listeria monocytogenes membrane trafficking and lifestyle: the exception or the rule?

ANRV389-CB25-26 ARI 4 September 2009 21:46

Listeria monocytogenesMembrane Trafficking andLifestyle: The Exceptionor the Rule?Javier Pizarro-Cerda1-3 and Pascale Cossart1-3

1Unite des Interactions Bacteries-Cellules, Institut Pasteur, Paris F75015, France2INSERM, U604, Paris F75015, France3INRA, USC2020, Paris F75015, France; email: [email protected],[email protected]

Annu. Rev. Cell Dev. Biol. 2009. 25:649–70

First published online as a Review in Advance onAugust 12, 2009

The Annual Review of Cell and DevelopmentalBiology is online at cellbio.annualreviews.org

This article’s doi:10.1146/annurev.cellbio.042308.113331

Copyright c© 2009 by Annual Reviews.All rights reserved

1081-0706/09/1110-0649$20.00

Key Words

endocytosis, autophagy, membrane microdomains, cytoskeleton,caveolae, clathrin

AbstractListeria monocytogenes is an intracellular bacterial pathogen that pro-motes its internalization within nonprofessional phagocytes by interact-ing with specific host cell receptors. L. monocytogenes resides transientlyin a membrane-bound compartment before escaping into the host cellcytosol where bacterial proliferation takes place. Actin-based motil-ity then promotes cell-to-cell pathogen spread. Extensive studies oncytoskeleton rearrangements, membrane trafficking, and other eventshave established this microorganism as an archetype of cellular func-tion subversion for intracellular parasitism. Here we discuss the mostsignificant membrane trafficking pathways hijacked by L. monocytogenesduring the host cell infection process and compare them to those ofother intracellular pathogens, in particular Shigella flexneri, Salmonellaenterica, and Mycobacterium tuberculosis.

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Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 650ENTRY INTO TARGET CELLS . . . . 650

Subversion of Host Cell Receptorsfor Pathogen Internalization . . . . . 650

Lipid Rafts and MembraneOrganization at PathogenEntry Sites . . . . . . . . . . . . . . . . . . . . . 655

Clathrin and Caveolin: New Playersand New Links with theCytoskeleton . . . . . . . . . . . . . . . . . . . 657

LIFE IN AND ESCAPE FROMTHE VACUOLE. . . . . . . . . . . . . . . . . . 659Maturation of the Vacuole . . . . . . . . . . 659Listeriolysin O and Vacuolar

Escape . . . . . . . . . . . . . . . . . . . . . . . . . 659INTRACYTOSOLIC SURVIVAL

AND AUTOPHAGY . . . . . . . . . . . . . . 661CELL-TO-CELL SPREAD . . . . . . . . . . 663CONCLUSIONS . . . . . . . . . . . . . . . . . . . . 664

INTRODUCTION

Invasin of Yersinia pseudotuberculosis was the firstreported bacterial gene product involved in theinvasion of host eukaryotic target cells (Isberget al. 1987), which opened the door to a newfield that emerged at the boundaries of classicalcell biology and microbiology: cellular micro-biology. During the same period, the hemolysingene of Listeria monocytogenes was characterizedand became not only the first L. monocytogenesvirulence factor to have its gene characterizedbut also the first bacterial gene product forwhich a function critical to bacterial survivalwithin host cells was attributed (Mengaud et al.1987). Since then, the molecular adaptationsof L. monocytogenes to the eukaryotic host cellenvironment have been studied extensively(Figure 1), leading to the identification of abroad range of new bacterial virulence factorsthat, similar to the Arp2/3-dependent actin-polymerizing ActA product or the InlB invasionprotein, have paved the road toward profounddiscoveries in the areas of cell and developmen-tal biology. Many other bacterial pathogens

have evolved fascinating adaptations to interactwith and hijack host cell functions, and in thefollowing sections we compare their molecularstrategies with those of L. monocytogenes withthe goal of drawing common themes in mem-brane trafficking subversion and intracellularlifestyle.

ENTRY INTO TARGET CELLS

Subversion of Host Cell Receptorsfor Pathogen Internalization

With the analysis of the L. monocytogenesgenome, it was highlighted that this pathogenpossesses an important arsenal of surface pro-teins that modulate bacterial interactions withthe environment, in particular with host eu-karyotic cells (Bierne & Cossart 2007). Amongthese bacterial surface molecules, the inter-nalin family is characterized by the presenceof amino-terminal leucine-rich repeat modulesimplicated in protein-protein interactions. Twomembers of this family are critical for the inter-nalization of L. monocytogenes within nonprofes-sional phagocytic cells through interaction withcell-specific receptors (Bierne et al. 2007).

The prototype internalin (also known asInlA) is a cell wall covalently anchored protein,which binds the cellular adhesion molecule E-cadherin and induces bacterial entry into polar-ized human epithelial cells (Gaillard et al. 1991,Mengaud et al. 1996) (Figure 2). E-cadherinis normally involved in homophilic interac-tions for the establishment and maintenanceof cell-to-cell contacts in epithelial tissues, andL. monocytogenes exploits the E-cadherin local-ization at the intestinal barrier to invade en-terocytes during the initial stages of host col-onization (Lecuit et al. 2001). E-cadherin isalso involved in the bacterial invasion of thesyncitio-trophoblast layer at later placental in-fection stages (Disson et al. 2008). Throughstudies in human epithelial cell lines, it hasbeen shown that, during host cell invasion,L. monocytogenes hijacks the molecular machin-ery associated with the cytoplasmic tail ofE-cadherin. β- and α-catenins, which are

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Entry: InlA & InlB

Vacuole lysis: LLO

Double-membranevacuole lysis:LLO, PlcA, PlcB

Intracellular movement andcell-to-cell spread: ActA

Listeria

Figure 1Model of the Listeria monocytogenes intracellular life cycle. Interaction of the bacterial surface proteins InlAand/or InlB with specific cellular receptors at the plasma membrane of host cells induces the internalizationof L. monocytogenes in a vacuole that is subsequently lysed by the pore-forming activity of the cholesterol-dependent cytolysin lysteriolysin O (LLO). Once in the host cell cytoplasm, L. monocytogenes proliferates andmoves via the polymerization of host actin (stippled regions) triggered by the bacterial surface protein ActA.Bacteria that reach the host plasma membrane induce the formation of membrane protrusions that invadeneighboring cells, and L. monocytogenes will be located in a double-membrane vacuole that is lysed by theactivity of LLO and two bacterial phospholipases, PlcA and PlcB. Redrawn with permission from the TheJournal of Cell Biology (Tilney & Portnoy 1989).

required to establish a functional link betweenE-cadherin and the actin cytoskeleton inadherens junctions (Imamura et al. 1999),are recruited by L. monocytogenes to entrysites and are required for actin remodel-ing during bacterial invasion (Lecuit et al.2000). The Arp2/3 complex and its acti-vator cortactin, which direct actin assemblyin nascent cellular adhesive contacts in aRac-dependent manner (Helwani et al. 2004,Kovacs et al. 2002), are also implicated duringL. monocytogenes internalization (Sousa et al.2007). Study of the InlA/E-cadherin invasionpathway allowed the identification of a novel

guanosine-activating protein for RhoA andCdc42 named ARHGAP10 that is involvedin the recruitment of α-catenin to theL. monocytogenes internalization site and neces-sary for α-catenin recruitment to cell-cell junc-tions (Sousa et al. 2005). The unconventionalmyosin VIIa and its ligand vezatin are also func-tionally involved in the formation of adherensjunctions and in the entry of L. monocytogenes inepithelial cells (Sousa et al. 2004).

The other major internalin-like moleculeinvolved in entry is InlB, a bacterial effec-tor loosely bound to the L. monocytogeneslipoteichoic acid, which can be released

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Listeria

Cell membrane

P

E–cadherin

Arp2

Arp3ActinCortactinCortactin

Hakai Ub

Clathrin

β-catenin

α-cateninArf6

Vezatin

Src

Src

MyoVIIA

InlA

ARHGAP10

Rac

P

Lipid raft

Caveolin-1

Figure 2Entry of Listeria monocytogenes in host polarized cells via the InlA invasion pathway. In polarized epithelialcells, the bacterial protein InlA interacts with its receptor E-cadherin located in lipid rafts to promotecellular invasion. Caveolin-1, associated with lipid rafts, is involved in the Src-dependent phosphorylation ofE-cadherin. This phosphorylation triggers the ubiquitination of E-cadherin by the ubiquitin ligase Hakaiand the recruitment of a clathrin coat. β-catenin interacts directly with the cytoplasmic tail of E-cadherin topromote recruitment of α-catenin, which in turn interacts with actin and with other effectors including theRho/Cdc42 guanosine activating protein ARHGAP10 and the small GTPase Arf6. Myosin VIIA providesthe tracking force along actin filaments to promote bacterial internalization while its ligand vezatin mediatesattachment to the membrane. Src is also involved in the phosphorylation of cortactin that promotes Arp2/3complex activation and actin polymerization. Rac is implicated in this signaling cascade at a still-unknownposition.

from the cell wall and interacts with threehost cell molecules to produce success-ful bacterial entry: Its C-terminal domainbinds extracellular matrix glycosaminoglycans( Jonquieres et al. 2001) and the receptorfor the globular part of the complement

molecule C1q (gC1q-R) (Braun et al. 2000),whereas its N-terminal leucine-rich repeats ac-tivate the hepatocyte growth factor receptorMet, a member of the receptor tyrosine ki-nase family (Shen et al. 2000) (Figure 3).Stimulation of Met with InlB mimics the


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Met

Cbl

ShcP

P

Clathrin

Sept2

PI4K

PI3K

Lipid raft

GAGs

Arp2

Arp3

Actin

Ub

InlB

P

PIP3

Wave/N–Wasp

Rac/Cdc42

PIP

Listeria

gC1q-R

CrkII

Gab1

P

Figure 3Entry of Listeria monocytogenes in host epithelial cells via the InlB invasion pathway. In a wide variety ofepithelial cells, the interaction between InlB and its main signaling receptor Met leads to bacterialinternalization. InlB interacts not only via its N-terminal domain with Met, but also through its C-terminaldomain with extracellular glycosaminoglycans (GAGs) and with the gC1q-R. Activation of the tyrosinereceptor kinase Met leads to its autophosphorylation and recruitment/phosphorylation of several proteinadaptors including CrkII, Gab1, Shc, and Cbl, which in turn are involved in the recruitment of the type I PI3-kinase (PI3K). Cbl is also a ubiquitin ligase required for Met ubiquitination and promoting clathrinrecruitment and receptor internalization via endocytosis. The type I PI 3-kinase produces PI(3,4,5)P3, andredistribution of this phosphoinositide within lipid rafts is involved in the activation of Rac (throughrecruitment of an as-yet-unidentified effector), which activates Wave and Arp2/3 for actin polymerization.Sept2, a new cellular effector required for InlB-mediated entry downstream of Met, is depicted (purple ovalnext to the plasma membrane) as well as the type II PI4Kα (red oval associated to the plasma membrane)involved in the production of PIP in a PI 3-kinase-independent signaling pathway.

physiological stimulation of the receptor withits natural ligand, the hepatocyte growth fac-tor: Met dimerizes, autophosphorylates, andtriggers the recruitment/phosphorylation ofseveral protein adaptors including Gab-1,Cbl, Shc, and CrkII (Dokainish et al. 2007,Ireton et al. 1999). These adaptors are in-volved in the recruitment to the bacterial entrysite of the phosphatidylinositol 3-kinase typeI (PI 3-kinase) (Ireton et al. 1996), which is

implicated in the activation of Rac-1 and the or-chestration of a complex signaling cascade thatleads not only to actin polymerization via theArp2/3 complex, WASP-related proteins, andEna/VASP (Bierne et al. 2005) but also actindepolymerization via LIM kinase and cofilin(Bierne et al. 2001). Recent research examiningthe InlB/Met signaling pathway has revealedthe role of novel molecular partners includ-ing two type II phosphatidylinositol 4-kinases


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Phase DAPI Septin 11 Actin Merge

Figure 4Septin 11 recruitment at the Listeria monocytogenes entry site in JEG-3 cells. The cytoskeletal protein septin 11 is recruited byL. monocytogenes during the very early stages of cellular infection, as revealed by immunofluorescence in cells infected for 5 minutes witha wild-type L. monocytogenes strain EGD expressing InlB covalently attached to the bacterial cell wall and labeled with DAPI (blue) toshow bacterial DNA, anti-SEPT11 antibodies (red ), and anti-actin antibodies ( green). Septin 11 forms characteristic rings around thebody of the internalized L. monocytogenes. Adapted from Mostowy et al. (2009).

implicated in a cascade independent of the PI3-kinase pathway (Pizarro-Cerda et al. 2007).Novel cytoskeletal proteins of the septin fam-ily also modulate L. monocytogenes entry via theInlB-dependent pathway (Mostowy et al. 2009)(Figure 4).

Subversion of surface cellular adhesion/attachment receptors for invasion has been re-ported for other bacterial pathogens. Invasin,an invasion protein of Yersinia enterocolitica andY. pseudotuberculosis (Isberg et al. 1987), pro-motes bacterial entry by binding members ofthe integrin family of extracellular matrix bind-ing receptors (Isberg & Leong 1990). Similarto the interaction between L. monocytogenes andE-cadherin, Y. pseudotuberculosis takes advantageof the molecular machinery associated to thecytoplasmic tail of β1 integrins to orchestrateactin rearrangements required for bacterial en-try: The kinases FAK and Src as well as thesmall GTPases Rac1 and Arf6 are subvertedfrom their normal integrin-mediated signalingto favor actin polymerization via the Arp2/3complex that induces pathogen internalization(Alrutz et al. 2001, Wong & Isberg 2003). Itis important to note that Y. pseudotuberculosis

proliferates in the host as an extracellularpathogen and that cellular invasion is relevantduring the early phases of host infection only(Bliska & Casadevall 2009). Probably as a strat-egy for persistence in host tissues, several otherextracellular pathogens that bind extracellularmatrix proteins access the intracellular space bystimulating the integrin-signaling pathway. Forexample, the Staphylococcus aureus fibronectin-binding adhesin A binds fibronectin to engageα5β1 integrin and induces endothelial cell inva-sion in a FAK-, tensin-, cortactin-, and Arp2/3-dependent manner (Agerer et al. 2005, Masseyet al. 2001). In a similar way, Streptococcus pneu-moniae binds vitronectin to promote entry inendothelial and epithelial cells via αVβ3 inte-grin engagement, integrin-linked kinase activa-tion, and PI 3-kinase recruitment (Bergmannet al. 2009).

Uropathogenic Escherichia coli (UPEC), abacterium responsible for human urinary in-fections, is another extracellular pathogen thatcan invade host cells facultatively to establisha bacterial reservoir involved in recurrent uri-nary infections (Anderson et al. 2003). TheUPEC adhesin FimH binds extracellular matrix


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components such as laminin (Kukkonen et al.1993); however, functional interaction betweenFimH and the monomannose moiety of thetetraspanin molecule uroplakin 1a leads to in-vasion of human bladder epithelial cells (Zhouet al. 2001). Bacterial entry requires the ac-tivation of small GTPases Cdc42 and Rac1,phosphorylation of FAK, PI 3-kinase recruit-ment, and actin binding by α-actinin/vinculin(Martinez & Hultgren 2002, Martinez et al.2000). The obligate intracellular pathogenRickettsia conorii, responsible for Mediterraneanspotted fever, also invades host cells in areceptor-dependent manner: R. conorii takesadvantage of its outer membrane protein B(rOmpB) to interact with the Ku70 subunit ofthe DNA-dependent protein kinase to invadehost epithelial cells (Chan et al. 2009, Martinezet al. 2005). Among the intracellular moleculesengaged by the rOmpB/Ku70 interaction arethe protein adaptor Cbl, the kinase Src, Cdc42,PI 3-kinase, cortactin, and the Arp2/3 complex(Martinez & Cossart 2004).

Molecular mimicry and subversion of nor-mal signaling cascades involved in the physi-ological function of surface receptors such astyrosine receptor kinases, cadherins, or inte-grins are thus a common feature of intracellularbacterial pathogens, which exploit these signal-ing pathways to reorganize the cortical actincytoskeleton and to produce membrane rear-rangements required for bacterial engulfment.It is important to mention that the dynamicsof actin filaments can be modulated by othercytoskeletal proteins including microtubules(Rodriguez et al. 2003), and disruption ofmicrotubules inhibits the entry of severalpathogens including L. monocytogenes andCampylobacter jejuni (Biswas et al. 2003, Kuhn1998). In these cases, however, the specificmolecular pathways involved in the potentialactin-microtubules cross talks have not beenexplored. Very recent work on UPEC showsthat histone deacetylase 6 (HDAC6), whichdeacetylates α-tubulin, can modify microtubulestability and affect kinesin-1 recruitment whileinhibiting UPEC entry into bladder cells. Thisfinding led Dhakal & Mulvey (2009) to propose

that HDAC6 can modulate directional traffick-ing of kinesin-1 and associated cargos such asWAVE2, highlighting a molecular pathway thatcould link actin and microtubules during bacte-rial entry. As mentioned above, septins, a fam-ily of small GTPases that have the property toform nonpolarized filaments, which associateto actin and microtubules and are increasinglyrecognized as new elements of the cytoskele-ton (Tooley et al. 2009), modulate the entry ofL. monocytogenes in host cells (Mostowy et al.2009) (Figure 4). These results should opennew avenues through which to study the inter-action of different cytoskeletal elements duringbacterial invasion.

Lipid Rafts and MembraneOrganization at Pathogen Entry Sites

Cellular receptors are located in membranes,and the physical environment of specificmembrane domains affects the behavior of thereceptors subverted by pathogens for entryinto cells. Lipid rafts are specialized mem-brane microdomains enriched in cholesteroland sphingolipids (Simons & Ikonen 1997).Protein-lipid and protein-protein interactionsparticipate in the formation of these dynamicstructures, which serve as platforms to clustersignaling molecules that participate in a widevariety of processes including phagocytosis, cellmigration, and immune responses (Lasserreet al. 2008). In many cases, their presence isrequired for bacterial entry into host cells.

The invasion of target cells by L. mono-cytogenes requires the integrity of membranemicrodomains (Seveau et al. 2004). Lipid raftmarkers such as glycosylphosphatidylinositol(GPI)-linked proteins, myristoylated andpalmitoylated peptides, and the gangliosideGM1 are detected by immunofluorescenceat bacterial entry sites (Seveau et al. 2004).Cholesterol depletion using the water-solublecyclic oligosaccharide methyl-β-cyclo-dextrin(MβCD) reversibly inhibits L. monocytogenesentry in mouse fibroblasts expressing thehuman E-cadherin or in green monkey Verokidney epithelial cells expressing a functional


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Met receptor (Seveau et al. 2004). The pres-ence of E-cadherin in lipid rafts is necessaryfor its initial clustering and interaction withInlA to promote bacterial entry (Seveau et al.2004). In contrast, the initial interaction ofInlB with Met does not require membranecholesterol, but downstream signaling thatleads to actin polymerization is cholesteroland lipid microdomain dependent (Seveauet al. 2004). PI 3-kinase activation downstreamof Met is not affected by cholesterol deple-tion using MβCD, but activation of Rac1downstream of PI 3-kinase is inhibited incholesterol-depleted cells, which suggests thatthe spatial distribution of 3′-phosphoinositidesproduced by the PI 3-kinase within membranemicrodomains is critical for Rac1 activationand consequently for actin polymerization atL. monocytogenes entry sites (Seveau et al. 2007).

Lipid rafts are required for the entry of manyother pathogens within target cells. As men-tioned above, FimH mediates UPEC internal-ization in human bladder cells by interactingwith uroplakin 1a (Zhou et al. 2001). This re-ceptor is associated with lipid rafts, and choles-terol depletion inhibits bacterial entry in hostcells (Duncan et al. 2004). FimH is also in-volved in the entry of UPEC in macrophagesand mast cells, and the fate of UPEC in thesecells depends on whether bacteria interact withthe GPI-anchored protein CD48 present inlipid rafts via FimH or whether bacteria are op-sonized: In the first case, bacteria replicate incompartments that do not fuse with lysosomes;in the second case, opsonized bacteria are ef-ficiently internalized but are degraded in lyso-somes (Baorto et al. 1997, Shin et al. 2000).

Shigella flexneri and Salmonella enterica, theagents of bacterial dysentery and systemic ty-phoid fever, respectively, are enteropathogensthat invade target cells by injecting bacterialeffectors into their host cell cytosol through amacromolecular syringe-like apparatus knownas the type 3 secretion system (T3SS) (Blockeret al. 2001, Kubori et al. 1998). At the S. flexnerientry site, GPI-anchored/lipid raft-associatedproteins distinctly accumulate around thebacteria during epithelial cell invasion (Lafont

et al. 2002). Initial binding of the S. flexneriT3SS effector IpaB to the cellular transmem-brane protein CD44 takes place in detergent-resistant microdomains and is cholesteroldependent (Lafont et al. 2002). It has been pro-posed that rafts activate the S. flexneri T3SSto functionally translocate effectors into targetcells (van der Goot et al. 2004). For S. enterica,its T3SS effector SipB, which is homologousto S. flexneri IpaB, binds cholesterol with highaffinity prior to effector delivery and cellularinvasion (Hayward et al. 2005).

Other bacterial species exploit lipid raftsduring infection: Mycobacterium tuberculosis, theagent of human tuberculosis, survives withinmacrophages if entry takes place in choles-terol microdomains that allow the recruitmentof coronin-1, a coat protein that may par-ticipate in the inhibition of lysosomal fusionwith M. tuberculosis-containing compartments(Ferrari et al. 1999, Gatfield & Pieters 2000).The disruption of lipid rafts also inhibits cel-lular invasion by R. conorii (Martinez et al.2005), Brucella abortus (Watarai et al. 2002),Campylobacter jejuni (Wooldridge et al. 1996),and some strains of Chlamydia (Stuart et al.2003), among others. Also interesting is thefact that the internalization of many bacterialtoxins from extracellular pathogens, includingVacA from Helicobacter pylori, cholera toxin fromVibrio cholerae, and anthrax toxin from Bacil-lus anthracis, takes place via lipid raft-mediatedendocytosis (Gupta et al. 2008, Saslowsky &Lencer 2008, Abrami et al. 2003).

The studies discussed above indicate overallthat the organization of the plasma membranein specialized microdomains is critical for theefficient orchestration of signaling during bac-terial invasion processes. However, the specificstructure of these specialized microdomains isfar from completely understood, and emphasison the role of cholesterol in the organization ofthese microdomains may have underscored theimportance of other mechanisms of lateral seg-regation for membrane constituents—for ex-ample, the underlying skeleton, as proposedin the fence-picket model (Kusumi & Suzuki2005), or the tetraspan web, which depends


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on the ability of tetraspanins to interact withvarious other surface proteins to form a net-work of molecular interactions clearly distinctfrom lipid raft microdomains (Espenel et al.2008). The next challenge is to identify pre-cisely the specificities of these different mem-brane microdomains in order to understandtheir unique functional contributions to the sig-naling cascades subverted by intracellular bac-terial pathogens.

Clathrin and Caveolin: New Playersand New Links with the Cytoskeleton

Endocytosis is the internalization of macro-molecules in cells, and several endocyticroutes have been described; the main andbest-characterized ones are clathrin- andcaveolin-dependent mechanisms (Conner &Schmid 2003). Caveolin-1 is a protein thatbinds cholesterol, inserts as a loop into theinner leaflet of the plasma membrane, andself-associates to form coats on the surface ofmembrane invaginations, i.e., caveolae (Glen-ney & Soppet 1992). Because caveolin-1 bindscholesterol, the functions of caveolae and lipidrafts are often associated (Head et al. 2006,Pelkmans et al. 2005). Clathrin-mediated en-docytosis requires the recruitment to endocyticsites of specific assembly proteins that recognizethe cargo to be internalized in some cases anddirect the targeting and assembly of clathrincoats to these internalization sites (Semerdjievaet al. 2008). The GTPase dynamin is involvedin the fission of vesicles from the plasma mem-brane both in clathrin- and caveolin-dependentprocesses (Yao et al. 2005, Rappoport et al.2008).

Analysis of the signaling cascade triggeredby the InlB/Met interaction led to the demon-stration that clathrin is required for cellularinvasion by L. monocytogenes (Veiga & Cossart2005, Veiga et al. 2007) (Figure 3). Indeed,the adaptor protein Cbl recruited by Met tothe bacterial entry site is a ubiquitin ligasethat is involved in the monoubiquitination ofMet to induce its endocytosis (Petrelli et al.2002). Met is also monoubiquitinated by Cbl

upon cellular stimulation by InlB, and shortinterfering RNA (siRNA) depletion of severalcomponents of the endocytic machinery, in-cluding dynamin, clathrin, and the clathrin-interacting protein eps15, blocks the entry ofL. monocytogenes in target cells (Veiga & Cossart2005). Clathrin, dynamin, and auxillin (this lat-ter protein is required for clathrin-coated vesi-cles disassembly) are detected by confocal mi-croscopy at the L. monocytogenes invasion foci,and real-time imaging analysis of the infectionprocess indicates that the dynamics of clathrinrecruitment at bacterial invasion sites differfrom the behavior observed during the forma-tion of classical clathrin-coated pits and vesiclesduring endocytosis of smaller cargo (Veiga et al.2007). Interestingly, depletion of clathrin or dy-namin by siRNA inhibits actin polymerization,suggesting that the recruitment of the clathrin-endocytic machinery precedes the actin re-arrangements required for InlB-mediated en-try (Veiga et al. 2007).

These results challenge the prevailingdogma establishing that the upper-limit size ofclathrin-coated vesicles is 150 nm (Cheng et al.2007), implying that larger particles cannotbe internalized through a clathrin-dependentmechanism. It is interesting to note, however,that large clathrin assemblies have been de-tected surrounding the base of nascent phago-somes containing opsonized beads (Aggeler &Werb 1982). Clathrin had also been previouslyobserved in human epithelial cells by trans-mission electron microscopy at the entry sitesof Chlamydia trachomatis (Wyrick et al. 1989)and E. coli expressing the adhesins AfaE/AfaD( Jouve et al. 1997), and use of the drug mono-dansylcadaverin, which acts as an inhibitorof the transglutaminase that participates inclathrin-dependent endocytosis, suggested thatS. aureus also requires clathrin for the invasionof cultured osteoblasts (Ellington et al. 1999).

The involvement of the endocytic machin-ery in bacterial entry is, in fact, a widespreadmechanism, and the recruitment of clathrin anddynamin also occurs for the Listeria innocuaInlA/E-cadherin-dependent invasion pathway,for other zippering bacteria that include


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S. aureus coated with fibronectin and E. coliexpressing the invasin protein of Y. pseu-dotuberculosis, and for latex beads of 1 or5 μm coated with epidermal growth factoror fibronectin, which suggests a specificityof the endocytic-machinery requirement forreceptor-dependent entry (Veiga et al. 2007).In agreement with this hypothesis, the entry ofpathogens that do not use a classical receptor-mediated entry mechanism, but use a T3SS-mediated entry as does S. flexneri or S. enter-ica, does not require clathrin (Green & Brown2006, Veiga et al. 2007). In addition, the FimH-dependent internalization of UPEC in bladdercells requires clathrin (Eto et al. 2008) and, in-terestingly, a noninvading pathogen such as en-teropathogenic E. coli (EPEC), which attachesto the extracellular surface and induces mem-brane reorganization and pedestal formationthrough the infection of T3SS effectors, re-cruits clathrin at its attachment site (Veiga et al.2007). However, how clathrin is assembled atthe EPEC attachment site or during the entryof zippering bacteria is still unknown.

Studies of the trophoblastic cell line JEG-3expressing the InlA receptor E-cadherinshowed that clathrin depletion impaired, butdid not completely block, the entry of thebacteria L. innocua (InlA), suggesting that othercellular internalization pathways should play arole in the InlA invasion pathway (Veiga et al.2007). In accordance with this hypothesis, ithas been recently shown that caveolin-1 is alsorequired for L. innocua (InlA) entry in JEG-3cells (Bonazzi et al. 2008) (Figure 2). Indeed,caveolin-1 is recruited to the L. innocua (InlA)entry sites with dynamics that reflect those of E-cadherin recruitment, and caveolin-1 depletionby siRNA prevents E-cadherin clustering atbacterial entry sites and bacterial entry (Bonazziet al. 2008). Interestingly, caveolin-1 recruit-ment occurs upstream of the tyrosine phospho-rylation of E-cadherin by the kinase Src, andthis posttranslation modification of E-cadherinis required for the recruitment of the ubiquitin-ligase Hakai, which is necessary for the ubiqui-tination and clathrin-mediated internalizationof E-cadherin (Bonazzi et al. 2008). Overall,

these results demonstrate that caveolin-1recruitment precedes clathrin recruitment.

Dependence on Cbl, clathrin, and caveolin-2 (but not caveolin-1) has been demonstratedrecently for the entry of E. coli expressing therOmpB protein of R. conorii in HeLa cells (Chanet al. 2009). However, the hierarchy of recruit-ment of clathrin and caveolin-2 following cel-lular stimulation with rOmpB as well as thestructural differences that caveolin-2 confers tothe bacterial invasion site in comparison withcaveolin-1 are not known. It is important alsoto note that Pseudomonas aeruginosa colocal-izes with caveolin-1 and caveolin-2 during theinvasion of type I pneumocytes. In addition,siRNA-mediated depletion of both caveolinsleads to a diminution in bacterial entry. How-ever, the selective depletion of caveolin-2 hasan identical susceptibility to P. aeruginosa infec-tion as the combined caveolin-1 and caveolin-2knock-down, and only tyrosine phosphoryla-tion of caveolin-2 is critical for P. aeruginosa in-vasion, which suggests a more important rolefor caveolin-2 in this process (Zaas et al. 2005).

As mentioned above, caveolin-1 bindscholesterol and hence associates with lipid rafts.During UPEC entry in the bladder epithe-lium, caveolin-1 is detected at bacterial en-try lipidic microdomains, and the reduction ofcaveolin-1 expression by siRNA inhibits bacte-rial invasion (Duncan et al. 2004). Lipid raft-associated caveolae and caveolin-1 act as entryports for Porphyromonas gingivalis in oral epithe-lial cells (Sukumaran et al. 2002) for Group AStreptococcus into epithelial and endothelial cells(Rohde et al. 2003), and they are requiredfor the transcytosis of meningitis-inducingE. coli K1 in brain microvascular endothelialcells (Sukumaran et al. 2002).

In summary, the traditional machinery asso-ciated with endocytosis appears critical for theinternalization of larger particles such as in-vading bacteria. The molecular pathways thatlink endocytosis and actin polymerization havebeen difficult to establish, but recent work fromseveral laboratories has identified connectionsbetween these different pathways (Galletta &Cooper 2009, Kaksonen et al. 2006). The study


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of bacterial internalization will probably helpus further understand how these connectionstake place (Veiga et al. 2007). Clathrin clearlyassembles in a nonclassical way at sites of bac-terial entry, and solving the structure of theseassemblies, as well as defining the cross talk be-tween caveolin and clathrin, will be an excitingtask for the near future.

LIFE IN AND ESCAPE FROMTHE VACUOLE

Maturation of the Vacuole

After the invasion of target cells, L. monocy-togenes is transiently trapped in a membrane-bound compartment before it destroys thevacuolar membrane to escape in the hostcell cytoplasm. L. monocytogenes was proposedto modify the vacuole maturation throughmodulation of the activity of the small GTPaseRab5a (Alvarez-Dominguez et al. 2008)(Figure 5). Rab5a (but not Rab5c) overexpres-sion increases the degradation of a hly mutantL. monocytogenes, which cannot escape from

the phagocytic vacuole (Alvarez-Dominguez& Stahl 1999). In macrophages infected withwild-type L. monocytogenes, Rab5a facilitatesthe translocation of the small Rho GTPaseRac to the bacterial-containing compartment,which in turn activates the NADPH oxidaseat the vacuolar membrane for bacterial killing(Prada-Delgado et al. 2001). However, L.monocytogenes inhibits the exchange activityof Rab5a from an inactive GDP- to an activeGTP-bound form by expressing a 40-kDaprotein (Lmo 2459) that ADP-ribosylatesRab5a in its inactive form (Alvarez-Dominguezet al. 2008, Prada-Delgado et al. 2005). Howthis enzyme is translocated across the vacuolarmembrane to interact with Rab5a is currentlyunknown.

Listeriolysin O and Vacuolar Escape

Lysis of the L. monocytogenes internalizationcompartment is mediated via the expressionand activity of listeriolysin O (LLO), whichis one of the major L. monocytogenes virulencefactors (Cossart et al. 1989, Kathariou et al.

GILTLLO

Lmo2459

GDP GTP

NADPHoxidase

O–

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Ca+2

Ca+2

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a b c

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PC-PLCListeria Listeria

Listeria

Listeria

Vacuole Vacuole Vacuole

Lmo2459

Figure 5Listeria monocytogenes residency in its internalization vacuole and escape to the host cytoplasm. After internalization in target cells,L. monocytogenes resides in a membrane-bound compartment before lysing the vacuole membrane to escape to the cytosol. (a) Activationof the small GTPase Rab5a is important for the Rac-dependent recruitment of the NADPH oxidase to the phagosomal membrane tofavor bacterial killing. L. monocytogenes counterbalances this signaling cascade by producing a 40-kDa protein Lmo 2459, which istransported through the vacuole membrane (by an unidentified translocation step) and ADP-ribosylates/blocks Rab5a in an inactiveGDP-bound form. (b) The host γ-interferon-inducible lysosomal thiol reductase (GILT) reduces the bacterial-produced listeriolysinO (LLO) and favors its oligomerization into an LLO pore, which is implicated in the reduction of vacuolar Ca2+ levels (inhibiting therecruitment of the lysosomal protein LAMP1). PI- and PC-PLC cooperate with LLO in the disruption of the vacuolar membrane.Acidification (H+) of the vacuole is important for LLO activity and PC-PLC maturation by a bacterial metalloprotease (not shown).(c) Disruption of the vacuolar membrane leads to bacterial translocation to the host cytoplasm and escape from phagosomal killing.


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1987). LLO belongs to the large familyof cholesterol-dependent cytolysins includingperfringolysin O from Clostridium perfringensand streptolysin O (SLO) from Streptococcuspyogenes, and monomers of these toxins bindto cholesterol-containing membranes in whichthey oligomerize to form 20–30-nm-diameterpores (Bhakdi et al. 1985, Sekiya et al. 1993).Despite several decades of research, LLO’s pre-cise mechanism of action on membranes andthe molecular events that lead to membraneperforation are still elusive. The initial hypoth-esis that cholesterol is the cellular receptorfor LLO, and therefore primarily required fortoxin binding to target membranes, was refutedby the observation that LLO monomers thatare preincubated with cholesterol can stillefficiently bind membranes and oligomerizebut are unable to form pores ( Jacobs et al.1998). Work performed with perfringolysin Oand SLO suggests that cholesterol is critical forthe prepore-to-pore transition (Giddings et al.2003). Acidification of the L. monocytogenes-containing vacuole is required for membranepermeabilization (Beauregard et al. 1997), inagreement with the observation that LLOpresents a higher hemolytic potential at acidicpH (Geoffroy et al. 1987) (Figure 5). However,acidification may be required for synthesis,release, or activation of other bacterial or hostfactors involved in phagosomal escape. Rup-ture of the phagosomal membrane is enhancedby a bacterial phosphatidylinositol-specificand a broad-range phospholipase C (PI- andPC-PLC, respectively) (Smith et al. 1995),and activation/release of the proPC-PLC tomature PC-PLC by a L. monocytogenes-encodedmetalloprotease is precisely dependent onacidic pH (Marquis & Hager 2000). LLOalso requires maturation through reductionof disulfide bonds, and full induction of theLLO lytic activity in vivo requires its reductionby the γ-interferon-inducible lysosomal thiolreductase (GILT), an enzyme that is deliveredfrom lysosomes to maturing compartments ofthe endocytic/phagocytic pathway in antigen-presenting cells: L. monocytogenes replication inGILT-negative macrophages is impaired owing

to delayed escape from the vacuole (Singh et al.2008) (Figure 5).

What are the effects of LLO insertion on theL. monocytogenes-containing vacuole? LLO in-duces small-membrane perforations, which al-low Ca2+ leakage from vacuoles shortly afterinfection and lead to an increase in the vac-uolar pH and inhibition of vacuolar matura-tion, as measured by the delayed acquisitionof the lysosomal-associated membrane pro-tein 1 (LAMP1) in wild-type L. monocytogenes-containing compartments compared with thoseof LLO-negative mutants (Henry et al. 2006,Shaughnessy et al. 2006). Ca2+ favors en-dosomal fusion with lysosomes (Pryor et al.2000), and calcium release could account forthe LLO-mediated alteration of vacuolar mat-uration (Shaughnessy et al. 2006). In addition,LLO is inefficient in lysis of late-stage vacuolesthat are positive for LAMP1 (Shaughnessy et al.2006). Overall, these observations indicate thatLLO favors the inhibition of vacuolar mat-uration and fusion with lysosomes in orderto protect L. monocytogenes from the destruc-tive effects of mature lysosomal enzymes. LLOmay also make pores to mediate the transloca-tion of PI- and PC-PLC to the cytosol, wherethey could access their substrate phospholipidspresent in the cytosolic leaflet for the bacteria-containing vacuole, thereby favoring vacuo-lar escape (Sibelius et al. 1996, Wadsworth &Goldfine 2002). This potential mechanism oftranslocation is reminiscent of that reported forthe SLO of S. pyogenes and may be similar tothe T3SS in gram-positive bacteria (Maddenet al. 2001). More recently, it has been pro-posed that bacterial vacuolar lysis that is depen-dent on LLO (and not on PI- and PC-PLC)leads to a host cell innate response that trig-gers the control of L. monocytogenes intracellulargrowth by autophagy (Py et al. 2007) (see nextsection).

Several other pathogens escape from theirinternalization compartments to replicate inthe host cytoplasm, but the molecular eventsleading to vacuolar escape are poorly under-stood. In the case of the Rickettsia species, phos-pholipase A2 (PLA2) activity was reported for


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Rickettsia prowazekii, and PLA2 was proposedto be involved in phagosomal escape (Ojciuset al. 1995). However, completion of the R.prowazekii and R. conorii genomes indicatesthat no such gene could be identified, buta gene that codes for a phospholipase D,which is expressed and detected in rickettsiallysates, could account for the previously de-scribed PLA2 activity (Renesto et al. 2003).Whether this enzyme plays a role in bacterialescape from the vacuole remains to be demon-strated. A similar situation has been reported forR. typhi because a hemolysin has been clonedand characterized in this pathogen (Radulovicet al. 1999), but its possible role in vivo has notbeen demonstrated.

Vacuolar escape of S. flexneri remains anelusive issue. In J774 murine macrophages,IpaB mutants remained trapped in a phagocyticvacuole, suggesting that IpaB plays a role in vac-uolar lysis (High et al. 1992). Subsequent com-parative studies of the S. flexneri IpaC proteinand its S. enterica homolog SipC showed thatS. enterica SipC mutants expressing IpaC werefound in the cytoplasm of host cells, which sug-gests a role for IpaC in S. flexneri escape fromthe vacuole (Osiecki et al. 2001). Subsequently,mutation of the leucine-rich repeat proteinIpaH7.8 was shown to slow down S. flexneri cyto-plasmic localization, implicating this protein inthe facilitation of vacuolar escape (Fernandez-Prada et al. 2000).

Recently, escape of M. tuberculosis andM. leprae from their phagosome to the hostcytoplasm of myeloid cells has been describedand proposed to depend on the type 7 secretionsystem ESX-1 (van der Wel et al. 2007); how-ever, these observations remain controversialin the field. M. marinum, a pathogen responsi-ble for tuberculosis in fish and also in humans,uses the ESX-1 system to secrete the proteinESAT-6, which has been directly implicated inthe formation of vacuolar pore and bacterialescape from phagocytic vacuoles (Smith et al.2008). Additionally, invasive S. pyogenes maybe able to escape phagosomes through SLO-induced membrane perforation and cytoplas-mic bacteria may be destroyed by innate defense

mechanisms (Nakagawa et al. 2004) (see nextsection), but the functional relevance of vacuo-lar escape for Group A Streptococcus has not beendiscussed.

Work concerning bacterial escape from vac-uoles has focused on the assumption that main-tenance of the vacuolar membrane is dependentonly on the activity of bacterial proteins or tox-ins that damage the vacuole physical integrity.It is important to mention that in the case ofS. enterica, an intracellular pathogen that nor-mally proliferates in a membrane-bound com-partment, deletion of the protein SifA leadsto bacterial localization in the host cell cyto-plasm (Beuzon et al. 2000), which indicatesthat bacteria can also actively participate in themaintenance of the vacuolar membrane. A sim-ilar situation is probably true in the case ofLegionella pneumophila (Isberg 2009), highlight-ing that bacterial interactions with host cellmolecular pathways are also implicated in acomplex balance between vacuolar membranemaintenance and disruption.

INTRACYTOSOLIC SURVIVALAND AUTOPHAGY

Autophagy is an important cellular processinvolved in the degradation of cellular compo-nents in the cytoplasm, which allows proteinturnover in starving cells or the removal of mal-formed or superfluous subcellular componentsby sequestration in a double-membrane com-partment termed the autophagosome, whichmay be derived from the rough endoplasmicreticulum (rER) (Klionsky 2007). Analysisof rER markers coupled with morphologicalanalyses permitted the initial characterizationof this pathway, and several bacterial speciesincluding Brucella abortus and Legionella pneu-mophila have been shown to interact withautophagosomes to establish a successfulintracellular replication niche within host cells(Pizarro-Cerda et al. 1998). Only recentlyhas the molecular machinery involved inautophagosome biogenesis been partially un-raveled, and it has been demonstrated that thisprocess can be used by the cell as an innate


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ER-derivedvesicles

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Actin

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ΔActA

VASP

a

bLLO

Pore

Listeria

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Figure 6Autophagy as a host innate defense mechanism against Listeria monocytogenes. Bacterial localization in thehost cytosol triggers the formation of autophagosomes to control bacterial proliferation. (a) The bacterialsurface protein ActA brings vasodilator-stimulated phosphoprotein (VASP) and, indirectly, profilin to thebacterial tail, which induces the recruitment of actin monomers that are polymerized by the Arp2/3 complexinto actin comet tails. This actin-based motility system allows L. monocytogenes to move in the host cytoplasmand to escape capture by autophagic vacuoles. (b) A �ActA L. monocytogenes mutant actively producing LLOwill be trapped by LC3/LAMP1-positive autophagosomes originally derived from the endoplasmicreticulum, thus inducing bacterial killing. Besides ActA expression, the secretion of phospholipases PI-PLCand PC-PLC seems important for wild-type bacteria to escape from autophagy.

mechanism to control intracellular pathogensurvival.

After vacuolar escape, the L. monocytogenessurface protein ActA promotes cellular actinpolymerization and bacterial movement inthe host cell cytoplasm (Kocks et al. 1992)(Figure 6). In macrophages infected with aL. monocytogenes �ActA mutant, chloram-phenicol treatment was found to lead tobacterial sequestration of previously cytoplas-mic bacteria into double-membrane organellesthat resembled autophagosomes, which weredecorated initially with the rER marker proteindisulfide isomerase and subsequently with thelysosomal LAMP1 marker. This promptedRich et al. (2003) to propose that the autophagicpathway could be used as a host defense mech-anism against invading pathogens. Yuan andcollaborators (Py et al. 2007) observed thatautophagy limits the L. monocytogenes intra-cellular growth during the early time pointsafter cellular invasion and vacuolar escape:Permeabilization of the vacuole by LLO (butnot by phospholipases) triggers the bacterialsequestering in autophagosomes labeled by the

microtubule-associated protein 1 light chain 3(LC3) autophagic degradation compartments(Py et al. 2007). ActA was proposed as the mainbacterial factor required for autophagosomeescape during the first 3 hours after infec-tion, but other virulence factors seem to beimportant to evade autophagy at later timepoints (Birmingham et al. 2007). In Drosophila,recognition of the L. monocytogenes peptidogly-can by the peptidoglycan-recognition protein(PGRP)-LE is crucial for the induction ofautophagy and the prevention of bacterial in-tracellular growth (Yano et al. 2008). In SCIDmice, LLO function has been highlightedagain as required for the formation of spaciousL. monocytogenes-containing phagosomes ofautophagic origin (Birmingham et al. 2008).

After the establishment of a functionallink between autophagy and control ofL. monocytogenes intracellular proliferation(Rich et al. 2003), other reports documentedthe role of autophagy as an innate host defensemechanism against bacterial pathogens such asM. tuberculosis (Gutierrez et al. 2004). Afterinternalization in macrophages, M. tuberculosis


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remains associated with a membrane-boundcompartment and blocks the maturation ofthis compartment at an early phagosomalstage (Sturgill-Koszycki et al. 1994, Via et al.1997). Autophagy stimulation by starvation orrapamycin treatment leads to M. tuberculosisphagosome colocalization with the LC3autophagosomal marker and inhibition ofmycobacterial intracellular survival (Gutierrezet al. 2004). The murine immunity-related p47guanosine triphosphatase Irgm1 and its humanortholog IRGM trigger autophagy to eliminateintracellular mycobacteria (Singh et al. 2006).And ubiquitin-derived peptides with bacteri-cidal activity against mycobacteria accumulatein autophagolysosomes and enhance bacterialkilling (Alonso et al. 2007).

Autophagy has been demonstrated asa defense mechanism against S. pyogenes: Inmouse embryonic autophagy-deficient Atg5−/−

fibroblasts, bacteria that escape from vacuolesvia an SLO-dependent mechanism are ableto multiply and be released from infectedcells (Nakagawa et al. 2004). The intercellularspread A (IcsA) protein of S. flexneri, requiredfor the actin-based motility of this bacteriumin the cytosol of host cells, is targeted by Atg5,thereby triggering autophagocytosis and bac-terial killing. However, the T3SS effector IcsBblocks the IcsA/Atg5 interaction by directly in-teracting with Atg5 and, in this way, S. flexneri isable to escape autopaghy (Ogawa et al. 2005). Inthe case of S. enterica, some bacteria-containingvacuoles that have been damaged by the activityof the T3SS, SPI-1 (normally required onlyfor cellular invasion and not for intracellularsurvival), are targeted by autophagosomes andcontrol potential bacterial replication in thehost cell cytoplasm (Birmingham et al. 2006).

Cytoplasmic and membrane-bound intra-cellular pathogens are thus targeted by au-tophagy for destruction. The deciphering of themolecular events that trigger this new innateimmune response will reveal whether a singlepathway is always activated or whether diversityalso exists in the formation of autophagosomesand different signaling cascades are activated ineach case.

CELL-TO-CELL SPREAD

As mentioned above, L. monocytogenes moves inthe host cell cytosol using an actin-based motil-ity process. In nonconfluent cells, bacteria thatreach the cellular plasma membrane induce theformation of long membrane protrusions thatextend with bacteria at their tips but, surpris-ingly, never lyse or break. Actin-based motilityis sufficient to promote protrusion formation,as revealed by an E. coli strain expressing invasinfrom Y. pseudotuberculosis for cellular invasion,LLO from L. monocytogenes to facilitate vacuo-lar escape, and IcsA of S. flexneri to produceactin polymerization (Monack & Theriot2001). In confluent monolayers, bacterialprotrusions penetrate in neighboring cells andlead to the formation of a double-membranecompartment from which L. monocytogenesescapes to initiate a new infection cycle. Theterm paracytophagy has been proposed todescribe a normal cellular mechanism thatallows epithelial cell engulfment of membranefragments from neighboring cells, which isexploited in part by L. monocytogenes to formproductive protrusions (Robbins et al. 1999).The inhibition of interaction between actin andmembers of the ezrin/radixin/moesin familyresults in the formation of short and collapsedprotrusions, suggesting that ezrin, in particular,is critical for cell-to-cell spread (Pust et al.2005). A recent study suggested that PI- andPC-PLC are required for the lysis of the innermembrane of the double-membrane vacuoleformed after protrusion formation in secondarycells and that LLO lyses the outer membraneof this compartment (Alberti-Segui et al.2007).

Few studies have been performed to addressthe cell-to-cell spread issue in other bacterialpathogens. In the case of S. flexneri, E-cadherinexpression seems to be required for a productiveprotrusion formation (Sansonetti et al. 1994).Clearly, cell-to-cell spread is a complex phe-nomenon that requires inventive cell manipu-lation techniques to be fully addressed, but itis worth investigating given its key role duringinfection.


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CONCLUSIONSSurvival and replication of bacterial pathogenswithin mammalian cells rely on a variety ofmolecular strategies. In this review, we describein detail the membrane trafficking pathwayshijacked by Listeria and highlight that otherbacteria, although using different effectors, can

target the same pathways to subvert cellularfunctions and to hamper deleterious host re-sponses. Knowledge of the specific cells or tis-sues in which these events occur in vivo will becritical for a full understanding of the infectionprocess and poses a real challenge for futureresearch.

DISCLOSURE STATEMENT

The authors are not aware of any biases that might be perceived as affecting the objectivity of thisreview.

ACKNOWLEDGMENTS

We apologize to colleagues whose work could not be included in this review owing to spacelimitations. Work in P.C.’s group received support from the Pasteur Institute, INSERM, INRA,and ANR. P.C. is also an international scholar from the Howard Hughes Medical Institute.

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AR389-FM ARI 14 September 2009 14:58

Annual Reviewof Cell andDevelopmentalBiology

Volume 25, 2009

ContentsChromosome Odds and Ends

Joseph G. Gall � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Small RNAs and Their Roles in Plant DevelopmentXuemei Chen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �21

From Progenitors to Differentiated Cells in the Vertebrate RetinaMichalis Agathocleous and William A. Harris � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �45

Mechanisms of Lipid Transport Involved in Organelle Biogenesisin Plant CellsChristoph Benning � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �71

Innovations in Teaching Undergraduate Biologyand Why We Need ThemWilliam B. Wood � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �93

Membrane Traffic within the Golgi ApparatusBenjamin S. Glick and Akihiko Nakano � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 113

Molecular Circuitry of Endocytosis at Nerve TerminalsJeremy Dittman and Timothy A. Ryan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 133

Many Paths to Synaptic SpecificityJoshua R. Sanes and Masahito Yamagata � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 161

Mechanisms of Growth and Homeostasis in the Drosophila WingRicardo M. Neto-Silva, Brent S. Wells, and Laura A. Johnston � � � � � � � � � � � � � � � � � � � � � � � � � 197

Vertebrate Endoderm Development and Organ FormationAaron M. Zorn and James M. Wells � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 221

Signaling in Adult NeurogenesisHoonkyo Suh, Wei Deng, and Fred H. Gage � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 253

Vernalization: Winter and the Timing of Flowering in PlantsDong-Hwan Kim, Mark R. Doyle, Sibum Sung, and Richard M. Amasino � � � � � � � � � � � � 277

Quantitative Time-Lapse Fluorescence Microscopy in Single CellsDale Muzzey and Alexander van Oudenaarden � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 301

Mechanisms Shaping the Membranes of Cellular OrganellesYoko Shibata, Junjie Hu, Michael M. Kozlov, and Tom A. Rapoport � � � � � � � � � � � � � � � � � � � � 329

The Biogenesis and Function of PIWI Proteins and piRNAs: Progressand ProspectTravis Thomson and Haifan Lin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 355

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Mechanisms of Stem Cell Self-RenewalShenghui He, Daisuke Nakada, and Sean J. Morrison � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 377

Collective Cell MigrationPernille Rørth � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 407

Hox Genes and Segmentation of the Hindbrain and Axial SkeletonTara Alexander, Christof Nolte, and Robb Krumlauf � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 431

Gonad Morphogenesis in Vertebrates: Divergent Means to aConvergent EndTony DeFalco and Blanche Capel � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 457

From Mouse Egg to Mouse Embryo: Polarities, Axes, and TissuesMartin H. Johnson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 483

Conflicting Views on the Membrane Fusion Machinery and the FusionPoreJakob B. Sørensen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 513

Coordination of Lipid Metabolism in Membrane BiogenesisAxel Nohturfft and Shao Chong Zhang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 539

Navigating ECM Barriers at the Invasive Front: The CancerCell–Stroma InterfaceR. Grant Rowe and Stephen J. Weiss � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 567

The Molecular Basis of Organ Formation: Insights from theC. elegans ForegutSusan E. Mango � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 597

Genetic Control of Bone FormationGerard Karsenty, Henry M. Kronenberg, and Carmine Settembre � � � � � � � � � � � � � � � � � � � � � � 629

Listeria monocytogenes Membrane Trafficking and Lifestyle:The Exception or the Rule?Javier Pizarro-Cerda and Pascale Cossart � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 649

Asymmetric Cell Divisions and Asymmetric Cell FatesShahragim Tajbakhsh, Pierre Rocheteau, and Isabelle Le Roux � � � � � � � � � � � � � � � � � � � � � � � � � � � 671

Indexes

Cumulative Index of Contributing Authors, Volumes 21–25 � � � � � � � � � � � � � � � � � � � � � � � � � � � 701

Cumulative Index of Chapter Titles, Volumes 21–25 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 704

Errata

An online log of corrections to Annual Review of Cell and Developmental Biology articlesmay be found at http://cellbio.annualreviews.org/errata.shtml

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listeria monocytogenes membrane trafficking and lifestyle: the exception or the rule?

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