From the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Previous studies have shown that Listeria monocytogenes (LM) modulates phagocytic membrane traffic. Here we explore whether Rab5a, a GTPase associated with phagosome-endosome fusion, is related to phagosome maturation and to the intracellular survival of LM. Stable transfection of Rab5a cDNA into macrophages accelerates intracellular degradation of LM. Morphological studies confirmed that phagosome maturation and phagosome-lysosome fusion is enhanced by overexpression of Rab5a.
Down-regulation experiments using antisense oligonucleotides targeted
to the Rab5a mRNA efficiently reduced Rab5a synthesis, reduced
phagosome-endosome traffic, blocked phagosome-lysosome fusion, and
extended intraphagosomal survival of LM. Down-regulation of Rab5a had
no effect on LM internalization. Down-regulation of Rab5c had no effect
on phagosome maturation and phagosome-lysosome fusion. The results
indicate that Rab5a controls early phagosome-endosome interactions and
governs the maturation of the early phagosome leading to
phagosome-lysosome fusion.
Phagocytosis is a complex process required for host defense and
tissue remodeling. The uptake of pathogens and the activation of
membrane trafficking and other events that lead to killing and disposal
is key to an efficient host defense strategy. Listeria monocytogenes (LM),1 a
human pathogen that infects a variety of cell types including macrophages (MØ), has served as an excellent model for examining membrane trafficking events involved in intracellular killing (1). MØ,
unlike other host cells, readily clear intracellular infections (2, 3).
Mounting a successful defense against internalized pathogens appears to
require maturation of the phagosome leading to phagosome-lysosome
fusion and the discharge of lysosomal contents into the phagosome. Thus
inhibition of phagosome-lysosome fusion represents a common strategy
for sustained intracellular growth. Variations of this strategy can be
found in Mycobacterium tuberculosis, Listeria
monocytogenes, and Salmonella typhimurium among others.
Virulent strains of LM have been shown to access the cytoplasm where
bacterial growth flourishes. However, using a nonhemolytic mutant of LM
(LMhly Rab5, the rate-limiting GTPase for endocytosis (5-7), is expressed as
three different isoforms (a, b, c) that appear to have overlapping
intracellular distributions (8). Rab5 isoforms in the pathogen
Trypanosoma brucei appear to have different localization and
functions (9). Although Rab5a has been localized on phagosomes containing different particles (10-13), its role in mediating
phagosome maturation has not been extensively investigated.
Our results indicate that Rab5a and Rab5c play different roles in the
phagocytic pathway.
Cells and Reagents--
HMØ from normal donors were cultured
and detached as described (14). J774 cells were cultured in RPMI 1640 medium, 5% fetal calf serum, 2 mM glutamine, 50 µg/ml
gentamicin. The nonhemolytic LM mutant strain (DP-L2161)
(LMhly Immunoprecipitations and PT-antisense Oligonucleotide
Treatment--
Immunoprecipitation was carried out as described (18).
PT-oligonucleotides (10 µg) were incubated with 10 µg/ml of
Lipofectin in 200 µl of Opti-MEM for 15 min at room temperature
before being added to Opti-MEM-treated HMØ for 4 h at 37° C.
For electroporation, HMØ (5 × 106/ml) were incubated
with 10 µg of PT-oligonucleotides on ice for 10 min. Electroporation
was performed with a Baxter BTX-600 electroporator using 2-mm gap
cuvette chambers with the following settings: 220 V, 800 microfarads,
72 Overexpression of Rab5a:wt in J774 Cells--
Rab5a:wt in
cDNA was subcloned into pcDNA3 using
EcoRI/BamHI sites. Cells (5 × 106) were transfected with (20 µg) Rab5a:wt/pcDNA3 or
with pcDNA3 vector (control cells) by electroporation (150 V, 800 microfarads, 129 LM Phagocytosis--
LM infection was performed according to
standard protocols (4, 11) at a 10:1 bacteria/cell ratio. Cells were
incubated at 37° C for 15 min to allow for bacterial uptake followed
by a 45-min incubation in medium containing gentamicin (5 µg/ml) to
kill extracellular bacteria. For kinetic experiments, this time period
was used as the zero time point. For other time points, infected cells
were incubated at 37° C in complete medium containing gentamicin (5 µg/ml) for the indicated time and then washed with phosphate-buffered
saline. Cells were fixed in 2.5% glutaraldehyde and processed for
electron microscopy. For kinetic studies, cells were lysed and plated
onto brain heart infusion agar plates (37° C, 24 h). The number
of live bacteria was estimated by counting cfu (colony forming units).
For phagosome-endosome or phagosome-lysosome fusion assays, infections
were allowed for only 10 min (1 h, 60° C (11)). Phagosomes from J774
cells were isolated as described with minor modifications (4, 11,
19).
LMhly Previous studies have shown that LMhly
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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), which is retained within the phagosome, we have
made several observations that are pertinent to intracellular survival
of the pathogen including delayed phagosome maturation and fusion with lysosomes. The choice of the LMhly
mutant was fortuitous,
since it allowed us to unmask bacterial targets, which modulate
intracellular trafficking, that would not have been possible using the
virulent organism (4).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
) derived from the wild type strain (10403S) (15),
was kindly provided by D. A. Portnoy (University of California).
The following antibodies were used: mouse monoclonal anti-Rab5a (4F11)
(1:300 dilution) (4, 8, 11, 16); polyclonal rabbit anti-Rab5c (1:50
dilution) was developed by immunizing rabbits with rat serum albumin
conjugated to the N terminus of a C-terminal peptide of Rab5c
(CAPSRNRGVDLQENSPASRSA). Phosphothioate hRab5a-antisense (5'-TGC GCC
TCG ACT AGC CAT GT-3') and sense (5'-ACA TGG CTA GTC GAG GCG CA-3')
oligonucleotides; Rab5c-antisense (5'-GC CTC CCC GAC CCG CCA TTG-3')
and sense (5'-CA ATG GCG GGT CGG GGA GGC-3') (PT-oligonucleotides)
(20-mer) purchased from Biosystems (Palo Alto, CA), included a pair of
bases before the ATG to maximize hybridization and specificity (17,
18). Phosphodiester oligonucleotides were also designed for polymerase
chain reaction purposes and used with Rab5a- and Rab5c-specific vectors
to confirm the specificity of the oligonucleotides. Lipofectin and
Opti-MEM medium were obtained from Life Technologies, Inc.
. Cells were placed on ice immediately, and complete medium was
added. Cells were set onto culture plates for 2 h at 37° C and
extensively washed before use.
) and selected with G418 (0.8 mg/ml). Cells
containing Rab5a:wt/pcDNA3 were cloned twice by limiting dilution
before using.
Uptake and Catabolism
Assays--
Phagocytosis and catabolism assays were performed as
described (4). Bacteria were labeled with Tran35S-label.
Dead (1 h, 60° C (11)) and live LM (3 × 105
cpm/well) were added to 2 × 106 HMØ pretreated with
PT-oligonucleotides as described above. After 20 min of
internalization, cells were washed and lysed to quantify LM uptake. To
measure LM catabolism, cells were incubated for 1.6 h before
lysis. Cells were solubilized in 1% Triton X-100 and proteins were
precipitated with 10% trichloroacetic acid.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
interferes
with phagocytic trafficking and phagosome maturation (4, 11). Here, we analyze the role of Rab5a in phagosome maturation. To demonstrate that
Rab5a, or a Rab5a regulatory factor plays a role in phagosome maturation, we stably expressed Rab5a in the mouse MØ cell line, J774,
and investigated whether elevated levels of functional Rab5a could
override the inhibitory effect of the bacterium. Stable transfection of
J774 with Rab5a cDNA cloned into a pcDNA3 vector (Fig.
1A) increased Rab5a levels by
approximately 5-10-fold. Intracellular killing of internalized
LMhly
in Rab5a-transfected cells was clearly higher than
in cells transfected with the vector alone or in nontransfected cells
(Fig. 1A). To confirm that overexpression of Rab5a overrides
the inhibition of phagosome maturation caused by LMhly
,
we recorded the interactions of phagosomes containing live
LMhly
with the endosomal and lysosomal compartments (Fig.
1B). Whereas LMhly
-containing phagosomes in
control cells interacted extensively with the endosomal compartment and
minimally with lysosomes, in Rab5a-overexpressing cells, phagosome
access to both endosomal and lysosomal markers was enhanced.
Phagosome-lysosome fusion was particularly enhanced in
Rab5a-transfected cells (from 5% in control cells to 45% in cells
overexpressing Rab5a). Phagosome maturation was also confirmed by the
acquisition of typical lysosomal proteins such as Lamp-1 and the mature
form of cathepsin-D (Ref. 4 and data not shown). Rab5a appears to
operate principally at the phagosomal compartment rather than in
phagosome formation, since overexpression of Rab5a caused only a
moderate increase in LMhly
uptake (1.2-1.5-fold) (Fig.
1C).
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Fig. 1.
Overexpression of Rab5a accelerates phagosome
maturation and enhances intracellular killing of LM. A,
Rab5a overexpression in J774 cells was confirmed by immunoprecipitation
with 4F11 antibody (A, upper panel). Infection
with LMhly was performed as described previously (4). The
ratio of live bacteria (i.e. cfu) recovered at time 0 divided by the cfu recovered at 8 h was used as an index of
intracellular killing. Cfu recovered at time 0 for control and for
Rab5a-transfected cells were 2.5 × 106 ± 267 cfu and
9 × 105 ± 189 cfu, respectively. At 8 h, cfu
recovered from control and from Rab5a-transfected cells was 1 × 106 ± 122 cfu and 2.7 × 104 ± 64 cfu,
respectively. Results are the mean ± S.D. of four different
experiments. B, J774 cells transfected with Rab5a or with
pcDNA3 vector alone (control cells) were incubated with BSA-gold
(10-nm particles, 1 mg/ml) for 10 min and chased overnight to label
lysosomes. Another set of cells was incubated the following day with
BSA-gold for 10 min to label endosomes. All cells were then infected
with LMhly
for 10 min. Total gold particles per cell were
quantified in each case from a total of 200 cells, as well as the
number of gold particles found in phagosomes. Results are expressed as
the percentage of gold particles found in phagosomes compared with
total gold. Results are representative of at least three different
experiments. C, cells were infected with radiolabeled live
LMhly
(200,000 cpm/well) as reported previously (4),
incubated at 37° C for 20 min, washed, and solubilized with 1%
Triton X-100. Proteins were precipitated from cell lysates with 10%
trichloroacetic acid on ice. Results correspond to bacteria uptake
(counts/min) after a 20-min incubation. Results are the mean of
triplicates ± S.D. of four different experiments.
The dramatic effect of Rab5a overexpression on phagosome maturation is
also reflected in the morphological characteristics of the
LMhly phagosomal compartment. LMhly
phagosomes in control cells appear as swollen, "endosomal-like" compartments (see Fig. 2a). In
Rab5a-transfected cells, LMhly
phagosomes display tightly
apposed limiting membranes with dense material and membrane inclusions,
typical of multivesicular and lysosomal compartments (BSA-gold-marked
lysosomes are shown in Fig. 2, b and c, and
BSA-gold marked endosomes are shown in Fig. 2d).
Interestingly, this compartment resembled phagolysosomes containing
dead bacteria (4). All of these findings point to a linkage between
phagosome maturation and LMhly
killing in MØ and to
Rab5a as an essential regulator of both processes.
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Conclusions based on overexpression of proteins alone may not rule out
compensatory expression of other factors that could account for the
dramatic effects observed above. To corroborate our findings with Rab5a
overexpression, we carried out experiments to determine whether
down-regulation of Rab5a would alter the killing capabilities of the
MØ and allow for LMhly survival.
Antisense phosphothioate oligonucleotides (PT-oligonucleotides) were
chosen due to their longer intracellular half-lives (17, 18, 20).
Transfection with PT-oligonucleotides was carried out by two methods
with similar results (Lipofectin treatment or electroporation) (21).
Transfection with antisense PT-oligonucleotides directed to the AUG
translation initiation codon of the mRNA sequence of Rab5a and
Rab5c was carried out on HMØ, because the sequences of human Rab5
isoforms (Rab5a and Rab5c) are known, whereas the mouse sequences are
not available. The intracellular degradation of LMhly in
HMØ resembles that found in the mouse MØ cell line, J774 (22-24). Several PT-oligonucleotides, designed to hybridize with specific Rab5a
mRNA sequences (e.g. effector domain, 3'-untranslated
regions, or translation initiation codon), were examined. However, only those oligonucleotides hybridizing with the translation initiation codon of Rab5a mRNA blocked Rab5a synthesis (approximately
85-90%, as determined by immunoprecipitation (Fig.
3A)). Rab5c, which shares more
than 80% identity with Rab5a (8, 25), was unaltered by Rab5a-antisense
treatment indicating that the PT-oligonucleotide effects were highly
specific. Rab5a-antisense-treated HMØ permitted intracellular growth
of LMhly
mutant, while in sense or control cells, the
bacteria were destroyed (Fig. 3B) (biosynthetic levels of
Rab5a are shown as an inset in Fig. 3B).
LMhly
growth occurred inside the phagosomes, since this
mutant lacks the protein necessary for lysis of the phagosomal
membranes (as detected by electron microscopy, data not shown). The
growth of LMhly
in Rab5a-antisense-treated cells was
heterogeneous (Table I). Approximately
35% of the cells were free of bacteria after a 2-h pulse. The
remaining 65% of the cells contained 1-2 bacteria per cell. Following
a 4-h incubation, 20% of the infected cells contained 1-2 bacteria
per cell, whereas nearly half of the cells contained 3-5 bacterial
profiles per cell. At 24 h, 15% of the infected cells contained
3-5 bacteria per cell, and 37% of the cells contained >5 bacteria
per cell, reflective of significant bacterial growth.
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It has been reported that the Rab5 isoforms (a, b, and c) share similar functions and colocalize to the same compartment (8,25-27). However, recent results demonstrating differential expression of Rab5a following lymphokine signaling in MØ suggests specialized functions for the Rab5 isoforms (19).
Rab5c-antisense treatment was efficient and selective in blocking Rab5c
synthesis (>88% inhibition, see Fig. 3A), since Rab5a synthesis remained unaltered. Analysis of LMhly infection
in Rab5c-antisense-treated cells revealed no differences in
LMhly
destruction (Fig. 3B). These findings
suggest that Rab5a is the predominant regulatory Rab GTPase in
the phagocytic pathway. Rab5c may play a minor role or function elsewhere.
Rab5a appears to play virtually no role in LMhly
internalization, since transport from the plasma membrane to the
phagosomes, as detected by following the internalization of
radiolabeled LMhly
in antisense-treated cells (both Rab5a
and Rab5c), was unaffected. However, Rab5a is clearly involved in the
degradation of pre-internalized dead LMhly
. The
percentage of radiolabeled bacteria remaining after a 20-min uptake and
100-min chase in Rab5a-antisense-treated cells was significantly higher
(33% higher) than in Rab5a-sense-treated cells or control cells (Fig.
3C).
Overexpression of Rab5a only marginally increased bacterial
phagocytosis. However, following phagosome formation, Rab5a appears to
play at least two roles: (i) by mediating fusion events within the
phagosomal-endosomal compartment and (ii) by facilitating or initiating
phagosome maturation culminating in phagosome-lysosome fusion. The
former is supported by in vitro reconstitution studies (11)
and by the observations presented here that the intermingling of
phagosomes and endosomes is reduced in Rab5a-antisense-treated cells.
The latter is supported by the observation that fusion of phagosomes,
containing dead LMhly bacteria, with lysosomes was
impaired in Rab5a-antisense treated cells. The finding that phagosomes
containing dead LMhly
are unable to mature in
Rab5a-antisense-treated cells and the observation that live
LMhly
phagosomes remain as immature endosomal-like
compartments point to key, perhaps common, regulatory steps that are
required to initiate phagosome maturation (28).
Phagosome maturation is a complex process, and results reported to date
suggest that the nature of the internalized particle plays a role in
modulating the rate and perhaps the quality of the process. For
example, a recent report using latex beads as model particles in J774
cells demonstrated that phagosomes, which had been internalized for
several hours, fused with early and late endosomes in vitro
in a Rab5-dependent manner (13). Indeed, earlier work (29)
demonstrated that the fusion capacity of phagosomes, containing
Staphylococcus A particles internalized via the Fc receptor, is
restricted to early endosomes in an in vitro
phagosome-endosome fusion. Thus, it is likely that both the receptor
that mediates particle internalization and the nature of the
internalized particle (e.g. digestible versus
nondigestible, live versus dead etc.) play important roles
in phagosome maturation and phagosome-lysosome fusion. Interestingly,
interferon-, a lymphokine known to accelerate intracellular killing
of pathogenic LM (LMhly+) and other intracellular pathogens
(30), specifically induces Rab5a biosynthesis and processing (18).
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ACKNOWLEDGEMENTS |
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The technical support of A. Zheleznyak, E. Peters, A. Monnaie, L. LaRose, and M. Levy is enormously appreciated. R. D. Schreiber, E. Carrasco-Marin, E. Brown, and D. Russell are acknowledged for critical advice and M. Zerial for kindly provided reagents.
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FOOTNOTES |
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* This work was supported by National Institutes of Health grants (to P. D. S.).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.
Recipient of a Postdoctoral Fellowship from the Formación de
Personal Investigador, Ministerio de Educación y Ciencia, Madrid, Spain.
§ To whom correspondence and reprint requests should be addressed. Tel.: 314-362-6950; Fax: 314-362-1490; E-mail: pstahl{at}cellbio.wustl.edu.
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ABBREVIATIONS |
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The abbreviations used are: LM, Listeria monocytogenes; PT, phosphothioate; cfu, colony forming units; BSA, bovine serum albumin.
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