Research Interest
Intracellular Trafficking
The scientific contributions of our research are primarily in the area of cell biology with special emphasis on the regulation of intracellular trafficking. Intracellular transport is mediated by vesicle fusion of one compartment to another specific compartment. The specificity of the fusion process is regulated by a family of ras-related GTPases called Rab, distinct members of which are specifically localized in particular compartments. Various Rabs in conjunction with other cellular factors mediate the fusion between two specific vesicles. As many as 70 members of this family have been identified in mammalian cells, but functions of all Rabs are not fully characterized yet.
SNAREs are other major players which regulate intracellular membrane trafficking by conferring specificity to vesicular fusion events along with Rab GTPases. Around 36 members of this family of proteins are known in mammalian cells. Most SNAREs have a membrane-spanning region and a conserved heptad repeat of SNARE motif which is required for SNARE complex formation. Depending on the conserved residue in the SNARE motif, SNAREs are also classified as R-SNAREs (arginine containing SNAREs) and Q-SNAREs (glutamine-containing SNAREs), which is again sub divided into Qa, Qb and Qc SNAREs on the basis of their N-terminus domain. Vesicular fusion events require one member each of the Qa-, Qb-, Qc- and one cognate R-SNAREs to form functional four-helix complex. This brings the donor and acceptor membranes close together to drive membrane fusion.
Hypothesis
Microorganisms are internalized by phagocytic cells and are targeted to the acidic lysosomes following endocytic pathway where it is degraded. Thus, the first challenge of intracellular pathogens is to evade the lysosomal transport in host cells. Successful intracellular pathogens like Mycobacterium, Salmonella, Legionella, Leishmania etc. have evolved various strategies to avoid their targeting to the lysosomes in the host cells. It is not very clearly established how they avoid lysosomal transport, therefore, current studies are focused to understand how these intracellular pathogens modulate endo-lysosomal pathway in the host cells to develop novel therapeutic target.
Modulation of host intracellular trafficking by pathogens
Our initial studies have shown that Rab7 acts downstream of Rab5 in regulating vesicular transport events from early endosomes to lysosomes. Using this knowledge of regulation of endocytosis by Rab GTPases, we initiated series of studies to understand the mechanism of survival of intracellular pathogens within the host cells. The broad objective of our studies is to understand the fundamental issues in host pathogens interactions with an aim to develop a potentially new therapeutic target for their intervention.
Salmonella
Our work has provided important insights into the molecular mechanisms of how pathogens modulate intracellular trafficking pathways to survive in phagocytes. We have demonstrated using an elegant in vitro reconstitution of transport assay and confocal analysis that live Salmonella produce a protein, SopE, that helps to recruit Rab5 and NSF on the phagosomes containing live bacteria. This promotes fusion of live Salmonella-containing phagosomes with early endosomes so that lysosomal targeting of such phagosomes is avoided. Thus, it is emerging from these studies that SopE could be a new and specific target for selective elimination of Salmonella from the host cells either by blocking the interaction of SopE with Rab5 or by modulating the content of endocytic GTPases in the host cells to divert trafficking of Salmonella to the lysosomes. Finally, we have tested these hypothesis and shown that it is possible to divert Salmonella to the lysosomes for selective killing by modulating the content of endocytic GTPases in the host cells. Interestingly, we have also shown how Salmonella recruits LAMP1, a lysosomal protein, by fusing wirh Golgi derieved LAMP1-containing vesicle through their effector molecule, SipC. Interestingly, we have identified another Salmonella protein, SipA which mimics as cognate host SNARE of Syntaxin 8 and help Salmonella to survive in the host cells.
Leishmania
Though intracellular pathogens avoid targeting to the lysosomes, nonetheless, Leishmania was predicted to reside in the phagolysosomal compartment of host macrophages. However, their mechanism of survival in the lysosomes is not known. Recently, we have shown 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. We have found that miR-494 negatively regulates the expression of Rab5a in cells, thereby, it upregulates the expression of Rab5a in Leishmania infected 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. 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 Leishmania-mediated remodeling of endo-lysosomal trafficking to reside and survive in a specialized early endocytic compartment.
Hemoglobin endocytosis in Leishmania: a novel target
Leishmania, lack the heme biosynthetic pathway and need to acquire heme from external sources for survival. This potential source may be available to the parasites if hemoglobin is degraded either in macrophages or if the parasites acquire Hb by some unknown mechanisms.
We have shown that endocytosis of Hb in Leishmania is mediated through receptors located in the flagellar pocket which is hexokinase. Subsequent studies have shown that Hb endocytosis and its transport to the lysosomes are regulated by Rab5 and Rab7 homologues in Leishmania where it is degraded to generate intracellular heme. Further, we have shown that newly synthesized Hb-receptor is trafficked to the cell surface via COPII dependent but Rab1 independent process. These results suggest that newly synthesized Hb receptor traffic to the cell surface via unconventional secretory pathway which needs to be characterized. Discovery of this new receptor system for acquiring heme by Leishmania is significant not only as a notable contribution to biology of this group of intracellular parasites but also as a potential new target against these parasites of paramount public health importance.
Recent studies from our group have shown that hemoglobin endocytosis in Leishmania is a clathrin dependent process and depolymerization of Leishmania clathrin by chloropromazine inhibits the growth of the parasites. In addition, we have found that chloroquine which is also a lysosomotropic agent blocks the degradation of Hb in parasites and parasite is growth is inhibited as it deprives of free heme. Chloropromazine and chloroquine are well characterized drugs, thus, it will be worth to evaluate these drugs or their derivatives as antileishmanial agent.
Taken together, these studies have shown that hemoglobin receptor in Leishmania is a novel target for drug development against this parasitic infection. Recently, we have validated that hemoglobin receptor in Leishmania is a potential vaccine candidate against visceral leishmaniasis. As no vaccine against visceral leishmaniasis is available currently, it has drawn worldwide attraction. As this vaccine candidate is already evaluated in two experimental animal models, it will worth to carry forward it to clinical trials and technology transfer.
Regulation of intracellular trafficking by cytokines
We are first to demonstrate the connection between cytokines and intracellular trafficking. We have found that IL-6 upregulates the expression of Rab5 whereas IL-12 induces the expression of Rab7. These studies suggest how different cytokines modulate the pathogen survival in the host cells by regulating the expression of endocytic Rab GTPases.
Site specifc drug delivery
We have also made seminal contributions to establish for the first time the feasibility of receptor-mediated enodocytosis as an alternative approach to deliver various drugs to macrophages using exquisite specificity and high efficiency process of scavenger receptor mediated endocytosis. We have shown that scavenger receptor-mediated intracellular delivery of various drugs to macrophages combat various infections as well as cancer cells both in vitro and in vivo.
