arial size=-1 color=blue>
« back to results for ""
Below is a cache of http://jcs.biologists.org/cgi/reprint/114/7/1331.pdf. It's a snapshot of the page taken as our search engine crawled the Web.
The web site itself may have changed. You can check the current page or check for previous versions at the Internet Archive. Yahoo! is not affiliated with the authors of this page or responsible for its content.
Rac1 regulates apical invasion by Salmonella INTRODUCTION
The bacterial pathogen Salmonella typhimurium invades its
animal hosts by entering and traversing the epithelial
monolayer lining the intestine. Salmonella achieve this end by
inducing epithelial cells, which are normally non-phagocytic,
to internalize them in a process resembling phagocytosis
(Francis et al., 1993; Takeuchi, 1967). Bacterial adhesion to the
tips of apical microvilli activates a type III, contact-dependent
secretory apparatus, through which bacterial gene products
essential to the invasion process are released into the host cell
cytosol (Collazo and Galan, 1997; Hueck et al., 1995; Kaniga
et al., 1995; Miller et al., 1989). Localized disassembly of
microvilli at these adhesion sites is followed by formation of
actin-rich membrane ruffles, which are ultimately responsible
for bacterial internalization (Finlay et al., 1991; Francis et al.,
1992). Since ruffle formation is essential to the invasion
process, understanding the development of these structures is
critical to understanding Salmonella pathogenesis as a whole.
The regulation of cortical actin dynamics in response to
extracellular stimuli has been ascribed to members of the Rho
family of small GTPases, which includes RhoA, Rac1, and
Cdc42 (Hall, 1998; Van Aelst and DSouza-Schorey, 1997).
As with other GTPases, Rho family members cycle between
active (GTP-bound) and inactive (GDP-bound) conformations.
Accessory factors modulate the nucleotide state of the
GTPases. Guanine nucleotide exchange factors (GEFs) activate
GTPases by facilitating the exchange of bound GDP for GTP.
GTPase activating proteins (GAPs) enhance the intrinsic
hydrolysis rate of the GTPases to promote their inactivation.
In broblasts, activation of RhoA promotes formation of stress
bers and focal contacts; Rac1 yields lamellipodia and dorsal
ruffles; and Cdc42 leads to the extension of lopodia (Kozma
et al., 1995; Nobes and Hall, 1995; Ridley and Hall, 1992;
Ridley et al., 1992). During cell spreading, Rho family
members function sequentially, with initial activation of
Cdc42, followed by Rac1 and RhoA (Nobes and Hall, 1995;
Ridley et al., 1992). In other actin-dependent processes,
distinct subsets of Rho GTPases become activated, often in a
cell-type specic manner.
The involvement of Rho GTPases in Salmonella
typhimurium invasion was initially examined in nonpolarized
cell lines of both epithelioid (HeLa, COS-1) and broblastic
(Rat-1) lineages. In these cells, Chen et al. demonstrated that
invasion of Salmonella was primarily dependent on Cdc42
(Chen et al., 1996). Expression of a point mutant of Cdc42
unable to bind GTP, which acts in a dominant-inhibitory
manner (Cdc42HsN17), prevented bacterial entry. Expression
1331
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 Rho 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 Rho
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 signicantly 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 ndings, which differ
signicantly 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.
Key words: Salmonella typhimurium, Actin, Epithelial cell, Rac1,
Cdc42
SUMMARY
The GTPase Rac1 selectively regulates
Salmonella
invasion at the apical plasma membrane of polarized
epithelial cells
Alison K. Criss
1,4
, Dawn M. Ahlgren
2
, Tzuu-Shuh Jou
3
, Beth A. McCormick
2
and James E. Casanova
4,
*
1
Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts 02115, USA
2
Department of Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA
3
Department of Internal Medicine, National Taiwan University Hospital, Taipei 100, Taiwan
4
Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908, USA
*Author for correspondence (e-mail: jec9e@virginia.edu)
Accepted 16 January 2001
Journal of Cell Science 114, 1331-1341 © The Company of Biologists Ltd
RESEARCH ARTICLE 1332
of dominant-negative Rac1 (Rac1N17) partially inhibited
internalization, but not as effectively as the Cdc42 mutant. In
addition, Cdc42 mediated the Salmonella-induced activation of
the signaling protein Jun kinase (JNK). It was subsequently
determined that this modulation of Rho GTPase activity was
dependent on two Salmonella typhimurium secreted proteins,
SopE and SptP. SopE was characterized as a GEF for Rho
GTPases by its ability to stimulate in vitro nucleotide exchange
on Cdc42, Rac1, and RhoA, and is required for entry. Ectopic
expression of SopE in mammalian cells was sufficient to
induce actin-dependent membrane ruffling (Hardt et al., 1998;
Rudolph et al., 1999; Wood et al., 1996). The recently
described effector SopE2, which has 69% sequence similarity
to SopE, possesses similar nucleotide exchange activity
(Bakshi et al., 2000; Stender et al., 2000). SptP has GAP
activity on Rho GTPases in vitro, and may aid in reassembly
of the actin cytoskeleton after bacterial entry (Fu and Galan,
1999).
Given the unique structure of the enterocyte brush border,
we hypothesized that the cytoskeletal regulatory factors co-
opted by Salmonella during invasion would differ from those
identied in studies with nonpolarized cells. We therefore
investigated the requirement for the GTPases Cdc42 and Rac1
during Salmonella entry into the polarizing epithelial MDCK
cell line. Surprisingly, dominant-negative Rac1, but not Cdc42,
signicantly inhibited bacterial entry at the apical aspect of
these cells. Further, bacterial invasion was associated with an
increase in endogenous Rac1 activation, an effect attributable
to the Salmonella type III secretion system and SopE. These
results suggest that the Rac1 GTPase is primarily responsible
for regulation of Salmonella entry at this physiologically
relevant site.
MATERIALS AND METHODS
Cell culture
The T23 clone of low-resistance MDCK strain II cells, which stably
express the tetracycline-repressible transactivator, was a gift from Y.
Altschuler and K. Mostov (Barth et al., 1997). T23-derived cell lines
expressing activated (G12V) and dominant-negative (T17N) Rac1 and
Cdc42 constructs under control of a tetracycline-responsive promoter
(Gossen and Bujard, 1992) were rst described by Jou et al. (Jou and
Nelson, 1998). Cells were grown in Dulbeccos modied Eagles
medium (DMEM) with 4.5 g/l glucose, 10% FBS, and antibiotics in a
37°C, 5% CO
2
incubator. Transfectants were additionally maintained
in 20 ng/ml doxycycline (Sigma, St Louis, MO) to repress expression
of the transgene.
Induction of gene expression in the mutant Rac1 and Cdc42 T23
cells was achieved in two ways. For invasion assays and biochemical
experiments, cells maintained in the presence of 20 ng/ml doxycycline
were seeded on Transwell lter supports (Costar, Cambridge, MA)
coated with rat tail collagen (Upstate Biotech, Lake Placid, NY).
48-72 hours later, cells were gently washed in Dulbeccos PBS
and replaced in complete DMEM lacking doxycycline to induce
expression. Monolayers were used for experimentation 48 hours after
washout. Cells maintained in doxycycline for the duration of the
experiment were used as controls. Under these conditions, V12Rac
was expressed approximately 3- to 4-fold over endogenous Rac1
levels, and N17Rac was expressed approximately 1- to 2-fold over
endogenous Rac1 (data not shown). Similar results were obtained
for the V12Cdc42 and N17Cdc42 mutants (data not shown).
Transmission electron microscopy conrmed that parental and