Review

Salmonella-induced tubular networksNina Schroeder1, Luıs Jaime Mota2 and Stephane Meresse3,4,5

1 Institute of Molecular Biology, Academia Sinica, Academia Rd. 128 Sec. 2, Nankang, Taipei 115, Taiwan, ROC2 Infection Biology Laboratory, Instituto de Tecnologia Quımica e Biologica, Universidade Nova de Lisboa,

Av. Republica, 2780-157 Oeiras, Portugal3 Centre d’Immunologie de Marseille-Luminy, Universite de la Mediterranee, Parc Scientifique de Luminy,

Case 906, 13288 Marseille Cedex 9, France4 Inserm, U631, Marseille, France5 CNRS, UMR6102, Marseille, France

Glossary

Brefeldin A (BFA): a drug produced by fungi that blocks the activation of the

host cell small GTPase Arf1 and thus prevents the formation of coat protein

complex I (COPI)-coated vesicles, leading to Golgi redistribution into the

endoplasmic reticulum.

LAMP1-negative tubules (LNTs): Salmonella-induced tubules enriched in

bacterial T3SS-2 effectors but lacking host cell LAMP1 and SCAMP3.

Rab-interacting lysosomal protein (RILP): a host cell protein that is an effector

of the host cell small GTPase Rab7.

Salmonella spp.: the Salmonella genus includes two species, Salmonella

bongori and Salmonella enterica. S. enterica comprises >2500 serovars,

including serovars Typhi and Typhimurium. Salmonella enterica serovar

Typhimurium (S. Typhimurium; previously known as S. typhimurium) has

been widely used as model for systemic infection in mice and to analyse

molecular and cellular mechanisms that underlie Salmonella virulence. For

simplicity, the generic term Salmonella is generally used instead of S.

Typhimurium.

Salmonella-containing vacuole (SCV): a membrane-bound compartment

where intracellular S. enterica resides and multiplies.

Salmonella-induced filaments (SIFs): Salmonella-induced tubules enriched in

host cell late endocytic proteins, such as LAMP1, and bacterial T3SS-2

effectors.

Salmonella-induced SCAMP3 tubules (SISTs): Salmonella-induced tubules

enriched in the host cell secretory carrier membrane protein 3 (SCAMP3) and

bacterial T3SS-2 effectors, but lacking LAMP1 and other late endocytic

markers.

Salmonella pathogenicity islands: a pathogenicity island is a large region of

DNA that encodes virulence genes and that is acquired by horizontal gene

transfer. Salmonella pathogenicity island-1 (SPI-1) encodes Salmonella T3SS-1

and Salmonella pathogenicity island-2 (SPI-2) encodes Salmonella T3SS-2.

SifA and Kinesin-interacting protein (SKIP): a host cell protein that interacts

both with the Salmonella effector SifA and with the host cell molecular motor

kinesin-1.

Spacious vacuole associated tubules (SVATs): Salmonella-induced tubules

enriched in the host cell protein sorting nexin 1.

Type III secretion system (T3SS): a mechanism used by several Gram-negative

Salmonella virulence relies on its capacity to replicateinside various cell types in a membrane-bound compart-ment, the Salmonella-containing vacuole (SCV). Aunique feature of Salmonella-infected cells is the pres-ence of tubular structures originating from and con-nected to the SCV, which often extend throughout thecell cytoplasm. These tubules include the well-studiedSalmonella-induced filaments (SIFs), enriched in lyso-somal membrane proteins. However, recent studiesrevealed that the Salmonella-induced tubular networkis more extensive than previously thought and includesthree types of tubules distinct from SIFs: sorting nexintubules, Salmonella-induced secretory carrier mem-brane protein 3 (SCAMP3) tubules and lysosome-associ-ated membrane protein 1 (LAMP1)-negative tubules. Inthis review, we examine the molecular mechanismsinvolved in the formation of Salmonella-induced tubularnetworks and discuss the importance of the tubules forSalmonella virulence and establishment of a Salmonellaintracellular replicative niche.

Salmonella infections and tubule formationSalmonella enterica is a Gram-negative facultative intra-cellular pathogen that causes disease in humans andanimals [1]. The diseases caused by Salmonella infectionsdepend both on host susceptibility and on the S. entericaserovar. For example, in humans, serovars Typhi or Para-typhi cause typhoid fever while serovars Typhimurium orEnteritidis usually cause a self-limiting gastroenteritis. Ingenetically susceptible mouse strains, S. enterica serovarTyphimurium (S. Typhimurium) induces a systemic illnesssimilar to typhoid fever [2].

Salmonella and other intracellular bacterial patho-gens (including Brucella, Chlamydia, Legionella andMycobacteria) have the ability to multiply inside a mem-brane-bound compartment called the vacuole [3]. Grow-ing within a vacuole presents important challenges tothe pathogen such as how to resist host defence mechan-isms, obtain nutrients and membrane for the expansionof the replicative niche and maintain the stability of thevacuole. To overcome these difficulties, pathogens oftenmanipulate eukaryotic host functions by translocatingbacterial virulence proteins (effectors) across their vacu-olar membrane.

Corresponding author: Meresse, S. (meresse@ciml.univ-mrs.fr).

268 0966-842X/$ – see front matter � 2011 Elsevier Ltd. All rights r

S. enterica use two distinct type III secretion systems(T3SSs) (see Glossary) encoded on Salmonella pathogenic-ity islands (SPIs) 1 and 2 to inject more than 30 effectorproteins [4]. The SPI-1-encoded T3SS (T3SS-1) is requiredfor the invasion of non-phagocytic cells [5]. T3SS-1 expres-sion is induced in the intestinal lumen and enables Salmo-nella to cross the epithelial gut barrier [6] and promoteintestinal inflammation [7]. The SPI-2-encoded T3SS(T3SS-2) mediates intracellular bacterial replication andis necessary for the establishment of systemic disease [8].However, the boundaries between T3SS-1 and T3SS-2functions are not very sharply defined because T3SS-2participates in intestinal inflammation [9] and T3SS-1effectors are involved in late stages of the intracellularlife style of Salmonella [10–12]. Further details on the

bacteria to translocate proteins (known as effectors) from the bacterial

cytoplasm into the cytosol and membranes of eukaryotic host cells.

eserved. doi:10.1016/j.tim.2011.01.006 Trends in Microbiology, June 2011, Vol. 19, No. 6

Review Trends in Microbiology June 2011, Vol. 19, No. 6

function of these effectors can be found below and inseveral comprehensive reviews on this subject [2,8,13,14].

S. Typhimurium induces the formation of tubular net-works, which extend from the Salmonella-containing vac-uole (SCV) and are coupled to the remodelling of host cellcompartments by translocated effectors [15–20]. Until veryrecently, the only known Salmonella-induced tubules werethe Salmonella-induced filaments (SIFs). SIFs were firstdescribed by Garcia-del Portillo and colleagues in 1993 [15]and have been extensively studied since then [21,22]. SIFsare rich in late endocytic markers, such as lysosome-asso-ciated membrane proteins (LAMPs). Recent studieshighlighted an unexpected level of complexity in the com-position and regulation of tubules associated to the SCV. Inaddition to SIFs, S. Typhimurium is able to induce sortingnexin (SNX) tubules [18,19], Salmonella-induced secretorycarrier membrane protein 3 (SCAMP3) tubules (SISTs)[16] and LAMP1-negative tubules (LNTs) [17].

In this review, we summarise and discuss the latestfindings on how S. Typhimurium modulates intracellularmembrane trafficking leading to the formation of distincttubular networks during host cell infection.

SCV biogenesis and maturationThe maturation of the SCV has been extensively studiedafter Salmonella T3SS-1-dependent invasion of epithelialcell lines. SCVmaturation is characterised by the transient[()TD$FIG]

+

Effectors

Time p.i.

EventSpacious SCVSVATSNX3 tubule

RepLNTSIF

10 min - 1 h

SopB Pip

1 h - 3 h

MaturatingSCV

+

L

Figure 1. Salmonella-induced tubular networks. After its internalisation, Salmonella (gre

post-invasion (p.i.), the SNX1-positive SVATs are formed, followed by the formatio

accumulation of the phosphoinositide PI(3)P necessary for the recruitment of SNX1

replication niche, close to the microtubule-organising centre (MTOC). The LNTs are form

lysosomal glycoproteins to the SCV. Next, SIFs start to form and SIST formation is del

presence of early endosomal markers followed by the ac-quisition of late endosomal markers including Rab7,LAMPs, and vacuolar adenosine triphosphatase (vATPase)[12,23]. Lysosomal hydrolases and their receptor, mannose6-phosphate receptor (M6PR), were initially reported to beexcluded from the SCV [24]. However, recent live cellimaging studies suggested that the SCV fuses with lyso-somes and continuously communicates with the endocyticpathway [25]. It could be that the amount of M6PR orlysosomal hydrolases recruited to the SCV is too low toallow their detection. Alternatively, S. Typhimuriummight selectively avoid the delivery of lysosomal hydro-lases by affecting trafficking of M6PR [12,18].

SCV maturation is accompanied by a spatial shift of thevacuole from the site of internalisation to the juxtanuclearregion adjacent to the microtubule-organising centre(MTOC) (Figure 1) [26]. Rab7 and its effector, Rab-interact-ing lysosomal protein (RILP), control the movement of theSCV. RILP binds dynactin, which results in recruitment ofthe dynein/dynactin motor complex to the SCV membrane,triggering centripetal movement of the vacuole [27] (Box 1).As a result, at �2 h post-invasion most SCVs localise to aperinuclear region within 5 mm of the MTOC [26].

Salmonella-induced tubular networksSCV maturation and intravacuolar bacterial replicationare both associated with the appearance of different

N

lication formation and vesicle recruitment

and SIST formation

3 h - 16 h

B2, SifA, SseJ, SopD2, SseG, SseF

GolgiTGN

SCAMP3

LAMPs

MTOC

T3SS-2effectors

SNX1

SNX3

E/LY

N

+

+

SIF

LNT

SIST

-

Key:

TRENDS in Microbiology

en rod) resides in a spacious vacuole that is positive for SNX1 and SNX3. At 10 min

n of SNX3 tubules at 30 min p.i. The Salmonella effector SopB mediates the

and SNX3 on the vacuolar membrane. Then, the SCV matures and reaches its

ed, contact late endosomal vesicles (LE/LY) and might support the recruitment of

ayed compared to LNT and SIF (4–5 h p.i.). Abbreviations: N, nucleus.

269

Box 1. Molecular motors and their cytoskeletal tracts

Molecular motors, such as myosin, dynein or kinesin, drive the

intracellular transport of vesicles, organelles and protein complexes.

These molecules move along tracks, actin filaments and micro-

tubules that, together with the intermediate filaments, constitute the

cytoskeleton of the cell. Myosins move on actin filaments, whereas

dyneins and kinesins travel along microtubules. Many pathogens

move inside their host cell by manipulating the actin and micro-

tubule networks [66,67]. Over the past 7 years, the interactions of

Salmonella with microtubule motors have been under intense

investigation [67]. Microtubules are polar filaments that have their

minus ends cluster around the microtubule organizing centre

(MTOC) and their plus ends pointing towards the cell periphery

[68]. Their surface is structurally asymmetric so that the motor can

read the direction toward which the filament is pointing. Motor-

mediated transport requires binding of the motor domain to the

cytoskeletal scaffold as well as the association of the tail domain

with the cargo. Movement along the cytoskeleton requires hydro-

lysis of ATP by the motor domain. Dyneins are minus end directed

motors whereas most kinesins transport to the plus ends of

microtubules [69]. Both dyneins and kinesins are members of two

large protein superfamilies [70,71]. Cytoplasmic dynein-1 is a large

complex of proteins [72] that associates with dynactin, which

mediates some interactions with cargo [73] and enhances motor

processivity, that is, its ability to move over long distances without

dissociating from the microtubule. Small GTPases of the Rab family

regulate the recruitment of this motor protein to membranes [69].

GTP-bound Rab7 induces the recruitment of dynein/dynactin on late

endosomal compartments through its effector, the Rab-interacting

lysosomal protein (RILP) [74]. Kinesins are found in all eukaryotic

organisms to mediate intracellular transport pathways [70]. Con-

ventional kinesin-1 is composed of two heavy chains (KHC) and two

light chains (KLC). The KHC contains the motor domain, which

comprises an ATP-binding sequence and a microtubule-binding

sequence. The KLC contains the cargo-binding domain, composed

of six tetratricopeptide repeats (TPRs) [75]. Rab GTPases are also

important mediators for the recruitment and/or regulation of

kinesins. For example, Rab5 regulates a kinesin-3-mediated trans-

port of early endosomes [76].

[()TD$FIG]

LAMP 1 PipB2 SCAMP3

Wild-type sifA- sifA-sopD2-

LAMP 1PipB2

(a)

(b) (c) (d)

TRENDS in Microbiology

Salmonella

Figure 2. Late tubules in Salmonella-infected cells. HeLa cells infected for 16 h with

S. Typhimurium show different kinds of tubules. (a) The infected cell is

characterised by the presence of LAMP1 (green) and/or SCAMP3 (blue) tubules

while a vesicular and peri-nuclear distribution of these molecules is observed in

the non-infected cell (left upper cell). SIFs (green arrows) are positive for LAMP1

and SCAMP3, whereas SISTs (blue arrows) are negative for LAMP1. PipB2 (red) is

present on every tubule and accumulates at their outward extremities (red arrows).

(b-d) HeLa cells infected with various Salmonella strains (blue) and

immunolabelled for LAMP1 (green) and PipB2 (red). (b) LAMP1- and/or PipB2-

positive tubules (green and red arrows, respectively) are observed in cells infected

with wild type Salmonella. (c) Effectors accumulate on sifA– SCVs (arrowheads).

(d) Effector-positive tubules form in the absence of SifA and SopD2. These tubules

are negative for LAMPs (white arrows). Scale bars, 20 mm.

Review Trends in Microbiology June 2011, Vol. 19, No. 6

tubular networks: first, the T3SS-1-dependent SNXtubules, and later the T3SS-2-dependent SIFs, SISTsand LNTs (Figures 1 and 2). S. Typhimurium inducesthe formation of SNX tubules 15–60 min post-invasionand before intracellular replication starts [18,19]. Theformation of early SNX tubules is dependent on the trans-location of the T3SS-1 effector SopB [18,19]. The bacteriastart to replicate after 3–4 h. This is concomitant withactivation of T3SS-2, formation of SIFs [15,21,22] andLNTs [17], and precedes formation of SISTs [16]. SeveralT3SS-2 effectors localise to the SCV and to the emergingtubules (Figures 1 and 2 and Table 1). SISTs containeffectors and SCAMP3, which concentrates on the trans-Golgi network (TGN) in uninfected cells, but do not containlate endosomal markers (LAMPs and vATPase); SIFs con-tain effectors, LAMPs and other late endocytic markers,and SCAMP3; LNTs contain effectors (Figures 1 and 2 andTable 2).

Vesicular tubulation is a phenomenon observed in cellendogenous processes such as post-Golgi trafficking [28],endosomal sorting [29], lysosomal movement [30], ortransport of major histocompability complex (MHC) classII molecules [31]. However, the Salmonella-inducedtubules are more stable and longer than the cell-intrinsictubules [21].

Salmonella-induced tubules have been mostly analysedin epithelial-like cell lines, such as HeLa [15–19,21,22].

270

However, SIFs can also form in interferon-g-activatedmacrophage-like RAW267.4 murine cells [21,32]. Further-more, Salmonella-induced tubules labelled with a fluid-phase fluorescent dextran marker, which were identifiedas SIFs, were detected in mouse peritoneal macrophagesand bone marrow-derived dendritic cells [21].

Next, we will discuss the features and mechanisms offormation of the different types of tubules. Thus far, Sal-monella-induced tubules were only observed in cellsinfected with serovar Typhimurium.

The early T3SS-1-dependent SNX tubulesSNXs are a family of over 30 proteins, which are involved inendocytic trafficking and signalling. All SNXs contain aphox (PX) domain, which binds phosphoinositides (PIs)[33]. Two recent studies analysed the role of SNX1 andSNX3 in SCV maturation in epithelial cells [18,19].

The PX domain of SNX1 binds to phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylinositol 3,5-bispho-sphate [PI(3,5)P2], which helps target SNX1 to early endo-somes. SNX1 also contains a Bin-amphiphysin-Rvs (BAR)domain that enables sensing of membrane curvature andinduces tubulation [33]. Within the first minutes of Sal-monella infection, SNX1 undergoes a rapid shift from itsendosomal localisation to the site of bacterial entry. Onceenriched on the nascent enlarged SCVs, SNX1 starts toform extensive and highly dynamic tubules called spacious

Table 1. Salmonella effectors located on or involved in the formation of Salmonella-induced tubules

Effector Intracellular localisation Function(s) Target(s)a Refsb

SopB/SigD Plasma membrane, SCV Phosphoinositide phosphatase PI(4,5)P2, Rab5 [2,12,23,77]

PipB SCV, SIFs n.d. n.d. [2,12,23,44]

PipB2 SCV, SIFs, SISTs, LNTs SIF and LNT extension,

recruitment of kinesin-1

to SCV

Kinesin-1 [2,12,17,23,44,45]

SifA SCV, SIFs, SISTs, LNTs SIF, SIST, LNT formation,

SCV integrity, SCV positioning,

redirection of post-Golgi trafficking,

putative GEF

SKIP, Rab7,

GDP-RhoA

[2,12,16,17,23,40–43,51–53]

SifB SCV, SIFs n.d. n.d. [2,12,23]

SopD2 SCV, SIFs, SISTs, LNTs,

late endosomes

SIF and SIST formation, inhibition

of LNT formation

n.d. [2,12,16,17,23,46]

SpvB n.d. ADP-ribosyltransferase, actin

depolymerisation, induction of

apoptosis, negative modulation

of SIF formation

Actin [2,12,23,38]

SseF SCV, SIFs, SISTs SIF and SIST formation, microtubule

bundling, SCV positioning, redirection

of post-Golgi trafficking, possible

recruitment of dynein

n.d. [2,12,16,23,48,49,61]

SseG SCV, SIFs SIF and SIST formation, microtubule

bundling, SCV positioning, redirection

of post-Golgi trafficking, possible

recruitment of dynein

n.d. [2,12,16,23,48,49,60]

SseJ SCV, SIFs, SISTs, LNTs Deacylase, phospholipase,

glycerophospholipid-cholesterol

acyltransferase (GCAT), SCV

dynamics, SIF formation

GTP-RhoA [2,12,17,23,38,39,53–55]

SseL SCV, SIFs Deubiquitinase, macrophage

cytotoxicity

Ubiquitin [2,12,23]

SteA trans-Golgi network (TGN) n.d. n.d. [20,62]

SteC SCV, SIFs Kinase, F-actin remodelling in

proximity to SCVs

n.d. [2,12,23]

aAbbreviations: n.d., not determined; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate.

bOwing to space limitations we cannot include all original references, which can be found in the cited reviews.

Review Trends in Microbiology June 2011, Vol. 19, No. 6

vacuole-associated tubules (SVATs) [18] (Figure 1).SNX1 localises to the SCV up to 3 h post-invasion butthe formation of SVATs peaks at 10–15 min after entry[18,19]. The formation of SVATs is concomitant with adecrease in vacuole size, suggesting membrane loss fromthe vacuoles [18]. SNX1 is part of the retromer complex andis involved in the retrieval of M6PR from endosomes tothe TGN [34]. Interestingly, depletion of SNX1 results inan accumulation of M6PR at the site of invasion [18]. Thissuggests that the M6PR is recruited onto the nascent SCVbut that SNX1 participates in its fast removal [18]. Fur-thermore, depletion of SNX1 leads to a slower progressionof SCVs towards the MTOC region and a delay in onset ofbacterial intracellular replication [18].

SNX3 also associates with early endosomes and is in-volved in the formation of multivesicular bodies [33,35].SNX3 was found to associate with the SCV as early as10 min after invasion and to localise to tubular structuresthat appear at 30 min, peak at 60 min and disappear 2 hafter invasion [19] (Figure 1). Interestingly, SNX3 does notpossess a tubulation-inducing BAR domain [33], and forma-tion of SNX3 tubules requires SNX1 and SNX2, which bothcontain BARdomains [19]. Depletion of SNX3 decreases therecruitment of Rab7 and LAMP1, and therefore SNX3 playsa role in the control of SCV maturation [19].

The T3SS-1 effector SopB, a phosphoinositide phospha-tase, is required for SCV biogenesis and maturation by

altering phosphoinositides and the surface charge at thevacuolar membrane [23,36]. SopB mediates the accumula-tion of PI(3)P on the vacuolar membrane, which is neces-sary for the recruitment of SNX1 and SNX3 [18,19].

In summary, upon Salmonella infection, SopBmediatesthe accumulation of PI(3)P at bacterial invasion sites andon the SCV. This promotes the recruitment of SNX1 andSNX3. Within the first minutes after invasion, SVATs areformed, which reduces vacuolar size and promotes M6PRremoval. After 30 min, SNX3 tubules start to form [23,36].These events contribute to recruitment of Rab7 andLAMP1 to the SCV and promote the movement of thevacuole towards the bacterial replication site.

The late T3SS-2-dependent tubular networks: SIFs,SISTs and LNTsSIFs

SIFs were recently analysed in detail by live cell imaging[21,22]. This revealed highly dynamic tubules extendingfrom the SCV 4–6 h after invasion. Later (>8 h), SIFs forma more complex but less dynamic network that oftenextends throughout the cell. SIFs form along a scaffoldof microtubules and they appeared to derive solely fromlate endocytic compartments. The composition of SIFs issimilar to that of the SCVmembrane: they contain LAMPs,Rab7, vATPase, lysobisphosphatidic acid, cholesterol andlow levels of cathepsin D [12,22]. However, recent data

271

Table 2. Main features of Salmonella-induced tubules

Name Time of formation

(post-invasion)

Known markers Susceptibilitya Other characteristics Refs

Spacious

vacuole-associated

tubules (SVATs)

10–60 min

(peaks at

10 min)

SNX1 n.d. Recruitment of SNX1

and tubule formation

involves a bacterial

effector (SopB)

[18,19]

SNX3 tubules 10–60 min

(peaks at

30–60 min)

SNX3 Nocodazole

sensitive, cyto

D resistant

Recruitment of SNX3

and tubule formation

involves a bacterial

effector (SopB) and

host cell factors

(PI3-kinases, PI(3)P,

Rab5, SNX1 and

SNX2)

[19]

SIFs 3–16 h (peaks

at 8–16 h)

LAMPs, vATPase,

Rab7, SCAMP3,

lysobisphosphatidic

acid (LBPA), cholesterol,

and T3SS-2 effectors

Nocodazole

sensitive,

cyto D resistant,

BFA resistant

Formation involves

bacterial effectors

(SifA, SopD2, SseF,

SseG, PipB2, SseJ,

and SpvB) and host

cell factors (Rab7,

Rab9, and SKIP)

[15,17,21,22,37,

40,44,46,49]

SISTs 8–14 h (peaks

at 14 h)

SCAMP3 and T3SS-2

effectors

Nocodazole

sensitive, cyto D

susceptibility n.d.,

BFA sensitive

Formation involves

bacterial effectors

(SifA, SopD2, SseF,

SseG, and PipB2)

[16]

LNTs 3–16 h (peaks

at 16 h)

T3SS-2 effectors,

cholesterol, and

vATPase

Nocodazole

sensitive, cyto D

resistant,

BFA resistant

Formation involves

bacterial effectors

(SifA, SopD2, and

PipB2) and host cell

factors (SKIP)

[17]

aAbbreviations: n.d., not determined; cyto D, cytochalasin D; BFA, brefeldin A.

Review Trends in Microbiology June 2011, Vol. 19, No. 6

showed that most SIFs are also positive for SCAMP3 [16](Figures 1 and 2). Depletion of SCAMP3 or inhibition of thesecretory pathway by treatment with brefeldin A (BFA)does not reduce the frequency of appearance of SIFs[16,37]. Hence, remodelling of endosomal compartmentsis sufficient to produce SIFs.

Infection of cells with single effector mutant strains(where the effector gene is deleted or disrupted) revealedthat formation of SIFs involves the T3SS-2 effectors SifA,PipB2, SopD2, SseG and SseF (functions and host targetsof these proteins are shown in Table 1). Similar experi-ments indicated that S. Typhimurium effectors SseJ andSpvB appear to antagonise SIF formation [38,39] (Table 1).By contrast, recent studies indicate that SseJ might play arole in initiating tubule formation (see below).

Cells infected with a sifA– mutant do not make any SIFs[40] (Figure 2). The same is observed in wild-type infectedcells depleted of thehost cell target of SifA, SifAandkinesin-interacting protein (SKIP), or cells infected with a strainexpressing point mutants of SifA that do not interactwith SKIP [41,42]. Therefore, the SifA–SKIP complexis essential for the formation of SIFs. SKIP also interactswith kinesin-1, thereby linking the SCV membrane to themicrotubule network [41]. SKIP and SifA promote the bud-ding of vesicles from the SCV [43], suggesting that, byinteracting with kinesin-1, SKIP activates the plus-enddirected motor to induce the fission of vesicles from theSCV and/or their centrifugal transport [43]. Hence, SifAandSKIP initiatemembrane transport fromtheSCV,whichmight triggerSIF formation.Furthermore, bybindingRab7,SifAmight displace RILP/dynein from SIFs, thus favouringthe extension of SIFs towards the cell periphery [27].

272

Cells infected with a pipB2– mutant have shorter SIFsthan those infected with wild-type Salmonella [44]. There-fore, PipB2 contributes to the centrifugal extension of SIFs.This probably occurs via kinesin-1, as PipB2 directly bindsthe light chain subunit of this plus-end directed motor [45].Cells infected with a sopD2– mutant have less SIFs thanthose infected with wild-type Salmonella [46]. Upon trans-location, SopD2 localises to the SCV membrane and asso-ciated tubules, and to late endosomes [16,17,46,47]. Thelatter association might contribute to SIF formation bytargeting endocytic vesicles towards the SCV and itsemerging tubules [17].

Cells infectedwith sseF– or sseG– mutant strains displayaggregated endosomes, but these stay in a punctuatedrather than continuous pattern [48]. SseF and SseG asso-ciate with microtubules and mediate their massive bun-dling close to the SCV [49]. Therefore, the fusion ofaggregated vesicles into tubules might depend on microtu-bule bundling by SseF and SseG, or by an SseF–SseGcomplex [50].

The ectopic expression of T3SS-2 effectors in host cellsprovided insights on the molecular mechanisms underly-ing the formation of endosomal tubules resembling SIFs.The ectopic expression of effectors can simplify the inter-pretation of the results or provide better-defined pheno-types. However, the levels of expression and the spatio-temporal specificity and complexity of T3SS-2-mediatedeffector translocation are lost, which might lead to arte-facts. Nevertheless, it has been shown that ectopic expres-sion of SifA induces swelling and fusion of late endocyticcompartments, leading to the formation of ‘SIF-like’tubules [46,51,52]. The frequency of formation of these

Review Trends in Microbiology June 2011, Vol. 19, No. 6

tubules increases after ectopic co-expression of SifAand SopD2 [46]. By contrast, recent data indicate thatco-expression of SifA and SseJ, but not SifA alone, inducesthe formation of ‘endosomal tubules’, supporting a role ofSifA and SseJ in mediating tubulation of endosomal com-partments and promoting SIF formation [53].

SseJ has deacylase, phospholipase and cholesterol-acyl-transferase activities, which are activated through itsinteraction with the GTP-bound form of Rho-GTPase RhoA[54–57]. Co-expression of SseJ and GTP-bound RhoAinduces the formation of endosomal tubules [53]. SifA alsobinds RhoA, but preferentially to the GDP-bound form[42,53]. Moreover, the C-terminus of SifA resembles aguanine nucleotide exchange factor (GEF) [53]. Therefore,SifA could indirectly activate SseJ through its putativeGEF activity on RhoA. Although it is still unclear if SifApossesses GEF activity [53,58], results of ectopic expres-sion suggest that SseJ, SifA and RhoA constitute a complexthat promotes tubulation of endosomal compartments.However, the situation inSalmonella-infected cells is morecomplex. The cellular levels and localisation of translo-cated proteins are different and other T3SS-2 effectorsmight coordinately modulate endosomal tubulation.

A current model of SIF formation (Figure 3) is that theGEF activity of SifA activates RhoA, which in turn bindsSseJ and activates its enzymatic activity. This alters thelipid composition of the SCV membrane, favouring theformation of nascent outward tubules. PipB2 binds kine-

[()TD$FIG]

(a) (

(c) (

SifA

RhoA

GDP

SseJ

+

SCV

Microtubule

Kinesin-1(active form)

GTP

RhoA

SifA

Skip PipB2SseJ

GTP

SseJ

Figure 3. The mechanisms of SIF formation. (a) Once Salmonella has reached its intrace

binds GDP-bound RhoA and might activate RhoA. (b) GTP-RhoA binds SseJ and activate

PipB2 recruits autoinhibited kinesin-1 to the SCV. (c) SifA interacts with the host protei

Microtubule binding and processive motility of the motor and the local lipid composi

toward the plus-end of microtubules. SseF, SseG and SopD2 contribute to SIF formatio

illustrated in this figure is hypothetical.

sin-1 and thus links the SCV and nascent tubules to themicrotubule network. The SifA–SKIP complex binds andactivates kinesin-1, thereby promoting the elongation ofnascent tubules along microtubules. Clearly, further stud-ies are required to validate this model and to understandhow the molecular function of SopD2, SseF and SseGcontributes to SIF formation.

SISTs

SCAMPs are integralmembrane proteins that reside in thecell recycling system, including the TGN and sorting andrecycling endosomes [59]. This family includes the ubiqui-tous isoforms SCAMP1-4 and the neuronal form SCAMP5[59]. SCAMP3 is present on the SCV membrane and is acomponent of the Salmonella-induced tubular networkformed in HeLa cells [16] (Figures 1 and 2). SCAMP3-tubules comprise not only SIFs but also tubules, namedSISTs, lacking late endosomal proteins [16]. The formationof SISTs is sensitive to treatment with BFA [16]. However,once formed, SISTs resist treatment with BFA, indicatingthat although SISTs originate from the secretory pathwaythey become functionally distinct from their compartmentof origin [16]. This shows that, as suggested in previousstudies [60,61], the SCV and associated tubules can incor-porate membrane originating from the secretory pathway.

The mechanisms that underlie the recruitment of TGN-derived SCAMP3 material to the SCV and its tubules areunknown. SCAMP3-containing vesicles might be recruited

Kinesin-1(autoinhibited form)

b)

d)

RhoA

GTPSifA

PipB2SseJ

RhoAGTP

SifA

SseJ

+

SopD2

Skip

PipB2

SseF

SseG

Nasce

nt tu

bule

/ves

icle

TRENDS in Microbiology

llular replication niche, the formation of late tubules is induced. SifA preferentially

s its enzymatic activity. SseJ modifies the lipid composition of the SCV membrane.

n SKIP. The SifA–SKIP complex binds kinesin-1 and relieves its autoinhibition. (d)

tion of the SCV membrane help the formation and elongation of nascent tubules

n but their mechanisms of action remain largely unclear. The timing of the events

273

Review Trends in Microbiology June 2011, Vol. 19, No. 6

directly from the TGN. Alternatively, Salmonella mightinterfere with trafficking between the TGN and late endo-somes. In this scenario, SCAMP3-containing membranesare recruited indirectly after being first re-directed to lateendocytic compartments [16]. Interestingly, a recent studybased on the tracking of Salmonella effectors in living cells[20] showed that SteA, a poorly characterised effector,localises on tubules that are enriched in a TGN markerbut is largely excluded from endolysosomal tubules. Inaddition, both bacterially translocated and ectopicallyexpressed SteA appear to localise to the TGN [62]. Thus,this effector might have a role in recruiting cargo from thesecretory pathway [20].

Formation of SCAMP3 tubules requires the T3SS-2effectors SifA, PipB2, SopD2 and, to a lesser extent, SseFand SseG [16]. It is quite possible that the mechanismsinvolved in the formation of SISTs are similar to those ofSIFs. For example, PipB2, via kinesin-1, also promotes thecentrifugal extension of SCAMP3 tubules [16]. However,themechanism bywhichmembranes from different originssegregate into distinct tubules remains unknown.

LNTs

The use of S. Typhimurium strains deficient for more thanone effector gene led to the discovery of LNTs. Infection ofHeLa cells with a sifA– sopD2– mutant, which resides in astable vacuole (see below), revealed the presence of tubulesemerging from the SCV that are LAMP1 negative butcontain T3SS-2 effectors [17] (Figures 1 and 2). Theseeffector-tubules, named LNTs, form along a scaffold ofmicrotubules in a kinesin-1-dependent manner and aresolely characterised by the presence of translocated effec-tors; with the exception of vATPase, they do not containany of the markers of SIFs or SISTs [17]. Another recentstudy reported similar effector tubules in Salmonella-infected cells [20].

A sifA– mutant strain is defective in LNT formation,suggesting that SifA and SopD2 activate and inhibit theformation of LNT, respectively. LNTs are also formed inwild-type infected cells indicating that the activation me-diated by SifA is dominant over the SopD2-directed inhi-bition [17]. Furthermore, PipB2 is involved in thecentrifugal extension of LNTs [17]. It is currently unknownwhat functions other effectors might have in the formationof LNTs.

Similar structures to LNTs have been observed previ-ously upon infection of HeLa cells with sopD2–, sseF– orsseG– mutants [46,48]. These strains are defective in SIFformation, arresting their formation at an intermediatestage. These structures, named ‘pseudo-SIFs’, are effector-positive tubules with punctuate LAMP1 staining, whichdiffer from the continuous LAMP1-tubules formed in wild-type infected cells [46,48].

Interestingly, nascent LNTs wrap around LAMP1-posi-tive vesicles [17], suggesting that they could act as pre-cursors of SIFs by supporting the recruitment of endocyticvesicles (Figure 1). LNTsmight also be a necessary scaffoldto help formation of SIFs and SISTs by promoting vesiclefusion and tubule continuity. T3SS-2 effectors on LNTscould mediate the interaction between Salmonella tubulesand both the endocytic and post-Golgi compartments. This

274

could explain why the sseF , sseG or sopD2 strains arrestSIF formation at an intermediate stage, although thesemutants are still able to form effector-positive tubules[46,48].

It remains to be elucidated how the LNT formation isinitiated and whether these tubules result from the remo-delling of a host cell compartment. It will be interesting toanalyse other effector mutants regarding formation ofLNT, SIF or SISTs. This might help to define more pre-cisely the molecular mechanisms of formation of the Sal-monella-induced tubules. In addition, it could providefurther insights into other effectors and host proteinsinvolved, as well as the role of these networks duringSalmonella infection.

Salmonella-induced tubular networks: a role in vacuole

membrane stability?

A S. Typhimurium sifA– mutant has an unstable vacuolethat eventually ruptures. This mutant has severe defectsfor replication within macrophages and for virulence inmice [40]. Strains that cannot assemble the T3SS-2, andtherefore do not translocate any effector, reside in a stablevacuole. This indicates that T3SS-2 effectors are responsi-ble for instability of the sifA– mutant vacuole [40]. BecausesifA– sseJ– or sifA– sopD2– mutants have a more stablevacuole than a sifA– single mutant [17,39], the activities ofSseJ and SopD2 contribute to SCV rupture.

The exact reasonwhy the sifA– mutant loses its vacuolarmembrane is unknown. It is also unclear how SseJ andparticularly SopD2 contribute to loss of the vacuolar mem-brane. SifA could mediate stability of the vacuolar mem-brane enclosing wild-type bacteria by controlling theactivation status of RhoA, the eukaryotic activator of SseJ.A second model could be that uncontrolled membranetransport and opposing motor forces of kinesin and dyneinon the sifA– mutant SCV lead to vacuole disruption[41,45,63]. Interestingly, characterisation of LNTs sug-gested that SCV tubulation could promote vacuolar stabil-ity. Although defective in the formation of SIFs and SISTs,sopD2– sifA– and sopD2– sifA– pipB2– mutant strains havea stable SCV and form LNTs [17]. The fact that sifA– orsifA– pipB2– mutant strains have an unstable SCV mem-brane and do not form LNTs suggests a direct relationshipbetween stabilisation of SCVs and formation of LNTs.

Physiological relevance of the Salmonella-inducedtubulesThere are no reports about the formation of Salmonella-induced tubules in vivo and one can only suggest whatmight be the physiological function(s) of these tubules. Asjudged by the consequences of SNX1 or SNX3 depletion inSalmonella-infected cells, the early SNX tubules might beimportant for SCV maturation and consequently for mi-gration of bacterial vacuoles towards the MTOC [18,19].Because SIFs, SISTs and LNTs appear at the onset ofbacterial intracellular replication, theymight be importantfor later events of host cell infection. It has been postulatedthat SIFs, SISTs or LNTs might satisfy membrane ornutritional requirements, mediate dilution of lysosomalenzymes, or promote the stability of the SCV membrane(see above) [2,16,17,21,22].

Review Trends in Microbiology June 2011, Vol. 19, No. 6

One thing is clear: mutants deficient in effectors re-quired for the formation of SIFs, SISTs or LNTs are alsoattenuated for virulence in different mouse models ofSalmonella infection [9,32,40,47,64]. Furthermore, al-though sifA– sseJ–, sifA– sopD2–, or sifA– sopD2– pipB2–

mutants have a stable SCV, only sifA– sopD2– and sifA–

sopD2– pipB2– strains form LNTs and are more virulentthan a sifA– mutant [17]. This suggests that SCV tubula-tion increasesSalmonella virulence. Overall, this indicatesthat the mechanisms underlying Salmonella-inducedtubulation are important for virulence.

Other intra-vacuolar pathogens have evolved ways totarget molecular motors as well as to intercept vesicletrafficking to ensure their intracellular survival andreplication [3]. Among these, Chlamydia trachomatisinduces the formation of effector-positive fibres that linkthe primary Chlamydia-containing vacuole (inclusion) to asecondary inclusion [65]. These fibres might promote C.trachomatis multiplication by inducing the formation of asecond replication site in the dividing cell [65]. The Chla-mydia-induced fibres are less dramatic than Salmonella-induced tubular networks. However, it is possible that C.trachomatis, or other intracellular bacterial pathogens, in-duce tubular networks similar to those seen in Salmonella-infected cells but that they remain undiscovered thus far.

Conclusions and future perspectivesThe regulation of membrane homeostasis is essential forthe intracellular survival and proliferation of Salmonella.In this review, we discussed the latest findings involvingthe regulation of the membrane exchanges of the SCV. It isnow obvious that Salmonella can induce distinct types oftubules at different times of infection. Although it is stillunclear why these tubular networks form, evidence ismounting that they could be important to insure intracel-lular survival and replication of the bacteria. Future workwill certainly clarify how the various tubules contribute tovacuole integrity and for intracellular replication of Sal-monella (Box 2). In this regard, it will also be important toanalyse whether these structures act separately or inconjunction with each other. The ultimate challenge willbe to detect the presence of the Salmonella-inducedtubules in vivo. This might become possible in the near

Box 2. Outstanding questions

� Which other effectors are localised to LNTs?

� Do they regulate their formation?

� Which other effectors are involved in the different types of tubule

formation?

� Are there effectors that specifically target a particular host cell

compartment?

� Do they have different ‘assignments’?

� Do LNTs derive from a host cell compartment?

� What are the mechanisms of their formation?

� What are the mechanisms of SIST formation?

� How do LNTs contribute to SCV stability?

� Do LNTs, SIFs and SISTs have different functions?

� Do LNTs constitute SIF and SIST precursors?

� How do SifA, SseJ and SopD2 influence SCV membrane

dynamics?

� How are the antagonistic activities of SopD2 and SifA regulated in

terms of SCV membrane dynamics?

future, given the huge advances that we are witnessing inmicroscopy-based techniques.

Regardless of the physiological significance of the Sal-monella-induced tubular networks, future studies willprobably focus on dissecting the molecular mechanismsinvolved in their formation (Box 2). It will be important todifferentiate between mutant strains that are able to formsome tubules but not others. Similarly, it will be importantto find host cell proteins and lipids that mediate theformation of such specific types of tubules. An underlyingquestion is how the membrane segregates at the SCV toyield SIFs, SISTs and LNTs. In summary, the tubularnetworks induced by Salmonella are excellent tools tounderstand the mode of action of bacterial effectors in-volved in their formation and how effectors help Salmonel-la to subvert the functions of host cells. Continuous effortsto understand how and why these tubules form might alsoreveal novel aspects of intracellular membrane trafficking.

AcknowledgementsWe thank Laurent Aussel for the critical review of this manuscript. N.S.was recipient of fellowships fromMarie Curie Microban, L’Association pourla Recherche sur le Cancer (ARC) and La Ligue Nationale contre le Cancer.This work was supported by grants from Equipe Federation pour laRecherche Medicale en France (FRM) (http://www.frm.org/) and l’AgenceNationale Recherche (ANR) (http://www.agence-nationale-recherche.fr/)(ANR-05-BLAN-0028-01) to S.M. and institutional grants from le CentreNational de la Recherche Scientifique (CNRS) and l’Institut National de laSante Et de la Recherche Medicale (INSERM). Research in the laboratoryof L.J.M. is funded by aMarie Curie EuropeanReintegrationGrant (PERG-GA-2008-230954) and by the Fundacao para a Ciencia e a Tecnologia (FCT)(http://alfa.fct.mctes.pt) (grant PTDC/SAU-MII/099623/2008).

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