Cellular Microbiology (2001) 3(8), 567±577

Remodelling of the actin cytoskeleton is essential forreplication of intravacuolar Salmonella

SteÂphane MeÂresse,1² Kate E. Unsworth,2² Anja

Habermann,3 Gareth Griffiths,3 Ferric Fang,4 MarõÂa

Jose MartõÂnez-Lorenzo,1 Scott R. Waterman,2 Jean-

Pierre Gorvel1 and David W. Holden2*1Centre d'Immunologie de Marseille-Luminy, INSERM-

CNRS-Univ.Med., Campus de Luminy, Case 906, 13288

Marseille Cedex 09, France. 2Department of Infectious

Diseases, Centre for Molecular Microbiology and

Infection, Imperial College School of Medicine, Armstrong

Road, London SW7 2AZ, UK. 3European Molecular

Biology Laboratory, Meyerhof Str. 1, 69012 Heidelberg,

Germany. 4University of Colorado Health Sciences

Center, 4200 E. Ninth Avenue, B168, Denver, CO 80262,

USA.

Summary

Maturation and maintenance of the intracellular

vacuole in which Salmonella replicates is controlled

by virulence proteins including the type III secretion

system encoded by Salmonella pathogenicity island

2 (SPI-2). Here, we show that, several hours after

bacterial uptake into different host cell types, Salmo-

nella induces the formation of an F-actin meshwork

around bacterial vacuoles. This structure is

assembled de novo from the cellular G-actin pool in

close proximity to the Salmonella vacuolar mem-

brane. We demonstrate that the phenomenon does

not require the Inv/Spa type III secretion system or

cognate effector proteins, which induce actin poly-

merization during bacterial invasion, but does require

a functional SPI-2 type III secretion system, which

plays an important role in intracellular replication and

systemic infection in mice. Treatment with actin-

depolymerizing agents significantly inhibited intra-

macrophage replication of wild-type Salmonella

typhimurium. Furthermore, after this treatment, wild-

type bacteria were released into the host cell

cytoplasm, whereas SPI-2 mutant bacteria remained

within vacuoles. We conclude that actin assembly

plays an important role in the establishment of an

intracellular niche that sustains bacterial growth.

Introduction

The process of phagocytosis is driven by the reorganiza-

tion of actin, which leads to engulfment and internalization

of large particles (. 0.5 mm). Phagosomes containing

non-pathogenic organisms and inert particles then inter-

act with components of the endocytic pathway and form a

mature phagolysosome, whose contents are usually

degraded. Although it is generally believed that actin is

depolymerized from the phagosome after internalization

(Aderem and Underhill, 1999), it has also been shown

recently that, in J774 macrophages, a proportion of

mature phagosomes containing inert particles is sur-

rounded by F-actin, and that the number of phagosomes

interacting with actin actually increases as they age

(Defacque et al., 2000).

Pathogenic intracellular bacteria either alter their

phagosomes in ways that avoid the terminal stages of

the degradative pathway or escape from this compart-

ment into the host cell cytosol (Meresse et al., 1999).

Several bacterial pathogens also modify the actin

cytoskeleton of host cells. These interactions can promote

uptake of bacteria into host cells, movement of bacterial

cells in the host cell cytoplasm or inhibition of phagocy-

tosis (Dramsi and Cossart, 1998; Donnenberg, 2000).

Modifications to the actin cytoskeleton by Gram-negative

bacteria are often controlled by effector proteins delivered

into the host cell through a type III secretion system

(TTSS) of the pathogen. The TTSS is a complex machine

comprising at least 20 proteins, some of which form a

secreton spanning the inner and outer membranes of the

bacterial cell. Transfer of effector proteins into the host

target cell is mediated by other secreted proteins, which

appear to form a pore in the host cell plasma membrane

(translocon) and connect this to the secreton (Hueck,

1998). In the case of Salmonella typhimurium, contact

with host cells activates a TTSS called Inv/Spa, which

secretes and translocates several effector proteins into

the host cell. One of these, SopE, activates Rho family

GTPases, which leads to actin cytoskeletal rearrange-

ments (Hardt et al., 1998). Another effector protein, SptP,

mediates the reversal of these rearrangements by acting

as a GTPase-activating protein (GAP) for Rac-1 and

Cdc42 (Fu and GalaÂn, 1999). Yet another effector, SipA,

complexes with the actin-bundling protein T-plastin, which

may stabilize actin filaments during invasion (Zhou et al.,

1999). A fourth SPI-1-secreted protein, SipC, nucleates

Q 2001 Blackwell Science Ltd

Received 18 June, 2001; accepted 18 June, 2001. ²The first twoauthors contributed equally to this work. *For correspondence. E-maild.holden@ic.ac.uk; Tel. (144) 20 7594 3073; Fax (144) 20 75943076.

and bundles actin in vitro, and these activities are

stimulated by SipA (Hayward and Koronakis, 1999;

McGhie et al., 2001).

Salmonella has a second type III secretion system

encoded by a pathogenicity island called SPI-2 (Ochman

et al., 1996; Shea et al., 1996). The SPI-2 TTSS is important

for systemic infection in mice (Hensel et al., 1995) and for

bacterial replication in macrophages (Ochman et al., 1996;

Cirillo et al., 1998; Hensel et al., 1998). The SPI-2 TTSS is

activated after bacteria enter into host cells and influences

the fate of the Salmonella-containing vacuole (SCV). The

SPI-2 effector protein SpiC inhibits interactions of the SCV

with late endosomes and lysosomes (Uchiya et al., 1999).

SPI-2 also inhibits trafficking of NADPH oxidase subunits to

the SCV, thereby avoiding exposure to the respiratory burst

(Vazquez-Torres et al., 2000; Gallois et al., 2001). The

vacuolar membrane surrounding intracellular S. typhimur-

ium is actively maintained by the pathogen, and this process

involves another SPI-2 effector, SifA (BeuzoÂn et al., 2000).

In this study, we have investigated the interaction between

intracellular S. typhimurium and the host cell actin cytoske-

leton. We found that replication of S. typhimurium within a

variety of host cell types was accompanied by F-actin

assembly in the vicinity of the SCV membrane, and this

phenomenon was dependent on the SPI-2 TTSS. Intrama-

crophage replication of wild-type S. typhimurium was

inhibited by actin-depolymerizing agents, which disrupted

the vacuolar membrane surrounding bacteria. SPI-2-

mediated assembly of F-actin on the SCV membrane

therefore represents a novel mechanism by which a

pathogen generates a specialized niche for intracellular

replication through manipulation of the host cell cytoskeleton.

Results

Actin reorganization during intracellular replication of wild-

type Salmonella

To determine whether Salmonella induces alterations to

the cytoskeleton during intracellular replication, we

examined the distribution of F-actin in a variety of cultured

cell types infected with S. typhimurium. Cells were

infected with wild-type S. typhimurium carrying a plasmid

constitutively expressing green fluorescent protein (GFP),

fixed at different time points and labelled with phalloidin±

Texas red to detect F-actin.

Confocal X/Y and X/Z imaging of infected HeLa cells

showed that, by 7 h after bacterial invasion, a meshwork of

F-actin was associated with SCVs (Fig. 1A). We next

examined actin localization in infected Swiss 3T3 fibroblasts,

as these cells have a well-studied cytoskeleton. Confocal X/

Z imaging showed that F-actin formed a dense nest-like

structuresurrounding clustersof replicating bacterial cells. In

X/Y sections, this often appeared as a ring (Fig. 1A). In the

Mel JuSo human melanoma cell line, S. typhimurium strain

SL1344 cell division is inhibited. As a result, multinucleoid

bacterial cells adopt a filamentous morphology (Martinez-

Lorenzo et al., 2001). F-actin was observed in tight

association with elongated bacterial cells at 24 h after

invasion (Fig. 1B). As macrophages are thought to be an

important site of intracellular replication by S. typhimurium

during systemic infection of mice, infected J774 murine

macrophage-like cells were also examined. In uninfected

cells, F-actin was localized mainly in the cortical region.

However, in infected cells, significant amounts of F-actin

were found associated with intracellular bacteria at16 h after

uptake (Fig. 1B). In addition to S. typhimurium strains 12023

and SL1344, similar F-actin rearrangements were induced

by Salmonella typhi, the causal agent of human typhoid

(Fig. 1C). Localized reorganization of the actin cytoskeleton

is therefore associated with intracellular replication of

different Salmonella serovars in representatives of four

differentiated cell types (macrophage, epithelial,melanocyte

and fibroblast), indicating that it is likely to be a general

characteristic of Salmonella during intracellular growth. As

the structure of the Salmonella-associated F-actin was

particularly well defined in Swiss 3T3 cells, these were used

for further characterization of the phenomenon.

The kinetics of F-actin accumulation in relation to

intracellular bacterial growth were examined over an 8 h

time course. Actin polymerization leading to membrane

ruffling is a characteristic response to S. typhimurium

invasion of host cells (GalaÂn and Zhou, 2000). However,

by 2 h after invasion, most intracellular bacteria had

moved away from the cell periphery and were no longer

associated with F-actin (Fig. 2A). By 4 h after invasion, F-

actin was detected in the vicinity of bacterial microcolo-

nies and continued to accumulate around clusters of

bacteria over the next 4 h. By 8 h after invasion, groups of

replicating bacteria were contained within a nest of

polymerized actin (Fig. 2A).

The proximity of F-actin to intracellular S. typhimurium

suggested that it might be intimately associated with the

vacuolar membrane surrounding the bacteria. To examine

this in more detail, infected cells were stained with

phalloidin and an antibody against LAMP-1, which is a

lysosomal membrane glycoprotein (lgp) also abundant in

the SCV membrane (BeuzoÂn et al., 2000). Confocal

microscopy showed a strong co-localization of F-actin and

LAMP-1 around bacterial cells (Fig. 2B). A similar pattern

of co-localization was observed in HeLa cells (unpub-

lished data). These results show that there is a close

association between F-actin and SCV membranes.

Actin reorganization requires the SPI-2 type III secretion

system

As interference with the actin cytoskeleton by Gram-negative

568 S. MeÂresse et al.

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bacteria is often mediated by TTSSs, we tested whether

actin reorganization during bacterial replication was

associated with either of the two TTSSs used by

Salmonella to secrete virulence proteins into the cytosol

of host cells. The prgH locus encodes an inner membrane

component of the Inv/Spa (SPI-1) secreton, and ssaV

encodes an essential component of the SPI-2 TTSS. Strains

carrying mutations in these genes are null for the functions of

Fig. 2. A. Assembly of F-actin during S.typhimurium replication in Swiss 3T3 cells.Confocal micrographs of cells infected withGFP-expressing wild-type S. typhimurium(green). After invasion, cells were fixed at thetimes shown, and F-actin was visualized withTexas red-conjugated phalloidin.B. Co-localization of F-actin and the Salmonellavacuolar membrane. Representative confocalmicrographs of Swiss 3T3 cells infected for16 h with wild-type S. typhimurium. F-actin wasvisualized by phalloidin staining, and antibodieswere used to label LAMP-1 and bacteria. Scalebar corresponds to 2 mm.

Fig. 1. Accumulation of F-actin during S. typhimurium intracellular replication. Confocal micrographs of representative infected cells. Bacterialstrains expressed GFP constitutively (green), and F-actin was visualized by phalloidin staining (red).A. HeLa and Swiss 3T3 cells were infected with wild-type S. typhimurium for 7 h and 8 h respectively. Right. XZ sections generated from a confocalZ-stack in the position indicated by the arrow.B. Mel JuSo cells and J774 macrophages were infected with wild-type S. typhimurium for 16 h and 24 h respectively.C. Swiss 3T3 cells were infected with S. typhi strain DTY8 for 8 h. Scale bar corresponds to 5 mm.

Actin remodelling by intravacuolar Salmonella 569

Q 2001 Blackwell Science Ltd, Cellular Microbiology, 3, 567±577

their respective secretion systems (Pegues et al., 1995;

BeuzoÂn et al., 1999; 2000). The ability of the prgH2 strain to

invade host cells was reduced compared with the wild-type

strain, but a small percentage of the inoculum was

internalized, and no defect in intracellular replication was

detected (by microscopy) among these bacteria (Fig. 3A).At

8 h after invasion, F-actin structures were associated with

clusters of wild-type or prgH2 mutant bacteria in over 90% of

infected host cells (Fig. 3A and B). Accumulation of F-actin

around intracellular bacteria was also consistently observed

in the small proportion of cells infected with strains carrying

mutations in the Inv/Spa effector genes sipA, sipC and sopE

(unpublished data). In contrast, the vast majority of ssaV2

mutant bacteria were not associated with any visible F-actin

(Fig. 3A and B). Similar results were obtained with S.

typhimurium strains carrying mutations in ssaJ or sseB,

which are required for SPI-2-mediated secretion and

translocation respectively (unpublished data).

Intracellular Salmonella-associated F-actin was also

examined by electron microscopy. Analysis of thin plastic

sections of infected cells revealed a meshwork of actin

filaments adjacent to vacuoles containing wild-type, but not

ssaV2 mutant bacteria (Fig. 4A). Furthermore, immunoe-

lectron microscopy using thawed cryosections labelled with

an anti-actin antibody revealed 47.0 ^ 8.1 gold parti-

cles mm22 in the area within 70 nm of the membrane

enclosing wild-type S. typhimurium. Only 7.1 ^ 2.2 parti-

cles mm22 were observed in the corresponding area

around the ssaV2 mutant (Fig. 4B). Therefore, reorganiza-

tion of actin during intracellular S. typhimurium replication

requires a functional SPI-2 secretion system.

Intracellular Salmonella induces de novo actin assembly

Reorganization of the actin cytoskeleton could occur by

recruitment of pre-existing F-actin to the SCV or

polymerization of actin monomers. To distinguish

between these possibilities, infected cells were incubated

with latrunculin B. Latrunculins form complexes with actin

Fig. 3. F-actin reorganization during S. typhimurium intracellularreplication requires a functional SPI-2 secretion system. Swiss 3T3cells were infected for 8 h with GFP-expressing wild-type, prgH2,ssaV2 or sifA2 S. typhimurium (green). F-actin was visualized byphalloidin staining (red).A. Representative confocal micrographs of cells infected with prgH2,ssaV2 or sifA2 bacteria. Scale bar corresponds to 2 mm.B. Percentage of infected cells in which bacterial microcolonies weresurrounded with F-actin. Only clusters of 8±20 bacteria were includedin the analysis. Results shown are the means ^ SEs of threeindependent experiments in which a total of 300 infected cells wasexamined for each strain.

Fig. 4. A. TEM of thin plastic sections. Arrowheads indicate actinfilaments.B. Actin immunogold labelling on thawed cryosections of Swiss 3T3cells infected for 8 h with wild-type or ssaV2 S. typhimurium.

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monomers, thereby excluding them from assembly into

filaments (Coue et al., 1987; Morton et al., 2000). The

dense ring of F-actin surrounding bacteria (Fig. 5A and B)

did not accumulate in cells treated with latrunculin

(Fig. 5A and B). However, if the drug was removed by

thorough washing of cells after 6 h of exposure, distinct

F-actin rings were detectable around 78 ^ 9.2% of wild-

type S. typhimurium microcolonies within 5 min (Fig. 5A

and B). This did not reflect non-specific assembly on

phagosomal membranes caused by a sudden increase in

available cellular G-actin, because only 5.3 ^ 2.3% of

Fig. 5. Intracellular S. typhimurium induces de novo polymerizationof actin. Swiss 3T3 cells were infected with GFP-expressing wild-type or ssaV2 S. typhimurium and fixed 9 h after invasion. F-actinwas visualized by phalloidin staining. Where indicated (1LB),latrunculin B (LB) was added (1 mg ml21) 3 h after invasion. Wherestated, 6 h after addition, the drug was removed by thoroughwashing, and cells were incubated for a further 5 min beforefixation (1wash). Scale bar corresponds to 2 mm.B. Percentage of infected cells in which bacteria were associated withF-actin after each treatment. Black and white bars indicate wild-typeand ssaV2 strains respectively. Results shown are the means ^ SEsof three independent experiments, in which a total of 70 infected cellswas examined for each condition.

Fig. 6. Effect of latrunculin B and cytochalasin D on intracellularreplication by S. typhimurium strains.A and B. Intracellular replication/survival of wild-type (black bars) andssaV2 (white bars) S. typhimurium in RAW (A) and periodate-elicited(B) macrophages. Where indicated, latrunculin B (LB) or cytochalasinD (CD) was added to a final concentration of 1 mg ml21 at 3 h afteruptake of bacteria. Fold increase represents the ratio of intracellularbacteria at 16 h and 2 h. Data are representative of threeindependent experiments.C. Association of F-actin with intracellular S. typhimurium inperiodate-elicited macrophages. Macrophages were infected for 16 hwith GFP-expressing wild-type or ssaV2 S. typhimurium. F-actin wasvisualized by phalloidin staining. Scale bar corresponds to 2 mm.

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Q 2001 Blackwell Science Ltd, Cellular Microbiology, 3, 567±577

ssaV2 mutant bacteria were associated with F-actin after

similar treatment (Fig. 5A and B). Therefore, S. typhimur-

ium induces SPI-2-dependent polymerization of actin from

the cellular G-actin pool.

Actin assembly is essential for intracellular replication of

S. typhimurium

Systemic growth of S. typhimurium is correlated with an

ability to replicate within macrophages (Fields et al., 1986).

When added to infected RAW macrophages 3 h after

bacterial uptake, latrunculin B or cytochalasin D had little

effect on the numbers of ssaV2 mutant bacteria 13 h later,

but significantly inhibited replication of wild-type S. typhimur-

ium (Fig. 6A). Furthermore, lactrunculin B significantly

affected the survival of wild-type S. typhimurium in murine-

elicited peritoneal macrophages (Fig. 6B). SPI-2-dependent

F-actinwasalso found tobeassociatedwithbacteria in these

cells (Fig. 6C). These data suggest that SPI-2-mediated

actin assembly plays an important role in intracellular

replication, and therefore virulence, of S. typhimurium.

Maintenance of the vacuolar membrane enclosing wild-

type S. typhimurium requires the actin cytoskeleton

Intracellular replication of Salmonella is dependent, in

part, on SPI-2-mediated maturation of the SCV (Uchiya

et al., 1999; BeuzoÂn et al., 2000). We therefore

investigated whether actin assembly is also involved in

this process. Infected RAW macrophages were incubated

Fig. 7. Effect of latrunculin B and cytochalasinD on trafficking of S. typhimurium strains inRAW macrophages. Where indicated,latrunculin B (LB) or cytochalasin D (CD) wasadded to a final concentration of 1 mg ml21 at3 h after uptake of bacteria.A. Confocal micrographs of representative cellsinfected with GFP-expressing wild-type orssaV2 bacteria. An antibody was used to labelLAMP-1. Scale bar corresponds to 2 mm.B. Co-localization of S. typhimurium wild-type(black bars) and ssaV2 (white bars) strains withLAMP-1 at 16 h after uptake. Results shownare the means ^ SEs of three independentexperiments.C. Streptolysin O permeabilization of cellsinfected with wild-type (black bars) or sifA2

(hatched bar) S. typhimurium strains. At 12 hafter uptake, cells were permeabilized withstreptolysin O, and bacteria exposed to thecytosol were labelled with anti-Salmonellaantibody in the absence of a permeabilizingagent. The vacuolar membrane around anintracellular sifA2 mutant strain of S.typhimurium is gradually lost (BeuzoÂn et al.,2000) and this strain is included as a control.Results are the means ^ SEs of threeindependent experiments. In each experiment,over 100 bacteria were examined for eachtreatment.

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with latrunculin B or cytochalasin D, then fixed and

stained for LAMP-1. This lgp was present on the majority

of SCVs containing wild-type or ssaV2 mutant bacteria

16 h after bacterial uptake (Fig. 7A and B). However, if

either actin-depolymerizing drug was added to cells 3 h

after bacterial uptake, less than 20% of wild-type bacteria

were associated with LAMP-1 at the 16 h timepoint

(Fig. 7A and B). In contrast, over 75% of vacuoles

containing ssaV2 mutant bacteria retained this marker

after the same treatments (Fig. 7A and B).

The reduced association between wild-type bacteria

and LAMP-1 in the presence of actin-depolymerizing

agents suggested that either the trafficking of LAMP-1 to

the SCV was altered or the bacteria were no longer within

a vacuole. The integrity of the vacuolar membrane was

investigated by testing the accessibility of intracellular S.

typhimurium to anti-Salmonella antibody in macrophages

treated with the pore-forming toxin streptolysin O (BeuzoÂn

et al., 2000). At 12 h after uptake, 71.3% of wild-type

bacteria in permeabilized cells were protected from the

antibody (Fig. 7C). However, incubation of infected cells

with latrunculin B or cytochalasin D resulted in a

significant reduction in the number of S. typhimurium

enclosed by an intact membrane (to 38.0% and 41.4%

respectively; Fig. 7C). This was not a non-specific effect

on the integrity of vesicular compartments, because the

membrane enclosing ssaV2 mutant bacteria was not

affected by the drugs (Fig. 7A and B). These experiments

show that maintenance of an intact vacuolar membrane

by intracellular wild-type S. typhimurium is dependent on

the actin cytoskeleton.

Discussion

In this paper, we have shown that intracellular Salmonella

induces the formation of a meshwork of F-actin in the

vicinity of replicating bacteria in a variety of host cell

types. The ability of extracellular Salmonella to induce

actin rearrangements via Rho subfamily GTPases is a

well-established activity of the Inv/Spa TTSS (GalaÂn and

Zhou, 2000). These rearrangements occur during the

process of cell invasion and are short-lived, with host cells

regaining a normal cytoskeleton shortly after bacterial

uptake (Takeuchi, 1967; Fu and GalaÂn, 1999). The

assembly of F-actin that we report here is independent

of this process and is more reminiscent of the de novo

assembly of actin on latex bead phagosomes shown by

Defacque et al. (2000). It first becomes detectable by

microscopy < 4 h after invasion of 3T3 cells and is

unaffected by mutation of a gene encoding a component

of the Inv/Spa secreton, but does require a functional SPI-

2 secretion system.

The G-actin-sequestering agent latrunculin B had a

significant inhibitory effect on the accumulation of F-actin

around intracellular S. typhimurium, suggesting that it is

polymerized in situ from the cellular G-actin pool. The

rapid accumulation of F-actin after the removal of

latrunculin also suggests that factors necessary for actin

polymerization are secreted by wild-type bacteria via the

SPI-2 TTSS even in the presence of the drug, and

assembly occurs as soon as actin monomers become

available. Latrunculin B and cytochalasin D also caused

the release of wild-type Salmonella into the cytosol of

macrophages. This was not a non-specific effect on the

integrity of vesicular compartments, because the mem-

brane enclosing ssaV2 mutant bacteria was insensitive to

the drugs. This differential effect may reflect the different

intracellular trafficking pathways of wild-type and SPI-2

mutant bacteria. Whereas the SPI-2 mutant vacuolar

membrane is probably derived from interactions with late

endocytic compartments (Uchiya et al., 1999), wild-type

SCVs become segregated from the endocytic pathway,

and their membrane is actively recruited and/or stabilized

by the bacteria (BeuzoÂn et al., 2000). Our experiments

show that SPI-2-mediated actin assembly is an integral

aspect of this process. Furthermore, the inhibitory effect

of actin-depolymerizing agents on intramacrophage repli-

cation shows that SPI-2-mediated actin assembly is likely

to play an important role in the virulence of S. typhimur-

ium.

We have shown recently that the S. typhimurium

protein SifA acts together with the SPI-2 TTSS to maintain

the integrity of the intracellular vacuolar membrane

(BeuzoÂn et al., 2000). Approximately 5 h after uptake,

intracellular sifA2 mutant bacteria begin to lose their

vacuolar membranes and are released into the cytosol of

host cells (BeuzoÂn et al., 2000). Although SifA was

therefore a likely candidate for an effector protein inducing

actin assembly, this can be ruled out because F-actin was

consistently present around sifA2 bacteria before the loss

of their vacuolar membrane (Fig. 3A and B). We conclude

from this result that, although SifA is involved in vacuolar

membrane recruitment, there is an additional SPI-2

effector that also plays an important role in this process

through the assembly of F-actin. There are numerous

candidates for this effector (Miao et al., 1999; Miao and

Miller, 2000; Worley et al., 2000). We propose that actin

assembly is essential for subsequent SifA-mediated

events that provide SCVs with the increasing membrane

surface area necessary to enclose replicating bacteria

(BeuzoÂn et al., 2000). Actin filaments could have a role in

the recruitment of specific subsets of membrane vesicles

or promote their selective fusion with the SCV. Prece-

dents for this type of activity include the involvement of the

actin cytoskeleton in the movement of LAMP-1-enriched

vesicles (Taunton et al., 2000) and interactions between

latex bead phagosomes and endocytic organelles (Defac-

que et al. 2000; Jahraus et al., 2001).

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Actin filaments assembled on the vacuolar membrane

may also physically block fusion with some endocytic

compartments (Valentijn et al., 1999; 2000). The SPI-2

TTSS is involved in the ability of Salmonella to prevent

trafficking of components of the NADPH oxidase to SCVs

(Vazquez-Torres et al. 2000; Gallois et al., 2001). The

membrane-bound and cytosolic components are

assembled into a complex mainly on phagocytic vacuoles

(DeLeo and Quinn, 1996; Vazquez-Torres et al., 2000),

and this process is known to involve the actin cytoskele-

ton (Dusi et al., 1996; Grogan et al., 1997). Deviation of

SCVs from the endocytic route and pathogen-driven

remodelling of the actin cytoskeleton may interfere with

this process, thereby inhibiting the formation of a

functional NADPH oxidase complex.

In addition to SPI-1- and SPI-2-mediated cytoskeletal

reorganization, it has recently been shown that SpvB, an

S. typhimurium virulence protein, specifically ADP-ribosy-

lates actin in infected cells (Lesnick et al., 2001; Tezcan-

Merdol et al., 2001). The physiological significance of the

resulting actin depolymerization is unknown, but is

unlikely to be directly related to the phenomenon

described here for three reasons. First, genetic studies

have demonstrated that the functions of SPI-2 and the spv

locus in vivo are unrelated (Shea et al., 1999). Secondly,

S. typhi, shown here to be proficient for intracellular F-

actin assembly (Fig. 1C), does not contain any homo-

logues of spvB within its genome sequence (unpublished

data). Thirdly, mutation of the active site of SpvB (Lesnick

et al., 2001) did not affect the ability of intracellular S.

typhimurium to assemble F-actin (data not shown).

Nevertheless, it is remarkable that three major indepen-

dent virulence functions of S. typhimurium involve

remodelling of the host cell cytoskeleton.

A variety of bacterial pathogens manipulate the actin

cytoskeleton in ways that promote or prevent the uptake

of extracellular bacteria into host cells or facilitate move-

ment through the host cell cytosol after escape from the

vacuole (Dramsi and Cossart, 1998; Donnenberg, 2000).

We have shown here that a bacterial pathogen can also

stimulate actin assembly from within a vacuole, and that

this activity is required for the recruitment or maintenance

of the vacuolar membrane. There is indirect evidence for

a role of cytoskeletal remodelling in the replication or

trafficking of several other intravacuolar bacterial patho-

gens. For example, a host protein called TACO is

recruited to and retained on J774 phagosomal mem-

branes enclosing Mycobacterium bovis (Ferrari et al.,

1999). TACO belongs to the WD repeat family, members

of which are involved in cytoskeletal organization and

vesicle fusion (Pryer et al., 1993; Salama et al., 1993),

and has significant similarity to the actin-binding protein

coronin (de Hostos et al., 1991). Replication of Mycobac-

terium avium within bone marrow-derived macrophages is

accompanied by disruption of the cells' actin filament

network (Guerin and de Chastellier, 2000). It has also

been demonstrated that intracellular growth of both

Legionella pneumophila and Chlamydia trachomatis is

inhibited by cytochalasin D (Elliott and Winn, 1986;

Schramm and Wyrick, 1995). Therefore, bacterial manip-

ulation of the actin cytoskeleton from within a vacuole may

represent a general theme in pathogen-controlled phago-

some maturation.

Experimental procedures

Bacterial strains, plasmids and growth conditions

Salmonella typhimurium NCTC 12023 was used as the wild-type strain in all experiments except for the infection of MelJuSo cells, in which SL1344 (Wray and Sojka, 1978) wasused. Strains HH109 (ssaV::aphT in 12023) (Hensel et al.,1998), P3H6 (sifA::mTn5 in 12023) (BeuzoÂn et al., 2000) andHH130 (prgH::TnphoA in 12023) (BeuzoÂn et al., 1999) havebeen described previously. Strain DTY8 (aroCD in S. typhiTy2) was a gift from D. Pickard, Imperial College of Science,Technology and Medicine, London, UK. Bacteria were grownin Luria±Bertani medium supplemented with ampicillin(50 mg ml21) or kanamycin (50 mg ml21) where appropriate.Strain DTY8 was grown in LB medium supplemented with40 mg ml21 phenylalanine, 40 mg ml21 tryptophan,10 mg ml21 para-aminobenzoic acid, 10 mg ml21 dihydrox-ybenzoic acid and 40 mg ml21 tyrosine.

Antibodies and reagents

Texas red-conjugated phalloidin was purchased from Mole-cular Probes and used at a dilution of 1:50. The mousemonoclonal antibody a-LAMP-1 H4A3 developed by J. T.August and J. E. K. Hildreth was obtained from theDevelopmental Studies Hybridoma Bank developed underthe auspices of the NICHD and maintained by the Universityof Iowa (Department of Biological Sciences, Iowa, IA, USA)and was used at a dilution of 1:2000. Rabbit polyclonalantibody a-LAMP-1 156 (Steele-Mortimer et al., 1999) wasused at a dilution of 1:1000. Texas red sulphonyl chloride(TRSC)-conjugated donkey a-mouse, a-rabbit and a-goatantibodies were purchased from Jackson ImmunoresearchLaboratories and used at a dilution of 1:400. Bacteria wereusually visualized by the introduction of plasmid pFVP25.1,carrying gfpmut3A under the control of a constitutivepromoter (Valdivia and Falkow, 1996). For the experimentsin Figs 2B and 7C, a-Salmonella goat polyclonal antibodyCSA-1 (purchased from Kirkegaard and Perry Laboratories)was used at a dilution of 1:400.

Cell culture

RAW 264.7 murine macrophage-like cells were obtained fromECACC (ECACC 91062702). J774A.1 murine macrophage-like cells were a gift from Dr V. Snewin (Department ofInfectious Diseases, Imperial College School of Medicine atSt Mary's, London, UK). HeLa (clone HtTA1) cells were kindly

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provided by Dr H. Bujard (Heidelberg, Germany). Swiss 3T3murine fibroblast cells and Mel JuSo cells were kindlyprovided by Dr G. Tran van Nhieu (Institut Pasteur, Paris,France) and Dr J. Neefjes (The Netherlands Cancer Institute,Amsterdam, The Netherlands) respectively. Cells were grownin Dulbecco's modified Eagle medium (DMEM) supplemen-ted with 10% fetal calf serum (FCS) and 2 mM glutamine at378C in 5% CO2.

Peritoneal cells were harvested 4 days after BALB/c micewere inoculated intraperitoneally with 5 mM periodate (DeGroote et al., 1997). Cells were plated at a density of5.5 � 105 cells per well in 24-well microtitre dishes andallowed to adhere for 2 h. Non-adherent cells were flushedout with prewarmed RPMI containing 10% FCS. Theadherent macrophages were incubated for a further 48 hbefore infection.

Bacterial infection of cultured cells

Macrophages were infected with opsonized, stationary phaseS. typhimurium as described previously (BeuzoÂn et al., 2000).HeLa, 3T3 and Mel JuSo cells were infected with exponentialphase S. typhimurium as described previously (BeuzoÂn et al.,2000). In order to follow a synchronized population ofbacteria, host cells were washed after 15 min of exposureto S. typhimurium and subsequently incubated in mediumcontaining gentamicin to kill extracellular bacteria. Thisprocedure was also used for HeLa cell infections with S.typhi DTY8, except that invasive bacteria were obtained byculturing in LB medium containing 3 M NaCl to an OD600 of1.5. During infection with this strain, cells were cultured inDMEM supplemented with 40 mg ml21 phenylalanine,40 mg ml21 tryptophan, 10 mg ml21 para-aminobenzoicacid, 10 mg ml21 dihydroxybenzoic acid and 40 mg ml21

tyrosine.Latrunculin B or cytochalasin D was added to infected cell

monolayers to a final concentration of 1 mg ml21 whereindicated. Macrophages rounded up shortly after exposure tothese drugs but, in control experiments, there was nodetectable increase in cytotoxicity (as measured by releaseof lactate dehydrogenase into the culture medium), and thedrugs did not cause detachment of cells from the plasticsurface (as determined microscopically). The drugs(1 mg ml21) had no detectable effects on the bacterial growthrate in DMEM (result not shown).

Immunofluorescence and electron microscopy

For immunofluorescence, cells were fixed in paraformalde-hyde, stained, mounted and analysed by confocal micro-scopy as described previously (BeuzoÂn et al., 2000). Imageswere processed using Adobe PHOTOSHOP 5.0. For transmis-sion electron microscopy, infected Swiss 3T3 cells were fixedin 1% glutaraldehyde and 1% OsO4 in phosphate buffer andprepared as described previously (Tilney et al., 1998). Forcryosections, cells were fixed in 4% paraformaldehyde and0.1% glutaraldehyde in PBS for 1 h. The samples wereprepared for cryosectioning and labelling with anti-actinantibody (kindly provided by Dr G. Gabbiani, Geneva,Switzerland) and quantified as described previously (Griffiths,

1993). Only gold particles within 70 nm of the vacuolarmembrane were counted, and results are expressed as goldparticles mm22.

Streptolysin O permeabilization of RAW cells

Streptolysin O was obtained from Corgenix UK. StreptolysinO permeabilization was performed as described previously(BeuzoÂn et al., 2000), except that goat a-Salmonella wasused to detect cytosolic bacteria. To prevent differences inpermeabilization efficiency from biasing results, only cells inwhich at least one bacterium was stained with anti-Salmonella antibody were included in the analysis.

Acknowledgements

We are indebted to Michael Way and Emmanuelle Caron for

providing materials and for valuable advice. We thank Carmen

BeuzoÂn and Christoph Tang for helpful discussions, Javier RuõÂz-

Albert and Sezgin Erdogan for practical assistance, andmembers of our laboratories for critical review. We are grateful

to Jorge GalaÂn for sipA2 and sopE2, and to Sam Miller for sipC±

and prgH2 mutant strains. This work was supported by grantsfrom the Medical Research Council (UK) to D.W.H. and from

CNRS and INSERM to S.M.

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