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In vitro reconstitution of Salmonella phagosome-endosome fusion |
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Principal Investigator : Amitabha Mukhopadhyay Project
Associates/Assistants
PhD
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. 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
reconstituion of in vitro fusion of Salmonella-containing
phagosomes with early endosome to understand the interaction between these two
compartments, modulaton of Salmonella phagosome and endosome fusion by
GTP binding proteins and determination of the role of various signal
transduction intermediates in the phagosome endosome fusion. In
the reporting year, we investigated the possibility of bacterial proteins that
might be involved in the recruitment of different factors from the host cell
cytoplasm. Our results showed that Salmonella specifically binds two
proteins from the macrophage cytosol of apparent molecular weight of 25kDa and
42kDa. In order to identify these proteins, western blot analysis were carried
out with antibodies against different endocytic Rab proteins, which are about
25kDa and regulate vesicular trafficking. Anti-Rab5 antibody specifically
detected the 25kDa protein bound to Salmonella, while anti-Rab7
antibody did not, demonstrating that Salmonella specifically recognized
Rab5 from the host cells. Similarly, the 42kDa protein bound to Salmonella was
identified as actin, which is consistent with the previous report that SipA, a
protein from Salmonella binds with actin to induce membrane ruffling
which facilitates the entry of the bacteria. To search for the bacterial
surface protein that interacts with Rab5, GST-Rab5 was incubated with Salmonella
lysate obtained after growing the cells in the presence of 35S-methionine. We demonstrated that GST-Rab5
specifically picked up two proteins of apparent molecular weight of 30kDa and
50kDa from the Salmonella lysate. In contrast, no proteins were
detected when GST or GST-Rab7 was used under the similar conditions. Western
blot analysis with anti-SopE antibody revealed the 30kDa protein as SopE.
However, we have not yet identified the 50kDa proteins, which also bind to
Rab5. Similar results were also obtained using WT S. dublin indicating
that SopE, a type III secretory protein of Salmonella, specifically binds with
Rab5. To determine the specificity of SopE mediated binding of Rab5, Salmonella
typhimurium was incubated with biotinylated GST-Rab5 in the presence and
absence of non-biotinylated Rab5 or SopE. Western blot analysis showed that
binding of biotinylated Rab5 with Salmonella is effectively competed by
both Rab5 and SopE. These results demonstrated that SopE specifically binds
with Rab5. In
order to determine whether the SopE produced by Salmonella is
transported on to the phagosomes, we purified phagosomes containing respective
wild type and mutant Salmonella and determined the presence of SopE on
phagosomes by immunogold localization. The immunolocalization studies
demonstrated that SopE is present on LSP containing either wild type Salmonella
typhimurium or wild type Salmonella dublin dublin. In
contrast, phagosomes containing dead Salmonella (DSP) do not show the
presence of SopE. Moreover, phagosomes containing live, SopE knockout mutant Salmonella
(S. dublin, strain SE1) were unable to recruit Rab5 on LSP. However,
phagosomes containing wild type Salmonella dublin (strain 2229)
recruited significant amounts of Rab5 on LSP comparable to that observed with
phagosomes containing wild type Salmonella typhimurium. These results
demonstrated that SopE is transported onto LSP and thereby possibly recruit
Rab5 on Salmonella-containing phagosomes. To
understand the mechanism of Rab5 recruitment by LSP, we studied the binding of
LSP with various mutant forms of Rab5 viz., Rab5:Q79L, a GTPase-defective
mutant; Rab5-S34N, a dominant-negative mutant locked in GDP conformation;
Rab5-DC4
where the isoprenylation motif is deleted and Rab5:WT. Respective Rab proteins
were preincubated with macrophage cytosol in the presence of ATP regenerating
system to allow in vitro prenylation. Phagosomes containing either Salmonella
typhimurium (WT), or Salmonella dublin (WT) or SopE knockout
mutant of S. dublin were treated with Rab-GDI to deplete the endogenous
Rabs and incubated in the presence of indicated GST-Rab5 mutant protein in
fusion buffer containing cytosol for 10 min at 37°C.
Our results showed that phagosomes containing WT bacteria bind with Rab5:WT,
Rab5:Q79L as well as Rab5-DC4 which are in the GTP
form. In contrast, Rab5: S34N, which is locked in GDP form did not bind with
LSP indicating that only the Rab5 in GTP form is recognized by LSP.
Significant binding of Rab5-DC4, a prenylation-defective Rab5
mutant, suggested that prenylation of Rab5 is not required for the binding of
Rab5 with LSP. No significant binding of Rab7: WT with LSP was observed under
similar conditions indicating that LSP specifically bind with Rab5.
Furthermore, phagosomes containing SopE knockout mutant Salmonella were
unable to bind any form of Rab5 under similar conditions demonstrating that
SopE present on LSP is responsible for the recruitment of Rab5 in GTP form. Our
results demonstrated that Salmonella-containing phagosomes specifically
bind Rab5 in its GTP form, the active form of the protein, which promotes
endosome-endosome fusion. Furthermore, SopE is also present in infected cell
cytosol, which prompted us to investigate the role of SopE in the activation
of Rab5. Our results showed that incubation of Rab5:WT and Rab5:Q79L in buffer
alone significantly induces the nucleotide exchange of GDP to GTP form over
that obtained with Rab5:S34N, a mutant which is unable to exchange GDP to GTP.
To determine the role of SopE, we measured the incorporation of 32P-GTP molecules into GDP-loaded Rab5 in the
presence of GST-SopE 78-240 .
Our results demonstrated that Rab5:WT and Rab5:Q79L incorporated more than 2
folds of GTP in the presence of GST-SopE 78-240
than Rab5 alone. Furthermore, SopE was unable to induce the nucleotide
exchange of Rab5:S34N which is locked in GDP form. Moreover, when Rab5 was
incubated with increasing concentrations of SopE in the similar assay, the
nucleotide exchange activity of Rab5 is proportional to the concentration of
SopE present in the reaction. These results demonstrated that SopE present in
the Salmonella infected cytosol acts as a nucleotide exchange factor of Rab5. To
determine whether nonprenylated Rab5, e.g. Rab5:DC4,
recruited on the phagosomes is functionally active, we used an in vitro
fusion assay. LSP (containing Salmonella typhimurium WT) were incubated
for 10 mins with endosomes loaded with avidin-HRP at 370C in the presence of cytosol and an
ATP-regenerating system. Our results showed that LSP efficiently fuse with
early endosomes in 10 min (Control). To establish the role of different forms
of Rab5 in this fusion event, the endogenous Rab5 from the phagosomes were
stripped off by Rab-GDI treatment and fusion was carried out in
Rab5-immunodepleted cytosol in the presence of indicated Rab5 mutant proteins.
We showed that fusion of phagosomes with endosomes is inhibited in Rab5
depleted condition. Addition of Rab5: WT and Rab5: Q79L restored the fusion of
the phagosomes with endosomes by more than 90% while Rab5: S34N, which is
locked in GDP form did not stimulate the fusion. Interestingly, Rab5:DC4
stimulated the fusion of phagosomes with endosomes by more than 70%. Our
finding that Rab5:DC4, which is identical with Rab5:WT
excepting for the deletion of the C-terminus cysteine motif that is essential
for prenylation; restored fusion demonstrating that nonprenylated Rab5 is
functionally active. However, when Rab-stripped LSP were pretreated with anti-SopE
antibody, no fusion of LSP with endosomes was detected. In
conclusion, our results demonstrated that SopE acts an a nucleotide exchange
factor for Rab5 and also mediates the specific recruitment of Rab5 in GTP form
on LSP, irrespective of prenylation, and thus promote fusion of LSP with early
endosomes and thereby inhibiting targeting of live Salmonella to the
lysosomes and their eventual destruction. Publications Original
peer-reviewed articles 1.
Mukherjee K, Parashuraman S, Raje M and Mukhopadhyay A (2001) SopE acts
as an Rab5-specific nucleotide exchange factor and recruits non-prenylated
Rab5 on Salmonella-containing phagosome to promote fusion with early
endosomes. J Biol Chem 276: 23607-23615. |