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In vitro reconstitution of Salmonella phagosome-endosome fusion |
| Principal Investigator : Amitabha
Mukhopadhyay
Project Associates
/ Assistants Ph D Students Pathogenesis
of typhoid fever is related to the survival of Salmonella sp. in
macrophages. The main goal of this project is to understand the mechanism of
survival of Salmonella in macrophages in the pathogenesis. We have also
initiated the studies to understand the hemoglobin trafficking in Leishmania
using in vitro reconstitution experiments. Mechanism
of survival of Salmonella in macrophages Phagocytosis
is an important process in host defense and is mediated by complex
interactions between defined intracellular compartments. The final fate of the
nascent phagosomes usually culminates with the fusion of lysosomes. But some
invading microorganisms modulate this central process for their survival in
the phagocytic cells. The major objectives of the present investigations are (i)
reconstituion of in vitro fusion of Salmonella-containing phagosomes
with early endosome to understand the interaction between these two
compartments (ii) modulation of Salmonella phagosome and endosome
fusion by GTP binding proteins and (iii) determination of the role of various
signal transduction intermediates in the phagosome endosome fusion. During
the past year, we have studied the intracellular route of wild type Salmonella
dublin (strain 2229) and SopE knockout mutant Salmonella dublin,
(strain SE1) in macrophages to determine role of SopE. Western blot analysis
of isolated phagosomes at different times of their maturation, using specific
antibodies revealed that by 5 min after internalization SopE-knock out mutant Salmonella-containing
phagosomes acquire transferrin receptors and Rab5 (a marker for early
endosomes) while by 30 min the these bacteria are found in vesicles carrying
the late endosomal marker Rab7 like dead Salmonella containing
phagosomes. In contrast, live Salmonella-containing phagosomes (WT)
retain significant amount of Rab5 and transferrin receptor till 30 min,
selectively deplete Rab7. These results demonstrated that SopE-knock out
mutant Salmonella is targeted to the late compartment and retention of
Rab5 on phagosomes by SopE in WT-Salmonella presumably enable their
transport to lysosomes. We have also shown that SopE-knock out mutant Salmonella
dublin colocalizes with gold-labelled lysosomes whereas WT Salmonella
dublin resides in a specialized compartment. These results unequivocally
demonstrated that SopE probably is the major player in the survival mechanism
of Salmonella in macrophages. We
have also cloned full length SopE from the Salmonella and expressed as
GST-SopE fusion protein. Our results demonstrated that recombinant GST-SopE
specifically binds with Rab5. In order to determine the specific domain of
SopE which is responsible for recognition of Rab5, we have also cloned and
expressed several truncated recombinant SopE proteins from Salmonella.
Studies are in progress to determine which region of SopE specifically
recognize Rab5. Mechanism
of hemoglobin trafficking in Leishmania Evidences
from a variety of sources, have established that transport of cargo along the
endocytic pathway requires a series of highly coordinated and specific vesicle
fusion events regulated by small GTP binding proteins of the Rab family. Not
much is known about the regulation of endocytosis and intracellular
trafficking in protozoan parasites. The major objective of the present project
is to understand the role of GTP binding protein in hemoglobin trafficking in Leishmania. Small
GTP binding proteins of Rab family regulates intercompartmental transport
through a series of vesicle fusions. The early events in endocytosis are
regulated by Rab5 whereas Rab7 serves as a targeting signal to late
compartment. However, the mechanism of endocytosis and their regulation in
protozoan parasites remains largely unknown. In order to understand the
intracellular trafficking of Hb in L. donovani promastigotes and
their sorting in early endocytic compartment, we have developed an in vitro
reconstitution assay to study the regulation of endosome fusion in this
protozoan parasite. We
have used a biochemical assay to determine the in vitro fusion of
endosomes isolated from Leishmania promastigotes using biotinylated
hemoglobin (BHb) and avidin-HRP (AHRP). Based on our previous findings, early
endocytic compartment in Leishmania were labeled by 5 min
internalization of BHb or AHRP. The cell fractionation data showed that
maximum AHRP activity as well as BHb are retained in the fractions positive
for Rab5 indicating that compartments labeled by these probes in Leishmania
are enriched in early endosomes. Our
results have shown that the requirements of endosome-endosome fusion in Leishmania
promastigotes have several similarities with fusion events described in
mammalian cells. Fusion between endosomes in Leishmania occurred in the
presence of an ATP regenerating system containing cytosol and is temperature
dependent. In contrast to the mammalian cells, significantly higher level of
fusion between the endosomes in Leishmania was observed at 23°C
than 37°C.
Moreover, our results demonstrated that both ATP and its hydrolysis is
required for endosome fusion in Leishmania which is consistent with ATP
requirement of endosome-endosome fusion, fusion between Golgi vesicle and ER
to Golgi transport in mammalian cells. Since Rab-GDI exhibits broad substrate
specificity even across the species and selectively stripped off Rab protein
in the presence of GDP, we have used mammalian GDI along with GDP in order to
determine the role of Rab GTPase in this fusion process. Under these
conditions as well as GTPgS
treatment fusion is inhibited between endosomes in Leishmania
demonstrating the requirement of Rab proteins in this fusion event. Previous studies in several mammalian cell free systems have shown that Rab5 regulates homotypic fusion between the early endosomes. In order to clone the Rab5 homologue from L. donovani, BLAST search was carried out using human Rab5 as a query. Five clones from different protozoa like Dictyostelium discoideum (88%), Toxoplasma gondii (75%), Plasmodium falciparum (78%), Entamoeba histolytica (78%) and Trypanosoma brucei (61%) showed significant similarity with human Rab5. Trypanosma being closest to Leishmania, a second BLAST search was carried out using Trypanosoma sequence as a query, which revealed a putative Rab5 like sequence from L. major with 72% homology. Putative start and stop codon were predicted and appropriate forward and reverse primers were used to amplify a fragment (~650bp) from L. donovani cDNA using PCR. The PCR product was cloned and sequenced. The nucleotide sequence was hypothetically translated (211 amino acids) and BLAST search revealed that cloned protein (LdRab5) has about 91% similarities with L. major putative Rab5, 65% with T. brucei Rab5, 66% with T. gondii Rab5, 62% with D. melanogaster Rab5 and 59% with Human, Mouse and D. discoideum Rab5. Alignment and comparison of LdRab5 sequence with other Rab5 sequences using CLUSTAL W software demonstrated the presence of conserved canonical Rab protein features like GTP-binding region, effector loop and C-terminal isoprenylation motif etc. The GTP binding ability and intrinsic GTPase activity are the key features of these proteins. The results presented in our study show that recombinant LdRab5 specifically binds 32P-GTP. But, the GTP hydrolysis of LdRab5 was found to be lower than the mammalian Rab5. It has been shown that a consensus sequence TIG(I/V/A)(D/E)F(K/G/L) in the effector region of Rab group of protein is involved in regulating the GTP hydrolysis and GDP/GTP exchange and this sequence is highly sensitive to alteration. The effector region of LdRab5 was found to consist of TVGASFF, which may possibly explain relatively low GTPase activity of the Leishmania protein in comparison to its mammalian equivalent. Though, LdRab5 has about 59% homology with human/mouse Rab5, but the antibody raised against the Leishmania protein did not react with the mouse protein or vice versa as revealed by Western blot analysis. Subsequently, the LdRab5 protein and specific antibodies were used to characterize the endosome fusion in Leishmania. Our results demonstrated that fusion of early endosomes containing BHb with AHRP containing early endosomes in Leishmania is regulated by Rab5. Publications Original
peer-reviewed articles 1.
Mukherjee K, Parashuraman S, Krishnamurthy G, Majumdar J, Yadav A,
Kumar R, Basu SK and Mukhopadhyay A (2002) Diverting intracellular trafficking
of Salmonella to the lysosomes through activation of the late endocytic Rab7
by intracellular delivery of muramyl dipeptide. J Cell Sci
115:3693-3701. Reviews/Proceedings 1. Mukhopadhyay A and Basu SK (2002) Scavenger receptor mediated drug delivery: a versatile approach for modulation of macrophage metabolism. Proc Ind Nat Sci Acad B68:361-370. |