig). Further quantitation revealed that more than 90% of Leishmania-PV recruits Rab5a and EEA1 whereas less than 10% PV recruits Rab7 (Fig 4D). In addition, our results also showed that L. donovani infection overexpresses Rab5a in human PBMCs (Fig 5A) by downregulating the expression of miR-494 (Fig 5B). Consequently, Rab5a was found to be recruited on Leishmania-PV in Leishmania infected human PBMCs (Fig 5C). In contrast, we observed that Leishmania infection in Raw 264. 7 mouse macrophages neither induces the expression of Rab5a (S6A Fig) nor recruits Rab5a on Leishmania-PV (S6B Fig). Taken together, these results demonstrated that Leishmania-PV specifically recruits and retains Rab5a and EEA1 in human macrophages to reside in an early endocytic compartment. In order to unequivocally prove that Leishmania inhibits its transport to lysosomes by recruiting Rab5a and EEA1 on PV, lysosomes of the human macrophages were labeled with the internalization of latex beads and infected with Leishmania. Accordingly, THP-1 differentiated human macrophages were incubated with Fluoresbrite-YG-latex beads (2 μm) for 3 h and chased for 24 h at 37°C and subsequently cells were immuno-stained with anti-Lamp1 or anti-CathepsinD antibody. As expected, our results showed that latex bead containing phagosomes after 24 h of internalization are positive for both Lamp1 and CathepsinD (Fig 6A). In addition, latex beads after 24 h of internalization in human macrophages were found to be transported to dextran-Texas Red prelabeled lysosomes (Fig 6A). These results clearly demonstrated that lysosomes in macrophages can be labeled with internalization of latex beads for 24 h. Therefore, THP-1 differentiated macrophages were co-infected with latex beads and live Leishmania for 3 h at 37°C, washed and chased for additional 24 h at 37°C. Our results showed that Leishmania-PVs are clearly separated from latex beads containing lysosomes in human macrophages after 24 h of incubation (Fig 6A, lower panel). In addition, we also found that latex beads containing phagosomes are clearly separated from Rab5a and EEA1 positive Leishmania-PV in macrophages after 24 h of infection (S7 Fig). Further quantitation revealed that more than 90% of the latex beads containing phagosomes are positive for Dextran-Texas Red, Lamp1 and CathepsinD whereas Rab5a and EEA1 were almost not detected on these phagosomes after 24 h of internalization in macrophages. Most interestingly, more than 95% of Leishmania-PVs were found to be separated from Latex beads containing lysosomes under same conditions (Fig 6B). To determine the proteolytic activity of the lysosomes of the L. donovani infected and uninfected human macrophages, cells were labeled with DQ-BSA Red which induces strong fluorescence upon hydrolysis by proteases [29]. Our results showed that most of the internalized latex beads in human macrophages are colocalized with DQ-BSA Red labeled proteolytically active lysosomes. Whereas, Leishmania failed to colocalize with DQ-BSA Red labeled proteolytically active lysosomes (Fig 6C). Most importantly, a significant reduction in the fluorescence of DQ-BSA Red and numbers of red puncta per cell were observed in Leishmania infected human macrophages in comparison to uninfected and latex beads infected cells (Fig 6C). These results indicated that Leishmania not only inhibits transport to lysosomes but also blocks the proteolytic activity of the lysosomes in infected macrophages. Previous studies demonstrated that Leishmania resides in the phagolysosomal compartment in mouse macrophages [30,31]. However, we found that Leishmania resides in Rab5a and EEA1 positive early endocytic compartment in human macrophages. Therefore, we tried to determine the localization of CathepsinD and Lamp1 on Leishmania-PV after 24 h of infection in human macrophages. Interestingly, we also found that Leishmania-PV acquires Lamp1 and CathepsinD after 24 h of infection in human macrophages (Fig 7A) as observed previously in mouse macrophages. This observation was puzzling; therefore, we tried to determine how Leishmania-PV recruits lysosomal markers like CathepsinD and Lamp1 when parasites reside in an early compartment positive for Rab5a and EEA1. Previous studies demonstrated that lysosomal enzymes like CathepsinD and Lamp1 are trafficked via early endosomes to their final destination of lysosomes [32]. Thus, we speculated that induced expression of Rab5a in Leishmania infected macrophages might promote the fusion of Golgi derived Lamp1 or CathepsinD containing vesicles with Leishmania PV and thereby, blocks the transport of lysosomal proteins in early endosomes. To test this hypothesis, we overexpressed GFP-Rab5a or its mutants in HeLa cells and determined the distribution of CathepsinD and Lamp1 by immune-staining. Our results showed that both CathepsinD (Fig 7B) and Lamp1 (Fig 7C) are localized into perinuclear lysosomal compartment in untransfected control cells as reported earlier [33]. In contrast, majority of CathepsinD (Fig 7B) and Lamp1 (Fig 7C) containing vesicles were found to be colocalized with GFP-Rab5a positive early endosomal compartments in GFP-Rab5a: WT and GFP-Rab5a: Q79L overexpressed cells. No apparent change in the distribution CathepsinD and Lamp1 was observed in GFP-Rab5a: S34N overexpressing HeLa cells in comparison to control cells. These results demonstrated that overexpression of Rab5a inhibits the transport of CathepsinD and Lamp1 to the lysosomes and retains them in the early endocytic compartment. Previous studies demonstrated that the lysosomal hydrolase CathepsinD is synthesized as a 52-kDa precursor protein which is cleaved from amino-terminus in acidified early endosome resulting in a 48-kDa intermediate enzyme form. Subsequently, further proteolytic cleavage of the protein in low pH of the lysosomes produced the mature active CathepsinD composed of heavy (34-kDa) and light (14-kDa) chains linked by non-covalent interactions [34]. Thus, size of CathepsinD was used as an indicator for their processing as well as for their localization in previous studies. Therefore, we determined the size of the CathepsinD in Leishmania infected human macrophages. We found significantly lower levels of the mature/activated form of CathepsinD in live parasite infected macrophages in comparison to dead parasite infected cells (Fig 7D). Further quantitation revealed that live Leishmania infection inhibits about 70% mature form of CathepsinD in infected human macrophages than dead parasite infected cells. Similar block in the processing of CathepsinD was found in Rab5a: WT, Rab5a: Q79L and Rab5a: S34N overexpressing HeLa cells (Fig 7E). Taken together, these results clearly demonstrated that Leishmania induces the expression of Rab5a in infected human macrophages and thereby blocks the trafficking of lysosomal proteins and retains them as inactive precursor form in early endosomes. To determine the function of Rab5a in the survival of L. donovani in human macrophages, Rab5a was knockdown in THP-1 differentiated macrophages by specific siRNA and these cells were infected with Leishmania. First, THP-1 differentiated macrophages were transfected with 50 nM of Rab5a specific siRNA and cells were incubated for 48 h at 37°C. Subsequently, levels of Rab5 isoforms in these cells were determined by Western blot analysis. The results presented in the Fig 8A showed that siRNA specifically downregulates the expression of Rab5a in macrophages. Finally, Rab5a knockdown cells were infected with Leishmania and the parasite load was determined at indicated time points. Our results showed that infection in Rab5a knockdown macrophages is not compromised as similar numbers of parasites are observed in both Rab5a knockdown and control cells at 0 h. However, more than 74% inhibition of parasite load was observed in Rab5a knockdown human macrophages in comparison to control cells after 96 h of infection (Fig 8B and 8C). Similar results were also obtained in miR-494 (50 nM) overexpressed macrophages under identical conditions (Fig 8B and 8C). Subsequently, our results showed that this is due to significant less recruitment of Rab5a on Leishmania-PV in miR-494 overexpressed human macrophages (Fig 8D). These results demonstrated that knocking down of Rab5a by siRNA or inhibiting the expression of Rab5a by miR-494 significantly inhibits the growth of parasite in macrophages.
One general strategy used by several intracellular pathogens is to avoid transport to lysosomes by modulating the intracellular trafficking pathway in the host cells to survive in modified phagosomes [11,12,13]. In contrast, previous studies using mainly mouse macrophages have shown that Coxiella [35,36] and Leishmania [2,3] survive in a phagolysosomal compartment decorated with Lamp1, vacuolar ATPase, and CathepsinD. As phagolysosomal compartment is detrimental for invading pathogen, it is puzzling how Coxiella and Leishmania are surviving in such a degradative compartment in the cell. Consequently, recent studies have shown that pathogenic C. burnetii blocks the recruitment of Vps41 and inhibits its transport to phagolysosomes [37,38]. However, not much is known about how Leishmania modulates the trafficking pathway in the host cells and the nature of the compartment they survive. Interestingly, we have found that Leishmania donovani infection specifically upregulates the expression of Rab5a in human macrophages. Subsequently, we have shown that upregulation of Rab5a expression is due to transcriptional activation as enhanced level of Rab5a transcript is detected in infected macrophages. To understand how Leishmania induces the expression of Rab5a in infected human macrophages, we have compared the miRNA profile of uninfected and infected macrophages as some of the current studies have shown that miRNA can also modulate the expression of Rab GTPases [39,40]. In addition, it has been shown that Leishmania infection downregulates miR-122 expression to lower serum cholesterol [41] and significantly enhances the miR-30A-3p expression to modulate autophagic pathway in macrophages [42]. Interestingly, we have also found that L. donovani infection modulates the expression of various miRNA in human macrophages. Subsequently, we have identified that miR-494 has a target site in 3/-regulatory region of Rab5a of human and hamster but not in mouse. As not much work has been done about regulation of expression of Rab GTPases, we have first validated that miR-494 specifically regulates the expression of Rab5a using a chimeric construct containing 3/-UTR of Rab5a with luciferase as heterologous reporter in mammalian cells. Our results have demonstrated that miR-494 binds with miR-494 recognition element present in the 3/-UTR of Rab5a to repress the expression of Rab5a in human cells. Most importantly, we have found that transfection of miR-494 specifically inhibits the expression of Rab5a protein in human macrophages and HeLa cells compared to the control mimic. This is the first demonstration that miR-494 negatively regulates the expression of Rab5a in HeLa cells and macrophages. Subsequently, we have shown that L. donovani infection downregulates the expression of miR-494. To determine how L. donovani infection downregulates the expression of miR-494 in macrophages, we have checked the expression of AP-1 transcription factor in infected human macrophages as it has been previously reported that AP-1 transcription factor is involved in the synthesis of miR-494 [25]. Interestingly, we have found that Leishmania degrades the c-Jun subunit of AP-1 transcription factor by gp63 in infected human macrophages using gp63 secretion deficient LdRab1: S22N transgenic parasites [28]. These results are supported by the fact that Leishmania major infection degrades AP-1 complex in infected mouse macrophages via their metalloprotease gp63 [26]. Consequently, we have also found that gp63 overexpression degrades c-Jun in Raw 264. 7 mouse macrophages. Taken together, our results have demonstrated that L. donovani infection degrades c-Jun by their metalloprotease, gp63 to inhibit the synthesis of miR-494 and thereby upregulates the expression of Rab5a in infected human macrophages. In contrast, Leishmania infection does not induce the expression of Rab5a in mouse macrophages as miR-494 target site is absent in the 3/-UTR of mouse Rab5a. In order to understand the significance of Rab5a upregulation in L. donovani infected human macrophages, we have checked the recruitment of Rab5a on Leishmania-PV. We have found that Leishmania not only specifically recruits Rab5a on PV but also retains it throughout the experimental period of 48 h. Similarly, Leishmania recruits and retains EEA1 on its PV which is a Rab5 effector molecule present in early endosomal compartment [43]. However, Leishmania containing PV does not acquire Rab7, Rab8 and Rab9 in human macrophages. It is also pertinent to mention that antibodies against several mammalian Rabs like Rab5, Rab7 and Rab1 do not cross react even with respective Leishmania proteins as it has been shown previously [28,44,45,46]. Therefore, our results unequivocally prove that Leishmania resides in the early endocytic compartment in human macrophages by selectively recruiting Rab5 and EEA1 as observed for Mycobacteriun and Salmonella -containing phagosomes [15,47,48]. As Rab5 is an early endocytic GTPase [49,50], retention of Rab5a on PV might promote the constitutive fusion of Leishmania-PV with early endosomes to inhibit its trafficking to lysosomes as it has been shown previously with Salmonella-containing phagosomes [51]. Therefore, we have checked whether Leishmania inhibits its transport to lysosomes. Indeed, we have found that Leishmania inhibits its transport to latex beads containing lysosomes even after 24 h of infection in human macrophages. Consequently, we have found that Leishmania-PV does not recruit Rab7 which is required for lysosomal targeting. Taken together, these results unambiguously prove that Leishmania inhibits its transport to the lysosomes to survive in human macrophages. However, our results have also shown that Leishmania containing PV in mouse macrophages does not recruit and retain Rab5a after 24 h of infection whereas Leishmania containing PV in mouse macrophages is shown to recruit Rab5 in early time point of infection [52]. This is possibly due to the fact that Leishmania infection does not overexpress Rab5a in mouse macrophages. Conversely, our results also suggest that Leishmania infection in hamster macrophages might induce the Rab5a expression to reside in early endosomes to produce persistent infection. Thus, it is tempting to speculate why among the two animal models of leishmaniasis [53], hamster model mimic human infection whereas Leishmania infection is self-healing in mouse. However, it has been previously shown that Leishmania-PV recruits lysosomal markers like CathepsinD and Lamp1 predominantly in mouse macrophages [30,31]. We have confirmed these observations and have also found that Leishmania-PVs are positive for CathepsinD and Lamp1 in human macrophages. This is puzzling how Leishmania-PV recruits lysosomal markers like CathepsinD and Lamp1 when they reside in an early compartment positive for Rab5a and EEA1. Thus, we have evaluated the trafficking of CathepsinD and Lamp1 in Rab5a overexpressed cells as Leishmania infection induces the expression of Rab5a in the infected cells. Our results have shown that both Lamp1 and CathepsinD are predominantly colocalized with GFP-Rab5a positive early endosomal compartments in GFP-Rab5a: WT and GFP-Rab5a: Q79L overexpressed cells in comparison to their perinuclear localization in control cells as reported earlier [33]. Thus, overexpression of Rab5a might promote the fusion of Golgi derived Lamp1 or CathepsinD containing vesicles with early endosomes and thereby, retain them in early endosomes as these proteins are trafficked via early endosomes to lysosomes [54]. Our results are also supported by the fact that overexpression of dominant active mutant of Rab5a redistributes the lysosomal enzymes in early endosomes and disturbs the lysosome biogenesis [55]. These results suggest that higher expression of Rab5a in L. donovani infected human macrophages blocks the trafficking of lysosomal enzymes and retains them in early endocytic compartment. Moreover, it has been shown that size of CathepsinD present in early endosomal compartment is 48kDa whereas the size of the mature protein in the lysosome is found to be around 34 kDa and 14 kDa [56]. Consequently, we have found that size of the CathepsinD is predominantly 48 kDa in L. donovani infected human macrophages as well as Rab5a and its mutants overexpressed cells indicating that Leishmania infection retains lysosomal enzymes in early compartment in an immature and inactive form via the overexpression of Rab5a. Though overexpression of Rab5a: S34N does not apparently alter the distribution of CathepsinD in HeLa cells, but our results have shown that overexpression of this mutant also blocks the processing of this enzyme. This might be due to the essential role of Rab5 in the biogenesis of the endolysosomal system [57]. In addition, we have shown that L. donovani infection blocks proteolytic activity of the lysosomes in infected human macrophages using DQ-BSA Red as a fluorogenic substrate for proteases [29]. Thus, Leishmania-PV recruits Lamp1 and CathepsinD as it has been shown earlier but these proteins are localized in immature and inactive form in early endosomes. Finally, we have shown that selective depletion of Rab5a in human macrophages by specific siRNA significantly inhibits the growth of the parasites. Similar results are also obtained in miR-494 overexpressed macrophages under identical conditions. These results suggest that knocking down of Rab5a by siRNA or inhibiting the expression of Rab5a by miR-494 possibly targets internalized parasites to lysosomes as they will not be able to promote Rab5a-mediated constitutive fusion with early endosomes. Thus, our results demonstrate that Rab5a function is essential for the survival of Leishmania in human macrophages. In conclusion, this is the first demonstration that Leishmania resides in Rab5a and EEA1 positive early endocytic compartment in human macrophages. To delineate the mechanism, we have shown that Leishmania upregulates the expression of Rab5a in infected macrophages by downregulating the synthesis of miR-494 by degrading c-Jun via gp63. Subsequently, parasites recruit Rab5a on PV and inhibit transport to lysosomes (Fig 9). However, Leishmania residing in early compartment also recruits lysosomal enzymes in immature and inactive form in human macrophages. Thus, blocking the processing of the lysosomal enzymes to the mature active form might also help the parasites to survive in macrophages. These results also indicate the possibility of modulating endo-lysosomal pathway in parasite infected cells by miR-494 or small molecules to divert trafficking of Leishmania probably to lysosome which might be useful for developing future therapeutic intervention.
Unless otherwise stated, all reagents were obtained from Sigma Chemical Co. (St. Louis, MO). Tissue culture supplies were obtained from the Grand Island Biological Co. (Grand Island, NY). Lipofectamine 2000 and Lipofectamine RNAi max reagent were purchased from Thermo Fisher Scientific. pmirGLO Dual-Luciferase miRNA Target Expression Vector and Dual-Luciferase Reporter Assay System were purchased from Promega Life Science (Madison, WI). microRNA mimics were obtained from Sigma Aldrich (St. Louis, MO). Antibodies against Rab5a and Rab5c were purchased from Abcam (Cambridge, England) whereas anti-Rab5b antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against Rab5, Rab7, Rab9, c-Jun, Lamp1 were obtained from Cell Signaling Technologies (Danver, MA). Anti-Rab8 and anti-Rab11 antibodies were purchased from BD Biosciences, USA. Anti-EEA1 antibody was received as kind gift from Dr. Marino Zerial (Max Planck Institute, Dresden, Germany). All HRP-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratory (West Grove, PA) and ECL was obtained from Amersham Biosciences, UK. All secondary antibodies used for immunofluorescence studies were purchased from Molecular Probes (Eugene, OR). All other reagents used were of analytical grade.
Leishmania donovani causes visceral leishmaniasis in human. This parasite is thought to reside and replicate in a phagolysosomal compartment in macrophages. But, how Leishmania survives in such a detrimental compartment in macrophages is not known. In contrast, most of the intracellular pathogens avoid targeting to lysosomes in the host cells. We have found that Leishmania upregulates the expression of Rab5a, an early endosomal protein, by downregulating the expression of miR-494 in infected human macrophage as a consequence of gp63-dependent degradation of c-Jun. Subsequently, parasites recruit and retain Rab5a and EEA1 on their parasitophorous vacuoles demonstrating that Leishmania resides in the early endosomal compartment and consequently inhibits their transport to the lysosomes. However, we have found that parasites also recruit Lamp1 and inactive pro-CathepsinD on PV which led to previous conclusion that Leishmania resides in the phagolysosomal compartment. We have also shown that parasites block the processing of the lysosomal enzymes in the early endosomal compartment by overexpressing Rab5a which also helps the parasites to survive in human macrophages. Finally, we have shown that overexpression of Rab5a by downregulating miR-494 in macrophages is essential for parasite survival. These results unequivocally prove that L. donovani resides in a modified early compartment in human macrophages.
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Leishmania donovani resides in modified early endosomes by upregulating Rab5a expression via the downregulation of miR-494