INFECTION AND IMMUNITY, Oct. 2006, p. 5964–5976
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 10
OspF and OspC1 Are Shigella flexneri Type III Secretion System
Effectors That Are Required for Postinvasion Aspects of Virulence
Daniel V. Zurawski,1Chieko Mitsuhata,1† Karen L. Mumy,2Beth A. McCormick,2
and Anthony T. Maurelli1*
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland,1and
Mucosal Immunology Laboratory, Massachusetts General Hospital, Charlestown, and Department of Microbiology
and Molecular Genetics, Harvard Medical School, Boston, Massachusetts2
Received 10 April 2006/Returned for modification 30 May 2006/Accepted 17 July 2006
Shigella flexneri is the causative agent of dysentery, and its pathogenesis is mediated by a type III secretion
system (T3SS). S. flexneri secretes effector proteins into the eukaryotic cell via the T3SS, and these proteins
usurp host cellular functions to the benefit of the bacteria. OspF and OspC1 are known to be secreted by S.
flexneri, but their functions are unknown. We transformed S. flexneri with a plasmid that expresses a two-
hemagglutinin tag (2HA) in frame with OspF or OspC1 and verified that these proteins are secreted in a
T3SS-dependent manner. Immunofluorescence of HeLa cells infected with S. flexneri expressing OspF-2HA or
OspC1-2HA revealed that both proteins localize in the nucleus and cytoplasm of host cells. To elucidate the
function of these T3SS effectors, we constructed ?ospF and ?ospC1 deletion mutants by allelic exchange. We
found that ?ospF and ?ospC1 mutants invade host cells and form plaques in confluent monolayers similar to
wild-type S. flexneri. However, in the polymorphonuclear (PMN) cell migration assay, a decrease in neutrophil
migration was observed for both mutants in comparison to the migration of wild-type bacteria. Moreover,
infection of polarized T84 intestinal cells infected with ?ospF and ?ospC1 mutants resulted in decreased
phosphorylation of extracellular signal-regulated kinase 1/2 in comparison to that of T84 cells infected with
wild-type S. flexneri. To date, these are the first examples of T3SS effectors implicated in mitogen-activated
protein kinase kinase/extracellular signal-regulated kinase pathway activation. Ultimately, OspF and OspC1
are essential for PMN transepithelial migration, a phenotype associated with increased inflammation and
bacterial access to the submucosa, which are fundamental aspects of S. flexneri pathogenesis.
Shigella species are responsible for dysentery (shigellosis) in
humans, which starts as an acute infection in the large intes-
tine, which is followed by cramps, diarrhea, and fever. The
infection is usually self-limiting, but it can also cause damage to
the colonic mucosa, intestinal bleeding, and death if untreated.
Worldwide, Shigella spp. infections are responsible for approx-
imately 163 million cases of dysentery and 1 million deaths
each year (23). The majority of infections occur in third-world
countries, where contaminated food and drinking water are
common (23); however, even developed nations still have mul-
tiple Shigella outbreaks every year (47). Therefore, studying
the pathogenesis of these gram-negative bacteria is of utmost
importance, particularly in light of emerging antibiotic resis-
tance, the lack of an appropriate vaccine, and the potential for
use of Shigella as a bioweapon (23, 33, 42).
Following ingestion, Shigella flexneri eventually reaches the
large intestine, the target site for infection, where access to the
basolateral membrane is a prerequisite for the invasion of
epithelial cells (see reference 48 for a review). M cells in the
large intestine phagocytose and subsequently transcytose the
bacteria from the lumen to the submucosal side of the epithe-
lial barrier (48). Once S. flexneri cells reach the submucosa,
they are engulfed by resident macrophages (48). S. flexneri cells
escape from the macrophage phagosome and kill the macro-
phage quickly, thus providing access to the basolateral side of
the epithelial barrier (22, 36, 53, 62). S. flexneri cells invade the
colonic epithelial cells at the basolateral membrane and, fol-
lowing invasion, are found inside a membrane-bound vacuole
(48). S. flexneri cells escape from this vacuole like they escape
from the macrophage phagosome; however, instead of killing
the epithelial cells, S. flexneri cells replicate in the cytoplasm
and spread cell to cell throughout the colonic epithelium (48).
Another aspect of S. flexneri pathogenesis is the disruption
of tight junctions, which leads to the loss of cell-to-cell contact
between polarized epithelial cells of the colon (45). One result
of this disruption is facilitation of the transepithelial migration
of polymorphonuclear leukocytes (PMN) to the luminal side of
the epithelial barrier (31, 41, 48). Lipopolysaccharide and un-
identified factors activate the mitogen-activated protein kinase
kinase/extracellular signal-regulated kinase signaling pathway
(MEK/ERK pathway), as measured by the phosphorylation of
ERK1/2 (21). In turn, the phosphorylation of ERK1/2 and its
localization to the nucleus result in the production of signaling
molecules required to recruit PMN into the lumen (21, 59).
PMN migration through the colonic epithelium may also pro-
vide additional openings to the submucosa for S. flexneri (41,
48). As infection progresses, increased tight junction disrup-
tion and PMN recruitment lead to severe inflammation and to
the destruction of the epithelial barrier associated with dysen-
tery (21, 31, 41, 45, 48).
S. flexneri possesses a 218-kb plasmid that harbors most of its
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, 4301 Jones Bridge Rd., Bethesda, MD 20814-
4799. Phone: (301) 295-3415. Fax: (301) 295-1545. E-mail: amaurelli
† Present address: Department of Pediatric Dentistry, Hiroshima
University, Hiroshima, Japan.
at WALTER REED ARMY MED CTR on April 23, 2008
virulence determinants (2, 49). A 31-kb region of this plasmid,
termed the mxi-spa locus, encodes the proteins necessary to
assemble a type III secretion system (T3SS) (40, 50). The
primary function of the T3SS is to secrete proteins, called
effectors, into the host to modify cell function and overcome
cell defenses (15, 34). In S. flexneri pathogenesis, the T3SS is
required for invasion, vacuolar escape, cell-to-cell spread, and
PMN transepithelial migration (5, 17, 31, 48).
The transcription and expression of the S. flexneri T3SS are
induced by the VirF/VirB system when the temperature is
shifted from 30°C to 37°C (54). The expression of the first set
of effector proteins secreted by S. flexneri is activated by VirB,
and these proteins are essential for invasion (25, 50). Secretion
from the S. flexneri T3SS is dependent on contact with the host
cell, specifically an interaction between IpaB and cholesterol
(4, 12, 57). However, secretion can also be artificially induced
in liquid growth media (1).
A second set of putative S. flexneri effectors are designated
OspB to OspG (outer Shigella protein) (2). These proteins are
secreted by S. flexneri into the growth medium in a ?ipaB
deletion mutant background (2), but most have undefined
functions. Some osp genes appear to be the result of gene
duplication during evolution, and each gene is at least ?70%
homologous to the other genes in the same group (2). Se-
quencing of the virulence plasmid from S. flexneri serotype 5
revealed three ospD genes (ospD1 to ospD3), four ospC genes
(ospC1 to ospC4), and two ospE genes (ospE1 and ospE2).
Interestingly, osp gene regulation varies, even between homo-
logues. For example, ospD1 and ospD2 are up-regulated solely
by VirB, while ospD3 is regulated solely by MxiE, a separate
transcription factor active only when S. flexneri is inside the
host cell (17, 25, 30). Finally, a third group of osp genes, which
includes ospF and ospC1, is regulated by both VirB and MxiE,
suggesting that these proteins are required throughout the
course of infection (25).
The purpose of this study was to identify the contribution of
the ospF and ospC1 genes to S. flexneri pathogenesis. We de-
termined that the OspF and OspC1 proteins are indeed se-
creted by the T3SS, and we elucidated their localization inside
the host cell. Furthermore, we generated ospF and ospC1 de-
letion mutants to examine the impact of these genes on phe-
notypes associated with virulence. While ospF and ospC1 de-
letion mutations did not obstruct invasion or cell-to-cell
spreading, these mutations did interfere with postinvasion as-
pects of S. flexneri pathogenesis. OspC1 was found to be re-
quired for the inflammation and swelling characteristics asso-
ciated with a positive Sere ´ny reaction in an animal model. Both
OspF and OspC1 were essential for Shigella-induced transepi-
thelial PMN migration and the up-regulation of the MEK/
ERK pathway that is required for PMN recruitment.
MATERIALS AND METHODS
Strains and growth conditions. All bacterial strains used in this study are listed
in Table 1. Escherichia coli and Salmonella enterica strains used for molecular
biology applications were routinely cultured in Luria-Bertani broth (LB). S.
flexneri was routinely cultured in tryptic soy broth. All bacteria grown overnight
were incubated at 37°C with aeration. Unless indicated otherwise, antibiotics
were used in growth media at the following concentrations: kanamycin, 50 ?g/ml;
chloramphenicol, 25 ?g/ml; spectinomycin, 100 ?g/ml; streptomycin, 25 ?g/ml;
and ampicillin, 200 ?g/ml.
Tissue culture. HeLa cells and L2 mouse fibroblast cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum (FBS). The J774.1 murine macrophage cell line was maintained in
DMEM containing 10% FBS supplemented with 2 mM glutamine and essential
amino acids. The human epithelial colon cancer-derived cell line T84 (passages
46 to 66) was maintained in DMEM/F-12 supplemented with 15 mM HEPES
(pH 7.5) and 10% FBS. To obtain polarized monolayers, T84 cells were grown
on 0.33- or 4.7-cm2collagen-coated permeable polycarbonate filters (Costar)
that had pore sizes of 5.0 and 3.0 ?m, respectively, and they were utilized after
they reached a confluent and differentiated state, as previously described (5, 21,
31). All tissue culture media were acquired from Invitrogen, and all cell lines
were maintained in the presence of 5% CO2at 37°C.
Plasmid construction. All plasmids and primers used in this study are de-
scribed in Tables 1 and 2. S. flexneri genes were amplified by PCR using Vent
polymerase (New England Biolabs), cloned into pGEM-T (Promega), and se-
quenced. ospF was amplified with BamHI and Acc65I sites engineered at the 5?
end of its forward primer and a BglII site engineered at the 3? end of the reverse
primer. ospC1 and ipaH9.8were cloned into pGEM-T with Acc65I and BglII
restriction enzyme sites engineered into the amplifying primers. To make the
pBAD constructs, both ospF and ospC1 were excised from pGEM-T by NcoI
digestion and BglII digestion and cloned into pBAD24 cut with the same en-
To make the tagged fusion plasmids, ospF was liberated from pGEM-T using
the SapI and BglII restriction enzymes and was cloned into pACB C2-HA that
was cut with SalI (blunted with T4 DNA polymerase) and BglII. The resulting
vector was designated pDZ1. The ospB promoter, at positions ?300 to ?1
upstream of ospB, was amplified with primers with BamHI ends and cloned into
the BamHI site engineered into pDZ1. The resulting plasmid was designated
pDZ2. ospC1 and ipaH9.8were subcloned into pDZ2 digested with Acc65I and
For cloning into pDsRed2-C1 or EGFP-C1, ospF or ospC1 was excised from
pGEM-T using the Acc65I and BglII restriction enzymes. The fragments were
ligated into the Acc65I and BamHI sites found in the multicloning site of both
red fluorescent protein (RFP) and green fluorescent protein (GFP) vectors. The
spvC gene from Salmonella enterica serovar Typhimurium 1344 was amplified by
PCR with Acc65I and BglII restriction sites engineered at the 5? and 3? ends as
well and was cloned into pGEM-T. spvC was digested with these restriction
enzymes and cloned into pDsRed2-C1 in the same way that ospF was cloned.
Mutant construction. The ?ospF and ?ospCI deletions in S. flexneri were
generated using a modification of the method of Datsenko and Wanner (3),
where the lambda red recombination genes, red and gam, were driven by the
PTACpromoter on the pKM208 plasmid (35). Briefly, PCR was used to generate
a chloramphenicol resistance cassette gene (cat) with sequences at the 5? and 3?
ends identical to sequences 20 bp internal to and 30 bp upstream of the gene
being deleted (Table 2). BS766 (S. flexneri carrying pKM208) was grown over-
night at 30°C, subcultured in LB without NaCl, and grown at 30°C. When late log
phase (optical density at 600 nm [OD600], 0.9 to 1.0) was reached, isopropyl-?-
D-thiogalactopyranoside (IPTG) (1 mM) was added to the medium to induce
expression of the lambda red recombination genes, and the bacteria were shifted
to 37°C for 30 min. After heat shock at 42°C and electroporation (35), bacteria
were recovered in SOC medium and plated on tryptic soy broth plates containing
Congo red (CR) and chloramphenicol at a concentration of 5 ?g/ml or 10 ?g/ml.
Cmrcolonies on these plates were purified and screened by PCR using three
different primer sets to identify the deletion mutants, and positive recombinants
were verified by sequencing. To remove cat from BS771 and BS772, these strains
were transformed with pCP20 and incubated at 42°C (3) to generate BS814 and
BS816. We verified that all mutations that were created did not have an effect on
S. flexneri growth.
Congo red secretion assay. Bacterial cultures following overnight incubation
were subcultured and grown at 37°C. Once late log phase (OD600, 0.7 to 0.8) was
reached, bacterial samples were normalized to the same OD600, and CR (0.7
?g/ml) was added. After 1 h, whole-cell lysates and supernatants were prepared.
Whole-cell lysates were generated by centrifugation of 1 ml of bacterial culture,
followed by one wash with ice-cold 1? phosphate-buffered saline (PBS). Cells
were centrifuged again and resuspended in equal amounts of Tris-glycine gel
sample buffer (24). CR supernatants were generated by centrifugation of 20 ml
of S. flexneri grown in liquid medium. The supernatants were removed, centri-
fuged again, and passed through a 0.45-?m-pore-size filter (Millipore). T3SS
secreted proteins were precipitated by addition of trichloroacetic acid (final
concentration, 10%), and this was followed by acetone washing and resuspension
of the pellet in 50 ?l gel sample buffer.
OspF antibody production. Glutathione S-transferase (GST)–OspF was gen-
erated by cloning the ospF gene from pGEM-T into the BamHI/XhoI sites of the
VOL. 74, 2006OspF AND OspC1 ARE REQUIRED FOR SHIGELLA VIRULENCE5965
at WALTER REED ARMY MED CTR on April 23, 2008
pGEX 6P-1 vector (Amersham) and transforming the preparation into BL21/
pLysS. Subsequently, GST-OspF was expressed by addition of IPTG (1 mM) and
was purified from bacterial lysates using glutathione beads (Amersham). OspF
was cleaved from GST using PreScission protease (Amersham), purified, and
dialyzed against 1? PBS. The molecular mass of this purified OspF was ?27.5
kDa, as determined by Coomassie blue staining (data not shown), and 200 ?g/ml
was injected into New Zealand White rabbits every other week for a total of 6
weeks. Bleeding of rabbits and serum purification were carried out 8 and 10
weeks after the initial injection (Spring Valley Antibodies).
Virulence assays. S. flexneri invasion assays were carried out as previously
described (9), and CFU were counted and compared to the amount of input
bacteria to calculate the invasion efficiency. Plaque assays were performed as
previously described (38). Briefly, L2 fibroblast monolayers were grown to con-
fluence and infected with S. flexneri strains whose concentrations were standard-
ized to an OD600of 0.30. Two dilutions of bacteria were used, and the assay was
performed at least twice in triplicate. Plaques were enumerated after 3 days. The
Sere ´ny test was used to assess invasion and the in vivo inflammatory response in
guinea pigs as previously described (52). Briefly, 2.5 ? 108CFU of wild-type S.
flexneri, BS771, or BS772 was used to infect one guinea pig eye. At least three
guinea pigs were used to evaluate each strain used for each experiment, and
symptoms were monitored for 4 days. Experiments were repeated at least twice.
To assess macrophage killing, ?5 ? 104J774.1 macrophages were seeded and
grown on acid-washed (0.1 N HCl) 12-mm coverslips overnight in 24-well tissue
culture plates. Bacteria were subcultured, grown to late log phase, and added to
wells at a multiplicity of infection (MOI) of ?200. After 1 h, macrophage death
was evaluated using a Live/Dead kit (Invitrogen), and the numbers of dead cells
(orange, ethidium) and live cells (green, fluorescein) per 100 total visible cells
were determined. This assay was done a minimum of two times in triplicate.
The PMN migration assay was performed as previously described (31). Shigella
cells were added to the basolateral side of 0.33-cm2transwells at an MOI of ?100
for 90 min. Cells were washed and then incubated in fresh media containing
gentamicin (50 ?g/ml) for 90 min. PMN were added to the basolateral compart-
ment, and transmigration to the apical compartment was quantified by assaying
for the PMN azurophilic granule marker myeloperoxidase. For inducible
complementation of deletion mutations, arabinose was added at a final concen-
tration of 0.6% to tissue culture media. Unpaired Student t tests were used to
analyze statistical significance in comparisons between the mutant strains and
TABLE 1. Strains and plasmids used in this study
Strain or plasmid Genotype and/or description
S. flexneri strains
Wild-type Shigella flexneri 2a
Virulence plasmid-cured derivative of 2457T
2457T/?spa47 (spa47::aadA), Specr
2457T transformed with pKM208 Ampr
2457T/?ospF (ospF::cat), Cmr
2457T/?ospC1 (ospC1::cat), Cmr
2457T transformed with pDZ2, Cmr
BS103 transformed with pDZ2, Cmr
2457T transformed with pDZ3, Cmr
BS103 transformed with pDZ3, Cmr
BS652 transformed with pDZ3, Cmr
2457T transformed with pDZ7, Cmr
BS771 with cat eliminated using FLP recombinase
BS814 transformed with pDZ2, Cmr
BS772 with cat eliminated using FLP recombinase
BS817 transformed with pDZ3, Cmr
E. coli strains
endA1 hsdR17 supE44 thi-1 recA1 gyrA96 relA1 (?lacZYA-argF)U169 deoR ?80dlacZ?M15
?) dcm gal ?(DE3) pLysS(Cmr) tonA
S. enterica 1344Wild-type S. enterica serovar Typhimurium 1344 hisG46 13
bla cat oriR6K
Temperature-sensitive red-, gam-, and lacI-expressing plasmid driven by PTacpromoter,
PipB2-2HA fusion, pACYC184 backbone, Cmr
OspF-2HA expression driven by POspB, Cmr
Protein expression vector results in GST fusion, Ampr
OspF cloned into pGEX 6P-1 with BamHI/XhoI, Ampr
OspC1 cloned into pGEX 6P-1 with BamHI/XhoI, Ampr
Arabinose-inducible vector, pBR322ori, Ampr
OspF cloned into NcoI/BamHI sites of pBAD24
OspC1 cloned into NcoI/BamHI sites of pBAD24
Red fluorescent protein vector, Kanr
Green fluorescent protein vector, Kanr
ospF N-terminal RFP fusion
ospC1 N-terminal GFP fusion
5966ZURAWSKI ET AL.INFECT. IMMUN.
at WALTER REED ARMY MED CTR on April 23, 2008
ERK1/2 phosphorylation was detected as previously described (21). Briefly,
polarized T84 monolayers on 4.7-cm2transwells were infected and harvested
with lysis buffer (1% Triton X-100, 100 mM NaCl, 10 mM HEPES, 2 mM EDTA,
4 mM Na3VO4, 40 mM NaF, 200 mM phenylmethylsulfonyl fluoride, protease
inhibitor cocktail [Roche]). Samples were centrifuged, and the supernatant rep-
resenting the cytosol was saved and stored at ?80°C until it was used.
Polyacrylamide gel electrophoresis and immunoblot analysis of proteins. For
protein analysis, samples were resolved by sodium dodecyl sulfate (SDS)-poly-
acrylamide gel electrophoresis (PAGE) on Tris-glycine gels (24). For immuno-
blotting, proteins were transferred to pure nitrocellulose membranes, and hem-
agglutinin (HA)-tagged proteins were detected by addition of mouse anti-HA
monoclonal antibody HA.11 (Covance). ERK1/2 samples were immunoblotted
using mouse monoclonal antibody against phosphorylated ERK1/2 and goat
polyclonal antibody against ERK1/2 (Santa Cruz). Bands were visualized using
sheep anti-mouse and sheep anti-goat secondary antibodies conjugated to horse-
radish peroxidase (Amersham). Rabbit anti-OspF antibody was detected using
donkey anti-rabbit polyclonal antibody conjugated to horseradish peroxidase
(Amersham). All primary and secondary antibodies were used at a 1:1,000
dilution. All blots were developed using Visualizer (Upstate), and images were
captured with a charge-coupled device camera from the LAS-3000 CH imaging
system (Fuji) or on film.
Immunofluorescence and transfection analysis. A total of ?5.0 ? 104HeLa
cells were seeded and grown on acid-treated, 12-mm coverslips overnight in
24-well tissue culture plates. S. flexneri was subcultured and grown at 37°C until
late log phase was reached. Bacterial cell numbers were normalized to an OD600
of 0.72, washed, and resuspended in 1 ml of DMEM. Bacteria were added to cells
at an MOI of ?200 bacteria/HeLa cell. The 24-well plates were centrifuged at
37°C for 10 min at 3,000 ? g and incubated at 37°C in the presence of 5% CO2
for 30 min. Then cells were washed twice with PBS, followed by the addition of
DMEM with gentamicin (50 ?g/ml). After 90 min, cells were washed three times
with PBS, and DMEM with gentamicin was added again. Cells were washed once
with PBS and fixed by addition of 3% paraformaldehyde for 10 min at 37°C for
After fixation, infected HeLa cells were analyzed by immunofluorescence.
Briefly, cells were washed twice with PBS and then blocked and permeabilized
with 10% natural goat serum (NGS) and 0.1% saponin, respectively, for 30 min
at room temperature. Cells were washed once with PBS and incubated in a PBS
solution containing mouse anti-HA monoclonal antibody HA.11 (Covance), 10%
NGS, and 0.1% saponin for 1 h. After three PBS washes, cells were incubated
with goat anti-mouse antibody conjugated to Alexa Fluor 488 (Invitrogen), 10%
NGS, and 0.1% saponin for 1 h. The primary antibody was used at a 1:1,000
dilution, and the secondary antibody was used at a 1:800 dilution. Cells were
washed three times with PBS and once with sterile distilled H2O before they
were mounted on slides using Anti-Fade reagent (Invitrogen).
For transfection experiments, HeLa cells were seeded as described above in
24-well plates. Then 1.0 ?g of RFP-OspF, GFP-OspC1, pDs-Red2, pEGFP-C1,
GFP-actin, or pEGFP-tub DNA was complexed with GeneJammer reagent and
incubated with cells according to the manufacturer’s specifications (Stratagene).
At 24 h posttransfection, cells were washed twice with PBS and fixed by addition
of 3% paraformaldehyde for 10 min at 37°C. HeLa cells were occasionally
stained with 4?,6?-diamidino-2-phenylindole (DAPI) (0.5 ?g/ml) for 20 min or
with anti-beta-tubulin antibody conjugated to fluorescein isothiocyanate (FITC)
(Sigma) at a 1:50 dilution for 1 h. All cell images were acquired with an Olympus
1X81 fluorescent microscope using a SensiCam charge-coupled device camera
Secretion of OspF and OspC1 is dependent on the S. flexneri
T3SS. While it was previously shown by N-terminal protein
sequencing that OspF and at least one OspC protein are se-
creted by S. flexneri (2), we wanted to determine if OspF and
OspC1 secretion is T3SS dependent. Therefore, we con-
structed a plasmid in which the ospF and ospC1 genes were
cloned in frame with a two-hemagglutinin (2HA) tag. Tran-
scription of ospF and ospC1 was driven by the ospB promoter
(POspB), which was cloned upstream of both genes. We chose
POspBto drive expression because this promoter is activated to
the highest degree inside eukaryotic cells compared to the
activation of other MxiE-regulated genes (17).
The OspF-2HA plasmid (pDZ2) and the OspC1-2HA plas-
mid (pDZ3) were transformed into 2457T to produce BS784
and BS792, respectively, and were transformed into BS103 (a
strain with a virulence plasmid-cured background) to produce
BS785 and BS793, respectively (Table 1). The pDZ3 plasmid
was also transformed into a ?spa47 mutant (BS652) to gener-
ate BS794 in order to evaluate the direct role of the T3SS in
OspC1 secretion. Spa47 is the ATPase of the S. flexneri T3SS
and is required for a functional T3SS (16). In order to evaluate
OspF secretion in a ?spa47 mutant background, a rabbit poly-
clonal antibody against recombinant OspF was generated.
However, cross-reactivity with other OspC proteins (OspC2 to
OspC4) prevented the use of anti-OspC1 antibody in a similar
2457T, BS784 (OspF-2HA in 2457T), BS103, and BS785
TABLE 2. Primers used in this study
Forward primer Reverse primer
Amplify the cassette for
deletion of ospF
Amplify the cassette for
deletion of ospC1
Clone POspBinto pDZ1 OspBProF AATAGGGGATCCTACCTGAC
Clone ospF into pGEM-T OspFF
Clone ospC1 into pGEM-T OspC1ForOspC1Rev
Clone ipaH9.8into pGEM-TIpaH9.8F
Clone spvC into pGEM-TSpvCF SpvCR
aUnderlining identifies an engineered Acc65I, BglII, or EcoRI restriction site.
VOL. 74, 2006 OspF AND OspC1 ARE REQUIRED FOR SHIGELLA VIRULENCE 5967
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(OspF-2HA in BS103) were grown separately in medium con-
taining CR to induce the T3SS. After 1 h of exposure to CR,
OspF-2HA (?29 kDa) was found in the whole-cell and super-
natant fractions of BS784; in contrast, no signal was detected in
the supernatant of BS785 (Fig. 1A). These results verify that
OspF secretion requires the virulence plasmid and that the
2HA tag does not inhibit the secretion of this protein.
To verify our 2HA results and to confirm the involvement of
the T3SS, 2457T, BS652 (?spa47), and BS103 were grown in
the presence of CR and analyzed using anti-OspF antibody.
BS771, a ?ospF deletion mutant, was used as a negative control
for the antibody. OspF was detected in only the supernatant of
the 2457T sample (Fig. 1B), and no OspF was detected in the
supernatant of BS652 (?spa47), which confirmed that the T3SS
is required for secretion of OspF.
Analysis of OspC1-2HA yielded results similar to the OspF
secretion profile. OspC1-2HA (BS792) was secreted only when
the T3SS was functional (Fig. 1C), and addition of the 2HA tag
to the C terminus also had no effect on OspC1 secretion. A
BS103 (virulence plasmid-cured) background (BS793) or a de-
letion in spa47 (BS794) abrogated the secretion of OspC1-
2HA (Fig. 1C), which implicated OspC1 as a T3SS effector as
well. It should be noted that the total protein secreted was
analyzed by Coomassie blue staining, and no discernible dif-
ference was observed between the overall secretion profiles of
2457T and the ?ospF and ?ospC1 mutants, which indicates
that mutations in ospF and ospC1 do not appear to affect the
secretion of other T3SS effectors (data not shown).
Virulence phenotypes of ?ospF and ?ospC1 mutants. (i)
Invasion assay. To gain insight into the function of the S.
flexneri effectors, OspF and OspC1, we used the lambda red
recombination system to generate allelic exchange mutants,
where the target gene was deleted and replaced with a Cmr
cassette. The resulting mutants, BS771 (?ospF) and BS772
(?ospC1), were then characterized to determine their S. flex-
neri virulence phenotypes. First, we compared the mutant
strains to wild-type parent strain 2457T with a standard inva-
sion assay using HeLa cells. In a typical experiment, 2457T
showed an invasion efficiency of 0.32% ? 0.04%. In the same
assay, BS771 had an invasion efficiency of 0.37% ? 0.06%, and
BS772 had an invasion efficiency of 0.33% ? 0.05%. We also
performed an invasion assay using polarized T84 cells, and no
difference in invasion efficiency was observed between BS771,
BS772, and 2457T (data not shown). Therefore, deletion of
either ospF or ospC1 did not alter the invasion of epithelial
cells by S. flexneri in either a nonpolarized or polarized tissue
(ii) Plaque assay. In order to assess the ability of the mutant
strains to invade, replicate, and spread cell to cell, BS771 and
BS772 were examined with the plaque assay (38). Again, no
significant differences were observed between the mutants and
2457T when the numbers of plaques or plaque sizes were
compared. MxiE regulates the transcription of ospF and ospC1,
and a ?mxiE mutant was previously shown to produce fewer
plaques and plaques having reduced sizes (17). Therefore, the
loss of OspF or OspC1 does not contribute to this ?mxiE
(iii) Macrophage killing. S. flexneri rapidly causes cell death
in infected macrophages (36, 62). As a model system, J774.1
murine macrophage cells in tissue culture are killed by S.
flexneri after 1 h of infection (36, 62). We incubated J774.1
macrophages for 1 h with BS771, BS772, and 2457T and mea-
sured cell death using fluorescent reporters. Both BS771 and
BS772 retained the ability to kill J774.1 macrophages similar to
wild-type bacteria (data not shown).
(iv) Sere ´ny test. The Sere ´ny test (52) was used to analyze the
effects of the ?ospF and ?ospC1 mutations on S. flexneri in-
fection in vivo. Guinea pig eyes were exposed to BS771, BS772,
and 2457T at a dose of 2.5 ? 108CFU. After 4 days, no
significant differences between the BS771- and 2457T-infected
guinea pigs were observed, as both groups developed conjunc-
tivitis and a strong inflammatory response (data not shown).
However, in all three guinea pigs infected with the BS772
strain the production of symptoms of conjunctivitis was de-
layed and there was less swelling and inflammation compared
to the guinea pigs infected with 2457T. In fact, one of the
BS772-infected guinea pigs cleared the bacterial infection after
2 days (data not shown). These results demonstrate that
OspC1 contributes to S. flexneri virulence when this animal
model of infection is used.
FIG. 1. T3SS-dependent secretion of OspF and OspC1. Congo red
was added to a final concentration of 7 ?g/ml to activate the secretion
of T3SS effectors. After 1 h, two sets of samples were saved: whole cell
fractions and supernatant. OspF samples were run on a 12% SDS–
PAGE gel, and OspC1 samples were run on a 10% SDS–PAGE gel.
(A) Samples were immunoblotted with anti-HA antibody to visualize
OspF-2HA (?29-kDa band). (B) Samples were immunoblotted with
anti-OspF antibody (?27.5-kDa band). (C) Samples were immuno-
blotted with anti-HA antibody to visualize OspC1-2HA (?54-kDa
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(v) PMN transepithelial migration. Not only does PMN
transepithelial migration provide a mechanism for S. flexneri
access to the basolateral side of the epithelial barrier, but
neutrophil recruitment is also responsible for the inflammation
associated with disease as modeled in the Sere ´ny test (41, 48).
The PMN transepithelial migration assay has been used pre-
viously to demonstrate that a functional T3SS of S. flexneri is
required for neutrophil migration (21, 31). In light of the
reduced inflammation with the ?ospC1 strain in the Sere ´ny test
and the fact that OspF and OspC1 are T3SS effectors, we
wanted to assess the ability of the ?ospF and ?ospC1 strains to
induce PMN transepithelial migration in a polarized tissue
BS771 and BS772 were used to infect a monolayer of polar-
ized T84 cells and compared to 2457T. BS103 served as a
negative control. After 180 min of infection, neutrophils were
added to the basolateral side of the transwell, and PMN trans-
epithelial migration was evaluated. We found that when both
deletion mutant strains were normalized to 2457T, there was a
statistically significant decrease (P ? 0.0001) in the amount of
neutrophils that migrated from the basolateral compartment to
the apical compartment of the polarized monolayer (Fig. 2).
We constructed plasmids with ospF or ospC1 cloned into the
pBAD24 vector (Table 1) and transformed these plasmids into
BS771 and BS772 to complement the deletion mutations. The
PMN migration assay was repeated in the presence of arabi-
nose to induce expression of the cloned ospF and ospC1 genes,
and the complementing strains (BS802 and BS803, respec-
tively) restored PMN migration to levels that were equal to or
greater than the wild-type levels. We also complemented ospF
and ospC1 deletion mutants (BS814 and BS816) with pDZ2
and pDZ3 (2HA-tagged proteins) and obtained similar results
(data not shown), suggesting that the tagged versions of these
proteins are still functional. These results suggest that OspF
and OspC1 are essential for the signaling events that stimulate
PMN transepithelial migration.
OspF and OspC1 contribute to Shigella-induced ERK1/2
phosphorylation. S. flexneri synthesizes factors during the
course of infection that initiate a host signaling cascade in
which a downstream target, the ERK1/2 protein, becomes
phosphorylated on threonine residues (5, 21). Phosphorylated
ERK1/2 (P-ERK) has at least 160 substrates in both the cytosol
and nucleus that regulate genes or proteins that are associated
with proliferation, differentiation, the cytoskeleton, and cell
cycle progression (59). Some S. flexneri mutants that show a
reduction in the PMN migration assay also have reduced
MEK/ERK pathway activation, as measured by the phosphor-
ylation of ERK1/2 (5, 21). Since both the ?ospF and ?ospC1
mutants displayed reduced activity in the PMN migration as-
say, we predicted that we would also observe a reduction in
MEK/ERK signaling in cells infected with these mutants.
Therefore, we infected polarized T84 cells with 2457T, BS771,
BS772, and BS103 and directly compared the amounts of P-
ERK by Western blotting using a monoclonal antibody specific
for the phosphorylated form of the ERK1/2 protein. Both
BS771- and BS772-infected cells showed a significant reduc-
tion in the amount of P-ERK (Fig. 3). Densitometric analysis
indicated that there were decreases of 62% and 43% in phos-
phorylated ERK1/2 for BS771- and BS772-infected cells, re-
spectively, compared to cells infected with wild-type S. flexneri.
Cells infected with BS103, the noninvasive mutant, displayed a
45% decrease in phosphorylated ERK1/2 compared to the
amount in wild-type infections. These results suggest that the
OspF and OspC1 proteins are secreted into the cytoplasm of
the host cell and play a role in MEK/ERK pathway activation.
FIG. 2. Transepithelial PMN migration induced by wild-type S.
flexneri. T84 polarized monolayers were infected with 2457T, BS771
(?ospF), BS772(?ospC1), BS802(pBAD-OspF),
OspC1), and BS103, and then PMN migration was evaluated. HBSS,
uninfected monolayers (buffer only). All strains were normalized to
wild-type strain 2457T. Experiments were performed three times in
triplicate. The data are means ? standard deviations (error bars) of
triplicate samples and represent one of the three experiments per-
formed in which similar results were obtained. Samples were com-
pared using an unpaired t test, and statistically significant differences
are indicated by an asterisk.
FIG. 3. ERK phosphorylation in S. flexneri-infected cells. T84 po-
larized monolayers were infected with 2457T, BS771 (?ospF), BS772
(?ospC1), and BS103, and then ERK phosphorylation was evaluated
180 min postinvasion. HBSS, uninfected monolayers (buffer only).
Lysates of monolayers were immunoblotted with monoclonal anti-P-
ERK antibody (anti-P-ERK), which recognized the phosphorylated
ERK1/2 proteins (p44 and p42), or anti-ERK1/2 antibody (anti-ERK1/
2), which was a total protein control and demonstrated equal loading.
The blot is representative of one experiment that was repeated three
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OspF and OspC1 T3SS effectors target the nucleus of the
host cell. Since the 2HA tag fusions of OspF and OspC1 were
secreted via the T3SS, we used BS784 and BS792 to determine
the intracellular localization of OspF and OspC1 in HeLa cells
infected with these strains. As a control, the gene encoding
IpaH9.8was also cloned into the pDZ2 vector (Table 1), gen-
erating a 2HA fusion. IpaH9.8is an MxiE-regulated T3SS ef-
fector of S. flexneri that localizes to the nucleus and cytoplasm
of HeLa cells (17, 25, 55). We confirmed that IpaH9.8was
secreted by the T3SS and that the 2HA tag did not disrupt its
secretion (data not shown).
Strains that harbored the plasmids with 2HA-tagged effec-
tors were used to infect HeLa cells, and immunofluorescence
using anti-HA antibody was used to evaluate protein localiza-
tion over time after invasion. Untransformed 2457T (no 2HA
plasmid) served as a negative control. The first time analyzed
was 4 h following invasion because IpaH9.8is found in the
nucleus and cytoplasm by this time (55). At 4 h postinvasion,
IpaH9.8-2HA was found in both the cytoplasm and the nucleus
(Fig. 4A), as previously shown for native IpaH9.8(55). OspC1-
2HA was found in the nucleus of host cells, similar to IpaH9.8,
as well as in the vicinity of invading bacteria (Fig. 4A). Simi-
larly, OspF-2HA also localized to the nucleus at this time (Fig.
4A), suggesting that the nucleus is a common destination for
some of the MxiE-regulated effectors of S. flexneri.
We also observed another signal with OspF-2HA that was
localized in the cytoplasm. Subsequently, the OspF-2HA signal
was evaluated at 2, 3, 4, and 5 h postinvasion. At 2 h postin-
vasion, we observed a signal with the anti-HA antibody only in
the cytoplasm, and no nuclear localization was observed (Fig.
4B). At 3, 4, and 5 h postinvasion, we observed that the nuclear
signal became more intense with greater time, suggesting that
there was accumulation of OspF in the nucleus (data not
shown). We did not observe any nuclear OspC1-2HA signal
before 4 h, suggesting that the majority of the protein was too
diffuse in the cytoplasm or that not enough protein was pro-
duced by this point to observe a signal (data not shown).
However, based on these results we believe that cytoplasmic
OspF is targeted to the nucleus, where it accumulates over
Ectopically expressed RFP-OspF and GFP-OspC1 display
nuclear localization. To confirm the nuclear localization of
OspF and OspC1 using a different tag, we cloned ospF and
ospC1 into RFP and GFP mammalian expression vectors to
generate N-terminal fusions of fluorescent proteins to OspF
and OspC1. Vectors were transiently transfected into a semi-
confluent monolayer of HeLa cells, and after 24 h, cells were
fixed and analyzed. RFP-OspF was found in the nucleus of
HeLa cells, and counterstaining with DAPI confirmed the nu-
clear localization (Fig. 5). The nuclear localization of the RFP-
OspF fusion not only supported the results obtained with Shi-
gella-infected cells but also demonstrated that additional S.
flexneri T3SS effectors are not required for OspF localization to
the nucleus. The same was true for OspC1 as the GFP-OspC1
signal was found throughout the transfected cell, including the
nucleus and cytoplasm (Fig. 5). We also observed that the
nuclear signals for both RFP-OspF and GFP-OspC1 increased
as the time posttransfection increased (data not shown), sug-
gesting that both proteins accumulate in the nucleus.
SpvC, a protein encoded on the virulence plasmid of S.
enterica serovar Typhimurium, exhibits 63% homology with
full-length OspF (2, 26). When the C termini (amino acids 100
to 237) of SpvC and OspF were compared, more than 80%
homology was detected (BLAST2). We postulated that SpvC
has a localization similar to that of OspF inside the host cell
because the N-terminal differences could most likely be attrib-
uted to secretion signals for the T3SS (34). Therefore, spvC
was cloned into the same mammalian expression vector as
OspF to generate an RFP-SpvC N-terminal fusion. Surpris-
ingly, RFP-SpvC did not target the nucleus and displayed a
punctate pattern in the cytoplasm of transfected HeLa cells
RFP-OspF colocalizes with microtubules in HeLa cells.
When analyzing the other focal planes of RFP-OspF-trans-
fected cells by microscopy, we noticed signals in the cytoplasm.
These signals had different characteristics, occasionally dis-
playing a punctate pattern of localization and sometimes re-
sembling cytoskeletal filaments (Fig. 6A). RFP-OspF signal
was also found on these structures before the nuclear localiza-
tion was observed, suggesting that there was initial targeting in
the cytoplasm before nuclear targeting. We wanted to identify
these structures because the localization could aid in discov-
ering a host cell mechanism that OspF exploits during S. flex-
Shigella, along with many other gram-negative pathogens,
modulates cytoskeletal components (actin and microtubules)
during infection (48, 60). Therefore, we cotransfected HeLa
cells with RFP-OspF and either GFP-actin or GFP-tubulin
plasmids. When signals were merged, RFP-OspF and GFP-
actin did not have similar localizations (Fig. 6B); however, the
GFP-tubulin signal did colocalize with the filament-like signal
of RFP-OspF (Fig. 6B). This result was verified by transfecting
HeLa cells with RFP-OspF and after 24 h fixing the cells and
staining them with anti-beta-tubulin conjugated to FITC.
Again, we observed colocalization of the RFP-OspF signal
with the anti-tubulin signal (Fig. 6C).
A multitude of T3SS effectors have been identified on the S.
flexneri virulence plasmid that contribute to invasion of the
host epithelial cell. However, only a few genes have been
FIG. 4. Localization of OspF-2HA and OspC1-2HA in infected host cells. (A) Strains 2457T, BS801 (IpaH9.8-2HA), BS792 (OspC1-2HA), and
BS784 (OspF-2HA) were used to infect semiconfluent monolayers of HeLa cells. After 4 h, cells were fixed and evaluated by immunofluorescence
using anti-HA antibody and a secondary goat anti-mouse antibody conjugated to Alexa Fluor 488. The images in the left panels are representative
of the immunofluorescence signals observed with each strain, and the images in the right panels are phase-contrast images of the same cells. The
nucleus is outlined by a dotted line in some images. (B) The experiment described above was repeated, but preparations were fixed and stained
at 2 h. The OspF-2HA signal (black arrows) did not colocalize with bacteria (white arrows). The panel on the left shows the immunofluorescence
signal. The panel on the right is a phase-contrast image of the same cells merged with the immunofluorescence signal.
VOL. 74, 2006 OspF AND OspC1 ARE REQUIRED FOR SHIGELLA VIRULENCE5971
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shown to play a role in the postinvasion aspects of S. flexneri
pathogenesis; these genes are icsA, mxiE, ospG, and ipaH (6,
17, 19, 39, 48). Recent studies have shown that a large number
of putative S. flexneri T3SS effectors encoded on the virulence
plasmid are regulated by MxiE, including many of the Osp
proteins (17, 25, 30). It appears that most of the Osp proteins
are secreted by the bacteria (2); however, the function of the
majority of these proteins is still unknown. It is clear from this
study that OspF and OspC1 function after invasion with re-
spect to S. flexneri pathogenesis similar to other MxiE-regu-
lated proteins (6, 19, 25, 39).
It was shown previously that the T3SS is essential for Shi-
gella-induced PMN transepithelial migration (31); however,
the effectors involved in this phenomenon were not identified.
In this study, we identified two T3SS effectors, OspF and
OspC1, that contribute to the PMN transepithelial migration
phenotype of Shigella virulence. Moreover, the deficiency in
PMN migration was directly correlated to a deficiency in acti-
vation of the MEK/ERK signaling pathway in cells infected
with ?ospC1 and ?ospF mutants. When we evaluated the
?ospC1 and ?ospF mutants with the Sere ´ny test, the ?ospC1
mutant caused reduced inflammation and swelling, and the
infection did not advance as quickly as a wild-type S. flexneri
infection. In fact, one guinea pig cleared the infection with the
?ospC1 mutant. These observations show that the S. flexneri
T3SS effectors OspF and OspC1 are required for essential
postinvasion aspects of virulence associated with S. flexneri
Modulation of the MEK/ERK pathway by T3SS effectors is
a common characteristic of infection by gram-negative patho-
gens. Salmonella spp. secrete SptP that interacts with the Raf
protein upstream of MEK and ERK1/2, resulting in down-
regulation of the MEK/ERK pathway during the early stages of
infection (27). Yersinia spp. secrete a protease called YopJ that
FIG. 5. Ectopic expression of RFP-OspF and GFP-OspC1 in HeLa cells. Semiconfluent monolayers of HeLa cells were transfected with
mammalian expression vectors RFP-OspF, GFP-OspC1, and RFP-SpvC. Following transfection, the nuclei were stained with DAPI (left panels),
and the GFP or RFP fluorescence of the fusion proteins was evaluated (right panels). In each panel the nucleus is outlined by a dotted line.
5972 ZURAWSKI ET AL.INFECT. IMMUN.
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deubiquinates a number of targets in the cytoplasm and, con-
sequently, leads to down-regulation of the MEK/ERK pathway
(61). Finally, a functional T3SS of Bordetella bronchiseptica is
required for down-regulation of the MEK/ERK pathway (43).
However, these are examples of MEK/ERK down-regulation.
The results that we report here suggest that the S. flexneri
effectors OspF and OspC1 are required for up-regulation of
the MEK/ERK pathway. While enteropathogenic E. coli
(EPEC) and enterohemorrhagic E. coli (EHEC) have been
shown to activate the MEK/ERK pathway, this has not be
attributed to T3SS effectors (51). It was recently shown that S.
enterica serovar Typhimurium macrophage infection activates
the MEK/ERK pathway in a Salmonella pathogenicity island
2-dependent manner; however, the T3SS effector responsible
FIG. 6. RFP-OspF localizes to a target in the cytoplasm. (A) HeLa cells were transfected with RFP-OspF. Each panel shows a different field
of cells. (B) HeLa cells were cotransfected with RFP-OspF and GFP-actin or with RFP-OspF and GFP-tubulin. The panels on the left show the
RFP-OspF signal (red), the panels in the middle show the GFP signal (green), and the panels on the right show the merged signals. (C) HeLa cells
were transfected with RFP-OspF, fixed, and stained with anti-beta-tubulin conjugated to FITC. The panel on the left shows the RFP-OspF signal
(red), the panel in the middle shows anti-beta-tubulin FITC signal (green), and the panel on the right shows the merged signals. The insets show
magnified areas of interest. The micrographs are representative of the results of experiments repeated at least three times.
VOL. 74, 2006 OspF AND OspC1 ARE REQUIRED FOR SHIGELLA VIRULENCE5973
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is unknown (56). Therefore, OspF and OspC1 represent the
first T3SS effectors shown to play a role in MEK/ERK pathway
Carboxy-terminal and amino-terminal protein tagging strat-
egies demonstrated that both OspF and OspC1 are T3SS-
dependent effectors that localize to the nucleus and cytoplasm
of HeLa cells. Nuclear localization is also not a novel pheno-
type for secreted bacterial proteins. There are two distinct
classes of proteins secreted by gram-negative bacteria that
localize and function within the nucleus of the host cell. First,
some bacteria secrete proteins that target the nucleus to mod-
ulate the cell cycle of the eukaryotic cell. These proteins have
been classified as cyclomodulins and include proteins such as
cytolethal distending toxin (secreted by many gram-negative
species) and Cif of EPEC and EHEC (37). While we have not
observed any evidence that OspF and OspC1 are cyclomodu-
lins (Zurawski and Maurelli, unpublished), we still cannot con-
clude that these proteins have an effect on cell division.
A second class of gram-negative secreted proteins that target
the nucleus of the host cell have a role in suppressing the
inflammatory response of the immune system typically caused
by lipopolysaccharide. For example, YopM of Yersinia pestis
localizes to the nucleus and reduces the amount of interleu-
kin-15 (IL-15) secreted by host cells (18). Xanthomonas
campestris pathovar vesicatoria, a pathogen of tomato and pep-
per plants, secretes XopD that targets the nucleus and hydro-
lyzes small ubiquitin-related modifier-conjugated proteins to
interfere with the plant defense response (14). Finally, IpaH9.8
from S. flexneri (55) and its homologue SspH1 from Salmonella
(11) also localize to the nucleus of mammalian cells. IpaH9.8
and SspH1 both down-regulate the inflammatory response
postinvasion (11). Specifically, IpaH9.8binds to the human
UAF35 splicing factor to interfere with the transcription of a
number of inflammatory response genes that are up-regulated
during Shigella invasion (39). These genes include the IL-8,
RANTES, colony-stimulating factor 1, and IL-1? genes. The
down-regulation of the innate immune response postinvasion
is also the function of another Shigella Osp protein, OspG.
OspG interacts with ubiquinated proteins to prevent phospho-
I?B? degradation and NF-?B activation induced by tumor
necrosis factor alpha stimulation (19). While it is possible that
OspF and OspC1 modulate cytokine production given their
nuclear localization, we did not see a decrease in IL-8 secretion
levels in cells infected with the ?ospF or ?ospC1 mutants
The ectopic expression of RFP-OspF allowed greater fluo-
rescence intensity to visualize OspF and its dynamic localiza-
tion inside HeLa cells, particularly its colocalization with mi-
crotubules. Cytoskeletal targets are a common target for
modulation by bacterial pathogens and for Shigella T3SS ef-
fectors. IpaH9.8requires microtubules for nuclear import, and
VirA disrupts microtubules to enhance invasion (55, 60). OspF
may use microtubules to mediate its transport to the nucleus,
like IpaH9.8. On the other hand, the localization of OspF to
microtubules could be the result of an interaction with a host
target protein(s). Many eukaryotic proteins involved in or-
ganelle transport and cell division interact with microtubules,
but the fact most relevant to this study is that MEK and
ERK1/2 interact with microtubules (44, 46). Because MEK/
ERK pathway components and OspF have a common local-
ization and because Shigella-induced ERK1/2 phosphorylation
requires OspF, it is possible that OspF directly interacts with
components of the MEK/ERK pathway. We are currently in-
vestigating this possibility.
Homologues of ospF are found in at least two other gram-
negative bacterial species (2, 26). One of these homologues is
spvC, which is found on the pSLT virulence plasmid of S.
enterica serovar Typhimurium and resides in an operon that
encodes the T3SS effector SpvB (28). SpvC is required for full
S. enterica serovar Typhimurium virulence in mice (28), but
nothing else is known about its function. Pseudomonas syringae
DC3000 also has an OspF homologue, HopAI1, which has
recently been shown to be involved in the down-regulation of
the innate immune response in the tomato plant, and overex-
pression of transfected copies of the hopAI1 gene generates
disease-like symptoms in plants (26). However, the contribu-
tion of HopAI1 is unclear and may be redundant, as a deletion
mutation does not result in a measurable difference in the
plant’s immune response (26). The immune response of a
guinea pig infected with the ?ospF mutant of S. flexneri also
displayed no discernible difference in the Sere ´ny test compared
to the immune response of a guinea pig infected with 2457T.
Therefore, OspF and HopAI1 may both have redundant func-
tions in their hosts, and a more sensitive assay to measure the
effect of mutation on virulence is required. For example, an
altered virulence phenotype for the ?ospF mutant was not
observed until we used the PMN migration assay. Additional
studies are required to characterize similar and different func-
tions of SpvC, HopAI1, and OspF.
OspC1 exhibits about 75% homology with the OspC2,
OspC3, and OspC4 proteins of S. flexneri (2). This level of
similarity also occurs for the OspC genes from other Shigella
spp. However, the contribution of the OspC2, OspC3, and
OspC4 proteins to virulence after invasion may be different
from the contribution of OspC1 for three reasons. First, ospC2,
ospC3, and ospC4 are regulated solely by VirB and not by
MxiE (25). Therefore, there should be more OspC1 than its
homologues inside the host cells because of the MxiE activa-
tion (17, 25, 30). Second, OspC2 to OspC4 are 96% homolo-
gous to each other, while OspC1 is more divergent evolution-
arily (2). Third, T84 cells infected with a ?ospC2 mutant do not
show a reduction in PMN transepithelial migration in the way
that the ?ospC1 strain does (unpublished observations). It is
conceivable that OspC proteins have similar but redundant
functions and that OspC1 has the greatest effect on the host.
Evidence that supports redundancy comes from the fact that
ospC4 found in S. flexneri serotype 5 M90T has a point muta-
tion that generates a stop codon, perhaps suggesting that S.
flexneri is evolving to remove the other OspC effectors (2).
In conclusion, in this study we found for the first time that
OspF and OspC1 are T3SS effectors secreted by S. flexneri and
that these effectors are required for Shigella-induced MEK/
ERK pathway activation and PMN transepithelial migration.
PMN transepithelial migration is an important aspect not only
of Shigella virulence but also of Salmonella, EPEC, and EHEC
virulence (32, 51). The results of this study also underscore the
fact that T3SS effectors can mediate virulence phenotypes
shared by enteric pathogens. Therefore, future studies of mo-
lecular mechanisms by which OspF and OspC1 induce host cell
signaling not only should improve our understanding of Shi-
5974ZURAWSKI ET AL.INFECT. IMMUN.
at WALTER REED ARMY MED CTR on April 23, 2008
gella virulence but also may highlight a paradigm that is shared
by other gram-negative, pathogenic bacteria.
We express our gratitude to the Olivia Steele-Mortimer laboratory
(National Institutes of Allergy and Infectious Diseases, Rocky Moun-
tain Laboratories, Montana), especially Leigh Knodler, for the gift of
the pACB C-2HA vector. We acknowledge the Giam laboratory (Uni-
formed Services University of the Health Sciences, Bethesda, MD) for
the gift of the GFP and RFP vectors and the Sanger laboratory (SUNY
Upstate Medical University, Syracuse, NY) for the gift of the GFP-
actin and GFP-tubulin vectors. We also thank Nancy Adams and
Reinaldo Fernandez for technical assistance and D. Scott Merrell for
critical reading of the manuscript.
This work was supported by National Institutes of Allergy and In-
fectious Diseases grant AI24656. B.A.M. was supported by National
Institutes of Health grants DK56754 and DK33506. K.L.M. was sup-
ported by a T32 training grant sponsored by Harvard Medical School
and the Division of Nuclear Medicine at Massachusetts General Hos-
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