|
In vitro reconstitution of intracellular transport: Role of GTPases |
| Principal Investigator : Amitabha Mukhopadhyay
Project
Associates/Assistants
Ph
D Students Pathogenesis
is related to the survival of pathogens in macrophages. The main goal of this
project is to understand the mechanism of survival of pathogens in
macrophages. We have also initiated the studies to understand the hemoglobin
trafficking in Leishmania using in vitro reconstitution
experiments. 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 this part of
present investigations are (i) reconstituion of in vitro fusion of
phathogen-containing phagosomes with early endosome to understand the
interaction between these two compartments, (ii) modulaton of phagosome and
endosome fusion by pathogens, and (iii) determination of the role of various
signal transduction intermediates in the phagosome endosome fusion. Secondly,
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 this
aspect of the project is to understand the role of GTP binding protein in
hemoglobin trafficking in Leishmania. Mechanism
of survival of Salmonella in macrophages In
the reporting year, we have further characterised 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. Initially,
we have shown that SopE mediated recruitment of Rab5 from the host cell on
phagosomes promotes fusion with early endosomes. Our recent results have shown
that SopE knock out mutant Salmonella-containing phagosomes unable to
fuse with early ensosomes and thereby targeted to compartment which is
positive for Lamp1 and contains mature cathepsin D, a lysosomal enzyme
suggesting that SopE knock out mutant is targeted to lysosomes like a dead
bug. These results unequivocally demonstrated that SopE probably is the major
player in the survival mechanism of Salmonella in macrophages. Recent
studies have shown that Rab GTPase, localized on specific compartment activate
a pair of SNARE, v-SNARE and t-SNARE which further regulate the transport
between the specific compartment. Accordingly, studies were initiated to
determine the role of SNAREs in the maturation of Salmonella in
macrophages. Earlier, we have shown that fusion between early endosomes with
early Salmonella-containing phagosomes are sensitive to ATP hydrolysis
and NEM treatment. However, recent results have shown that fusion of early
endosome with late phagosomes is not inhibited by ATPgS
and NEM treatment suggesting SNAREs are possibly activated on phagosome during
maturation. To determine the role of different SNAREs, we have cloned and
expressed different early and late endosomal v-SNARE and t-SNAREs, namely
Syntaxin 7, Syntaxin 13, VAMP 7, VAMP 8. We have used the specific antibodies
against these and other SNAREs to determine the relative level of these
regulatory molecules on early and late Salmonella-containing phagosomes.
Our preliminary results suggest that Syntaxin 6 and Syntaxin 13 are recruited
more efficiently on late-Salmonella containing phagosome in comparison
to early phagosomes. Mechanism
of hemoglobin trafficking in Leishmania Small
GTP binding proteins of Rab family regulate 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. In the reporting period, we have cloned and expressed Rab7
homologue from Leishmania. Using specific antibodies against Ld Rab7,
we have shown that Rab7 in Leishmania promastigotes localizes in
perinuclear late endosomal compartment. To determine the role of Rab5 and Rab7
in hemoglobin trafficking in Leishmania, we have compared the fusion of
Hb-containing early endosomal compartment with early or late compartment
containing fluid phase marker. Our
results showed that fusion between early endosomes was inhibited by about 75%
in Rab5 depleted cytosol, whereas Rab7 depletion reduced fusion by only about
25%. Addition of in vitro prenylated LdRab5 to the Rab5 depleted system
completely restored fusion to the control level. Previous studies have shown
that early endosomes can fuse with early endosomes whereas in vitro
fusion between early and late endosomes does not occur. This prompted us
to test heterotypic fusion between early and late endosomes in Leishmania.
Accordingly, late endosomal fractions were prepared by 5 min internalization
of AHRP followed by 15 min chase at 23°C as described previously and
separated by sucrose gradient. Partial characterization of fractions
containing maximum HRP activity revealed that these vesicles predominantly
contain Rab7, a late endosomal marker, but no Rab5 or transferrin receptor,
whereas, 5 min internalized AHRP vesicles possess early endosomal markers like
Rab5 and transferrin receptor. In contrast to the previous study, about 80%
fusion between BHb-loaded early endosomes and late endosomes containing AHRP
was observed in Leishmania. However, consistent with the earlier
report, that early endosomes containing fluid phase markers like avidin and
BHRP fuse efficiently whereas fusion of early endosomes containing avidin with
late endosome containing BHRP is significantly inhibited. Moreover, the
observed heterotypic fusion between BHb-loaded early endosomes and late
endosome containing AHRP is significantly inhibited in the presence of Rab7
depleted cytosol but not by Rab5 depleted cytosol. Addition of in vitro prenylated
LdRab7 to Rab7 depleted system significantly restored fusion. These results
indicate that the cytoplasmic tail of HbR from early endosomes containing BHb
may possibly promote this heterotypic fusion. To
determine the role of cytoplasmic tail of HbR in heterotypic fusion, we have
cloned and expressed different deletion mutants of the receptor as GST fusion
proteins : the N-terminus (HbR-DC),
middle region (HbR-DNC)
and C-terminus (HbR-DN).
Our results have shown that polyclonal antibody against Hb receptor purified
from Leishmania (PHbR) recognized HbR-DNC
and HbR-DN
while a monoclonal antibody (1B6), specifically recognized HbR-DN.
In addition, HbR-DC
predominantly bound with Hb in comparison to other fragments. When BHb-loaded
early endosomes were pretreated with 1B6, heterotypic fusion with late
endosomes containing AHRP was significantly inhibited. Moreover, addition of
HbR-DN
in the fusion assay inhibited about 80% of fusion, whereas no significant
inhibition was observed with HbR-DC
or HbR-DNC.
Publications
Original
peer-reviewed articles 1.
Singh SB, Tandon R, Krishnamurthy G, Vikram R, Sharma N, Basu SK and
Mukhopadhyay A (2003) Rab5 mediated endosome-endosome fusion regulates
hemoglobin endocytosis in Leishmania donovani. EMBO J 22:5712-5722.
Reviews/Proceedings 1. Mukhopadhyay A and Basu SK (2003) Intracellular delivery of drugs to macrophages. Adv Biochem Eng Biotechnol 84:183-209. |