Asembly and Function of Type III Secretion Pathway

8/2/2019 Asembly and Function of Type III Secretion Pathway

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Annu. Rev. Microbiol. 2000. 54:73574Copyright c 2000 by Annual Reviews. All rights reserved

ASSEMBLY AND FUNCTION OF TYPE IIISECRETORY SYSTEMS

Guy R. Cornelis1 and Frederique Van Gijsegem21Microbial Pathogenesis Unit, Christian de Duve Institute of Cellular Pathology and

Facult e de M edecine, Universit e Catholique de Louvain, B-1200 Brussels, Belgium;

e-mail: [email protected]; 2 Laboratoire Associ e de lINRA (France),

Department of Plant GeneticsVIB, Universiteit Gent, B-9000 Gent, Belgium;

e-mail: [email protected]

Key Words microbial pathogenesis, plant pathogens, secretion, translocation,effector, hypersensitive response

s Abstract Type III secretion systems allow Yersinia spp., Salmonella spp.,Shigella spp., Bordetella spp., and Pseudomonas aeruginosa and enteropathogenic

Escherichia coli adhering at the surface of a eukaryotic cell to inject bacterial proteinsacross the two bacterial membranes and the eukaryotic cell membrane to destroy or

subvert the target cell. These systems consist of a secretion apparatus, made of25proteins, and an array of proteins released by this apparatus. Some of these releasedproteins are effectors, which are delivered into the cytosol of the target cell, whereasthe others are translocators, which help the effectors to cross the membrane of the eu-karyotic cell. Most of the effectors act on the cytoskeleton or on intracellular-signalingcascades. A protein injected by the enteropathogenic E. coli serves as a membranereceptor for the docking of the bacterium itself at the surface of the cell. Type III se-cretion systems also occur in plant pathogens where they are involved both in causingdisease in susceptible hosts and in eliciting the so-called hypersensitive response inresistant or nonhost plants. They consist of 1520 Hrp proteins building a secretionapparatus and two groups of effectors: harpins and avirulence proteins. Harpins arepresumably secreted in the extracellular compartment, whereas avirulence proteins arethought to be targeted into plant cells. Although a coherent picture is clearly emerging,basic questions remain to be answered. In particular, little is known about how the typeIII apparatus fits together to deliver proteins in animal cells. It is even more mysteriousfor plant cells where a thick wall has to be crossed. In spite of these haunting questions,type III secretion appears as a fascinating trans-kingdom communication device.

CONTENTS

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736 CORNELIS VAN GIJSEGEM

From the Yersinia Ysc Secretion Apparatus to the Salmonella and

Shigella Needles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738

Type III Pili from Plant Pathogens and Enteropathogenic E. coli . . . . . . . . . . . . . 741

Translocation of Effectors Across Animal Cell Membranes . . . . . . . . . . . . . . . . . 742A Pore Formed by Translocators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 742

Relation Between the Pore and the Needle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 743

Harpins and Avr Proteins from Plant Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . 744

The Cytosolic Chaperones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748

Recognition of the Transported Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751

Control of the Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752

EFFECTOR PROTEINS AND HOST RESPONSES . . . . . . . . . . . . . . . . . . . . . . . 752

A Panoply of Effectors and Avirulence Proteins . . . . . . . . . . . . . . . . . . . . . . . . . 752

Enzymic Activities of Effectors and Avirulence Proteins . . . . . . . . . . . . . . . . . . . 753

The Cytoskeleton Is a Major Target in Animal Cells . . . . . . . . . . . . . . . . . . . . . . 753Modulation of Inflammation and Signaling Interference . . . . . . . . . . . . . . . . . . . 757

Signaling Interference in Plant Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 758

The Tir Protein, a Unique Case of Translocated Receptor . . . . . . . . . . . . . . . . . . 759

The YopM and AvrBs3/Pth Families: Effectors Targeted to the Nucleus . . . . . . . . 760

Role of Avr Proteins in Virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760

Intracellular Action of Translocators in Animal Cells . . . . . . . . . . . . . . . . . . . . . 761

The Enigmatic Role of Harpins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 761

COMPARISON OF THE VARIOUS TYPE III SYSTEMS . . . . . . . . . . . . . . . . . . . 762

Three Major Groups of Systems Among the Animal Pathogens . . . . . . . . . . . . . . 762

Two Different Groups ofhrp Gene Clusters in Plant Pathogens . . . . . . . . . . . . . . 763Exchangeability Between the Effectors of the Different Systems . . . . . . . . . . . . . 763

GENETIC BASES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 764

PROSPECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 764

INTRODUCTION

For a rather long period, it was assumed that gram-negative bacteria do not se-

crete proteins into their environment but only export proteins in their strategic pe-riplasm. However, research in the last two decades has revealed that gram-negative

bacteria do indeed transfer proteins across their sophisticated outer membrane, and

they do this by a variety of systems that are now classified into four major types

and several minor ones. Type I, exemplified by the hemolysin secretion system of

Escherichia coli, is a rather simple exporter that is based on only three proteins,

one of which belongs to the ABC transporters. Type II is a very complex apparatus

that extends the general secretory pathway and transfers fully folded enzymes or

toxins from the periplasm to the extracellular medium, across the outer membrane.

Type IV, another complex system that transfers pertussis toxin among others, isrelated to the apparatus of Agrobacterium spp. that transfers DNA to plant cells.

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TYPE III SECRETION 737

proteins across the plasma membrane. This system probably also allows bacteria

residing in vacuoles to inject proteins across the vacuolar membrane. The injected

proteins subvert the functioning of the aggressed cell or destroy its communica-

tions, favoring the entry or survival of the invading bacteria. Type III is thus not asecretion apparatus in the strict sense of the term but rather a complex weapon for

close combat. It contributes to a number of totally different animal diseases with

a variety of symptoms and severities, from fatal septicemia to mild diarrhea and

from fulgurant diarrhea to chronic infection of the lung. Type III secretion has

been extensively studied in Yersinia spp. (reviewed in 25), in Salmonella spp. (re-

viewed in 47), in Shigella spp. (reviewed in 138), and in enteropathogenic E. coli

(EPEC) and enterohemorrhagic E. coli (EHEC) (40, 50, 72). It has also been de-

scribed in Pseudomonas aeruginosa (TL Yahr & DW Frank, Genbank PAU56077),

Chlamydia trachomatis and Chlamydia pneumoniae (73a),Bordetella bronchisep-tica (MH Yuk, ET Harvill, JF Miller, Genbank AFO49488), Bordetella pertussis

(78a) and in Burkholderia pseudomallei (The Sanger Center, Cambridge, UK). It

is surprising that Salmonella typhimurium and Yersinia spp. have not only one

type III system but two (61, 104; S Carlson & DE Pierson, Genbank AFO055744;

The Sanger Center, Cambridge, UK), presumably playing their role at different

stages of the infection (Figure 1).

Type III secretion systems are also encountered in most gram-negative phy-

topathogenic bacteria. Agrobacterium is about the only gram-negative phyto-

pathogenic genus in which this system has not been found. Type III systemsare well documented in Erwinia amylovora, Pseudomonas syringae, Ralstonia

solanacearum, and Xanthomonas spp. (3, 47). These systems are involved not

only in causing diseases, which can be as different as localized lesions or sys-

temic wilting or blights, but also in the elicitation of plant defense mechanisms

that lead to resistance. Very often, resistance is accompanied by the occurrence of

the so-called hypersensitive response (HR), which is defined as the rapid death of

plant cells at the infection site leading to restricted colonization of the potential

pathogen.

The genes encoding type III pathways have thus been named hrp, becausemutants were impaired both in the elicitation of the HR and in pathogenicity (87).

Although there is no direct proof yet for protein injection by Hrp type III systems,

there is indirect evidence and a strong consensus that type III systems from plant

pathogens, besides secreting proteins in the extracellular medium, also deliver

proteins into plant cells. Finally, the type of intercellular communication that type

III secretion allows is not restricted to pathogenesis; it also modulates the host

range of some Rhizobium spp. in symbiosis (141).

A secretion system that is very similar to the various type III systems is also

dedicated to the export of the components of the flagellum (reviewed in 96). Itis interesting that a Yersinia enterocolitica phospholipase involved in virulence

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Figure 1 Type III systems in animal pathogens. Illustrated are the various bacterial pathogensendowed with type III secretion, injecting effectors into the cytosol of a eukaryotic target cell. See

Table 3 for references.

reviews for more detailed information and references (18, 25, 47, 66, 87). We

apologize to the authors of many important relevant studies that could not be

cited here.

A DEVICE TO INJECT BACTERIAL PROTEINS ACROSSEUKARYOTIC CELL MEMBRANES

From the YersiniaYsc Secretion Apparatusto the Salmonellaand ShigellaNeedles

The first observation of type III secretion was made with Yersinia in ca 1990.

It was the first major outcome of long and tenacious research by a few groups

trying to understand the mysterious phenomenon of Ca2+-dependency: when in-

cubated at 37C in the absence of Ca2+ ions, Yersinia bacteria no longer grow, butinstead release large amounts of proteins called Yops in the culture supernatant

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leakage is presumably artefactual, this observation turned out to be of paramount

importance because it allowed workers to carry out the genetic analysis that led

to the identification of 29 genes involved in this process of Yops release and

called ysc for Yop secretion. Only a minority of the Ysc proteins have beencharacterized so far, but the Ysc system remains the archetype of the type III

secretion systems. The ysc gene nomenclature has been transposed in P. aerug-

inosa (psc genes), in Bordetella spp. (bsc genes), and in EPECs (esc genes),

but also in plant pathogens (hrc genes for HR conserved) and in Rhizobium spp.

(rhc genes for Rhizobium conserved) for the type III genes that are conserved:

the psc, bsc, esc, hrc, and rhc genes thus carry the same letter code as their ysc

homolog.

We first briefly describe the 11 Ysc proteins (YscC, -D, -J, -L, -N, -Q, -R, -S,

-T, -U, and -V) that have counterparts in almost every type III secretion apparatusand then mention a few of the other Ysc components (Figure 2; see References 25

and 66 for the complete lists and references). YscC, one of the best known Ysc

proteins, belongs to the family of secretins, a group of outer membrane proteins

involved in the transport of various macromolecules and filamentous phages across

the outer membrane. As the other secretins, it forms a ring-shaped structure with

an external diameter of about 200 A and an apparent central pore of about 50 A

(82). As a matter of comparison, the PIV secretin of phage f1 has an internal

diameter of about 80 A, allowing the passage of the filamentous capsid with a

diameter of 65 A (86). Four proteins (YscD, -R, -U and -V, formerly called LcrD)have been shown and two other proteins (YscS and -T) have been predicted to

span the inner membrane. YscN is a 47.8-kDa protein with ATP-binding motifs

(Walker boxes A and B) resembling the catalytic subunit of F0F1 proton translo-

case and related ATPases. It probably energizes the secretion process. YscJ is a

lipoprotein that has not been localized yet but its counterpart in P. syringae has

been shown to span the inner and the outer membranes (30). Little is known

about YscL and YscQ but SpaO, the Salmonella counterpart of YscQ is itself

secreted (24).

Little is also known about the Ysc proteins that are less conserved. YscW isa lipoprotein that serves for the proper insertion of YscC in the outer membrane

(82). Finally, the two proteins YscO and YscP are themselves released upon Ca2+

chelation, suggesting that they belong to the external part of the apparatus (106;

127a).

In the plant pathogen R. solanacearum, only 4 extra proteins are required for

secretion of PopA, in addition to the 11 conserved proteins that have been men-

tioned (136). These include HrpY, the major component of the Hrp pilus (see

below), HrpV, and the two small proteins HrpK and HrpX (F Van Gijsegem,

J Vasse, P Castello, C Boucher, unpublished data).In the flagellum, there is no secretin, but there are counterparts to the 10 other

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YscN

OM

IM

YopB, YopD

LcrV

YopB, YopD

ATP

ADP

Effector Yops

YopE

YopH

YopMYopT

YopP/JYopO/YpkA

Macrophage

YscC

YscD, R, S, T, U, V

YscW

YopN, TyeA, LcrG ?

Syc chaperone

Yop effector

Inhibition of phagocytosis

apoptosis,inhibition cytokines release

Yersinia200 A

50 Ainner

outer

YscC secretin

YopBD

YscJ

YopO/YpkA

YopTYopM

YopEYopH YopP/YopJFocal adhesion plaque

Rac

RhoA

MKKsIKK

NFB

cytoskeleton dynamics

YopBD pore 16 - 23 A

Contact

Figure 2 The Yersinia Yop virulon. When Yersinia isolates are placed at 37C into a

rich environment, the Ysc secretion apparatus is installed, and a stock of Yop proteins is

synthesized. As long as there is no contact with a eukaryotic cell, a stop-valve, possibly

made of YopN, TyeA, and LcrG, blocks the Ysc secretion channel. Upon contact with the

eukaryotic target cell, a sensor interacts with a receptor on the cell surface, which results in

the opening of the secretion channel at the zone of contact. The Yops are then transported

through the secretion channels, and the Yop effectors are translocated across the plasma

membrane, guided by YopB and -D. During their intrabacterial stage, Yops are capped with

theirspecific chaperone, presumably to prevent prematureassociations. TranslocatedYopH,

YopE,and YopT block the cytoskeletondynamics, which blocks phagocytosis. TranslocatedYopP/YopJ induces macrophage apoptosis by a mechanism involving caspase activation.

It also down-regulates mitogen-activated protein kinases and impairs NF-B activation by

inhibiting IKK, two effects that could explain the YopP/YopJ-induced reduction of tumor

necrosis factor- production and apoptosis. See text for details and references.

the localization proposed for the homologous Ysc proteins. Thus, the similarity

between type III secretion apparatus and the flagellum export apparatus resides in

their most inner parts. This similarity prompted the groups led by I Aizawa (TeikyoUniversity, Japan) and J Galan (SUNY, Stony Brook, NY) to apply visualization

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that strikingly resembles a needle (83). This needle complex is a long hollow

structure 1200 A long and composed of two clearly identifiable domains: a

needlelike portion that projects outwards from the surface of the bacterial cell and

a cylindrical base that anchors the structure to the inner and outer membranes.The base closely resembles the flagellar basal body, in good agreement with the

sequence similarity data. N-terminal sequencing of proteins present in the purified

needle complex revealed that it is composed of at least three proteins: the secretin

homologous to YscC and two lipoproteins, one of which resembles YscJ. More

recently, a similar needlelike structure could be seen on the surface of plasmolyzed

Shigella flexneri (13).

Little is known about the actual mechanism of export. The structure of the

needlelike complex suggests that it serves as a hollow conduit through which the

exported proteins cross the two membranes and the peptidoglycan barrier, in onestep, taking its energy from the hydrolysis of ATP. Whether proteins travel folded

or unfolded has not been demonstrated yet, but, given the size of channel, it is

likely that they travel at least partially unfolded.

Type III Pili from Plant Pathogensand Enteropathogenic E. coli

Apart from the needle, described in Salmonella spp. and in Shigella spp., otherstructural components have been found to be associated with other type III ma-

chineries. P. syringae pv. tomato andR. solanacearum both produce a filamentous

surface appendage that is 6080 A in diameter and3m in length, called the Hrp

pilus, the formation of which is dependent on a functional Hrp secretion apparatus

(114, 136a; F Van Gijsegem, J Vasse, P Castello, C Boucher, unpublished data).

The major structural protein of this Hrp pilus is encoded by hrpA in P. syringae and

hrpY in R. solanacearum, both genes that are essential for the type III-mediated

HR and pathogenicity. In both bacteria, the Hrp pilus is required for secretion

of type III-secreted proteins (136a, 148a). In R. solanacearum, the Hrp pili areproduced at one pole of the bacterium. R. solanacearum can attach in a polar

manner to plant cells, both in cell culture and in planta, but the hrp genes are not

required for attachment.

A filamentous organelle is also associated with the type III system of EPECs

and EHECs (33, 81). It has a diameter of7080 A and a length of2 m. It

contains EspA (33, 81), one of the proteins secreted by the Esc type III secretion

system of EPECs. In contrast to the hrp pilus mutants, espA mutants are not

deficient in type III secretion. However, they are deficient for the delivery of

EspB into host cells (81). It seems likely that these EspA filaments play a role inthe translocation process and possibly act as molecular go-betweens, transporting

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Translocation of Effectors Across Animal Cell Membranes

Purified secreted Yops have no cytotoxic effect on cultured cells, although live

extracellular Yersinia spp. have such an activity. Cytotoxicity was nevertheless

found to depend on the capacity of the bacterium to secrete YopE and YopD. More-

over, YopE alone was found to be cytotoxic when microinjected into the cells. This

observation led to the hypothesis that YopE is a cytotoxin that needs to be injected

into the eukaryotic cells cytosol by a mechanism involving YopD to exert its effect

(116). In 1994, this hypothesis was demonstrated by two different approaches.

Rosqvist et al used immunofluorescence and confocal laser scanning microscopy

(118), whereas Sory & Cornelis introduced a reporter enzyme strategy based on

the calmodulin-activated adenylate cyclase (127). Infection of a monolayer of

eukaryotic cells by a recombinant Y. enterocolitica, producing a hybrid proteinmade of the N terminus of YopE and the catalytic domain of the adenylate cy-

clase ofBordetella pertussis (YopE-Cya protein), led to an accumulation of cyclic

AMP (cAMP) in the cells. Because the cyclase is not functional in the bacterial

cell and in the culture medium owing to a lack of calmodulin, this accumulation

of cAMP signified the internalization of YopE-Cya into the cytosol of eukary-

otic cells (127). Thus, extracellular Yersinia spp. inject YopE into the cytosol

of eukaryotic cells by a mechanism that involves at least one other Yop protein,

YopD. YopB was shown later to be also required for delivery of YopE and YopH,

like YopD (17, 53). These observations led to the present concept that Yops are acollection of intracellular effectors (including YopE) and proteins that are required

for translocation of these effectors across the plasma membrane of eukaryotic cells

(including YopB and YopD; 26). Delivery of effector Yops into eukaryotic cells

appears to be a directional phenomenon in the sense that the majority of the Yop

effector molecules produced are directed into the cytosol of the eukaryotic cell and

not to the outside environment (110, 118).

This model of intracellular delivery of Yop effectors by extracellular adhering

bacteria is now largely supported by a number of other results, including im-

munological observations. Whereas antigens processed in phagocytic vacuoles ofphagocytes are cleaved and presented by major histocompatibility complex class II

molecules, epitope 249257 of YopH produced by Y. enterocolitica during a mouse

infection is presented by major histocompatibility complex class I molecules, like

cytosolic proteins (128).

A Pore Formed by Translocators

As mentioned previously, translocation of the effector Yops across the cell mem-

brane requires YopB and YopD (17, 110, 118, 127; Figure 2). These two Yops havehydrophobic domains, suggesting that they could act as transmembrane proteins.

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by which the Yop effectors pass through into the cytosol. This YopB- and YopD-

dependent lytic activity is higher when the effectoryop genes are deleted, suggest-

ing that the pore is normally filled with effectors (53). The idea of a translocation

pore is further supported by the observation that the membrane of macrophage-likecells infected with an effector polymutant Y. enterocolitica becomes permeable to

small dyes (101). If the macrophages are preloaded, before the infection, with a

low-molecular-weight fluorescent marker, they release the fluorescent marker but

not cytosolic proteins, indicating that there is no membrane lysis but rather inser-

tion of a small pore (diameter, 1623 A) into the macrophage plasma membrane

(101). The hypothesis of a channel was recently reinforced by the observation that

artificial liposomes that have been incubated with Yersinia spp. also contain chan-

nels that are detectable by electrophysiology (133). All these events are dependent

on translocators YopB and YopD. These two hydrophobic Yops seem thus to becentral for the translocation of the effectors and for the formation of a channel in

lipid membranes. Whether the two events are linked is very likely but not formally

proven, so far.

Translocation of the effectors also requires the secreted LcrV protein, which

interacts with YopB and YopD (121) and is surface exposed before target cell

contact (111). Finally, the 11-kDa LcrG protein is also required for efficient

translocation of Yersinia Yop effector proteins into the eukaryotic cells, but it

is not required for pore formation. LcrG was shown to bind to heparan sulfate

proteoglycans (19), suggesting that it could play a role in the control of releaseby contact, but its exact localization in the bacterium remains elusive. These four

proteins are encoded by the same large operon, lcrGVsycDyopBD, which reinforces

the idea that YopB, YopD, LcrV, and LcrG act together as translocators. This

does not necessarily exclude that some of them could themselves end up in the

eukaryotic cytoplasm, as was shown for YopD (39).

P. aeruginosa has a translocation apparatus consisting of PcrG, PcrV, PopB,

and PopD, which are very similar to the LcrG, LcrV, YopB, and YopD proteins of

Yersinia spp. Shigella spp. and Salmonella SPI1 share a very similar translocation

apparatus made of IpaB, -C, and -D and SipB, -C, and -D, respectively. IpaB andSipB could be considered as the counterparts of YopB, but IpaC and -D and SipC

and -D are not similar to either YopD or LcrV. Like Yersinia spp., Shigella spp.

also have a contact-dependent hemolytic activity that requires the presence of IpaB

and IpaC. This hemolysis has been shown to result from the appearance of a 25-A

pore and the insertion of IpaB and IpaC in the membranes of erythrocytes (13).

Finally, EPECs are also hemolytic and this property is dependent on the presence

of at least EspA, EspB, and EspD, the latter being considered as a counterpart of

YopB (143, 144a).

Relation Between the Pore and the Needle

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been seen yet, but there are clues in Shigella spp. Blocker et al (13) used the

electron microscope to examine the needle of a mutant that was deficient in IpaB,

and they found it to be undistinguishable from that of the wild type, suggesting

that the needle probably does not comprise the translocators or, at least, that thetranslocators are not an abundant element of the needle.

Harpins and Avr Proteins from Plant Pathogens

In plant pathogens, the first proteins that were shown to be transported by the Hrp

type III pathways were called harpins (Figure 3). These include HrpN (Erwinia

spp.), HrpW ( E. amylovora and P. syringae), HrpZ (P. syringae), and PopA

( R. solanacearum) (8, 10, 21, 48, 60, 79, 148; Table 1). These proteins have

in common that they are heat stable, acidic, glycine rich, and devoid of cysteines.When infiltrated into plant leaves, they elicit a nonspecific hypersensitivity re-

sponse in some plants. These proteins are well secreted in vitro in synthetic

growth media, inducing expression of hrp genes. HrpN has even recently been

shown to be secreted in planta, where it appears to be mainly localized in the

surroundings of the bacteria outside the plant cells (108).

Other proteins secreted via the Hrp system do not exhibit HR-elicitor activity.

This is the case for DspA/E (for disease specific), which is required for viru-

lence of E. amylovora (14, 15, 49). It is also the case for PopB and PopC from

R. solanacearum (51a).The avirulence proteins (Avr) represent another class of type III-secreted pro-

teins (Figure 3). They have been known for a long time to be keys in the interactions

between plants and bacterial pathogens, in the sense that they are responsible for

host-pathogen specificity. They are mainly encountered in Pseudomonas spp. and

Xanthomonas spp., where they are responsible for the limited host range of these

bacteria (84, 142). Indeed, interactions between a plant and a pathogen only rarely

turn to disease; the most common issue is the HR response and resistance. This re-

sistance is often determined by the match between an avrgene from the incoming

pathogen and a resistance gene from the plant. This observation, known as thegene-for-gene theory, implies that resistance genes in the plant encode proteins

(R proteins) that recognize signals associated with avr gene products (18, 38).

To be functional in their interaction with plants, the avrgenes need the presence

of a functional Hrp secretion system (51, 113), which suggests that they must be

transported somewhere. Moreover, like the effector Yops, most Avr proteins do not

possess transmembrane segments or signal peptides, and they have no effect when

applied to plant cells or infiltrated in planta. The type III secretion-translocation

concept derived from the observations in Yersinia spp. prompted the hypothesis

that Avr proteins are intracellular effectors that are delivered by extracellular plantpathogens. However, the situation is somewhat different in that plant cells have

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1

Pro

teinstraffickingthroughHrptypeIIIsecretionsystemsa

Biochemical

activityor

n

Effector

characteristics

Similarity

E

ffectonhost

References(s)

proteins

aamylovora

HrpN

HrpNEch,

H

R

148

HrpNEcc

HrpW

Dualproteinwiththe

HrpWPss

H

R

48,79

Ndomainstructurallys

imilar

toPopAorharpins

andexhibitingHR-elicitor

activityandtheC-domain

similartopectatelyases

DspA/E

Similartoand

AvrE

V

irulencefactor

14,15,49

functionallyinterchangeable

withAvrE

omonassyringae

HrpZ

H

R

60

HrpW

Dualproteinwiththe

HrpWEa

H

R

21

N-domainstructurally

similartoPopAor

harpinsandexhibiting

HR-elicitoractivity

andtheC-domain

similartopectate

lyases,whichis

abletobindpectin

butdoesnothave

pectinaseactivity

(

)

Continued

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AvrRpt2

HRandresistance

98

HrmA(HopPsyA)

Temperature-and

HR

135

pH-dependentsecretion

AvrPto

Bindstoand

HRandresistance

135

activatestheSer/Thr

kinasePtoresistancegene

product;temperature-and

pH-dependentsecretion

asolan

acearum

PopA

HR

8

PopB

NLS,localizedto

51a

plantnuclei

PopC

LRRprotein

HrpA

Majorcomponentof

114,

148a

Hrppilus

HrpY

Majorcomponentof

Requiredforsecretion

136a

Hrppilus

secretedbyheterologousorenginee

redtypeIIIsystems

ngae

AvrPto

Seeabove

HRandresistance

54

AvrB

1

Biochemical

activityor

n

Effector

characteristics

Similarity

E

ffectonhost

Refe

rences(s)

Co

ntinued

(

)

HRandresistance

54

R

equiredforsecretion

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onas

AvrBs3

NLS,localized

H

Randresistance

119

tris

inplantnuclei,

transcriptional

activationdomain

AvrRxv

AvrA,AvrBsT,H

Randresistance

119

YopJ/P

activewhenexpressedinsidetheplantcell

ngae

AvrRpt2

H

Randresistance

85,91

AvrB

H

Randresistance

51,85

HrmA

H

R

4

AvrPto

H

Randresistance

124,132

AvrPphB

H

Randresistance

129

AvrPphE

H

Randresistance

129

pestris

AvrBs3

Seeabove

H

Randresistance

137

AvrB4

AvrBs3family

H

Randresistance

28

Avrb6

AvrBs3family

H

Randresistance

28

Avrb7

AvrBs3family

H

Randresistance

28

AvrBln

AvrBs3family

H

Randresistance

28

AvrB102

AvrBs3family

H

Randresistance

28

PthA

AvrBs3family

C

anker-associated

31

symptomsincitrus,

HRinotherplants

ons:HR,hypersensitiveresponse;NLS,nuclea

rlocalizationsignals;LRR,leucine-richrepeats.

y

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to it. First, with the exception of Xa21, most plant R proteins that recognize

bacterial pathogens are predicted to be cytoplasmic (reviewed in 35) or located at

the cytoplasmic face of the plant plasma membrane (20; see Figure 3 for details).

Furthermore, in one instance, direct interaction between the plant R protein andthe bacterial Avr protein could even be demonstrated (124, 132). Second, some

Avr proteins possess nuclear localization signals (NLS) that direct them to plant

nuclei (18, 46). Third, out of the 40 avr genes identified so far (84, 142), >10

were actually shown to induce plant cell death when expressed directly in plant

cells (Table 1). A lot of effort was put into trying to demonstrate Hrp-mediated

in vitro secretion of Avr proteins. One of the key parameters appears to be pH

control. Indeed, two effector proteins, namely HrmA, also called HopPsyA, and

AvrPto are secreted by P. syringae after growth at low temperature and low pH

(135). Similarly, some AvrRpt2 secretion was detected by P. syringae cells grownin minimal hrp-inducing medium at low pH (98). Because the pH of the plant

intercellular apoplast is acidic, such a pH dependency for secretion might have

a biological significance. It is interesting that such a pH dependency was also

shown for the type III system of EPECs and for the SPI2-encoded system of

S. typhimurium, which is active in the acidic vacuole (11, 32). Also, a heterologous

system expressing the hrp gene cluster ofE. chrysanthemi in E. coli allowed Ham

et al to detect the secretion of AvrB and AvrPto (54). In X. campestris, the

conjunction of a minimal medium and low pH with mutations up-regulating the

hrp genes allowed secretion of AvrBs3 and AvrB proteins (119). These systemsare, however, not very efficient because only minute amounts (a few percent) of the

total Avr proteins produced are secreted. However, as artefactual as this may be, it

will help in the identification of new proteins in transit through the Hrp systems. In

conclusion, the type III Hrp secretion system of plant pathogens secretes proteins

that appear to play different roles in the interactions with plants. Some of these

proteins are called harpins, others are Avr proteins and some do not fit in any of

these categories. The role of harpins is not very well defined while the role of Avr

can be understood in the gene-for-gene concept. However, avr genes occur not

only in bacteria but also in other plant pathogens, which means that Avr proteinsare not necessarily related to type III secretion. Thus, one could wonder whether a

new term should not be coined to call specifically all the proteins that are trafficking

through the Hrp type III systems. The generic name Hop (for Hrp outer protein),

inspired from the name Yop has been proposed (135).

The Cytosolic Chaperones

A hallmark of type III secretion is that normal secretion of some substrate proteins

requires the presence of a new type of small cytosolic chaperones (92, 146, 147;Figure 2). In general, these chaperones are encoded by a gene located close to the

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SycE, the chaperone of YopE, is the archetype of the first family (147). There

are four representatives of this family in Yersinia spp. (SycE, SycH, SycT and

SycN), one in Salmonella spp. (SicP), one in P. aeruginosa (SpcU), one in EPECs

(CesT), and two in the Proteus flagellum assembly system (Table 2). All of thesechaperones are small (14- to 15-kDa) proteins with a putative C-terminal am-

phiphilic -helix, and most of them are acidic (pI 4.45.2). They specifically

bind to their partner Yops. The main feature is that, in the absence of these chap-

erones, secretion of their cognate protein is severely reduced, if not abolished.

However, the exact role of these chaperones remains elusive. Research on Syc

chaperones has focused so far on SycE and SycH. They both bind to their part-

ner Yop at a unique site spanning roughly residues 2070 (126). It is surprising

that, when this site is removed, the cognate Yop is still secreted, although maybe

in reduced amounts, and the chaperone becomes dispensible for secretion (149).This suggests that it is the binding site itself that creates the need for the chaperone

and thus that the chaperone somehow protects this site from premature associa-

tions, which would lead to degradation. In agreement with this hypothesis, SycE

has an antidegradation role; the half-life of YopE is longer in wild-type bacte-

ria than in sycE mutant bacteria (23, 43). Woestyn et al (149) suggested that the

chaperone-binding site could be a site that is also involved in interaction between

YopE and the translocators, YopB, YopD, or LcrV, and thus that SycE would

prevent premature interactions between effectors and translocators. Although ap-

pealing, this hypothesis is not sufficient to explain the need of SycE. Indeed, YopEcan be secreted by the plant pathogen X. campestris (see below) and, although

X. campestris does not synthesize proteins resembling the Yersinia transloca-

tors, SycE is necessary to ensure intrabacterial stability of YopE in X. campestris

(119).

In addition to this putative role of bodyguard, SycE has also been claimed to act

as a secretion pilot, leading the YopE protein to the secretion locus (see below).

Finally, both SycE and SycH are required for efficient translocation of their partner

Yop into eukaryotic cells (126). However, when YopE is delivered by a Yersinia

polymutant strain that synthesizes an intact secretion and translocation apparatusbut no other effector, it appears that YopE is delivered even in the absence of its

chaperone and chaperone-bindingsite (18a). This shows that the chaperone-binding

domain of the effectors does not interact with translocators. This also shows that

the SycE chaperone appears to be needed only when YopE competes with other

Yops for delivery. It suggests that the Syc chaperones could be involved in some

kind of hierarchy for delivery. This new hypothesis about the role of the Syc chap-

erones fits quite well with the observation that only a subset of the effectors seems

to have a chaperone. Little is known about the role of SycT and SycN. However,

there is an unexpected complexity for the latter; SycN apparently requires YscBworking as a cochaperone (27).

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BLE2

Type-IIIcytosolicchaperonesa

mily

Protein

kDa

pl

Assistedprotein

Strongsimilarit

ies

Reference(s)

cEfam

ily

SycE

14.7

4.55

YopE(Yersiniaspp.)binds

ORF1(P.aeruginosa)

147

toaa1550

Scc1(Chlamydia

psittaci)

SycH

14.7

4.88

YopH(Yersiniaspp.)binds

146

toaa2070

SycT

15.7

4.4

YopT(Yersiniaspp.)

68

SycN

15.1

5.2

YopN(Yersiniaspp.)

Pcr2(P.aeruginn

osa)

27,69

YscB

15.4

9.3

YopN(Yersiniaspp.)(cochaperone)

27,70

SicP

13.6

4.0

SptP(Salmonellaspp.)

44

SpcU

4.4

ExoU(P.aeruginosa)

37

CesT

7.1

Tir(EPECsandEHECs)

1,34

FlgN

16.5

FlgKandFlgL(Proteusflagellum)

41

FliT

14.0

HAP2(Proteusflagellum)

41

cDfam

ily

SycD

19.0

4.53

YopBandYopD

PcrH(P.aerugin

osa

Yersinia

spp.)

146

CesD

(EPEcs)

144

IpgC

18.0

IpaBandIpaC(Shigellasp

p.)

92

SicA

19.0

4.61

SipBandSipC(Salmonellaspp.)

74

breviations:kDa,molecularmass(Kilodaltons

);pI,isolectricpoint;aa,aminoacid(s);EPEC,enteropathogenicE.coli;EH

EC,enterohemorrhagicE.coli.

(

)

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that it binds to several domains on YopB, which instead evokes SecB, a molecular

chaperone in E. coli that is dedicated to the export of proteins and has multiple

binding sites on its targets. IpgC, the related chaperone from S. flexneri, has been

shown to prevent the intrabacterial association between translocators IpaB andIpaC (92). The similarity between IpgC and SycD suggested that SycD could play

a similar role and could thus prevent the intrabacterial association of YopB and

YopD, but this turned out not to be the case. Because YopB and YopD also have

the capacity to bind to LcrV, one could speculate that SycD prevents a premature

association, not between YopB and YopD but rather between YopB, YopD, and

LcrV, but this has not been shown yet. CesD, the homolog from the EPECs, has

also been shown to be required for full secretion of the translocators EspB and

EspD, but it was shown to bind only to EspD, the translocator that is the most

similar to YopB and IpaB. Like SycD and IpgC, CesD is present in the bacterialcytosol, but a substantial amount of this protein was also found to be associated

with the inner membrane of the bacterium (144).

In plant pathogens, the only protein sharing the structural characteristics of

the chaperones described here is DspB, which is needed for the secretion of the

E. amylovora DspA protein (49).

Recognition of the Transported Proteins

Effectors delivered by type III secretion systems have no classical cleavedN-terminal signal sequence (94). However, it appeared very early that Yops are

recognized by their N terminus, but that no N-terminal sequence is cleaved off

during Yop secretion (94). The minimal region shown to be sufficient for secre-

tion was gradually reduced to 17 residues of YopH (126), to 15 residues of YopE

(126), and to 15 residues of YopN (6).

A systematic mutagenesis of the secretion signal by Anderson & Schneewind

(6, 7) led to doubts about the proteinic nature of this signal. No point muta-

tion could be identified that specifically abolished secretion of YopE, YopN, and

YopQ. Moreover, some frameshift mutations that completely altered the peptidesequences of the YopE and YopN signals also failed to prevent secretion. Anderson

& Schneewind (6, 7) concluded from these observations that the signal leading to

the secretion of these Yops could be in the 5 end of the mRNA rather than in the

peptide sequence. Translation ofyop mRNA might be inhibited either by its own

RNA structure or as a result of its binding to other regulatory elements. If this is

correct, one would expect that no Yop could be detected inside bacteria. However,

although this absence is reported to be true for YopQ (7), it is certainly not true for

other Yops such as YopE. By using similar approaches, Anderson et al (5) showed

that the signals necessary and sufficient for the secretion of AvrB and AvrPto, bothbyE. coli carrying theE. chrysanthemi hrp cluster and by Yersinia spp., are located

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To determine whether this N-terminal (or 5-terminal) signal is absolutely re-

quired for YopE secretion, Cheng et al (23) deleted codons 215 from YopE-npt

hybrid genes, and they observed that 10% of the hybrid proteins that were deprived

of the N-terminal secretion signal were still secreted. They inferred from this thatthere is a second secretion signal, and they showed that this second, weaker se-

cretion signal corresponds to the SycE-binding site. It is not surprising that this

secretion signal is functional only in the presence of the SycE chaperone (23).

Whether this signal plays a role in vivo remains to be elucidated.

Control of the Injection

We have seen that type III secretion systems can secrete their substrate in vitro

under artificial conditions, such as Ca2+ chelation for instance. What is the trig-

gering signal in vivo? Most probably it is contact with a eukaryotic cell. Several

reports of results in Yersinia spp. have shown that Yops delivery is a directional

phenomenon, in the sense that most of the load is delivered inside the eukaryotic

cell and there is little leakage (110). Based on the assays used, there is some

discrepancy in the degree of directionality (17), but there is no doubt that the

majority of the released Yops load ends up in the eukaryotic cell and thus that

contact must be the signal. Pettersson et al (112) provided a nice visual demon-

stration of the phenomenon. By expressing luciferase under the control of a yop

promoter, they showed that active transcription of yop genes is indeed limited to

bacteria that are in close contact with eukaryotic cells. Release of Ipa proteins from

Shigella spp. was also shown to depend on contact between bacteria and epithelial

cells (145).

EFFECTOR PROTEINS AND HOST RESPONSES

A Panoply of Effectors and Avirulence Proteins

Delivery of effector proteins across the plasma or vacuolar membrane appears tobe the object of type III secretion. More than 20 effectors have been described in

the various animal pathogens systems, and this relatively large list is increasing

very quickly. The effectors and their activity are detailed in Table 3, including

references. Six effectors have been characterized in Yersinia spp.: YopE, YopH,

YopM, YopJ/P, YopO/YpkA, and YopT (Figure 2). Eight effectors are delivered

by the Salmonella SPI1-encoded apparatus (AvrA, SipA, SipC, SopB, SopD,

SopE, SptP, and SspH1), and two, SpiC and SspH2, have been identified for

SPI2. Four are delivered by the Psc apparatus of Pseudomonas aeruginosa: ExoS,

ExoT, ExoU, and ExoY; Shigella spp. deliver IpaA and IpaC. Finally, EPECs orEHECs deliver their own receptor, Tir or EspE. In addition to these effectors, it

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pathogens,>10 secreted proteins (Table 1) and as many as 40 avrgenes have been

characterized.

Enzymic Activities of Effectors and Avirulence ProteinsFive different enzymatic activities could be identified so far in the panoply of

type III effectors from animal pathogens: phosphotyrosine phosphatase (YopH

and SptP), serine-threonine kinase (YpkA, also called YopO), inositol phosphate

phosphatase (SopB), ADP-ribosyltransferase (ExoS and ExoT), and adenylate

cyclase (ExoY). The two latter activities are classical in A-B toxins, but, un-

like the adenylate cyclases from B. pertussis and Bacillus anthracis, ExoY does

not require calmodulin for its activity. The similarity between activities of type

III effectors and A-B toxins suggests that these type III effectors could be con-

sidered as some kind of toxins that need a very sophisticated apparatus for their

delivery. Some of the type III effectors are hybrid proteins composed of two do-

mains that display different activities. SptP from S. enterica appears to be a hybrid

between YopE and YopH; the C-terminal part is a phosphotyrosine phosphatase

that is homologous to YopH, whereas the N-terminal part is homologous to YopE

(75). This YopE-like domain also occurs in the N-terminal part of ExoS from

P. aeruginosa (97).

In plant pathogens, of40 avrgenes characterized thus far, an enzymatic ac-

tivity could be assigned only to the products of two genes. The AvrD protein is

involved in the biosynthesis of syringolides, small diffusible elicitors specifically

recognized by the soybean Rpg4 disease-resistance gene. Overexpression ofavrD

in P. syringae or in E. coli is sufficient for the elicitation of the HR on Rpg4

plants. In conditions where avrD is overexpressed, this elicitation does not re-

quire a functional Hrp system (77, 84). We cannot, however, exclude that the

AvrD protein might be translocated to the plant cell by P. syringae where it may

synthesize the syringolide elicitors from plant precursors. It is clear that under-

standing what really happens in vivo awaits further investigation. The other Avr

protein that resembles proteins found in databases is the X. campestris AvrBs2.

This protein shares homologies with both theA. tumefaciens agrocinopine synthase

and the E. coli glycerophosphoryl diester phosphodiesterase UgpQ, two enzymes

involved in the synthesis or hydrolysis of phosphodiester linkages between car-

bohydrates or phospholipids (131). It is interesting that this gene is required

not only for the production of an avirulence signal, which is detected by pep-

per harboring the Bs2 resistance gene, but also for promoting pathogen virulence

(76, 131).

The Cytoskeleton Is a Major Target in Animal CellsThere is a great diversity among the targets and the effects induced by the type-III

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20/42Type

IIIeffectorsofanimalpathog

ensa

Effector

Enzymaticactivity

Target

Similarity

b

Effect

Reference(s)

p.

YopE

Likely:GTPase-activating

Likely

ExoS,SptP

Cytotoxin,actin

94,118,127

protein(seeSptP)

Cdc42,Rac

filamentsdisruption,

antiphagocytic

YopH

PTPase

P130(Cas),

SptP

Disruptionof

12,109

FAK,paxillin

peripheralfocal

complexes,antiphagocy

tic

YopM

Unknown

Unknown

IpaH

Migratestothe

17,125

nucleus

YpkA/YopO

Serine,threonine

Unknown

Unknown,routedto

52

kinase

surfaceof

inner

plasmamembrane

YopP/YopJ

Unknown

MAKKs,

AvrA

InhibitionofTNF-

16,95,97,

IKK

AvrRxv

releaseinhibitionof

105,120,

activationof

122,123

NF-B,proapoptotic

YopT

Unknown

RhoA

Cytotoxin,actin

68,156

filamentsdisruption,

antiphagocytic

(secretedbySPI1)

AvrA

Unknown

Unknown

YopP/Yop

J

56

AvrRxv

SipA

Unknown

Actin

IpaA

Enhances

154

actin

polymerization,

macropinocytosis

SipB

Caspase-1

Inductionofapoptosis

63

SipC

Actin

Nucleationand

59

polymerizationofactin

AvrBst

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SopB/SigD

InsPphosphatase

Various

Intestinalchloride

73,103

secretion

SopE

GDP-GTPexchange

Cdc42,Rac

55

factor

SptP

GTPase-activating

Cdc42,Rac

YopE,YopH

Deactivationofactin

45,75

protein,PTPase

polymerization

SopD

73

onella(secretedbySPI2)

SpiC

Inhibitionoffusion

134

SspH2

betweenphagosomes

andlysosomes

onellaSPI1

SspH1

YopM

93

SPI2

IpaH

SI2P

onellaSPI1and/or

Si2P

YopM

133a

ipaH

2

SspH1

SspH2

ruginosa

ExoS

ADP-ribosyltransferase

Ras

YopE,ExoT

36,42,90,

ExoT

ADP-ribosyltransferase

Unknown

ExoS

107

36

ExoU/PepA

Cytotoxin

36,58

ExoY

Adenylatecyclase

151

llaspp.

IpaA

Unknown

Vinculin

SipA

139

IpaB

Caspase-1

SipB

Inductionofapoptosis

64

IpaC

Activationof

140

Cdc42,entryof

Shigellaspp.

C/EHEC

Tir/EspE

Receptor

Receptorforintimin

29,78

viations:

MKK:MAPK(mitogen-activatedprote

inkinase)kinasesIKK:iB(inhibitor

B)kinase;NF-B:nuclearfactorkappaB.TNF-,tumornecrosisfactor-.

XXX.

93

YopM

ipaHSIrPSspH2

SspH2

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systems either promote macropinocytosis by nonphagocytic cells (Shigella and

Salmonella spp.) or block phagocytosis (Yersinia spp. and P. aeruginosa). Some

effectors act directly on the cytoskeleton components, whereas others interfere

with the control exerted on actin polymerization by small GTPases, which act asmolecular switches, cycling between a GDP-bound (inactive) and a GTP-bound

(active) state.

SipA and SipC from Salmonella spp. bind directly to actin. Purified SipC

nucleates actin polymerization and bundles actin into cables (59), whereas SipA

inhibits depolymerization of actin filaments (154). By doing so, both contribute to

host cell membrane ruffling and entry ofSalmonella spp. into nonphagocytic cells.

In addition, Salmonella spp. inject SopE, an effector that amplifies this membrane

ruffling by stimulating GDP/GTP nucleotide exchanges and thus stimulating the

small GTPases Rac-1 and Cdc42 (55). It is interesting that Salmonella spp. alsoinject SptP, which acts as a GTPase-activating protein for Rac-1 and Cdc42 and

thus can reverse the up-regulation of these small GTPases after bacterial internal-

ization (45). Because the C-terminal part of SptP is also homologous to the YopH

phosphatase (see below), one can expect an even stronger reverse effect (see be-

low for Yersinia spp.). The subversion of the cytoskeleton activity in a sequential

manner clearly implies that there is some order or hierarchy in the delivery of

the effectors. This field has not been investigated very much yet, but, from our

observations in Yersinia spp., we would suspect that chaperones play a role in this

process (18a).Two effectors from Shigella spp. have been shown to promote bacterial entry

into nonphagocytic cells, by acting on the cytoskeleton. IpaA has been shown to

bind to vinculin, which initiates the formation of focal adhesion-like structures

required for Shigella invasion (139). IpaC, the homolog of SipC, induces poly-

merization of actin and formation of lamellipodes, presumably via activation of

Cdc42 (140).

Yersinia spp. inject three effectors that interfere with the cytoskeleton dynamics.

YopH dephosphorylates three proteins from the focal adhesions, which leads to

the disassembly of these complexes and reorganization of the cytoskeleton, inparticular of the stress fibers (12, 109). YopE is a homolog of the N-terminal

domain of SptP from Salmonella spp., which acts as a GTPase-activating protein

for Rac-1 and Cdc42. It is thus likely that it exerts the same negative action

on membrane ruffling as SptP does in Salmonella spp. (see above). Finally,

YopT modifies and inactivates RhoA, a GTPase that regulates the formation of

stress fibers (156). Thus, of six proteins delivered by Yersinia spp., three exert a

negative role on cytoskeleton dynamics and contribute to the strong resistance of

Yersinia spp. to phagocytosis by macrophages (115; N Grosdent & GR Cornelis,

unpublished data).In P. aeruginosa, the N-terminal domain of ExoS is also a homolog to YopE

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Modulation of Inflammation and Signaling Interference

The second theme for the action of type III effectors in animal cells is the mod-

ulation of inflammation and cell signaling. Cytokines and chemokines are key

elements in the induction of the inflammatory response. Central to their synthesis

are mitogen-activated protein kinases (MAPK) and the transcriptional activator

NF-B. In the absence of stimulation, the latter is held in the cytosol by its in-

hibitor IB. Phosphorylation of IB targets it for degradation, allowing migration

of NF-B to the nucleus.

Yersinia spp. provide an example of down-regulation of the inflammatory

response by the action of YopP (YopJ in Y. pestis and Y. pseudotuberculosis)

(Figure 2). YopP/J blocks the release of tumor necrosis factor- by macrophages

and of interleukin (IL)-8 by epithelial cells, which leads to a significant reduc-tion in inflammation (16, 122, 123). These events result from the inhibition of the

activation of the transcription factor NF-B (120, 122). This inhibition is in turn

presumably caused by the inhibitory effect of YopP/J on the MAPK and on the

IB kinase (IKK) (105). In addition to this, YopP/J also induces apoptosis in

macrophages (95, 97). This apoptosis is accompanied by cleavage of the cytosolic

protein BID, the release of cytochrome c, and the cleavage of caspase-3 and -7.

The release of cytochrome c and the cleavage of BID can both be inhibited by cas-

pase inhibitors, suggesting that YopP/J interferes with a signaling pathway that is

upstream of the mitochondria (C Geuijen, W Declerq, A Boland, P Vandenabeele,GR Cornelis, manuscript in preparation). One could wonder whether reduction in

the release of tumor necrosis factor- is not simply the consequence of apoptosis.

This is, however, not the case, because it occurs even if apoptosis is prevented by

inhibiting the activity of caspases (120). One could even suspect that apoptosis re-

sults from the loss of the antiapoptotic factor NF-B (120). Alternatively, the two

events could be the consequence of the same early event in a common signaling

cascade. It is interesting that YopP/J shares a high level of similarity with AvrRxv

and AvrBsT from X. campestris and a protein from the nitrogen-fixing Rhizobium

spp. Because of the similarity with Avr proteins, the Salmonella counterpart ofYopP/J was called AvrA (56).

In contrast to the Yersinia Ysc system, which down-regulates the inflammatory

response, the Shigella system tends to induce a profound inflammatory response

in the intestinal epithelium. The resulting influx of circulating phagocytes leads

to the opening of the intercellular junctions of the epithelium, which favors the

progress of the infection. This response results from the activation by IpaB of

the ICE cysteine protease, which converts pro-IL-1 to the mature proinflamma-

tory cytokine IL-1 (reviewed in 138). Induction of an inflammatory response

is also a feature of Salmonella infection. SipB, like IpaB, activates ICE (63),which leads to the release of IL-1. In addition, there is an activation of NF-

B which also leads to the release of proinflammatory cytokines such as IL 8

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Signaling Interference in Plant Cells

In plant pathogens, resistance to the disease involves active processes that require

plant metabolism. These processes are accompanied by the activation of so-called

defense-related genes, often leading to a programmed cell death culminating in the

HR. As seen above, this plant defense program is often activated by the specific

recognition of bacterial signals encoded by avr genes, by the matching plant re-

sistance gene product. More than 20 such resistance genes have already been

cloned. Although involved in resistance to many different pathogens, including

viruses, bacteria, fungi, insects, and nematodes, they share common motifs (re-

viewed in 35). Seven R genes confer resistance to bacterial diseases (reviewed in

18; Figure 3). Two of them, Xa21 and Pto, encode active protein kinases. In only

one case, AvrPto/Pto, a direct interaction between the Avr and the R proteins hasbeen demonstrated by the yeast two-hybrid system (124, 132). To decipher further

the signal transduction pathway, plant proteins interacting with Pto were isolated

by a two-hybrid screening. Two of these (Pti1 and Pti4, called Pti for Pto interac-

tors) were shown to be phosphorylated by Pto. Pti1 is a serine-threonine kinase

involved in the development of the HR. Pti4/5/6 are putative transcription factors

that bind to the GCC box found upstream of several pathogenesis-related (PR)

genes whose expression is induced after pathogen attack. Pti4/5/6 also contain

NLS sequences and were shown to be localized to the nucleus (reviewed in 89).

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These elegant studies show that signal transduction from recognition of the Avr

protein to gene induction can be quite short.

The Tir Protein, a Unique Case of Translocated ReceptorTir from EPECs (EspE in EHECs) is particularly interesting because it inserts into

the plasma membrane of the target enterocytes and serves as a receptor for intimin,

a powerful adhesin of EPECs. Thus, EPECs and EHECs insert their own receptor

into mammalian cell surfaces, to which they then adhere to trigger additional host

Figure 3 A model Hrp type III system. A. Proteins secreted by the Hrp secretion ap-

paratus. Only the Hrc proteins for which a localization has been shown or predicted aredepicted. Localization of proteins in the extracelular medium does not preclude delivery of

some of them into plant cells (as, for example, PopB from R. solanacearum). The scheme

represents a polar Hrp pilus as is the case for R. solanacearum, in which it is required for

PopA secretion. The product of the rice Xa21 resistance gene spans the plant plasma mem-

brane. It contains leucine-rich repeats that are exposed in the extracellular compartment

and may react with the matching Avr gene product. The arrow nearby HrpW indicates the

possible interaction between this protein and the cell wall pectate. B. Protein delivery into

plant cells. Such an injection has not been directly proven so far, but considerable indirect

evidence points to such a mechanism. Of the seven resistance genes involved in bacterial

disease resistance, six were shown or predicted to be intracellular (18, 35). The RPM1 geneproduct was shown to be located at the cytoplasmic face of the plant plasma membrane.

Because RPM1 does not have characteristics of membrane proteins, it could interact with a

membrane-anchored docking protein (20). The Pto resistance gene encodes a protein kinase

that interacts with the matching AvrPto avirulence protein (124, 132) and triggers a trans-

duction cascade that leads to activation of plant defense-related genes (see text for details;

89). For other Avr/R pairs, recognition may be indirect or more complex as indicated by the

question marks. Several bacterial proteins trafficking through the Hrp systems possess NLS

that allow them to be routed to the plant nucleus. Proteins of the AvrBs3/Pth family also

have a putative transcription activation domain. In the presence of the matching plant resis-

tance gene, they could thus directly activate defense genes but the nature of the Bs3/AvrBs3interaction has not been defined yet. In the absence of matching plant resistance genes, they

could alter expression of as yet unknown genes to promote parasitism and disease. The role

of the Hrp pilus in this process is unknown. Is it a go-between allowing the proteins to be

delivered to reach the plant membrane, or is it a conduit directly injecting proteins into the

plant cell? Harpins: HrpN, HrpW, HrpZ, and PopA; avirulence/virulence bacterial proteins:

AvrB, AvrPto, AvrBs3, Pth, DspA, putative avirulence/virulence bacterial proteins: HpaA,

PopB, and PopC; plant resistance gene products: RPM1, Pto, Xa21, and Bs3; elements of

plant signaling cascades: Pti1 and Pti4, 5, and 6; P: phosphate group; PK: protein kinase.

Leucine-rich repeats are represented by laddered rectangles. Question marks indicate some

of the unanswered questions: What is the nature of the interaction between Bs3 and AvrBs3

or between other R and Avr proteins? What are the genes activated by Avr/Pth proteins

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signaling events and actin nucleation (29, 78).

The YopM and AvrBs3/Pth Families: EffectorsTargeted to the Nucleus

YopM from Yersinia spp. is a strongly acidic protein containing leucine-rich

repeats whose action and target remain unknown. However, it has been shown

to traffick to the cells nucleus by means of a vesicle-associated pathway that

is strongly inhibited by brefeldin A and perturbed by monensin or bafilomycin

(125). It has several homologs in Shigella (ipaHmultigene family) and Salmonella

(93) spp.

Avr proteins of the AvrBs3/Pth family from Xanthomonas spp. contain rep-

etitions of a 34-amino-acid motif with only two hypervariable codons. It is thenumber and order of these repeats that confer the recognition specificity by the

plant (62; reviewed in 46). It is interesting that some members of this gene family,

such as the pthA gene, were first identified as virulence factors but were subse-

quently shown to have a dual rolebeing involved in virulence on some plants and

having an avirulence function on others (130). In these proteins, both virulence

and avirulence are determined by the repeats (130; reviewed in 46). Two other

structural features, localized in the conserved C terminus, are important for the

function of most members of the AvrBs3/Pth family. The first one is the presence

of NLS, which allow the routing of these proteins to the plant cell nucleus, andthese NLS are required for expression of virulence/avirulence (137; reviewed in

46). A domain that is structurally similar to the acidic activation domain of many

eukaryotic transcription factors is present in several AvrBs3/Pth family members.

For three Avr proteins, deletion of this domain results in the loss of the avirulence

function. Fusion of AvrXa10 with the Gal4 DNA-binding domain allows tran-

scription activation in yeasts and Arabidopsis thaliana (155). Collectively these

data suggest that the Avr proteins of this family are transported into the host nu-

cleus, where they alter transcription. Thus, the similarity between proteins of the

YopM family and the AvrBs3/Pth could be functional as well as structural. It isinteresting that expression of the pthA gene in plant cells is sufficient to induce,

in a host-specific manner, cancer-like lesions reminiscent of disease symptoms

provoked by the pathogen (31).

Functional NLS are also present in other effector proteins from plant pathogens

like PopB (51a) or HpaA from X. campestris (67).

Role of Avr Proteins in Virulence

Although avrgenes were first identified as key determinants in race-specific resis-tance, there is increasing evidence of a dual role for these genes both in avirulence

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P. syringae to tomato (88). Quite often the role ofavrgenes in virulence was ob-

scured by the presence of many different avrgenes in the same bacterium, and the

respective role of these genes was highlighted only after the isolation of multiple

mutants (reviewed in 142). Proteins of the AvrBs3/Pth family are a good examplein this respect. The pthA gene may function either as a virulence determinant

or as an avirulence gene, depending on the interacting plant. Moreover, some

Xanthomonas strains carry up to a dozen avrgenes that belong to this family, and

they have an additive effect in virulence (152; reviewed in 46). Another striking

example of such a dual role for type III effectors came from the identification of the

E. amylovora DspA/E protein (14, 15, 49). This protein, required for virulence in

E. amylovora, shows similarities to the P. syringae AvrE protein and acts as an avir-

ulence determinant when expressed in P. syringae. Reciprocally, the P. syringae

avrE gene can at least partially complement a dspE E. amylovora mutant.A new and promising approach was recently developed to analyze the effects of

Avr proteins in planta. Plants either carrying or lacking theRPS2 gene were stably

transformed with the avrRpt2 gene under the control of a glucocorticoid-inducible

promoter, allowing modulation of avrRpt2 expression. This allowed McNellis

et al to visualize clear effects of AvrRpt2 even in the absence of the matching

R gene (91).

The most favored actual model that integrates all of these data is that the Avr

proteins are virulence determinants that were subverted by the plant to become

signals to elicit defense pathways.

Intracellular Action of Translocators in Animal Cells

We have seen above that IpaB and SipB, two translocators, have a proinflammatory

action by reacting with the cytosolic macrophage protein ICE. This also leads to

apoptosis of the macrophage, because ICE is also a caspase (Casp-1). Inhibition

of Casp-1 activity by a specific inhibitor blocks macrophage cytotoxicity, and

macrophages lacking Casp-1 are not susceptible to Salmonella-induced apoptosis

(22, 63, 64). Shigella-and Salmonella-induced apoptosis is thus distinct from other

forms of apoptosisincluding that induced by Yersiniain that it is uniquely

dependent on Casp-1.

Similarly, translocators YopD and EspB, the last of which somehow resem-

bles YopD, were both shown to be translocated themselves into eukaryotic cells

(39, 150). Finally, as mentionedpreviously, IpaC and SipC, which arealso involved

in translocation, interact with actin (59, 140). Taken together, these observations

indicate that the translocators are not restricted to the area of contact between bac-

teria and eukaryotic cells but that they are themselves trafficking in the eukaryotic

cell, possibly in association with membranes, but this has not been determined yet.

The Enigmatic Role of Harpins

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resistance and the way that they interact with plants are, however, still enigmatic.

In E. amylovora, hrpN mutants are much less virulent, and they have a variable

ability to elicit the HR on tobacco (9, 148). In other members of the Erwiniae,

the harpins have no role or only a marginal one in virulence (10, 99). The roleof the P. syringae HrpZ protein is even more controversial. Although infiltration

of the purified protein in tobacco leaves elicits the HR, a P. fluorescens saprophytic

bacterium carrying the P. syringae hrp gene cluster and hrpZ failed to cause this

reaction, even when HrpZ was secreted via the Hrp pathway. In the same way,

conflicting results have been reported for the requirement of a functional hrpZ

gene for elicitation of the HR by saprophytic bacteria (P. fluorescens or E. coli)

carrying diverse avrgenes (2, 51, 113). A P. syringae mutant deleted of the hrpZ

gene is still able to elicit the HR on tobacco, albeit at a higher inoculum level (2).

Another type of harpins encoded by the hrpW genes has been identified both inE. amylovora (48, 79) and P. syringae (21). The HrpW proteins are modular in

the sense that they consist of an N-terminal domain that is sufficient for eliciting

the HR and shows structural similarities with harpins. The C-terminal domain is

homologous to a certain class of pectate lyases. In P. syringae, the pectate lyase

domain was shown to bind to pectate, but no pectinase activity was detected. hrpW

mutants were still able to provoke disease and to elicit the HR. In E. amylovora,

an hrpW mutant is even able to elicit the HR or to provoke electrolyte leakage

on tobacco at a lower level of bacterial inoculum than the wild type. This led

Gaudriault et al (48) to propose that HrpW might act as a negative effector of HRmechanisms. On the contrary, in P. syringae, a double hrpW hrpZmutant is more

affected in HR elicitation than the single mutants (21).

Similarly, the role of the PopA protein secreted by R. solanacearum is unclear.

A popA mutant is as virulent as the wild type on tomato, and it elicits the HR

on tobacco at the same level (8). Petunia, however, is highly reactive to low

amounts of PopA, and this reactivity is genotype dependent, indicating that, in

this plant, the PopA protein might be involved in specificity (8). In conclusion, we

are facing the striking situation that these proteins, which are widely distributed

in plant pathogens, seem to have in most cases only a marginal role in interactionswith plants. Is that the result of some redundancy, or do they have more subtle

functions which are not highlighted by the currently used assays? Harpins may

actually function as ancillary translocators rather than effectors. However, since

the translocation of effectors into plant cells could not be formally demonstrated,

it is, of course, impossible, so far, to demonstrate this hypothesis.

COMPARISON OF THE VARIOUS TYPE III SYSTEMS

Three Major Groups of Systems Among the Animal Pathogens

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lated to the Ysc system of Yersinia spp., which is quite surprising given the

long evolutionary distance between these two bacterial species. Second, the

SPI1 system of S. typhimurium and the Mxi/Spa system of Shigella spp.

are also very similar. Finally, the SPI2 system of S. typhimurium (61, 104) isrelated to the systems found in EPECs and EHECs and in the recently dis-

covered chromosome-encoded system of Y. pestis (The Sanger Center,

Cambridge, UK).

Two Different Groups ofhrp Gene Clusters in Plant Pathogens

According to their genetic organization, the hrp gene clusters can be divided

into two classes, one comprising the P. syringae and the different Erwiniaclusters and the second one including the R. solanacearum and X. campestris

clusters (3). Here again the two classes are not related to phylogenetic

proximity.

Exchangeability Between the Effectorsof the Different Systems

Are the various type III systems functionally interchangeable in the sense thateffectors from one system could be secreted or even delivered intracellularly by

another system? We have seen that the N-terminal domain of ExoS from P. aerug-

inosa is similar to YopE and that the protein encoded by the gene next to exoS

(ORF1) is very similar to SycE. These observations prompted Frithz-Lindsten

et al (42) to introduce the exoS gene and ORF1 in a noncytotoxic, double yopE

yopH mutant of Y. pseudotuberculosis and to infect HeLa cells. The result was

clear cytotoxicity, indicating that ExoS is translocated across the HeLa cell plasma

membrane. Rosqvist et al also observed that Y. pseudotuberculosis can secrete

IpaB from S. flexneri and that S. typhimurium can secrete YopE (117). The YopE-producing Salmonella is also cytotoxic for HeLa cells, suggesting that YopE could

even be translocated across the cell plasma membrane. As already mentioned,

some Avr proteins can be secreted by heterologous Hrp secretion systems. The

interchangeability even passes the interkingdom barrier because Avr proteins are

also secreted by Yersinia spp., and the type III secretion systems of E. chrysan-

themi or X. campestris can secrete YopQ and/or YopE (5, 119). Thus, the effectors

from a given type III system are generally recognized by other systems, although

sometimes not very efficiently. In contrast, the elements of the various secretion

systems themselves seem not to be interchangeable, at least in between type IIIsystems that do not belong to the same group. No heterologous complementation

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GENETIC BASES

Comparison of the secretion apparatus and the phylogenic analyses suggest that

these systems must have been transfered hortizontally during evolution. It is notsurprising that the genes encoding these systems have been found to be part of

elements that are more mobile than most of the other bacterial genes. The Yersinia

ysc and yop genes and the Shigella mxi-spa genes are plasmid borne. The two

systems ofSalmonella spp. and the EPECs system are encoded by pathogenicity

islands. In general, the genes encoding the secretion-translocation systems appear

to be part of large, compact operons, whereas the genes encoding effectors are more

scattered. Pathogenicity islands are sometimes considered as vestigial phages. It

is interesting that, in S. typhimurium, Hardt et al (57) observed that SopE, one

of the substrates of the system encoded by SPI1, is encoded outside the SPI buton a cryptic P2-like phage. This observation suggests that the effectors could be

horizontally transferred independently from the secretion-translocation systems.

In plant pathogens also, hrp genes are organized in clusters that may also

contain genes encoding effectors, but other effector genes are scattered either

on the chromosome or on plasmids that may be conjugative. hrp gene clusters

themselves are sometimes carried on by plasmids as in R. solanacearum and in

Erwinia herbicola pv. gypsophilae (102; reviewed in 142). Some avr genes are

flanked by sequences related to transposable elements or bacteriophages (80) or

are part of pathogenicity islands (71).

PROSPECTS

Since its discovery in 1994, type III secretion has expanded very rapidly to become

a whole field. Study of the type III systems allowed a better understanding of the

pathogenesis of gram-negative bacteria, and the discoveries made with the different

pathogens benefited from a constant cross-feed. The recent very fast progress

made with P. aeruginosa, taking advantage of its similarity with Yersinia spp., isa spectacular example for such cross-feeding. From a medical point of view, the

discovery andunderstanding of type III systems will lead to the development of new

vaccines and new antigen delivery systems. It could also lead to the development

of antipathogenicity drugs, but whether these drugs could be active against all

type III pathogens is uncertain. Moreover, the presence of type III systems in

only a small number of animal pathogens would probably also represent a serious

handicap. From a more basic point of view, the discovery and understanding of

type III systems could also be beneficial to eukaryotic cell biology, by providing

new tools if not new concepts. In particular, type III systems can be used tointroduce proteins of interest in almost any type of cultured cell.

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emerging between avirulence and virulence, with more and more avrgenes having

a dual function, give a rationale for the persistence ofavrgenes in pathogens, even

if this is accompanied by restriction in host range. The next challenge is to under-

stand the role and the plant targets of these virulence proteins in the establishmentof diseases. From a more practical point of view, the deciphering of the signal

transduction cascades leading to resistance might open the way to new strategies

for increasing disease resistance in plants.

ACKNOWLEDGMENTS

We acknowledge J Alfano, M Barny, S Bleves, A Collmer, B Finlay, A Gauthier,

and S Totemeyer for comments and suggestions. Thanks are also due to M-A

Barny, A Collmer, and C Boucher for providing unpublished data. We are grateful

to M Monteforte for handling the reference list. Work in Guy Corneliss laboratory

is supported by the Belgian Fonds National de la Recherche Scientifique Medicale

(Convention 3.4595.97), the Direction Generale de la Recherche Scientifique-

Communaute Francaise de Belgique (Action de Recherche Concertee 99/03.), and

the Interuniversity Poles of Attraction Program-Belgian State, Prime Ministers

Office, Federal Office for Scientific, Technical and Cultural affairs (PAI 4/03).

Visit the Annual Reviews home page at www.AnnualReviews.org

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