From the Centro de Biología Molecular
"Severo Ochoa," Universidad Autónoma de Madrid, 28049 Madrid,
Spain and the ¶ Department of Immunology-IMM14/R221, The Scripps
Research Institute, La Jolla, California 92037, and the
Cell Biology Department, Washington University,
St. Louis, Missouri 63110
Received for publication, February 21, 2001, and in revised form, March 21, 2001
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ABSTRACT |
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Control and clearance of Listeria
monocytogenes infection is an
interferon- Listeria monocytogenes is an intracellular
facultative bacterium able to invade phagocytic cells and is
responsible for severe pathologies in immunocompromised people,
newborns and pregnant women (1). L. monocytogenes entry into
the host cell is an active process involving several protein
components. After a short phagosomal period (~30 min), L. monocytogenes escapes to the cytosol, avoids intracellular
killing, and replicates (reviewed in Ref. 2). The L. monocytogenes survival mechanism involves two steps: (i) live
bacteria avoid phagosome maturation by inactivation of the endosomal
trafficking regulator Rab5a, which blocks the recruitment of lysosomal
proteins to the phagosomes (Lamp-1 and cathepsin-D) (3) and (ii)
secretion by L. monocytogenes of listeriolysin and
PI-PLC lyses the phagosomal membrane, translocates L. monocytogenes to the cytoplasm, and consequently, allows for
L. monocytogenes intracellular survival (4).
Control of L. monocytogenes infection and clearance is an
interferon- Recently, we have shown that in resting MØs the inhibition of Rab5a
synthesis allows for intracellular survival of a
listeriolysin-defective L. monocytogenes mutant, that under
normal Rab5a levels is unable to grow and fails to escape from the
phagosome (12). Furthermore, we have also described that IFN- Cells and Reagents--
J774 cells and proteose peptone-elicited
peritoneal MØs from Balb/c mice were cultured in Dulbecco's modified
Eagle's medium, 5% fetal calf serum, 2 mM
L-glutamine, and 50-µg/ml gentamicin. Phosphothioate
Rab5a antisense (5'-TGC GCC TCG ACT AGC CAT GT-3') and sense (5'-ACA
TCG CTA GTC GAG GCG CA-3') oligonucleotides (20-mer) were from
Isogen Bioscience BV (Maarseen, Holland). Mouse recombinant IFN- Transient Overexpression of Rab5 Constructs in J774
Cells--
Rab5a:Q79L, Rab5a:S34N, and Rab5c:Q80L cDNAs were
subcloned into pcDNA3 using EcoRI/BamHI
sites. Cells (5 × 106) were transfected by
electroporation (150 V, 800 microfarads, 129 ohms) for 24 h.
Overexpression was checked on cell lysates with specific antibodies at
8 and 24 h, respectively.
Antibodies and Proteins--
The following antibodies were used.
Mouse monoclonal anti-Rab5a (4F11) was described in Ref. 3.
Polyclonal rabbit anti-Rab5c was a gift from M. Zerial (EMBL,
Heidelberg, Germany). Rabbit anti-Rab7 was a generous gift from A. Wandinger-Ness (University of New Mexico, Alburquerque, NM). Rabbit
anti-Rac2 was developed in rabbits (R786/9) (14), and rabbit
anti-cathepsin-D (3) was a gift from P. D. Stahl (Washington
University. St. Louis, MO). Rabbit anti-Limp-II was a gift from I. V. Sandoval (Centro de Biología Molecular "Severo Ochoa,"
Madrid, Spain). Rat anti-mouse Lamp-1 monoclonal antibody (1G11) was a
gift from D. G. Russell, (Washington University, St. Louis, MO).
Biotinylated rat anti-mouse TfR (CD71) was purchased from Caltag, and
secondary peroxidase-conjugated antibodies (goat anti-mouse,
anti-rabbit, or anti-rat) were from Amersham Pharmacia Biotech.
Streptoavidin-peroxidase-conjugated antibody was purchased from Roche
Molecular Biochemicals.
GST-PBD, the p21-activated kinase-derived binding domain for activated
Rac2 proteins, was expressed in E. coli BL-21 strain. Recombinant proteins were induced with 5 mM
isopropyl- Antisense and IFN- Bacterial Infection and Intracellular Assays--
D. A. Portnoy (University of California, CA) kindly provided the pathogenic
L. monocytogenes strain (10403S). L. monocytogenes infection was performed according to standard
protocols (3) at a 10:1 bacteria/cell ratio. After 15 min of uptake,
cells were incubated for 45 min in medium containing 5 µg/ml
gentamicin to kill extracellular bacteria. This time period was
considered 0 h. Infected cells were then incubated at 37 °C in
complete medium containing 5 µg/ml gentamicin for 16 h in the
presence or absence of 100 units/ml IFN- Isolation of Phagosomes--
Phagosomes were purified from
30-min (Western experiments) or 1-h (viability experiments) infection
protocols. L. monocytogenes-infected cells were pretreated
or not with IFN- Western Blot Assays--
30 µg of phagosomal proteins per lane
were loaded for Western blots. Phagosomes were normalized using
anti-Rab5c marker as a standard, since this marker does not vary upon
IFN- Immunoprecipitation--
Cells (5 × 106/ml)
were labeled with 50 µCi of [35S]Met/Cys promix for
2 h. Immunoprecipitations were performed as described (13). Beads
were washed three times with radioimmune precipitation buffer (1% PBS,
0.1% Triton X-100, 0.5% SDS), radioimmune precipitation buffer plus
500 mM NaCl, and PBS. Elution was performed with 1× Laemmli sample buffer for 1 h at room temperature.
Membrane and Cytosolic Distribution of Rab5a--
J774 cells
treated with antisense/sense oligonucleotides, incubated with (+) or
without ( Assays for Rac2 Activation in J774 Cells--
These assays were
performed as previously reported for Rac2 (15). In brief, cells (2 × 107 cells/assay) were treated with Rab5a antisense/sense
oligonucleotides for 6 h and incubated with 100 units/ml IFN- O L. monocytogenes is a facultative intracellular
parasite able to infect and replicate inside nonactivated MØs. Once in
the phagosome, the bacterium secretes listeriolysin and PI-PLC
to lyse the phagosome and escape to the cytoplasm, where it replicates (4). The activation of MØs by IFN- Analysis of phagosomes from infected MØs pulsed with IFN- At the molecular level, IFN- IFN- IFN-
Analysis of phagosomes from MØs treated with Rab5a antisense
oligonucleotide (A) showed that this treatment effectively
blocked Rab5a expression on phagosomes (A
Parallel studies of Rab5c and Rab7 levels showed that their expression
in phagosomes was not affected by the Rab5a antisense treatment, either
alone or in combination with a pulse of IFN-
These observations highlighted two important conclusions: (i)
Rab5a acquires a novel function upon IFN-
IFN- Rab5a Mediates the IFN-
We also estimated lysosomal protein synthesis after antisense or sense
treatment in the presence or absence of IFN-
Taken together, these results show that IFN- Rab5a Regulates the IFN-
In summary, the experiments shown in Figs. 2-4 indicated that IFN- Rab5a Acts Upstream of Rac2 and ROI Production in the IFN-
Activation of the phagocyte NADPH oxidase leads to a functional enzyme
able to produce ROI toxic molecules (31). Next, we studied the role of
Rab5a on the IFN-
To address whether Rab5a synthesis alone was sufficient to trigger the
effects observed on phagosome-lysosome fusion (Fig. 4) and Rac2-GTP
recruitment (Fig. 5), we transiently overexpressed both the active and
inactive forms of Rab5a into J774 cells; we also included the active
form of another Rab5 isoform expressed onto L. monocytogenes
phagosomes, Rab5c, as a control (5a:Q79L, 5a:S34N, and 5c:Q80L, respectively).
Overexpression protocols after 24 h of transfection gave 5-7-fold
increased levels above controls (cells transfected with vector alone)
(Fig. 6A). Phagosomes (after
30 min of infection) from these cells showed that transfer of HRP from
lysosomes was particularly enhanced in Rab5a:Q79L-transfected cells
(Fig. 6B). These results were in accordance with those
previously reported for Rab5a:WT-transfected cells (13). Overexpression
with Rab5a-inactive form (5a:S34N) inhibited the phagosome-lysosome
fusion enhancement, even below control levels. However, overexpression
with the active form of Rab5c, Q80L, showed a very low levels of
phagosome-lysosome fusion, similar to control cells and to results
reported previously (13). These data argue that Rab5a synthesis and its
activation in the GTP form were sufficient to promote
phagosome-lysosome fusion.
We also checked whether Rab5a synthesis was able to control the
observed rac2-GTP recruitment to phagosomes (Fig. 5B). To do
this, we isolated phagosomes from cells overexpressed with Rab5a
or Rab5c cDNAs, as in Fig. 6A, and analyzed the
induction of Rac2 activation and translocation to the phagosomes, using the same protocol as used in Fig. 5B. As shown in Fig.
6C, Rac2 recruitment was promoted in
Rab5a:Q79L-overexpressed cells and was diminished below control levels
in Rab5a:S34N-overexpressing cells. It should be noted that cells
overexpressing the active Rab5c:Q80L form showed levels similar to the
controls. These results clearly indicate that Rab5a-GTP, exclusively,
controls the recruitment of Rac2-GTP and that Rab5a action is upstream
of Rac2.
It is also interesting that another cytokine, the granulocyte
colony-stimulating factor, could control the growth of the pathogen Brucella abortus also by regulating the interactions with
the endosomal compartment (35). This interaction may transfer bacteria from a relative nonhostile environment to one that contains reducing agents, acid hydrolases, and oxygen radicals (35). It can be speculated
that these ROI, elements of the respiratory burst, also present in the
endocytic compartments, can then reach both the Listeria-
and Brucella-containing phagosomes under each cytokine situation and compromise the bacterial growth inside the cells.
In summary, our results are the first to implicate a small GTPase,
Rab5a, in pathogen clearance by phagocytes and to show that this
function is induced by IFN--dependent process. The listericidal
mechanism of action involves activation of NADPH oxidase and inducible
nitric-oxide synthase to produce reactive oxygen and nitrogen
intermediate radicals, respectively. Recently, we have described in a
nonpathogenic model of L. monocytogenes (hemolysin negative
mutant strain) that the interferon-
-inducible GTPase Rab5a
contributed to Listeria destruction in resting macrophages.
Here, we report in a pathogenic model of L. monocytogenes
(hemolysin-positive strain) that Rab5a plays a central role in
Listeria destruction induced by interferon-
and within
the phagosomal environment. These findings reveal the importance of
Rab5a as the responsible factor mediating the listericidal action of
interferon-
. Active Rab5a causes remodeling of the phagosomal
environment, facilitates the translocation of Rac2 to LM
phagosomes, and regulates the activity of this GTPase. Rac2 activation
and translocation governs the phagocyte NADPH oxidase activity and the
consequent reactive oxygen intermediate production that leads to
killing of the pathogen.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(IFN-
)1-dependent
process. IFN-
priming of macrophages (MØs) recruited at the
inflammatory site triggers their listericidal abilities (5). IFN-
signaling modulates the expression and activation of more than 200 proteins (6). However, to date, only a few of these molecules have been
shown to exert a direct role in pathogen elimination (7). Among these
are (i) IGTP, a GTP-binding protein relevant for Toxoplasma
clearance (8) and (ii) Nramp1, a MØ-restricted lysosomal protein
involved in Leishmania, Salmonella, and
Mycobacterium spp. clearance (9). In addition, IFN-
induces the production of reactive oxygen (ROI) and nitrogen (RNI)
intermediates with microbicidal activity (10). From this set of
molecules, only ROI and RNI have been shown to restrict L. monocytogenes growth (10, 11), while the other two molecules
(i.e. IGTP or Nramp1) play no role at all in L. monocytogenes clearance (8, 9).
signaling up-regulates Rab5a function (13). However, at this stage, no
correlation between the induction of ROI and RNI by IFN-
and the
Rab5a function has been established. Here, we show that Rab5a is a key
molecule for the IFN-
promoted clearance of a pathogenic L. monocytogenes strain at the phagosomal stage. We show that Rab5a,
in the GTP form, controls the recruitment of active Rac2 to the
transformed L. monocytogenes phagolysosome and the assembly
of the phagocyte NADPH oxidase with the production of toxic radicals.
These Rab5a-mediated actions compromise Listeria viability
within the phagolysosomes and further L. monocytogenes
intracellular survival.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
was purchased from Roche Molecular Biochemicals; 35S
translabel (10 mCi/ml) was from Amersham Pharmacia Biotech; horseradish
peroxidase (HRP), superoxide dismutase, and ferricytochrome c were from Sigma; and brain heart infusion was from Difco.
-D-thiogalactoside for 3 h at 37 °C
and purified with glutathione-Sepharose according to the
manufacturer's instructions (CLONTECH, Palo Alto, CA).
Treatment of Cells--
Introduction
of antisense and sense phosphothioate oligonucleotides onto J774 cells
or peritoneal MØs (5 × 106/ml) was performed as
described (12) with a Baxter BTX-603 electroporator and the following
settings: 220 V, 800 microfarads, 75 ohms. Cells were set onto culture
plates for 6 h at 37 °C and washed and incubated (+) or not
(
) with 100 units/ml of IFN-
for 16 h.
, washed, lysed, and plated
onto brain heart infusion agar plates (37 °C, 36 h). The number
of live bacteria was estimated by counting CFU. To estimate the
percentage of growth, the ratio of CFU recovered at 16 h divided
by the CFU recovered at 0 h was expressed as the replication index.
(16 h). For phagosome-lysosome fusion assays, cells
were offered HRP (100 µg/ml, 5 min, washed and chased for 2 h).
Cells were infected with L. monocytogenes for 30 min.
Postnuclear supernatants from these cells were applied to a
8.8-20-40% discontinuous sucrose gradient and L. monocytogenes phagosomes recovered from the 20-40% interface and
lower 20% sucrose fraction as previously reported (13). Phagosomes
were solubilized with PBS-0.05% Triton X-100 and plated onto brain
heart infusion-agar plates to count CFU or added to 4× Laemmli sample
buffer for Western blots. For phagosome-lysosome fusion assays, HRP
reaction was evaluated with o-dianisidine as a substrate.
HRP was measured in postnuclear supernatants and considered the total
HRP. Results were expressed as the percentage of total absorbance
values of HRP activity per mg of protein.
or antisense treatment. Normalization was also performed after
analysis with a rabbit anti-Listeria protein antiserum, to
check the same level of Listeria proteins onto
phagosomes. Antibody dilutions were as follows: mouse anti-Rab5a and
rabbit anti-Rab5c, anti-Rac2, and anti-Listeria antibodies
(1:1000); rabbit anti-cathepsin-D and anti-Limp-II and rat anti-mouse
Lamp-1 and anti-mouse TfR (1:500); and rabbit anti-Rab7 (1:300).
) IFN-
for 16 h and labeled with 50 µCi of
[35S]Met/Cys promix as above, were homogenized in HBE
buffer (250 mM sucrose, 0.5 mM EGTA, 20 mM Hepes-KOH, pH 7.2) with a ball bearing homogenizer.
Homogenates were spun down at 2000 × g to remove
nuclei and large organelles, and supernatants were centrifuged at
100,000 × g for 60 min to obtain total membranes
(M) and cytosols (C) (supernatants) (see Fig. 2).
Pellets were resuspended in HBE buffer, and supernatants were
precipitated with 10% trichloroacetic acid and resuspended in the same
volume as pellets. All samples were quickly frozen in liquid nitrogen,
and immunoprecipitations were performed as above.
16 h. Accordingly, similar assays were performed in transient
overexpressed cells with Rab5a:Q79L, Rab5a:S34N, Rab5c:Q80L, or
pcDNA3 cDNAs. L. monocytogenes phagosomes or whole
cells were then washed in HBSS-g and lysed in a 2× lysis buffer (50 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, 5 mM EDTA, 200 mM NaCl, 2%
Nonidet P-40, 10% glycerol, 2 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 2 mM sodium orthovanadate). Lysates placed on ice were
clarified by 20,000 × g centrifugation at 4 °C, and
8 µg of GST-PBD was added. Binding buffer (25 mM
Tris-HCl, pH 7.5, 1 mM dithiothreitol, 30 mM
MgCl2, 40 mM NaCl, 0.5% Nonidet P-40), and
glutathione-Sepharose were added for 90 min at 4 °C with shaking (a
total of 300 µl). Washings were performed with binding buffer. The
bead pellets were resuspended in 1× Laemmli sample buffer. Proteins
were separated by 15% SDS-PAGE, transferred to membranes, and blotted
for the appropriate GTPase using antibody R786/9 (Rac2). ECL detected
immunoblots. Total putative activated protein from each sample
was obtained by treatment with 100 µM GTP
S for 15 min
at 30 °C before GST-PBD incubation.
). Cells
were incubated for 60 min at 37 °C. The change in absorbance
(A550 nm) obtained in well samples was
subtracted from those wells incubated with the same stimulus in the
presence of superoxide dismutase (10 µg/ml). Results were expressed
as nmol of O
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
provokes an increase in the
phagocytic rate, partially prevents the escape of L. monocytogenes to the cytosol (17), and triggers the production of
toxic radicals (i.e. ROI and RNI), which lead to the
elimination of the microorganism (10).
has
established that this lymphokine promotes phagosome maturation (18,
19). These observations show that phagosome maturation is an active
process that involves the interaction of phagosomes with several
organelles (reviewed in Ref. 20).
signaling up-regulates the levels of
endosomal/lysosomal proteins, such as cathepsin-D (21) and Rab5a (13),
and down-regulates endosomal markers, such as the mannose and the
transferrin receptors, TfR (22, 23), while other Rabs remain unaffected
(i.e. Rab5b, Rab5c, Rab7, or Rab11) (13). However, until
now, the link between the IFN-
-induced microbicidal effects and the
up-regulation of Rab5a has remained elusive.
Reduces Intraphagosomal Listeria Viability and Promotes
Phagosome Maturation--
Since IFN-
triggers the listericidal
activity of MØs, we first investigated its action in infected MØs at
the phagosomal stage. As shown in Fig. 1,
among the typical endosomal markers analyzed in L. monocytogenes isolated phagosomes, Rab5a was clearly increased
after IFN-
treatment (+), whereas Rab5c or Rab7 was unmodified and
TfR was down-regulated. Similar data have been reported in studies of
whole cell extracts (13, 23). Isolated phagosomes from MØs not treated
with IFN-
(
) lacked lysosomal markers such as cathepsin-D, Lamp-1
and Limp-II. In contrast, all of these markers were increased in
phagosomes from MØs previously pulsed with IFN-
(+). These results
suggested that IFN-
up-regulated the interactions of phagosomes with
late endosomes and lysosomes rather than with early endosomes. IFN-
promotion of phagosome maturation was accompanied by a significant
reduction of the pathogen viability inside the phagosomes (1-h
phagosomes in Fig. 1, bar graphs). These results
argue that a more rapid entry of this lysosomal environment into the
phagosomal space contributes to the killing of the pathogen.
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Fig. 1.
Effects of IFN- on
L. monocytogenes survival and phagosomal
composition. J774 cells were incubated (+) or not (
) with 100 units/ml IFN-
for 16 h before infection with L. monocytogenes (1 h). Phagosomes were isolated, and viable bacteria
(CFU) were determined after plating onto brain heart infusion-agar
plates. Results shown are the mean ± S.D. of triplicate cultures
(A, graphic bar). Lanes
corresponded to Western blots of solubilized isolated phagosomes
(30-min infection) and incubated with the following antibodies:
monoclonal anti-Rab5a antibody; rabbit anti-Rab5c, anti-Rab7,
anti-Limp-II, and anti-cathepsin-D (cat-D); and rat anti-mouse TfR and
anti-mouse Lamp-1.
Promotion of Phagosomal Maturation and Lysosomal Protein
Transport Is Regulated by Rab5a--
Our earlier data showed that
within the phagosome, Listeria avoided phagosome maturation
by blocking the Rab5a activity (3, 12). Since IFN-
signaling
increases the synthesis and enhances the activity of this small GTPase
(13), we next studied whether promotion of phagosome maturation by
IFN-
was mediated by Rab5a and designed the following experimental
strategy. Rab5a synthesis was first blocked using phosphothioate
antisense oligonucleotides targeted to the Rab5a translation initiation
codon, and then an IFN-
pulse was given (12).
versus S
cells
in Fig. 2A). Moreover, Rab5a
antisense treatment also blocked Rab5a expression even in phagosomes of
MØs pulsed with IFN-
(+) (compare A+ with S+ cells and S
cells
with S+ cells, respectively; Fig. 2A). The inhibition was
thus effective at the level of protein synthesis; as shown in
Fig. 2B, this reduction mainly affected membrane-bound Rab5a.
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Fig. 2.
Effect of Rab5a antisense/sense and
IFN- treatment onto L. monocytogenes intracellular survival, phagosomal viability,
and composition. A, J774 were treated with Rab5a
antisense oligonucleotide (A) or with Rab5a sense
oligonucleotide (S) for 6 h. The cells were pulsed (+)
or not (
) with 100 units/ml IFN-
for 16 h and then infected
with L. monocytogenes for 30 min. Finally, phagosomes
(Phg) were isolated and solubilized, and material was
assayed for the presence of different proteins: Rab5a, Rab5c, Rab7,
TfR, Lamp-1, Limp-II, and cathepsin-D. B, J774 cells treated
as above were metabolically labeled for 2 h. After that, cytosol
(C) and membrane extracts (M) were obtained, and
Rab5a was immunoprecipitated (IP) to test the distribution
of newly synthesized Rab5a in both fractions. C, L. monocytogenes viability was analyzed on J774 cells treated as
before. Results shown are the mean of triplicates ± S.D. and
expressed as the replication index after 16 h of infection for
total viability of L. monocytogenes (white
bars) or as CFU in 1-h obtained phagosomes
(filled bars).
(Fig.
2A). On the other hand, the increase in the levels of
Lamp-1, Limp-II, and cathepsin-D in phagosomes induced by IFN-
in
control cells (S+), were almost completely abolished in cells treated with Rab5a antisense oligonucleotides (A+). Experiments performed with
lysosensor green as a pH indicator showed that IFN-
treatment decreased MØ vesicle pH, but independently of Rab5a antisense or sense
treatment (data not shown).
stimulation that affects
the transport of lysosomal proteins to phagosomes, and (ii) the normal
L. monocytogenes strategy of blocking transport of newly
synthesized lysosomal proteins to phagosomes (3) can be overcome by the
increase in Rab5a function promoted by IFN-
.
induction of Rab5a synthesis has been shown to promote the
binding of the GTP form of Rab5a to membranes (13). This argues that
the Rab5a form that controls the transport of lysosomal proteins to the
phagosomes should be the GTP form. Our data are also in agreement with
a recent report showing a role for rabenosyn-5, a Rab5-GTP-interacting
effector protein, in the transport of cathepsin-D from the Golgi
complex to lysosomes (24).
-induced Listericidal Abilities at the
Phagosomal Stage--
When intracellular growth of L. monocytogenes was studied, there was an inverse correlation
between viability, expressed as the replication index, and Rab5a
levels. The highest replication index corresponded to Rab5a
antisense-treated cells (A
) (Fig. 2C) that showed no
detectable Rab5a levels (Fig. 2B, Rab5a-IP lanes). A lower replication index was found in Rab5a
antisense-treated cells also pulsed with IFN-
(A+) relative to the
controls (S
); the replication index values were 22.5 in A
cells, 9 in A+ cells, and 6.2 in S
cells (Fig. 2C, white
bars). Interestingly, these Rab5a antisense-treated cells
pulsed with IFN-
(A+) expressed detectable Rab5a protein
levels (Fig. 2B, Rab5a-IP lanes). The Rab5a
protein expressed in these A+ cells efficiently bound to intracellular
membranes, with no detectable pool in the cytosolic fraction (see
M/C distributions on IP-Rab5a lanes;
Fig. 2B). Rab5a antisense treatment blocks the listericidal
effect of IFN-
as shown by the replication index values of 9 in A+
cells compared with a replication index value of 0.18 in S+ cells (Fig.
2C, white bars). This Rab5a antisense
treatment also prevented the expression and induction of Rab5a observed
in control cells (Fig. 2B, Rab5a-IP lanes). These
results cannot be explained by different L. monocytogenes ingestion rates on Rab5a antisense- or Rab5a sense-treated cells (12). Similarly, intraphagosomal L. monocytogenes viability, expressed as CFU from isolated phagosomes after 1 h of infection, inversely correlated with Rab5a expression on L. monocytogenes phagosomes. The highest viability corresponded to
A
phagosomes with the lowest Rab5a levels. The lowest viability
corresponded to S+ phagosomes with the highest Rab5a levels, as shown
by the viability values ranging from 29 × 104 CFU in
A
cells to 16 × 104 CFU in A+ cells, 9 × 104 CFU in S
cells, and 2.7 × 104 CFU
in S+ cells (Fig. 2C, filled
bars).
. As shown in Fig.
3, synthesis of Lamp-1 or Limp-II was not
affected by any of the treatments. IFN-
increased the synthesis of
cathepsin-D, as previously reported (21), independently of Rab5a
antisense or sense treatments. These results confirmed that Rab5a
regulated the transport of lysosomal proteins to phagosomes induced by
IFN-
but not the synthesis of lysosomal proteins.
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Fig. 3.
Rab5a antisense/sense treatment did not
affect lysosomal protein synthesis. J774 cells were treated with
Rab5a antisense oligonucleotide (A) or with Rab5a sense
oligonucleotide (S) for 6 h. Cells were then pulsed (+)
or not ( ) with 100 units/ml IFN-
for 16 h. Cells were
metabolically labeled for 2 h, and then cell lysates were
immunoprecipitated with 1G11 (anti-Lamp-1), rabbit anti-Limp-II, or
rabbit anti-cathepsin-D antibody.
-promoted phagosomal
maturation depends on Rab5a that more importantly mediated the
listericidal effect of IFN-
on phagosomes, leading to a significant decrease in L. monocytogenes viability.
-promoted Interactions of L. monocytogenes Phagosomes with Lysosomes--
Until now, transport from
late endosomes/lysosomes to phagosomes has been suggested to be
Rab5a-mediated in resting phagosomes (25). However, no report has
implicated Rab5a in these transport events from activated MØs;
nor has the effect of IFN-
in stimulating this process been
shown. To analyze the role of Rab5a and IFN-
in this event,
we studied the transfer of HRP from preloaded early endosomes (100 µg/ml, HRP uptake for 5 min) or lysosomes (100 µg/ml, HRP uptake
for 5 min and 2 h chase) into L. monocytogenes phagosomes. L. monocytogenes phagosomes were isolated from
cells treated with antisense/sense oligonucleotides and pulsed with IFN-
as above. The results shown in Fig.
4 indicated that the Rab5a antisense
treatment significantly blocked the transfer of HRP from early
endosomes to phagosomes. However, under these conditions, IFN-
treatment did not promote the fusion between early endosomes and
L. monocytogenes phagosomes (Fig. 4, white
bars). In contrast, IFN-
clearly promoted the transfer of
lysosomal HRP to phagosomes as shown by the increase in HRP values that
ranged from 1.8 in S
cells to 4.9 in S+ cells. The most striking
finding was that Rab5a antisense treatment strongly blocked the ability
of IFN-
to enhance the transfer of lysosomal HRP to L. monocytogenes phagosomes, as shown by the decrease in HRP values
from 4.9 in S+ cells to 2.5 in A+ cells (Fig. 4, filled
bars).
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Fig. 4.
Rab5a regulates the IFN-
promoted interactions of L. monocytogenes
phagosomes with lysosomes. For phagosome-endosome fusion
experiments (P-E fusion), cells were treated with Rab5a antisense/sense
oligonucleotides and IFN-
as above and then offered HRP (100 µg/ml, 5 min) and infected with L. monocytogenes for 15 min. For phagosome-lysosome fusion experiments (P-L fusion), cells
treated as above were offered HRP (100 µg/ml, 5 min, washed and
chased for 2 h). Cells were infected with L. monocytogenes for 15 min, and phagosomes were isolated,
solubilized, and HRP reaction-evaluated with o-dianisidine
as a substrate. Results are expressed as the ratio of absorbance value
per mg of protein. Results are the mean ± S.D. of
triplicates.
promoted the delivery of lysosomal proteins to L. monocytogenes phagosomes via a process that was clearly dependent
on Rab5a. These results link Rab5a action to the clearance of L. monocytogenes by phagocytes. Moreover, the simplest model to
explain the IFN-
effect on L. monocytogenes phagosome
maturation is that it promotes the Rab5a-mediated fusion of phagosomes
with late endosomes/lysosomes. These data were in accordance with those
reported with the yeast homologue, Ypt51p, or the allelic form,
Vps21p, on late transport events (26-28) as well as in fusion events
of latex bead phagosomes with lysosomes from resting MØs (25).
Signaling Pathway--
The results presented above clearly indicate
that Rab5a regulated the IFN-
-induced listericidal abilities of MØs
at the phagosomal stage. To date, the listericidal mechanisms induced
by different signals including IFN-
rely on the production of
ROI and RNI toxic molecules (5, 11, 29, 30). Production of ROI radicals requires translocation of Rac2 to the membranes to assemble an active
phagocyte NADPH oxidase complex (31), a process triggered by IFN-
.
Only active Rac2 (GTP form) is known to be required to activate the
phagocyte NADPH oxidase to produce ROI (32). We next analyzed whether
Rab5a was involved in the activation of Rac2 and production of ROI
radicals regulated by IFN-
. First, we observed that IFN-
promoted
the translocation of Rac2 to the phagosomes, and this was almost
completely abolished by treatment of MØs with Rab5a antisense (Fig.
5A). These results strongly suggested that in the pathway of IFN-
signaling, Rab5a action acted
upstream of rac2. For ROI production, the NADPH oxidase enzyme needs to
bind and activate onto the phagosomal membranes (31). NADPH oxidase
activation correlates with the recruitment of the active form of Rac2
to the membranes (32). Recently, a protocol has been described to
quantify activated Rac2 in whole cells using the binding domain of the
p21-activated kinase 1 that exclusively binds Rac2-GTP (Fig.
5B, GST-PBD lanes) (15). Using this protocol, we
observed that the amount of activated Rac2 in Rab5a antisense-treated
cells was significantly lower than in sense-treated cells, both in the
absence and presence of IFN-
(Fig. 5B, GST-PBD-IP
lanes), while the total Rac2 levels remained constant (Fig.
5B, +GTP
s lanes and
Rac2 lanes). The same results were observed when
the study was repeated in isolated L. monocytogenes phagosomes after Rab5a antisense/sense and IFN-
treatment (data not
shown). This observation suggests that measurement of Rac2 activation
on whole cell extracts was a valid indicator of the translocation of
GTP-active Rac2 to the phagosomes. More interestingly, these findings
indicate that Rab5a mediates the IFN-
-induced Rac2 activation and
translocation to the phagosomes. With respect to this, it is not
inconceivable that Rab5a-Rac2 may act in conjunction. In fact, Rab5a
and the Rac family have been reported to act together in coordinating
the process of (re)assembly of stress fibers and focal adhesions (33)
as well as in coordinating EGF receptor signaling and trafficking
(34).
View larger version (18K):
[in a new window]
Fig. 5.
Rab5a acts upstream of Rac2 and ROI
production in the IFN- signaling pathway.
A, J774 cells were treated with Rab5a antisense/sense and
IFN-
as above. Isolated phagosomes were solubilized, and Western
blots were developed with rabbit R786/9 anti-Rac2 antibody.
B, J774 cells treated as above and assayed for Rac2
activation (GST-PBD lanes) (lanes labeled as
GTP
S). Controls (lanes labeled as
+GTP
S) corresponded to total Rac2 protein able
to be activated. Whole cell Rac2 levels are shown in lanes labeled as
rac2. C, proteose peptone-elicited peritoneal
MØs (or J774 cells) were treated with Rab5a antisense/sense
oligonucleotides as above and assayed for O
/well for 60 min at 37 °C. Absorbance
(A550) of each value was subtracted from
superoxide dismutase values, and results are expressed as nmol
of O
-induced production of ROI radicals by a functional
phagocyte NADPH oxidase. For this purpose, we used the same Rab5a
antisense/sense strategy in the presence and absence of an IFN-
pulse as above. For ROI production, we used peritoneally
elicited MØs due to their higher ROI production levels. Nonetheless,
experiments performed in the J774 MØ cell line showed
similar results (an average of five different assays were performed
(data not shown). As shown in Fig. 5C, which shows one
representative experiment (out of seven), production of ROI correlated
perfectly with Rac2 activation and was inhibited by Rab5a antisense
treatment. A 1.4-fold inhibition was observed in A
cells compared
with S
cells. As expected, IFN-
treatment induced ROI production
in control cells, but interestingly, this ROI induction was 1.6-fold
inhibited by the Rab5a antisense treatment. The effect of Rab5a
antisense treatment on ROI production correlated well with the effect
shown on Rac2 activation (Fig. 5B) and translocation to the
phagosomes (Fig. 5A). The data show that Rab5a regulates the
IFN-
-mediated Rac2 activation, both by enhancing its translocation from the cytosol to the phagosomes and by locking Rac2 in its active
GTP conformation.
View larger version (16K):
[in a new window]
Fig. 6.
Rab5a-GTP form promoted the interactions of
L. monocytogenes phagosomes with lysosomes and the
recruitment of active Rac2-GTP. J774 cells were transiently
transfected as described under "Experimental Procedures" with
Rab5a:Q79L, Rab5a:S34N, and Rab5c:Q80L subcloned into pcDNA3 vector
or with vector alone for 24 h. A, cells were
metabolically labeled for 2 h, and then lysates were
immunoprecipitated with 4F11 (anti-Rab5a) or rabbit anti-Rab5c
antibody, respectively. Controls corresponded to Rab5a levels. Rab5c
levels were similar to Rab5a levels in control cells (data not shown).
B, phagosome-lysosome fusion was performed after offering
HRP to the cells (100 µg/ml, 5 min, washed and chased for 2 h).
Cells were infected with L. monocytogenes for 30 min, and
phagosomes were isolated, solubilized, and HRP reaction-evaluated with
o-dianisidine as a substrate. HRP was also analyzed in
postnuclear supernatants to give total values. Results are expressed as
the percentage of total absorbance values per mg of protein. Results
are the mean ± S.D. of triplicates. C, phagosomes from
transfected cells as in A were solubilized and assayed for
Rac2 activation and translocation (GST-PBD/IP lanes). After
immunoprecipitation, Westerns blots were developed with rabbit R786/9
anti-Rac2 antibody (whole cell extracts from these cells showed similar
results; data not shown).
action. The novelty of this
Rab5a-IFN-
-mediated function resides in the regulation by this
GTPase of two sequential processes in the phagosomes. First, Rab5a-GTP
promotes phagosomal maturation by regulating the transport of lysosomal
proteins to the phagosomes. Second, it regulates Rac2 activation and
the assembly of the phagocyte NADPH oxidase to produce toxic free
radicals. The combined effects of these Rab5a actions are a more
effective destruction of pathogens. Finally, these Rab5a novel
functions acquired by the IFN-
treatment acted together with another
IFN-
-mediated feature on L. monocytogenes phagosome
(i.e. the blockage of the action of the two membrane lytic
L. monocytogenes proteins, listeriolysin and
PI-PLC).
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ACKNOWLEDGEMENTS |
---|
We are indebted to P. D. Stahl, A. Wandinger-Ness, and M. Zerial for generous gifts of reagents. We especially acknowledge the critical review and suggestions of G. Griffiths, J. P. Gorvel, and G. Li and the encouragement, research facilities, financial support and critical reading of the manuscript by I. V. Sandoval.
![]() |
FOOTNOTES |
---|
* This was supported in part by Spanish DGCICYT Grant PB94-0035, INCO-DEV program of the European Union Grant ICA4-CT-10001, and National Institutes of Health Grant GM44428.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a contract from the MEC-Universidad Autónoma de Madrid program to reincorporate doctors.
** Supported by a contract from the MEC-Consejo Superior de Investigaciones Científicas program to reincorporate doctors. To whom correspondence should be addressed: Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Tel.: 34-91-3978455; Fax: 34-91-3974799; E-mail: calvarez@cbm.uam.es.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M101639200
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ABBREVIATIONS |
---|
The abbreviations used are:
IFN-, interferon-
;
CFU, colony-forming units;
GST, glutathione
S-transferase;
HBE, homogenization buffer;
HBSS-g, Hank's balanced salt solution containing 10 mM glucose;
RNI, reactive nitrogen intermediates;
ROI, reactive oxygen
intermediates;
MØ, macrophage;
HRP, horseradish peroxidase;
PBD, p21-activated kinase-derived binding domain;
GTP
S, guanosine
5'-3-O-(thio) triphosphate or guanosine
5'-O-(3-thiotriphosphate);
PI-PLC, phosphatidyl inositol
phospholipase C.
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