REVIEW ARTICLE
Functional domains and motifs of bacterial type III e¡ector proteins
and their roles in infection
Paul 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: p.dean@ncl.ac.uk
Received 13 February 2011; revised 5 April
2011; accepted 7 April 2011.
DOI:10.1111/j.1574-6976.2011.00271.x
Editor: Miguel Camara
MICROBIOLOGY REVIEWS
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, Salmonella, Shigella, Yersinia, Pseudomonas, Xanthomonas, Ralstonia 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 (Coburn 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
FEMS Microbiol Rev ]] (2011) 1–26
in host cell physiology compared with the more overt effects
of bacterial exotoxins. The effector protein family is evolutionarily 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.
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P. Dean
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
Xanthamonas spp.
Wide range of plant diseases, e.g. rice
bacterial blight and citrus canker
Ralstonia solancearum
Plant wilt on many host species
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)
AvrBs1, AvrBs2, AvrBs3, AvrRxo1, AvrRxv, AvrXccC, AvrXv3, Ecf,
HpaA, XopJ, XopX, XopB, XopC, XopD, XopE, XopF, XopN,
XopO, XopP, XopQ
GALA1-7, SKWP1-6, HLK1-3, RipB, PopW, PopP, PopC, RipT,
AvrA, PopB, PopA, RipA (many others predicted)
DspE, HrpN, HrpW, HopPtoC, AvrRpt2, EopB
Erwinia amylovora
Animal
host
Causes fire blight on a range of plant
species
Rhizobium spp.
Symbiont; forms nodules on legumes
Pantoea spp.
Bacterial wilt on corn and maize
Pseudomonas aeruginosa
Opportunist pathogenic. Can cause
pneumonia
Escherichia coli (EPEC and
Diarrhoea (EPEC) or haemorrhagic colitis
EHEC), Cirobacter rodentium (EHEC). Cattle commensal (EHEC)
Salmonella enterica serovars
Gastroenteritis; typhoid fever
Shigella spp.
Bacillary dysentery; shigellosis
Yersinia spp.
Bubonic plague (pestis); Gastrointestinal
disease (enterocolitica)
Yersinia enterolytica biovar 1B Severe gastrointestinal disease
Photorhabdus spp.
Opportunistic pathogen (asymbiotica);
insect pathogen (luminescens)
Chlamydia spp.
Obligate intracellular parasites, sexually
transmitted disease; can cause blindness
Burkholderia spp.
Melioidosis (B. pseudomallei); glanders
(B. mallei)
Vibrio spp.
Gastroenteritis, wound infections
(parahaemolyticus); secretory diarrhoea
(cholerae)
Bordetella spp.
Whooping cough
Aeromonas spp.
Opportunistic pathogen; fish/humans
NopL, NopP, NopJ, NopM, NopT, NopB, NopN
WtsE, PthG, HsvG, HsvB
ExoU, ExoY, ExoS, ExoT
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
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
IpaA, IpaB, IpaC, IpaD, IpaJ, IpgD, IpgB, IcsB, OspC, OspD, OspZ,
OspB, OspF, VirA, OspE, OspG, IpaH family
YopE, YopH, YopP/J, YopE, YopM, YopT, YpkA/YopO
YspA, YspL, YspP, YspF, YspE, YspI, YspK, YspM
LopT
CADD, CT847, tarp, IncA, IncG, CT229, CT813, Cpn0585,
Cpn0909, Cpn1020
CHBP, BopE
VopL, VopA, VPA450, VopT, VopF, VopS, VopQ
BopC/BteA, BopN
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.
Although there are many excellent publications on the functions 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 in
bacterial 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 with2011 Federation of European Microbiological Societies
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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
FEMS Microbiol Rev ]] (2011) 1–26
3
Functional domains and motifs of bacterial effector proteins
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.
domains (Fig. 2) that confer a subversive function. Strikingly, the individual modules within an effector often
mediate very different, unrelated functions (see Fig. 2),
strongly suggesting that they evolved independent of each
FEMS Microbiol Rev ]] (2011) 1–26
other and subsequently combined to form the chimeric
effector (Stavrinides et al., 2006). This process of chimerization is a common theme shared by many effectors, as
exemplified by the Salmonella effector SptP, which shares a
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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., chaperonebinding domain; PRR, proline-rich repeats. Abbreviations of common motifs are given in the
text. Effectors are not drawn to scale.
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 acquisition, 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
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below), the new protein is likely to be secreted and translocated 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 homologous N-termini, but different C-terminal regions (Hansen-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, suggesting that this process is important for the evolution of these
virulence proteins (Stavrinides et al., 2006).
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5
Functional domains and motifs of bacterial effector proteins
Table 2. Proven type three effector motifs
Subcellular
targeting
Subcellular
target/activity
Effector examples
Description of responsible motifs
Intracellular membrane
PipP2, SopD2, YopE, ExoS
Nucleus (NLS)
PopB, PopP2, XopD, AvrBs3
Nucleolus
Plasma membrane
EspF
ExoU, HopZ, AvrPto, AvrPphB
Mitochondrial MTS
Map, EspF, SopA
LFNEF (PipP2), WEK(I/M)xxFF (SopD2); N-terminal Lx2Rx4L (YopE, ExoS);
CLCCFL prenylation motif (SifA)
Bipartite NLS: KRKR and KKKKKR (PopB); RRRR and RRQRQ (PopP2).
Monopartite NLS: XopD, TAL effectors such as AvrBs3, e.g. KRPR
Atypical nuclear/nucleolar targeting motif
C-terminal MLD containing an essential KAWRN motif (ExoU); PM
targeting mediated by Myr and Palm motifs (several plant pathogen
effectors)
N-terminal domain rich in arginine and leucine (Map and EspF); a critical
Leu16 (EspF); internal atypical domain (SopA)
WEK(I/M)xxFF motif alone; internal golgi targeting domain (SseG)
Large domain similar to Ser/Thr kinases with critical Asp287 and Lys269
residues
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
Catalytic loop containing the GAGA motif
Catalytic loop containing the consensus (D/P/E)x3AQ; a conserved WxxxE
structural motif
C-terminal domain with a GDSL lipase motif and a SHD catalytic triad
Large cPLA2 domain. SD catalytic dyad of – GxSx(G/S) hydrolase motif and
a Dx(G/A) active site motif. An essential GGGxxG motif
Catalytic HEC triad: HX18EX44C (YopJ); catalytic CHD triad (YopT, AvrPphB)
and N-terminal Rho-binding domain (YopT), autoproteolytic site GDKG
(AvrPphB)
Active site HECT motif – Lx4TC (SopA); CxD motif (IpaH and SspH1)
ADPRT conserved region I – invariant arginine; region II – Gx9ST(S/T);
region III – NAD-binding ExE motif
Kinase subdomain I: nucleotide positioning motif – GxGxxG (SteC);
subdomain II: invariant lysine residue; subdomain III: invariant glutamic
acid
PTPase motif – HC(x)5R(S/T); essential WPD loop
PIPase domain – FN(F/V)G(V/I)NE (motif 1) and CKSxKDRTxM (motif 2)
Src kinase phosphorylation motif – ExLYxx(I/V)
The LPx6I type F box (all GALA effectors)
Lyase motif – GDKxH – essential for lyase activity
Homology to WASP WH2; contains the LKKV-like motif
Docking motif required for specific binding to MAPKs
Highly homologous to mammalian formins (VopF)
Phosphotyrosine Nck-binding YDEV motif (Tir), vinculin-binding domain
(IpaA), N-WASP-binding motif (EspF)
Mediates binding to the 14-3-3 protein FAS to activate the ADPRT domain
Repeated motif; binds sorting nexin 9 via a conserved RxAPxxP motif
Hsp70 interaction domain with an invariant HPD motif
Extreme C-terminal motif: -SKI (NleH1), -TRL (Map), -TRV (NleA); mediates
effector sorting and protein–protein interaction
Far C-terminal region of the TAL effectors. Functions in conjunction with the
central DNA-binding repeat region
Processing site containing the motif DEVD
Required for AMPylation of Rho GTPases. C-terminal motif: HxFx(D/
E)GNGR
Golgi
Subversive
Ser/Thr kinase
domain/motif
RhoGAP
STE motif, SseG
YpkA
AexT, ExoS, ExoT, YopE, SptP
RhoGEF
RhoGEF (WxxxE family)
SopE, SopE2, BopE
Map, SifA, EspM2
Lipase
Phospholipase
SseJ
ExoU
Cysteine protease
YopJ, YopT, AvrPphB
E3 Ub ligase
ADP-ribosyltransferase
SopA, IpaH, SspH1
HopU1, SpvB, ExoS, ExoT
Kinase
SteC, YpkA, OspG
Protein phosphatase
PIP phosphatase
pY motifs
F boxes
Lyase
WH2
D motif
FH1
Actin-related protein
binding
14-3-3-binding site
SH3-binding motif
J domain
PDZ1-binding motif
YopH, SptP
IpgD, SopB
Tir
GALA family
OspF, SpvC HopAI1
VopL, VopF
SpvC, OspF
VopF
IpaA, Tir, EspF
Transcription activation
domain
Caspase-3 site
Fic domain
ExoS
EspF
HopI1
Map, NleA, NleH1
AvrBs3 effector family
SipA, SopA
VopS
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.
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Table 3. Bacterial effectors and their domains/motifs with proven roles in virulence
Bacterial effector
Domain or motif with a virulence role
References
SipA
SseI
SseL
YopM
ExoS
HopF2Pto
AvrPto
WtsE and AvrE1
SifA
GALA effectors
SseJ
SpvB
NleA
YpkA
YopE
DEVD – caspase 3 cleavage site
Cys178 – unknown function
Cysteine protease site
Leucine-rich repeats (6–15)
ADP-ribosyltransferase domain
Myristoylation motif
Ser149 phosphorylation
WxxxE motif
CAAX box
F-box
Lipase domain – SHD triad
ADP-ribosyltransferase domain
PDZ domain
Kinase domain
GAP domain
Srikanth et al. (2010)
McLaughlin et al. (2009)
Rytkonen et al. (2007)
McPhee et al. (2010)
Shaver & Hauser (2004)
Wilton et al. (2010)
Anderson et al. (2006)
Ham et al. (2009)
Boucrot et al. (2003)
Angot et al. (2006)
Ohlson et al. (2005)
Lesnick et al. (2001)
Lee et al. (2008)
Wiley et al. (2006)
Black & Bliska (2000)
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 chaperonebinding domain located closely downstream (between
residues 50 and 150; for a review, see Ghosh, 2004). The
N-terminal secretion signal, while being functionally interchangeable 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 dependent 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
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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 & Donnenberg, 2004; Papatheodorou et al., 2006). Like many eukaryotic 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 important 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 mitochondrial 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 (residues 1–50) (Layton et al., 2005). Likewise, Pseudomonas
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7
Functional domains and motifs of bacterial effector proteins
syringae HopG1 does not have a predicted MTS, but green
fluorescent protein (GFP) fusions to the C-terminus indicated 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 anomalous localization patterns. This is exemplified with the
E. coli effector Tir, which normally targets the plasma membrane, 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 membranetargeting 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 superhelical 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 including 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 translocation 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. Interestingly, 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
FEMS Microbiol Rev ]] (2011) 1–26
(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 differently 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 phospholipase, 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). Importantly, 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 Cterminal 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
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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 (Boucrot 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 -- myristoylation
and 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 Nterminus 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 anchoring (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 conforms 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
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P. Dean
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) and
transcription-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 bipartite NLS in the centre of the protein, with the sequences
KRKR and KKKKKR separated by 13 amino acids (Gueneron 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-dependent 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 interaction 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
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9
Functional domains and motifs of bacterial effector proteins
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 Cterminal 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 conservation across the TAL effector, but do not confer lineagespecific 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 Cterminus (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 nucleartargeted LPX effectors YopM, SspH1 and IpaH9.8 share the
conserved repeating motif NxLTxLPELP, unlike the nonnuclear 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/nucleolar localization sequence (NoLS) of EspF resides in the
N-terminal region (residues 21–74) between two other cell
FEMS Microbiol Rev ]] (2011) 1–26
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 cytoplasmic 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 ubiquitin 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 effectors 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 residues) 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).
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Effectors that mimic host E3 ubiquitin ligases
The P. syringae effector AvrPtoB suppresses plant programmed cell death (PCD), allowing the pathogen to multiply 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 sequence 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 Nterminal 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 proteins 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
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P. Dean
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 polyubiquitinated such as Salmonella SopE (Kubori & Galan, 2003),
Yersinia YopE (Ruckdeschel et al., 2006) and P. aeruginosa
ExoU (Stirling et al., 2006). In most cases, polyubiquitination 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 phosphatases. Yersinia YopH is a protein tyrosine phosphatase
(PTPase) that exhibits among the highest-recorded PTPase
activity (Zhang et al., 1992). YopH prevents bacteria internalization into macrophages by dephosphorylating a large
number of proteins involved in integrin-mediated phagocytosis, such as FAK and paxillin (Persson et al., 1997). The Nterminal region of YopH contains a phosphotyrosine-binding domain that directly targets host tyrosine phosphorylated 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 (Ploop) motif – HC(x)5R(S/T) – that is highly conserved in
lipid and protein phosphatases (Stuckey et al., 1994). The Ploop is critical for phosphate binding as mutation of the
nucleophilic cysteine within this motif abolishes the enzymatic 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 sequence 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 (Kaniga 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, modulates actin rearrangements in host cells (Fu & Galan, 1999).
A substrate of SptP is the ATPase protein VCP (valosincontaining 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 Aeromonas AopH, although these have not been studied in detail.
FEMS Microbiol Rev ]] (2011) 1–26
11
Functional domains and motifs of bacterial effector proteins
Phosphoinositide phosphatases (PiPases)
Phosphoinositides are lipid molecules that regulate eukaryotic 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 phosphatidylinositol-4,5-biphosphate to generate the novel lipid phosphatidylinositol 5-monophosphate (Niebuhr et al., 2002).
Salmonella SopB/SigD is another effector PiPase, which, like
IpgD, perturbs the actin cytoskeleton and causes the depletion 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 mammalian 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).
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 phosphorylation (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–kinase 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 (Anderson et al., 2006).
Effector lipases
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 Factin 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
FEMS Microbiol Rev ]] (2011) 1–26
Lipases are a large group of functionally diverse molecules
that include several bacterial effector proteins. The Salmonella 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 positions 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
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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 activation (Sato et al., 2003; Lossi et al., 2008), possibly representing 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 antiinflammatory 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 experiments 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) (CollierHyams 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
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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 Cterminal 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 (BrugirardRicaud 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 autoproteolysis between residues K62 and G63, converting it from a 35to a 28-kDa protein, dependent on each residue of the CHD
triad (Shao et al., 2002; Dowen et al., 2009). A proteaseinactive 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).
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13
Functional domains and motifs of bacterial effector proteins
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 eukaryotic 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
FEMS Microbiol Rev ]] (2011) 1–26
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-ribosylates 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 ADPribosylates 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 (Henriksson 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, resulting 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 selfregulation 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 virulence 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 mechanisms of bacterial effector proteins is that they sometimes
reveal themselves as highly novel proteins, mediating previously 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
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14
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 conformation (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 PawelRammingen 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 consensus 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 believed 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
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P. Dean
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 polymerization 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 Cterminal 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? -- the
WxxxE 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 magnetic 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
FEMS Microbiol Rev ]] (2011) 1–26
15
Functional domains and motifs of bacterial effector proteins
(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 alanine 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 maintaining 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 (Matsuzawa 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 disruption 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 inhibitors, suggesting that VirA may not have direct proteolytic 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
FEMS Microbiol Rev ]] (2011) 1–26
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 nucleation 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 (Liverman 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 nucleation 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
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16
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 vinculinbinding site of IpaA was mapped to its last 74 residues
(Ramarao et al., 2007), which contains at least two vinculinbinding motifs (Hamiaux et al., 2006). These motifs bind to
the N-terminal VD1 domain of vinculin, inducing a conformational change that promotes vinculin binding to Factin. 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 depolymerization by binding actin directly through its C-terminal
domain.
Other effectors with proven actin modulatory domains
include SipC, a Salmonella translocator protein that nucleates 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 nucleation 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
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P. Dean
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 membrane 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
FEMS Microbiol Rev ]] (2011) 1–26
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Functional domains and motifs of bacterial effector proteins
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 PDZbinding 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 PDZbinding motif of NleA was shown to be essential for its
ability to disrupt intestinal barrier function (Thanabalasuriar 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 (Srikanth et al., 2010). Thus, caspase-3 cleavage sites may
represent a common mechanism to regulate effector function in host cells.
FEMS Microbiol Rev ]] (2011) 1–26
YpkA -- a shining example of
effector 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-terminaltargeting 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 proposed 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 abolishing 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
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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 organizing all the reading material used in this review. P.D. is
supported by a Medical School Faculty Fellowship, University of Newcastle.
References
Abramovitch RB & Martin GB (2005) AvrPtoB: a bacterial type
III effector that both elicits and suppresses programmed cell
death associated with plant immunity. FEMS Microbiol Lett
245: 1–8.
Abramovitch RB, Janjusevic R, Stebbins CE & Martin GB (2006)
Type III effector AvrPtoB requires intrinsic E3 ubiquitin ligase
activity to suppress plant cell death and immunity. P Natl Acad
Sci USA 103: 2851–2856.
Adam T, Arpin M, Prevost MC, Gounon P & Sansonetti PJ (1995)
Cytoskeletal rearrangements and the functional role of Tplastin during entry of Shigella flexneri into HeLa cells. J Cell
Biol 129: 367–381.
Aepfelbacher M, Trasak C, Wilharm G, Wiedemann A, Trulzsch
K, Krauss K, Gierschik P & Heesemann J (2003)
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
P. Dean
Characterization of YopT effects on Rho GTPases in Yersinia
enterocolitica-infected cells. J Biol Chem 278: 33217–33223.
Ahuja N, Kumar P & Bhatnagar R (2004) The adenylate cyclase
toxins. Crit Rev Microbiol 30: 187–196.
Aitio O, Hellman M, Kazlauskas A, Vingadassalom DF, Leong JM,
Saksela K & Permi P (2010) Recognition of tandem PxxP
motifs as a unique Src homology 3-binding mode triggers
pathogen-driven actin assembly. P Natl Acad Sci USA 107:
21743–21748.
Akoh CC, Lee GC, Liaw YC, Huang TH & Shaw JF (2004) GDSL
family of serine esterases/lipases. Prog Lipid Res 43: 534–552.
Aleman A, Rodriguez-Escudero I, Mallo GV, Cid VJ, Molina M &
Rotger R (2005) The amino-terminal non-catalytic region of
Salmonella typhimurium SigD affects actin organization in
yeast and mammalian cells. Cell Microbiol 7: 1432–1446.
Allen-Vercoe E, Toh MC, Waddell B, Ho H & DeVinney R (2005)
A carboxy-terminal domain of Tir from enterohemorrhagic
Escherichia coli O157:H7 (EHEC O157:H7) required for
efficient type III secretion. FEMS Microbiol Lett 243: 355–364.
Alto NM, Shao F, Lazar CS et al. (2006) Identification of a
bacterial type III effector family with G protein mimicry
functions. Cell 124: 133–145.
Alto NM, Weflen AW, Rardin MJ et al. (2007) The type III effector
EspF coordinates membrane trafficking by the spatiotemporal
activation of two eukaryotic signaling pathways. J Cell Biol
178: 1265–1278.
Anderson DM, Fouts DE, Collmer A & Schneewind O (1999)
Reciprocal secretion of proteins by the bacterial type III
machines of plant and animal pathogens suggests universal
recognition of mRNA targeting signals. P Natl Acad Sci USA
96: 12839–12843.
Anderson JC, Pascuzzi PE, Xiao F, Sessa G & Martin GB (2006)
Host-mediated phosphorylation of type III effector AvrPto
promotes Pseudomonas virulence and avirulence in tomato.
Plant Cell 18: 502–514.
Angot A, Peeters N, Lechner E, Vailleau F, Baud C, Gentzbittel L,
Sartorel E, Genschik P, Boucher C & Genin S (2006) Ralstonia
solanacearum requires F-box-like domain-containing type III
effectors to promote disease on several host plants. P Natl Acad
Sci USA 103: 14620–14625.
Angot A, Vergunst A, Genin S & Peeters N (2007) Exploitation of
eukaryotic ubiquitin signaling pathways by effectors
translocated by bacterial type III and type IV secretion
systems. PLoS Pathog 3: e3.
Arbeloa A, Garnett J, Lillington J, Bulgin RR, Berger CN, Lea SM,
Matthews S & Frankel G (2009) EspM2 is a RhoA guanine
nucleotide exchange factor. Cell Microbiol 12: 654–664.
Arnold R, Brandmaier S, Kleine F, Tischler P, Heinz E, Behrens S,
Niinikoski A, Mewes HW, Horn M & Rattei T (2009)
Sequence-based prediction of type III secreted proteins. PLoS
Pathog 5: e1000376.
Ashida H, Toyotome T, Nagai T & Sasakawa C (2007) Shigella
chromosomal IpaH proteins are secreted via the type III
secretion system and act as effectors. Mol Microbiol 63:
680–693.
FEMS Microbiol Rev ]] (2011) 1–26
19
Functional domains and motifs of bacterial effector proteins
Axtell MJ, Chisholm ST, Dahlbeck D & Staskawicz BJ (2003)
Genetic and molecular evidence that the Pseudomonas syringae
type III effector protein AvrRpt2 is a cysteine protease. Mol
Microbiol 49: 1537–1546.
Backert S & Selbach M (2005) Tyrosine-phosphorylated bacterial
effector proteins: the enemies within. Trends Microbiol 13:
476–484.
Bai C, Sen P, Hofmann K, Ma L, Goebl M, Harper JW & Elledge
SJ (1996) SKP1 connects cell cycle regulators to the ubiquitin
proteolysis machinery through a novel motif, the F-box. Cell
86: 263–274.
Barz C, Abahji TN, Trulzsch K & Heesemann J (2000) The
Yersinia Ser/Thr protein kinase YpkA/YopO directly interacts
with the small GTPases RhoA and Rac-1. FEBS Lett 482:
139–143.
Bateman A, Murzin AG & Teichmann SA (1998) Structure and
distribution of pentapeptide repeats in bacteria. Protein Sci 7:
1477–1480.
Benabdillah R, Mota LJ, Lutzelschwab S, Demoinet E & Cornelis
GR (2004) Identification of a nuclear targeting signal in YopM
from Yersinia spp. Microb Pathogenesis 36: 247–261.
Beuzon CR, Meresse S, Unsworth KE, Ruiz-Albert J, Garvis S,
Waterman SR, Ryder TA, Boucrot E & Holden DW (2000)
Salmonella maintains the integrity of its intracellular vacuole
through the action of SifA. EMBO J 19: 3235–3249.
Black DS & Bliska JB (2000) The RhoGAP activity of the Yersinia
pseudotuberculosis cytotoxin YopE is required for
antiphagocytic function and virulence. Mol Microbiol 37:
515–527.
Block A, Guo M, Li G, Elowsky C, Clemente TE & Alfano JR
(2009) The Pseudomonas syringae type III effector HopG1
targets mitochondria, alters plant development, and
suppresses plant innate immunity. Cell Microbiol 12: 318–330.
Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S,
Lahaye T, Nickstadt A & Bonas U (2009) Breaking the code of
DNA binding specificity of TAL-type III effectors. Science 326:
1509–1512.
Bos JL, Rehmann H & Wittinghofer A (2007) GEFs and GAPs:
critical elements in the control of small G proteins. Cell 129:
865–877.
Boucrot E, Beuzon CR, Holden DW, Gorvel JP & Meresse S
(2003) Salmonella typhimurium SifA effector protein requires
its membrane-anchoring C-terminal hexapeptide for its
biological function. J Biol Chem 278: 14196–14202.
Bourdet-Sicard R, Rudiger M, Jockusch BM, Gounon P,
Sansonetti PJ & Nhieu GT (1999) Binding of the Shigella
protein IpaA to vinculin induces F-actin depolymerization.
EMBO J 18: 5853–5862.
Brown NF, Szeto J, Jiang X, Coombes BK, Finlay BB & Brumell JH
(2006) Mutational analysis of Salmonella translocated effector
members SifA and SopD2 reveals domains implicated in
translocation, subcellular localization and function.
Microbiology 152: 2323–2343.
Brugirard-Ricaud K, Duchaud E, Givaudan A, Girard PA, Kunst
F, Boemare N, Brehelin M & Zumbihl R (2005) Site-specific
FEMS Microbiol Rev ]] (2011) 1–26
antiphagocytic function of the Photorhabdus luminescens type
III secretion system during insect colonization. Cell Microbiol
7: 363–371.
Brumell JH, Goosney DL & Finlay BB (2002) SifA, a type III
secreted effector of Salmonella typhimurium, directs
Salmonella-induced filament (Sif) formation along
microtubules. Traffic 3: 407–415.
Brumell JH, Kujat-Choy S, Brown NF, Vallance BA, Knodler LA &
Finlay BB (2003) SopD2 is a novel type III secreted effector of
Salmonella typhimurium that targets late endocytic
compartments upon delivery into host cells. Traffic 4: 36–48.
Brumlik MJ & Buckley JT (1996) Identification of the catalytic
triad of the lipase/acyltransferase from Aeromonas hydrophila.
J Bacteriol 178: 2060–2064.
Buchanan SG & Gay NJ (1996) Structural and functional
diversity in the leucine-rich repeat family of proteins. Prog
Biophys Mol Bio 65: 1–44.
Chang J, Chen J & Zhou D (2005) Delineation and
characterization of the actin nucleation and effector
translocation activities of Salmonella SipC. Mol Microbiol 55:
1379–1389.
Chisholm ST, Dahlbeck D, Krishnamurthy N, Day B, Sjolander K
& Staskawicz BJ (2005) Molecular characterization of
proteolytic cleavage sites of the Pseudomonas syringae effector
AvrRpt2. P Natl Acad Sci USA 102: 2087–2092.
Coaker G, Falick A & Staskawicz B (2005) Activation of a
phytopathogenic bacterial effector protein by a eukaryotic
cyclophilin. Science 308: 548–550.
Coburn B, Sekirov I & Finlay BB (2007) Type III secretion systems
and disease. Clin Microbiol Rev 20: 535–549.
Collier-Hyams LS, Zeng H, Sun J, Tomlinson AD, Bao ZQ, Chen
H, Madara JL, Orth K & Neish AS (2002) Cutting edge:
Salmonella AvrA effector inhibits the key proinflammatory,
anti-apoptotic NF-kappa B pathway. J Immunol 169:
2846–2850.
Cunnac S, Lindeberg M & Collmer A (2009) Pseudomonas
syringae type III secretion system effectors: repertoires in
search of functions. Curr Opin Microbiol 12: 53–60.
Davis J, Wang J, Tropea JE, Zhang D, Dauter Z, Waugh DS &
Wlodawer A (2008) Novel fold of VirA, a type III secretion
system effector protein from Shigella flexneri. Protein Sci 17:
2167–2173.
Dean P, Muhlen S, Quitard S & Kenny B (2010a) The bacterial
effectors EspG and EspG2 induce a destructive calpain activity
that is kept in check by the co-delivered Tir effector. Cell
Microbiol 12: 1308–1321.
Dean P, Scott JA, Knox AA, Quitard S, Watkins NJ & Kenny B
(2010b) The enteropathogenic E. coli effector EspF targets and
disrupts the nucleolus by a process regulated by mitochondrial
dysfunction. PLoS Pathog 6: e1000961.
Demali KA, Jue AL & Burridge K (2006) IpaA targets beta1
integrins and rho to promote actin cytoskeleton
rearrangements necessary for Shigella entry. J Biol Chem 281:
39534–39541.
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
20
Deslandes L, Olivier J, Peeters N, Feng DX, Khounlotham M,
Boucher C, Somssich I, Genin S & Marco Y (2003) Physical
interaction between RRS1-R, a protein conferring resistance to
bacterial wilt, and PopP2, a type III effector targeted to the
plant nucleus. P Natl Acad Sci USA 100: 8024–8029.
Diao J, Zhang Y, Huibregtse JM, Zhou D & Chen J (2008) Crystal
structure of SopA, a Salmonella effector protein mimicking a
eukaryotic ubiquitin ligase. Nat Struct Mol Biol 15: 65–70.
Domenighini M, Magagnoli C, Pizza M & Rappuoli R (1994)
Common features of the NAD-binding and catalytic site of
ADP-ribosylating toxins. Mol Microbiol 14: 41–50.
Dominguez R (2009) Actin filament nucleation and elongation
factors – structure–function relationships. Crit Rev Biochem
Mol 44: 351–366.
Dowen RH, Engel JL, Shao F, Ecker JR & Dixon JE (2009) A
family of bacterial cysteine protease type III effectors utilizes
acylation-dependent and -independent strategies to localize to
plasma membranes. J Biol Chem 284: 15867–15879.
Dukuzumuremyi JM, Rosqvist R, Hallberg B, Akerstrom B, WolfWatz H & Schesser K (2000) The Yersinia protein kinase A is a
host factor inducible RhoA/Rac-binding virulence factor. J Biol
Chem 275: 35281–35290.
Espinosa A, Guo M, Tam VC, Fu ZQ & Alfano JR (2003) The
Pseudomonas syringae type III-secreted protein HopPtoD2
possesses protein tyrosine phosphatase activity and suppresses
programmed cell death in plants. Mol Microbiol 49: 377–387.
Evdokimov AG, Tropea JE, Routzahn KM & Waugh DS (2002)
Crystal structure of the Yersinia pestis GTPase activator YopE.
Protein Sci 11: 401–408.
Fehr D, Burr SE, Gibert M, d’Alayer J, Frey J & Popoff MR (2007)
Aeromonas exoenzyme T of Aeromonas salmonicida is a
bifunctional protein that targets the host cytoskeleton. J Biol
Chem 282: 28843–28852.
Fu Y & Galan JE (1999) A Salmonella protein antagonizes Rac-1
and Cdc42 to mediate host-cell recovery after bacterial
invasion. Nature 401: 293–297.
Fu H, Coburn J & Collier RJ (1993) The eukaryotic host factor
that activates exoenzyme S of Pseudomonas aeruginosa is a
member of the 14-3-3 protein family. P Natl Acad Sci USA 90:
2320–2324.
Fu ZQ, Guo M, Jeong BR, Tian F, Elthon TE, Cerny RL, Staiger D
& Alfano JR (2007) A type III effector ADP-ribosylates RNAbinding proteins and quells plant immunity. Nature 447:
284–288.
Galan JE (2009) Common themes in the design and function of
bacterial effectors. Cell Host Microbe 5: 571–579.
Galyov EE, Hakansson S, Forsberg A & Wolf-Watz H (1993) A
secreted protein kinase of Yersinia pseudotuberculosis is an
indispensable virulence determinant. Nature 361: 730–732.
Gamblin SJ & Smerdon SJ (1998) GTPase-activating proteins and
their complexes. Curr Opin Struc Biol 8: 195–201.
Gao J, Liao J & Yang GY (2009) CAAX-box protein, prenylation
process and carcinogenesis. Am J Transl Res 1: 312–325.
Garcia-Mata R & Burridge K (2007) Catching a GEF by its tail.
Trends Cell Biol 17: 36–43.
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
P. Dean
Garmendia J, Phillips AD, Carlier MF et al. (2004) TccP is an
enterohaemorrhagic Escherichia coli O157:H7 type III effector
protein that couples Tir to the actin-cytoskeleton. Cell
Microbiol 6: 1167–1183.
Garrity-Ryan L, Shafikhani S, Balachandran P et al. (2004) The
ADP ribosyltransferase domain of Pseudomonas aeruginosa
ExoT contributes to its biological activities. Infect Immun 72:
546–558.
Germane KL, Ohi R, Goldberg MB & Spiller BW (2008)
Structural and functional studies indicate that Shigella VirA is
not a protease and does not directly destabilize microtubules.
Biochemistry 47: 10241–10243.
Ghosh P (2004) Process of protein transport by the type III
secretion system. Microbiol Mol Biol R 68: 771–795.
Gruenheid S, DeVinney R, Bladt F, Goosney D, Gelkop S, Gish
GD, Pawson T & Finlay BB (2001) Enteropathogenic E. coli Tir
binds Nck to initiate actin pedestal formation in host cells. Nat
Cell Biol 3: 856–859.
Gruenheid S, Sekirov I, Thomas NA et al. (2004) Identification
and characterization of NleA, a non-LEE-encoded type III
translocated virulence factor of enterohaemorrhagic
Escherichia coli O157:H7. Mol Microbiol 51: 1233–1249.
Gueneron M, Timmers AC, Boucher C & Arlat M (2000) Two
novel proteins, PopB, which has functional nuclear
localization signals, and PopC, which has a large leucine-rich
repeat domain, are secreted through the hrp-secretion
apparatus of Ralstonia solanacearum. Mol Microbiol 36:
261–277.
Gurlebeck D, Szurek B & Bonas U (2005) Dimerization of the
bacterial effector protein AvrBs3 in the plant cell cytoplasm
prior to nuclear import. Plant J 42: 175–187.
Hakansson S, Galyov EE, Rosqvist R & Wolf-Watz H (1996) The
Yersinia YpkA Ser/Thr kinase is translocated and subsequently
targeted to the inner surface of the HeLa cell plasma
membrane. Mol Microbiol 20: 593–603.
Ham JH, Majerczak DR, Nomura K, Mecey C, Uribe F, He SY,
Mackey D & Coplin DL (2009) Multiple activities of the plant
pathogen type III effector proteins WtsE and AvrE require
WxxxE motifs. Mol Plant Microbe In 22: 703–712.
Hamiaux C, van Eerde A, Parsot C, Broos J & Dijkstra BW (2006)
Structural mimicry for vinculin activation by IpaA, a virulence
factor of Shigella flexneri. EMBO Rep 7: 794–799.
Hanks SK, Quinn AM & Hunter T (1988) The protein kinase
family: conserved features and deduced phylogeny of the
catalytic domains. Science 241: 42–52.
Hansen-Wester I, Stecher B & Hensel M (2002) Analyses of the
evolutionary distribution of Salmonella translocated effectors.
Infect Immun 70: 1619–1622.
Haraga A & Miller SI (2003) A Salmonella enterica serovar
typhimurium translocated leucine-rich repeat effector protein
inhibits NF-kappa B-dependent gene expression. Infect Immun
71: 4052–4058.
Hardt WD, Chen LM, Schuebel KE, Bustelo XR & Galan JE
(1998) S. typhimurium encodes an activator of Rho GTPases
FEMS Microbiol Rev ]] (2011) 1–26
21
Functional domains and motifs of bacterial effector proteins
that induces membrane ruffling and nuclear responses in host
cells. Cell 93: 815–826.
Hayward RD & Koronakis V (1999) Direct nucleation and
bundling of actin by the SipC protein of invasive Salmonella.
EMBO J 18: 4926–4934.
Hennessy F, Nicoll WS, Zimmermann R, Cheetham ME & Blatch
GL (2005) Not all J domains are created equal: implications for
the specificity of Hsp40–Hsp70 interactions. Protein Sci 14:
1697–1709.
Henriksson ML, Francis MS, Peden A, Aili M, Stefansson K,
Palmer R, Aitken A & Hallberg B (2002a) A
nonphosphorylated 14-3-3 binding motif on exoenzyme S that
is functional in vivo. Eur J Biochem 269: 4921–4929.
Henriksson ML, Sundin C, Jansson AL, Forsberg A, Palmer RH &
Hallberg B (2002b) Exoenzyme S shows selective ADPribosylation and GTPase-activating protein (GAP) activities
towards small GTPases in vivo. Biochem J 367: 617–628.
Henry T, Couillault C, Rockenfeller P et al. (2006) The Salmonella
effector protein PipB2 is a linker for kinesin-1. P Natl Acad Sci
USA 103: 13497–13502.
Hernandez LD, Pypaert M, Flavell RA & Galan JE (2003) A
Salmonella protein causes macrophage cell death by inducing
autophagy. J Cell Biol 163: 1123–1131.
Holmes A, Muhlen S, Roe AJ & Dean P (2010) The EspF effector,
a bacterial pathogen’s Swiss army knife. Infect Immun 78:
4445–4453.
Hotson A, Chosed R, Shu H, Orth K & Mudgett MB (2003)
Xanthomonas type III effector XopD targets SUMOconjugated proteins in planta. Mol Microbiol 50: 377–389.
Hu X & Stebbins CE (2006) Dynamics of the WPD loop of the
Yersinia protein tyrosine phosphatase. Biophys J 91: 948–956.
Huang Z, Sutton SE, Wallenfang AJ, Orchard RC, Wu X, Feng Y,
Chai J & Alto NM (2009) Structural insights into host GTPase
isoform selection by a family of bacterial GEF mimics. Nat
Struct Mol Biol 16: 853–860.
Huibregtse JM, Scheffner M, Beaudenon S & Howley PM (1995)
A family of proteins structurally and functionally related to the
E6-AP ubiquitin-protein ligase. P Natl Acad Sci USA 92:
2563–2567.
Humphreys D, Hume PJ & Koronakis V (2009) The Salmonella
effector SptP dephosphorylates host AAA1ATPase VCP to
promote development of its intracellular replicative niche. Cell
Host Microbe 5: 225–233.
Hung AY & Sheng M (2002) PDZ domains: structural modules
for protein complex assembly. J Biol Chem 277: 5699–5702.
Ivanov MI, Stuckey JA, Schubert HL, Saper MA & Bliska JB
(2005) Two substrate-targeting sites in the Yersinia protein
tyrosine phosphatase co-operate to promote bacterial
virulence. Mol Microbiol 55: 1346–1356.
Iwai H, Kim M, Yoshikawa Y et al. (2007) A bacterial effector
targets Mad2L2, an APC inhibitor, to modulate host cell
cycling. Cell 130: 611–623.
Janjusevic R, Abramovitch RB, Martin GB & Stebbins CE (2006)
A bacterial inhibitor of host programmed cell death defenses is
an E3 ubiquitin ligase. Science 311: 222–226.
FEMS Microbiol Rev ]] (2011) 1–26
Jelenska J, Yao N, Vinatzer BA, Wright CM, Brodsky JL &
Greenberg JT (2007) A J domain virulence effector of
Pseudomonas syringae remodels host chloroplasts and
suppresses defenses. Curr Biol 17: 499–508.
Jelenska J, van Hal JA & Greenberg JT (2010) Pseudomonas
syringae hijacks plant stress chaperone machinery for
virulence. P Natl Acad Sci USA 107: 13177–13182.
Jewett TJ, Fischer ER, Mead DJ & Hackstadt T (2006) Chlamydial
TARP is a bacterial nucleator of actin. P Natl Acad Sci USA
103: 15599–15604.
Juris SJ, Rudolph AE, Huddler D, Orth K & Dixon JE (2000) A
distinctive role for the Yersinia protein kinase: actin binding,
kinase activation, and cytoskeleton disruption. P Natl Acad Sci
USA 97: 9431–9436.
Juris SJ, Shao F & Dixon JE (2002) Yersinia effectors target
mammalian signalling pathways. Cell Microbiol 4: 201–211.
Kaniga K, Uralil J, Bliska JB & Galan JE (1996) A secreted protein
tyrosine phosphatase with modular effector domains in the
bacterial pathogen Salmonella typhimurium. Mol Microbiol 21:
633–641.
Kay S, Hahn S, Marois E, Hause G & Bonas U (2007) A bacterial
effector acts as a plant transcription factor and induces a cell
size regulator. Science 318: 648–651.
Kelley WL (1998) The J-domain family and the recruitment of
chaperone power. Trends Biochem Sci 23: 222–227.
Kenny B & Jepson M (2000) Targeting of an enteropathogenic
Escherichia coli (EPEC) effector protein to host mitochondria.
Cell Microbiol 2: 579–590.
Kenny B & Valdivia R (2009) Host–microbe interactions:
bacteria. Curr Opin Microbiol 12: 1–3.
Kenny B, DeVinney R, Stein M, Reinscheid DJ, Frey EA & Finlay
BB (1997) Enteropathogenic E. coli (EPEC) transfers its
receptor for intimate adherence into mammalian cells. Cell 91:
511–520.
Kenny B, Ellis S, Leard AD, Warawa J, Mellor H & Jepson MA
(2002) Co-ordinate regulation of distinct host cell signalling
pathways by multifunctional enteropathogenic Escherichia coli
effector molecules. Mol Microbiol 44: 1095–1107.
Kim DW, Lenzen G, Page AL, Legrain P, Sansonetti PJ & Parsot C
(2005a) The Shigella flexneri effector OspG interferes with
innate immune responses by targeting ubiquitin-conjugating
enzymes. P Natl Acad Sci USA 102: 14046–14051.
Kim HS, Desveaux D, Singer AU, Patel P, Sondek J & Dangl JL
(2005b) The Pseudomonas syringae effector AvrRpt2 cleaves its
C-terminally acylated target, RIN4, from Arabidopsis
membranes to block RPM1 activation. P Natl Acad Sci USA
102: 6496–6501.
Kim BH, Kim HG, Kim JS, Jang JI & Park YK (2007) Analysis of
functional domains present in the N-terminus of the SipB
protein. Microbiology 153: 2998–3008.
Kinch LN, Yarbrough ML, Orth K & Grishin NV (2009) Fido, a
novel AMPylation domain common to fic, doc, and AvrB.
PLoS One 4: e5818.
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
22
Knodler LA & Steele-Mortimer O (2005) The Salmonella effector
PipB2 affects late endosome/lysosome distribution to mediate
Sif extension. Mol Biol Cell 16: 4108–4123.
Knodler LA, Vallance BA, Hensel M, Jackel D, Finlay BB & SteeleMortimer O (2003) Salmonella type III effectors PipB and
PipB2 are targeted to detergent-resistant microdomains on
internal host cell membranes. Mol Microbiol 49: 685–704.
Knodler LA, Winfree S, Drecktrah D, Ireland R & SteeleMortimer O (2009) Ubiquitination of the bacterial inositol
phosphatase, SopB, regulates its biological activity at the
plasma membrane. Cell Microbiol 11: 1652–1670.
Krall R, Zhang Y & Barbieri JT (2004) Intracellular membrane
localization of Pseudomonas ExoS and Yersinia YopE in
mammalian cells. J Biol Chem 279: 2747–2753.
Kubori T & Galan JE (2003) Temporal regulation of Salmonella
virulence effector function by proteasome-dependent protein
degradation. Cell 115: 333–342.
Kuhle V, Jackel D & Hensel M (2004) Effector proteins encoded
by Salmonella pathogenicity island 2 interfere with the
microtubule cytoskeleton after translocation into host cells.
Traffic 5: 356–370.
Layton AN, Brown PJ & Galyov EE (2005) The Salmonella
translocated effector SopA is targeted to the mitochondria of
infected cells. J Bacteriol 187: 3565–3571.
Lee SF, Kelly M, McAlister A, Luck SN, Garcia EL, Hall RA,
Robins-Browne RM, Frankel G & Hartland EL (2008) A Cterminal class I PDZ binding motif of EspI/NleA modulates
the virulence of attaching and effacing Escherichia coli and
Citrobacter rodentium. Cell Microbiol 10: 499–513.
Lesnick ML, Reiner NE, Fierer J & Guiney DG (2001) The
Salmonella spvB virulence gene encodes an enzyme that ADPribosylates actin and destabilizes the cytoskeleton of
eukaryotic cells. Mol Microbiol 39: 1464–1470.
Letzelter M, Sorg I, Mota LJ, Meyer S, Stalder J, Feldman M, Kuhn
M, Callebaut I & Cornelis GR (2006) The discovery of SycO
highlights a new function for type III secretion effector
chaperones. EMBO J 25: 3223–3233.
Lewis JD, Abada W, Ma W, Guttman DS & Desveaux D (2008)
The HopZ family of Pseudomonas syringae type III effectors
require myristoylation for virulence and avirulence functions
in Arabidopsis thaliana. J Bacteriol 190: 2880–2891.
Li H, Xu H, Zhou Y, Zhang J, Long C, Li S, Chen S, Zhou JM &
Shao F (2007) The phosphothreonine lyase activity of a
bacterial type III effector family. Science 315: 1000–1003.
Liverman AD, Cheng HC, Trosky JE, Leung DW, Yarbrough ML,
Burdette DL, Rosen MK & Orth K (2007) Arp2/3-independent
assembly of actin by Vibrio type III effector VopL. P Natl Acad
Sci USA 104: 17117–17122.
Lloyd SA, Norman M, Rosqvist R & Wolf-Watz H (2001) Yersinia
YopE is targeted for type III secretion by N-terminal, not
mRNA, signals. Mol Microbiol 39: 520–531.
Lossi NS, Rolhion N, Magee AI, Boyle C & Holden DW (2008)
The Salmonella SPI-2 effector SseJ exhibits eukaryotic
activator-dependent phospholipase A and
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
P. Dean
glycerophospholipid: cholesterol acyltransferase activity.
Microbiology 154: 2680–2688.
Lutter M, Fang M, Luo X, Nishijima M, Xie X & Wang X (2000)
Cardiolipin provides specificity for targeting of tBid to
mitochondria. Nat Cell Biol 2: 754–761.
Malish HR, Freeman NL, Zurawski DV, Chowrashi P, Ayoob JC,
Sanger JW & Sanger JM (2003) Potential role of the EPEC
translocated intimin receptor (Tir) in host apoptotic events.
Apoptosis 8: 179–190.
Marcus SL, Wenk MR, Steele-Mortimer O & Finlay BB (2001) A
synaptojanin-homologous region of Salmonella typhimurium
SigD is essential for inositol phosphatase activity and Akt
activation. FEBS Lett 494: 201–207.
Marcus SL, Knodler LA & Finlay BB (2002) Salmonella enterica
serovar Typhimurium effector SigD/SopB is membraneassociated and ubiquitinated inside host cells. Cell Microbiol 4:
435–446.
Martinez E, Schroeder GN, Berger CN et al. (2010) Binding to
Na(1)/H(1) exchanger regulatory factor 2 (NHERF2) affects
trafficking and function of the enteropathogenic Escherichia
coli type III secretion system effectors Map, EspI and NleH.
Cell Microbiol 12: 1718–1731.
Matsumoto H & Young GM (2006) Proteomic and functional
analysis of the suite of Ysp proteins exported by the Ysa type
III secretion system of Yersinia enterocolitica Biovar 1B. Mol
Microbiol 59: 689–706.
Matsumoto H & Young GM (2009) Translocated effectors of
Yersinia. Curr Opin Microbiol 12: 94–100.
Matsuzawa T, Kuwae A, Yoshida S, Sasakawa C & Abe A (2004)
Enteropathogenic Escherichia coli activates the RhoA signaling
pathway via the stimulation of GEF-H1. EMBO J 23:
3570–3582.
Maurer-Stroh S & Eisenhaber F (2004) Myristoylation of viral
and bacterial proteins. Trends Microbiol 12: 178–185.
McCoy MW, Marre ML, Lesser CF & Mecsas J (2010) The Cterminal tail of Yersinia pseudotuberculosis YopM is critical for
interacting with RSK1 and for virulence. Infect Immun 78:
2584–2598.
McDermott JE, Corrigan A, Peterson E, Oehmen C, Niemann G,
Cambronne ED, Sharp D, Adkins JN, Samudrala R & Heffron
F (2011) Computational prediction of type III and IV secreted
effectors in gram-negative bacteria. Infect Immun 79: 23–32.
McLaughlin LM, Govoni GR, Gerke C et al. (2009) The
Salmonella SPI2 effector SseI mediates long-term systemic
infection by modulating host cell migration. PLoS Pathog 5:
e1000671.
McPhee JB, Mena P & Bliska JB (2010) Delineation of regions of
the Yersinia YopM protein required for interaction with the
RSK1 and PRK2 host kinases and their requirement for
interleukin-10 production and virulence. Infect Immun 78:
3529–3539.
Miao EA, Scherer CA, Tsolis RM, Kingsley RA, Adams LG,
Baumler AJ & Miller SI (1999) Salmonella typhimurium
leucine-rich repeat proteins are targeted to the SPI1 and SPI2
type III secretion systems. Mol Microbiol 34: 850–864.
FEMS Microbiol Rev ]] (2011) 1–26
23
Functional domains and motifs of bacterial effector proteins
Montagna LG, Ivanov MI & Bliska JB (2001) Identification of
residues in the N-terminal domain of the Yersinia tyrosine
phosphatase that are critical for substrate recognition. J Biol
Chem 276: 5005–5011.
Moscou MJ & Bogdanove AJ (2009) A simple cipher governs
DNA recognition by TAL effectors. Science 326: 1501.
Mukherjee S, Keitany G, Li Y, Wang Y, Ball HL, Goldsmith EJ &
Orth K (2006) Yersinia YopJ acetylates and inhibits kinase
activation by blocking phosphorylation. Science 312:
1211–1214.
Nagai T, Abe A & Sasakawa C (2005) Targeting of
enteropathogenic Escherichia coli EspF to host mitochondria is
essential for bacterial pathogenesis: critical role of the 16th
leucine residue in EspF. J Biol Chem 280: 2998–3011.
Nagamatsu K, Kuwae A, Konaka T, Nagai S, Yoshida S, Eguchi M,
Watanabe M, Mimuro H, Koyasu S & Abe A (2009) Bordetella
evades the host immune system by inducing IL-10 through a
type III effector, BopN. J Exp Med 206: 3073–3088.
Navarro L, Koller A, Nordfelth R, Wolf-Watz H, Taylor S & Dixon
JE (2007) Identification of a molecular target for the Yersinia
protein kinase A. Mol Cell 26: 465–477.
Neupert W (1997) Protein import into mitochondria. Annu Rev
Biochem 66: 863–917.
Niebuhr K, Giuriato S, Pedron T, Philpott DJ, Gaits F, Sable J,
Sheetz MP, Parsot C, Sansonetti PJ & Payrastre B (2002)
Conversion of PtdIns(4,5)P(2) into PtdIns(5)P by the
S.flexneri effector IpgD reorganizes host cell morphology.
EMBO J 21: 5069–5078.
Nimchuk Z, Marois E, Kjemtrup S, Leister RT, Katagiri F & Dangl
JL (2000) Eukaryotic fatty acylation drives plasma membrane
targeting and enhances function of several type III effector
proteins from Pseudomonas syringae. Cell 101: 353–363.
Norris FA, Wilson MP, Wallis TS, Galyov EE & Majerus PW
(1998) SopB, a protein required for virulence of Salmonella
dublin, is an inositol phosphate phosphatase. P Natl Acad Sci
USA 95: 14057–14059.
Nougayrede JP & Donnenberg MS (2004) Enteropathogenic
Escherichia coli EspF is targeted to mitochondria and is
required to initiate the mitochondrial death pathway. Cell
Microbiol 6: 1097–1111.
Ohlson MB, Fluhr K, Birmingham CL, Brumell JH & Miller SI
(2005) SseJ deacylase activity by Salmonella enterica serovar
Typhimurium promotes virulence in mice. Infect Immun 73:
6249–6259.
Ohlson MB, Huang Z, Alto NM, Blanc MP, Dixon JE, Chai J &
Miller SI (2008) Structure and function of Salmonella SifA
indicate that its interactions with SKIP, SseJ, and RhoA family
GTPases induce endosomal tubulation. Cell Host Microbe 4:
434–446.
Orth K, Xu Z, Mudgett MB, Bao ZQ, Palmer LE, Bliska JB,
Mangel WF, Staskawicz B & Dixon JE (2000) Disruption of
signaling by Yersinia effector YopJ, a ubiquitin-like protein
protease. Science 290: 1594–1597.
Papatheodorou P, Domanska G, Oxle M, Mathieu J, Selchow O,
Kenny B & Rassow J (2006) The enteropathogenic Escherichia
FEMS Microbiol Rev ]] (2011) 1–26
coli (EPEC) Map effector is imported into the mitochondrial
matrix by the TOM/Hsp70 system and alters organelle
morphology. Cell Microbiol 8: 677–689.
Passmore LA & Barford D (2004) Getting into position: the
catalytic mechanisms of protein ubiquitylation. Biochem J 379:
513–525.
Pederson KJ, Pal S, Vallis AJ, Frank DW & Barbieri JT (2000)
Intracellular localization and processing of Pseudomonas
aeruginosa ExoS in eukaryotic cells. Mol Microbiol 37: 287–299.
Persson C, Carballeira N, Wolf-Watz H & Fallman M (1997) The
PTPase YopH inhibits uptake of Yersinia, tyrosine
phosphorylation of p130Cas and FAK, and the associated
accumulation of these proteins in peripheral focal adhesions.
EMBO J 16: 2307–2318.
Phillips RM, Six DA, Dennis EA & Ghosh P (2003) In vivo
phospholipase activity of the Pseudomonas aeruginosa
cytotoxin ExoU and protection of mammalian cells with
phospholipase A2 inhibitors. J Biol Chem 278: 41326–41332.
Pickart CM (2001) Mechanisms underlying ubiquitination. Annu
Rev Biochem 70: 503–533.
Poh J, Odendall C, Spanos A, Boyle C, Liu M, Freemont P &
Holden DW (2008) SteC is a Salmonella kinase required for
SPI-2-dependent F-actin remodelling. Cell Microbiol 10:
20–30.
Prehna G, Ivanov MI, Bliska JB & Stebbins CE (2006) Yersinia
virulence depends on mimicry of host Rho-family nucleotide
dissociation inhibitors. Cell 126: 869–880.
Preston GM (2007) Metropolitan microbes: type III secretion in
multihost symbionts. Cell Host Microbe 2: 291–294.
Price CT, Al-Quadan T, Santic M, Jones SC & Abu Kwaik Y
(2010) Exploitation of conserved eukaryotic host cell
farnesylation machinery by an F-box effector of Legionella
pneumophila. J Exp Med 207: 1713–1726.
Rabin SD & Hauser AR (2005) Functional regions of the
Pseudomonas aeruginosa cytotoxin ExoU. Infect Immun 73:
573–582.
Rabin SD, Veesenmeyer JL, Bieging KT & Hauser AR (2006) A Cterminal domain targets the Pseudomonas aeruginosa
cytotoxin ExoU to the plasma membrane of host cells. Infect
Immun 74: 2552–2561.
Ramarao N, Le Clainche C, Izard T, Bourdet-Sicard R, Ageron E,
Sansonetti PJ, Carlier MF & Tran Van Nhieu G (2007) Capping
of actin filaments by vinculin activated by the Shigella IpaA
carboxyl-terminal domain. FEBS Lett 581: 853–857.
Reinicke AT, Hutchinson JL, Magee AI, Mastroeni P, Trowsdale J
& Kelly AP (2005) A Salmonella typhimurium effector protein
SifA is modified by host cell prenylation and S-acylation
machinery. J Biol Chem 280: 14620–14627.
Resh MD (2006) Trafficking and signaling by fatty-acylated and
prenylated proteins. Nat Chem Biol 2: 584–590.
Riese MJ, Goehring UM, Ehrmantraut ME, Moss J, Barbieri JT,
Aktories K & Schmidt G (2002) Auto-ADP-ribosylation of
Pseudomonas aeruginosa ExoS. J Biol Chem 277: 12082–12088.
Robert-Seilaniantz A, Shan L, Zhou JM & Tang X (2006) The
Pseudomonas syringae pv. tomato DC3000 type III effector
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
24
HopF2 has a putative myristoylation site required for its
avirulence and virulence functions. Mol Plant Microbe In 19:
130–138.
Rohde JR, Breitkreutz A, Chenal A, Sansonetti PJ & Parsot C
(2007) Type III secretion effectors of the IpaH family are E3
ubiquitin ligases. Cell Host Microbe 1: 77–83.
Rohmer L, Guttman DS & Dangl JL (2004) Diverse evolutionary
mechanisms shape the type III effector virulence factor
repertoire in the plant pathogen Pseudomonas syringae.
Genetics 167: 1341–1360.
Rosebrock TR, Zeng L, Brady JJ, Abramovitch RB, Xiao F &
Martin GB (2007) A bacterial E3 ubiquitin ligase targets a host
protein kinase to disrupt plant immunity. Nature 448:
370–374.
Rossman KL, Der CJ & Sondek J (2005) GEF means go: turning
on RHO GTPases with guanine nucleotide-exchange factors.
Nat Rev Mol Cell Bio 6: 167–180.
Rottger S, Frischknecht F, Reckmann I, Smith GL & Way M
(1999) Interactions between vaccinia virus IEV membrane
proteins and their roles in IEV assembly and actin tail
formation. J Virol 73: 2863–2875.
Ruckdeschel K, Pfaffinger G, Trulzsch K, Zenner G, Richter K,
Heesemann J & Aepfelbacher M (2006) The proteasome
pathway destabilizes Yersinia outer protein E and represses its
antihost cell activities. J Immunol 176: 6093–6102.
Rudolph MG, Weise C, Mirold S, Hillenbrand B, Bader B,
Wittinghofer A & Hardt WD (1999) Biochemical analysis of
SopE from Salmonella typhimurium, a highly efficient
guanosine nucleotide exchange factor for RhoGTPases. J Biol
Chem 274: 30501–30509.
Ruiz-Albert J, Yu XJ, Beuzon CR, Blakey AN, Galyov EE &
Holden DW (2002) Complementary activities of SseJ and SifA
regulate dynamics of the Salmonella typhimurium vacuolar
membrane. Mol Microbiol 44: 645–661.
Rytkonen A, Poh J, Garmendia J, Boyle C, Thompson A, Liu M,
Freemont P, Hinton JC & Holden DW (2007) SseL, a
Salmonella deubiquitinase required for macrophage killing and
virulence. P Natl Acad Sci USA 104: 3502–3507.
Salcedo SP & Holden DW (2003) SseG, a virulence protein that
targets Salmonella to the Golgi network. EMBO J 22:
5003–5014.
Sato H, Frank DW, Hillard CJ et al. (2003) The mechanism of
action of the Pseudomonas aeruginosa-encoded type III
cytotoxin, ExoU. EMBO J 22: 2959–2969.
Schlumberger MC, Friebel A, Buchwald G, Scheffzek K,
Wittinghofer A & Hardt WD (2003) Amino acids of the
bacterial toxin SopE involved in G nucleotide exchange on
Cdc42. J Biol Chem 278: 27149–27159.
Schmiel DH, Wagar E, Karamanou L, Weeks D & Miller VL
(1998) Phospholipase A of Yersinia enterocolitica contributes
to pathogenesis in a mouse model. Infect Immun 66:
3941–3951.
Shan L, Thara VK, Martin GB, Zhou JM & Tang X (2000) The
Pseudomonas AvrPto protein is differentially recognized by
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
P. Dean
tomato and tobacco and is localized to the plant plasma
membrane. Plant Cell 12: 2323–2338.
Shao F, Merritt PM, Bao Z, Innes RW & Dixon JE (2002) A
Yersinia effector and a Pseudomonas avirulence protein define a
family of cysteine proteases functioning in bacterial
pathogenesis. Cell 109: 575–588.
Shao F, Vacratsis PO, Bao Z, Bowers KE, Fierke CA & Dixon JE
(2003) Biochemical characterization of the Yersinia YopT
protease: cleavage site and recognition elements in Rho
GTPases. P Natl Acad Sci USA 100: 904–909.
Shaver CM & Hauser AR (2004) Relative contributions of
Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in
the lung. Infect Immun 72: 6969–6977.
Singer AU, Rohde JR, Lam R et al. (2008) Structure of the Shigella
T3SS effector IpaH defines a new class of E3 ubiquitin ligases.
Nat Struct Mol Biol 15: 1293–1301.
Skrzypek E, Cowan C & Straley SC (1998) Targeting of the
Yersinia pestis YopM protein into HeLa cells and intracellular
trafficking to the nucleus. Mol Microbiol 30: 1051–1065.
Sorg I, Hoffmann C, Dumbach J, Aktories K & Schmidt G (2003)
The C terminus of YopT is crucial for activity and the N
terminus is crucial for substrate binding. Infect Immun 71:
4623–4632.
Sorg JA, Miller NC & Schneewind O (2005) Substrate recognition
of type III secretion machines – testing the RNA signal
hypothesis. Cell Microbiol 7: 1217–1225.
Srikanth CV, Wall DM, Maldonado-Contreras A, Shi HN, Zhou
D, Demma Z, Mumy KL & McCormick BA (2010) Salmonella
pathogenesis and processing of secreted effectors by caspase-3.
Science 330: 390–393.
Stavrinides J, Ma W & Guttman DS (2006) Terminal reassortment
drives the quantum evolution of type III effectors in bacterial
pathogens. PLoS Pathog 2: e104.
Steele-Mortimer O, Knodler LA, Marcus SL, Scheid MP, Goh B,
Pfeifer CG, Duronio V & Finlay BB (2000) Activation of Akt/
protein kinase B in epithelial cells by the Salmonella
typhimurium effector sigD. J Biol Chem 275: 37718–37724.
Stein MA, Leung KY, Zwick M, Garcia-del Portillo F & Finlay BB
(1996) Identification of a Salmonella virulence gene required
for formation of filamentous structures containing lysosomal
membrane glycoproteins within epithelial cells. Mol Microbiol
20: 151–164.
Stirling FR, Cuzick A, Kelly SM, Oxley D & Evans TJ (2006)
Eukaryotic localization, activation and ubiquitinylation of a
bacterial type III secreted toxin. Cell Microbiol 8: 1294–1309.
Stuckey JA, Schubert HL, Fauman EB, Zhang ZY, Dixon JE &
Saper MA (1994) Crystal structure of Yersinia protein tyrosine
phosphatase at 2.5 Å and the complex with tungstate. Nature
370: 571–575.
Sun J & Barbieri JT (2003) Pseudomonas aeruginosa ExoT ADPribosylates CT10 regulator of kinase (Crk) proteins. J Biol
Chem 278: 32794–32800.
Szurek B, Rossier O, Hause G & Bonas U (2002) Type IIIdependent translocation of the Xanthomonas AvrBs3 protein
into the plant cell. Mol Microbiol 46: 13–23.
FEMS Microbiol Rev ]] (2011) 1–26
25
Functional domains and motifs of bacterial effector proteins
Takemoto D & Jones DA (2005) Membrane release and
destabilization of Arabidopsis RIN4 following cleavage by
Pseudomonas syringae AvrRpt2. Mol Plant Microbe In 18:
1258–1268.
Tam VC, Serruto D, Dziejman M, Brieher W & Mekalanos JJ
(2007) A type III secretion system in Vibrio cholerae
translocates a formin/spire hybrid-like actin nucleator to
promote intestinal colonization. Cell Host Microbe 1: 95–107.
Terebiznik MR, Vieira OV, Marcus SL, Slade A, Yip CM, Trimble
WS, Meyer T, Finlay BB & Grinstein S (2002) Elimination of
host cell PtdIns(4,5)P(2) by bacterial SigD promotes
membrane fission during invasion by Salmonella. Nat Cell Biol
4: 766–773.
Tezcan-Merdol D, Nyman T, Lindberg U, Haag F, Koch-Nolte F &
Rhen M (2001) Actin is ADP-ribosylated by the Salmonella
enterica virulence-associated protein SpvB. Mol Microbiol 39:
606–619.
Thanabalasuriar A, Koutsouris A, Weflen A, Mimee M, Hecht G
& Gruenheid S (2010) The bacterial virulence factor NleA is
required for the disruption of intestinal tight junctions by
enteropathogenic Escherichia coli. Cell Microbiol 12: 31–41.
Thieme F, Szczesny R, Urban A, Kirchner O, Hause G & Bonas U
(2007) New type III effectors from Xanthomonas campestris pv.
vesicatoria trigger plant reactions dependent on a conserved Nmyristoylation motif. Mol Plant Microbe In 20: 1250–1261.
Tobe T, Beatson SA, Taniguchi H et al. (2006) An extensive
repertoire of type III secretion effectors in Escherichia coli
O157 and the role of lambdoid phages in their dissemination.
P Natl Acad Sci USA 103: 14941–14946.
Toyotome T, Suzuki T, Kuwae A, Nonaka T, Fukuda H, ImajohOhmi S, Toyofuku T, Hori M & Sasakawa C (2001) Shigella
protein IpaH(9.8) is secreted from bacteria within mammalian
cells and transported to the nucleus. J Biol Chem 276:
32071–32079.
Tran EJ & Wente SR (2006) Dynamic nuclear pore complexes: life
on the edge. Cell 125: 1041–1053.
Tran Van Nhieu G, Ben-Ze’ev A & Sansonetti PJ (1997)
Modulation of bacterial entry into epithelial cells by
association between vinculin and the Shigella IpaA invasin.
EMBO J 16: 2717–2729.
Trasak C, Zenner G, Vogel A et al. (2007) Yersinia protein kinase
YopO is activated by a novel G-actin binding process. J Biol
Chem 282: 2268–2277.
Troisfontaines P & Cornelis GR (2005) Type III secretion: more
systems than you think. Physiology 20: 326–339.
Trosky JE, Mukherjee S, Burdette DL, Roberts M, McCarter L,
Siegel RM & Orth K (2004) Inhibition of MAPK signaling
pathways by VopA from Vibrio parahaemolyticus. J Biol Chem
279: 51953–51957.
Tzfira T, Vaidya M & Citovsky V (2004) Involvement of targeted
proteolysis in plant genetic transformation by Agrobacterium.
Nature 431: 87–92.
Underwood W, Zhang S & He SY (2007) The Pseudomonas
syringae type III effector tyrosine phosphatase HopAO1
FEMS Microbiol Rev ]] (2011) 1–26
suppresses innate immunity in Arabidopsis thaliana. Plant J 52:
658–672.
Upadhyay A, Williams C, Gill AC, Philippe DL, Davis K, Taylor
LA, Stevens MP, Galyov EE & Bagby S (2004) Biophysical
characterization of the catalytic domain of guanine nucleotide
exchange factor BopE from Burkholderia pseudomallei.
Biochim Biophys Acta 1698: 111–119.
Vingadassalom D, Kazlauskas A, Skehan B, Cheng HC, Magoun
L, Robbins D, Rosen MK, Saksela K & Leong JM (2009) Insulin
receptor tyrosine kinase substrate links the E. coli O157:H7
actin assembly effectors Tir and EspF(U) during pedestal
formation. Proc Natl Acad Sci USA 106: 6754–6759.
Von Pawel-Rammingen U, Telepnev MV, Schmidt G, Aktories K,
Wolf-Watz H & Rosqvist R (2000) GAP activity of the Yersinia
YopE cytotoxin specifically targets the Rho pathway: a
mechanism for disruption of actin microfilament structure.
Mol Microbiol 36: 737–748.
Weber SS, Ragaz C & Hilbi H (2009) Pathogen trafficking
pathways and host phosphoinositide metabolism. Mol
Microbiol 71: 1341–1352.
Wiley DJ, Nordfeldth R, Rosenzweig J, DaFonseca CJ, Gustin R,
Wolf-Watz H & Schesser K (2006) The Ser/Thr kinase activity
of the Yersinia protein kinase A (YpkA) is necessary for full
virulence in the mouse, mollifying phagocytes, and
disrupting the eukaryotic cytoskeleton. Microb Pathogenesis
40: 234–243.
Wilton M, Subramaniam R, Elmore J, Felsensteiner C, Coaker G
& Desveaux D (2010) The type III effector HopF2Pto targets
Arabidopsis RIN4 protein to promote Pseudomonas syringae
virulence. P Natl Acad Sci USA 107: 2349–2354.
Wood MW, Jones MA, Watson PR, Siber AM, McCormick BA,
Hedges S, Rosqvist R, Wallis TS & Galyov EE (2000) The
secreted effector protein of Salmonella dublin, SopA, is
translocated into eukaryotic cells and influences the induction
of enteritis. Cell Microbiol 2: 293–303.
Wurtele M, Wolf E, Pederson KJ, Buchwald G, Ahmadian MR,
Barbieri JT & Wittinghofer A (2001) How the Pseudomonas
aeruginosa ExoS toxin downregulates Rac. Nat Struct Biol 8:
23–26.
Xie HX, Yu HB, Zheng J, Nie P, Foster LJ, Mok YK, Finlay BB &
Leung KY (2010) EseG, an effector of the type III secretion
system of Edwardsiella tarda, triggers microtubule
destabilization. Infect Immun 78: 5011–5021.
Yahr TL, Vallis AJ, Hancock MK, Barbieri JT & Frank DW (1998)
ExoY, an adenylate cyclase secreted by the Pseudomonas
aeruginosa type III system. P Natl Acad Sci USA 95:
13899–13904.
Yarbrough ML, Li Y, Kinch LN, Grishin NV, Ball HL & Orth K
(2009) AMPylation of Rho GTPases by Vibrio VopS disrupts
effector binding and downstream signaling. Science 323:
269–272.
Yoshida S, Katayama E, Kuwae A, Mimuro H, Suzuki T &
Sasakawa C (2002) Shigella deliver an effector protein to
trigger host microtubule destabilization, which promotes Rac1
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
26
activity and efficient bacterial internalization. EMBO J 21:
2923–2935.
Yoshida S, Handa Y, Suzuki T, Ogawa M, Suzuki M, Tamai A, Abe
A, Katayama E & Sasakawa C (2006) Microtubule-severing
activity of Shigella is pivotal for intercellular spreading. Science
314: 985–989.
Zhang Y, Higashide WM, McCormick BA, Chen J & Zhou D
(2006) The inflammation-associated Salmonella SopA is a
HECT-like E3 ubiquitin ligase. Mol Microbiol 62: 786–793.
Zhang ZY, Clemens JC, Schubert HL, Stuckey JA, Fischer MW,
Hume DM, Saper MA & Dixon JE (1992) Expression,
purification, and physicochemical characterization of a
recombinant Yersinia protein tyrosine phosphatase. J Biol
Chem 267: 23759–23766.
Zhang ZY, Wang Y & Dixon JE (1994) Dissecting the catalytic
mechanism of protein-tyrosine phosphatases. P Natl Acad Sci
USA 91: 1624–1627.
Zhou D, Mooseker MS & Galan JE (1999) Role of the S.
typhimurium actin-binding protein SipA in bacterial
internalization. Science 283: 2092–2095.
Zhu M, Shao F, Innes RW, Dixon JE & Xu Z (2004) The crystal
structure of Pseudomonas avirulence protein AvrPphB: a
papain-like fold with a distinct substrate-binding site. P Natl
Acad Sci USA 101: 302–307.
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
P. Dean
Zhu W, Yang B, Chittoor JM, Johnson LB & White FF (1998)
AvrXa10 contains an acidic transcriptional activation domain
in the functionally conserved C terminus. Mol Plant Microbe In
11: 824–832.
Zhu Y, Li H, Long C, Hu L, Xu H, Liu L, Chen S, Wang DC &
Shao F (2007) Structural insights into the enzymatic
mechanism of the pathogenic MAPK phosphothreonine lyase.
Mol Cell 28: 899–913.
Zhu Y, Li H, Hu L, Wang J, Zhou Y, Pang Z, Liu L & Shao F
(2008) Structure of a Shigella effector reveals a new
class of ubiquitin ligases. Nat Struct Mol Biol 15:
1302–1308.
Zurawski DV, Mitsuhata C, Mumy KL, McCormick BA &
Maurelli AT (2006) OspF and OspC1 are Shigella flexneri
type III secretion system effectors that are required for
postinvasion aspects of virulence. Infect Immun 74:
5964–5976.
Zurawski DV, Mumy KL, Faherty CS, McCormick BA & Maurelli
AT (2009) Shigella flexneri type III secretion system effectors
OspB and OspF target the nucleus to downregulate the host
inflammatory response via interactions with retinoblastoma
protein. Mol Microbiol 71: 350–368.
FEMS Microbiol Rev ]] (2011) 1–26