Article Images (0)
×![]()
featured_play_listSave article to folder
X
Create new folder
Full Text
PDF
Table of Contents
Abstract
Requirement Of Rho-Family GTPases In The Invasion Of
Juan J. Martinez† And Scott J. Hultgren*
Department Of Molecular Microbiology And Microbial Pathogenesis, Washington University School Of Medicine, St. Louis, MO 63110, USA.
Summary
Introduction
Results
Discussion
Experimental Procedures
Gentamicin Protection (Invasion) Assays
Immunoprecipitations
Acknowledgements
Abstract
flexible tip fibrillum, containing the FimH adhesin, joined to the rod by FimG and FimF (Russell and Orndorff, 1992; Jones et al., 1995). FimH mediates attachment to mannose-containing receptors on a variety of different host cells (Baorto et al., 1997; Malaviya and Abraham, 1998; Mulvey et al., 1998;Martinez et al., 2000). The ability of UPEC to adhere to the luminal surface of the bladder epithelium is a critical event in the colonization and subsequent establishment of disease (Connell et al., 1996; Langermann et al., 1997; Thankavel et al., 1997; Mulvey et al., 1998). UPEC has long been considered to be, primarily, an extracellular pathogen; however, recent studies have demonstrated that UPEC can induce their own internalization into bladder epithelial cells both in vivo and in vitro, and that the invasion process is triggered by FimH (Mulvey et al., 1998; Martinez et al., 2000). Interestingly, the host–pathogen interactions mediated by FimH in the bladder also activate a cascade of innate defences including the induction of cytokines and the recruitment of neutrophils into the bladder (Schilling et al., 2001). In addition, these interactions also trigger an apoptotic-like cascade that leads to the exfoliation of the superficial umbrella cells lining the luminal surface of the bladder (Mulvey et al., 1998). The exfoliation of the superficial umbrella cells is thought to be part of a defence mechanism to eliminate bacteria from the bladder (Mulvey et al., 1998). However, the ability of UPEC to invade into the bladder epithelium has been shown to be part of a mechanism that allows the bacteria to subvert these innate defences and establish persistent urinary tract infections, even in the face of antibiotic treatments that are used commonly to treat UTIs (Mulvey et al., 1998; 2001). Taken together, these data argued that adherence and invasion mediated by FimH are critical events in the pathogenesis of UTIs. Morphologically, the invasion of type 1-piliated E. coli into bladder epithelial cells resembles that of invasion by Listeria and Yersinia species into a variety of nonphagocytic, cultured cell lines. In these cases, uptake is mediated by interactions between adhesin molecules and host receptors that trigger complex signal transduction cascades, ultimately leading to the ‘zippering’ of plasma membrane around the bacterium and the subsequent internalization of the microbe. Activation of PI 3-kinase (Ireton et al., 1996;1999) and focal adhesion kinase (FAK) (Alrutz and Isberg, 1998) are important events in the uptake of Listeria monocytogenes, Yersinia pseudotuberCellular Microbiology (2002) 4(1), 19–28
© 2002 Blackwell Science Ltd
Requirement of Rho-family GTPases in the invasion of
Type 1-piliated uropathogenic Escherichia coli flexible tip fibrillum, containing the FimH adhesin, joined to the rod by FimG and FimF (Russell and Orndorff, 1992; Jones et al., 1995). FimH mediates attachment to mannose-containing receptors on a variety of different host cells (Baorto et al., 1997; Malaviya and Abraham, 1998; Mulvey et al., 1998;Martinez et al., 2000). The ability of UPEC to adhere to the luminal surface of the bladder epithelium is a critical event in the colonization and subsequent establishment of disease (Connell et al., 1996; Langermann et al., 1997; Thankavel et al., 1997; Mulvey et al., 1998). UPEC has long been considered to be, primarily, an extracellular pathogen; however, recent studies have demonstrated that UPEC can induce their own internalization into bladder epithelial cells both in vivo and in vitro, and that the invasion process is triggered by FimH (Mulvey et al., 1998; Martinez et al., 2000). Interestingly, the host–pathogen interactions mediated by FimH in the bladder also activate a cascade of innate defences including the induction of cytokines and the recruitment of neutrophils into the bladder (Schilling et al., 2001). In addition, these interactions also trigger an apoptotic-like cascade that leads to the exfoliation of the superficial umbrella cells lining the luminal surface of the bladder (Mulvey et al., 1998). The exfoliation of the superficial umbrella cells is thought to be part of a defence mechanism to eliminate bacteria from the bladder (Mulvey et al., 1998). However, the ability of UPEC to invade into the bladder epithelium has been shown to be part of a mechanism that allows the bacteria to subvert these innate defences and establish persistent urinary tract infections, even in the face of antibiotic treatments that are used commonly to treat UTIs (Mulvey et al., 1998; 2001). Taken together, these data argued that adherence and invasion mediated by FimH are critical events in the pathogenesis of UTIs. Morphologically, the invasion of type 1-piliated E. coli into bladder epithelial cells resembles that of invasion by Listeria and Yersinia species into a variety of nonphagocytic, cultured cell lines. In these cases, uptake is mediated by interactions between adhesin molecules and host receptors that trigger complex signal transduction cascades, ultimately leading to the ‘zippering’ of plasma membrane around the bacterium and the subsequent internalization of the microbe. Activation of PI 3-kinase (Ireton et al., 1996;1999) and focal adhesion kinase (FAK) (Alrutz and Isberg, 1998) are important events in the uptake of Listeria monocytogenes, Yersinia pseudotuber-
Juan J. Martinez† and Scott J. Hultgren*
Department of Molecular Microbiology and Microbial Pathogenesis, Washington University School of Medicine, St. Louis, MO 63110, USA.
Summary
Bladder infections caused by uropathogenic Escherichia coli (UPEC) depends on the ability of E. coli to express type 1 pili. The adhesive component of the pilus, FimH, mediates the invasion of E. coli into the bladder epithelium, a mechanism that facilitates the survival and persistence of E. coli in the bladder. The invasion mechanism requires actin polymerization, focal adhesion kinase phosphorylation and PI 3-kinase activation as well as the formation of FAK/PI 3-kinase and downstream vinculin/a-actinin complexes. In this study, we report a role for RhoGTPase family members, namely RhoA, Cdc42 and Rac1, in the invasion process. Internalization of type 1-piliated E. coli (fimH +) and FimH-coated microspheres was inhibited by compactin, a pan-RhoGTPase inhibitor and dominant negative isoforms of Rac1 and Cdc42. Expression of active Rac1 induced an internalization of E. coli that was insensitive to wortmannin and genistein. Expression of constitutively active Cdc42 induced the formation of FAK/PI 3-kinase and vinculin/a-actinin complexes whereas active Rac1 induced only a vinculin/aactinin complex. Taken together, these data suggest that FimH-mediated invasion is dependent on GTPbinding protein activity that involves Cdc42 and PI 3-kinase activation probably upstream of Rac1.
Introduction
Escherichia coli is the aetiological agent in more than 80–90% of all urinary tract infections (UTIs). The majority of uropathogenic E. coli (UPEC) isolates express composite, adhesive organelles called type 1pili (Langermann et al., 1997). Type 1pili are composed of a thick, helical rod comprising repeating FimA subunits joined to a thin, Received 20 July, 2000; revised 11 October, 2001; accepted 17 October, 2001. *For correspondence. E-mail hultgren@ borcim.wustl.edu; Tel. (+1) 314 747 3627; Fax (+1) 314 362 1998. †Present address: Institut Pasteur, 25–28 Rue du Dr. Roux, Paris, France 75724 Cedex 15. culosis and UPEC, three pathogens that use the ‘zipper’ mechanism for entry. In some cells, activation of PI 3- kinase and downstream actin polymerization can be mediated by the binding of the p85a subunit of PI 3-kinase to tyrosine-phosphorylated proteins including FAK (Shoelson et al., 1993; Chen and Guan, 1994; Chen et al., 1996a). Infection of bladder epithelial cells with type 1- piliated (fimH+) E. coli led to the transient tyrosine phosphorylation of FAK at Y397, and complex formation with the p85a subunit of PI 3-kinase, as well as the transient formation of a downstream, actin-stabilizing a-actinin– vinculin complex. Inhibitors that blocked uptake also blocked complex formation suggesting that these signalling intermediates were critical in the reorganization of the host actin cytoskeleton leading to bacterial invasion (Martinez et al., 2000). Much work has focused on the role of small GTPbinding proteins, namely Rho-family members RhoA, Cdc42 and Rac, and related members of the Rassuperfamily of GTPases, which act as guanine nucleotide exchange switches to regulate diverse cellular processes; (Ridley and Hall, 1992; Ridley et al., 1992; Olson et al., 1995) including actin cytoskeletal reorganization (Hall, 1998a; 1998b). For example, microinjection of Rac and Cdc42 into host cells induced distinct, actin-rich extensions called lamellipodia (or ruffles) and filopodia respectively (Ridley and Hall, 1992; Nobes and Hall, 1995; Nobes et al., 1995; Hall, 1998a). Interestingly, the Gram-negative invasive pathogens Shigella flexneri and Salmonella typhimurium induce distinct changes in host cells leading to internalization (termed ‘trigger’ mechanism), which resemble those mediated by Rho-family members (Adam et al., 1996; Watarai et al., 1997) but, for Salmonella, are independent of PI 3-kinase activity (Chen et al., 1996b). These actin rearrangements are distinct from cytoskeletal changes associated with the ‘zipper’ mechanism of invasion (Finlay and Cossart, 1997), suggesting that the two pathways may possess distinct mechanisms to polymerize host actin leading to bacterial invasion. In the case of the ‘trigger’ mechanism, invasive Salmonella and Shigella species utilize a secretion machinery, termed the type III secretion apparatus, to inject bacterial proteins (effectors) into the host cell cytoplasm (Galan, 1996; Sansonetti and Egile, 1998; Tran Van Nhieu and Sansonetti, 1999). Some of these effectors, such as SopE and SptP for Salmonella (Fu and Galan, 1999), and IpaC for Shigella (Tran Van Nhieu and Sansonetti, 1999) are used to modulate the activity of Rac and Cdc42, leading to actin reorganization and uptake of bacteria. Though a role for Cdc42, Rac1 and RhoA in the uptake of Shigella and Salmonella species has been established, the role of these proteins in the uptake of pathogens using the ‘zipper’ mechanism for invasion was not clear. We report here that a pan-GTP-binding protein inhibitor, compactin, and expression of dominant negative Cdc42 and Rac1, inhibits invasion of FimH-expressing, type 1- piliated E. coli. Expression of constitutively active Cdc42 induced both p85a–FAK and a-actinin–vinculin complexes, whereas active Rac1 expression induced only the downstream a-actinin–vinculin complex, signalling intermediates that have been shown previously to be part of the uptake pathway (Martinez et al., 2000). Results presented in this study demonstrate that Rac1 activation is probably downstream of tyrosine kinase and PI 3-kinase activity during FimH-mediated invasion. More importantly, these results establish a role for Rho-family GTPases in the ‘zipper’ mechanism of invasion used by UPEC.
Results
Pan-GTPase inhibitor, compactin, blocks the uptake of type 1-piliated E. coli (fimH+) and adhesin-coated beads To determine the roles of Rho-family GTPase members in the invasion process, we used compactin, a paninhibitor of GTP-binding proteins (Ben-Ami et al., 1998) in conjunction with a gentamicin resistance (invasion) assay (Elsinghorst, 1994). Confluent monolayers of 5637 human bladder epithelial cells were pretreated with different concentrations of compactin for 16 h to block endogenous GTP-binding proteins before infection with type 1-piliated E. coli K12 strain AAEC185/pSH2 (fimH+). As demonstrated in Fig. 1A, invasion is diminished in a concentration-dependent fashion, with maximal inhibition occurring at 25 mM. Similar results were observed when FimHcoated microspheres (~1.5 mm diameter) were used in a fluorescent-based uptake assay (Fig. 1B and Martinez et al., 2000). Compactin had no effect on bacterial viability or on FimH-mediated adherence of bacteria and protein-coated beads (Fig. 1C–D). Rac1 and Cdc42 are involved in the FimH-mediated uptake process The experiments with compactin indicated that GTPbinding proteins, such as the Rho-family GTPase members, are probably involved in FimH-mediated uptake of type 1-piliated E. coli. However, it was unclear as to the identity of the GTPases involved. To test the role of RhoA, Cdc42 and Rac1 in the invasion process, we transiently transfected 5637 bladder cells with c-Myc epitope-tagged cDNAs encoding dominant negative (N19 RhoA, N17Rac1 and N17Cdc42) forms of RhoA, Rac1 and Cdc42, and determined the efficiency of type 1-piliated E. coli (fimH+) invasion using the gentamicin protection assay. Transfection of dominant negative RhoA © 2002 Blackwell Science Ltd, Cellular Microbiology, 4, 19–28 into 5637 cells caused a cytotoxic effect, causing the cells to round up and lift off the dish (data not shown), presumably because of other effects mediated by high expression levels of this protein in the cell. However, transfection of C3 transferase, which targets and inactivates endogenous RhoA (Watarai et al., 1997), efficiently blocked bacterial uptake as shown in Fig. 2A. Both N17Rac1 and N17Cdc42 effectively blocked the uptake of AAEC185/pSH2 (type 1+, fimH+) (Fig. 2B–C). The transfection efficiencies varied from 15–40% of the total cell population (data not shown), depending on the assay; therefore, next we examined only transfected cells. We used a fluorescent-based FimH-microsphere uptake assay (described in Experimental Procedures) to determine the effects of expressing c-Myc epitope-tagged N17Rac1 and N17Cdc42 on the uptake of the FimHcoated beads. As shown in Fig. 2D–I, both N17Rac1 and N17Cdc42 effectively blocked the uptake of FimH-coated microspheres (89.2% and 75.5% of the mock-transfected control respectively), but did not block adherence (Fig. 2J and K). Taken together, these data demonstrate Rhofamily members as having an important role in the uptake process of type 1-piliated UPEC. Inhibitors of invasion do not block Rac1-mediated uptake We noticed that tranfection of L61Rac and V12Cdc42 could enhance the ability of 5637 cells to internalize FimH-coated microspheres (Fig. 3A). Therefore, we utilized the active forms of Cdc42 and Rac1 as tools to stimulate the internalization pathway at different points along the pathway, so as to determine the probable temporal activation of Rac1 and Cdc42. We had demonstrated previously that inhibition of protein tyrosine kinases (PTKs) and PI 3-kinase blocks FimH-mediated uptake in 5637 cells (Martinez et al. 2000). Therefore, by inhibiting these processes in V12Cdc42 and L61Rac1transfected cells, we would be able to determine whether or not Cdc42 and Rac1 activities are downstream or © 2002 Blackwell Science Ltd, Cellular Microbiology, 4, 19–28 upstream of PI 3-kinase and PTK activity. Pre-incubation of V12Cdc42-transfected cells with either wortmannin or genistein drastically decreased the anchorage of the cells to the tissue culture plate, but had no effect on L61Rac1transfected cells (data not shown). Genistein and wortmannin failed to block L61Rac1-mediated uptake (Fig. 3B); therefore, PI 3-kinase and tyrosine kinase activities involved in the uptake pathway are probably upstream of Rac1 activity. Activated Rac1 and Cdc42 stimulate signals involved in FimH-mediated invasion Previous work had demonstrated that FimH-mediated invasion of bladder epithelial cells was dependent on PI 3-kinase and protein-tyrosine kinase activation, and that invasion was correlated with the formation of p85a–FAK and vinculin–a-actinin complexes. Inhibitors that blocked invasion also blocked the formation of these complexes, © 2002 Blackwell Science Ltd, Cellular Microbiology, 4, 19–28 suggesting that these proteins played an important role in the invasion pathway (Martinez et al., 2000). To determine where along the invasion pathway Cdc42 and Rac1 exerted their effects, we transiently transfected 5637 bladder epithelial cells with dominant negative and constitutively active forms of Cdc42 and Rac1. At 48h post transfection, soluble proteins were harvested and immunoprecipitated with anti-p85a and anti-vinculin antisera to determine whether or not transfection in the absence of bacterial invasion was sufficient to induce p85a–FAK and a-actinin–vinculin complexes. As shown in Fig. 4A and B), transfection of L61Rac1 and V12Cdc42, but not N17Rac1 or N17Cdc42, induced the formation of a vinculin–a-actinin complex; however, transfection of V12Cdc42, but not L61Rac1, induced p85a–FAK complex formation. Thus, stimulation of Cdc42 and Rac1 activity, in the absence of bacterial infection, induced the same signals involved in the uptake of type 1-piliated E. coli (fimH+). Surprisingly, N17Rac1 expression slightly induced FAK/p85a, suggesting a possible regulatory loop between Rac1 and PI 3-kinase activation. These results demonstrate that Cdc42 is probably upstream of PI 3- kinase activation, which is, in turn, upstream of Rac1 activity along the FimH-mediated invasion pathway.
Discussion
Rho-family members, including Rac, Cdc42 and RhoA, are small, GTP-binding proteins that, when activated, induce a variety of host cell responses; (Ridley and Hall, 1992; Ridley et al., 1992; Nobes et al., 1995; Hall, 1998b). These Rho-family GTPases serve as guanine nucleotideregulated switches that transduce external stimuli to modulate different cellular functions; (Ridley et al., 1992; Olson et al., 1995). Among these functions is the induction of distinct changes in the host cell actin cytoskeleton, which include formation of lamellopodia mediated by Cdc42, filopodia mediated by Rac and stress fibres induced by RhoA (Nobes and Hall, 1995; Hall, 1998a). The membrane changes generated by active Cdc42 and Rac have been shown to be associated with the uptake of the invasive pathogens, S. flexneri (Tran Van Nhieu and Sansonetti, 1999) and S. typhimurium (Fu and Galan, 1999), into non-phagocytic mammalian cells in a process termed ‘trigger mechanism.’ In contrast, very little was known about the roles of these proteins in the invasion of pathogens that use the ‘zipper’ mechanism, namely invasive strains of Yersinia, Listeria and UPEC. We have demonstrated that FimH-mediated invasion is dependent on the activation of RhoA, Rac1 and Cdc42, in addition to the activation of actin polymerization, tyrosine kinase and PI 3-kinase activation (Martinez et al., 2000). Inhibition of endogenous Rho-family members by compactin and the transient transfection of dominantnegative Rac (N17Rac) and Cdc42 (N17Cdc42), as well as the C3 transferase, effectively reduced the uptake of type 1-piliated E. coli and FimH-coated microspheres, demonstrating the importance of these proteins in the invasion process. Interestingly, a recent study has demonstrated that Yersinia adherence to host cells triggers a signalling cascade, involving Rac1, which is critical to bacterial uptake, further highlighting the importance of GTP-binding proteins in the ‘zipper’ mechanism (Weidow et al., 2000). Internalization of type 1-piliated E. coli and FimH-coated microspheres by 5637 human bladder epithelial cells was induced by the transient expression of © 2002 Blackwell Science Ltd, Cellular Microbiology, 4, 19–28 constitutively active Cdc42 (V12Cdc42) and Rac1 (L61Rac1). The induced internalization mediated by L61Rac1 was insensitive to PTK inhibitor, genistein and PI 3-kinase inhibitors, wortmannin and LY294002, arguing that Rac1 activation is downstream of protein tyrosine kinase and PI 3-kinase activation. To invade, pathogens often trigger pre-existing host–signal transduction cascades, often involving tyrosine phosphorylation of host proteins (Bliska et al., 1993; Finlay and Cossart, 1997; Finlay and Falkow, 1997; Palmer et al., 1997) and activation of lipid kinases, such as PI 3-kinase (Ireton et al., 1996;Schulte et al., 1998; Ireton et al., 1999; Martinez et al., 2000). We have demonstrated previously that FimH-mediated invasion of bladder epithelial cells in vitro was dependent on actin polymerization, tyrosine kinase and PI 3-kinase activation. Inva- sion of type 1-piliated E. coli is correlated with the formation of a p85a–FAK complex (Martinez et al., 2000), and it has been demonstrated that this interaction stimulates PI 3-kinase activity (Chen and Guan, 1994; Chen et al., 1996a) and localized changes in actin polymerization (Shoelson et al., 1993). In addition, invasion of type 1- piliated E. coli was correlated with the formation of an actin filament-stabilizing, a-actinin–vinculin complex that was downstream of protein tyrosine kinase and PI 3- kinase activity. Inhibitors that blocked uptake also blocked the formation of these complexes, arguing that they played key roles in the invasion pathway (Martinez et al., 2000). We have shown that in the absence of bacterial infection, transient expression of active Cdc42 (V12Cdc42), but not active Rac1 (L61Rac1), induced the formation of a FAK–p85 complex. Blocking downstream Rac1 activation may result in a build-up of activated PI 3- kinase as a result of a regulatory loop between Rac1 and PI 3-kinase. However, it is unclear as to why expression of N17Rac1 slightly induced FAK–p85a formation. As expected, both constitutively active, but not dominantnegative Cdc42 and Rac1 induced the formation of aactinin–vinculin complexes, again demonstrating the involvement of Rac1 and Cdc42 in the invasion pathway. These data, coupled with the invasion inhibition data, strongly argue that Cdc42 acts upstream of PI 3-kinase, which, in turn, is probably upstream of Rac1 activation. It is unclear exactly how Cdc42 could potentially regulate the activity of PI 3-kinase during the invasion pathway; however, several upstream and downstream effectors have been identified; (Manser et al., 1998; Obermeier et al., 1998; Bagrodia and Cerione, 1999; Scita et al., 2000). For example, binding of p85a to Rho-family GTPase members, namely Rac1 and Cdc42, can serve as an additional mechanism to stimulate PI 3-kinase activity in vitro (Zheng et al., 1994; Tolias et al., 1995). In some cells, the 3,4,5-phosphoinositides generated by PI 3- kinase activation can directly uncap barbed ends of actin filaments leading to polymerization (Hartwig et al., 1995) whereas in other cells, these secondary messengers can either directly or indirectly stimulate Rac1 activation by binding and activating guanine nucleotide exchange factors (GEFs) (Han et al., 1998; Nimnual et al., 1998). It is possible that upon bacterial invasion, Cdc42 and tyrosine-phosphorylated FAK can act synergistically to bind to and activate PI 3-kinase. The generation of 3,4,5- phosphoinostides could directly stimulate actin polymerization, in addition to stimulating Rac1 activity. All of these processes could potentially be involved in the localized re-arrangements of the actin cytoskeleton involved in bacterial uptake (see Fig. 5). Whether or not p85a and activated Cdc42 interact upon bacterial infection of bladder cells is currently under investigation. These results further highlight the importance of Rho-family GTPases in the © 2002 Blackwell Science Ltd, Cellular Microbiology, 4, 19–28 internalization of pathogens, and demonstrate that their function may be modulated in a distinct manner than previously described, depending on the routes of activation in different cell types.
Experimental procedures
Bacterial strains and cell lines The E. coli K12 strains AAEC185/pSH2 (type1+, fimH+) and AAEC185/pUT2002 (type 1+, fimH-) have been described previously (Martinez et al., 2000). To induce expression of type 1 pili, bacterial strains were grown in 20 ml static Luria–Bertani (LB) broth with appropriate antibiotics at 37∞C, for 24 h, before use in experiments. Type 1 pilus expression was confirmed by mannose-sensitive agglutination of 1% baker’s yeast or of guinea pig erythrocytes (A640~1.9) (Colorado Serum Company) suspended in phosphate-buffered saline (PBS). The human bladder epithelial cell line 5637 (ATCC HTB-9) was maintained in RPMI 1640 (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Sigma), 2.0 g l–1 sodium bicarbonate (Sigma), and 0.3 g l–1 L-glutamine. Cells were grown at 37∞C with 5% CO2 and utilized between passages 10–24. Antibodies, plasmids and other reagents Rabbit polyclonal antisera against FimH was a kind gift from MedImmune and has been described previously (Martinez et al., 2000). Rabbit polyclonal antisera against human p85a and monoclonal antibodies against vinculin (clone V284), a-actinin (clone AT6/172), Rac1 and Cdc42 were obtained from Upstate Biotech- nology (UBI). Rabbit polyclonal antibodies against focal adhesion kinase (FAK) and monoclonal antibodies against the c-Myc epitope tag (clone 9E10) were obtained from Santa Cruz Biotechnology. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and anti-mouse IgG antibodies used for immunoblotting were purchased from Sigma. Anti-goat IgG-HRP antibodies were obtained from Santa Cruz Biotechnology. For immunofluorescence studies, Oregon Green 488-conjugated goat anti-mouse and goat anti-rabbit IgG antisera were obtained from Molecular Probes. Cy3-conjugated goat anti-rabbit IgG and goat anti-mouse IgG antisera were obtained from Jackson ImmunoResearch Laboratories. The cDNAs encoding the C3 transferase, dominant-negative Rac1 (N17Rac1) and constitutively active Rac1 (L61Rac1) in pRK5-cMyc were a kind gift from Dr Alan Hall and have been described previously (Ridley et al., 1992; Lamarche et al., 1996; Watarai et al., 1997). The cDNAs encoding dominant-negative Cdc42 (N17Cdc42), constitutively active Cdc42 (V12Cdc42) and dominant-negative RhoA (N19RhoA) in pRK5-cMyc were kindly provided by Dr Coumaran Egile (Harvard Medical School) and have been described previously (Mounier et al., 1999). Complete protease inhibitor cocktail was purchased from Boehringer Mannheim. The construction of FimCH-coated microspheres has been described previously (Martinez et al., 2000). Compactin, wortmannin, genistein and LY294002 were purchased from Sigma. Transient transfections and bead uptake assay Plasmid DNA used for transient transfection into 5637 bladder cells was purified using the ENDOFree plasmid Maxi kit (Qiagen) according to the manufacturer’s directions. Bladder cells were © 2002 Blackwell Science Ltd, Cellular Microbiology, 4, 19–28 seeded 2.0 ¥ 105 cells per well onto 12 mm sterile glass coverslips in RPMI 1640/10%FBS 24 h before transfection. Cells were transfected with 0.5 mg DNA per well using the TransIT LT1 transfection reagent (Mirus), according to the manufacturer’s directions, for 24–48 h. Transfection efficiencies typically ranged from 15–40%, depending on the plasmid used. After transfection, cells were washed three times with PBS + Mg2+/Ca2+ and incubated for 30–60 min with FimCHcoated beads in RPMI 1640 + 40 mg ml–1 BSA as described (Martinez et al., 2000). In some cases, cells were pre-incubated with 200 nM wortmannin or 100 mM genistein for 30 min before the addition of beads. In some experiments, mock-transfected (control) and transfected cells were pre-incubated for 18 h in RPMI 1640/10%FBS + 50 mM compactin to inhibit endogenous Rho-family GTPases as described by Ben Ami and colleagues (Ben-Ami et al., 1998) before incubation with FimCH beads. Cells were washed in PBS and fixed in 2.5% paraformaldehyde in PBS for 20 min at room temperature. Fixed cells were then processed for the immunofluorescence-based uptake assay as described (Martinez et al., 2000) with slight modifications. Briefly, before permeabilization, extracellular FimCH-beads were labelled with rabbit polyclonal anti-FimH antisera (1:500) and Oregon Green 488 conjugated-goat anti-rabbit IgG (1:500). After permeabilization, transfected cells were identified using anti-cMyc epitope antisera (1:100) and Cy3-conjugated anti-mouse IgG antisera (1:500). The percentage of internalized beads was calculated by determining the total number of intracellular FimCH-beads associated with the transfected cells and control (mock-transfected) cells. Data are presented as the mean of three separate assays in which at least 100 cells were counted.
Gentamicin protection (invasion) assays
Bacterial invasion assays were performed essentially as described (Elsinghorst, 1994; Martinez et al., 2000). Briefly, 2.0 ¥ 105 bladder cells were seeded into 24-well plates and grown for 24 h. In some experiments, cells were transfected with cDNAs encoding dominant-negative forms of Rac1 and Cdc42 for 48 h. Before the addition of bacteria, cells were washed with PBS+Mg2+/Ca2+ and then 960 ml of prewarmed media was added. In three sets of triplicate wells, bladder cells were infected with 20 ml of a bacteria diluted in LB broth, A600~ 0.5). Bacterial contact with host cells was expedited by centrifugation of plates at 600 g for 5 min. After 1 h of incubation at 37∞C, one set of triplicate wells was lysed by the addition of 10 ml of 10% Triton X100. Bacteria present in these lysates, representing the total number of bacteria present both intra- and extracellularly, were titred. To determine invasion frequencies, after the initial 1h incubation, an additional set of wells were washed twice with PBS (with Mg2+/Ca2+) and then incubated for another 2 h in media containing 100 mg ml–1 of the membrane-impermeable, bactericidal antibiotic gentamicin (Sigma) to kill any extracellular bacteria. Cells were then washed three times with PBS, lysed in 1 ml of 0.1% Triton X-100 in ddH2O, and plated on LB-agar plates. Invasion frequencies were calculated as the number of bacteria surviving incubation with gentamicin divided by the total number of bacteria present just before addition of gentamicin. To test the effects of compactin on bacterial invasion, compactin was added 18 h before bacterial infection. As a control, DMSO was added to cells to a final concentration of 0.1%. Bacterial and FimH-coated bead adherence assays were performed as described (Martinez et al., 2000). Inhibitors used did not have an effect on bacterial viability or on bacterial adherence during the infection period. Data for protein coated-bead binding to 5637 cells are presented as an average of the number of beads per cell and are from at least two independent assays.
Immunoprecipitations
A total of 5637 cells were seeded at a density of 8.0 ¥ 105 cells per well in 6-well plates in RPMI 1640/10% FBS. Then, 24 h after plating (~60–70% confluent) cells were transfected for 48 h with the indicated DNAs (2.0 mg per well) using the TransIT LT1 transfection reagent (Mirus) according to the manufacturer’s directions. On the day of the experiment, cells were washed three times with ice-cold PBS, lysed with 1 ¥ magnesium-containing lysis buffer (MCB) [25 mM Hepes pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 0.5 mM EDTA, 2 mM NaF, 2 mM Na3VO4 and 1 ¥ Complete Protease Inhibitor Cocktail (Boehringer Mannheim, Germany)], and then centrifuged at 12000 g. Cleared lysates were adjusted for equal protein content and immunoprecipitated overnight at 4∞C with either anti-FAK antisera (1:100) or antivinculin (2 mg ml–1). Immune complexes were captured with 50 ml of 50% Protein G or Protein A-sepharose in PBS for 1 h at 4∞C, washed three times with ice-cold lysis buffer and then boiled for 8 min in SDS sample buffer. Proteins were resolved by SDS–PAGE using 10% polyacrylamide gels, transferred to nitrocellulose, and immunoblotted with anti-p85a antisera (1 mg ml–1) or antia-actinin (2 mg ml–1), as indicated. Proteins were visualized with Super Signal WestDura-enhanced chemiluminescence system (Pierce) and exposure to film. In some cases, blots were stripped with IgG elution buffer (Pierce) according to the manufacturer’s instructions and reprobed with the appropriate antisera to demonstrate that similar amounts of proteins were present in each lane.
Acknowledgements
We would like to thank S. Langermann and MedImmune for providing anti-FimH antibodies; T. Stappenback and A. Barbieri for providing reagents and assistance in transfections; and M. Mulvey and members of the Hultgren lab for helpful discussions and suggestions. This work was supported by NIH grants R01AI29549, R01DK51406 and R01AI48689.
Article Images (0)
×![]()
Bioz is the world’s most comprehensive AI search engine for scientific experimentation. The Bioz search engine offers researchers billions of data-driven product, technique, and protocol recommendations. Bioz taps into the latest advances in Artificial Intelligence (AI) – including Natural Language Processing (NLP), Machine Learning (ML) and Generative AI to mine and structure hundreds of millions of pages of complex and unstructured scientific papers. By harnessing the power of AI, Bioz provides researchers with an unprecedented amount of summarized scientific experimentation knowledge right at their fingertips, ultimately helping to speed up drug discovery and the rate of success in finding cures for diseases. Bioz is used by over 15 Million researchers from over 17,000 different universities and companies in 196 countries.
