From the Centre d'Immunologie de Marseille-Luminy,
Case 906, 13288 Marseille Cedex 9, France and the
§ Department of Infectious Diseases, Centre for Molecular
Microbiology and Infection, Imperial College School of Medicine,
London SW7 2AZ, United Kingdom
Received for publication, August 2, 2002, and in revised form, January 20, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SifA is a Salmonella
typhimurium effector protein that is translocated across the
membrane of the Salmonella-containing vacuole by
the Salmonella pathogenicity island 2-encoded type III
secretion system. SifA is necessary for the formation of
Salmonella-induced filaments and for the maintenance of the
vacuolar membrane enclosing the pathogen. We have investigated the role
of the C-terminal hexapeptide of SifA as a potential site for membrane
anchoring. An S. typhimurium
strain carrying a deletion of the sequence encoding this hexapeptide
(sifA Salmonellae are the etiological agents of a variety of diseases
ranging from gastroenteritis to enteric fever. Salmonella typhimurium is the principal agent of food poisoning in humans and
causes a typhoid fever-like disease in mice and immuno-compromised humans (1, 2). Ingested S. typhimurium crosses
the intestinal epithelial barrier through M cells of Peyer's patches
(3). Following invasion, the bacterium survives and replicates within macrophages of the spleen and the liver (4, 5). Bacterial multiplication within host cells is essential for virulence, because mutants defective for intracellular replication are attenuated in mice
(6, 7). Intracellular replication of Salmonella takes place
in a membrane-bound compartment, the
Salmonella-containing vacuole
(SCV).1 Maintenance of the
bacteria within this vacuolar enclosure is a key aspect of the
virulence process (8, 9).
The nascent vacuole resulting from the bacterium internalization is
diverted from the phagocytic pathway and undergoes a specific biogenesis process. The mature SCV is highly enriched in vacuolar ATPase and lysosomal membrane glycoproteins (Lgps) such as Lamp1, Lamp2, and CD 63 (10, 11). However, SCVs are essentially devoid of
soluble lysosomal contents (10, 12).
Intravacuolar bacterial replication initiates 3-5 h after infection
and correlates with the formation in epithelial cells of unusual
tubular membranous structures termed Sifs (for
Salmonella-induced filaments) (13). Sifs extend
from and connect SCVs and are characterized by a high enrichment in
Lgps. The formation of Sifs requires a functional type III secretion
system (TTSS) encoded by the Salmonella pathogenicity island-2 (SPI-2)
(8).
Several Gram-negative pathogens use TTSS to inject proteins, termed
bacterial effectors, into the host cell, were they subvert specific
host functions. SifA is a SPI-2 TTSS effector (14) which is essential
for Sif formation (15). In addition, sifA Other SPI-2 TTSS-translocated proteins, SseF, SseG and SpiC, have also
been found to be necessary for the formation of Sifs (17). SpiC was
originally described as an inhibitor of interactions between SCV and
late endocytic compartments (18). SPI-2-encoded SseF and SseG are
present on SCVs and Sifs (19, 20). The role of these proteins in the
biogenesis of Sifs remains undetermined. PipB, SseJ, and SifB are other
effector proteins secreted by the SPI-2 TTSS that have been found to
localize to Sifs and SCVs (20-22). Transfection of HeLa cells with a
plasmid encoding SifA fused to GFP complements the defect of a
sifA To gain some insight in the molecular mechanisms of action of SifA, we
analyzed the role of a C-terminal, cysteine-rich, hexapeptide in the
subcellular localization and biological function of this virulence
factor. In the present paper, we show that this domain is necessary for
systemic virulence in mice, intracellular replication, and
reorganization of the host endocytic compartment. Collectively, our
results indicate that SifA requires its C-terminal hexapeptide to
achieve its function.
Antibodies and Reagents--
The rabbit antiserum against the
Rab7 GTPase has been described previously (23). The mouse anti-Lamp1
H4A3 and rat anti-Lamp2 ABL-93 monoclonal antibodies, both developed by
J. T. August, were obtained from the Developmental Studies
Hybridoma Bank developed under the auspices of the NICHD, National
Institutes of Health, and maintained by the University of Iowa
(Department of Biological Sciences, Ames, IA). Dr. S. Kornfeld (St.
Louis, MO) kindly provided rabbit polyclonal antibody against cathepsin
D. Mouse monoclonal anti-GFP antibody JL-8 was purchased from
Clontech. Polyclonal rabbit anti-S.
typhimurium lipopolysaccharide was purchased from Difco
Laboratories. Goat anti-mouse peroxidase was purchased from Sigma. The
secondary antibodies Goat anti-rabbit, anti-rat or anti-mouse IgG
conjugated to Alexa 488, Alexa 594, or Alexa 680 were purchased from
Molecular Probes.
Cell Lines and Culture Conditions--
HeLa human epithelial
cells and RAW 264.7 mouse macrophage cells were routinely grown in
Dulbecco's modified Eagle's medium (DMEM; Invitrogen),
supplemented with 10% fetal calf serum (FCS, Life Technologies, Inc.),
2 mM glutamine, and non-essential amino acids at 37 °C
in 5% CO2.
Bacterial Strains--
The S. typhimurium strains
used in this work were 12023 wild-type strain (NTCC), HH109
(ssaV::aphT) (24), P3H6
(sifA::mTn5) (8), and HH215
(sifA
Plasmid pFVP25.1, carrying gfpmut3A under the control of a
constitutive promoter, was introduced into bacterial strains for fluorescence visualization (26). Bacteria were grown in Luria-Bertani (LB) medium supplemented with ampicillin (50 µg/ml), kanamycin (50 µg/ml), or chloramphenicol (50 µg/ml) as appropriate.
Mouse Mixed Infections--
Mice were inoculated
intraperitoneally with 105 colony-forming unit per
mouse, as described previously (27). The spleens were aseptically
removed 48 h after inoculation, and bacteria recovered and
enumerated after plating a dilution series onto LB agar and LB agar
with the appropriate antibiotics. Each competitive index (CI) value is
the mean of three independent mice infections and is defined as the
ratio between the mutant and wild-type strains within the output
(bacteria recovered from the mouse after infection) divided by their
ratios within the input (initial inoculum) (28, 29).
Bacterial Infection of HeLa Cells--
HeLa cells were seeded
onto glass coverslips (12-mm diameter) in 10-cm dishes at a density of
106 cells per dish 24 h before infection. Bacteria
were incubated overnight at 37 °C with shaking, diluted 1:33 in
fresh LB broth and incubated in the same conditions for 3.5 h. The
cultures were diluted in Earle's buffered salt solution, pH 7.4 and
added to the HeLa cells at a multiplicity of infection of ~100:1. The
infection was allowed to proceed for 10 min at 37 °C in 7%
CO2. Cells were washed three times with DMEM containing
fetal calf serum (FCS) and 100 µg·ml Bacterial Infection of Macrophages and Survival
Assays--
Macrophages were seeded at a density of
105 cells per well in 24-well tissue culture plates 24 h before use. Bacteria were cultured overnight at 37 °C with
shaking. The cultures were opsonized in DMEM containing FCS and 10%
normal mouse serum for 20 min. Bacteria were added to the cells at a
multiplicity of infection of ~100:1 and incubated for 20 min at
37 °C in 7% CO2. Macrophages were washed three times
with DMEM containing FCS and 100 µg·ml Expression of GFP·SifA in HeLa Cells--
The full
sifA ORF was amplified from S. typhimurium
genomic DNA by PCR using the primer 1 (5'-AAA AAA GAA TTC CAC CAC CAT GCC GAT TAC TAT AGG GAA TGG-3') and primer 2 (5'-AAA AAA CCC GGG TTA
TAA AAA ACA ACA TAA ACA-3'). The PCR product was subcloned into the
unique EcoR1 and XmaI sites of pEGF-C1 vector
(Clontech) into the same reading frame as EGFP,
generating pgfp::sifA. The C-terminal
deletion or substitution mutants were generated using the primer 1 and
second primers designed so that they correspond to the different
C-terminal sequences. For fusing the 11 C-terminal residues of SifA to
GFP, the following primers (5'-AATTCTGAACAACAAAGCGGCTGTTT ATGTTGTTTTTTATAG-3' and 5'-GATCCTATAAAAAACAACATAAACAGC
CGCTTTGTTGTTCAG-3') were annealed and cloned into the EcoR1
and BamH1 sites of pEGFP-C1 generating
GFP·SifA-(326-336). HeLa cells were transfected with FuGENE 6 (Roche Molecular Biochemicals) following the manufacturer's instructions. Cells were further incubated for 24 h.
Immunofluorescence--
Cells grown on coverslips were fixed
with 3% paraformaldehyde, pH 7.4, in PBS at room temperature for 10 min. Fixed cells were washed three times in PBS and permeabilized by
incubating in PBS containing 0.1% saponin. Saponin 0.1% was included
in all subsequent incubation steps. Primary and secondary antibodies were diluted in PBS containing 0.1% saponin and 5% normal horse serum. Coverslips were incubated with primary antibodies for 20 min at
room temperature, washed in PBS containing 0.1% saponin, and then
incubated with appropriate Alexa goat secondary antibodies. Coverslips
were mounted onto glass slides using Mowiol (Aldrich). Cells were
observed with a Leica epifluorescence microscope or a confocal
laser-scanning microscope LSM510 Zeiss.
Subcellular Fractionation--
For each chimeric GFP a 10-cm
dish of transfected HeLa cells was chilled on ice and washed three
times in ice-cold PBS. Scraped cells were pelleted for 5 min at
100 × g in a clinical centrifuge, overlaid with 3 ml
of homogenization buffer (250 mM sucrose, 3 mM
imidazole, pH 7.4) and centrifuged for 5 min at 1800 × g. Cells were resuspended in 0.4 ml of homogenization buffer
containing 1 mM phenylmethylsulfonyl fluoride and
homogenized by three passages through a 22-gauge needle. After
centrifugation for 10 min at 1800 × g, the
post-nuclear supernatant was collected and centrifuged at 100,000 × g for 20 min. The soluble fraction was saved, and the
pellet was resuspended in a volume of homogenization buffer. For high
pH extraction, the pellet was resuspended in a volume of 100 mM NaOH, pH 11, 50 mM NaCl, incubated for 15 min on ice, and centrifuged at 100,000 × g for 20 min.
Fractions were loaded on a 12% SDS-PAGE and transferred on Immobilon-P
for Western blotting.
A sifA A SifA Loss of Vacuolar Membrane--
SifA is essential to maintain the
integrity of the vacuolar membrane, as sifA
A drastic reorganization of Lgps to the vicinity of vacuoles occurs in
epithelial cells infected by S. typhimurium. This process is
dependent on the function of SifA (15, 22). In Raw 264.7 macrophages, a
similar SifA-dependent phenomenon was observed. Alteration
of the distribution of Lamp2 was detected in 65 ± 6 or 5.5 ± 0.7% of macrophages infected with wild-type or
sifA The C-terminal Domain of SifA Plays a Role in the Formation of
Sifs--
The presence of Sifs is the hallmark of S. typhimurium infection of epithelial cells (13), and the
sifA gene is essential for their formation (15). At 10 h after bacterial entry, Sifs were detected in more than 65% of
epithelial cells infected with the wild-type strain, whereas the
sifA GFP·SifA but Not GFP·SifA
Transfection of epithelial cells with a vector encoding GFP·SifA
induces the formation of large Lgp-enriched vesicles and filamentous
structures resembling Sifs (16). Compared with non-transfected cells
(star symbol in Fig.
4A), we observed similar large
Lamp1-positive vesicles and tubules in cells expressing GFP·SifA
(Fig. 4A). Tubules were more numerous but thinner than Sifs
in infected cells (compare inset in Fig. 4A with
Fig. 2B) and will thereafter be referred to as Sif-like
tubules. Large Lgp-enriched vesicles and Sif-like tubules were observed
in 65 ± 13% and 15 ± 5%, respectively, of GFP·SifA
transfected cells. GFP·SifA co-localized with Lamp1 on vesicles and
tubules (Fig. 4A), and appeared essentially membrane bound.
To investigate the composition of the SifA-induced structures in more
detail, we examined the effect of ectopic expression of these chimeras
on the distribution of several other markers of the endocytic pathway.
EEA1, a marker for early endosomes, was unaffected by the cellular
expression of SifA (data not shown). We also analyzed the effects on
the distribution of late endosomal (Rab7 and the cation-independent
mannose 6-phophate receptor) and lysosomal (lysobisphosphatidic acid,
Lamp2, CD63, vATPase and cathepsin D) markers. In non-transfected cells
these two groups of markers were distributed to distinct compartments,
and no co-localization of late endosomal and lysosomal markers could be
detected by confocal microscopy (data not shown). In contrast, cells
expressing GFP·SifA were characterized by the presence of both Rab7
and cathepsin D in the large vesicular compartments in which they
co-localized with the over-expressed protein (Fig. 4B). This
was also the case for other late endosomal and lysosomal protein and
lipid markers (data not shown). Therefore, ectopic expression of SifA
induces a profound reorganization of late endocytic structures and
results in the formation of an unusual vesicular and tubular compartment.
We next examined the intracellular localization and the consequences of
the expression of GFP·SifA The C-terminal Domain of SifA Contains a Signal for Its Association
to Membranes--
To gain insight in the function of the C-terminal
motif, the distribution of wild-type and mutant versions of the
GFP-tagged version of SifA between soluble and membrane fractions of
transfected cells was analyzed (Fig. 6).
GFP·SifA was found to associate with the membrane fraction, whereas
the majority of GFP·SifA
The C-terminal hexapeptide of SifA contains three cysteine residues,
two of them being removed in the GFP·SifA
Cysteine residues in position 333 and 334 could act as isoprenylation
or palmitoylation sites. Such protein lipidations stably anchor
proteins to membranes, rendering them resistant to extraction by
alkali. To investigate the possibility that SifA is tightly associated
with membranes, membrane fractions from cells expressing GFP·SifA
were treated with 0.1 M sodium hydroxide. As shown in Fig.
6C, the membrane association of GFP·SifA resisted such
high pH treatment. As expected, similar results were obtained for Rab7, which associates with membranes by virtue of isoprenylation, whereas calreticulin, which associates with membranes by binding to the KDEL
receptor, was extracted by this treatment (data not shown). These
results suggest that SifA undergoes lipidation involving its cysteine residues.
The C-terminal Domain of SifA Is Sufficient to Target GFP to
Membranes--
SifB is another S. typhimurium SPI-2 TTSS
effector (20). In contrast to GFP·SifA, GFP·SifB was found to be
cytosolic upon expression in HeLa cells (data not shown). SifB exhibits
26.4% identity to SifA over the length of the proteins. However the last 11 amino acid residues of the C-terminal domain of SifA are absent
in SifB. To examine the effectiveness of this domain as a membrane
targeting signal, we fused the 11 C-terminal residues of SifA to GFP or
GFP·SifB, generating GFP·SifA-(326-336) (see Fig. 3A)
or GFP·SifB·SifA-(326-336), respectively. As shown in Fig.
7A, a fluorescence microscopic
observation of transfected HeLa cells revealed that
GFP·SifA-(326-336) appeared to be essentially membrane associated
contrasting with the cytosolic distribution of GFP. Biochemical
analysis of fractionated transfected cells confirmed that
GFP·SifA-(326-336) (Fig. 7B) and
GFP·SifB·SifA-(326-336) (data not shown) partitioned to the
membrane fraction and resisted to high pH extraction (data not shown).
These experiments demonstrate that the eleven C-terminal residues of
SifA are sufficient to target cytosolic proteins to membranes.
Membrane Association of SifA Is Required for Its Biological
Activity--
Removal of the last or two last residues only slightly
reduced the biological activity of SifA, as measured by its ability to
induce formation of Lgp-enriched vesicles and Sif-like tubules in
transfected cells (Fig. 3, B and C). This ability
was greatly decreased when the C-terminal 5 residues were removed.
Interestingly, some residual activity was still detected in cells
transfected with GFP·SifA In the present paper we have investigated the structure-function
relationship of SifA. This Salmonella protein is synthesized after bacterial entry into host cells and is translocated across the
vacuolar membrane into host cell membranes by the SPI-2-encoded TTSS
(8, 33). SifA was originally described as essential for the formation
of Sifs in epithelial cells (15). We have previously shown that, in
absence of this effector protein, S. typhimurium is unable
to preserve the integrity of the vacuolar membrane in which it is
enclosed and is progressively released into the host cytoplasm (8).
We are interested in understanding how SifA interferes with the host
cellular machinery. We hypothesize that SifA directly competes with a
host component. However, neither a BLASTp against protein databases nor
a search for proteins signatures revealed significant similarities with
eukaryotic proteins. PSORT analysis of SifA revealed the presence, at
the C terminus, of a putative isoprenylation motif similar to those
found on Rab proteins (CAAX boxes are CC, CXC,
CCXX, CCXXX; C is cysteine, A is aliphatic and
X can be any amino acid residue). Rab GTPases are
important regulators of different steps of vesicular transport in
eukaryotic cells (for review see Refs. 34-36). Isoprenylation is
necessary for membrane binding and biological activities of Rab
proteins (37). Therefore, we were interested in exploring the
significance of this motif on a prokaryotic protein, SifA, which
affects intracellular trafficking in eukaryotic cells.
The functional importance of the C-terminal hexapeptide is illustrated
by the virulence attenuation of the sifA In both macrophages and epithelial cells the late phase of infection is
marked by a dramatic redistribution of Lgps toward the SCVs, as well as
a disappearance of the vesicular pattern of lysosomes. This
SifA-dependent event is rarely observed in macrophages
infected with the sifA The functional defects observed with the sifA GFP·SifA is essentially membrane bound, whereas the functionally
inactive GFP·SifA To investigate further the contribution of the C-terminal domain of
SifA as a membrane targeting signal, its 11 C-terminal residues were
fused to the cytosolic proteins GFP and GFP·SifB. The rationale for
this experiment is based on the observation that the similarity between
SifA and SifB excludes the last 11 amino acids residues of SifA, which
are absent in SifB. The membranous localization of both GFP·SifA
(326-336) and GFP·SifB·SifA (326-336) demonstrates that the 11 C-terminal amino acid residues of SifA contain a signal that is
sufficient to target this protein to membranes. One can hypothesize
that this peptide sequence is either recognized by a membrane receptor
or contains a consensus sequences for lipidation. Very short peptides
containing consensus lipidation sequences (4-16 amino acid residues)
have been used to target various GFP variants to membranes (38). The
resistance of GFP·SifA and GFP·SifA-(326-336) to alkali extraction
and the specific role of cysteine residues 333 and 334 in the partition
of SifA are consistent with the lipidation hypothesis. Indeed, the
myristoylation of type III effector proteins from Pseudomonas
syringae (a plant pathogen) by the host cell has been already
reported (39). Interestingly, myristoylation targets Avr proteins to
membranes, enhances their functions, and is required for virulence.
However, lipidation of SifA by the host cells remains to be demonstrated.
Overall, our analysis of mutants shows that SifA must be membrane-bound
to be fully functional and has revealed a crucial role of its
C-terminal domain as a membrane-targeting signal. Further work is
needed to establish the molecular function of SifA and to understand
how this function confers a selective advantage to S. typhimurium within the host cell.
6) was found to be attenuated for systemic virulence in mice. In mouse macrophages, sifA
6 mutant
bacteria displayed a reduced association with vacuolar markers, similar to that of sifA null mutant bacteria, and exhibited a
dramatic replication defect. Expression of SifA in epithelial cells
results in the mobilization of lysosomal glycoproteins in large
vesicular structures and Sif-like tubules. This process requires the
presence of the C-terminal hexapeptide domain of SifA. Ectopic
expression of truncated or mutated versions of SifA affecting the
C-terminal hexapeptide revealed a strong correlation between the
membrane binding capability and the biological activity of the protein. Finally, the eleven C-terminal residues of SifA are shown to be sufficient to target the Aequorea green
fluorescent protein to membranes. Altogether, our results indicate
that membrane anchoring of SifA requires its C-terminal hexapeptide
domain, which is important for the biological function of this
bacterial effector.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutant bacteria progressively lose their vacuolar membranes in cultured
macrophages and epithelial cells, as well as in splenocytes in
vivo (5, 8, 9). Mutation of sifA causes virulence attenuation in mice and a strong replication defect in macrophages (8,
15-17). It is not known whether the absence of Sifs and vacuolar
membrane result from the same loss of function.
strain to induce Sif formation and
maintain the vacuolar membrane (8). Furthermore, expression of a
GFP·SifA fusion protein in non-infected cells induces the
vacuolation of Lamp1-positive compartments and the appearance of
tubular structures that resemble Sifs (16).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6). HH215 was constructed following the method described in Ref. 25, using pKD4 as a template for PCR amplification with SIFAD1 (5'-GTT AAC CAC GCT ACA CGT TCG CTC AGA ACA
ACA AAG CGG CTA ATA AGT GTA GGC TGG AGC TGC TTC-3') and SIFAD2 (5'-GAC
CGA TCA TTC AAG TTC CAC CTT CTT ATT CAG AGG ATG GGG CAT ATG AAT ATC CTC
CTT AG-3') as primers, to generate a chromosomal deletion of
sifA that lacks the sequence encoding the C-terminal last
six amino acids of the resulting protein. The mutated gene was
amplified by PCR, and the deletion confirmed by sequencing.
1 gentamicin and
incubated in this medium for 1 h, after which the gentamicin con
centration was decreased to 10 µg·ml
1.
1 gentamicin
and incubated in this medium for 1 h. The medium was replaced with
DMEM containing FCS and 10 µg·ml
1 gentamicin for the
remainder of the experiment. For enumeration of intracellular bacteria,
macrophages were washed three times with PBS and lysed with 0.1%
Triton X-100 for 10 min, and a dilution series was plated onto LB agar.
Plates were incubated overnight at 37 °C. Colonies were counted.
Each time point was performed in triplicate, and each individual
experiment was performed three times or more.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6 Mutant Strain Is Attenuated in Mouse Virulence--
The
C-terminal hexapeptide of SifA (CLCCFL) contains three cysteine
residues that may serve as recognition sites for lipidations. These
post-translational modifications are important for membrane attachment
and the biological functions of many proteins. To investigate the role
of the C-terminal hexapeptide in the function of SifA, a mutant strain
(HH215) was constructed carrying a deletion of sifA
(sifA
6) in the chromosome. The truncated gene lacks the sequence encoding the C-terminal last six amino acids of SifA. The
virulence of this strain was tested by comparing it with the wild-type
strain in mixed infections of mice. Bacteria from infected spleen were
recovered 48 h after intra-peritoneal inoculation, and the
competitive index was determined as described previously (30). The CI
is a sensitive measure of the relative degree of virulence attenuation
of a given mutant (27). The CI of the sifA
6 mutant
versus the wild-type strains was significantly lower than
1.0 (CI = 0.226 ± 0.08) showing that, in the mouse, the net growth of the sifA
6 mutant strain was considerably less
than that of the wild-type strain. This result indicates that the
C-terminal hexapeptide motif of SifA is required for full virulence in
mice and, therefore, SifA function.
6 Mutant Strain Has a Replication Defect in
Macrophages--
Because the sifA
6 mutant strain is
attenuated in the systemic phase of the infection, and S. typhimurium systemic virulence is correlated with its ability to
replicate inside host macrophages, this mutant strain was tested for
replication over 16 h in the macrophage-like RAW 264.7 cell line.
As a control we used an ssaV
mutant strain
that is completely defective for SPI-2-mediated secretion and, thus,
defective for intracellular replication (31, 32). We observed that the
sifA
6 mutant has a replication defect similar to that of
sifA
and ssaV
mutant
strains (Fig. 1A). This
indicates that SifA-mediated proliferation in macrophages requires its
C-terminal hexapeptide.
View larger version (37K):
[in a new window]
Fig. 1.
A sifA 6 mutant strain is
defective for replication and for association with Lamp2 in RAW 264.7 macrophages. Macrophages were allowed to phagocytose either
opsonized wild-type (12023), a SPI-2 secretion defective mutant
(ssaV
), sifA
, or
sifA
6 bacteria and were either lysed for enumeration of
intracellular bacteria or fixed and examined by epi or confocal
fluorescence microscopy. A, this graph shows the fold
increase of various strains at 16 h post-uptake. The fold increase
was calculated as the number of intracellular bacteria at 16 h
after uptake divided by their number at 2 h. B, the
graph shows the percentage of bacteria associating with Lamp2 at
10 h post-uptake. Results in panels A and B
are the means ± S.E. of three independent experiments.
C, confocal microscopic images of macrophages infected with
various strains expressing GFP (green) and
immuno-labeled for Lamp2 (red) show recruitment of cellular
Lamp2 to vacuoles containing wild-type but not to vacuoles containing
sifA
mutant bacteria. sifA
6
mutant bacteria show an intermediate phenotype. Bar, 10 µm.
mutant bacteria are gradually released into the host cell cytosol (8).
The cytosol of macrophages does not support S. typhimurium replication (9). Therefore it is possible that the failure of the
sifA
6 mutant strain to replicate inside these cells is due to a failure to maintain the vacuolar membrane. To address this
question, we compared the level of association with an SCV membrane
marker, Lamp2, of sifA
6 mutant bacteria to that of the wild-type and sifA
strain by
immunofluorescence analysis. In macrophages, 95% of wild-type and 47%
of sifA
bacteria were decorated by the
anti-Lamp2 antibody 10 h after bacterial uptake. The
sifA
6 mutant displayed an intermediate phenotype with
70% of the bacteria found in association with the membrane marker
(Fig. 1B). Similar results were obtained in bone marrow-derived macrophages (data not shown).
mutant bacteria, respectively (Fig.
1C). 27 ± 3% of macrophages infected with the
sifA
6 mutant strain exhibited modified Lamp2 distribution
(Fig. 1C). We concluded that the maintenance of bacteria inside a vacuolar membrane and the recruitment of Lgp-containing compartments require a full-length SifA.
mutant strain was unable to induce the
formation of such structures (Fig.
2A). The sifA
6
mutant strain exhibited an intermediate phenotype, as Sifs were found
in ~30% of infected cells (Fig. 2A). Where Sifs were
observed their morphology was altered, appearing shorter and thinner
(Fig. 2B). Therefore, we conclude that the C-terminal
hexapeptide motif of SifA contributes to the function of this SPI-2
effector in remodeling intracellular compartments.
View larger version (21K):
[in a new window]
Fig. 2.
A sifA 6 mutant strain is
partially defective for Sif formation in HeLa cells. Cells were
infected with either wild-type (12023), sifA
,
or sifA
6 strains expressing GFP, fixed, and examined by
epi or confocal fluorescence microscopy. A, each S. typhimurium strain was scored for its ability to induce Sif
formation 10 h after bacterial entry. Values are given as
percentage of infected cells containing Sifs. Results are the
means ± S.E. of three independent experiments. B,
confocal microscopic images of representative examples of cells
infected with either wild-type or sifA
6 strains. Sifs in
cells infected with the sifA
6 mutant bacteria are shorter
and thinner (arrows) than in cells infected with wild-type
strain. Bar, 10 µm.
6 Induces the Formation of
Enlarged, Late Endosomal Vesicles and Sif-like Structures--
Ectopic
expression of SifA in epithelial cells complements the failure of a
sifA
mutant strain to induce Sif formation (8)
and results, in non-infected cells, in the reorganization of late
endocytic compartments (16). To investigate the specific contribution
of each of the six C-terminal residues to SifA function, we generated a
series of sifA alleles carrying deletions or substitutions
in the sequence encoding the C-terminal region of the protein and
ligated them into a vector for transfection from which GFP-tagged
versions of each proteins were expressed (Fig.
3A).
View larger version (22K):
[in a new window]
Fig. 3.
C-terminal deletion and substitution mutants
and their effects on SifA activities. A, schematic
representation of deletion and substitution mutants of GFP·SifA. The
positions of cysteine-333 and cysteine-334 and terminal leucine-336
residues are indicated. The number following the symbol indicates
the number of C-terminal residues deleted. For GFP·SifA(326-336),
residues 326 to 336 corresponding to the eleven C-terminal residues of
SifA were fused to GFP. B and C, mean values ± S.E. of three independent experiments where HeLa cells
(n = 100 for each experiment) transfected with the
various constructs were scored for the presence of large Lamp1-positive
vesicles (B) or the formation of Sif-like tubules
(C). Values are given as the percentage of transfected cells
displaying the phenotype.
View larger version (48K):
[in a new window]
Fig. 4.
Ectopic expression of GFP·SifA in HeLa cell
induces the reorganization of late endocytic compartment. HeLa
cells transiently expressing GFP·SifA were fixed, immuno-labeled for
various markers, and observed by confocal microscopy. A, the
color image shows GFP·SifA (green) transfected cells
immuno-labeled for Lamp1 (red). Gray scale images
present GFP·SifA and Lamp1 labeling of insets magnified
twice. GFP·SifA induces the formation of large Lamp1-positive
vesicles (top left inset) and the
formation of Sif-like tubules (bottom middle
inset). For comparison, a non-transfected cell with a
typical lysosomal pattern for Lamp1 is marked (*).
B, HeLa cells expressing or not expressing GFP·SifA and
labeled for Rab7 or cathepsin D. Both Rab7 and cathepsin D are present
in GFP·SifA-induced vesicles. Bar, 10 or 20 µm for
magnified insets.
6 in transfected cells. Compared with
cells expressing the full-length version of SifA, a drastic reduction
in the frequency of redistribution of Lamp1-containing structures was
observed, and Sif-like tubules were very rarely seen (Fig. 3,
B and C). Confocal imaging revealed that the
GFP·SifA
6 fusion protein was localized both in the nucleus and the
cytoplasm (Fig. 5), contrasting with the
membrane distribution of GFP·SifA (Fig. 4). Therefore, we conclude
that both membrane association and SifA activity in transfected cells
are dependent on its C-terminal hexapeptide motif.
View larger version (30K):
[in a new window]
Fig. 5.
GFP·SifA 6 does not
alter the distribution of Lamp1 in transfected cells. HeLa cells
transiently expressing GFP·SifA
6 were fixed, immuno-labeled for
Lamp1, and observed by confocal microscopy. Bar, 10 µm.
6 was found in the cytosol (Fig.
6A), consistent with fluorescence microscopic observations
(Fig. 5). Removal of the last or the last two amino acid residues did
not result in a significant change in the distribution of the protein.
By contrast, the deletion of the five C-terminal amino acid residues
rendered the protein mostly cytosolic (Fig. 6A).
View larger version (18K):
[in a new window]
Fig. 6.
The C-terminal domain of SifA contains a
signal for membrane attachment. A and
B, HeLa cells expressing either GFP·SifA or various
deletion or substitution mutant in the C-terminal domain of SifA were
homogenized. Post-nuclear supernatants were fractionated into membrane
(M) and soluble (S) fractions, run on a SDS-PAGE,
and Western blotted with the use of a mouse monoclonal anti-GFP
antibody. C, total membranes of HeLa cells expressing
GFP·SifA were extracted at high pH and fractionated again into
extracted membrane (M ext) and extract
fractions.
5 mutant (Cys-333 and
Cys-334, see Fig. 3A). To determine the relative importance of each of these residues, we substituted each cysteine with serine, either individually or two at a time, and tested their ability to
mediate association with membranes in transfected cells. We found that
the membrane association of the double C333S/C334S and both of the
individual C333S and C334S mutants was strongly reduced and comparable
with that of the GFP·SifA
5 mutant protein (Fig. 6C). We
conclude from these results that both Cys-333 and Cys-334 are required
for the membrane association of SifA.
View larger version (74K):
[in a new window]
Fig. 7.
The eleven C-terminal residues of SiFA are
sufficient to target GFP to membranes. Intracellular distribution
of ectopically expressed GFP or GFP·SifA-(326-336) was observed by
microscopy or cellular fractionation. A, typical
fluorescence microscopy images of HeLa cells expressing each construct.
Bar, 10 µm. B, transfected cells were
homogenized. Post-nuclear supernatants were fractionated into membrane
(M) and soluble (S) fractions, run on a SDS-PAGE,
and Western blotted using a mouse monoclonal anti-GFP antibody.
GFP·SifA-(326-336) is essentially membranous, whereas GFP is
cytosolic.
5 that was consistently higher than that
detected in cells transfected with GFP·SifA
6. Ectopic expression
of SifA alleles carrying mutations in either Cys-333, Cys-334, or both, induced residual reorganization of Lgp-compartments in a manner similar
to that induced in cells expressing the GFP·SifA
5 mutant protein.
These results establish a direct correlation between the membrane
association of the various deletion or substitution mutants and their
ability to reorganize late endocytic compartments. We conclude from
these results that the biological activity of SifA depends on its
membrane binding capacity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6 mutant strain in the mouse model of systemic infection. In macrophages, the sifA
6 mutant strain displayed a replication defect
comparable with that of the ssaV
and
sifA
strains. In the case of the
sifA
strain, this defect has been shown to be
mainly due to its inability to replicate into the macrophage cytosol
(9). Despite its strong replication defect, only 30% of
sifA
6 mutant bacteria lost their vacuolar membranes.
sifA
mutant bacteria that remain enclosed
within a vacuole are still not capable of replicating in either
macrophages or epithelial cells (9), suggesting that SifA function is
also necessary for the maturation of the SCV into a compartment that
allows bacterial replication. Our results show that, beyond the
phenotype of vacuolar membrane loss, the sifA
6 mutant
strain has an additional defect for replication inside the vacuole,
suggesting that the C-terminal hexapeptide is also necessary for SCV
maturation into a compartment permissive for replication.
6 mutant strain. It suggests that,
in the absence of its C-terminal hexapeptide, SifA is functionally defective. The low number of cells displaying Sifs and their altered morphology in cells infected with the sifA
6 mutant is
likely to result from the same loss of function.
6 mutant
strain, are correlated with the failure of ectopically expressed
SifA
6 to induce the reorganization of late endocytic compartments
that is observed in cells expressing the full protein. GFP·SifA
induces the formation of large vesicles or large clusters of vesicles that exhibit both late endosomal and lysosomal markers. In contrast to
what has been observed previously with GFP·SifA (16) we found that
cathepsin D is present in GFP·SifA-induced vesicles. GFP·SifA also
induces the formation of Sif-like tubules. These are more numerous but
thinner than the Sifs found in infected epithelial cells. In cells
expressing Lamp1·GFP we have observed that lysosomes are very dynamic
organelles from which tubules are constantly forming, growing,
shrinking, and contacting other tubular or vesicular structures. In
contrast, in cells expressing Lamp1·GFP and infected with wild-type
S. typhimurium, Sifs are essentially non
dynamic.2 The function of
SifA could be to stabilize tubular structures, therefore favoring
membrane exchanges between the SCV and host compartments. Large
vesicles in SifA-expressing cells may result from unbalanced membrane
exchanges between late endocytic structures.
6 is cytosolic. Deletion mapping of the C-terminal domain showed that a SifA mutant protein with a deletion of
the last two amino acid residues (GFP·SifA
2) retains its membrane binding ability as well as full activity. In contrast, deletion of the
pentapeptide motif LCCFL as well as the cysteine to serine substitution
of residues 333 and 334 abolished both membrane association and
biological functions of SifA. Considering the homology of SifA
C-terminal domain with eukaryotic CAAX motifs, the crucial role of cysteine residues, and the ability of SifA to resist extraction by alkali, it seems very likely that the cysteine residues 333 and 334 of SifA are sites of lipidation.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Jörg Deiwick and Thomas Henry for critically reading the manuscript and Javier Ruiz-Albert for helpful suggestions.
![]() |
FOOTNOTES |
---|
* This work was supported by institutional grants from the CNRS and INSERM, Association pour la Recherche sur le Cancer (ARC) Grant 7541, and a grant from the Medical Research Council, United Kingdom (to D. W. H.).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.
¶ Present address: Departamento de Biología Celular, Genética y Fisiología, Facultad de Ciencias, Universidad de Málaga, Campus Teatinos, Málaga 29071, Spain.
To whom correspondence should be addressed: Centre
d'Immunologie de Marseille Luminy, INSERM, CNRS, Université de
la Méditerranée, Case 906, 13288 Marseille Cedex 9, France.
Tel.: 330-4926-9315; Fax: 330-4926-9430; E-mail:
meresse@ciml.univ-mrs.fr.
Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M207901200
2 S. Méresse and J.-P. Gorvel, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: SCV, Salmonella-containing vacuole; Lgp, lysosomal membrane glycoprotein; Sif, Salmonella-induced filament; TTSS, type III secretion system; SPI2, Salmonella pathogenicity island 2; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; LB, Luria-Bertani (medium); CI, competitive index; FCS, fetal calf serum; PBS, phosphate-buffered saline.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Levine, W. C., Buehler, J. W., Bean, N. H., and Tauxe, R. V. (1991) J. Infect. Dis. 164, 81-87[Medline] [Order article via Infotrieve] |
2. | Gordon, M. A., Walsh, A. L., Chaponda, M., Soko, D., Mbvwinji, M., Molyneux, M. E., and Gordon, S. B. (2001) J. Infect. 42, 44-49[CrossRef][Medline] [Order article via Infotrieve] |
3. | Jensen, F. B., Wang, T., Jones, D. R., and Brahm, J. (1998) Am. J. Physiol. 274, R661-R671[Medline] [Order article via Infotrieve] |
4. |
Richter-Dahlfors, A.,
Buchan, A. M. J.,
and Finlay, B. B.
(1997)
J. Exp. Med.
186,
569-580 |
5. | Salcedo, S. P., Noursadeghi, M., Cohen, J., and Holden, D. W. (2001) Cell. Microbiol. 3, 587-597[CrossRef][Medline] [Order article via Infotrieve] |
6. | Fields, P. I., Swanson, R. V., Haidaris, C. G., and Heffron, F. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5189-5193[Abstract] |
7. | Leung, K. Y., and Finlay, B. B. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11470-11474[Abstract] |
8. |
Beuzon, C. R.,
Méresse, S.,
Unsworth, K. E.,
Ruiz-Albert, J.,
Garvis, S.,
Waterman, S. R.,
Ryder, T. A.,
Boucrot, E.,
and Holden, D. W.
(2000)
EMBO J.
19,
3235-3249 |
9. |
Beuzon, C. R.,
Salcedo, S. P.,
and Holden, D. W.
(2002)
Microbiology
148,
2705-2715 |
10. | Garcia-del Portillo, F., and Finlay, B. B. (1995) J. Cell Biol. 129, 81-97[Abstract] |
11. | Steele-Mortimer, O., Meresse, S., Gorvel, J. P., Toh, B. H., and Finlay, B. B. (1999) Cell. Microbiol. 1, 33-49[CrossRef][Medline] [Order article via Infotrieve] |
12. | Rathman, M., Barker, L. P., and Falkow, S. (1997) Infect. Immun. 65, 1475-1485[Abstract] |
13. | Garcia-del Portillo, F., Zwick, M. B., Leung, K. Y., and Finlay, B. B. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10544-10548[Abstract] |
14. | Brumell, J. H., Tang, P., Mills, S. D., and Finlay, B. B. (2001) Traffic 2, 643-653[CrossRef][Medline] [Order article via Infotrieve] |
15. | Stein, M. A., Leung, K. Y., Zwick, M., Garcia-del Portillo, F., and Finlay, B. B. (1996) Mol. Microbiol. 20, 151-164[Medline] [Order article via Infotrieve] |
16. | Brumell, J. H., Rosenberger, C. M., Gotto, G. T., Marcus, S. L., and Finlay, B. B. (2001) Cell. Microbiol 3, 75-84[CrossRef][Medline] [Order article via Infotrieve] |
17. | Guy, R. L., Gonias, L. A., and Stein, M. A. (2000) Mol. Microbiol. 37, 1417-1435[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Uchiya, K.,
Barbieri, M. A.,
Funato, K.,
Shah, A. H.,
Stahl, P. D.,
and Groisman, E. A.
(1999)
EMBO J.
18,
3924-3933 |
19. |
Hansen-Wester, I.,
Stecher, B.,
and Hensel, M.
(2002)
Infect. Immun.
70,
1403-1409 |
20. |
Freeman, J. A.,
Ohl, M. E.,
and Miller, S. I.
(2003)
Infect. Immun.
71,
418-427 |
21. | Knodler, L. A., Celli, J., Hardt, W. D., Vallance, B. A., Yip, C., and Finlay, B. B. (2002) Mol. Microbiol. 43, 1089-1103[CrossRef][Medline] [Order article via Infotrieve] |
22. | Ruiz-Albert, J., Yu, X. J., Beuzon, C. R., Blakey, A. N., Galyov, E. E., and Holden, D. W. (2002) Mol. Microbiol. 44, 645-661[CrossRef][Medline] [Order article via Infotrieve] |
23. | Méresse, S., André, P., Mishal, Z., Barad, M., Brun, N., Desjardins, M., and Gorvel, J. P. (1997) Electrophoresis 18, 2682-2688[Medline] [Order article via Infotrieve] |
24. | Deiwick, J., Nikolaus, T., Erdogan, S., and Hensel, M. (1999) Mol. Microbiol. 31, 1759-1773[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Datsenko, K. A.,
and Wanner, B. L.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
6640-6645 |
26. |
Valdivia, R. H.,
and Falkow, S.
(1997)
Science
277,
2007-2011 |
27. | Beuzon, C. R., and Holden, D. W. (2001) Microbes Infect. 3, 1345-1352[CrossRef][Medline] [Order article via Infotrieve] |
28. | Taylor, R. K., Miller, V. L., Furlong, D. B., and Mekalanos, J. J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2833-2837[Abstract] |
29. | Freter, R., O'Brien, P. C., and Macsai, M. S. (1981) Infect. Immun. 34, 234-240[Medline] [Order article via Infotrieve] |
30. |
Beuzon, C. R.,
Unsworth, K. E.,
and Holden, D. W.
(2001)
Infect. Immun.
69,
7254-7261 |
31. | Beuzon, C. R., Banks, G., Deiwick, J., Hensel, M., and Holden, D. W. (1999) Mol. Microbiol. 33, 806-816[CrossRef][Medline] [Order article via Infotrieve] |
32. | Hensel, M., Shea, J. E., Waterman, S. R., Mundy, R., Nikolaus, T., Banks, G., Vazquez-Torres, A., Gleeson, C., Fang, F. C., and Holden, D. W. (1998) Mol. Microbiol. 30, 163-174[CrossRef][Medline] [Order article via Infotrieve] |
33. | Brumell, J. H., Goosney, D. L., and Finlay, B. B. (2002) Traffic 3, 407-415[CrossRef][Medline] [Order article via Infotrieve] |
34. | Zerial, M., and McBride, H. (2001) Nat. Rev. Mol. Cell Biol. 2, 107-117[CrossRef][Medline] [Order article via Infotrieve] |
35. | Pfeffer, S. R. (2001) Trends Cell Biol. 11, 487-491[CrossRef][Medline] [Order article via Infotrieve] |
36. | Seabra, M. C., Mules, E. H., and Hume, A. N. (2002) Trends Mol. Med. 8, 23-30[CrossRef][Medline] [Order article via Infotrieve] |
37. | Hancock, J. F., Magee, A. I., Childs, J. E., and Marshall, C. J. (1989) Cell 57, 1167-1177[Medline] [Order article via Infotrieve] |
38. |
Zacharias, D. A.,
Violin, J. D.,
Newton, A. C.,
and Tsien, R. Y.
(2002)
Science
296,
913-916 |
39. | Nimchuk, Z., Marois, E., Kjemtrup, S., Leister, R. T., Katagiri, F., and Dangl, J. L. (2000) Cell 101, 353-363[Medline] [Order article via Infotrieve] |