effector review

R E V I E W A R T I C L E

Functional domains andmotifs of bacterial type III e¡ector proteinsand their roles in infectionPaul Dean

Institute of Cell and Molecular Bioscience, Medical School, University of Newcastle, Newcastle Upon Tyne, UK

Correspondence: Paul Dean, Institute of Cell

and Molecular Bioscience, Medical School,

University of Newcastle, Newcastle Upon Tyne

NE2 4HH, UK. Tel.: 1191 222 3481; fax:

1191 222 7424; e-mail: [email protected]

Received 13 February 2011; revised 5 April

2011; accepted 7 April 2011.

DOI:10.1111/j.1574-6976.2011.00271.x

Editor: Miguel Camara

Keywords

T3SS; TTSS; type three secretion system;

pathogens; disease; pathogenesis.

Abstract

A key feature of the virulence of many bacterial pathogens is the ability to deliver

effector proteins into eukaryotic cells via a dedicated type three secretion system

(T3SS). Many bacterial pathogens, including species of Chlamydia, Xanthomonas,

Pseudomonas, Ralstonia, Shigella, Salmonella, Escherichia and Yersinia, depend on

the T3SS to cause disease. T3SS effectors constitute a large and diverse group of

virulence proteins that mimic eukaryotic proteins in structure and function. A

salient feature of bacterial effectors is their modular architecture, comprising

domains or motifs that confer an array of subversive functions within the

eukaryotic cell. These domains/motifs therefore represent a fascinating repertoire

of molecular determinants with important roles during infection. This review

provides a snapshot of our current understanding of bacterial effector domains

and motifs where a defined role in infection has been demonstrated.

Introduction

Several of the world’s most important diseases are caused

by bacterial pathogens that deliver effector proteins into

eukaryotic host cells using a type three secretion system

(T3SS) (Troisfontaines & Cornelis, 2005; Table 1). Bacteria

that possess a T3SS cause a wide range of diseases in plants,

animals and humans (Troisfontaines & Cornelis, 2005),

while others have symbiotic relationships with plant or

animal hosts (Preston, 2007; Table 1). The best-studied

pathogens to date, which have provided the most knowledge

about bacterial effectors, are species of Chlamdyia, Salmo-

nella, Shigella, Yersinia, Pseudomonas, Xanthomonas, Ralsto-

nia and pathogenic Escherichia coli. In all of these organisms,

the T3SS is an essential virulence factor, highlighting the

central importance of the effector proteins in disease (Co-

burn et al., 2007).

Up to 100 different effector proteins may be delivered

into individual host cells by a single bacterium (see Kenny &

Valdivia, 2009; Table 1). Effectors are often multifunctional

proteins with many overlapping properties and may also

cooperate with each other to orchestrate specific responses

in the host cell (see Galan, 2009; Fig. 1). In general, although

not always, type three effectors display subtle functions

inside host cells, often causing more restrained alterations

in host cell physiology compared with the more overt effects

of bacterial exotoxins. The effector protein family is evolu-

tionarily diverse and exhibits a range of functions within

host cells (Fig. 1), targeting most aspects of eukaryotic

physiology (Fig. 1). Work over the last decade has been

instrumental in defining the commonalities between the

functions, molecular mechanisms and structure of bacterial

effectors. Indeed, most effectors are viewed as modular

proteins, composed of functionally distinct domains or

motifs that range from an enzymatic active site to a

protein–protein interaction or even an organelle-targeting

motif. Despite the large size of the bacterial effector family

(over 400 predicted proteins), they collectively possess a

relatively small subset of domains and motifs that have been

shown to play a role in infection (Fig. 2 and Table 2). This

review provides a detailed assessment and classification of

the effector domains/motifs that have been demonstrated to

play a role in the infection process. Where possible, their role

in disease has been discussed and is presented in Table 3,

although most of the functional studies on effectors to date

have been performed on cultured host cells. Throughout the

review, motifs are given using the standard amino acid

nomenclature, with ‘x’ referring to any amino acid residue.

For brevity, the bacterial origin of the effectors is most often

designated according to the genus name of the pathogen.

FEMS Microbiol Rev ]] (2011) 1–26 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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Although there are many excellent publications on the func-

tions of effector proteins, this review specifically focuses on

examples where effector domains/motifs have a proven role

within the host cell during infection, as summarized in Table 2.

Evolution of modularity inbacterial effectors

Where do bacterial effectors come from? The evolutionary

origin of effector genes is open to debate as most bacterial

effectors lack significant overall homology to proteins with-

in sequenced genomes. Bacterial effector genes typically

exhibit a G/C base composition that is distinctly different

from the overall bacterial genome, suggesting that effectors

have been acquired by horizontal gene transfer, and much

evidence suggests that this is the driving force behind

effector evolution (Rohmer et al., 2004). Once acquired by

a bacterial strain, effector evolution is then driven by

lineage-specific effector functions, dependent on specific

protein interactions within the host (Rohmer et al., 2004).

A prominent feature shared by bacterial effectors is their

modular architecture – comprising well-defined regions or

Table 1. Bacterial pathogens that utilize the T3SS

Host Bacterial species Disease caused Repertoires of effectors proven to be secreted or translocated

Plant

host

Pseudomonas syringae,

numerous pathovars

Range of plant diseases, e.g. tomato speck HopK1, HopY1, HopAS1, HopU1, HopF2, HopH1, HopC1,

HopAT1, HopG1, HopD1, HopQ1, HopR1, HopAM1, HopN1,

HopM1, AvrE, AvrB3, HopB1, HopX1, HopZ3, HopAb2, AvrPto,

HopE1, HopV1, HopAQ1, HopG1, HopI1, HopA1, HopX1,

HopO1, HopT1, AvrRpt2, AvrA, HopW1, HopD1, HopQ1,

AvrD1, AvrB2, HopAR1 (see Cunnac et al., 2009 for

nomenclature)

Xanthamonas spp. Wide range of plant diseases, e.g. rice

bacterial blight and citrus canker

AvrBs1, AvrBs2, AvrBs3, AvrRxo1, AvrRxv, AvrXccC, AvrXv3, Ecf,

HpaA, XopJ, XopX, XopB, XopC, XopD, XopE, XopF, XopN,

XopO, XopP, XopQ

Ralstonia solancearum Plant wilt on many host species GALA1-7, SKWP1-6, HLK1-3, RipB, PopW, PopP, PopC, RipT,

AvrA, PopB, PopA, RipA (many others predicted)

Erwinia amylovora Causes fire blight on a range of plant

species

DspE, HrpN, HrpW, HopPtoC, AvrRpt2, EopB

Rhizobium spp. Symbiont; forms nodules on legumes NopL, NopP, NopJ, NopM, NopT, NopB, NopN

Pantoea spp. Bacterial wilt on corn and maize WtsE, PthG, HsvG, HsvB

Animal

host

Pseudomonas aeruginosa Opportunist pathogenic. Can cause

pneumonia

ExoU, ExoY, ExoS, ExoT

Escherichia coli (EPEC and

EHEC), Cirobacter rodentium

Diarrhoea (EPEC) or haemorrhagic colitis

(EHEC). Cattle commensal (EHEC)

Tir, Map, EspF, EspB, EspZ, EspH, EspG, NleA, NleB, NleC, NleD,

NleE, NleF, NleG, NleH, NleK, NleL, EspJ, EspK, EspL, EspM, EspY,

EspX, EspO, EspW

Salmonella enterica serovars Gastroenteritis; typhoid fever AvrA, SipA, SipB, SipC, SipD, SopA, SopB, SopE, SopE2, SptP,

SlrP, SopD, SspH1, SteA, SteB, GogB, PipB, SifA, SifB, SopD,

SpiC, SseF, SseG, SseI, SseJ, SseK, SspH, SteC, SpvB, SpvC

Shigella spp. Bacillary dysentery; shigellosis IpaA, IpaB, IpaC, IpaD, IpaJ, IpgD, IpgB, IcsB, OspC, OspD, OspZ,

OspB, OspF, VirA, OspE, OspG, IpaH family

Yersinia spp. Bubonic plague (pestis); Gastrointestinal

disease (enterocolitica)

YopE, YopH, YopP/J, YopE, YopM, YopT, YpkA/YopO

Yersinia enterolytica biovar 1B Severe gastrointestinal disease YspA, YspL, YspP, YspF, YspE, YspI, YspK, YspM

Photorhabdus spp. Opportunistic pathogen (asymbiotica);

insect pathogen (luminescens)

LopT

Chlamydia spp. Obligate intracellular parasites, sexually

transmitted disease; can cause blindness

CADD, CT847, tarp, IncA, IncG, CT229, CT813, Cpn0585,

Cpn0909, Cpn1020

Burkholderia spp. Melioidosis (B. pseudomallei); glanders

(B. mallei)

CHBP, BopE

Vibrio spp. Gastroenteritis, wound infections

(parahaemolyticus); secretory diarrhoea

(cholerae)

VopL, VopA, VPA450, VopT, VopF, VopS, VopQ

Bordetella spp. Whooping cough BopC/BteA, BopN

Aeromonas spp. Opportunistic pathogen; fish/humans AexT, AopB

Diseases caused by bacteria that are dependent on a T3SS are shown along with a snapshot of the current repertoires of proven effector proteins.

Putative effectors based on predictive methods are not given.

EPEC, enteropathogenic Escherichia coli; EHEC, enterohaemorrhagic E. coli.

FEMS Microbiol Rev ]] (2011) 1–26c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

2 P. Dean

domains (Fig. 2) that confer a subversive function. Strik-

ingly, the individual modules within an effector often

mediate very different, unrelated functions (see Fig. 2),

strongly suggesting that they evolved independent of each

other and subsequently combined to form the chimeric

effector (Stavrinides et al., 2006). This process of chimeriza-

tion is a common theme shared by many effectors, as

exemplified by the Salmonella effector SptP, which shares a

Fig. 1. The cellular functions of bacterial effectors. Activities of bacterial effectors following their translocation into the host cell from a wide range of

animal and plant pathogens. Many of the effector functions have been attributed to the domains and motifs described in this review. Major functional

classes are subdivided by dotted lines. Effectors are colour-coded depending on species origin (bottom), with host factors given in orange. P, phosphate

group; ADPr, ADP-ribose; Ac, acetyl group; Ub., ubiquitin; PI, phosphoinositide. Other common abbreviations are given in the text.


3Functional domains and motifs of bacterial effector proteins

homologous N-terminal domain with Pseudomonas ExoS

and Yersinia YopE, while its C-terminal region is highly

homologous to that of another effector, YopH (Kaniga et al.,

1996; Fig. 2). This apparent design-by-recombination forms

the basis of an attractive hypothesis termed ‘terminal

reassortment’, proposed by Guttman and colleagues to

explain the diversity of bacterial effectors (Stavrinides et al.,

2006). In line with this hypothesis, horizontal gene acquisi-

tion, followed by genomic shuffling results in the fusion of

functional effector modules to the C-termini of different

effectors. Providing the newly formed chimeric effector

contains an intact N-terminal secretion domain (described

below), the new protein is likely to be secreted and translo-

cated into the host cell – giving rise to a quick ‘one-step’

evolutionary mechanism to yield new effectors (Stavrinides

et al., 2006). This is evident with the four Salmonella

effectors SifA, SspH1, SseJ and SseI, which all share homo-

logous N-termini, but different C-terminal regions (Han-

sen-Wester et al., 2002). The terminal reassortment

hypothesis is strengthened by the finding that 32% of all

type three effector families contain chimeric effectors, far

greater than that in other analysed protein families, suggest-

ing that this process is important for the evolution of these

virulence proteins (Stavrinides et al., 2006).

Fig. 2. The modular architecture of type

three effector proteins. Examples of effectors

that exhibit high modularity that contain the

domains and motifs described in this review.

Sec., secretion domain; Chap., chaperone-

binding domain; PRR, proline-rich repeats. Ab-

breviations of common motifs are given in the

text. Effectors are not drawn to scale.


4 P. Dean

Table 2. Proven type three effector motifs

Subcellular

target/activity Effector examples Description of responsible motifs

Subcellular

targeting

Intracellular membrane PipP2, SopD2, YopE, ExoS LFNEF (PipP2), WEK(I/M)xxFF (SopD2); N-terminal Lx2Rx4L (YopE, ExoS);

CLCCFL prenylation motif (SifA)

Nucleus (NLS) PopB, PopP2, XopD, AvrBs3 Bipartite NLS: KRKR and KKKKKR (PopB); RRRR and RRQRQ (PopP2).

Monopartite NLS: XopD, TAL effectors such as AvrBs3, e.g. KRPR

Nucleolus EspF Atypical nuclear/nucleolar targeting motif

Plasma membrane ExoU, HopZ, AvrPto, AvrPphB C-terminal MLD containing an essential KAWRN motif (ExoU); PM

targeting mediated by Myr and Palm motifs (several plant pathogen

effectors)

Mitochondrial MTS Map, EspF, SopA N-terminal domain rich in arginine and leucine (Map and EspF); a critical

Leu16 (EspF); internal atypical domain (SopA)

Golgi STE motif, SseG WEK(I/M)xxFF motif alone; internal golgi targeting domain (SseG)

Subversive

domain/motif

Ser/Thr kinase YpkA Large domain similar to Ser/Thr kinases with critical Asp287 and Lys269

residues

RhoGAP AexT, ExoS, ExoT, YopE, SptP Critical arginine finger motif GxLRx3T and an essential downstream loop

containing the consensus QWGTxGG (AexT, ExoS, ExoT, YopE, SptP); Tir

also possesses the arginine finger GxLR

RhoGEF SopE, SopE2, BopE Catalytic loop containing the GAGA motif

RhoGEF (WxxxE family) Map, SifA, EspM2 Catalytic loop containing the consensus (D/P/E)x3AQ; a conserved WxxxE

structural motif

Lipase SseJ C-terminal domain with a GDSL lipase motif and a SHD catalytic triad

Phospholipase ExoU Large cPLA2 domain. SD catalytic dyad of – GxSx(G/S) hydrolase motif and

a Dx(G/A) active site motif. An essential GGGxxG motif

Cysteine protease YopJ, YopT, AvrPphB Catalytic HEC triad: HX18EX44C (YopJ); catalytic CHD triad (YopT, AvrPphB)

and N-terminal Rho-binding domain (YopT), autoproteolytic site GDKG

(AvrPphB)

E3 Ub ligase SopA, IpaH, SspH1 Active site HECT motif – Lx4TC (SopA); CxD motif (IpaH and SspH1)

ADP-ribosyltransferase HopU1, SpvB, ExoS, ExoT ADPRT conserved region I – invariant arginine; region II – Gx9ST(S/T);

region III – NAD-binding ExE motif

Kinase SteC, YpkA, OspG Kinase subdomain I: nucleotide positioning motif – GxGxxG (SteC);

subdomain II: invariant lysine residue; subdomain III: invariant glutamic

acid

Protein phosphatase YopH, SptP PTPase motif – HC(x)5R(S/T); essential WPD loop

PIP phosphatase IpgD, SopB PIPase domain – FN(F/V)G(V/I)NE (motif 1) and CKSxKDRTxM (motif 2)

pY motifs Tir Src kinase phosphorylation motif – ExLYxx(I/V)

F boxes GALA family The LPx6I type F box (all GALA effectors)

Lyase OspF, SpvC HopAI1 Lyase motif – GDKxH – essential for lyase activity

WH2 VopL, VopF Homology to WASP WH2; contains the LKKV-like motif

D motif SpvC, OspF Docking motif required for specific binding to MAPKs

FH1 VopF Highly homologous to mammalian formins (VopF)

Actin-related protein

binding

IpaA, Tir, EspF Phosphotyrosine Nck-binding YDEV motif (Tir), vinculin-binding domain

(IpaA), N-WASP-binding motif (EspF)

14-3-3-binding site ExoS Mediates binding to the 14-3-3 protein FAS to activate the ADPRT domain

SH3-binding motif EspF Repeated motif; binds sorting nexin 9 via a conserved RxAPxxP motif

J domain HopI1 Hsp70 interaction domain with an invariant HPD motif

PDZ1-binding motif Map, NleA, NleH1 Extreme C-terminal motif: -SKI (NleH1), -TRL (Map), -TRV (NleA); mediates

effector sorting and protein–protein interaction

Transcription activation

domain

AvrBs3 effector family Far C-terminal region of the TAL effectors. Functions in conjunction with the

central DNA-binding repeat region

Caspase-3 site SipA, SopA Processing site containing the motif DEVD

Fic domain VopS Required for AMPylation of Rho GTPases. C-terminal motif: HxFx(D/

E)GNGR

The effector activities and subcellular targets are listed together with examples of the effector domains and motifs that have been demonstrated to be

responsible. Abbreviations for the domains and motifs are given in the text of the review. Motifs are given in bold. The motif sequence conforms to the

annotation used throughout the review.



Effector secretion and translocation --the N-terminus

For the majority of effector proteins, the N-terminal region

(first 15–25 residues) encodes the signal sequence required

for type three secretion with a more expansive chaperone-

binding domain located closely downstream (between

residues 50 and 150; for a review, see Ghosh, 2004). The

N-terminal secretion signal, while being functionally inter-

changeable between many effectors (Anderson et al., 1999),

does not exhibit a discernable consensus sequence. Some

evidence suggests that the effector mRNA sequence may be

important (Sorg et al., 2005), although this premise is

somewhat controversial and appears to depend on the

effector in question (Lloyd et al., 2001; Ghosh, 2004).

Interestingly, a recently documented prediction method,

using a machine-learning approach, analysed the N-termini

of 100 effectors and was able to identify putative effectors

with over 70% accuracy (Arnold et al., 2009). This was based

on the amino acid frequency and the physicochemical

properties of the N-terminal residues, suggesting that the

amino acid sequence, and not the mRNA signal, is most

important for secretion (Arnold et al., 2009). Similar

approaches have been published recently that collectively

show that the amino acid composition of the N-terminal

region can be used successfully for effector prediction

(reviewed in McDermott et al., 2011).

For some effectors, secretion/translocation is also depen-

dent on the C-terminal region. For example, the Salmonella

effector SipB requires both the N- and the C-terminal

regions for type three secretion as the first 160 residues of

SipB are secreted through the flagella secretory system. Only

when the C-terminal region of SipB was fused with this

N-terminal domain did the protein target the T3SS (Kim

et al., 2007). Other examples of effectors that require the

C-terminal regions for secretion include SifA (Brown et al.,

2006), SipC (Chang et al., 2005) and the E. coli effector Tir

(Allen-Vercoe et al., 2005).

Subcellular-targeting domains/motifs

Mitochondrial-targeting sequences (MTSs)

Only a few bacterial effectors are reported to target the

mitochondria (Fig. 1). These include Map (Kenny & Jepson,

2000), EspF (Nougayrede & Donnenberg, 2004), SopA

(Layton et al., 2005), SipB (Hernandez et al., 2003) and

HopG1 (Block et al., 2009). Most mitochondrial proteins in

eukaryotes are cytoplasmic and must be imported into the

mitochondria via an N-terminal MTS that is cleaved in the

mitochondrial matrix (Neupert, 1997). The E. coli effectors

EspF and Map bear canonical MTS within their N-termini,

with the first 41 and 24 residues of Map and EspF sufficient

to target these effectors to host the mitochondria (Fig. 2)

(Nagai et al., 2005), where they are subsequently cleaved in

the matrix (Kenny & Jepson, 2000; Nougayrede & Donnen-

berg, 2004; Papatheodorou et al., 2006). Like many eukar-

yotic mitochondrial proteins, the MTS of EspF contains

essential arginine and leucine residues, such as the 16th

leucine, which is critical for mitochondrial import (Nagai

et al., 2005), and mutation of this residue has been im-

portant in identifying the cytoplasmic functions of EspF

(Holmes et al., 2010). The two Salmonella effectors SipB and

SopA target the mitochondria, but do not have a canonical

N-terminal MTS (Hernandez et al., 2003; Layton et al.,

2005), although other mechanisms do exist for mitochon-

drial import (Lutter et al., 2000). Indeed, SopA targets the

mitochondria dependent on an internal sequence (residues

100–347) with no apparent role for the N-terminus (resi-

dues 1–50) (Layton et al., 2005). Likewise, Pseudomonas

Table 3. Bacterial effectors and their domains/motifs with proven roles in virulence

Bacterial effector Domain or motif with a virulence role References

SipA DEVD – caspase 3 cleavage site Srikanth et al. (2010)

SseI Cys178 – unknown function McLaughlin et al. (2009)

SseL Cysteine protease site Rytkonen et al. (2007)

YopM Leucine-rich repeats (6–15) McPhee et al. (2010)

ExoS ADP-ribosyltransferase domain Shaver & Hauser (2004)

HopF2Pto Myristoylation motif Wilton et al. (2010)

AvrPto Ser149 phosphorylation Anderson et al. (2006)

WtsE and AvrE1 WxxxE motif Ham et al. (2009)

SifA CAAX box Boucrot et al. (2003)

GALA effectors F-box Angot et al. (2006)

SseJ Lipase domain – SHD triad Ohlson et al. (2005)

SpvB ADP-ribosyltransferase domain Lesnick et al. (2001)

NleA PDZ domain Lee et al. (2008)

YpkA Kinase domain Wiley et al. (2006)

YopE GAP domain Black & Bliska (2000)


6 P. Dean

syringae HopG1 does not have a predicted MTS, but green

fluorescent protein (GFP) fusions to the C-terminus indi-

cated that the N-terminal residues 1–13 were essential for

mitochondrial targeting (Block et al., 2009). However, it is

unclear whether native HopG1 targets the mitochondria as

ectopic expression of effector fusions can result in anom-

alous localization patterns. This is exemplified with the

E. coli effector Tir, which normally targets the plasma mem-

brane, but when fused to GFP and expressed in host cells,

reportedly targets the mitochondria (Malish et al., 2003).

Effector-specific membrane-targeting motifs

While some effectors encode eukaryotic-like membrane-

targeting regions, others possess novel sites with no obvious

similarity to known eukaryotic proteins. The Salmonella

homologues PipB and PipB2 target intracellular membrane

compartments and the membranous Salmonella-containing

vesicle (SCV) (Knodler et al., 2003). These two effectors

share homology mainly due to over 20 pentapeptide repeats

(PPR) in their C-terminal domain, bearing the consensus

sequence A(N/D)(L/M/F)xx (Knodler et al., 2003). PPR are

widely distributed across both pro- and eukaryotes and

confer specific structural characteristics such as a super-

helical structure (Bateman et al., 1998), but do not confer a

common biological function and are likely to be involved in

protein–protein interactions (Bateman et al., 1998). PipB

and PipB2 are considerably less conserved outside of their

PPR region, with the C-terminal domain (last 38 residues)

of PipB2, but not PipB, targeting this effector to peripheral

vesicular compartments (Knodler & Steele-Mortimer,

2005). A C-terminal motif LFNEF of PipB2 is essential for

vesicular targeting as alanine substitutions revealed an

essential role for each residue in this motif. Subsequent

studies have also shown that the LFNEF motif is important

for PipB2 function, essential for the recruitment of kinesin-1

to the membrane of the SCV (Henry et al., 2006).

SopD2 belongs to a family of Salmonella effectors includ-

ing SifA, SopD, SseJ and SspH2 termed STE (Salmonella

translocated effector) that share a conserved (�140 residue)

N-terminal region containing the consensus sequence

WEK(I/M)xxFF. This motif is required for effector translo-

cation from the bacterium (Miao et al., 1999), but also plays

a functional role inside the host cell. Indeed, the STE motif

of SopD2 is essential for targeting this effector to late

endocytic vesicles as point mutations in this motif (W37P

and F44R) abolished endosome targeting of SopD2-GFP

(Brown et al., 2006). This indicates that the STE motif is

bifunctional, and has been exploited by Salmonella to

mediate two distinct functions in different cell types. Inter-

estingly, the WEK(I/M)xxFF motif alone, from a range of

STE effectors, is not sufficient to target GFP to endocytic

compartments, but instead targets it to the Golgi apparatus

(Brown et al., 2006). In addition, the SopD effector, a

homologue of SopD2 that also carries the STE motif, was

found to reside in the cytoplasm, suggesting that the

organelle-targeting function of this motif has been altered

in this effector (Brumell et al., 2003). These studies elegantly

demonstrate that an effector motif may behave very differ-

ently depending on its context and highlight the importance

of the entire effector sequence organization in influencing

motif function.

Pseudomonas aeruginosa ExoS and Yersinia YopE, which

share a RhoGAP domain in their C-terminal region (Fig. 2),

possess an N-terminally located membrane localization

domain (MLD) that targets these effectors to perinuclear

vesicles (Pederson et al., 2000; Krall et al., 2004). Although

the MLD in YopE shares little similarity to the MLD of ExoS

(Krall et al., 2004), both domains are highly hydrophobic

and can functionally substitute for each other. Of note, the

MLD of YopE and ExoS bear the consensus sequence

Lx2Rx4L (Krall et al., 2004), suggesting possible functional

significance, although whether this motif is critical for

membrane targeting is unknown.

Other effectors that target host cell membranes include

SseG, a Salmonella effector that targets the Golgi apparatus

dependent on an internal localization domain (residues

87–143) (Salcedo & Holden, 2003). This Golgi-targeting

region of SseG has no obvious similarity to eukaryotic

proteins, but does share high regional homology to the

Edwardsiella effectors EseG and EseF, suggesting functional

similarities between them. The E. coli effector NleA also

targets the Golgi apparatus, but a targeting motif has not

been defined for this protein (Gruenheid et al., 2004).

Finally, the P. aeruginosa effector ExoU, a potent phospho-

lipase, targets the plasma membrane via a C-terminal MLD

(Fig. 2) containing the essential pentapeptide motif KAWRN

(Rabin et al., 2006). This motif was demonstrated to be

essential for ubiquitination of this effector on a distal lysine

residue, suggesting that membrane targeting of ExoU is a

prerequisite for ubiquitination (Stirling et al., 2006). Im-

portantly, the KAWRN motif was shown to be essential for

the phospholipase activity of ExoU and its subsequent

toxicity in the host cell (Stirling et al., 2006).

Membrane-targeting motifs -- prenylation

The Salmonella effector SifA is required for the formation of

intracellular membranous tubules called Sifs along host cell

microtubules (Stein et al., 1996; Beuzon et al., 2000; Brumell

et al., 2002). SifA targets intracellular membranes via a C-

terminal hexapeptide motif – CLCCFL – that conforms to

the canonical CAAX prenylation box (where AA represents

two aliphatic residues) (Reinicke et al., 2005). CAAX boxes

target eukaryotic proteins to the plasma membrane by

mediating the covalent attachment of a lipid isoprenyl group



to the target CAAX cysteine residue (Gao et al., 2009).

Fusion of the last 11 C-terminal residues of SifA to GFP is

sufficient to direct GFP to membrane compartments (Bou-

crot et al., 2003). The removal of the CAAX box from SifA

abolished its cellular functions and virulence properties,

implying that membrane localization is essential for this

effector (Boucrot et al., 2003). Interestingly, the N-terminal

region of SifA was also shown to be essential for its function,

but only when it was fused to the C-terminal prenylation

motif (Boucrot et al., 2003). In addition to prenylation, the

cysteine residue adjacent to the SifA CAAX box (CLCCFL)

was shown to be acylated following prenylation (Reinicke

et al., 2005), although the relevance of this is unclear, but

may be linked to protein sorting such as targeting to lipid

rafts (Resh, 2006).

Membrane-targeting motifs -- myristoylationand palmitoylation

Myristoylation (Myr) serves to target proteins to the plasma

membrane via the irreversible attachment of a myristoyl

group (14-carbon fatty acid) to an extreme N-terminal

glycine, which then acts as the membrane anchor. In most

cases, the N-terminal methionine must be cleaved off to

expose the second residue glycine and therefore almost all

Myr motifs begin with a methionine and glycine at the N-

terminus and are typically short, approximately six to eight

residues in length. In the majority of proteins, Myr sites are

also closely associated with a palmitoylation (Palm) site that

contains a cysteine at position 3 or 5 to which palmitate or

other long-chain fatty acids are attached. Myr and Palm

often act in combination to strengthen membrane anchor-

ing (Maurer-Stroh & Eisenhaber, 2004) and both are specific

to eukaryotes (Maurer-Stroh & Eisenhaber, 2004), but have

also been found in bacterial effectors.

All bacterial effectors that have defined Myr and Palm

sites are from plant pathogens, particularly P. syringae, and

include the HopZ family, HopF2 (Lewis et al., 2008),

AvrPhB, AvrRpm1, AvrB, AvrC, AvrPto (Nimchuk et al.,

2000; Shan et al., 2000; Maurer-Stroh & Eisenhaber, 2004;

Robert-Seilaniantz et al., 2006) and Xanthomonas XopE1/2

(Thieme et al., 2007). Almost all these effectors encode a

Myr motif at their extreme N-terminus that targets them to

the plant plasma membrane. The consensus motif is not

straightforward, but the P. syringae effectors generally con-

forms to MG(N/C)(I/V)(C/S) – noting the palmitoylation

cysteines at positions 3 and 5 and the critical Myr glycine

residue at position 2. The HopX effector family is slightly

different as it displays a highly conserved N-terminal

MGLCxSKP Myr motif that differs from the typical pattern

(Thieme et al., 2007), but is still crucial in plasma membrane

targeting and the subsequent function of the effector (Lewis

et al., 2008). Interestingly, the cysteine protease effector

AvrPphB from P. syringae undergoes autoproteolysis to

expose a new, embedded Myr and Palm site (Nimchuk

et al., 2000) that mediates plasma membrane targeting

(Dowen et al., 2009). It seems likely that other effectors,

with no obvious myristoylation motifs, may utilize similar

internal cleavage reactions to promote plasma membrane

targeting similar to AvrPphB.

Nuclear localization sequences (NLSs) andtranscription-related domains

A growing number of bacterial effectors (see Fig. 1) target

the nucleus including BopN (Nagamatsu et al., 2009),

PopP2 (Deslandes et al., 2003), the AvrBs3 family (Szurek

et al., 2002), PopB (Gueneron et al., 2000), OspF & OspC

(Zurawski et al., 2006), IpaB (Iwai et al., 2007), OspB

(Zurawski et al., 2009), YopM (Skrzypek et al., 1998), XopD

(Hotson et al., 2003), IpaH9.8 (Toyotome et al., 2001) and

SspH1 (Haraga & Miller, 2003). Nuclear import occurs

through the nuclear pore complex and may be an active

process, dependent on a NLS, or a passive one if the proteins

are o 40 kDa (Tran & Wente, 2006). Canonical nuclear

localization signals are mono- or bipartite, composed of

short stretches of basic positively charged amino acids

(especially lysine and arginine), and in the case of the

bipartite signal, are separated by a stretch of about 9–12

amino acids. The Ralstonia effector PopB contains a bipar-

tite NLS in the centre of the protein, with the sequences

KRKR and KKKKKR separated by 13 amino acids (Guener-

on et al., 2000). The deletion of either motif prevented PopB

from localizing to the nucleus and instead it targeted the

cytoplasm (Gueneron et al., 2000). The Ralstonia PopP2 also

has a functional bipartite NLS in its N-terminus with the

sequence RRRR and RRQRQ separated by 11 amino acids

(Deslandes et al., 2003). By contrast, XopD carries a single

putative NLS in its C-terminus, although functional studies

suggest that this may not be important for its nuclear

import, at least when fused to EYFP (Hotson et al., 2003).

Interestingly, in addition to being a translocator that inserts

into the host plasma membrane, the Shigella effector IpaB

has been found to target the nucleus in a cell-cycle-depen-

dent manner, where it interacts with the cell-cycle-regulated

protein Mad2L2 (Iwai et al., 2007). IpaB does not possess an

NLS, and due to its rather large size (�62 kDa), has been

proposed to enter the nucleus by piggy-back, via its interac-

tion with Mad2L2 (Iwai et al., 2007). This mode of nuclear

import may be utilized by other nuclear-targeted effectors

that do not possess an NLS.

The Xanthomonas AvrBs3 effector families (termed the

TAL effectors for transcription activator-like) are highly

homologous proteins from plant pathogens that include

AvrBs3, PthA, Avrb6, AvrXa7, AvrBs4, PthXo1, AvrHah,

Avrxa5 and AvrXa10. These fascinating effectors all mimic


8 P. Dean

eukaryotic transcription factors and, despite their sequence

similarity, induce a diverse array of phenotypes in plants

(Kay et al., 2007). The functions of the TAL effectors are

dependent on three defined regions (Fig. 2) that are related

to their nuclear function: (1) interchangeable NLSs in the C-

terminal region (Szurek et al., 2002), (2) highly conserved

tandem repeats in the centre of the effector that facilitate

effector dimerization – essential for nuclear import and

DNA binding (Gurlebeck et al., 2005; Kay et al., 2007) and

(3) a C-terminal transcription activation domain (TAD)

(Zhu et al., 1998). The functional specificity of each

TAL effector is determined by the central DNA-binding

region, with subtle amino acid changes responsible for

binding different host DNA sites (Boch et al., 2009; Moscou

& Bogdanove, 2009). By contrast to the central region,

the TAD and NLS domains retain functional conserva-

tion across the TAL effector, but do not confer lineage-

specific functions to the effector as they are functionally

interchangeable.

Several effectors from different bacterial species display

long homologous stretches of leucine-rich repeats (LRR)

that bear the consensus sequence Lx6Lx2I/LPx3P. Although

LRR are widely distributed in nature, this ‘LPX’ subtype of

LRR is specific to bacterial effectors (Buchanan & Gay, 1996)

and is found in YopM, the IpaH family (Fig. 2), SlrP, SspH1

and SspH2 (Miao et al., 1999). Notably, YopM, IpaH9.8 and

SspH1 all target the nucleus (Toyotome et al., 2001)

(Skrzypek et al., 1998) (Haraga & Miller, 2003), whereas

SlrP and SspH2 do not (Haraga & Miller, 2003), suggesting

that the LPX domain does not confer this ability alone. The

NLS of the LPX effectors has not been studied in depth, with

only YopM found to contain an NLS embedded towards the

end of its LRR, between the 8th and the 15th LRR, although

conflicting reports suggest that a second NLS exists in the C-

terminus (Benabdillah et al., 2004; McCoy et al., 2010).

Thus, it seems likely that specific residues in the LPX

domain of the nuclear-located effectors mediate nuclear

targeting. It is therefore interesting to note that the nuclear-

targeted LPX effectors YopM, SspH1 and IpaH9.8 share the

conserved repeating motif NxLTxLPELP, unlike the non-

nuclear effectors SlrP and SspH2. Whether this motif is

directly related to nuclear targeting or to a common

biological function remains to be clarified. Recently, the

LRRs of YopM were shown to mediate binding to the host

kinase PRK2 and were also shown to be essential for the

virulence properties of YopM (McPhee et al., 2010),

although the role of nuclear targeting in virulence remains

undetermined.

Recently, the E. coli effector EspF was found to target the

nucleolus, a subnuclear structure involved in ribosome

biosynthesis (Dean et al., 2010b). The nuclear/nucleo-

lar localization sequence (NoLS) of EspF resides in the

N-terminal region (residues 21–74) between two other cell

sorting domains (the MTS and SNX9 motif) and is highly

conserved in E. coli strains (Holmes et al., 2010). The NoLS

of EspF is unlike other typical nuclear/nucleolar-targeting

domains in eukaryotes and viruses, which contain an

abundance of arginines and lysines, suggesting that it may

represent a novel targeting motif. Alternatively, the NoLS

region of EspF may mediate an interaction with a cytoplas-

mic host protein that then carries EspF into the nucleus/

nucleolus, as suggested above for the nuclear import of IpaB

(Iwai et al., 2007).

Ubiquitination

Ubiquitination is an important eukaryotic process that

involves the covalent attachment of ubiquitin to lysine

residues of a target protein (Passmore & Barford, 2004).

The process occurs in three steps involving (1) activation of

ubiquitin by E1-activating enzyme, (2) transferral of ubi-

quitin to an E2 ubiquitin-conjugating enzyme and (3) E3

ubiquitin ligase catalysing the covalent reaction between

ubiquitin and a lysine residue on the substrate (Passmore &

Barford, 2004). E3 ligases may bind ubiquitin directly

(HECT E3 ligases) or act as a scaffold between E2 and the

substrate (RING E3 ligases). As ubiquitination serves to

target proteins for degradation by the host proteosome or

regulate their function (Passmore & Barford, 2004), it is an

obvious target for bacterial pathogens and numerous effec-

tors have evolved mechanisms to manipulate it (Angot et al.,

2007; Fig. 1).

Effectors that exploit E3 ligases via an F-box

F-boxes are well-conserved structural domains (�50 resi-

dues) in eukaryotic proteins that recruit target proteins

to the ubiquitin-conjugating enzyme (Bai et al., 1996).

The Agrobacterium effector VirF, although delivered by

the type four secretion system, was the first prokaryotic

protein found to contain an F-box, which was shown

to be essential for virulence (Tzfira et al., 2004). F-boxes

have been found in other effectors including Legionella

type four effectors (Price et al., 2010) and the Ralstonia type

three GALA effector family (Tzfira et al., 2004). GALA

effectors, so called because they contain a leucine-rich

GAxALA repeat, bear a functional N-terminal F-box that is

important for virulence in plant hosts (Angot et al., 2006).

Although there is no strict consensus sequence for the

F-box, there are several conserved residues along its length

as most eukaryotic F-box proteins contain an invariant

LPx10L motif at the N-terminal region of the F-box that is

also conserved in VirF (Tzfira et al., 2004), while the GALA

effectors exhibit a less typical LPx6I motif (Angot et al.,

2006).



Effectors that mimic host E3 ubiquitin ligases

The P. syringae effector AvrPtoB suppresses plant pro-

grammed cell death (PCD), allowing the pathogen to multi-

ply and cause disease (Abramovitch & Martin, 2005). This

inhibitory process is conveyed through a large C-terminal

domain (residues 387–553) that has been found to mimic

the structure of eukaryotic U-box and RING-finger E3

ubiquitin ligases (Janjusevic et al., 2006), despite no se-

quence similarity between them. Within the host cell,

AvrPtoB interacts with ubiquitin directly (Abramovitch

et al., 2006) and causes the ubiquitination of the plant

kinase Fen, resulting in its proteosomal degradation

(Rosebrock et al., 2007). Remarkably, Fen recognizes the

N-terminal region of AvrPtoB leading to PCD in resistant

plants, thus revealing that by acquiring the C-terminal E3

ligase domain, AvrPtoB has evolved an effective mechanism

to combat its own detection.

The Salmonella effector SopA is an E3 ubiquitin ligase

(Zhang et al., 2006) with a key role in the induction of

inflammation (Wood et al., 2000). Although SopA has weak

sequence homology to eukaryotic HECT E3 ligases, it has a

highly conserved C-terminal Lx4TC motif that is found in

the active site of all HECT E3 ligase domains (Huibregtse

et al., 1995) (Zhang et al., 2006). The active site cysteine is

essential for the E3 ligase activity of SopA (Zhang et al.,

2006) and also dictated the ability of SopA to induce

inflammation (Zhang et al., 2006). Like AvrPtoB, SopA

mimics HECT E3 ligases through convergent evolution,

adopting several key structural elements in its C-terminal

region in addition to the active site (Diao et al., 2008).

The Shigella IpaH effector family defines a new class of E3

ligases (Singer et al., 2008; Zhu et al., 2008), distinct from

that mimicked by SopA and AvrPtoB. All IpaH effectors

possess a variable N-terminal region containing LRRs and a

highly conserved C-terminal region that contains the novel E3

ligase domain (Ashida et al., 2007). An invariant cysteine

within this region becomes exposed on an eight-residue loop

(Rohde et al., 2007; Singer et al., 2008) that bears the

conserved CxD motif with both the cysteine and the aspartic

acid residues critical for E3 ligase activity (Singer et al., 2008).

IpaH has numerous homologues in other bacterial species,

including the Salmonella effector SspH1 that possesses the N-

terminal LRR, the C-terminal CxD loop and has been

demonstrated to possess E3 ligase activity (Rohde et al., 2007).

Effectors that are targets for ubiquitination

Ubiquitination increases the molecular mass of target pro-

teins by approximately 8 kDa, enabling straightforward

identification of many effectors that are ubiquitinated

(Marcus et al., 2002; Kubori & Galan, 2003; Ruckdeschel

et al., 2006; Stirling et al., 2006). Although single lysines are

the target of the ubiquitination reaction, no consensus

ubiquitination motifs have been identified to date, and so

the preference for the target lysine is difficult to predict

(Pickart, 2001). Effectors may be monoubiquitinated such

as Salmonella SopB (Marcus et al., 2002) or polyubiquiti-

nated such as Salmonella SopE (Kubori & Galan, 2003),

Yersinia YopE (Ruckdeschel et al., 2006) and P. aeruginosa

ExoU (Stirling et al., 2006). In most cases, polyubiquitina-

tion targets proteins to the proteosome for degradation

while monoubiquitination may modulate protein function,

as demonstrated for the SopB effector (Knodler et al., 2009).

Phosphorylation

Protein phosphatases

Some of the best-studied bacterial effectors are phospha-

tases. Yersinia YopH is a protein tyrosine phosphatase

(PTPase) that exhibits among the highest-recorded PTPase

activity (Zhang et al., 1992). YopH prevents bacteria inter-

nalization into macrophages by dephosphorylating a large

number of proteins involved in integrin-mediated phagocy-

tosis, such as FAK and paxillin (Persson et al., 1997). The N-

terminal region of YopH contains a phosphotyrosine-bind-

ing domain that directly targets host tyrosine phosphory-

lated proteins (Montagna et al., 2001; Ivanov et al., 2005). A

proline-rich stretch separates this substrate-binding region

from a C-terminal domain that has homology to the

catalytic sites of eukaryotic PTPases, with a signature (P-

loop) motif – HC(x)5R(S/T) – that is highly conserved in

lipid and protein phosphatases (Stuckey et al., 1994). The P-

loop is critical for phosphate binding as mutation of the

nucleophilic cysteine within this motif abolishes the enzy-

matic activity of YopH and results in a substrate-trapping

protein (Persson et al., 1997). YopH contains a second, more

divergent loop (the ‘WPD loop’) found in PTPases that is a

�10-residue motif bearing the well-conserved WPD se-

quence that is also essential for catalysis (Zhang et al., 1994;

Hu & Stebbins, 2006).

The Salmonella effector SptP, another potent PTPase,

shares homology with YopH in its C-terminal region (Kani-

ga et al., 1996) containing the catalytical HC(x)5R(S/T)

motif and the WPD loop (Kaniga et al., 1996). Unlike YopH,

SptP contains an N-terminal GTPase-activating protein

(GAP) domain that, along with the PTPase domain, mod-

ulates actin rearrangements in host cells (Fu & Galan, 1999).

A substrate of SptP is the ATPase protein VCP (valosin-

containing protein), which is dephosphorylated by SptP,

promoting bacterial intracellular replication (Humphreys

et al., 2009). In addition to YopH and SptP, other effectors

that also possess the signature PTPase motifs include the

Pseudomonas HopAO1 (Espinosa et al., 2003; Underwood

et al., 2007), Chromobacterium CV0974 protein and Aero-

monas AopH, although these have not been studied in detail.


10 P. Dean

Phosphoinositide phosphatases (PiPases)

Phosphoinositides are lipid molecules that regulate eukar-

yotic signalling involved in cytoskeletal reorganization and

membrane dynamics (Weber et al., 2009). Because of their

pivotal role in these events, phosphoinositide metabolism is

a logical target of intracellular bacterial pathogens (Weber

et al., 2009). The IpgD effector of the intracellular bacterium

Shigella is a PiPase that dephosphorylates phosphatidylino-

sitol-4,5-biphosphate to generate the novel lipid phosphati-

dylinositol 5-monophosphate (Niebuhr et al., 2002).

Salmonella SopB/SigD is another effector PiPase, which, like

IpgD, perturbs the actin cytoskeleton and causes the deple-

tion of phosphatidylinositol-4,5-biphosphate (Terebiznik

et al., 2002). Both IpgD and SopB possess two domains in

their C-terminal region that are conserved in mammalian

PiPases (Norris et al., 1998) bearing the consensus sequences

FN(F/V)G(V/I)NE (motif 1) and CKSxKDRTxM (motif 2)

(see Fig. 2). The conserved cysteine residue in motif 2, which

is essential for mammalian inositol 4-phosphatase activity, is

also required for the phosphatase activity of SopB and SopB

activation of Akt (Steele-Mortimer et al., 2000). A similar

finding was obtained when the arginine residue of motif 2

was substituted for an alanine, revealing a critical role for

both motifs (Aleman et al., 2005). In addition, SopB also has

a C-terminal domain with close homology to the mamma-

lian inositol 5-phosphatase protein synaptojanin and this

domain has been shown to play a role in the full phosphatase

activity of SopB (Marcus et al., 2001).

Effector kinases

Effectors that have been reported to possess kinase activity

include Salmonella SteC, Yersinia YpkA and Shigella OspG.

Like eukaryotic kinases, these effectors contain a series of

well-conserved kinase subdomains bearing invariant motifs

(see Hanks et al., 1988). SteC for example, which induces F-

actin meshwork formation, has the nucleotide positioning

motif GxGxxG of kinase subdomain I that has a crucial role

in ATP binding (Hanks et al., 1988), an invariant lysine of

kinase subdomain II that anchors ATP and an invariant

glutamic acid residue in subdomain III that stabilizes the

lysine–ATP interaction (Poh et al., 2008). Despite SteC

containing these subdomains and exhibiting kinase activity

in vitro (Poh et al., 2008), it lacks the central catalytic core of

many kinases (subdomains VI–IX). Other effector kinases,

including YpkA (discussed below) and OspG (Kim et al.,

2005a), possess the invariant Glu and Lys in subdomains II

and III, while the GxGxxG motif of subdomain I is only

partially conserved in these effectors. OspG belongs to a

larger family of effectors that includes E. coli NleH1/2 (Tobe

et al., 2006) and Yersinia YspK (Matsumoto & Young, 2006)

that all have the GxGxxG motif and are predicted kinases

(Poh et al., 2008). SteC, on the other hand, is not part of the

OspG family, but instead shows greater similarity to the

eukaryotic kinase Raf-1, which, like SteC, is known to

modulate the actin cytoskeleton (Poh et al., 2008).

Tyrosine phosphorylated effector proteins

A number of effectors, such as Tir and Tarp, are themselves

substrates for host kinases (Backert & Selbach, 2005), often

evidenced by shift changes in their apparent molecular

weight (Kenny et al., 1997; Anderson et al., 2006). The

phosphorylation of E. coli Tir on tyrosine 474 is essential for

the interaction of Tir with the host protein Nck, leading to

actin polymerization (Gruenheid et al., 2001). Interestingly,

despite no significant homology between them, the type

three effectors Tarp and Tir, and the type four effectors CagA

and BepD, share a remarkably similar tyrosine phosphoryla-

tion (pY) motif with the consensus ExLYxx(I/V) – which

suggests that they are Src kinases substrates (Backert &

Selbach, 2005). The C-terminal residues upstream of the

pY motif are more divergent between Tarp, Tir, CagA and

BepD, and this likely contributes to their different substrate

specificities as this region is known to mediate substrate–ki-

nase interaction (Backert & Selbach, 2005). It should be

noted that bacterial effectors may also be phosphorylated on

nontyrosine residues (Anderson et al., 2006) as with the

serine phosphorylation of P. syringae AvrPto – a step that is

required for the virulence function of this effector (Ander-

son et al., 2006).

Effector lipases

Lipases are a large group of functionally diverse molecules

that include several bacterial effector proteins. The Salmo-

nella effector SseJ, a member of the Salmonella STE effector

family that shares similar N-terminal translocation domains

(Miao et al., 1999), has a C-terminus that is 29% identical to

several members of the GDSL lipase family (Akoh et al.,

2004). These lipases are characterized by a GDSL motif

(Akoh et al., 2004) and a downstream catalytic triad of SDH

residues that are essential for lipolytic activity (Brumlik &

Buckley, 1996). SseJ bears both these features, with the

catalytic triad spread across the C-terminal region at posi-

tions S151, D274 and H384. The SHD triad of SseJ is

essential for lipase activity in host cells (Ohlson et al., 2005;

Lossi et al., 2008) and contributes to the function of SseJ in

Sif formation (Ruiz-Albert et al., 2002). The importance of

the lipase activity was revealed in animal studies as the SHD

triad played a significant role in SseJ contribution to

virulence (Ohlson et al., 2005).

The P. aeruginosa effector ExoU is a potent phospholipase

with cytotoxic activity and is responsible for epithelial

damage in vivo (Phillips et al., 2003; Sato et al., 2003). ExoU

shares sequence similarity to a family of widely distributed

phospholipases (Schmiel et al., 1998) that include human



cytosolic PLA2 (cPLA2) (Sato et al., 2003), which all have a

serine–aspartic acid (SD) dyad that is crucial for enzyme

activity. Like other bacterial effectors, the overall homology

of ExoU to eukaryotic proteins is weak, but there are three

well-conserved motifs in ExoU that are important for the

activity of many phospholipases (Phillips et al., 2003; Sato

et al., 2003). This includes (1) a glycine-rich N-terminal

motif that is responsible for stabilizing the tetrahedral

intermediate in cPLA2, (2) a serine hydrolase motif

GxSx(G/S) and (3) the active site Asp (D) motif Dx(G/A).

In addition to the essential requirement of the SD dyad

(Sato et al., 2003) that make up the latter two motifs

(underlined), the glycines of GxSxG were also essential for

ExoU cytotoxicity (Rabin, 2005). Notably, both effector

lipases SseJ and ExoU require host factors for their activa-

tion (Sato et al., 2003; Lossi et al., 2008), possibly represent-

ing an avoidance strategy to restrict the toxicity of these

effectors to the host cell.

Effector proteases

The YopJ effector (also called YopP) is Yersinia’s primary anti-

inflammatory effector (Matsumoto & Young, 2009) that acts

by directly binding to mitogen-activated protein kinase

(MAPK) kinases and preventing their phosphorylation. It

was originally found that the predicted secondary structure

of the YopJ family resembled the cysteine protease AVP of

adenoviruses (Orth et al., 2000), which also weakly resembled

yeast ubiquitin-like protease 1, and it was proposed that

YopJ is a ubiquitin-like protein protease. Subsequent experi-

ments revealed that an invariant catalytic triad of histidine,

glutamic acid and cysteine residues (H(x)18E(x)44C) was

essential for NFkB inhibition by YopJ (Orth et al., 2000).

Several other effectors including VopA, SseL, XopJ, AvrA and

AvrBst display variations of the catalytic triad (H-(E/N/D)-

C), which, at least for AvrA, SseL and VopA, have been shown

to be essential for activity (Trosky et al., 2004) (Collier-

Hyams et al., 2002; Rytkonen et al., 2007). Indeed, SseL

exhibits deubiquitinase activity that is dependent on the

catalytic cysteine and was also important for the virulence

properties of the effector in vivo (Rytkonen et al., 2007).

Recently, YopJ was shown to be an acetyltransferase that

acetylates the serine and threonine residues in the activation

loop of MAPKK or IKKs – preventing their activation by

phosphorylation (Mukherjee et al., 2006). Intriguingly, the

acetylation activity of YopJ depends on the cysteine protease

catalytic triad (Mukherjee et al., 2006).

Another family of bacterial effectors, defined by Yersinia

YopT and Pseudomonas AvrPphB (Shao et al., 2002), shares

an invariant Cys-His-Asp (CHD) triad found in the catalytic

core of CA clan cysteine proteases (Zhu et al., 2004). YopT is

known to target and cleave prenylated (membrane-bound)

Rho GTPases, dependent on its C-terminal cysteine protease

domain, preventing them from modulating the cytoskeleton

(Aepfelbacher et al., 2003; Shao et al., 2003). In addition to

its CHD triad, the proteolytic activity of YopT also depends

on its first hundred N-terminal residues and its last eight C-

terminal residues. Neither of these regions contains the

catalytic triad, but residues 75–100 of YopT were found to

be important in binding to RhoA (Sorg et al., 2003), while

the C-terminal domain was proposed to play a direct role in

protease activity (Sorg et al., 2003). Although the CHD

catalytic residues are invariant between YopT and AvrPphB,

the substrate-binding sites are highly divergent and account

for the different substrate specificities (Zhu et al., 2004).

Other homologues of YopT include the LopT effector from

the insect pathogen Photorhabdus, which is known to inhibit

phagocytosis (Brugirard-Ricaud et al., 2005). Like YopT,

LopT maintains the invariant CHD residues and releases

RhoA and Rac from the host plasma membrane (Brugirard-

Ricaud et al., 2005).

AvrPphB is an interesting effector protease, which, like

YopT, contains the catalytic CHD triad, but, when delivered

into host cells, is processed to reveal a novel N-terminus

(Shao et al., 2002). This exposes embedded Myr and Palm

motifs that play an important role in the function of this

effector (described above). AvrPphB undergoes autoproteo-

lysis between residues K62 and G63, converting it from a 35-

to a 28-kDa protein, dependent on each residue of the CHD

triad (Shao et al., 2002; Dowen et al., 2009). A protease-

inactive mutant of the processed form of AvrPphB (28 kDa)

was unable to induce a plant hypersensitive response,

demonstrating the importance of protease activity for this

effector (Shao et al., 2002). Interestingly, the autoproteolytic

site (GDK-G) of AvrPphB is very similar to the processing site

(GDK-S) of its substrate the plant serine threonine kinase

PBS1 (Shao et al., 2002). The crystal structure of AvrPphB

(Zhu et al., 2004) shows that it resembles the papain-like

superfamily of cysteine proteases and as all YopT family

members have similar secondary structures, it is likely that

they also adopt a similar papain-like tertiary conformation

and this is supported by inhibitor studies (Zhu et al., 2004).

Finally, the plant pathogen effector, AvrRpt2 from P.

syringae, shares structural similarity to staphopain, a CA

clan cysteine protease from Staphylococcus (Axtell et al.,

2003). AvrRpt2 is known to target the plant host protein

RIN4 and cause its elimination (Kim et al., 2005b),

dependent on the CA clan catalytic triad of CHD as

described above (Axtell et al., 2003). AvrRpt2 requires

activation by host cyclophilin (Coaker et al., 2005) and

undergoes autocleavage at a VPAFGGW motif that is similar

to the two cleavage sites in RIN4 (VPKFGNW and

VPKFGDW) (Chisholm et al., 2005; Takemoto & Jones,

2005). Like AvrPphB, mutation of the catalytical triad

residues abolished the proteolytic activity of AvrRpt2 (Axtell

et al., 2003).


12 P. Dean

Effector lyases

Lyases are a diverse group of enzymes that are able to cleave

chemical bonds by mechanisms other than hydrolysis or

oxidation and have a wide range of substrates. It was

originally reported that the Shigella effector OspF removed

a phosphate group from phosphothreonine in MAPK,

suggesting that it was acting as a threonine-specific MAPK

phosphatase (Li et al., 2007). However, Li et al. (2007)

revealed that OspF (and the effectors SpvC and HopAI1)

functions as a phosphothreonine lyase by mediating the

irreversible removal of phosphate from MAPK (Li et al.,

2007), thus representing a novel type of lyase. OspF shares

considerable regional homology with SpvC (and HopAI1)

including a critical GDKxH motif in the central region of the

proteins that is required for phosphothreonine lyase activity

(Li et al., 2007). In addition, the N-termini of both SpvC

and OspF possess a short canonical D motif found in many

MAPK substrates that is required for MAPK docking (Zhu

et al., 2007) and is the likely mechanism for MAPK binding

by these effectors.

Effector adenylate cyclases

Cyclic AMP is an important secondary messenger in eukar-

yotic cells, involved in the regulation of several cellular

processes. Numerous pathogens disrupt the levels of cAMP

inside host cells via the deployment of toxins that possess

adenylate cyclase activity (Ahuja et al., 2004). ExoY is an

effector produced by some strains of P. aeruginosa that

shares homology to the calmodulin-activated adenylate

cyclase toxins CyaA (Bordetella) and EF (Bacillus). Although

ExoY possesses several regions conserved in these toxins,

including an ATP/GTP-binding site (Yahr et al., 1998), it

does not possess the binding site for calmodulin (Yahr et al.,

1998). The ATP-binding site of ExoY is essential for its in

vitro adenylate cyclase activity and its ability to cause cell

rounding in vivo (Yahr et al., 1998). The absence of the

calmodulin-binding site suggests that unlike other bacterial

adenylate cyclases, ExoY is not activated by calmodulin and

this was indeed the case, although ExoY still requires a host

cytosolic factor for activation (Yahr et al., 1998).

ADP-ribosyltransferases (ADPRTs)

ADP-ribosylation, the transfer of ADP-ribose to a target

protein, is used by several bacterial exotoxins and type three

effector proteins. HopU1, an effector from P. syringae, is a

mono-ADPRT that suppresses plant immunity dependent

on ADP-ribosylation of host RNA-binding proteins (Fu

et al., 2007). Like several bacterial ADPRTs (including the

toxins VIP2, LT and CT and the effectors ExoS, ExoT and

SpvB), HopU1 has three distinct regions with an invariant

arginine in region one, a Gx9ST(S/T) motif in region two

and an ExE biglutamic acid motif in region three (Fu et al.,

2007) that is typical of arginine-specific mono-ADPRTs

(Domenighini et al., 1994). The SpvB effector ADP-ribosy-

lates actin in infected cells, preventing the conversion of

G- into F-actin (Tezcan-Merdol et al., 2001). Importantly,

this ADPRT activity of SpvB plays a significant role in

disease as mutagenesis of the biglutamic motif attenuated

Salmonella virulence in mice (Lesnick et al., 2001).

The best-studied ADPRT effectors are the P. aeruginosa

homologues ExoT and ExoS. Both proteins are bifunctional

due to an N-terminal RhoGAP domain (described below)

and a C-terminal ADPRT domain that also bears the critical

biglutamic ExE motif described above (Garrity-Ryan et al.,

2004). Alanine substitutions revealed that the first glutamic

acid in the ExE motif is the active site residue, while the

second one mediates the transfer of ADP-ribose to the

targeted protein (Garrity-Ryan et al., 2004). ExoT ADP-

ribosylates the SH2 domain of host cytosolic Crk proteins,

which are involved in focal adhesion and phagocytosis (Sun

& Barbieri, 2003), while ExoS ADP-ribosylates multiple

eukaryotic Ras family members such as RhoA, Rac1 and

Cdc42 (Henriksson et al., 2002b). ExoS was also shown to

depend on the host 14-3-3 protein FAS (factor activating

ExoS), which bound ExoS through its extreme C-terminal

14-3-3-binding site (Fu et al., 1993), and the importance of

ExoS activation was demonstrated by removing the last 27

residues of ExoS, which abolished its cytotoxicity (Henriks-

son et al., 2002a). Interestingly, the arginine finger residue

(R146) of the N-terminal GAP domain of ExoS is itself

ADP-ribosylated by the C-terminal ADPRT domain, result-

ing in the inhibition of GAP activity during infection (Riese

et al., 2002). This autoregulatory mechanism is an excellent

example of how effector domains have coevolved to control

effector functions within the host cell. The beauty of self-

regulation by ExoS is even more impressive when one

considers that the ADPRT activity is itself autoregulated by

the 14-3-3 domain (Fu et al., 1993) – providing a multitiered

level of regulation for this effector. Finally, ExoS is an

important virulence factor for P. aeruginosa and the viru-

lence properties of ExoS have been assessed by mutational

analysis (Shaver & Hauser, 2004), revealing an essential role

for the ADPRT domain with a much lesser role in vivo for

GAP activity.

AMPylation -- a novel effector function

One of the benefits of understanding the molecular mechan-

isms of bacterial effector proteins is that they sometimes

reveal themselves as highly novel proteins, mediating pre-

viously unseen biochemical properties. VopS of Vibrio

parahaemolyticus is one such effector as it was shown to

inactivate host Rho, Rac and Cdc42 by the covalent transfer

of AMP to a conserved threonine residue on these Rho



GTPases (Yarbrough et al., 2009). The region of VopS that

was responsible for AMPylation was a C-terminal region

that contained the Fic domain that is widespread in nature.

The Fic domain contains an invariant histidine (H) residue

within a conserved motif – HxFx(D/E)GNGR – that was

essential for AMPylation by VopS (Yarbrough et al., 2009).

Interestingly, a domain with a structure similar to the Fic

domain has also been identified in the P. syringae effector

AvrB (Kinch et al., 2009); hence, it is likely that AMPylation

may be utilized by multiple bacterial effectors.

G protein regulatory domains

Eukaryotic G proteins bind GTP and cycle between an

inactive GDP-bound and an active GTP-bound conforma-

tion (Bos et al., 2007). This process is tightly regulated in the

host cell by guanine nucleotide exchange factors (GEFs) that

activate the G protein, and GAPs that inactivate them (Bos

et al., 2007). Several effectors act as GEFs (e.g. SopE, Map

and EspM) or GAPs (e.g. AexT, ExoS, ExoT, YopE and SptP)

that particularly target G proteins mainly involved in

cytoskeletal regulation.

Effector GAPs

Several well-studied effectors disrupt the actin cytoskeleton

via RhoGAP activity (see Fig. 2), including AexT (Fehr et al.,

2007), ExoS (Fu & Galan, 1999), ExoT, YopE (Von Pawel-

Rammingen et al., 2000) and SptP (Fu & Galan, 1999).

Eukaryotic Rho GTPase GAP proteins possess a catalytic

arginine carried on an arginine finger that bears the con-

sensus motif GxxRxSG (Juris et al., 2002). The bacterial

effector GAPs also possess this critical arginine finger motif

with the consensus sequence GxLRx3T (Wurtele et al.,

2001), for example SptP (GPLRSLMT), ExoS (GALRSLAT),

YopE (GPLRGSIT) and AexT (GPLRSLCT). Mutation of the

catalytic arginine in this motif abolishes GAP activity for

each effector (Fu & Galan, 1999; Black & Bliska, 2000; Von

Pawel-Rammingen et al., 2000; Fehr et al., 2007) and was

shown to be important in disease as Yersinia carrying a YopE

GAP mutant (R144A) was avirulent in mice (Black & Bliska,

2000). Although the arginine finger motif is typical of

eukaryotic GAP proteins (Gamblin & Smerdon, 1998), the

bacterial GAPs – ExoS, ExoT, YopE and SptP – all possess a

second conserved region comprising a loop �40 residues

downstream of the arginine finger. This conserved loop

contains the invariant sequence QWGTxGG, which is be-

lieved to facilitate hydrogen bond formation between the

hydroxyl group of the nucleotide ribose and the effector

(Wurtele et al., 2001). Structural elucidation of the GAP

domains of SptP, ExoS and YopE reveals that despite

diverging at a rapid rate, they share a strikingly high

structural similarity (Evdokimov et al., 2002), implying that

they have a similar molecular mechanism. This study by

Evdokimov et al. (2002) also showed that with the exception

of the arginine finger, bacterial GAPs bear little resemblance

to their eukaryotic counterparts, suggesting that they arose

by convergent evolution.

The E. coli Tir effector, while inducing actin polymeriza-

tion through the Nck/Arp2/3 complex, also possesses GAP

activity to downregulate filopodia formation induced by

another E. coli effector, Map (Kenny et al., 2002). Like the

bacterial GAPs described above, Tir possesses an arginine

finger motif, containing the GxLR sequence in its C-

terminal region, which was shown to be essential for the

downregulation of Map-induced actin polymerization

(Kenny et al., 2002). However, unlike the bacterial GAPs,

Tir does not possess the second downstream signature motif

described above.

Effector GEFs

The Salmonella effector SopE is a potent GEF protein that

directly binds to the Rho GTPases Cdc42 and Rac1 to

stimulate GDP/GTP nucleotide exchange (Hardt et al.,

1998; Rudolph et al., 1999). SopE is a new type of RhoGEF

(Rudolph et al., 1999) as it lacks sequence similarity to

known GEF proteins and does not contain the signature DH

(Dbl homology) or PH (pleckstrin homology) motifs that

are required for catalysis by eukaryotic RhoGEFs (Rossman

et al., 2005). Instead, SopE and its effector homologues

SopE2 and BopE (Burkholderia) (Upadhyay et al., 2004),

which are all confirmed GEFs, share a unique GAGA motif

on a catalytic loop that inserts itself between the switch I/II

regions of Cdc42 (Schlumberger et al., 2003). Strikingly, the

SopE GEF activity is counteracted within the host cell by the

GAP activity of the Salmonella effector SptP, providing a

fascinating example of the intermolecular crosstalk between

these effector domains (Fu & Galan, 1999).

G protein mimics or bacterial GEFs? -- theWxxxE family

A family of type three effectors that includes EspM, EspT,

Map, IpgB1, IpgB2, SifA and SifB were initially believed to

be G protein mimics dependent on a WxxxE motif (Alto

et al., 2006). However, subsequent work on the crystal

structures of Map and SifA revealed a different story –

implicating these effectors as GEFs that structurally mimic

the Salmonella effector SopE, despite sharing no similarity at

the sequence level (Ohlson et al., 2008; Huang et al., 2009).

In support of this, molecular modelling and nuclear mag-

netic resonance revealed that EspM2 adopts a SopE-like

conformation (Arbeloa et al., 2009). In addition, the ability

of Map to induce actin polymerization was dependent on

Cdc42 (Kenny et al., 2002) and Map also binds directly to

nucleotide-free Cdc42 to promote GTP incorporation


14 P. Dean

(Huang et al., 2009), strongly suggesting that Map is a

RhoGEF protein.

The crystal structure of Map in complex with Cdc42

reveals that Map is composed of two bundles of a-helices,

forming a V-shaped structure that presents a long catalytic

loop (Huang et al., 2009) containing the general consensus

sequence (D/P/E)x3AQ. Substitution of the conserved ala-

nine or glutamine residues (underlined) abolishes GEF

activity (Huang et al., 2009). The essentiality of the WxxxE

motif that defines this effector family appears to be a

structural one, as this motif was found at the interface of

the a-helical bundles, and may thus be essential in main-

taining the conformation of the catalytic loop (Huang et al.,

2009). Interestingly, despite Map and SopE being unrelated

at the sequence levels, these two bacterial GEFs have adopted

a conserved functional mechanism for nucleotide exchange

and also induce similar conformation changes in Cdc42

(Huang et al., 2009).

Several plant pathogen effectors, including most of the

AvrE effector family, have recently been reported to contain

at least one WxxxE motif (Ham et al., 2009). Indeed, WtsE

from Pantoea stewartii and the AvrE1 effector of P. syringae

both contain the WxxxE motif, which has been shown

to play a central role for the virulence functions of

these effectors (Ham et al., 2009). Thus, the WxxxE motif,

whether playing a structural role or not, is clearly an

important feature of the WxxxE effector family.

Microtubule-disrupting effectors

Effectors that interfere with microtubules include EseG (Xie

et al., 2010), SseG, SseF (Kuhle et al., 2004), EspG (Matsu-

zawa et al., 2004) and VirA (Yoshida et al., 2002). With the

exception of VirA and EspG, little progress has been made

regarding the molecular mechanisms of microtubule dis-

ruption by these effectors. The Shigella effector VirA binds

tubulin subunits directly through a �90 residue region in

the centre of the effector (residues 224–315) (Yoshida et al.,

2002), resulting in the destabilization of host microtubules

(Yoshida et al., 2002). The secondary structure of VirA

resembles CA clan cysteine proteases, and Yoshida et al.

(2006) showed that degradation of tubulin monomers by

VirA is dependent on a critical Cys-34 residue (Yoshida

et al., 2006). However, more recent structural data suggest

that VirA and its E. coli homologue EspG has no protease

active site, but conversely resembles cysteine protease in-

hibitors, suggesting that VirA may not have direct proteoly-

tic activity (Davis et al., 2008) (Germane et al., 2008). Also,

the N-terminal region of VirA that contains the proposed

catalytic Cys34 residue (Yoshida et al., 2006) was found to be

disordered, like the N-termini of many effectors, making it

unlikely to be the active site (Davis et al., 2008). The fact that

the VirA structure resembled cysteine protease inhibitors

raises the possibility that it may bind to cysteine proteases

and, indeed, recent work on the VirA homologue EspG

suggests that it can induce the activity of host cysteine

proteases (Dean et al., 2010a). These studies on VirA clearly

show that although the primary and secondary sequences of

an effector may display clues on its identity, structural

studies may provide a very different or even a contrary

elucidation.

Actin nucleation motifs

WH2 motifs

With the exception of eukaryotic formins, all actin nuclea-

tion proteins interact with actin via their WH2 (WASP

homology 2) domains (Dominguez, 2009). WH2 domains

are short (17–22 residues) regions nearly always found in

tandem and forming an N-terminal helix with a conserved

downstream LKKV motif (Dominguez, 2009). VopL of

Vibrio contains three closely spaced WH2 domains (Liver-

man et al., 2007) bearing the LKKV-like residues. It induces

actin stress fibre formation by directly nucleating actin

filaments in vitro, dependent on its three WH2 domains

(Liverman et al., 2007). Indeed, mutation of the WH2

domains considerably reduces the actin nucleation activity

of VopL, although a single WH2 domain is sufficient to

induce this activity (Liverman et al., 2007). The WH2

domains of VopL have high sequence similarity to the

WH2 domains of actin-binding proteins WASP, WAVE,

Drosophila Spire and the bacterial effectors, Tarp and VopF,

which all maintain the LKKV-like motif that is essential for

actin binding (Liverman et al., 2007).

Of the WH2-containing actin nucleation proteins, the

WH2 domain of Chlamydia effector Tarp is the most poorly

conserved and it does not encode the LKKV motif, but a more

divergent LDDV motif. Tarp binds and nucleates actin directly

via a C-terminal actin-binding domain that contains a single

WH2 motif that is sufficient for actin nucleation (Jewett et al.,

2006). This is unlike all other WH2-containing actin nuclea-

tion proteins, which require multiple WH2 motifs, although

the finding that Tarp oligomerizes to nucleate actin possibly

explains this deviation (Jewett et al., 2006).

FH1 domains

The Vibrio cholerae effector VopF, like VopL, is able to

nucleate actin directly in vitro in the absence of other cellular

components (Tam et al., 2007). VopF contains three WH2

domains (discussed above), but also contains a formin

homology 1 (FH1) domain, which is typically found in

formin family proteins to interact with and nucleate actin

(Dominguez, 2009). FH1 domains are highly variable in

size, ranging from 15 to 229 residues, and have a high

proline content. The VopF FH1 domain is strikingly similar



along its length to the FH1 domain of mammalian formins

(Tam et al., 2007) and, while most formins possess both FH1

and FH2 domains, VopF lacks the FH2 domain. It is likely

that the FH2 domain of VopF has been substituted by its

WH2 domains, and indeed, both types of domains are

required for efficient VopF-mediated actin nucleation (Tam

et al., 2007).

Other actin modulatory domains

IpaA is an important Shigella effector that recruits the focal

adhesion protein vinculin, an interaction that is believed to

promote cell invasion (Tran Van Nhieu et al., 1997). It has

been shown that upon binding vinculin, IpaA increases the

association of vinculin with F-actin, leading to F-actin

depolymerization (Bourdet-Sicard et al., 1999), although

this is controversial (Demali et al., 2006). The vinculin-

binding site of IpaA was mapped to its last 74 residues

(Ramarao et al., 2007), which contains at least two vinculin-

binding motifs (Hamiaux et al., 2006). These motifs bind to

the N-terminal VD1 domain of vinculin, inducing a con-

formational change that promotes vinculin binding to F-

actin. The IpaA–VD1 interaction is very similar to that

between VD1 and the host proteins talin and a-actinin,

suggesting that IpaA structurally mimics these eukaryotic

proteins (Hamiaux et al., 2006). Notably, while the

C-terminal region of IpaA shares partial similarity to its

Salmonella homologue SipA, SipA does not bind vinculin

(Zhou et al., 1999), suggesting no mechanistic relatedness

between the two domains (Zhou et al., 1999). Indeed, SipA

has a very different function, preventing actin depolymer-

ization by binding actin directly through its C-terminal

domain.

Other effectors with proven actin modulatory domains

include SipC, a Salmonella translocator protein that nucle-

ates and bundles actin through its C- and N-terminal

domains, respectively (Hayward & Koronakis, 1999). This

dual functionality of SipC is a clear example of functional

complementation between effector domains (Hayward &

Koronakis, 1999). Interestingly, the SipC homologue

IpaC from Shigella possesses the C-terminal actin nuclea-

tion domain, but lacks the N-terminal bundling domain,

which may explain why Shigella requires the host

actin-bundling protein T-fimbrin for efficient uptake

(Adam et al., 1995).

Finally, the enteropathogenic E. coli effector protein Tir

induces actin polymerization following insertion into the

host plasma membrane (Kenny et al., 1997). This activity is

dependent on Tir binding the eukaryotic adapter protein

Nck through a C-terminal Nck-binding site (Gruenheid

et al., 2001). The Nck-binding site of Tir bears a conserved

YDEV motif, with phosphorylation of the tyrosine essential

for Nck binding (Kenny et al., 1997). Nck itself possesses a

single SH2 domain and three src homology 3 (SH3)

domains, with the former binding to the YDEV Tir motif,

while the latter binds to and recruits proteins involved in

actin polymerization. Intriguingly, the Nck-binding

site of Tir is homologous to that of the vaccinia virus protein

A36R that is also tyrosine phosphorylated and is known to

recruit Nck (Rottger et al., 1999), implying clear functional

similarities between these unrelated proteins. In contrast,

the Tir homologue in enterohaemorrhagic E. coli does not

possess the Nck-binding site, but instead recruits its own

type three effector called Tccp, which substitutes for the Nck

protein (Garmendia et al., 2004). The recruitment of Tccp

by Tir requires a C-terminal NPY sequence that was

found to mediate binding to the host protein IRTKS (insulin

receptor tyrosine kinase substrate), which then acts as

an adapter between the two effectors (Vingadassalom et al.,

2009).

Protein--protein interaction elements

The J domain

J domains are highly conserved modules of about 70 amino

acids that are found in Hsp40 and Hsp40-like proteins,

including the E. coli chaperone DnaJ (Hennessy et al., 2005).

They mediate the interaction of Hsp40-like proteins with

their chaperone Hsp70 partner and regulate Hsp70 function

(Hennessy et al., 2005). All J domains contain four a-helices

with a loop, exposed between helices II and III, encoding a

highly conserved HPD motif that is essential for Hsp70

interaction (Kelley, 1998; Hennessy et al., 2005). The P.

syringae effector HopI1 contains a C-terminal region with

homology to the J domain, containing the invariant HPD

motif (Jelenska et al., 2007). HopI1 localizes to the plant

chloroplasts, where it suppresses salicylic acid synthesis and

disrupts the thylakoid stack structure (Jelenska et al., 2007).

These functions are dependent on a functional J-domain as

mutation of the HPD motif abolished HopI1 activity

(Jelenska et al., 2007). Recently, HopI1 was shown to

interact directly with plant Hsp70 proteins via its J domain

and cause an increase in Hsp70 abundance and its targeting

to the chloroplast (Jelenska et al., 2010). Although Hsp70

was found to be required for HopI1 virulence function, the

reason it targets Hsp70 has not yet been determined

(Jelenska et al., 2010).

SH3-binding motifs

The E. coli effector EspF has been shown to recruit and bind

the host protein sorting nexin 9 (SNX9), leading to mem-

brane remodelling (Alto et al., 2007). Phage display revealed

that EspF binds to the SH3 domain of SNX9 via a repeated

motif of RxAPxxP in the C-terminal proline-rich repeat

region of EspF (Fig. 2). This repeating motif contains


16 P. Dean

the canonical PxxP sequence that is known to bind the

SH3 domains of eukaryotic proteins (Alto et al., 2007).

Interestingly, SH3 motifs are found in numerous effectors,

but there are very few documented examples of a functional

role for these motifs, the exception being EspF and the E. coli

effector Tccp (Aitio et al., 2010).

PDZ-binding motifs

PDZ (PSD-95/Disk-large/ZO-1) domains are widespread

protein–protein recognition modules (80–90 residues)

found in proteins mainly from multicellular organisms

(Hung & Sheng, 2002). They are important in mediating

multiprotein complexes at specific subcellular locations

within eukaryotic cells. The PDZ domain typically binds to

proteins that contain an extreme C-terminal PDZ-binding

motif that conforms to the consensus (S/T)x(I/V/L). Several

bacterial effectors bear a C-terminal PDZ-binding motif

including the E. coli effectors NleH1 (-SKI), Map (-TRL)

and NleA (-TRV). All three of these motifs have been shown

to be functionally important, acting as cell-sorting signals

and mediating the interaction with the host PDZ-containing

protein NHERF2 (Martinez et al., 2010). Indeed, the PDZ-

binding motif of Map allows it to target the host apical

plasma membrane while that of NleA mediates trafficking to

the Golgi apparatus (Lee et al., 2008; Martinez et al., 2010).

This cell sorting function of the PDZ domain was found to

be essential for the GEF function of Map (described above)

and follows a trend similar to that found in human

RhoGEFs, as over 35% possess a C-terminal PDZ-binding

motif (Garcia-Mata & Burridge, 2007). Recently, the PDZ-

binding motif of NleA was shown to be essential for its

ability to disrupt intestinal barrier function (Thanabalasur-

iar et al., 2010) and was also shown to play a role in virulence

(Lee et al., 2008).

Caspase 3 processing sites

Recently, McCormick and colleagues revealed that the

Salmonella typhimurium effector SipA possesses a functional

caspase-3 cleavage site, containing a DEVD sequence, which

lies between the two major functional domains of this

effector (Srikanth et al., 2010). Intriguingly, SipA itself

induces the activity of caspase-3 within host cells infected

with S. typhimurium and mutation of its caspase-3 cleavage

site of SipA revealed an important role for this motif in vivo

(Srikanth et al., 2010). Remarkably, many other Salmonella

effectors possess a centrally located caspase-3 cleavage site,

including AvrA, SopB, SifA, SipB and SopA, which, for the

latter, has been shown to be functionally important (Sri-

kanth et al., 2010). Thus, caspase-3 cleavage sites may

represent a common mechanism to regulate effector func-

tion in host cells.

YpkA -- a shining example ofeffector modularity

The Yersinia effector YpkA (also called YopO) was purposely

excluded from the sections in this review to provide a fitting

epilogue of type three effector modularity, although many

of the effectors described in this review would have been as

suitable. YpkA is an essential Yersinia virulence factor

(Galyov et al., 1993) that disrupts the cytoskeleton and

inhibits phagocytosis (Wiley et al., 2006). It was originally

characterized as a Ser/Thr kinase (Galyov et al., 1993), but

subsequent work has shown that it is composed of several

important functional domains along its length (Fig. 2),

responsible for (1) secretion and chaperone binding, (2)

plasma membrane targeting, (3) kinase activity, (4) RhoGDI

mimicking and (5) actin binding.

YpkA targets the cytoplasmic face of the host plasma

membrane (Hakansson et al., 1996) via an N-terminal-

targeting domain (residues 20–90) that is essential for the

morphological changes induced by this effector (Letzelter

et al., 2006). In the same study, the chaperone-binding site

(residues 20–77) of YpkA, which directly correlates with the

MLD, played no role in bacterial secretion, but was pro-

posed to mask the MLD within the bacteria (Letzelter et al.,

2006). Downstream of the MLD is a large kinase domain

(residues 150–400; Fig. 2) with homology to eukaryotic Ser/

Thr kinases and in vitro kinase assays have revealed that

YpkA autophosphorylates itself (Galyov et al., 1993) with

mutation of two critical residues Asp287 and Lys269 abol-

ishing its kinase activity (Juris et al., 2000). The specific

kinase activity of YpkA is important for Yersinia disease

(Wiley et al., 2006) and plays an intermediate role in

cytoskeletal disruption within infected host cells (Juris

et al., 2000). YpkA is also known to phosphorylate actin

(Juris et al., 2000) and the heterotrimeric G protein Gaq

(Navarro et al., 2007), although why YpkA targets Gaq is

currently unknown (Navarro et al., 2007).

YpkA is synthesized as an inactive kinase, unable to

autophosphorylate itself unless specifically activated by

monomeric G-, but not F-actin inside the host cell (Trasak

et al., 2007), providing an effective mechanism to prevent

kinase activity within the bacterium itself. The actin-binding

domain of YpkA is found at its extreme C-terminus (Fig. 2)

and is critical for the activity of the effector as the removal of

the last 20 amino acids abolished YpkA kinase activity

(Trasak et al., 2007).

The finding that a kinase-deficient YpkA mutant was still

able to cause morphological changes in the host cell, similar

to the wild-type protein (Dukuzumuremyi et al., 2000), led

to the discovery of a second subversive domain of YpkA, in

its C-terminal region. This region was found to directly bind

the small GTPases RhoA and Rac, but not Cdc42 (Barz et al.,

2000; Dukuzumuremyi et al., 2000). The crystal structure of



the C-terminal domain of YpkA in complex with Rac1

finally revealed that this domain of YpkA mimics guanidine

nucleotide dissociation inhibitors (GDI) that bind to and

lock Rac and RhoA in a GDP-bound off-state (Prehna et al.,

2006). Mutations that interfered with GTPase binding

completely abolished cytoskeletal disruption by YpkA in

infected host cells, demonstrating the importance of this

activity during infection (Prehna et al., 2006). Taken

together, YpkA is a good example of the multidomain

structure of bacterial effector proteins: possessing two

prominent, but very different, functional domains that

mimic eukaryotic proteins and supported by a smaller cast

of essential regulatory or accessory domains.

Conclusion

The ability of bacterial effectors to take control of their host

cell is truly remarkable. These sophisticated molecules are

responsible for some of our most devastating diseases, and

yet depend on a relatively small set of domains and motifs to

do so. As more effectors are uncovered and characterized,

this family of molecular determinants will continue to

enhance our understanding of host–pathogen interactions.

I hope this review has provided a glimpse into the ingenuity

of bacterial pathogens in evolving such a diverse and

fascinating group of virulence proteins.

Acknowledgements

I am grateful to Profs Brendan Kenny and Martin Embley,

Drs David Bulmer, Anjam Khan, Sabrina Muelen and Tom

Williams for assistance with this review. I would also like to

thank Mrs Philippa Breakspear for collating and organiz-

ing all the reading material used in this review. P.D. is

supported by a Medical School Faculty Fellowship, Univer-

sity of Newcastle.

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26 P. Dean

effector review

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