1 Department of Medical Microbiology, Faculty of Health Science, Linköping
University, SE-581 85 Linköping, Sweden
2 Department of Pathology II, Faculty of Health Science, Linköping
University, SE-581 85 Linköping, Sweden
* Author for correspondence (e-mail: naspe{at}ihm.liu.se )
Accepted 27 November 2001
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Neutrophil, Mycobacterium tuberculosis, Rab5, Phagocytosis, Fusion
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fusion of these compartments with the phagosomes is controlled by various
signals and proteins, including calcium, actin, annexins and Src kinases
(Stendahl et al., 1994;
Diakonova et al., 1997
;
Korade-Mirnics and Corey,
2000
). Indeed more than 140 proteins have been found in isolated
phagosomes from macrophages (Garin et al.,
2001
). In particular, small GTP-binding proteins of the Rab family
regulate the docking and fusion of endocytic organelles. Rab5 is a member of
this family, and it is expressed in three different isoforms (designated a, b
and c) that appear to exhibit overlapping intracellular distributions
(Bucci et al., 1995
). Studies
of macrophages have shown that Rab5a regulates events in the fusion of
bacteria-containing vacuoles and early endosomes
(Bucci et al., 1995
;
Sturgill-Koszycki et al.,
1996
; Alvarez-Dominguez and
Stahl, 1999
) and that the arrest of maturation can be linked to a
block between Rab5- and Rab7- controlled steps
(Via et al., 1997
). In
neutrophils, as well as in other eukaryotic cells, this protein participates
in intracellular trafficking (Vita et al.,
1996
). At present, no information is available about the
involvement of Rab5 in phagolysosome biogenesis in neutrophils. In addition to
Rab GTPases, a second family of vesicle-targeting molecules, the SNARE
proteins, seems to convey another element of specificity to Rab-dependent
membrane fusion (Brennwald,
2000
). The differential subcellular distribution (plasma membrane
versus granules) of SNAREs together with their in vitro interaction suggests a
role for SNAREs in docking granules with the neutrophil plasma membrane during
exocytosis (Ligeti and Mócsai,
1999
). Experiments on these cells have shown that the t-SNARE,
syntaxin-4, accumulates in or near the lamellipodium, which is the region of
the cell that is involved in phagocytosis
(Brumell et al., 1995
).
Moreover, microorganisms have also developed various methods to avoid being
ingested and killed. For example, the intracellular bacteria Mycobacterium
tuberculosis can actually survive and multiply within macrophages
(Sturgill-Koszycki et al.,
1996
). Extracellular bacteria can interfere with the phagocytic
capacity of the host cell and some of them, such as Staphylococcus
aureus, produce toxins that destroy phagocytes
(Thelestam, 1983
). Despite
this, these microorganisms are usually efficiently phagocytosed by and killed
within neutrophils.
Despite the obvious importance of the functions of neutrophils and the
complexity of intracellular trafficking, the molecular mechanisms underlying
phagosome biogenesis are poorly understood. To explore these mechanisms, we
used human neutrophils and the following bacteria: a virulent and attenuated
strain of M. tuberculosis (H37Rv, and H37Ra respectively), to
represent an intracellular parasite that infects a variety of cell types
(Hernandez-Pando et al., 2000)
including neutrophils (Perskvist et al.,
2000
), and S. aureus, to represent an extracellular
pathogen. We characterized the molecular mechanisms governing fusion between
phagosomes containing M. tuberculosis (H37Rv or H37Ra) or S.
aureus and various granules and endosomal compartments. Using isolated
bacteria-laden phagosomes, we found that Rab5a is an essential molecule that
controls fusion between phagosomes containing bacteria and intracellular
compartments, thereby regulating the ability of neutrophils to restrict the
spreading of the pathogens. Whereas phagosomes containing S. aureus
display the complex of Rab5a and syntaxin-4 briefly, phagosomes containing
either strains of M. tuberculosis bind to the complex for an extended
period of time, potentially leading to delayed phagolysosome maturation.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bacterial preparations
The virulent M. tuberculosis strain H37Rv was obtained from the
Swedish Institute of Infection Laboratory (Stockholm, Sweden), and the
attenuated strain H37Ra (ATCC 25177) was obtained from the American Type
Culture Collection (Manassas, VA). Early passages of mycobacteria were
prepared and maintained in Middlebrook broth (Difco Laboratories, Detroit, MI)
supplemented with OADC (oleic acid, albumin, dextrose and NaCl; Difco).
Initial colonies of H37Rv were expanded and frozen in aliquots
(Perskvist et al., 2000).
Because continuous passage of mycobacteria in liquid culture is associated
with loss of virulence, freshly thawed aliquots were not passaged more than
once before use in the experiments. Single-cell mycobacterial suspensions were
prepared using a syringe equipped with a 27 G 3/4 0.4x19 needles. After
several passages through the needle, the bacteria were filtered in a sterile
Pasteur pipette equipped with cotton wool
(Perskvist et al., 2000
).
Microscopic examination confirmed that a significant portion (>95%) were
present as single bacteria. The S. aureus were of the
catalase-positive WOOD 46 strain that is devoid of protein A. These bacteria
were stored at -70°C and prior to experiments, were cultured at 37°C
for 18 hours in Luria broth (LB) with constant shaking (300 rpm) and then
transferred to fresh medium and cultured for another 3 hours
(Wilsson et al., 1996
).
C3b/bi-opsonized bacteria were obtained by exposure to 20% normal human serum,
as previously described (Perskvist et al.,
2000
). The viability of the bacteria was assessed at each step by
comparing bacterial counts determined by microscopy and assays of colony
forming units (CFU). All bacterial were replenished after one passage. The
integrity of the cell wall of the bacilli was confirmed by electron
microscopy.
Neutrophils preparation and phagocytosis
Neutrophils were isolated by subjecting buffy coat from healthy blood
donors to dextran sedimentation and Ficoll-Paque gradient centrifugation
(Böyum, 1968). A brief
hypotonic lysis was performed to remove contaminating red blood cells,
resulting in >90% neutrophil purity. The neutrophils were subsequently
resuspended (1x107 cells/ml) in Krebs-Ringer glucose (KRG)
buffer containing 1 mM Ca2+ and Mg2+. The cells were
routinely pretreated with 5 mM diisopropyl fluorophosphate (DFP) for 15
minutes on ice to minimize proteolysis.
To obtain phagosomes, neutrophils were allowed to ingest opsonised bacteria
for the amount of time indicated in the figure legends. The cells
(4x108 in 40 ml of KRG) were exposed to H37Rv or H37Ra (at a
ratio of one neutrophil to 20 mycobacteria) or S. aureus (one
neutrophil to 10 bacteria) at 37°C. Adding ice-cold KRG stopped
phagocytosis; thereafter the neutrophils were washed and the phagosomes were
isolated. The suitable neutrophil-to-bacteria ratio was determined by
incubating the cells for 15, 30 and 60 minutes with FITC-conjugated
(Majeed et al., 1997) and
opsonised bacteria and utilizing the trypan blue exclusion test
(Jaconi et al., 1990
). The
results showed that the percentages of neutrophils ingesting H37Rv were
23%±5, 55%±7 and 70%±9 (means±s.e.m.
n=5), and the corresponding values for S. aureus-ingesting
neutrophils were 32%±3, 60%±8 and 85%±11. These results
gave roughly equivalent quantities of bacteria ingested by neutrophils, thus
these proportions were used in subsequent experiments. It is plausible that
the difference in the nature of the phagosomes between mycobacteria and S.
aureus relates to differences in the rate and amount of phagocytosis
between these two bacterial species within the time frame of the assay. To
eliminate this probability, in some experiments, we utilised synchronised
phagocytosis. Briefly the bacteria were added to the neutrophils and incubated
for 10 minutes at 37°C for mycobacteria and 5 minutes for S.
aureus. The non-adherent bacteria were removed by centrifugation at
4°C, and internalisation of the bound bacteria was stimulated by rapidly
warming the cells to 37°C. After 0 to 60 minutes, cells were processed for
isolation of the phagosomes.
Preparation of phagosomes containing bacteria
The M. tuberculosis strains H37Rv or H37Ra and S.
aureus-containing phagosomes (respectively designated MCPv, MCPa and SCP)
were isolated using a procedure modified from two previously described methods
(Desjardins et al., 1994;
Chakraborty et al., 1994
). The
neutrophil pellets were resuspended in 2 ml of homogenisation buffer (250 mM
sucrose, and 3 mM imidazole, pH 7.4), containing 1 mM
Na3VO4 and 10 µg/ml aprotinin, leupeptin and
pepstatin. Passing the suspension through a tuberculin syringe with a 23-gauge
needle disrupted the cells. The disruption was monitored in a light microscope
and stopped before there was any major damage to the nuclei. The intact
neutrophils and nuclei were sedimented by centrifugation at 300
g for 10 minutes; the pellet was discarded, and the
postnuclear supernatant was loaded onto 12% sucrose in 3 mM imidazole (w/w)
and centrifuged at 800 g for 45 minutes. This yielded a pellet
containing bacteria-laden phagosomes, which was gently resuspended in 4 ml of
homogenisation buffer without inhibitors. This suspension was run (by gravity)
through a 47 mm, 3 µm pore Nucleopore filter followed by an additional 3 ml
volume of homogenisation buffer, and the flow-through was loaded onto 12%
sucrose and centrifuged at 800 g for 45 minutes to collect the
phagosomes in the bottom of the tubes. All procedures were performed at
4°C.
The viability of the bacteria in the phagosomes was determined by selective lysis of the phagosomal membranes using KRG containing 0.5% Triton X-100 followed by comparison of bacterial counts and CFU results. The integrity of the phagosomal membrane was confirmed by exposing the phagosomes containing FITC-labelled bacteria to trypan blue. This test showed that >90% of the FITC bacteria were not quenched by this dye. Staining of these phagosomes by LAMP-1 verified that the bacteria were retained inside phagosomes. The amount of phagosomal protein loaded for SDS-PAGE was first adjusted using colorimetric measurement of the protein concentration and DC protein reagents (Bio-Rad) and thereafter ascertained as the level of LAMP-1 expression. We found that LAMP-1 is a consistent marker that is expressed on the isolated vacuoles containing mycobacteria or S. aureus.
The level of contamination of the phagosome preparations with other
cellular components was analysed as described elsewhere
(Russell et al., 1996). In
short, we prepared four tubes of neutrophils (in each there were
1x108 cells in 10 ml of KRG), two of which were metabolically
labelled with 15 µCi/ml [35S] methionine for 30 minutes on ice.
Bacteria were added to one labelled and one unlabelled tube. After 30 minutes
of phagocytosis, the labelled neutrophils that contained bacteria were
combined with unlabelled cells without ingested bacteria, and the unlabelled
bacteria-containing neutrophils were combined with the labelled bacteria-free
cells. After the isolation procedures were completed, equal aliquots of the
two samples were measured for radioactivity. To calculate the percentage of
contamination the cpm from the bacteria in unlabelled cells was divided by a
value representing the combined cpm values from bacteria in labelled and
unlabelled cells multiplied by 100. In the protocols given, the levels of
contaminations varied between 5 and 7% for SCP and between 10 and 12% for MCP.
The purity of the MCPv, MCPa and SCP was further analysed by electron
microscopy and two-dimensional gel electrophoresis.
Transmission electron microscopy
Both intact neutrophils with ingested bacteria and isolated phagosomes were
prepared for transmission electron microscopy. Specimens were fixed by adding
2% glutaraldehyde in 100 mM sucrose-sodium cacodylate-HCl buffer (pH 7.2), and
post-fixed in 2% osmiumtetroxide and then centrifuged in 2% agar (2500
g, 2 minutes). Pieces of the resulting agar pellet were
stained on block with 2% uranyl acetate in 50% ethanol and subsequently
dehydrated and embedded in Epon-812. Ultrathin sections were cut with a
diamond knife (DIATOME, Bienne, Switzerland), stained with lead citrate and
examined and photographed in a JEOL 1200 EX electron microscope (Tokyo,
Japan).
Protein gel electrophoresis and immunoblotting
Proteins were separated according to their isoelectric point and molecular
weight using high-resolution two-dimensional gel electrophoresis based on the
method of O'Farrel (O'Farrel, 1975). For each sample in a given experiment,
the same amount of proteins was solubilised in urea sample buffer (9 M urea,
0.8% [w/v] pharmalyte (pH 3-10), 1% [w/v] DTT, 2% [w/v] CHAPS and 0.01% [w/v]
bromophenole blue). Separation in the first dimension was done with an
Immobiline DryStrip Kit (Pharmacia Biotech. Uppsala, Sweden) using a
non-linear pH gradient from 3 to 10. After focusing, the strips were
positioned over a vertical 8-18% Exel SDS-PAGE slab gel (Pharmacia Biotech.)
and subjected to electrophoresis according to the instructions of the
manufacturer. The gels were then silver stained for protein pattern
analysis.
SDS-PAGE was performed using a 12% separating gel as described by Laemmli
(Laemmli, 1970). Isolated
phagosomes were lysed for 30 minutes at 4°C with 100 µl of RIPA buffer
(pH 7.5) containing 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 9.1
mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, 150 mM NaCl
and the same protease inhibitors as in the homogenisation buffer. Equal
amounts of protein were separated by SDS-PAGE and then electrophoretically
transferred onto nitrocellulose membranes. The membranes were blocked with 5%
BSA in PBS, and the phagosomal proteins on the blots were detected with either
1 µg/ml mAbs (1:500 dilution) or 0.5 µg/ml polyclonal Abs (1:1000) in 3%
BSA in PBS for 60 minutes at room temperature (RT) followed by HRP-conjugated
anti-mouse or anti-rabbit Ab (1:3000 dilution) and then visualized with a
commercial ECL kit. To confirm that the lanes received equal amounts of
proteins, the blots were stripped and reprobed with anti-LAMP-1 Ab. The
intensity of the bands was measured by densitometric assay using a Howtek
scanner and Quantity One Software (Advanced American Biotechnology, Fullerton
CA)
Coimmunoprecipitation and GTP-binding overlay assay
MCPv, MCPa and SCP (750 µg protein each) were lysed in 100 µl of 20
mM Tris-HCl (pH 8.0) containing 1 mM EDTA, 1 mM DTT, 5 mM Mg Cl2
and 0.6% CHAPS and then incubated for 30 minutes at 4°C and subsequently
centrifuged at 35,000 g for 60 minutes
(Mukherjee et al., 2000). The
supernatants were precleared once with protein-A-agarose and then incubated at
4°C with 2 µg of anti-Rab5a Ab for 60 minutes and thereafter with 30
µl of 50% (v/v) protein A-agarose for an additional 60 minutes. The
concentration of the protein eluted from beads was measured by colorimetric
assay as above and the same amounts of proteins were separated by 12% SDS-PAGE
and transferred to a nitrocellulose membrane. Essentially the same procedure
was used to precipitate syntaxin-4, except that the phagosome fractions were
lysed in RIPA buffer without inhibitors and centrifuged at 15,000
g for 15 minutes. After lyses, the protein was
immunoprecipitated with 2.5 µg of anti-syntaxin-4 mAb. An overlay assay
utilizing 1 µCi/ml of [
-32P] GTP (3.000 Ci/mM, DuPont
NEN) in 50 mM phosphate buffer (pH 7.5) containing 5 mM MgCl2, 1 mM
EGTA and 0.3% Tween 20 and 1 µM ATP as competing substrate
(Deretic et al., 1995
) was
used to determine the GTP-binding state of the Rab5a, which was then
visualized by autoradiography and GTP binding was quantified in a
PhosphorImager. The same membranes were washed thoroughly with 0.5% triton
X-100 in PBS and then stripped and immunoblotted with Abs as specified in the
figure legends. Duplicate gels were silver stained to establish whether other
proteins coimmunoprecipitated with anti-Rab5a Ab. To confirm the molecular
mass of GTP-bound Rab5a, the same membrane was washed and then immunoblotted
with anti-Rab5a Ab. The specificity of the [
-32P]
GTP-binding was examined by addition of 10 µM GTP
s, a
nonhydrolysable analogue of GTP, to the various phagosome fractions
Treatment of neutrophils with antisense oligonucleotides
We used the antisense oligonucleotide sequences Rab5a-antisense (AS),
5'-TGC GCC TCG ACT AGC CAT GT-3' and Rab5a-sense (S), 5'-ACA
TGG CTA GTC GAG GCG CA-3' (20 mer), which were chosen in view of results
published by Alvarez-Dominguez and Stahl
(Alvarez-Dominguez and Stahl,
1999) and were purchased from Life Technology, Inc. A pair of
bases was included before the ATG to maximize hybridisation and specificity.
In all oligonucleotides, the internucleoside linkages were completely
phosphorothioate-modified to activate RNAse H. 5' and 3' terminal
bases were also modified to resist nuclease attack. Equal enhancement of
delivery of the oligonucleotides was achieved with the cationic lipid DMRIE-C
(1,2-dimyristoyloxypropyl-3-dimethyl-hyroxyethyl ammonium bromide/cholesterol,
1:1, M/M) (Life Technology, Inc.). We chose DMRIE-C because it lacks
dioctanoyl phosphatidylethanolamine, which can be toxic to phagocytic cells
(Korchak et al., 1998
).
Neutrophils (7x107 cells) were preincubated in 10 ml of
Opti-MEM I-reduced serum medium (Life Technology, Inc) in 160 ml tissue
culture flasks for 30 minutes at 37°C in a humidified CO2
incubator. Rab5a-AS or -S oligonucleotide (120 µg) was suspended in
Opti-MEM and incubated with 360 µl of DMRIE-C at room temperature for 30
minutes and thereafter added to neutrophils for 4 hours. Following incubation,
the cells were exposed to the opsonised H37Rv or S. aureus for 30
minutes at the same ratio as previously mentioned, and the induction and
isolation of phagosomes were carried out as described above. This treatment
did not affect the viability of the cells, as assessed by the trypan blue
test.
To determine whether downregulation of Rab5a affected the phagocytic uptake of bacteria, neutrophils were treated with Rab5a-antisense or -sense oligonucleotide as described above. The cells (106) were allowed to ingest FITC-conjugated and opsonised bacteria at a ratio of one neutrophil to 20 H37Rv or to 10 S. aureus for 15, 30 and 60 minutes. The percentage of ingesting neutrophils was determined by the trypan blue exclusion test.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
MCPv, MCPa and SCP acquire different endosome and granule markers and
cytosolic proteins
We used the following proteins to investigate the interaction between
different neutrophil granules and newly formed phagosomes: hck, a src-kinase
associated with the membrane of azurophil granules
(N'Diaye et al., 1998);
lactoferrin, which is found in secondary granules; and LAMP-1, a marker of
late endosomal compartments. (Cieutat et
al., 1998
; Bainton et al., 1999). In initial experiments,
neutrophils were allowed to phagocytose H37Rv or H37Ra or S. aureus
for 60 minutes prior to isolation of phagosomes. The same amount of protein
found associated with the phagosomes was subjected to SDS-PAGE and subsequent
immunoblot analysis. Other investigators
(N'Diaye et al., 1998
) have
used immuonoelectron microscopy to study neutrophils and observed that
phagosomes containing mycobacteria fail to acquire myeloperoxidase (MPO) and
hck. Our results showed that the amount of hck protein associated with the
membrane of MCPv and MCPa was markedly reduced compared with that of the SCP,
indicating a limitation of fusion between MCP and azurophil granules
(Fig. 3). Azurophil granules
contain numerous lysosomal enzymes and bactericidal factors
(Bainton, 1999
). Thus the
fusion incompetence of azurophil granules indicates a mechanism by which
mycobacteria avoid the toxicity of these factors. Moreover, both lactoferrin
and LAMP-1 were associated with the membranes of MCPv, MCPa and SCP,
suggesting that the vacuoles remained accessible to the specific granules and
late endosomal organelles (Fig.
3).
|
The differences we observed between the MCP and SCP regarding fusion with
azurophil granules prompted us to examine which cytosolic proteins are
involved in phagolysosome fusion. We analysed the ability of isolated
phagosomes to bind to annexins, proteins that are implicated in fusion of
intracellular membranes (Diakonova et al.,
1997). Western blotting of isolated phagosomes showed that annexin
I, III and V were present in SCP, whereas only annexins I and III were
detected in lysates of MCP preparation
(Fig. 3). Whole neutrophil
lysates also contained all three kinds of the annexins (data not shown). The
anti-annexin I Ab reproducibly recognized two distinct bands in western blots,
which reflected post-translation modification by proteases
(Movitz et al., 1999
). The
level of expression of LAMP-1 was also used as an internal control of loading
of the phagosomal proteins.
Recruitment of Rab5a and syntaxin-4 to MCPv, MCPa and SCP
Rab GTPases are known to be key regulators of vesicular transport in
mammalian cells. There is evidence that Rab5a is present in the cytosol of
human neutrophils and that upon cell activation with PMA Rab5a translocates to
the membrane (Vita et al.,
1996). Roberts et al. (Roberts
et al., 2000
) found that Rab5a associated transiently with newly
formed phagosomes in macrophages. Thus we studied the kinetics of interaction
of Rab5a with the MCPv, MCPa or SCP to determine whether this protein is
involved in the maturation of these phagosomes. The results showed that MCPv
and MCPa acquired Rab5a within 15 minutes and the amount of the protein
increased slightly to a level that was maintained up to 60 minutes, whereas
SCP recruited Rab5a within 5 minutes but the protein was selectively depleted
after 30 minutes (Fig. 4). Densitometric quantification of Rab5a at 15, 30 and 60 minutes indicated
245±10, 251±9 and 268±12 arbitrary units of Rab5a in the
MCPv, and at 5, 15, 30 and 60 minutes the corresponding values for SCP were
52±11, 230±10, 213±13 and 25±9 arbitrary units.
These data demonstrated that SCP transiently recruited Rab5a during fusion
with endosomes and granules, whereas MCP retained Rab5a throughout a longer
period of time.
|
As previously mentioned, proteins of the t-SNARE family ensure that
vesicles are delivered to their correct target membrane, and Rab GTPases
appear to act upstream of SNARE molecules during membrane docking/fusion
(Cao et al., 1998). Syntaxin-4
is a t-SNARE protein in neutrophils, thus we investigated the possibility that
it is also involved in fusion of endosomes and/or granules with neutrophils
phagosomes. The same kinetics of association were observed for syntaxin-4 and
Rab5a (Fig. 4). Densitometric
measurements confirmed this result. In our assay, an insufficient amount of
MCP was recovered after less than 15 minutes of phagocytosis, hence the
kinetic analyses were done from 15 to 60 minutes of exposure to mycobacteria.
The expression levels of LAMP-1 were determined to verify that the same amount
of each protein was loaded. Synchronised phagocytosis was initially performed
using FITC-conjugated and opsonized bacteria. The results showed that
75%±4 or 83%±6 (mean±s.e.m., n=3) of neutrophils
bound to mycobacteria or S. aureus, whereas the percentage of
neutrophils ingesting these bacteria was 3%±1.3 or 5%±2,
respectively. Thus these short periods of time allowed for adequate numbers of
bacteria to attach to the cell surface before the warming period, with a low
percentage of ingested neutrophils. Consequently, the synchronised
phagocytosis assay was used to verify the kinetics of Rab5a and syntaxin-4
recruitment to phagosome-containing mycobacteria or S. aureus.
Similar data were obtained compared to the unsynchronised phagocytosis
assay.
Interaction between GTP-bound Rab5a and syntaxin-4 on the membrane of
MCP and SCP
The assembly of Rab5a and syntaxin-4 on the membrane of phagosomes showed
that the fusion events in neutrophils occur through a highly controlled
mechanism. Similar to other low molecular weight GTPases, Rab5a is active in
its GTP-bound form (Barbieri et al.,
1994; Alvarez-Dominguez et al.,
1996
); thus, we investigated whether Rab5a in its GTP-bound form
can interact with syntaxin-4 on the phagosomal membrane of the bacteria during
the fusion process. We extended the phagocytic time of the mycobacteria to
examine the later time points of association of Rab5a and syntaxin-4 on the
phagosomes and allowed neutrophils to ingest H37Rv or H37Ra for 120 minutes or
S. aureus for 60 minutes. Lysates of the phagosomes were precipitated
with anti-Rab5a Ab, subjected to SDS-PAGE and transferred to a nitrocellulose
membrane. The GTP-binding state of Rab5a was detected using
[
-32P] GTP, and the same membrane was then blotted to detect
possible coprecipitation of syntaxin-4 with Rab5a. We found a sustained
association between GTP-bound Rab5a and syntaxin-4 on the MCPv
(Fig. 5A) and MCPa
(Fig. 5B) up to 90 minutes
after phagocytosis, and then it gradually declined. SCP retained the
Rab5a-synatxin-4 complex for up to 30 minutes; thereafter the SCP was
completely depleted from the complex (Fig.
5C). The kinetics of association of Rab5a-GTP and syntaxin-4
revealed that although these proteins are absent from SCP, functional
Rab5a-GTP and syntaxin-4 persist on MCPv
(Fig. 5D). Next we confirmed
that Rab5a in its GTP-bound state coimmunoprecipitated with anti-syntaxin-4 in
the phagosomal fractions obtained after 30 minutes of phagocytosis
(Fig. 5E). There was no such
interaction in lysates of non-ingesting neutrophils at 4°C
(Fig. 5A,B,C), thus confirming
that the association of syntaxin-4 with GTP-bound Rab5a occurred in
neutrophils activated by phagocytosis. The results from silver staining showed
that Rab5a also coprecipitated with other regulatory proteins than syntaxin-4.
The nature of these proteins remains to be determined.
|
Downregulation of Rab5a reduced the capacity of MCP and SCP to fuse
with intracellular organelles
To elucidate the functional mechanism(s) of the interaction between Rab5a
and syntaxin-4 on the phagosomal membrane, we examined the effects of
downregulation of Rab5a on the properties of fusion between pathogen-laden
phagosomes and intracellular endosomes and granules. Initial experiments
demonstrated that treatment of the neutrophils with antisense Rab5a markedly
decreased the expression of Rab5a (Fig.
6A). Actin was used to confirm that equal amounts of the proteins
were loaded. Expression of both Rab5a and syntaxin-4 declined on the isolated
phagosomes (Fig. 6B). In light
of the reduction of Rab5a and syntaxin-4, we considered the possibility that
intracellular membrane trafficking had been altered. We thus evaluated the
translocation of the endosomes and granule markers in antisense-treated
neutrophils. We found that downregulation of Rab5a impaired the ability of the
MCPv and SCP to incorporate hck, lactoferrin and LAMP-1 into their membranes
(Fig. 6B). Finally,
downregulation of Rab5a had no effect on the phagocytic capacity of
neutrophils (Fig. 7). Together,
these findings demonstrated that Rab5a did not play a role in bacterial uptake
but was clearly involved in degranulation and intracellular trafficking in
neutrophils. To rule out the non-specific effect of the Rab5a-antisense
treatment, the whole cell lysate from treated or untreated neutrophils was
subjected to SDS-PAGE and immunoblotted with antibodies to lactoferrin, hck
and LAMP-1. The results showed that the levels of these proteins were not
affected by the antisense treatment (Fig.
6A).
|
|
MCPv and MCPa but not SCP displayed similar fusion properties
In this study we analysed the maturation of MCPv, MCPa and SCP in
neutrophils. The results clearly confirmed that MCPv and MCPa exhibited
similar fusion patterns to granules and/or endosomal organelles, which
differed from that of SCP. Accordingly, we used 2D gel electrophoresis to
compare the protein compositions of these phagosomes and found that there are
indeed differences between MCPv and MCPa. For example, the following sequences
were reproducibly detected in MCPv: 23 kDa, pI 5-5.5; 29 kDa, pI 6.3-6.5; 44
kDa pI 6.5-6.8 (for a, b, and c, respectively, in
Fig. 2B), and the following
proteins were found only in MCPa: 24 kDa, pI 6-6.5; 40 kDa, pI 5.5-5.8; 80
kDa, pI 5.3-5.6; and 98 kDa, pI 5-5.5 (d, e, f, and g, respectively, in
Fig. 2C). The different protein
composition may reflect different intraphagosomal milieus. Further studies are
needed to identify both these and other unknown proteins. However, we conclude
that, despite the differing polypeptide compositions of MCPv and MCPa, these
phagosomes exhibited similar intracellular fusion capacities to the
endosomal/granule compartments.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
According to previous studies of neutrophils performed in our laboratory,
elevation of intracellular calcium does not occur during phagocytosis of
mycobacteria (Majeed et al.,
1997), although it does occur during ingestion of S.
aureus (Wilsson et al.,
1996
). The absence of fusion of MCP with azurophil granules may in
fact be due to a transient lack of intracellular calcium, because it is known
that these granules require higher concentrations of intracellular calcium
than the others to fuse with the membranes
(Nüsse et al., 1998
). It
is also tempting to speculate that the decreased ability of MCPv and MCPa to
fuse with azurophil granules may be related to the lack of translocation of
certain calcium-regulated cytosolic proteins, for example, annexin V, to the
membrane of these phagosomes and that such a translocation does occur in SCP.
Extensive studies of vesicle trafficking along the endocytic pathway have
indicated that various annexins regulate vesicle fusion in a calcium-dependent
manner (Diakonova et al.,
1997
; Collins et al.,
1997
), hence the differential assembly of annexins in proximity to
phagosomal membranes may influence the binding capacity of various
intracellular organelles. It should be noted that our finding of annexin I on
the MCPv and MCPa are not in line with our previously published results
(Majeed et al., 1997
). A
possible explanation for this discrepancy is that the immunofluorescence
staining performed in the cited study was not sensitive enough to detect the
limited amount of annexin I present on mycobacterial phagosomes. Inasmuch as
all three types of phagosomes we studied exhibited characteristics of the late
endocytic pathway, the presence of LAMP-1 on these structures suggests that
the MCPv and MCPa bypass the normal endocytic route, implying that the
mycobacteria interfere with maturation of theirs phagosomes. By comparison, in
human macrophages LAMP-1 translocates to the mycobacteria-containing phagosome
(Zimmerli et al., 1996
), they
exclude the vacuolar ATPase in bone marrow-dived mouse macrophages and
consequently do not become acidified
(Sturgill-Koszycki et al.,
1994
). In neutrophils, maturation of bacterial vacuoles into
phagolysosomes occurs through fusion with azurophil granules, thus the
differential fusion competence of the phagosomes with azurophil granules is
related to the maturation process.
We found that, in neutrophils, Rab5a in GTP-bound form was expressed on
MCPv and MCPa for a longer period of time during phagosome-granule fusion
while targeted briefly to the membrane of the SCP. These results are in
agreement with the findings of Clemens and co-workers
(Clemens et al., 2000). Like
the M. tuberculosis strains in our experiments, the M.
tuberculosis strain Erdman used by Clemens et al.
(Clemens et al., 2000
) blocks
phagosomal maturational in macrophages through persistent expression of Rab5a
on the vacuole. The fact that Rab5a did not persist on the neutrophil
phagosome and dissociated after 120 minutes may reflect the rapid and
effective phagolysosome fusion of neutrophils. Alvarez-Dominguez and Stahl
(Alvarez-Dominguez and Stahl,
1999
) studying human macrophages reported that overexpression of
Rab5a accelerates maturation of the phagosomes and enhances intracellular
killing of Listeria monocytogenes, whereas the downregulation of
Rab5a selectively impairs maturation of the phagosomes. We have found that in
neutrophils downregulation of Rab5a by its antisense oligonucleotide reduced
expression of Rab5a and syntaxin-4 on the phagosome of the ingested bacteria
and consequently reduced the fusion capacity of the phagosomes with the
intracellular compartments. The results underscore the importance of Rab5a in
mediating phagosome-endosomal/granule interaction in neutrophils. This is in
accordance with the results showing that Rab5a is a key molecule regulating
phagolysosome biogenesis in macrophages
(Duclos et al., 2000
). In our
study, the recruitment and maintenance of GTP-bound Rab5a in association with
syntaxin-4 on the mycobacterial phagosomes correlated with exclusion of fusion
with azurophil granule and thereby impairment of the phagosome maturation. In
contrast, a brief expression of Rab5a GTP and syntaxin-4 on the SCP related to
the complete phagolysosome maturation. In all likelihood the Rab proteins also
cooperate with other regulatory molecules to create a platform for membrane
organization. A direct interaction between the Rab GTPase Ypt1p and the SNARE
Sed5p has been detected in yeast (Lupashin
and Waters, 1997
). Furthermore, Pep120, which is a yeast homologue
of syntaxin-6, interacts with the Rab5 homologue Vps21p
(Peterson et al., 1999
). Hence
it is conceivable that Rab5a interacts with syntaxin-4 in neutrophils. We
noted that the translocation of syntaxin-4 has kinetics identical to the Rab5a
and that it interacts with GTP-bound Rab5a. Thus the combination of these
proteins involves the fusion events in neutrophils. Owing to the complexity of
the conglomerate of effector proteins at the site of fusion, we expect that
other effecter molecules are also integrated into the Rab5a/syntaxin-4
complex. In that context, Simonsen et al.
(Simonsen et al., 1999
)
recently observed that Rab5 and syntaxin-6 bound competitively to the
membrane-proximal C-terminus of EEA1, a Rab5 effector protein. This effector
protein was recently shown to be targeted for mycobacterial phagosome
maturation arrest (Fratti et al.,
2001
).
Bacterial factors can affect the state of Rab proteins in phagosomes. For
example, within a phagosome, Neisseria gonorrhoeae continuously
synthesizes porin, which is translocated into the phagosomal membrane and
influences the association of Rab5a
(Mosleh et al., 1998). Also it
has been shown that SopE, a protein secreted by Salmonella, interacts
with Rab5 in GTP bound forms (Hardt et
al., 1998
). This result suggests that mycobacteria may secrete a
similar protein that affects the recruitment of Rab5, which in turn activates
SNARE proteins and triggers vesicle fusion. After such recruitment,
GTPase-activating protein (GAP) could increase the GTPase activity of the Rab
protein, converting it into its GDP form, thereby initiating GDI-mediated
release of the Rab to the cytosol,
(Pfeffer, 1994
). Thus the task
of a secreted mycobacterial protein would be to inhibit GAP activity and
thereby maintain Rab on the phagosomal membrane. In fact, Mukherjee and
co-workers (Mukherjee et al.,
2001
) recently showed that SopE mediates recruitment of
non-pernylated Rab5-GTP on Salmonella-containing phagosomes and acts
as Rab5 specific exchange factor converting Rab GDP to GTP without the
prerequisite of prenylation.
Neutrophils are quintessential phagocytes that contribute to the restriction of the extracellular and intracellular pathogens. Phagolysosome biogenesis involves the fusion of the phagosome-containing pathogens with various neutrophil granule and/or endosomal compartments. We conclude that whereas phagolysosome fusion in neutrophils occurs in a Rab5a-dependent pathway, the fusion of M. tuberculosis phagosomes with neutrophil organelles is different from that of the S. aureus phagosomes.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alvarez-Dominguez, C. and Stahl P. D. (1999).
Increased expression of Rab5a correlates directly with accelerated maturation
of Listeria monocytogenes phagosomes. J. Biol.
Chem. 274,11459
-11462.
Alvarez-Dominguez, C., Barbieri A. M., Beron, W.,
Wandinger-Ness, A. and Stahl, P. D. (1996). Phagocytosed live
monocytogenes Listeria influences Rab5-regulated in vitro
phagosome-endosome fusion. J. Biol. Chem.
271,13834
-13843.
Bainton, D. F. (1999). Distinct granule population in human neutrophils and lysosomal organelles identified by immuno-electron microscopy. J. Immunol. Methods 232,153 -168.[Medline]
Barbieri, M. A., Li, G., Colombo, M. I. and Stahl, P. D.
(1994). Rab5, an early acting endosomal GTPase, supports in vitro
endosome fusion without GTP hydrolysis. J. Biol. Chem.
269,18720
-18722.
Brennwald, P. (2000). Reversal of fortune: Do
Rab GTPases act on the target membrane? J. Cell Biol.
149, 1-3.
Brown, A. E., Holzer, T. J. and Andersen, B. R. (1987). Capacity of human neutrophils to kill Mycobacterium tuberculosis. J. Infect. Dis. 156,985 -989[Medline]
Brumell, J. H., Volchuk, A., Sengelov, H., Borregaard, N., Cieutat, A. M., Bainton, D. F., Grinstein, S. and Klip, A. (1995). Subcellular distribution of docking/fusion proteins in neutrophils, secretory cells with multiple exocytic compartments. J. Immunol. 155,5750 -5759.[Abstract]
Bucci, C., Lutcke, A., Steele-Mortimer, O., Olkkonen, V. M., Dupree, P., Chiariello, M., Bruni, C. B., Simons, K. and Zerial M. (1995). Co-operative regulation of endocytosis by three Rab5 isoforms. FEBS Lett. 366, 65-71.[Medline]
Böyum, A. (1968). Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Invest. 97,77 -89.
Cao, X., Ballew, N. and Barlowe, C. (1998).
Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is
independent of SNARE proteins. EMBO J.
17,2156
-2165.
Chakraborty, P., Sturgill-Koszycki, S. and Russell, D. G. (1994). Isolation and characterization of pathogen-containing phagosomes. Methods Cell Biol. 45,261 -276.[Medline]
Cieutat, A. M., Lobel, P., August, J. T., Kjeldsen, L.,
Sengelov, H., Borregaard, N. and Bainton, D. F. (1998).
Azurophilic granules of human neutrophilic leukocytes are deficient in
lysosme-associated membrane proteins but retain the mannose 6-phosphate
recognition marker. Blood
91,1044
-1058.
Clemens, D. L., Lee, B-Y. and Horwitz, M. A.
(2000). Deviant expression of Rab5 on phagosomes containing the
intracellular pathogens Mycobacterium tuberculosis and Legionella
pneumophila is associated with altered phagosomal fate.
Infect. Immun. 68,2671
-2684.
Collins, H. L., Schaible, U. E., Ernst, J. D. and Russell, D.
G. (1997). Transfer of phagocytosed particles to the
parasitophorous vacuole of Leishmania mexicana is a transient
phenomenon preceding the acquisition of annexin I by the phagosome.
J. Cell Sci. 110,191
-200.
Deretic. D., Huber, L. A., Ransom, N., Mancini, M., Simons, K.
and Papermaster, D. S. (1995). rab8 in retinal photoreceptors
may participate in rhodopsin transport and in rod outer segment disk
morphogenesis. J. Cell Sci.
108,215
-224.
Desjardins, M., Huber, L. A., Parton, R. G. and Griffiths, G. (1994). Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus. J. Cell Biol. 124,677 -688.[Abstract]
Diakonova, M., Gerke, V., Ernst, J., Liautard, J. P., van der
Vusse, G. and Griffiths, G. (1997). Localization of five
annexins in J774 macrophages and on isolated phagosomes. J. Cell
Sci. 110,1199
-1213
Duclos, S., Diez, R., Garin, J., Papadopoulou, B., Descoteaux, A., Stenmark, H. and Desjardins, M. (2000). Rab5 regulates the kiss and run fusion between phagosomes and endosomes and the acquisition of phagosome leishmanicidal properties in RAW 264.7 macrophages. J. Cell Sci. 19,3531 -3541.
Fratti, R. A., Backer, J. M., Gruenberg, J., Corvera, S. and
Deretic, V. (2001). Role of phosphatidylinositol 3-kinase and
Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation
arrest. J. Cell Biol.
154,631
-644.
Garin, J., Diez, R., Kieffer, S., Dermine, J. E., Duclos, S.,
Gagnon, E., Sadoul, R., Rondeau, C. and Desjardins, M.
(2001). The phagosome proteome: Insight into phagosome functions.
J. Cell Biol. 152,165
-180.
Hardt, W. D., Chen, L. M., Schuebel, K. E., Bustelo, X. R. and Galan, J. E. (1998). S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells. Cell. 93,815 -826.[Medline]
Hernandez-Pando, R., Jeyanathan, M., Mengistu, G., Aguilar, D., Orozco, H., Harboe, M., Rook, G. A. and Bjune G. (2000). Persistence of DNA from Mycobacterium tuberculosis in superficially normal lung tissue during latent infection. Lancet 356,2133 -2138.[Medline]
Jaconi, M. E. E., Lew, D. P., Carpentier, J-L., Magnusson, K. L., Sjögren, M. and Stendahl, O. (1990). Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils. J. Cell Biol. 110,1555 -1564.[Abstract]
Joiner, K. A., Ganz, T., Albert, J. and Rotrosen, D. (1989). The opsonizing ligand on Salmonella typhimurium influences incorporation of specific, but not azurophil granule constituents into neutrophil phagosomes. J. Cell Biol. 109,2771 -2782.[Abstract]
Korade-Mirnics, Z. and Corey, S. G. (2000). Src
kinase-mediated signaling in leukocytes. J. Leukoc.
Biol. 68,603
-613.
Korchak, H. M., Rossi, M. W. and Kilpatrick, L. E.
(1998). Selective role for ß-protein kinase C in signaling
for O-2 generation but not degranulation or adherence in
differentiated HL60 cell. J. Biol. Chem.
273,27292
-27299.
Laemmli, U. K. (1970). Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227,680 -685.[Medline]
Ligeti, E. and Mocsai, A. (1999). Exocytosis of neutrophil granulocytes. Biochem. Pharmacol. 57,1209 -1214.[Medline]
Lupashin, V. V. and Waters, M. G. (1997).
t-SNARE activation through transient interaction with a rab-like guanosine
triphosphatase. Science
276,1255
-1258.
Majeed, M., Perskvist, N., Ernst, J. D., Orselius, K. and Stendahl, O. (1997). Roles of calcium and annexins in phagocytosis and elimination of an attenuated strain of Mycobacterium tuberculosis in human neutrophils. Microb. Pathog. 24,309 -320.
Mosleh, I. M., Huber, L. A., Steinlein, P., Pasquali, C.,
Gunther, D. and Meyer, T. F. (1998). Neisseria
gonorrhoeae porin modulates phagosome maturation. J. Biol.
Chem. 273,35332
-35338.
Movitz, C., Sjölin, C. and Dahlgren, C. (1999). Cleavage of annexin I in human neutrophils is mediated by a membrane-localized metalloprotease. Biochim. Biophys. Acta. 1416,101 -108.[Medline]
Mukherjee, K., Siddiqi, S. A., Hashim, S., Raje, M., Basu, S. K.
and Mukhopadhyay, A. (2000). Live Salmonella
recruits N-ethylmaleimide-sensitive fusion protein on phagosomal membrane and
promotes fusion with early endosome. J. Cell Biol.
148,741
-753.
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
phagosomes to promote fusion with early endosomes. J. Biol.
Chem. 276,23607
-23615.
N'Diaye, E. N., Darzacq, X., Astarie-Dequeker, C., Daffe, M.,
Calafat, J. and Maridonneau-Parini, I. (1998). Fusion of
azurophil granules with phagosomes and activation of the tyrosine kinase Hck
are specifically inhibited during phagocytosis of mycobacteria by human
neutrophils. J. Immunol.
161,4983
-4991.
Nüsse, O., Serrander, L., Lew, D. P. and Krause, K. H.
(1998). Ca2+-induced exocytosis in individual human
neutrophils: high- and low-affinity granule populations and submaximal
responses. EMBO J. 17,1279
-1288.
O'Farrell, P. A. (1975). High resolution two-dimensional electrophoresis. J. Biol. Chem. 250,4007 -4021.[Abstract]
Perskvist, N., Zheng, L. and Stendahl, O.
(2000). Activation of human neutrophils by Mycobacterium
tuberculosis H37Ra involves phospholipase C gamma 2, Shc adapter protein,
and p38 mitogen-activated protein kinase. J. Immunol.
164,959
-965.
Peterson, M. R., Burd, C. G. and Emr, S. D. (1999). Vac1p coordinates Rab and hosphatidylinositol 3-kinase signaling in Vps45p-dependent vesicle docking/fusion at the endosome. Curr. Biol. 9,159 -162.[Medline]
Pfeffer, S. R. (1994). Rab GTPases: master regulators of membrane trafficking. Curr. Opin. Cell Biol. 6,522 -526.[Medline]
Ricevuti, G., Mazzone, A., Fossati, G., Mazzucchelli, I., Cavigliano, P. M., Pascotti, D. and Notario, A. (1993). Assay of phagocytic cell functions. Allerg. Immunol. 25, 55-66.
Roberts, R. L., Barbieri, M. A., Ullrich, J. and Stahl, P.
D. (2000). Dynamics of rab5 activation in endocytosis and
phagocytosis. J. Leukoc. Biol.
68,627
-632.
Russell, D. G., Dant, J. and Sturgill-Koszycki, S.
(1996). Mycobacterium avium- and Mycobacterium
tuberculosis-containing vacuoles are dynamic, fusion-competent vesicles
that are accessible to glycosphingolipids from the host cell plasmalemma.
J. Immunol. 156,4764
-4773.
Sengelov, H., Follin, P., Kjeldsen, L., Lollike, K., Dahlgren,
C. and Borregaard, N. (1995). Mobilization of granules and
secretory vesicles during in vivo exudation of human neutrophils.
J. Immunol. 154,4157
-4165.
Simonsen, A., Gaullier, J. M., D'Arrigo, A. and Stenmark, H.
(1999). The Rab5 effector EEA1 interacts directly with
syntaxin-6. J. Biol. Chem.
274,28857
-28860.
Stendahl, O., Krause, K. H., Krischer, J., Jerstrom, P., Theler, J. M., Clark, R. A., Carpentier, J. L. and Lew, P. D. (1994). Redistribution of intracellular Ca2+ stores during phagocytosis in human neutrophils. Science 265,1439 -1441.[Medline]
Sturgill-Koszycki, S., Schlesinger, P. H., Chakraborty, P., Haddix, P. L., Collins, H. L., Fok, A. K., Allen, R. D. Gluck, S. L., Heuser, J. and Russell, D. G. (1994). Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science 263,678 -681.[Medline]
Sturgill-Koszycki, S., Schaible, U. E. and Russell, D. G. (1996). Mycobacterium-containing phagosomes are accessible to early endosomes and reflect a transitional state in normal phagosome biogenesis. EMBO J. 15,6960 -6968.[Abstract]
Thelestam, M. (1983). Membrane damage by staphylococcal alpha-toxin to different types of cultured mammalian cell. Biochim. Biophys. Acta. 762,481 -488.[Medline]
Via, L. E., Deretic, D., Ulmer, R. J., Hibler, N. S., Huber, L.
A. and Deretic, V. (1997). Arrest of mycobacterial phagosome
maturation is caused by a block in vesicle fusion between stages controlled by
rab5 and rab7. J. Biol. Chem.
272,13326
-13331.
Vita, F., Soranzo, M. R., Borelli, V., Bertoncin, P. and Zabucchi, G. (1996). Sucellular localization of the small GTPase Rab5a in resting and stimulated human neutrophils. Exp. Cell. Res. 227,367 -373.[Medline]
Wilsson, A., Lundqvist, H., Gustafsson, M. and Stendahl, O. (1996). Killing of phagocytosed Staphylococcus aureus by human neutrophils requires intracellular free calcium. J. Leukoc. Biol. 59,902 -907.[Abstract]
Zimmerli, S., Majeed, M., Gustavsson, M., Stendahl, O., Sanan, D. A. and Ernst, J. D. (1996). Phagosome-lysosome fusion is a calcium-independent event in macrophages. J. Cell Biol. 132,49 -61.[Abstract]