National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India
* Author for correspondence (e-mail: amitabha{at}nil.res.in)
Accepted 27 June 2002
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Summary |
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Key words: Endocytosis, Phagocytosis, Salmonella, Rab GTPases, Fusion, Lysosomes
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Introduction |
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Materials and Methods |
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Antibodies and recombinant proteins
Monoclonal antibody (mAb) 4F11, a mouse IgG2ak mAb specific for
the C-terminus of mouse Rab5, and an affinity-purified rabbit polyclonal
antibody that recognizes the C-terminal domain of Rab7 were generously
provided by A. Wandinger-Ness (University of New Mexico, Albuquerque, NM).
Recombinant GDI and different constructs of Rab5 and Rab7 were kindly provided
by Philip Stahl (Washington University School of Medicine, St Louis, MO).
Anti-vacuolar ATPase antibody was a kind gift from Andrea Jahraus (EMBL,
Heidelberg, Germany). V. Bal (National Institute of Immunology, New Delhi)
kindly provided the virulent strain of Salmonella typhimurium.
GFP-Salmonella was obtained as a gift from A. Aballay (Massachusetts
General Hospital, Boston). Anti-cathepsin D and all the second antibodies
labelled with HRP were purchased from Santa Cruz Biotechnology, Santa Cruz,
CA. Mouse anti-actin and anti-Rab6 antibodies were purchased from Calbiochem
(La Jolla, CA), and the anti-transferrin receptor antibody was obtained from
Zymed Laboratories.
Preparation of MBSA-MDP conjugates
MDP was conjugated to BSA by using water-soluble carbodiimide in the
presence of N-hydroxysulfosuccinimide as described previously
(Srividya et al., 2000).
Briefly, 10 mg of the MDP was incubated in the presence of 3.6 mg of EDC and
3.2 mg of sulfo-NHS in 0.1 ml of water for 10 minutes at room temperature to
generate an active ester of MDP. Subsequently, activated MDP was reacted with
10 mg of BSA in 0.2 M sodium-carbonate buffer, pH 9.5, for 1 hour at room
temperature. BSA-MDP conjugate was purified through Sephadex G25 column
chromatography, and the purified conjugate was maleylated with maleic
anhydride at a pH of 8.0. A conjugate with 15:1 molar ratio of MDP to protein
was used in these studies.
MBSA-MDP-mediated killing of Salmonella in J774E
macrophages
The virulent Salmonella typhimurium strain was a clinical isolate
from Lady Harding Medical College, New Delhi. Bacteria were grown overnight in
Luria broth (LB) at 37°C with constant shaking (300 rpm). The bacteria
were harvested in the stationary phase and were washed twice in
phosphate-buffered saline (PBS) and used for infection. To determine the
effect of MDP in the free or conjugated form on the microbicidal properties of
macrophages, J774E macrophages (1x106 cells/well) were
cultured in RPMI-1640 medium containing 10% FCS in the presence of MBSA-MDP or
free MDP for 12 hours. Cells were washed twice with PBS, and
1x107 Salmonella typhimurium were added to each well
and centrifuged at a low speed (500 g, 5 minutes at 4°C)
to synchronize the infection. Subsequently, cells were incubated for 20
minutes at 37°C for infection, and then the cells were washed five times
with PBS to remove uninternalized bacteria. The infected macrophages were
incubated in the respective drug-containing medium at 37°C. After
incubation for 12 hours, the macrophages were lysed in solubilization buffer
(SB, PBS containing 0.5% Triton X-100), and an aliquot of the cell lysates was
plated on Salmonella-Shigella agar plates to determine the number of
viable Salmonella present in the lysate in terms of colony-forming
units.
Treatment of Salmonella-infected mice with MBSA-MDP
To determine the efficacy of MBSA-MDP for treatment of Salmonella
infection in vivo, C57B1-6 mice (6 week's old, weighing about 20 g each) were
injected intraperitoneally with 1x103 Salmonella (in
100 µl of PBS) as described (Pashine et
al., 1999) on day 0. Subsequently, Salmonella-infected
animals received intraperitoneal injections of free MDP or MBSA-MDP (1 µg/
mouse of MDP equivalent; 50 µg/kg body weight) or ciprofloxacin (1.4
mg/mouse; 70 mg/kg body weight) daily for four consecutive days. Finally, mice
were sacrificed on day 10, and the spleen was dissected out from each animal.
A portion of the spleen (50 mg) from each animal was teased apart in SB, and
aliquots of the cell lysates were plated on Salmonella-Shigella agar
plates to determine the number of viable Salmonella present in the
spleen in terms of colony-forming units.
Detection of Rab proteins in MBSA-MDP-treated cells
To determine whether MBSA-MDP treatment induced the expression of endocytic
Rabs, J774E cell monolayers were incubated with free or conjugated MDP (MDP
equivalent, 1 µg/ml) for 12 hours at 37°C in RPMI-1640 medium with 10%
FCS. Cells were washed and 80 µg of the cellular proteins were analysed by
12% SDS-PAGE. The proteins were transferred onto nitrocellulose membranes and
checked for the presence of actin, Rab5, Rab7 and Rab6 using the respective
antibodies. Proteins were visualized by appropriate HRP-labelled second
antibodies and ECL.
Purification of phagosomes
Salmonella-containing phagosomes were purified from untreated
cells as well as from MDP- and MBSA-MDP-treated cells as described previously
(Mukherjee et al., 2000;
Hashim et al., 2000
). Briefly,
J774E macrophages were pretreated with MDP in the free or conjugated form (MDP
equivalent, 1 µg/ml) for 12 hours at 37°C. Subsequently, macrophages
were allowed to internalize Salmonella (1 macrophage: 10
Salmonella) for 5 minutes to restrict their entry primarily to the
early compartment. Cells were washed five times with PBS and resuspended
(2x108 cells/ml) in homogenization buffer (HB: 250 mM
sucrose, 0.5 mM EGTA and 20 mM Hepes-KOH, pH 7.2 containing protease
inhibitors) and homogenized in a ball-bearing homogenizer. Homogenates were
centrifuged at a low speed (400 g for 5 minutes) at 4°C to
remove nuclei and unbroken cells. The post-nuclear supernatants were
centrifuged at 12,000 g for 6 minutes at 4°C to pellet the
phagosomal fraction. Finally, the pellets were resuspended in 100 µl of HB
and loaded on a 12% sucrose cushion. Samples were centrifuged at 1,700
g for 45 minutes at 4°C, and the purified phagosomes were
recovered from the bottom of the tube. The phagosomes, thus purified, were
free of plasma membrane, endosomes, lysosome, Golgi and endoplasmic reticulum
contamination (Hashim et al.,
2000
). To determine the expression of endocytic Rabs on purified
phagosomes, 40 µg of the respective phagosomes were subjected to 12%
SDS-PAGE. The proteins on the gels were transferred onto nitrocellulose
membranes and checked for the presence of transferrin receptor, Rab5 and Rab7
as described above.
Preparation of endosome
Early endosomes containing avidin-HRP were prepared as described previously
(Mukherjee et al., 2000).
Briefly, J774E macrophages were incubated with avidin-HRP (1 mg/ml) in
internalization medium (MEM containing 10 mM HEPES and 5 mM glucose, pH 7.4)
at 4°C for 1 hour to allow cell-surface binding. Internalization was
carried out by the addition of prewarmed medium and incubated for 5 minutes at
37°C to label the early endosomal compartment, and uptake was stopped by
the addition of ice-cold medium. Cells were washed with ice-cold medium and
homogenized in HB at 4°C, and post nuclear supernatants (PNS) were
prepared and quickly frozen in liquid nitrogen. To prepare the enriched
endosomal fraction, thawed PNS was diluted with HB (1:3) and centrifuged at
37,000 g for 1 minute at 4°C. The supernatant was again
centrifuged at 50,000 g for 5 minutes at 4°C. The
resultant pellet enriched in early endosomal vesicles was used for the in
vitro fusion assay.
In vitro fusion assay
In vitro fusion of phagosomes containing the biotinylated
Salmonella with early endosomes containing avidin-HRP was carried out
using the procedure described previously
(Mukherjee et al., 2000).
Briefly, phagosomes isolated from untreated or treated cells were mixed with
early endosomes in fusion buffer (250 mM sucrose, 0.5 mM EGTA, 20 mM
HEPES-KOH, pH 7.2, 1 mM dithiothreitol, 1.5 mM MgCl2, 100 mM KCl,
including an ATP regenerating system, 1 mM ATP, 8 mM creatine phosphate, 31
units/ml creatine phosphokinase and 0.25 mg/ml avidin as scavenger)
supplemented with gel-filtered cytosol prepared from untreated or MBSA-MDP
treated cells. Fusion was carried out for 10 minutes at 37°C, and the
reaction was stopped by chilling on ice. The HRP-avidin-biotin bacterial
complex was recovered by centrifugation (10,000 g for 5
minutes) after solubilization of the membrane in solubilization buffer (SB,
PBS containing 0.5% Triton X-100 with 0.25 mg/ml avidin as scavenger). The
enzymatic activity of avidin-HRP associated with the biotinylated bacteria was
measured as a fusion unit. The maximum fusion between endosomes and phagosomes
isolated from untreated control cells was observed at 0.5 mg/ml of normal
cytosol concentration, which was expressed as 1 unit of relative fusion. HRP
activity corresponding to 1 unit is mentioned in the figure legends.
Assay for transport to lysosomes
To determine the transport of Salmonella to the lysosomes in
MBSA-MDP-treated cells, J774E cells (1x106 cells) were
treated with MDP in the free or conjugated (MDP equivalent, 1 µg/ml) form
for 12 hours at 37°C as described in a previous section. Transport of
Salmonella from early to late lysosomes was detected using an assay
described previously (Hashim et al.,
2000). Briefly, J774E cells were incubated in the presence of
avidin-HRP (200 µg/ml) at 4°C to allow binding. Subsequently,
avidin-HRP was chased for appropriate time (90 minutes) at 37°C to label
the lysosomes. After washing, cells were allowed to bind to biotinylated live
or dead Salmonella (1x107 cells) at 4°C for 1
hour. Cells were resuspended in prewarmed medium and uptake was carried out
for 5 minutes at 37°C to restrict internalization predominantly to the
early compartment. Cells were washed three times to remove unbound bacteria by
centrifugation at low speed (300 g for 6 minutes).
Uninternalized surface-bound biotinylated bacteria were quenched by adding
free avidin (0.25 mg/ml). Cells were washed twice and chased for the indicated
time at 37°C. The reaction was stopped by chilling on ice, and the cells
were solubilized in solubilization buffer (SB, PBS containing 0.5% Triton
X-100 with 0.25 mg/ml avidin as scavenger). The HRP-avidin-biotin bacterial
complexes in the lysates were recovered by centrifugation (10,000
g for 5 minutes). The enzymatic activity of avidin-HRP
associated with the biotinylated bacteria was measured in relative transport
units.
Reconstitution of phagosome-lysosome transport in permeabilized
cells
To directly demonstrate the role of altered Rab5 and Rab7 content of
MBSA-MDP-treated cells in lysosomal targeting of Salmonella,
reconstitution of phagosome-lysosomes transport assay was carried out in
permeabilized cells using a similar assay to one described previously
(Funato et al., 1997).
Briefly, lysosomes of J774E cells (1x106 cells) were loaded
with avidin-HRP, and subsequently cells were allowed to bind to biotinylated
live or dead Salmonella (1x107 cells) at 4°C for
1 hour followed by 5 minutes uptake at 37°C to restrict internalization
predominantly to the early compartment as mentioned in a previous section
(Hashim et al., 2000
). Cells
were washed three times to remove unbound bacteria. In order to permeabilize
the cells using the freeze-thaw method
(Klenchin et al., 1998
), cells
were resuspended in ice-cold permeabilzation buffer (10 mM K-phosphate, 120 mM
KCl, 0.5 mM EGTA, pH 7.2 containing 50 µg/ml avidin) and quickly frozen in
liquid nitrogen. Subsequently, cells were kept at -80°C for 12 hours. The
cell suspension was thawed by warming the tubes at room temperature. Under
these conditions more than 80% of the cells were permeabilized, as measured by
the release of lactate dehydrogenase, a cytosolic marker. However, less than
5% of the HRP was released from the avidin-HRP-loaded lysosomes. After
permeabilization, cells were incubated for 30 minutes at 4°C to deplete
cytosol and gently washed twice with HB. Subsequently, endogenous Rab proteins
from the cells were depleted using Rab-GDI along with GDP, as described
earlier (Funato et al., 1997
).
Loading of reconstituted cytosol was carried out by incubating the cells at
4°C for 30 minutes in 40 µl of fusion buffer containing an ATP
regenerating system in the presence of indicated cytosol (4 mg/ml)
reconstituted with respective Rab protein (300 ng). Rab5 or Rab7 was
immunodepleted from the respective macrophage cytosol as described previously
(Mukherjee et al., 2000
), and
Rab proteins were preincubated with cytosol in the fusion buffer at room
temperature for 30 minutes for in vitro prenylation
(Lombardi et al., 1993
).
Finally, cells were incubated for 60 minutes at 37°C to allow transport to
the lysosomes. The reaction was stopped by chilling on ice, and the cells were
solubilized in solubilization buffer (SB, PBS containing 0.5% Triton X-100
with 0.25 mg/ml avidin as scavenger). The HRP-avidin-biotin bacterial
complexes in the lysates were recovered by centrifugation (10,000
g for 5 minutes). The enzymatic activity of avidin-HRP
associated with the biotinylated bacteria was measured in relative transport
units.
Determination of Salmonella transport to lysosome in
MBSA-MDP-treated cells by confocal microscopy
To confirm the transport of Salmonella to the lysosomes in
MBSA-MDP-treated cells, J774E cells (1x106) were plated on
sterile glass coverslip placed in six-well tissue culture plate and treated
with MBSA-MDP (MDP equivalent, 1 µg/ml) for 12 hours at 37°C as
described in the previous section. Subsequently, treated or untreated J774E
cells (1x106 cells) were incubated with GFP-Salmonella
typhimurium (1x107 cells) for 10 minutes at 37°C to
restrict their entry to the early compartment. Cells were washed three times
to remove unbound bacteria and chased for another 90 minutes at 37°C.
During the last 30 minutes of the chase, Lysotracker Red (100 nM) was added to
GFP-Salmonella-infected macrophages to label the lysosomes. Cells
were washed three times with cold PBS, and confocal microscopy was carried out
using a Zeiss, LSM 510 confocal microscope using an oil immersion
objective.
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Results and Discussion |
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|
To determine the efficacy of MBSA-MDP for treatment of Salmonella
infection in vivo, C57B1-6 mice were infected with 103
Salmonella per mouse as described previously
(Pashine et al., 1999). The
animals received intraperitoneal injections of free or MBSA-MDP (1 µg/
mouse of MDP equivalent; 50 µg/kg body weight) or ciprofloxacin (1.4 mg/
mouse; 70 mg/kg body weight) daily for four consecutive days. On day 10, the
mice were sacrificed to determine the content of viable Salmonella in
the spleen (Pashine et al.,
1999
). Data in Fig.
1B show that administration of MBSA-MDP (50 µg/kg body weight)
eliminated about 96% of the bacteria from the infected spleen, whereas free
MDP at the same dose eliminated only 60% of the Salmonella. In
comparison, the therapeutic dose (70 mg/kg body weight) of ciprofloxacin,
which is about 1500-fold higher than the MDP concentration in the conjugate,
removed about 90% of the Salmonella from the mice. These results are
consistent with the previous observations that macrophages activated by MDP
augment the host defense against various infections
(Koff et al., 1985
; Sarkar et
al., 1997), but the mechanism of killing of intracellular pathogens by
MDP-mediated macrophage activation is not known.
Recently, we reported that intracellular delivery of MDP to macrophages
through scavenger receptor (SCR)-mediated endocytosis triggers the secretion
of different cytokines (Srividya et al.,
2000). Several independent studies have shown that activation of
macrophages through different cytokines alone or in concert can contribute to
the antimycobacterial potential of these cells, resulting in the control of
infection in vitro (Rook et al.,
1986
; Chan et al.,
1992
; Flynn et al.,
1995
; Schaible et al.,
1998
; Via et al.,
1998
). However, the mechanism of killing of Mycobacterium
by cytokine-activated macrophages is not known. In view of the reports that
IFN
alters the expression of Rab5 (Alvarez-Dominguez et al., 1998), and
Rabs are the major regulators of intracellular transport, we investigated
whether the intracellular delivery of MDP, which induces the secretion of
different cytokines, modulates the level of endocytic Rabs. Western blot
analysis of the cell lysate with specific antibodies after 12 hours of
treatment with MDP or MBSA-MDP revealed that MBSA-MDP treatment resulted in a
significant increase in the level of Rab7 and a decrease in the content of
Rab5 in the macrophages compared with free MDP treatment
(Fig. 2). By contrast, content
of the Golgi-associated Rab6 (Mallard et
al., 2002
) remained unaltered with MBSA-MDP treatment, suggesting
that the effects seen with MBSA-MDP might be restricted to endocytic Rabs like
Rab5 and Rab7. However, treatment with MBSA alone did not alter the level of
endocytic Rab proteins in the cells, suggesting that the observed enhanced
effect of MDP in MBSA-MDP is caused by SCR-mediated uptake of more MDP than
fluid phase uptake of free MDP (Fig.
2). As Rab7 is the targeting signal to the late lysosomal
compartment (Mukhopadhyay et al.,
1997a
), enhanced expression of Rab7 in MBSA-MDP-treated cells
probably explains the enhanced killing of Salmonella in macrophages
by MBSA-MDP treatment.
|
To investigate the consequences of the altered cellular levels of Rab5 and
Rab7 in the maturation of Salmonella-containing phagosomes, we
purified early (5 minutes) LSP from untreated cells
(Mukherjee et al., 2000) as
well as from MBSA, MDP and MBSA-MDP-treated cells and measured their levels of
Rab5 and Rab7 by western blot analysis. Data in
Fig. 3A show that LSP from
untreated control cells display significant levels of Rab5 and little Rab7,
whereas those from MBSA-MDP-treated cells primarily display Rab7 and almost no
Rab5. LSP isolated from free MDP-treated cells, however, showed substantially
less Rab7 than those from MBSA-MDP-treated cells. The higher content of Rab5
on LSP compared with the total Rab5 content in MDP-treated cells is due to
SopE, a secretory protein of Salmonella, which mediates recruitment
of Rab5 on LSP (Mukherjee et al.,
2000
; Mukherjee et al.,
2001
). Since the contents of viable bacteria were similar in early
LSP isolated from the control cells and cells treated with MBSA, MDP or
MBSA-MDP (data not shown), enhanced recruitment of Rab7 by LSP in
MBSA-MDP-treated cells is not caused by the differential viability of the
bacteria. Possibly, higher expression of Rab7 in MBSA-MDP-treated cells than
in untreated or MDP-treated cells correlates with the SCR-mediated enhanced
uptake of MDP by macrophages (Mukhopadhyay
et al., 1989
; Majumdar et al., 1991). It is tempting to speculate
that the enhanced secretion of various cytokines by MBSA-MDP- treated cells
(Srividya et al., 2000
)
regulates the expression of the Rab proteins. This is supported by a recent
finding that macrophages treated with IFN-
induced Rab5 expression
(Alvarez-Dominguez et al., 1998). However, macrophages treated with MBSA-MDP
do not secrete IFN-
(data not shown), which is in agreement with the
fact that IFN-
is exclusively secreted by NK cells and certain
subpopulations of T lymphocytes (Billiau et al., 1996). Furthermore, reduced
Rab5 content in MBSA-MDP-treated macrophages did not alter the uptake of
Salmonella (data not shown), suggesting that the increased Rab7
content of MBSA-MDP-treated cells presumably compensates for the endocytic
function of Rab5, which is consistent with the report that injection of Rab7
alone into frog oocytes stimulated HRP uptake
(Mukhopadhyay et al.,
1997a
).
|
Macrophages are phagocytic cells that usually target the invading
microorganisms to the lysosomes where the lysosomal hydrolases in the acidic
compartment degrade them. By contrast, morphological studies have shown that
Salmonella bypass the M-6-P-receptor-positive compartment
(Portillo and Finlay, 1995)
and reside in a unique compartment that clearly diverges from the degradation
pathway of the macrophages (Rathman et
al., 1997
; Buchmeier and
Heffron, 1991
; Alpuche-Aranda
et al., 1992
). But, the molecular mechanism for inhibition of
Salmonella transport to lysosomes was not known. Recently, we have
shown that live Salmonella-containing phagosomes (LSP) transport a
bacterial protein, SopE, on the surface of phagosomes and thereby recruit the
early acting Rab5 and fusion factors like
-SNAP and NSF to promote
fusion with early endosomes (Mukherjee et
al., 2000
; Mukherjee et al.,
2001
). Salmonella persist in a specialized low-acidity
compartment lacking active lysosomal enzymes and transferrin receptors but
retaining Rab5 and Rab18 (Hashim et al.,
2000
). Thus, SopE-mediated recruitment of Rab5 on LSP, which
promotes fusion with early endosomes, is the major mechanism by which
Salmonella survive in macrophages. Therefore, the downregulation of
Rab5 expression in MBSA-MDP-treated cells might prevent the interaction of
Salmonella-containing phagosomes with early endosomes, whereas
simultaneous upregulation of Rab7 could induce the transport of
Salmonella-containing phagosomes to the lysosomes. The data presented
in Fig. 3B show that the extent
of in vitro fusion of early endosomes with LSP isolated from MBSA-MDP-treated
cells in the presence of cytosol prepared from MBSA-MDP-treated cells is
significantly lower than that observed with LSP isolated from untreated
control cells in the presence of normal cytosol. This suggests that the
reduced content of Rab5 in MBSA-MDP-treated cells inhibits fusion with early
endosomes, thus subverting the primary survival mechanism of
Salmonella (Mukherjee et al.,
2000
; Hashim et al.,
2000
; Mukherjee et al.,
2001
).
To determine whether the enhanced content of Rab7 in macrophages induced by
MBSA-MDP leads to enhanced lysosomal targeting of live Salmonella, we
studied the transport of biotinylated live or dead bacteria to the lysosomes
preloaded with avidin-HRP as described previously
(Hashim et al., 2000). The
results presented in Fig. 3C
show that in MBSA-MDP-treated macrophages live biotinylated
Salmonella colocalized with avidin-HRP-loaded lysosomes at a rate
similar to that of dead biotinylated bacteria in untreated macrophages,
whereas transport of live biotinylated Salmonella to the lysosomes is
inhibited in untreated macrophages (Hashim
et al., 2000
). In free MDP-treated cells, by contrast, only
20% of the live biotinylated Salmonella were transported to the
lysosomes during the same interval (90 minutes).
Intracellular delivery of MDP may modulate several signal transduction
molecules. To directly demonstrate that the altered Rab5 and Rab7 content of
MBSA-MDP-treated cells is sufficient to drive the killing of
Salmonella by lysosomal targeting, reconstitution of the
phagosome-lysosome transport assay was carried out in permeabilized cells
(Klenchin et al., 1998) in the
presence of different combinations of in vitro prenylated Rab5 and Rab7
proteins. The results presented in Fig.
4 show that dead biotinylated Salmonella are transported
to avidin-HRP-loaded lysosomes, whereas transport of live Salmonella
is significantly inhibited in the presence of cytosol prepared from normal
macrophages. Interestingly, when a similar assay was carried out in the
presence of Rab5-immunodepleted cytosol, transport of the live bacteria to the
lysosomes was partially induced in comparison with that in normal cytosol.
These results suggest that the reduced content of Rab5 in the depleted cytosol
might inhibit the fusion of LSP with the endosome, and endogenous Rab7 is
probably responsible for the partial transport of Salmonella to the
lysosomes. However, the transport of Salmonella to the lysosome in
Rab5-depleted cytosol was relatively low in comparison with MBSA-MDP-treated
cytosol mainly because of the enhanced content of Rab7 in treated cells. This
is further supported by the fact that addition of Rab7:WT in Rab5-depleted
cytosol induced more transport of Salmonella to the lysosome.
Moreover, transport of the live Salmonella to the lysosomes was
significantly induced, almost to the extent observed in the presence of
cytosol prepared from MBSA-MDP-treated cells, when reconstitution of transport
was carried out in the presence of cytosol containing Rab5-S34N, a negative
mutant of Rab5 locked in GDP form (Li and
Stahl, 1993
) along with Rab7:WT protein
(Fig. 4). These results
indicate that the reduced content of Rab5 and enhanced level of Rab7 in
MBSA-MDP-treated cells are probably responsible for transport of
Salmonella to the lysosomes. By contrast, transport of live
Salmonella to the lysosomes was significantly blocked when
reconstitution was carried out in the presence of cytosol containing Rab5:WT
and Rab7:T22N, a negative mutant of Rab7 locked in the GDP form (Mukhopdhyay
et al., 1997a). In addition, transport of Salmonella to the lysosome
was significantly blocked in comparison with MBSA-MDP-treated cytosol when the
transport assay was carried out in the presence of Rab7-immunodepleted cytosol
supplemented with Rab5:WT protein (Fig.
4). These results unequivocally prove that enhanced content of
Rab7 and reduced level of Rab5 in MBSA-MDP-treated cells is responsible for
the induced transport of Salmonella to the lysosomes. Although,
intracellular delivery of MDP may activate different signalling cascades, our
results clearly demonstrate that downregulation of Rab5 in parallel with
upregulation of Rab7 triggered by intracellular delivery of MDP is sufficient
to target LSP to the lysosomes.
|
LSP isolated from MBSA-MDP-treated macrophages 90 minutes after
internalization showed enhanced levels of cathepsin D and vacuolar ATPase in
comparison with LSP isolated 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
(Fig. 5A). However, in HeLa
cells overexpression of Rab7 alone was reported to target Salmonella
to a compartment that is positive for LAMP-1 and Rab7 but lacks lysosomal
enzymes like cathepsin D (Meresse et al.,
1999). Whereas, Rab7 overexpression in HeLa cells failed to target
M. Tuberculosis and L. pneumophila to a LAMP1-containing
compartment (Clemens et al.,
2000
). Although Rab 7 is the signal to transport the cargo to the
lysosomes, the overexpression of Rab7 alone is unable to transport these
microorganisms to fully competent lysosomes. This may be because of the
bacteria-driven mechanism, which arrests their transport to the lysosomes.
Finally, the results presented in Fig.
5B clearly demonstrate that GFP-Salmonella colocalized
with the Lysotracker-Red-labelled lysosomal compartment in MBSA-MDP-treated
cells (Fig. 5Bf), whereas
GFP-Salmonella in untreated cells resides in a compartment that is
not labelled with Lysotracker Red (Fig.
5Bc). It is pertinent to mention that more than 80% of the
GFP-Salmonella colocalized with the Lysotracker-Red-labelled
lysosomal compartment in MBSA-MDP-treated cells, whereas less than 10% of the
GFP-Salmonella colocalized with the Lysotracker-Red-labelled
compartment in untreated cells. Lysotracker Red was shown to accumulate in the
compartment that labelled with Rab7 and LAMP1 antibodies, which is a
characteristic feature of the late lysosomal compartment
(Schaible et al., 1998
;
Magez et al., 1997
;
Wubbolts et al., 1996
).
Therefore, our data suggest that Salmonella is transported to fully
competent lysosomes in MBSA-MDP-treated macrophages for efficient killing.
|
In conclusion, this is the first demonstration that intracellular delivery of MDP leads to simultaneous reduction of Rab5 and enhancement of Rab7. Reduction in Rab5 levels inhibits the fusion of LSP with early endosomes, subverting the mechanism by which Salmonella resist transport to the lysosomes, whereas Rab7, being involved in the transport of vesicles towards the late/lysosomal compartments, enhanced the content of Rab7 in the cells and presumably targets the Salmonella to the lysosomes for eventual destruction. Currently, we are trying to understand the mechanism by which MBSA-MDP-mediated enhanced secretion of different cytokines regulates the expression of Rab proteins. This novel approach of modulating the cellular contents of endocytic Rabs to ensure lysosomal targeting and destruction might be generally useful in combating intracellular pathogens, such as Salmonella typhi, M. tuberculosis, L. pneumophilia and Toxoplasma gondii that normally survive in the host cell by resisting transport to lysosomes.
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Acknowledgments |
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