The bacterial pathogen Salmonella typhimurium colonizes its animal hosts by inducing its internalization into intestinal epithelial cells. This process requires reorganization of the actin cytoskeleton of the apical plasma membrane into elaborate membrane ruffles that engulf the bacteria. Members of the Ρ family of small GTPases are critical regulators of actin structure, and in nonpolarized cells, the GTPase Cdc42 has been shown to modulate Salmonella entry. Because the actin architecture of epithelial cells is organized differently from that of nonpolarized cells, we examined the role of two ‘Rgr; family GTPases, Cdc42 and Rac1, in invasion of polarized monolayers of MDCK cells by S. typhimurium. Surprisingly, we found that endogenous Rac1, but not Cdc42, was activated during bacterial entry at the apical pole, and that this activation required the bacterial effector protein SopE. Furthermore, expression of dominant inhibitory Rac1 but not Cdc42 significantly inhibited apical internalization of Salmonella, indicating that Rac1 activation is integral to the bacterial entry process. In contrast, during basolateral internalization, both Cdc42 and Rac1 were activated; however, neither GTPase was required for entry. These findings, which differ significantly from previous observations in nonpolarized cells, indicate that the host cell signaling pathways activated by bacterial pathogens may vary with cell type, and in epithelial tissues may further differ between plasma membrane domains.

REFERENCES

Adam
T.
,
Giry
M.
,
Boquet
P.
,
Sansonetti
P.
(
1996
).
Rho-dependent membrane folding causes Shigella entry into epithelial cells.
EMBO J
15
,
3315
3321
Bakshi
C. S.
,
Singh
V. P.
,
Wood
M. W.
,
Jones
P. W.
,
Wallis
T. S.
,
Galyov
E. E.
(
2000
).
Identification of SopE2, a Salmonella secreted protein which is highly homologous to SopE and involved in bacterial invasion of epithelial cells.
J. Bacteriol
182
,
2341
2344
Barth
A. I.
,
Pollack
A. L.
,
Altschuler
Y.
,
Mostov
K. E.
,
Nelson
W. J.
(
1997
).
NH2-terminal deletion of beta-catenin results in stable colocalization of mutant beta-catenin with adenomatous polyposis coli protein and altered MDCK adhesion.
J. Cell Biol
136
,
693
706
Benard
V.
,
Bohl
B. P.
,
Bokoch
G. M.
(
1999
).
Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases.
J. Biol. Chem
274
,
13198
13204
Braga
V. M.
,
Machesky
L. M.
,
Hall
A.
,
Hotchin
N. A.
(
1997
).
The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts.
J. Cell Biol
137
,
1421
1431
Caron
E.
,
Hall
A.
(
1998
).
Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases.
Science
282
,
1717
1721
Chen
L.-M.
,
Hobbie
S.
,
Galan
J. E.
(
1996
).
Requirement of CDC42 for Salmonella -induced cytoskeletal and nuclear responses.
Science
274
,
2115
2118
Chen
L. M.
,
Bagrodia
S.
,
Cerione
R. A.
,
Galan
J. E.
(
1999
).
Requirement of p21-activated kinase (PAK) for Salmonella typhimurium -induced nuclear responses.
J. Exp. Med
189
,
1479
1488
Cherayil
B. J.
,
McCormick
B. A.
,
Bosley
J.
(
2000
).
Salmonella enterica serovar typhimurium -dependent regulation of inducible nitric oxidesynthase expression in macrophages by invasins SipB, SipC, and SipD and effector SopE2.
Infect. Immun
68
,
5567
5574
Collazo
C. M.
,
Galan
J. E.
(
1997
).
The invasion-associated type-III protein secretion system in Salmonella — a review.
Gene
192
,
51
59
Cox
D.
,
Chang
P.
,
Zhang
Q.
,
Reddy
P. G.
,
Bokoch
G. M.
,
Greenberg
S.
(
1997
).
Requirements for both Rac1 and Cdc42 in membrane ruffling and phagocytosis in leukocytes.
J. Exp. Med
186
,
1487
1494
Crago
A. M.
,
Koronakis
V.
(
1998
).
Salmonella InvG forms a ring-like multimer that requires the InvH lipoprotein for outer membrane localization.
Mol. Microbiol
30
,
47
56
Daefler
S.
,
Russel
M.
(
1998
).
The Salmonella typhimurium InvH protein is an outer membrane lipoprotein required for the proper localization of InvG.
Mol. Microbiol
28
,
1367
1380
Finlay
B. B.
,
Gumbiner
B.
,
Falkow
S.
(
1988
).
Penetration of Salmonella through a polarized Madin-Darby Canine Kidney epithelial cell monolayer.
J. Cell Biol
107
,
221
230
Finlay
B. B.
,
Falkow
S.
(
1990
).
Salmonella interactions with polarized human intestinal Caco-2 epithelial cells.
J. Infect. Dis
162
,
1096
1106
Finlay
B. B.
,
Ruschkowski
S.
,
Dedhar
S.
(
1991
).
Cytoskeletal rearrangements accompanying Salmonella entry into epithelial cells.
J. Cell Sci
99
,
283
296
Francis
C. L.
,
Starnbach
M. N.
,
Falkow
S.
(
1992
).
Morphological and cytoskeletal changes in epithelial cells occur immediately upon interaction with Salmonella typhimurium grown under low oxygen conditions.
Mol. Microbiol
6
,
3077
3087
Francis
C. L.
,
Ryan
T. A.
,
Jones
B. D.
,
Smith
S. J.
,
Falkow
S.
(
1993
).
Ruffles induced by Salmonella and other stimuli direct macropinocytosis of bacteria.
Nature
364
,
639
642
Fu
Y.
,
Galan
J. E.
(
1999
).
A Salmonella protein antagonizes Rac-1 and Cdc42 to mediate host-cell recovery after bacterial invasion [see comments].
Nature
401
,
293
297
Gewirtz
A. T.
,
Siber
A. M.
,
Madara
J. L.
,
McCormick
B. A.
(
1999
).
Orchestration of neutrophil movement by intestinal epithelial cells in response to Salmonella typhimurium can be uncoupled from bacterial internalization.
Infect. Immun
67
,
608
617
Gossen
M.
,
Bujard
H.
(
1992
).
Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.
Proc. Nat. Acad. Sci. USA
89
,
5547
5551
Hall
A.
(
1998
).
Rho GTPases and the Actin Cytoskeleton.
Science
279
,
509
514
Handler
J. S.
(
1989
).
Overview of epithelial polarity.
Annu. Rev. Physiol
51
,
729
740
Hardt
W. D.
,
Chen
L. M.
,
Schuebel
K. E.
,
Bustelo
X. R.
,
Galan
J. E.
(
1998
).
S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells.
Cell
93
,
815
826
Hueck
C. J.
,
Hantman
M. J.
,
Bajaj
V.
,
Johnston
C.
,
Lee
C. A.
,
Miller
S. I.
(
1995
).
Salmonella typhimurium secreted invasion determinants are homologous to Shigella Ipa proteins.
Mol. Microbiol
18
,
479
490
Jones
B. D.
,
Paterson
H. F.
,
Hall
A.
,
Falkow
S.
(
1993
).
Salmonella typhimurium induces membrane ruffling by a growth factor-receptor-independent mechanism.
Proc. Nat. Acad. Sci. USA
90
,
10390
10394
Jones
B. D.
,
Ghori
N.
,
Falkow
S.
(
1994
).
Salmonella typhimurium initiates murine infection by penetrating and destroying the specialized epithelial M cells of the Peyer's patches [see comments].
J. Exp. Med
180
,
15
23
Jou
T. S.
,
Nelson
W. J.
(
1998
).
Effects of regulated expression of mutant RhoA and Rac1 small GTPases on the development of epithelial (MDCK) cell polarity.
J. Cell Biol
142
,
85
100
Jou
T. S.
,
Schneeberger
E. E.
,
Nelson
W. J.
(
1998
).
Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases.
J. Cell Biol
142
,
101
115
Kaniga
K.
,
Tucker
S.
,
Trollinger
D.
,
Galan
J. E.
(
1995
).
Homologsof the Shigella IpaB and IpaC invasins are required for Salmonella typhimurium entry into cultured epithelial cells.
J. Bacteriol
177
,
3965
3971
Kozma
R.
,
Ahmed
S.
,
Best
A.
,
Lim
L.
(
1995
).
The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts.
Mol. Cell. Biol
15
,
1942
1952
Lee
C. A.
,
Falkow
S.
(
1990
).
The ability of Salmonella to enter mammalian cells is affected by bacterial growth state.
Proc. Nat. Acad. Sci. USA
87
,
4304
4308
Lee
D. J.
,
Cox
D.
,
Li
J.
,
Greenberg
S.
(
2000
).
Rac1 and Cdc42 are required for phagocytosis, but not NF-kappaB-dependent gene expression, in macrophages challenged with Pseudomonas aeruginosa.
J. Biol. Chem
275
,
141
146
Massol
P.
,
Montcourrier
P.
,
Guillemot
J. C.
,
Chavrier
P.
(
1998
).
Fc receptor-mediated phagocytosis requires CDC42 and Rac1.
EMBO J
17
,
6219
6229
Miller
S. I.
,
Kukral
A. M.
,
Mekalanos
J. J.
(
1989
).
A two-component regulatory system (phoP phoQ) controls Salmonella typhimurium virulence.
Proc. Nat. Acad. Sci. USA
86
,
5054
5058
Mounier
J.
,
Vasselon
T.
,
Hellio
R.
,
Lesourd
M.
,
Sansonetti
P. J.
(
1992
).
Shigellaflexneri enters human colonic Caco-2 epithelial cells through the basolateral pole.
Infect. Immun
60
,
237
248
Nobes
C. D.
,
Hall
A.
(
1995
).
Rho, Rac and cdc42 GTPases regulate the assembly of multimolecular focal adhesion complexes associated with actin stress fibers, lamellipodia and filopodia.
Cell
81
,
53
62
Ridley
A. J.
,
Hall
A.
(
1992
).
The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.
Cell
70
,
389
399
Ridley
A. J.
,
Paterson
H. F.
,
Johnston
C. L.
,
Diekmann
D.
,
Hall
A.
(
1992
).
The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.
Cell
70
,
401
410
Rohatgi
R.
,
Ma
L.
,
Miki
H.
,
Lopez
M.
,
Kirchhausen
T.
,
Takenawa
T.
,
Kirschner
M. W.
(
1999
).
The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly.
Cell
97
,
221
231
Rudolph
M. G.
,
Weise
C.
,
Mirold
S.
,
Hillenbrand
B.
,
Bader
B.
,
Wittinghofer
A.
,
Hardt
W. D.
(
1999
).
Biochemical analysis of SopE from Salmonella typhimurium, a highly efficient guanosine nucleotide exchange factor for RhoGTPases.
J. Biol. Chem
274
,
30501
30509
Sander
E. E.
,
van Delft
S.
,
ten Klooster
J. P.
,
Reid
T.
,
van der Kammen
R. A.
,
Michiels
F.
,
Collard
J. G.
(
1998
).
Matrix-dependent Tiam1/Rac signaling in epithelial cells promotes either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase.
J. Cell Biol
143
,
1385
1398
Simons
K.
,
Wandinger-Ness
A.
(
1990
).
Polarized sorting in epithelia.
Cell
62
,
207
210
Stender
S.
,
Friebel
A.
,
Linder
S.
,
Rohde
M.
,
Mirold
S.
,
Hardt
W. D.
(
2000
).
Identification of SopE2 from Salmonella typhimurium, a conserved guanine nucleotide exchange factor for Cdc42 of the host cell.
Mol. Microbiol
36
,
1206
1221
Symons
M.
,
Derry
J. M.
,
Karlak
B.
,
Jiang
S.
,
Lemahieu
V.
,
McCormick
F.
,
Francke
U.
,
Abo
A.
(
1996
).
Wiskott-Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs, is implicated in actin polymerization.
Cell
84
,
723
734
Takeuchi
A.
(
1967
).
Electron microscope studies of experimental Salmonella infection. I. Penetration into the intestinal epithelium by Salmonella typhimurium.
Am. J. Pathol
50
,
109
136
Van Aelst
L.
,
D'Souza-Schorey
C.
(
1997
).
Rho GTPases and signaling networks.
Genes Dev
11
,
2295
2322
Wood
M. W.
,
Rosqvist
R.
,
Mullan
P. B.
,
Edwards
M. H.
,
Galyov
E. E.
(
1996
).
SopE, a secreted protein of Salmonella dublin, is translocated into the target eukaryotic cell via a sip-dependent mechanism and promotes bacterial entry.
Mol. Microbiol
22
,
327
338
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