Project Assistant
Ganga Krishnamurthy

PhD Students
Vikram
Nitin Patel

The programme aims at developing systems for intracellular delivery of drugs or pharmacologically active agents selectively to specific cell types by exploiting the efficiency of the process of receptor-mediated endocytosis. The objectives of the project are (i) exploration of heme acquisition mechanism in Leishmania donovani, (ii) drug targeting for overcoming multidrug resistance in cancer chemotherapy, (iii) modulation of macrophage metabolism through receptor-mediated delivery of biological response modifiers and (iv) therapeutic strategies based on scavenger receptor-mediated delivery of antisense oligonucleotides.

A.    Exploration of heme acquisition mechanism in L. donovani

In the reporting year, we developed an in vitro assay to determine the binding of purified Leishmania protein with hemoglobin. Briefly, the purified protein was immobilised in the well of ELISA plates, blocked and incubated with biotinylated hemoglobin. Binding of biotinylated hemoglobin with the purified protein was detected by avidin-HRP. Using this assay, we showed that biotinylated hemoglobin binds with the purified protein and maximum binding was observed at pH 6.5. We also showed that binding of biotinylated hemoglobin is specifically competed by hemoglobin but not by other proteins like myoglobin, transferrin, hemocyanin, etc. Commercially done amino acid sequencing of the purified protein provided seventeen peptide sequences. BLAST search with some of these peptide sequences revealed that purified hemoglobin-binding protein could be a hexokinase. When we compared the known hexokinase sequence of Trypanosoma, we found that eight of the peptides generated from the purified Leishmania protein were identical with Trypanosoma hexokinase. An in vitro binding assay showed that yeast hexokinase could bind biotinylated hemoglobin and this binding was competed by the purified 46 kDa hemoglobin-binding protein from Leishmania. Using a specific antibody against the purified protein, the hemoglobin-binding protein was localized in the flagellar pocket of Leishmania. In order to measure the uptake of hemoglobin by Leishmania, the promastigotes were incubated in the presence of hemoglobin for 10 min at 25°C, permeabilized and the presence of internalized hemoglobin was detected by incubation with mouse anti-hemoglobin antibody followed by rabbit anti-mouse IgG labeled with Texas Red. We found that under these conditions hemoglobin was localized in the perinuclear compartment and the uptake of hemoglobin could be blocked by preincubating the cells with yeast hexokinase or an antibody against the purified 46 kDa hemoglobin-binding protein.

B.    Modulation of macrophage metabolism through receptor-mediated delivery of biological response modifiers

Previously, we reported that treatment of macrophages with MBSA-MDP resulted in an increase in the level of Rab7 and decrease in the content of Rab5 in the macrophages. In the reporting year, we investigated whether transcriptional or posttranscriptional events regulated the observed modulation of Rab content in the treated cells. We compared the Rab5 and Rab7 mRNA levels in untreated control cells with that of MBSA-MDP treated cells at different time intervals by limiting dilution RT-PCR using specific primers for Rab5 and Rab7. We showed that the Rab7 mRNA was induced after 1hr treatment with MBSA-MDP and remained higher in the treated cells for up to 12 hrs. Thus, the increased content of the Rab7 protein in the treated cells after 12 hrs presumably reflects the combination of a rapidly responsive but short-lived mRNA and long-lived protein. However, the level of Rab5 specific mRNA was reduced over time while the levels of b-actin specific mRNA remained unaltered with MBSA-MDP treatment. These results suggest that the contents of Rab7 and Rab5 in MBSA-MDP treated cell are regulated transcriptionally.

Intracellular delivery of MDP may modulate several signal transduction molecules. To directly determine if the altered Rab5 and Rab7 contents of MBSA-MDP treated cells is sufficient to drive the observed killing of Salmonella by lysosomal targeting, we reconstituted the phagosome-lysosome transport process in permeabilised cells using different combinations of in vitro prenylated Rab5 and Rab7 proteins. The results showed that dead biotinylated Salmonella is transported to avidin-HRP loaded lysosomes whereas transport of live Salmonella is inhibited in the presence of cytosol prepared from normal macrophages. When a similar assay was carried out in the presence of Rab5 immunodepleted cytosol, live bacteria were transported to the lysosomes. Further, when reconstitution of transport was carried out in presence of cytosol containing Rab5:S34N (a negative mutant of Rab5 locked in GDP form) along with Rab7:wild type (WT) protein, transport of the live Salmonella to the lysosomes was comparable to that observed in presence of cytosol prepared from MBSA-MDP treated cells. In contrast, transport of live Salmonella to the lysosomes was significantly blocked when reconstitution was carried out in presence of cytosol containing Rab5:WT and Rab7:T22N (a negative mutant of Rab7 locked in GDP form). Thus, although intracellular delivery of MDP may activate different signaling cascades, our results strongly indicate that downregulation of Rab5 concurrent with upregulation of Rab7 triggered by intracellular delivery of MDP is sufficient to target live Salmonella-containing phagosomes (LSP) to the lysosomes.

In order to characterize the nature of the compartment in which Salmonella is targeted after MBSA-MDP treatment, LSP were isolated from MBSA-MDP treated macrophages 90 min after internalization. LSP isolated from MBSA-MDP treated cells showed higher levels of cathepsin D and vacuolar ATPase than those from untreated cells indicating that in MBSA-MDP treated cells the bacteria are transported to a fully competent lysosomal compartment containing lysosomal enzymes and vacuolar ATPase. We further demonstrated that in MBSA-MDP treated cells, Salmonella tagged with green fluorescent protein (GFP-Salmonella) co-localized with Lysotracker Red labeled lysosomal compartment whereas GFP-Salmonella in untreated cells reside in a compartment which is not labeled with Lysotracker Red. Lysotracker Red was shown to accumulate in the compartment that labeled with Rab7 and Lamp1 antibodies, characteristic features of late/lysosomal compartment. Therefore, our data demonstrate that in MBSA-MDP treated macrophages live Salmonella is transported to fully competent lysosomes.

Publications

Original peer-reviewed articles

1.     Bhatia S, Mukhopadhyay S, Jarman E, Hall G, George A, Basu SK, Rath S, Lamb JR and Bal V (2002) Scavenger receptor-specific allergen delivery elicits IFN-g-dominated immunity and directs established TH2-dominated responses to a non-allergic phenotype J Allergy Clin Immunol 109:321-328.