Arp2/3-independent assembly of actin
by Vibrio type III effector VopL
Amy D. B. Liverman†, Hui-Chun Cheng‡, Jennifer E. Trosky†, Daisy W. Leung‡, Melanie L. Yarbrough†, Dara L. Burdette†,
Michael K. Rosen‡, and Kim Orth†§
†Department of Molecular Biology and‡Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical
Center, 6000 Harry Hines Boulevard, Dallas, TX 75390
Edited by Thomas D. Pollard, Yale University, New Haven, CT, and approved September 6, 2007 (received for review April 5, 2007)
Microbial pathogens use a variety of mechanisms to disrupt the actin
cytoskeleton during infection. Vibrio parahaemolyticus (V. para) is a
Gram-negative bacterium that causes gastroenteritis, and new pan-
demic strains are emerging throughout the world. Analysis of the V.
para genome revealed a type III secretion system effector, VopL,
encoding three Wiskott–Aldrich homology 2 domains that are inter-
spersed with three proline-rich motifs. Infection of HeLa cells with V.
para induces the formation of long actin fibers in a VopL-dependent
manner. Transfection of VopL promotes the assembly of actin stress
fibers. In vitro, recombinant VopL potently induces assembly of actin
filaments that grow at their barbed ends, independent of eukaryotic
factors. Vibrio VopL is predicted to be a bacterial virulence factor that
disrupts actin homeostasis during an enteric infection of the host.
actin assembly ? microbial pathogenesis ? virulence ? stress fibers ?
or raw shellfish. New pandemic strains of this pathogen are emerg-
ing throughout the world (1). Sequencing of the V. para genome
identified two pathogenicity islands encoding type III secretion
systems, TTSS1 and TTSS2, the latter associated with virulent,
clinical isolates of V. para (2). Iida and colleagues (3, 4) have
to cytotoxicity in infected tissue culture cells, whereas the TTSS on
chromosome two (TTSS2) is associated with enterotoxicity in the
rabbit ileal loop model.
TTSSs transfer virulence factors (also called effectors) from the
bacterial cell to the cytoplasm of the infected cell, resulting in
disruption of a variety of eukaryotic signaling pathways (5). Each
effector mimics or captures the activity of a eukaryotic protein and
to the pathogen during infection. The effectors remain quiescent
inside the pathogen because of the presence of a chaperone or
because the bacterium lacks a specific substrate or activator (6, 7).
Ultimately, utilization of TTSS effector proteins to manipulate
eukaryotic signaling systems ensures the bacterium’s survival.
Two major pathways targeted by these pathogens are the innate
immune system and the actin cytoskeleton (6, 8). The innate
immune system is a key target for bacterial pathogens because
attenuating this system provides an obvious advantage for the
pathogen in the initial stages of infection. Bacterial pathogens also
use and manipulate the actin cytoskeleton, but for diverse reasons.
They can manipulate the actin cytoskeleton to prevent (5, 6) or
induce (9) their own phagocytosis. Other pathogens manipulate
actin assembly to facilitate their movement into, out of, and within
infected host cells (10).
Actin plays a key role in cellular motility and is a major
determinant of the shape of a eukaryotic cell (11). Actin is bound
to ATP or ADP and is found as a monomer (G-actin) or as a
filamentous polymer (F-actin) in a cell. The actin cytoskeleton in a
cell is highly dynamic with continuous assembly and disassembly of
in response to external cues (12). The assembly of actin fibers is a
ibrio parahaemolyticus (V. para) is a Gram-negative bacterium
that causes gastroenteritis after consumption of undercooked
complex process that involves an initial nucleation step requiring
three or more actin monomers that then serve as a priming site for
further polymerization of an actin filament (12). The actin filament
is polarized, with a slow-growing ‘‘pointed’’ end and a fast-growing
‘‘barbed’’ end. A variety of proteins manipulate the actin assembly
process, resulting in an acceleration or inhibition of each of the
several steps involved in actin polymerization. One of these pro-
teins, profilin, is extremely abundant and associated with the
majority of G-actin in a eukaryotic cell (13). Profilin-bound actin
cannot spontaneously nucleate, nor can it be added to the pointed
end of a filament (13).
A key step in actin dynamics is nucleation of new filaments (12).
Eukaryotic cells have specialized machineries to accelerate this
process. The Arp2/3 complex nucleates filaments that grow from
the side of existing filaments, creating branched networks, whereas
formins and SPIRE nucleate unbranched filaments (14–18). A
common mechanistic feature of all three systems is the ability to
assemble actin or actin-like proteins into an arrangement that can
serve as a template for growth of a new filament. The Arp2/3
complex contains two actin-related subunits, which form a pseudo-
actin trimer with an actin monomer provided by activators in the
Wiskott–Aldrich syndrome protein (WASP) family. Formins bind
for filament growth (14). SPIRE proteins have multiple repeats of
Wiskott–Aldrich homology 2 (WH2) domains that bind actin and
appear to create a three-actin template for filament extension (17).
All three of these systems manipulate the structure and dynamics
of the actin cytoskeleton.
Because of the complexity of this system, pathogens are able to
target and manipulate the formation and dissolution of the actin
cytoskeleton using diverse mechanisms. By influencing these sys-
tems, bacteria are able to survive an infection and ensure their
transmission to another host. Herein we describe VopL, a type III
is a bacterial effector that mimics a eukaryotic actin-nucleating
protein by containing all of the necessary domains to enable it to
potently and directly facilitate the assembly of actin. VopL-
mediated actin assembly occurs in a manner that is independent of
any other eukaryotic factor.
Author contributions: A.D.B.L., H.-C.C., J.E.T., D.W.L., M.L.Y., D.L.B., M.K.R., and K.O.
designed research; A.D.B.L., H.-C.C., J.E.T., D.W.L., M.L.Y., and D.L.B. performed research;
M.K.R. contributed new reagents/analytic tools; A.D.B.L., H.-C.C., J.E.T., D.W.L., M.L.Y.,
D.L.B., M.K.R., and K.O. analyzed data; and A.D.B.L., H.-C.C., M.K.R., and K.O. wrote the
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviations: PRM, proline-rich motif; WH2, Wiskott–Aldrich homology 2; V. para, Vibrio
parahaemolyticus; TTSS, type III secretion system; WASP, Wiskott–Aldrich syndrome protein.
§To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
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Vibrio Effector VopL Contains WH2 Domains and Proline-Rich Repeats.
We used a bioinformatic approach to identify effectors on TTSS2
that encode domains similar to those found in other effectors or
was identified as a YopJ-like protein and, subsequently, was shown
to inhibit MAPK signaling pathways (19). Another gene, VopL
(Vibrio outer protein L, VPA1370, 483 aa), was also identified as a
candidate TTSS2 effector protein. It encodes three closely spaced
been shown to promote actin nucleation (17, 20) (Fig. 1A). Inter-
spersed with the WH2 domains are three proline-rich motifs
in VopL closely resemble those found in the FH1 domains of
formins, which are known to bind profilin and profilin–actin
VopL Is Secreted in a TTSS2-Dependent Manner from V. para. To
determine whether VopL is a candidate TTSS effector protein, we
of strains (POR-1, POR-2, and POR-3) that have been character-
ized previously by Iida and colleagues (22). POR-1 is a derivative
of the pathogenic KP-positive RIMD2210633 strain with deletions
The second strain (POR-2) is a derivative of the POR-1 strain that
encodes an additional deletion in vcrD1, an essential inner-
membrane component of TTSS1 (22). The third strain (POR-3) is
also a derivative of POR-1 but encodes a deletion in the vcrD2, an
these two strains encode a functional TTSS2. Analysis of proteins
secreted from the three strains reveals that POR-1 and POR-2, but
not POR-3, secrete VopL (Fig. 1B). These data support the
hypothesis that VopL is secreted from V. para in a TTSS2-
dependent manner (Fig. 1B).
Induction of Actin Stress Fibers During Infection with V. para Is
VopL-Dependent. Using the POR strains to infect HeLa cells, we
dramatic changes in the actin cytoskeleton, inducing long actin
filaments that traverse the cell (Fig. 1 G and H). These structures
have similar morphology to actin stress fibers. We hypothesized,
based on its domain architecture, that VopL might be the effector
that induced the observed changes in the actin cytoskeleton.
Therefore, we deleted VopL from the POR-2 strain and infected
HeLa cells with the mutant POR-2 strain [supporting information
(SI) Fig. 7]. After infection of HeLa cells with the POR-2-?VopL
when compared with POR-2-infected cells (SI Fig. 7 E and F and
SI Fig. 7 C and D, respectively). We predict that this is due to a
combination of confounding effects caused by the presence of
additional effectors/virulence factors and generalized cellular tox-
icity caused by exposure of the cells to endotoxin (23, 24). Inter-
7) and more cells to die, relative to the POR-2-infected cells. When
we reconstituted POR-2-?VopL with pLM1877-VopL and used
this strain to infect HeLa cells, the phenotype of the infected cells
reverted to that of the POR-2-infected cells, with both increased
stress fibers and cell viability (SI Fig. 7 G and H). No change in
infection phenotype was observed with the POR-2-?VopL strain
containing an empty vector (SI Fig. 7 I and J). Based on the
molecular Koch’s postulate, it appears that VopL is a Vibrio TTSS
effector that contributes to changes in cellular morphology, viabil-
ity, and the cytoskeleton (25).
Transfection of VopL Induces Actin Stress Fibers in HeLa Cells. Many
bacterial pathogens use TTSS virulence factors to manipulate the
structure and dynamics of actin filament networks (10). We spec-
ulated that VopL may be like the Drosophila protein SPIRE, which
similarly contains multiple WH2 domains and induces the forma-
tion of actin clusters when transfected into NIH 3T3 cells (17).
VopL, like SPIRE, causes an increase in cellular F actin upon
transfection of eukaryotic cells (17). However, the actin morphol-
ogy induced in NIH 3T3 cells by the two factors is quite different.
dependent manner from V. para. (A) Schematic diagram and amino acid se-
quence of VopL indicating the three PRMs (blue) and the three WH2 domains
(red). (B) Secretion of VopL by the V. para strains POR-1, POR-2, and POR-3
HeLa cells were either mock-infected (C and D) or infected with POR-1 (E and F),
stain nuclei and bacteria. (Magnification: C–J, ?100.).
VopL, a WH2- and PRM-containing protein, is secreted in a TTSS2-
www.pnas.org?cgi?doi?10.1073?pnas.0703196104 Liverman et al.
SPIRE creates actin clusters, whereas VopL creates long parallel
fibers that span transfected NIH 3T3 cells (SI Fig. 8). This is not
cell-type-specific because we also see the induction of long actin
actin and vinculin reveals that VopL induces actin fibers, the ends
distributed evenly around the periphery of the cells rather than
concentrated at opposite ends of the cell as is seen for VopL-
transfected cells. In addition, staining with anti-VopL antibody
reveals localization of VopL along the length of these actin struc-
myosin IIA antibody shows that myosin localizes to the actin fibers
induced by VopL (Fig. 2 M, N, and P) and that VopL localizes to
these same actin structures (Fig. 2 L, N, and O). Myosin colocalizes
to actin structures in vector-transfected cells as well (Fig. 2 I–K).
VopL Induces Stress Fibers in the Presence of Dominant Negative
Small Rho-Like GTPases. Many bacterial virulence factors target the
small Rho family of GTPases including RhoA, Rac, and Cdc42,
which are involved in the formation of stress fibers, lamellipodia,
and filapodia, respectively (11). Bacterial pathogens manipulate
these proteins so that infected cells exhibit either a dominant active
phenotype (constitutive induction of stress fiber or membrane
fibers or membrane ruffling) (26). In HeLa cells, RhoA activation
is associated with the induction of actin stress fibers; therefore, we
hypothesized that VopL might use RhoA to induce changes in the
cytoskeleton of a transfected cell. We cotransfected VopL with
dominant negative RhoA-T19N (RhoDN), which is known to
prevent stress fiber formation induced by upstream stimuli (26). In
(Fig. 3C). Upon coexpression of VopL with RhoDN, transfected
cells contain long actin fibers, similar to those observed in cells
transfected with VopL alone (Fig. 3 B and D). VopL activity is also
observed in cells cotransfected with dominant negative Rac1 or
Cdc42 and VopL (SI Fig. 9). We also analyzed the ability of VopL
fibers. (A–H) HeLa cells were transfected with either empty vector (A–C) or
pSFFV-VopL-FLAG (D–H). The cells were then analyzed by confocal microscopy
using rhodamine–phalloidin to stain for actin (A, D, and G), mouse anti-vincullin
with a FITC-conjugated secondary antibody (B, E, G, and H), and anti-VopL with
using rhodamine–phalloidin to stain for actin (I, L, and O), rabbit polyclonal
for VopL-FLAG (N and P). (Magnification: ?100.)
Transfection of VopL into HeLa cells induces formation of actin stress
images from confocal microscopy using rhodamine–phalloidin to stain for
actin in HeLa cells cotransfected with peGFP-N1 (GFP) and empty vector (A),
pSFFV-VopL-Flag (B), pcDNA3-(HA)3-RhoA T19N (dominant negative) (C), and
RhoA T19N with VopL-Flag (D). (Magnification: ?100.) (E) Quantitation of
transfected cells with increased actin fibers in a double blind study.
VopL activity in the presence of dominant negative RhoA. Shown are
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to induce the formation of stress fibers in the presence of Clostrid-
ium C3 exoenzyme (C3), which potently inactivates RhoA, RhoB,
and RhoC by ADP-ribosylating an asparagine residue located
adjacent to the switch I region (27, 28). Cells expressing C3 alone
lack stress fibers and have only weak actin staining at the cell
periphery. In contrast, coexpression of VopL and C3 produces a
profound phenotype wherein actin is assembled around the inner
rim of the cell membrane (SI Fig. 10). Interestingly, VopL-induced
actin fibers that stretch across the cell are not observed, indicating
that localization of RhoA may play a role in this activity. These
observations support the hypothesis that VopL acts independent of
the Rho family GTPases to induce actin assembly, but the presence
of endogenous wild-type RhoA may play a role in VopL-induced
stress fibers that extend cross the cell.
VopL-Induced Actin Assembly Depends on the WH2 Domains.Because
WH2 domains are linked with actin assembly, we assessed whether
the WH2 domains of VopL are necessary for the formation of the
actin fibers in VopL-transfected cells. We mutated the WH2
domains by substituting four highly conserved amino acids with
alanine residues, as indicated by asterisks below mutated residues
in Fig. 4B. These amino acids have been identified as being
important for actin binding to WH2 domains (17, 20). When all
three WH2 domains are mutated, the resulting mutant VopL-
WH2*?3 has no effect on the actin cytoskeleton (Fig. 4A and SI
Fig. 11G). However, mutation of any single WH2 domain has only
the most C-terminal WH2 mutant (WH2–3*) showing the greatest
decrease in activity (Fig. 4A and SI Fig. 11 C–E). Mutation of two
WH2 domains (VopL-WH2–1*?2*) further decreases actin fiber
formation in transfected HeLa cells (Fig. 4A and SI Fig. 11F),
demonstrating that one WH2 domain is sufficient, although not as
potent as molecules with two or more functional WH2 domains.
Interestingly, VopL does not contain a linker-3 sequence like that
found in SPIRE, nor does it have similarity with Arp2/3 or its
related subunits. Fig. 4B shows conservation in the WH2 domains
from VopL compared with WH2 domains from the human WASP
and WAVE proteins, Drosophila SPIRE, Chlamydia TARP, and
Vibrio cholerae VopF. The WH2 domains in VopL maintain most
of the important hydrophobic residues in the N-terminal ?-helical
region of WH2, and all have the downstream LKKV motif, both of
which are important for actin binding (Fig. 4B) (29).
VopL Nucleates New Actin Filaments That Grow from Their Barbed
Ends. To test whether VopL is able to directly induce the assembly
of actin monomers into filaments, we expressed and purified
recombinant wild-type VopL and the VopL-WH2?3* mutant and
analyzed their activity in an in vitro actin assembly assay (Fig. 5A)
(30). When increasing amounts of purified rVopL (0.1–5 nM) are
added to 4 ?M 5% pyrene-labeled actin, the rate of actin assembly,
indicated by an increase in pyrene fluorescence, is correspondingly
and greatly accelerated over the spontaneous rate (Fig. 5A) (30).
complex maximally activated by the VCA domain of WASP (Fig.
5 A and B). Identical rates of actin assembly are observed with a
truncated, more soluble form of VopL, VopL-WH2C (90–483 aa),
(data not shown).
The effect of VopL on filament assembly could arise from
increased filament nucleation or an increased elongation rate of
existing filaments. However, at concentrations up to 3 nM, VopL-
WH2C has no effect on the initial rate of barbed-end elongation of
actin filament seeds (Fig. 5D). Thus, VopL most likely accelerates
novo. The VopL-WH2C-nucleated filaments grow at their barbed
ends, because assembly of 0.5 ?M actin is substantially blocked by
5 nM capping protein (Fig. 5C). Inhibition is identical whether
capping protein is added to the assay before or after assembly is
induced by VopL-WH2C (data not shown). At higher actin con-
centrations, where filaments can also grow at their pointed ends,
capping protein also substantially inhibits VopL-WH2C-induced
assembly (SI Fig. 12). However, inhibition is not complete under
these conditions because of the filament-nucleating activity of
capping protein at concentrations ?5 nM (31). Because of this
complication, we do not yet know whether VopL-nucleated fila-
ments can grow from their pointed ends, or whether the pointed
ends are blocked as in Arp2/3-nucleated filaments (16). As a
positive control in these assays, we used purified Arp2/3 complex
maximally activated by the VCA domain of WASP (Fig. 5B) (32,
33). For similar maximal rates, VopL assembly shows a shorter lag
phase relative to Arp2/3 complex maximally activated by the VCA
domain of WASP, suggesting a different dependence on actin
filaments of the two nucleation systems (Fig. 5 A and B) (32, 33).
vitro (Fig. 5A) consistent with the idea that the WH2 domains are
required for effects of VopL on the cytoskeleton in vivo (Fig. 4 and
SI Fig. 11). Finally, we found that VopL-WH2C binds to actin
filaments sedimented by centrifugation (Fig. 5E). However, com-
pared with capping protein, which binds only the filament barbed
end, the stoichiometry of VopL-WH2C binding is much higher,
suggesting that VopL can bind filament sides (Fig. 5E). We do not
yet know the relationship, if any, of this filament side binding to
We have identified and characterized VopL, a V. para TTSS
effector protein. The VopL gene is encoded within a pathogenicity
island that correlates with both the virulent strains of V. para and
the emerging pandemic strains of V. para. We observe VopL-
dependent formation of actin stress fibers in infected and trans-
fected HeLa cells. In vitro, the assembly of actin by VopL is direct
Quantitation of transfected cells with increased actin fibers in a double blind
study. (B) An alignment of the WH2 domains (with National Center for Biotech-
(D88460), WAVE1 (D87459), WAVE2 (AB026542), WAVE3 (AB020707), Drosoph-
ila melanogaster Spire (AF184975), Chlamydia TARP (YP?328278), V. cholerae
the WH2 domain mutants are indicated by asterisks below the consensus.
Mutation of the WH2 domains affects VopL activity in HeLa cells. (A)
www.pnas.org?cgi?doi?10.1073?pnas.0703196104Liverman et al.
and extremely potent. Interestingly, strains of another pathogenic
bacterium, V. cholerae, possess a TTSS that encodes a protein with
significant similarity to VopL (34). Tam et al. (35) recently dem-
onstrated that this effector from V. cholerae is able to induce actin
polymerization in vivo and in vitro but that the phenotype in cells is
distinct from that observed with VopL.
Vibrio appears to have usurped eukaryotic WH2 and PRM
domains to create VopL, a TTSS effector that can nucleate actin
filaments in vitro and generate actin stress fibers in vivo. The
phenotype of cells transfected with VopL supports the hypothesis
that VopL induces the formation of unbranched actin structures
observe a similar rate of actin assembly when using 0.5 nM VopL
and 5 nM maximally activated Arp2/3 complex, indicating that
VopL is a more potent activator than the Arp2/3 complex (33).
VopL is also more potent than SPIRE, which requires a ?500 nM
concentration for efficient actin assembly (17). VopL, Arp2/3
complex, and SPIRE all require WH2 domains for activity. In
SPIRE, a minimum of one WH2 domain plus a small peptide
sequence called ‘‘linker 3’’ is needed in vitro, but a nucleation
mechanism has been proposed in which three of SPIRE’s WH2
domains organize actin monomers along a long pitch actin strand
(17). In Arp2/3 complex, only a single WH2 domain, provided by
that during nucleation this WH2 serves to recruit the first actin
monomer to the pseudoactin dimer created by the actin-related
subunits of Arp2/3 complex (18). Like SPIRE, VopL contains
SPIRE-like fashion to assemble an actin trimer representing one
strand of an actin filament (17). However, our in vivo data indicate
that only one WH2 domain of VopL is necessary to induce actin
stress fiber formation, albeit with reduced efficiency relative to the
wild-type protein. Additional studies are needed to elucidate the
molecular mechanism of VopL-mediated actin assembly.
including mimicking or capturing a eukaryotic activity, higher
partners that are found only in eukaryotes (37). First, VopL utilizes
multiple eukaryotic WH2 domains to manipulate the actin cy-
toskeleton. Second, VopL encodes an activity that is orders of
(A) A rVopL (0.1–5 nM) or rVopL-WH2?3* mutant was
incubated with 4 ?M actin (5% pyrene-labeled), and
changes in fluorescence were measured over time. (B)
Comparison of rVopL (5 nM) and Arp2/3 (5 nM) maxi-
mally activated with WASP VCA (500 nM). The proteins
were incubated with 4 ?M actin (5% pyrene-labeled),
and changes in fluorescence were measured over time.
‘‘Control’’ refers to the spontaneous assembly of 4 ?M
grow at their barbed ends. Shown are pyrene actin po-
pyrene), 5 nM VopL-WH2C (WH2C), and mouse capping
protein ?1?2 (CP) from 0.2 nM to 10 nM. At 10 nM, CP
completely inhibits barbed-end filament growth. (D)
VopL has no effect on filament elongation. Shown is
elongation of 1 ?M phalloidin-stabilized actin filaments
with 0.5 ?M actin monomer (40% pyrene) in the pres-
binds filament sides. Shown are filament binding assays
stabilized actin filaments. In this assay, GST-VopL-WH2C
is used so that it can be distinguished from actin by
SDS/PAGE. Lane 1, 1 ?M GST-VopL-WH2C alone; lanes
2–6, 5 ?M actin filaments and 1, 2.5, 5, 7.5, and 10 ?M
GST-VopL-WH2C, respectively; lane 7, 5 ?M actin fila-
1 ?g) and rVopL-WH2?3* (mt, 1 ?g).
volved in assembly of actin filaments. The formation of actin filaments can be
nucleation factors, which control the rate-limiting step. Bacterial effectors (red)
manipulate the state of GTPases by mimicking GTPase-activating proteins (Sal-
proteolytic (Yersinia YopT) or kinase activity (Yersinia YpkA). Effectors (Listeria
ActA, Rickettsia RickA, and Shigella IscA) can also hijack the Arp2/3 nucleation
induce actin assembly, albeit inefficiently, and others induce the formation of
to have usurped all of the domains necessary to be an extremely efficient
nucleation factor for actin and profilin-bound actin.
Bacterial virulence factors that manipulate eukaryotic machinery in-
Liverman et al.PNAS ?
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magnitude more efficient than its predicted eukaryotic counter- Download full-text
parts, thereby enabling the pathogen to tip the balance in a
host/microbe interaction in favor of the invading pathogen (6, 38).
Third, VopL uses a substrate (actin) that is found only in the
eukaryotic host, which supports the hypothesis that this extremely
active protein is quiescent in the pathogen because of lack of
substrate (5, 6).
Bacterial pathogens use a number of mechanisms to manipulate
the actin cytoskeleton, including GTPase exchange factors and
GTPase-activating proteins, proteases, and kinases (Fig. 6). Still
other bacterial pathogens use virulence factors, such as ActA from
Listeria, which encodes multiple PRMs to manipulate the eukary-
otic nucleating factor Arp2/3 (39, 40). The TARP protein from
Chlamydia contains only one WH2 domain and is less potent than
VopL in in vitro actin assembly assays (41). In addition, a family of
bacterial G protein mimics have been identified that replicate
signaling properties of Rho-like GTPase in manipulating the actin
Herein we present studies on VopL, a nucleating factor func-
tioning independent of other eukaryotic proteins that promotes
actin assembly in a manner even more efficient than its eukaryotic
counterparts. We propose that V. para uses VopL to disrupt actin
homeostasis in the epithelial cells of the gut during infection,
thereby initiating an enterotoxic effect in the intestine. Support for
this hypothesis comes from observations by Nelson and colleagues
integrity of the monolayers.
Identifying and characterizing bacterial effector proteins is im-
portant, not only for our understanding of new and emerging
pandemic bacterial strains, but also for identifying and character-
izing key components and mechanisms of eukaryotic signaling
systems. Future microbial, biochemical, and biophysical studies are
aimed at elucidating the mechanism of actin assembly that VopL
has so efficiently usurped.
Materials and Methods
Details. Methods for the following are in SI Text: bioinformatics,
Vibrio secretion, infections, gene disruption, confocal micros-
copy, protein purification, quantitation of transfections, pyrene
actin assembly assays, and barbed-end elongation assays.
Vectors and Strains.pSFFV-VopL-Flag,pSFFV-VopL-WH2C(90–
483 aa), pGEX-rTEV-VopL, pGEX-rTEV VopL-WH2C, and
WH2 mutants (SI Figs. 11 C–G and 12) were made by using
standard molecular biology techniques (44, 45). pCMV5-C3 was a
kind gift from P. C. Sternweis (University of Texas Southwestern
Medical Center). V. para strains POR-1, POR-2, and POR-3 were
obtained from T. Iida and T. Honda (Osaka University, Osaka,
Japan) (3, 22). Expression plasmids containing dominant negative
[pcDNA3-(HA)3-RhoA T19N] RhoA, Rac1 T17N, and CDC42
T17N were obtained from the Guthrie cDNA Resource Center.
was obtained from L. McCarter (University of Iowa, Ames, IA)
(46). See SI Text for specifics.
Cell Culture and Transfections. HeLa and NIH 3T3 cells were
passaged under standard conditions, and transfected with Fu-
GENE (Roche Diagnostics). See SI Text for specifics.
Pyrene Actin Assembly Assays. Actin polymerization assays were
performed as described previously (36). See SI Text for specifics.
Actin Filament Binding Assays. Filament binding assays were con-
ducted as described previously (47, 48). Briefly, phalloidin-
stabilized actin filaments were incubated with various concentra-
tions of GST-VopL-WH2C, GST, or capping protein in KMEI
buffer for 30 min before centrifugation at 200,000 ? g for 20 min.
Supernatants and pellets were analyzed by SDS/PAGE and Coo-
We thank Drs. T. Iida and T. Honda for their generosity in supplying
Vibrio strains. For their kind support and assistance, we thank Dr.
Michelle Laskowski-Arce and other members of the K.O. laboratory,
Drs. Linda McCarter and Igor Rybkin. K.O., D.L.B., and J.E.T. are
supported by National Institute of Allergy and Infectious Diseases
Grants R01-AI056404 and R21-DK072134 and Welch Research Foun-
dation Grant I-1561. A.D.B.L. and M.L.Y. are supported by National
Institutes of Health Training Grant T32-GM08203-18. H.-C.C. and
D.W.L. are supported by National Institutes of Health Grants R01-
GM056322 and P01-GM066311 and Welch Research Foundation Grant
I-1544. M.K.R. is a Howard Hughes Medical Institute Investigator. K.O.
is a Burroughs Wellcome Investigator in Pathogenesis of Infectious
Disease and a C. C. Caruth Biomedical Scholar.
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