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0019-9567/92/062211-07$02.00/0 Copyright X) 1992, American Society for Microbiology
Tyrosine Protein Kinase Inhibitors Block Invasin-Promoted Bacterial Uptake by Epithelial Cells ILAN ROSENSHINE,1'2 VINCENT DURONIO,3 AND B. BRETT FINLAY' 2* Biotechnology Laboratory, Departments of Biochemistry and Microbiology,1 Canadian Bacterial Diseases Network 2 and Biomedical Research Center,3 University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3 Received 9 October 1991/Accepted 10 March 1992
The ability to enter into (invade) mammalian cells is an essential virulence determinant of many pathogenic bacteria and intracellular parasites. These organisms are internalized by host cells upon attachment to their surface. However, the mechanisms used by intracellular parasites to induce internalization into host cells have not been defined. We found that the protein kinase inhibitor staurosporine blocks invasion by some pathogenic bacteria, including Yersinia enterocolitica and Yersinia pseudotuberculosis. Using Escherichia coli containing the cloned Y. enterocolitica invasion gene inv (which codes for invasin, an integrin-binding protein), we found that staurosporine inhibits invasion by blocking bacterial internalization. Two specific tyrosine protein kinase inhibitors, genistein and tyrphostin, also block the internalization but not the binding of bacteria, suggesting that bacterial uptake may be dependent on the activity of this enzyme class in host HeLa cells. In contrast to invasion promoted by invasin, the invasion of HeLa cells by Salmonella typhimurium is not inhibited by any of these drugs.
Invasion into host epithelial cells is an important virulence factor for many pathogenic bacteria. Some Shigella, Salmonella, Listeria, and Yersinia species are facultative intracellular parasites, while some Chlamydia and Rickettsia species are obligatory intracellular parasites (10, 27). Once in the intracellular environment, bacteria are protected from the immune system and many chemotherapeutic agents. Salmonella species also use invasion to penetrate through monolayers of polarized epithelial cells (12). The entry of the pathogen into the host cell is initiated by bacterial binding to the host cell surface, which is followed by internalization. The internalization step is facilitated by host cell actin filaments, since pathogen internalization is inhibited by agents, such as cytochalasin D, which disrupt actin filaments (4, 9, 11). The binding of bacteria to the cell surface is not necessarily followed by internalization, as many bacterial species bind to eucaryotic cells but do not enter them (19, 26). Given the active involvement of the host cytoskeleton, it appears that invasive bacteria generate an "uptake signal" which induces the host cell to internalize them. To gain a better understanding of invasion processes, we have begun to characterize the nature of the uptake signals and the mechanism of their transduction by using several invasive pathogens and the cloned invasion genes of Yersinia enterocolitica. The Y enterocolitica and Yersinia pseudotuberculosis inv invasion genes are highly homologous and code for invasin, a 92- or 103-kDa outer membrane protein (21, 28, 37). Invasin promotes invasion by interacting with eucaryotic transmembrane proteins belonging to the integrin superfamily, including the a3,1, a4l, a51, and a6,13 integrins (19, 20). Integrins bind to various basal membrane proteins, such as fibronectin, collagen, and laminin, and are localized in focal contacts. They also participate in transducing extracellular signals and are components of the apparatus that anchors the cytoskeleton to the membrane (18). In eucaryotic cells, different classes of protein kinases *
(PK) play a major role in transducing extracellular signals. We examined the involvement of these enzymes in transducing signals needed for bacterial internalization by epithelial cells. In this paper we present evidence that invasion promoted by Yersinia invasin can be blocked by tyrosine PK (TPK) inhibitors. MATERIALS AND METHODS Bacterial strains and plasmid. Salmonella typhimurium SL1344, Y enterocolitica 8081c, Y pseudotuberculosis YPIIIp-, and Escherichia coli HB101 have been described elsewhere (9). Plasmid pVM101 (28) was obtained from J. Bliska, V. Miller, and S. Falkow (Stanford University). The bacteria were grown and plated in LB broth or on LB agar. When needed, ampicillin (Sigma) was added to a final concentration of 50 ,ug/ml. Eucaryotic cells. HeLa cells (ATCC CCL2) were grown and assayed at 37°C with 5% CO2 in minimal essential medium (MEM) supplemented with 10% (vol/vol) fetal calf serum. Kinase inhibitors. All inhibitors were dissolved in dimethyl sulfoxide, and stock solutions were divided into aliquots and stored at -20°C. The stock concentrations were as follows: staurosporine (Boehringer Mannheim), 1 mM; genistein (ICN), 100 mM; tyrphostin-AG34 (obtained from A. Levitzki, The Hebrew University of Jerusalem; AG34 is com-
pound 9 in reference 35), 50 mM; calphostin C (Kamiya Biomedical), 10 mM; 1-(5-isoquinolinesulfonyl)-2-methyl piperazine (H7 [Sigma]), 300 mM; N- (2- aminoethyl) -5isoquinolinesulfonamide dihydrochloride (H9 [Calbiochem]) 300 mM; and 12-O-tetradecanoylphorobol-13-acetate (TPA [Sigma]), 10 mM. Prior to use, the inhibitors were diluted in MEM supplemented with 10% (vol/vol) fetal calf serum, and the mixture was added to the cultured epithelial cells. Effect of PK inhibitors on bacterial viability. Fresh overnight bacterial cultures (10-,ul aliquots) were inoculated into 1 ml of MEM supplemented with 10% (vol/vol) fetal calf serum with or without a PK inhibitor. The cultures were
Corresponding author. 2211
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incubated (37°C, 5% C02) for various times. The number of CFU was compared for treated and untreated bacteria. Effect of PK inhibitors on the phosphorylation of a major PKC substrate, p80. The method used for detecting p80 phosphorylation is a slight modification of the procedure described by Rosengurt et al. (29). HeLa cells were seeded at 5 x 105 cells per dish in 35-mm tissue culture petri dishes. On the next day, the cultures were washed twice with phosphate-free MEM and the cells were incubated (37°C, 5% C02) with this medium containing carrier-free 32p; (ICN) at 290 RCi/ml to label the endogenous ATP pool. After 1.5 h, the different inhibitors were added as needed (to the following final concentrations: staurosporine, 0.5 ,uM; calphostin C, 0.35 ,uM; and H7, 300 ,uM), the cells were incubated as before for an additional 1 h, and then protein kinase C (PKC) was induced by the addition of TPA at 0.1 ,uM for 5 min. The reaction was stopped by removal of the medium and rapid washing of the cultures twice with ice-cold 0.15 M NaCl-20 mM Tris-HCl (pH 7.5). The cells were immediately extracted by scraping into 0.5 ml of lysis solution (1% Triton X-100, 50 mM Tris-HCI [pH 7.6], 1 mM EDTA, 0.4 mM V04, 1 mM NaF, 0.1 mg of phenylmethylsulfonyl fluoride per ml, 10 ,ug of leupeptin per ml). The extracts were cleared by centrifugation (1 min at 14,000 x g) and removed to a fresh tube containing 130 ,ul of Sx loading buffer (0.5 M Tris-HCl [pH 6.8], 10% sodium dodecyl sulfate [SDS], 0.5 M dithiothreitol, 50% glycerol). Aliquots were heated at 98°C for 10 min and cleared as before prior to resolution by gel electrophoresis. The samples were adjusted to 1.4 x 106 cpm/20 ,ul, and electrophoresis was performed with an 8% SDS-polyacrylamide gel as described previously (24). The gel was dried and used for radioautography. Determination of PKC activity in staurosporine-treated cells. Subconfluent cultures of HeLa cells in 175-cm2 flasks were treated for 1 h with or without 1 ,uM staurosporine. The HeLa cells were washed twice with phosphate-buffered saline (PBS), scraped from the flasks into 1.4 ml of PBS, centrifuged, resuspended in sonication buffer, and sonicated so that they would lyse (6). The supernatant was cleared by centrifugation, and PKC activity was assayed immediately. PKC activity was determined by comparing the activity of histone Hi phosphorylation in the presence or absence of PKC activators and cofactors (Ca2+, diolein, and phosphatidylserine) as described elsewhere (6). The specific activities of PKC in treated and untreated cells were compared. Bacterial internalization. Bacterial internalization was determined as described elsewhere (9) with slight modifications. In brief, 10 RI of a fresh overnight bacterial culture was added to a HeLa cell culture that had been seeded the previous day in a 24-well plate at a density of 105 cells per well, and the mixture was centrifuged for 5 min at 800 x g and incubated for 90 min (37°C, 5% CC2). The cells were washed twice and incubated for an additional 90 min in medium containing 100 ,ug of gentamicin per ml; this antibiotic kills extracellular bacteria. The monolayers were washed and lysed with 1% Triton X-100, and appropriate dilutions were plated to determine the number of viable intracellular bacteria. When inhibitors were used, they were added to MEM and were present throughout the invasion assay. RESULTS The PK inhibitor staurosporine blocks the invasion of HeLa cells by Yersinia species. In eucaryotic cells, protein phosphorylation is often involved in the transduction of
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FIG. 1. Effect of staurosporine treatment on invasion of HeLa cells by Y. enterocolitica 8081c (-), Y pseudotuberculosis YPIIIp(0), S. typhimunum (A), and E. coli HB101 containing plasmid pVM101 (inv gene) (O). Values are averages of duplicates. Staurosporine in MEM was added to HeLa cells 15 min prior to infection.
extracellular signals. If PK activity is necessary for transducing a bacterial uptake signal, inhibitors of these enzymes should also inhibit bacterial invasion. To test this hypothesis, we used staurosporine to inhibit PK activity. Staurosporine is an extremely potent PK inhibitor, having 50% inhibitory concentrations in the nanomolar range for PKC, cAMP-dependent PK, and TPKs such as pp60vsrc (33). However, staurosporine is a poor inhibitor of other TPKs, such as those of the epidermal growth factor receptor and the insulinlike growth factor receptor (33). In vivo, 1 ,uM staurosporine is needed to inhibit PKC almost completely (7, 34). Staurosporine exhibited dose-dependent inhibition of invasion of HeLa cells by Y enterocolitica and Y pseudotuberculosis (Fig. 1). Staurosporine did not have any inhibitory effect on the invasion rates of S. typhimurium (Fig. 1). Staurosporine inhibits invasion promoted by the Yersinia inv gene. Y enterocolitica and Y pseudotuberculosis possess multiple invasion systems; the most efficient invasion is promoted by invasin (19). To avoid the complications of multiple invasion systems, we examined the effect of staurosporine on the invasiveness of E. coli carrying the inv invasion gene of Y enterocolitica. Although E. coli HB101 does not normally invade eucaryotic cells, introduction of the inv invasion gene into this bacterium is sufficient to promote its binding to and invasion of various eucaryotic cell lines (37). Invasion by E. coli HB101 carrying the inv gene of Y. enterocolitica on plasmid pVM101 (HB101/pVM101) was also inhibited by staurosporine (Fig. 1). At a concentration of 1 ,M, this drug inhibited 99.8% of the invasion promoted by this gene. Staurosporine also had a similar effect on invasion by E. coli carrying the inv gene of Y pseudotuberculosis (data not shown). Staurosporine inhibits invasion by blocking bacterial internalization by the host cell. Staurosporine may block invasion or may affect the results of the invasion assay by a variety of mechanisms, including (i) inhibiting some bacterial function, such as a kinase activity; (ii) affecting bacterial viability; (iii) triggering some antibacterial activity in the host cell; (iv) reducing the ability of invasin to bind to its receptors; or (v) preventing treated HeLa cells from internalizing bacteria by various mechanisms, including blockage of the transduction of an uptake signal. We examined each of these possibilities to identify the mechanism involved. Exposure to 1 puM staurosporine for 2 h did not have any
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FIG. 2. Binding of HB101/pUC19 (control) (A to C) and HB101/pVM101 (inv) (D to F) to HeLa cells. HeLa cells were seeded on coverslips, grown overnight, and pretreated with 1 pLM staurosporine for 30 min (B and E) or 0.1 ,uM TPA for 24 h (C and F) or left untreated (A and D). The cells were infected for 1 h, washed four times to remove the unbound bacteria, and fixed with 2% paraformaldehyde in PBS before being photographed.
significant effect on the viability of any of the bacterial species used in this study. The effect of this drug on the viability of bacteria in the intracellular environment was tested by first infecting HeLa cells with HB101/pVM101 for 60 min. The cells were then washed and incubated for 2 h in medium containing 100 ,ug of gentamicin per ml and with or without 1 ,uM staurosporine. Staurosporine did not have any effect on the viability of intracellular E. coli. The inhibitory effect of staurosporine on PK is only very slowly reversed after the drug is removed. We used this property to exclude the possibility that staurosporine inhibited invasion by affecting some bacterial function. HeLa cells were pretreated for 60 min with 1 ,uM staurosporine, washed several times to remove the drug, and infected with HB101/pVM101. This staurosporine pretreatment still reduced the invasion rate by 98%. Microscopic studies of the effect of staurosporine on the binding of HB101/pVM101 to HeLa cells demonstrated that staurosporine does not inhibit the binding of this bacterium to the epithelial cell surface (Fig. 2), indicating that this drug acts by preventing internalization of the bacteria rather than by blocking their binding to the host cell surface. To examine the effect of staurosporine on internalization, we performed an experiment in which the initial binding was uncoupled from the subsequent internalization event. Binding of HB101/pVM101 to HeLa cells without staurosporine was accomplished by incubation at 4°C for 60 min. (At this temperature, HB101/pVM101 binds to the epithelial cell surface but is not internalized [21].) The cells were washed five times with cold medium to remove the unbound bacteria. Medium with or without 1 ,uM staurosporine was added,
and the temperature was shifted to 37°C to allow internalization of the bound bacteria. Internalization of HB1O1/ pVM101 was reduced by 1,000-fold in the staurosporinesupplemented cultures. The effect of staurosporine on the shape of HeLa cells (Fig. 2) and on the cell cytoskeleton (16) might suggest that it inhibits internalization by paralyzing nonspecifically the phagocytic capability of these cells. However, our results indicated that this is not the case, since HeLa cells treated with 1 puM staurosporine still internalized S. typhimurium (which enters cells by an actin-requiring process similar to phagocytosis [11]) as efficiently as nontreated cells. Moreover, 1 ,uM staurosporine did not have any effect on the efficiency of nonspecific uptake of E. coli HB101 by the murine macrophagelike J774 cell line. All the above-described results indicate that staurosporine does not affect bacterial binding but prevents specifically the internalization promoted by invasin. Staurosporine is a potent inhibitor of different PK classes, including PKC, cAMP-dependent PK, and some TPKs. Of these various enzymes, TPK and PKC are the most likely candidates to participate in transducing the internalization signal that is generated upon binding of invasin to its P, integrin receptor. Both enzymes (PKC and TPKs such as pp6O-src) are localized in focal contacts associated with integrins (22, 23). Moreover, integrins and proteins which are believed to be involved in anchoring integrins to actin filaments, such as vinculin and talin, have been shown to be potential substrates for both enzymes (2, 23, 25, 30). Therefore, we attempted to identify which of these kinase activities is needed for invasion promoted by invasin.
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A 2 18.3
00.5
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4X.2 FIG. 3. Effect of PK inhibitors on TPA-induced phosphorylation of p80. HeLa cells were labeled with 32Pi and pretreated with different PK inhibitors. PKC activity was then induced by the addition of TPA to a concentration of 0.1 ,uM and, after 5 min of induction, the reaction was stopped and the cells were extracted.
Electrophoresis was performed with an 8% SDS-polyacrylamide gel. The location of the major PKC substrate, p80, is indicated by the arrowhead, and the locations of other proteins whose phosphorylation was affected by the inhibitors are indicated by arrows. The extracts that were analyzed were as follows: lane A, cells induced with TPA and treated with 300 ,uM H7; lane B, cells induced with TPA and treated with 0.35 ,uM calphostin C; lane C, cells induced with TPA and treated with 0.5 ,uM staurosporine; lane D, cells induced with TPA; and lane E, cells that were not induced with TPA. Numbers at left are in kilodaltons.
Role of PKC in the internalization of HB101/pVM101. To examine whether PKC activity is needed for invasion promoted by invasin, we tested the effect of inhibitors which are known to have a higher specificity for PKC than for TPK (3, 33). Calphostin C (at a final concentration of 0.35 jiM) and H7 and H9 (both at a final concentration of 300 jiM) did not block the invasion of HeLa cells by HB101/pVM101 or S.
typhimunium.
These drugs were toxic for HeLa cells at
concentrations higher than those used, as determined by trypan blue staining of the treated cells. The effect of these inhibitors on PKC activity in intact cells was examined by analyzing the effect of the inhibitors on the phosphorylation of the major PKC substrate, p80 (29, 32). The cells were preincubated for 1 h with PKC inhibitors (0.5 jiM staurosporine, 0.35 FM calphostin C, or 300 jiM H7). PKC was then induced for 5 min by the addition of TPA to a final concentration of 0.1 jiM. The cells were lysed, and the cells extracts were analyzed by SDS-polyacrylamide gel electrophoresis. While staurosporine strongly inhibited TPA-induced p80 phosphorylation, H7 and calphostin C had a less inhibitory effect on p80 phosphorylation (Fig. 3), confirming other studies that showed that calphostin C and H7 are not as potent PKC inhibitors as staurosporine (the 50% inhibitory concentrations for PKC are as follows:
staurosporine, 0.003 jiM; calphostin C, 0.05 jiM; H7, 6 jiM; and H9, 15 ,uM [3, 33]). Interestingly, calphostin C and H7 inhibited the phosphorylation of proteins other than p80, whose phosphorylation was only moderately induced by TPA (Fig. 3). These results suggest that H7 and calphostin C
are not suitable for examining the correlation between PKC activity and other processes in intact cells. An alternate approach was used to determine whether PKC activity is correlated with invasion efficiency. PKC is activated in vivo by the short-living second messenger diacylglycerol. Similarly, PKC can be activated, as shown earlier, by phorbol esters, such as TPA, which act as stable analogs of diacylglycerol. However, long-term exposure to TPA down-regulates the enzyme activity by sensitizing the enzyme to proteolytic degradation and by specifically inhibiting its transcription by an unknown mechanism (6, 36). We hypothesized that if PKC is needed for invasin-mediated uptake of E. coli, it would be possible to block this uptake by using TPA to down-regulate PKC. For determination of the TPA concentration and incubation time needed to down-regulate PKC in HeLa cells, PKC from untreated cells and from cells which had been incubated with TPA for 24, 48, and 72 h was partially purified and its activity was assayed. We found that 24 h of incubation with 0.1 ,uM TPA was sufficient to reduce the PKC activity to its minimum level, which was 5.8% of the activity in nontreated cells (Fig. 4A and B). This 24-h pretreatment also reduced invasion by HB101/pVM101 to 17.7% ± 10.9% (an average of five different experiments) of the invasion of nontreated cells (Fig. 4C). To examine whether TPA inhibited invasion by a mechanism consistent with down-regulation of PKC, we tested the invasion of cells which had been pretreated with 0.1 ,uM TPA for 30 min (under these conditions, PKC is activated rather than down-regulated). In contrast to 24 h of pretreatment, 30 min of pretreatment did not inhibit invasion (Fig. 4C). The possibility that TPA inhibited invasion by affecting bacterial viability, reducing the binding of the bacteria to the host cells, or paralyzing the phagocytic machinery was also examined. Incubation for 2 h with concentrations of TPA as high as 1 ,uM did not have any effect on bacterial viability. To examine whether this drug affected the viability of the intracellular bacteria, we infected HeLa cells with HB1O1/ pVM101 for 90 min and then washed and incubated them in medium containing 100 ,ug of gentamicin per ml to kill extracellular bacteria. After 2 h of incubation, this medium was replaced by a medium containing 10 jig of gentamicin per ml and 0.1 jiM TPA, and the cells were incubated for an additional 24 h before being lysed to determine the number of viable bacteria inside them. This 24-h TPA treatment did not affect the viability of the intracellular E. coli. We also determined whether TPA had any effect on the binding of the bacteria to the HeLa cells or on the phagocytic capability of these cells, since TPA, like staurosporine, induces actin filament rearrangement and cell rounding (31) (Fig. 2). We used S. typhimurium to test the capability of TPA-treated cells to internalize bacteria. As seen with staurosporine, down-regulation of PKC with TPA did not inhibit the uptake of S. typhimurium. In addition, TPA treatment did not reduce the binding of HB101/pVM101 to HeLa cells (Fig. 2). These results demonstrate that TPA treatment inhibits the internalization of HB101/pVM101 by HeLa cells. Although TPA treatment inhibited invasin-mediated bacterial uptake by HeLa cells, staurosporine was a much more efficient inhibitor (Fig. 1 and 4C). Thus, to correlate PKC activity and invasion efficiency, we determined the residual PKC activity in staurosporine-treated HeLa cells and compared it with the residual PKC activity in HeLa cells treated for 24 h with TPA. The PKC specific activity in cells treated for 1 h with 1 jiM staurosporine was 6.1% of the specific activity in nontreated cells. While both TPA and staurospo-
BLOCKAGE OF BACTERIAL UPTAKE BY TPK INHIBITORS
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FIG. 5. Effect of the TPK inhibitors genistein and tyrphostinAG34 on invasion promoted by invasin (0) and on invasion by S. typhimurium (O) of HeLa cells. (A) Genistein was added 15 min before infection. (B) Tyrphostin-AG34 was added 1 h before infection.
T 10
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FIG. 4. Effect of TPA on PKC activity and efficiency of bacterial invasion. PKC was partially purified from HeLa cells which had been left untreated (A) or pretreated for 24 h with 0.1 p.M TPA (B). Fractionation of the cell extracts with a MonoQ column and assays of PKC activity were carried out as described elsewhere (6). Histone Hi phosphorylation activity in the presence (-) or absence (E) of added Ca2 diolein, and phosphatidylserine is shown. (C) To determine invasion levels, we preincubated HeLa cells with 0.1 P.M TPA for 30 min or 24 h before infecting them with HB101/pVM101 and compared invasion levels with those for untreated cells.
rine treatments reduced PKC activity to similar extents (5.8 and 6.1%, respectively), staurosporine blocked invasion much more efficiently than TPA, suggesting that the inhibition of PKC is not the major reason for the inhibition of invasion by staurosporine. Internalization of HB101/pVM101 by HeLa cells can be blocked by specific TPK inhibitors. Since staurosporine also
inhibits some TPK activities, we examined the effect of two specific TPK inhibitors, tyrphostin-AG34 and genistein, on invasin-mediated bacterial uptake. These two drugs inhibit TPK by different mechanisms: tyrphostin-AG34 competes with the tyrosine residue binding to the kinase, and genistein inhibits the binding of ATP to the enzyme (1, 35). However, both drugs are highly specific TPK inhibitors (1, 14, 35). Both genistein and tryphostin-AG34 strongly inhibited the invasion of HeLa cells by HB101/pVM101 (98.5 and 93.2% inhibition, respectively), but they did not have a similar effect on invasion by S. typhimurium (Fig. 5A and B). We also examined, as described for staurosporine, the possibility that these drugs affect the invasion assay not by blocking internalization but by some other mechanism. The exposure of extracellular HB101/pVM101 or S. typhimurium for 3 h to either 250 ,uM genistein or 1 mM tyrphostin-AG34 in MEM did not affect bacterial viability. Genistein at 250 ,uM also did not affect the viability of intracellular HB101/pVM101 after 5 h of incubation. Microscopic examination revealed that these drugs do not reduce the efficiency of binding of the bacteria to the cells. Moreover, neither drug affects the morphology of the HeLa
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FIG. 6. Invasion of HeLa cells by HB101/pVM101 after genistein removal. The bacteria adhered to but did not invade HeLa cells when the medium was supplemented with 250 ,uM genistein. The rate of internalization after removal of the drug was determined as described in the text and compared with that for untreated cells (100% invasion).
cells, suggesting that they do not
cause
major cytoskeleton
rearrangement.
The inhibition of TPK by genistein is known to be highly reversible (1, 17). We predicted that if genistein blocked invasion by inhibiting TPK activity, then the inhibition of invasion should also be reversible. To examine this possibility, we infected HeLa cells in medium containing 250 ,uM genistein with HB101/pVM101 for 90 min to allow binding. At this point, the cells were washed five times with PBS to remove the drug and the unbound bacteria, and prewarmed MEM was added instead to allow bacterial internalization. To monitor the internalization rate, we killed extracellular bacteria at different times after genistein removal by the addition of gentamicin to a final concentration of 100 ,ug/ml and incubation for an additional 90 min. The number of internalized bacteria was determined and compared with the invasion efficiency for nontreated cells and for cells from which genistein was not removed. The bound bacteria were internalized rapidly after genistein removal (Fig. 6). Less than 5 min after genistein removal, there was more than a 100-fold increase in the number of intracellular bacteria, and 20 min after genistein removal, the number of intracellular bacteria was the same as in nontreated cells (Fig. 6). These results suggest that the internalization of HB101/pVM101 may be dependent on some genistein-sensitive TPK activity in the host cell. DISCUSSION In this study, we used the PK inhibitor staurosporine to demonstrate a correlation between the efficiency of invasion promoted by the Yersinia inv gene product, invasin, and host cell PK activity. Further analysis with the specific TPK inhibitors genistein and tyrphostin-AG34 indicated that TPK activity in host cells may be essential for invasin-mediated bacterial internalization. Staurosporine and long-term TPA treatments reduced PKC activity to similar extents (6.1 and 5.8%, respectively), but the staurosporine treatment inhibited bacterial uptake about 90-fold more than the TPA treatment. Therefore, it seems that the major mechanism of the effect of staurospo-
rine on bacterial internalization is the inhibition of a PK other than PKC, possibly TPK inhibition. The PKC down-regulation experiment also suggested that PKC plays some role in the process of bacterial uptake by epithelial cells. However, the inhibitory effect of TPA on invasion may also result from some secondary effect of long-term PKC activation and down-regulation or, alternatively, TPA may interact directly with different unknown targets to inhibit invasion. The inhibition of invasion of several invasive bacteria after 1 h of TPA treatment has been described (8). However, under the conditions used by Ewanowich et al. (8), PKC was probably activated rather than down-regulated, and different bacterial strains were used. Nevertheless, analyses of the TPA effects in both studies may be interpreted in several different ways. Therefore, it is not yet clear whether PKC activity is needed for invasin-promoted bacterial uptake. The potential involvement of TPK in regulating the function of the invasin receptor integrin and of focal contact has been suggested by several studies (5, 23). However, in normal epithelial cells integrin has not been shown to be functionally affected by TPK activity. In this study, we showed that integrin-mediated bacterial internalization can be blocked by TPK inhibitors. However, it is not yet clear whether binding of the bacteria to integrin induces TPK activity to trigger internalization or, alternatively, whether some protein that is involved in this internalization must be phosphorylated to function. We are currently trying to examine these possibilities, but we have not yet detected the induction of specific tyrosine protein phosphorylation by invasin. Further investigation of how the binding of invasin to integrin induces internalization might provide new insights into how integrins interact with the cytoskeleton, how these interactions are regulated, and how they mediate signal transduction. Interestingly, one of the Yersinia virulence genes (the Y. enterocolitica gene yop-51 or its homologous gene in Y. pseudotuberculosis, yopH) encodes a specific tyrosine phosphatase (15). It is not clear how this enzyme contributes to bacterial virulence or whether it has anything to do with the correlation of invasin-mediated invasion with tyrosine phosphorylation. It will be interesting to determine whether the expression of the tyrosine phosphatase by intracellular yersiniae inhibits further infection of host cells by extracellular yersiniae. The results presented in this study substantiate previous observations that the mechanism of S. typhimunium invasion is different from that of Yersinia species. In a previous study, we reported that anti-integrin antibodies that inhibit invasinmediated invasion do not affect S. typhimurium invasion (13). We have found that, in contrast to S. typhimunum, Y. enterocolitica and HB101/pVM101 do not induce any major cytoskeleton rearrangement when they invade (13). Furthermore, the different profiles of the sensitivities of the two invasion systems to PK inhibitors indicate that S. typhimurium and Yersinia species are internalized by different pathways. Further studies of signaling between different invasive bacteria and their host cells will contribute to an understanding of these virulence factors. They may also shed new light on the dynamics of the cytoskeleton-membrane interaction and some aspects of endocytosis. In addition, these studies may lead to the identification and development of new chemotherapeutic agents that block host cell invasion by pathogens.
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BLOCKAGE OF BACTERIAL UPTAKE BY TPK INHIBITORS ACKNOWLEDGMENTS
We thank E. Lim and H. D. Fong for technical assistance. We also thank J. Bliska, V. Miller, and S. Falkow for plasmid pVM101 and A. Levitzki for tyrphostin-AG34. This work was supported by an operating grant to B.B.F. from the Medical Research Council of Canada and the NCE as part of the Canadian Bacterial Diseases Network Research Program. I.R. is a recipient of a long-term fellowship from the European Molecular Biology Organization. 1.
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15. 16.
17.
REFERENCES Akiyama, T., J. Ishida, S. Nakagawa, H. Ogawara, S. Watanabe, N. Itoh, M. Shibuya, and Y. Fukami. 1987. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J. Biol. Chem. 262:5592-5595. Burn, P., A. Kupfer, and S. J. Singer. 1988. Dynamic membrane-cytoskeletal interactions: specific association of integrin and talin arises in vivo after phorbol ester treatment of peripheral blood lymphocytes. Proc. Natl. Acad. Sci. USA 85:497501. Calbiochem Co. 1990-1991. Biochemical/immunological catalog. Calbiochem Co., San Diego, Calif. Clerc, P., and P. J. Sansonetti. 1987. Entry of Shigella fle-xneri into HeLa cells: evidence for directed phagocytosis involving actin polymerization and myosin accumulation. Infect. Immun. 55:2681-2688. Davis, S., M. L. Lu, S. H. Lo, S. Lin, J. A. Butler, B. J. Druker, T. M. Roberts, Q. An, and L. B. Chen. 1991. Presence of an SH2 domain in the actin-binding protein tensin. Science 252:712-715. Duronio, V., B. E. Huber, and S. Jacobs. 1990. Partial downregulation of protein kinase C reverses the growth inhibitory effect of phorbol esters on HepG2 cells. J. Cell. Physiol. 145:381-389. Duronio, V., and S. Jacobs. 1990. The effect of protein kinase-C inhibition on insulin receptor phosphorylation. Endocrinology 127:481-487. Ewanowich, C. A., and M. S. Peppler. 1990. Phorbol myristate acetate inhibits HeLa 229 invasion by Bordetella pertussis and other invasive bacterial pathogens. Infect. Immun. 58:31873193. Finlay, B. B., and S. Falkow. 1988. Comparison of the invasion strategies used by Salmonella cholerae-suis, Shigella flexneri and Yersinia enterocolitica to enter cultured animal cells: endosome acidification is not required for bacterial invasion or intracellular replication. Biochimie 70:1089-1099. Finlay, B. B., and S. Falkow. 1989. Common themes in microbial pathogenicity. Microbiol. Rev. 53:210-230. Finlay, B. B., J. Fry, E. P. Rock, and S. Falkow. 1989. Passage of Salmonella through polarized epithelial cells: role of the host and bacterium. J. Cell Sci. Suppl. 11:99-107. Finlay, B. B., B. Gumbiner, and S. Falkow. 1988. Penetration of Salmonella through a polarized Madin-Darby canine kidney epithelial cell monolayer. J. Cell Biol. 107:221-230. Finlay, B. B., S. Ruschkowski, and S. Dedhar. 1991. Cytoskeletal rearrangements accompanying Salmonella entry into epithelial cells. J. Cell Sci. 99:283-292. Gazit, A., P. Yaish, C. Gilon, and A. Levitzki. 1989. Tyrphostins. I. Synthesis and biological activity of protein tyrosine kinase inhibitors. J. Med. Chem. 32:2344-2352. Guan, K. L., and J. E. Dixon. 1990. Protein tyrosine phosphatase activity of an essential virulence determinant in Yersinia. Science 249:553-556. Hedberg, K. K., G. B. Birrell, D. L. Habliston, and 0. H. Griffith. 1990. Staurosporine induces dissolution of microfilament bundles by a protein kinase C-independent pathway. Exp. Cell Res. 188:199-208. Hill, T. D., N. M. Dean, L. J. Mordan, A. F. Lau, M. Y. Kanemitsu, and A. L. Boynton. 1990. PDGF-induced activation
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of phospholipase C is not required for induction of DNA synthesis. Science 248:1660-1663. Hynes, R. 0. 1987. Integrins: a family of cell surface receptors. Cell 48:549-554. Isberg, R. R. 1991. Discrimination between intracellular uptake and surface adhesion of bacterial pathogens. Science 252:934938. Isberg, R. R., and J. M. Leong. 1990. Multiple beta 1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells. Cell 60:861-871. Isberg, R. R., D. L. Voorhis, and S. Falkow. 1987. Identification of invasin: a protein that allows enteric bacteria to penetrate cultured mammalian cells. Cell 50:769-778. Jaken, S., K. Leach, and T. Klauck. 1989. Association of type 3 protein kinase C with focal contacts in rat embryo fibroblasts. J. Cell Biol. 109:697-704. Kellie, S., A. R. Horvath, and M. A. Elmore. 1991. Cytoskeletal targets for oncogenic tyrosine kinases. J. Cell Sci. 99:207-211. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. Litchfield, D. W., and E. H. Ball. 1986. Phosphorylation of the cytoskeletal protein talin by protein kinase C. Biochem. Biophys. Res. Commun. 134:1276-1283. Miller, V. L., and S. Falkow. 1988. Evidence for two genetic loci in Yersinia enterocolitica that can promote invasion of epithelial cells. Infect. Immun. 56:1242-1248. Moulder, J. W. 1985. Comparative biology of intracellular parasitism. Microbiol. Rev. 49:298-337. Pepe, J. C., and V. L. Miller. 1990. The Yersinia enterocolitica inv gene product is an outer membrane protein that shares epitopes with Yersinia pseudotuberculosis invasin. J. Bacteriol.
172:3780-3789. 29. Rosengurt, E., P. M. Rodriguez, and K. A. Smith. 1983. Phorbol esters, phospholipase C, and growth factors rapidly stimulate the phosphorylation of a Mr 80,000 protein in intact quiescent 3T3 cells. Proc. Natl. Acad. Sci. USA 80:7244-7248. 30. Shaw, L. M., J. M. Messier, and A. M. Mercurio. 1990. The activation dependent adhesion of macrophages to laminin involves cytoskeletal anchoring and phosphorylation of the alpha 6 beta 1 integrin. J. Cell Biol. 110:2167-2174. 31. Shiba, Y., Y. Sasaki, and Y. Kanno. 1988. 12-0-tetradecanoylphorbol-13-acetate disrupts actin filaments and focal contacts and enhances binding of fibronectin-coated latex beads to 3T3-L1 cells. Exp. Cell Res. 178:233-241. 32. Stumpo, D. J., J. M. Graff, K. A. Albert, P. Greengard, and P. J. Blackshear. 1989. Molecular cloning, characterization, and expression of a cDNA encoding the "80- to 87-kDa" myristoylated alanine-rich C kinase substrate: a major cellular substrate for protein kinase C. Proc. Natl. Acad. Sci. USA 86:4012-4016. 33. Tamaoki, T., and H. Nakano. 1990. Potent and specific inhibitors of protein kinase C of microbial origin. Bio/Technology 8:732-735. 34. Watson, S. P., J. McNally, L. J. Shipman, and P. P. Godfrey. 1988. The action of the protein kinase C inhibitor, staurosporine, on human platelets. Evidence against a regulatory role for protein kinase C in the formation of inositol trisphosphate by thrombin. Biochem. J. 249:345-350. 35. Yaish, P., A. Gazit, C. Gilon, and A. Levitzki. 1988. Blocking of EGF-dependent cell proliferation by EGF receptor kinase inhibitors. Science 242:933-935. 36. Young, S., J. Rothbard, and P. J. Parker. 1988. A monoclonal antibody recognising the site of limited proteolysis of protein kinase C. Inhibition of down-regulation in vivo. Eur. J. Biochem. 173:247-252. 37. Young, V. B., V. L. Miller, S. Falkow, and G. K. Schoolnik. 1990. Sequence, localization and function of the invasin protein of Yersinia enterocolitica. Mol. Microbiol. 4:1119-1128.
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0019-9567/92/062211-07$02.00/0 Copyright X) 1992, American Society for Microbiology
Tyrosine Protein Kinase Inhibitors Block Invasin-Promoted Bacterial Uptake by Epithelial Cells ILAN ROSENSHINE,1'2 VINCENT DURONIO,3 AND B. BRETT FINLAY' 2* Biotechnology Laboratory, Departments of Biochemistry and Microbiology,1 Canadian Bacterial Diseases Network 2 and Biomedical Research Center,3 University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3 Received 9 October 1991/Accepted 10 March 1992
The ability to enter into (invade) mammalian cells is an essential virulence determinant of many pathogenic bacteria and intracellular parasites. These organisms are internalized by host cells upon attachment to their surface. However, the mechanisms used by intracellular parasites to induce internalization into host cells have not been defined. We found that the protein kinase inhibitor staurosporine blocks invasion by some pathogenic bacteria, including Yersinia enterocolitica and Yersinia pseudotuberculosis. Using Escherichia coli containing the cloned Y. enterocolitica invasion gene inv (which codes for invasin, an integrin-binding protein), we found that staurosporine inhibits invasion by blocking bacterial internalization. Two specific tyrosine protein kinase inhibitors, genistein and tyrphostin, also block the internalization but not the binding of bacteria, suggesting that bacterial uptake may be dependent on the activity of this enzyme class in host HeLa cells. In contrast to invasion promoted by invasin, the invasion of HeLa cells by Salmonella typhimurium is not inhibited by any of these drugs.
Invasion into host epithelial cells is an important virulence factor for many pathogenic bacteria. Some Shigella, Salmonella, Listeria, and Yersinia species are facultative intracellular parasites, while some Chlamydia and Rickettsia species are obligatory intracellular parasites (10, 27). Once in the intracellular environment, bacteria are protected from the immune system and many chemotherapeutic agents. Salmonella species also use invasion to penetrate through monolayers of polarized epithelial cells (12). The entry of the pathogen into the host cell is initiated by bacterial binding to the host cell surface, which is followed by internalization. The internalization step is facilitated by host cell actin filaments, since pathogen internalization is inhibited by agents, such as cytochalasin D, which disrupt actin filaments (4, 9, 11). The binding of bacteria to the cell surface is not necessarily followed by internalization, as many bacterial species bind to eucaryotic cells but do not enter them (19, 26). Given the active involvement of the host cytoskeleton, it appears that invasive bacteria generate an "uptake signal" which induces the host cell to internalize them. To gain a better understanding of invasion processes, we have begun to characterize the nature of the uptake signals and the mechanism of their transduction by using several invasive pathogens and the cloned invasion genes of Yersinia enterocolitica. The Y enterocolitica and Yersinia pseudotuberculosis inv invasion genes are highly homologous and code for invasin, a 92- or 103-kDa outer membrane protein (21, 28, 37). Invasin promotes invasion by interacting with eucaryotic transmembrane proteins belonging to the integrin superfamily, including the a3,1, a4l, a51, and a6,13 integrins (19, 20). Integrins bind to various basal membrane proteins, such as fibronectin, collagen, and laminin, and are localized in focal contacts. They also participate in transducing extracellular signals and are components of the apparatus that anchors the cytoskeleton to the membrane (18). In eucaryotic cells, different classes of protein kinases *
(PK) play a major role in transducing extracellular signals. We examined the involvement of these enzymes in transducing signals needed for bacterial internalization by epithelial cells. In this paper we present evidence that invasion promoted by Yersinia invasin can be blocked by tyrosine PK (TPK) inhibitors. MATERIALS AND METHODS Bacterial strains and plasmid. Salmonella typhimurium SL1344, Y enterocolitica 8081c, Y pseudotuberculosis YPIIIp-, and Escherichia coli HB101 have been described elsewhere (9). Plasmid pVM101 (28) was obtained from J. Bliska, V. Miller, and S. Falkow (Stanford University). The bacteria were grown and plated in LB broth or on LB agar. When needed, ampicillin (Sigma) was added to a final concentration of 50 ,ug/ml. Eucaryotic cells. HeLa cells (ATCC CCL2) were grown and assayed at 37°C with 5% CO2 in minimal essential medium (MEM) supplemented with 10% (vol/vol) fetal calf serum. Kinase inhibitors. All inhibitors were dissolved in dimethyl sulfoxide, and stock solutions were divided into aliquots and stored at -20°C. The stock concentrations were as follows: staurosporine (Boehringer Mannheim), 1 mM; genistein (ICN), 100 mM; tyrphostin-AG34 (obtained from A. Levitzki, The Hebrew University of Jerusalem; AG34 is com-
pound 9 in reference 35), 50 mM; calphostin C (Kamiya Biomedical), 10 mM; 1-(5-isoquinolinesulfonyl)-2-methyl piperazine (H7 [Sigma]), 300 mM; N- (2- aminoethyl) -5isoquinolinesulfonamide dihydrochloride (H9 [Calbiochem]) 300 mM; and 12-O-tetradecanoylphorobol-13-acetate (TPA [Sigma]), 10 mM. Prior to use, the inhibitors were diluted in MEM supplemented with 10% (vol/vol) fetal calf serum, and the mixture was added to the cultured epithelial cells. Effect of PK inhibitors on bacterial viability. Fresh overnight bacterial cultures (10-,ul aliquots) were inoculated into 1 ml of MEM supplemented with 10% (vol/vol) fetal calf serum with or without a PK inhibitor. The cultures were
Corresponding author. 2211
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incubated (37°C, 5% C02) for various times. The number of CFU was compared for treated and untreated bacteria. Effect of PK inhibitors on the phosphorylation of a major PKC substrate, p80. The method used for detecting p80 phosphorylation is a slight modification of the procedure described by Rosengurt et al. (29). HeLa cells were seeded at 5 x 105 cells per dish in 35-mm tissue culture petri dishes. On the next day, the cultures were washed twice with phosphate-free MEM and the cells were incubated (37°C, 5% C02) with this medium containing carrier-free 32p; (ICN) at 290 RCi/ml to label the endogenous ATP pool. After 1.5 h, the different inhibitors were added as needed (to the following final concentrations: staurosporine, 0.5 ,uM; calphostin C, 0.35 ,uM; and H7, 300 ,uM), the cells were incubated as before for an additional 1 h, and then protein kinase C (PKC) was induced by the addition of TPA at 0.1 ,uM for 5 min. The reaction was stopped by removal of the medium and rapid washing of the cultures twice with ice-cold 0.15 M NaCl-20 mM Tris-HCl (pH 7.5). The cells were immediately extracted by scraping into 0.5 ml of lysis solution (1% Triton X-100, 50 mM Tris-HCI [pH 7.6], 1 mM EDTA, 0.4 mM V04, 1 mM NaF, 0.1 mg of phenylmethylsulfonyl fluoride per ml, 10 ,ug of leupeptin per ml). The extracts were cleared by centrifugation (1 min at 14,000 x g) and removed to a fresh tube containing 130 ,ul of Sx loading buffer (0.5 M Tris-HCl [pH 6.8], 10% sodium dodecyl sulfate [SDS], 0.5 M dithiothreitol, 50% glycerol). Aliquots were heated at 98°C for 10 min and cleared as before prior to resolution by gel electrophoresis. The samples were adjusted to 1.4 x 106 cpm/20 ,ul, and electrophoresis was performed with an 8% SDS-polyacrylamide gel as described previously (24). The gel was dried and used for radioautography. Determination of PKC activity in staurosporine-treated cells. Subconfluent cultures of HeLa cells in 175-cm2 flasks were treated for 1 h with or without 1 ,uM staurosporine. The HeLa cells were washed twice with phosphate-buffered saline (PBS), scraped from the flasks into 1.4 ml of PBS, centrifuged, resuspended in sonication buffer, and sonicated so that they would lyse (6). The supernatant was cleared by centrifugation, and PKC activity was assayed immediately. PKC activity was determined by comparing the activity of histone Hi phosphorylation in the presence or absence of PKC activators and cofactors (Ca2+, diolein, and phosphatidylserine) as described elsewhere (6). The specific activities of PKC in treated and untreated cells were compared. Bacterial internalization. Bacterial internalization was determined as described elsewhere (9) with slight modifications. In brief, 10 RI of a fresh overnight bacterial culture was added to a HeLa cell culture that had been seeded the previous day in a 24-well plate at a density of 105 cells per well, and the mixture was centrifuged for 5 min at 800 x g and incubated for 90 min (37°C, 5% CC2). The cells were washed twice and incubated for an additional 90 min in medium containing 100 ,ug of gentamicin per ml; this antibiotic kills extracellular bacteria. The monolayers were washed and lysed with 1% Triton X-100, and appropriate dilutions were plated to determine the number of viable intracellular bacteria. When inhibitors were used, they were added to MEM and were present throughout the invasion assay. RESULTS The PK inhibitor staurosporine blocks the invasion of HeLa cells by Yersinia species. In eucaryotic cells, protein phosphorylation is often involved in the transduction of
1000
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0.4 0.6 0.8 Staurosporine ,LM
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FIG. 1. Effect of staurosporine treatment on invasion of HeLa cells by Y. enterocolitica 8081c (-), Y pseudotuberculosis YPIIIp(0), S. typhimunum (A), and E. coli HB101 containing plasmid pVM101 (inv gene) (O). Values are averages of duplicates. Staurosporine in MEM was added to HeLa cells 15 min prior to infection.
extracellular signals. If PK activity is necessary for transducing a bacterial uptake signal, inhibitors of these enzymes should also inhibit bacterial invasion. To test this hypothesis, we used staurosporine to inhibit PK activity. Staurosporine is an extremely potent PK inhibitor, having 50% inhibitory concentrations in the nanomolar range for PKC, cAMP-dependent PK, and TPKs such as pp60vsrc (33). However, staurosporine is a poor inhibitor of other TPKs, such as those of the epidermal growth factor receptor and the insulinlike growth factor receptor (33). In vivo, 1 ,uM staurosporine is needed to inhibit PKC almost completely (7, 34). Staurosporine exhibited dose-dependent inhibition of invasion of HeLa cells by Y enterocolitica and Y pseudotuberculosis (Fig. 1). Staurosporine did not have any inhibitory effect on the invasion rates of S. typhimurium (Fig. 1). Staurosporine inhibits invasion promoted by the Yersinia inv gene. Y enterocolitica and Y pseudotuberculosis possess multiple invasion systems; the most efficient invasion is promoted by invasin (19). To avoid the complications of multiple invasion systems, we examined the effect of staurosporine on the invasiveness of E. coli carrying the inv invasion gene of Y enterocolitica. Although E. coli HB101 does not normally invade eucaryotic cells, introduction of the inv invasion gene into this bacterium is sufficient to promote its binding to and invasion of various eucaryotic cell lines (37). Invasion by E. coli HB101 carrying the inv gene of Y. enterocolitica on plasmid pVM101 (HB101/pVM101) was also inhibited by staurosporine (Fig. 1). At a concentration of 1 ,M, this drug inhibited 99.8% of the invasion promoted by this gene. Staurosporine also had a similar effect on invasion by E. coli carrying the inv gene of Y pseudotuberculosis (data not shown). Staurosporine inhibits invasion by blocking bacterial internalization by the host cell. Staurosporine may block invasion or may affect the results of the invasion assay by a variety of mechanisms, including (i) inhibiting some bacterial function, such as a kinase activity; (ii) affecting bacterial viability; (iii) triggering some antibacterial activity in the host cell; (iv) reducing the ability of invasin to bind to its receptors; or (v) preventing treated HeLa cells from internalizing bacteria by various mechanisms, including blockage of the transduction of an uptake signal. We examined each of these possibilities to identify the mechanism involved. Exposure to 1 puM staurosporine for 2 h did not have any
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FIG. 2. Binding of HB101/pUC19 (control) (A to C) and HB101/pVM101 (inv) (D to F) to HeLa cells. HeLa cells were seeded on coverslips, grown overnight, and pretreated with 1 pLM staurosporine for 30 min (B and E) or 0.1 ,uM TPA for 24 h (C and F) or left untreated (A and D). The cells were infected for 1 h, washed four times to remove the unbound bacteria, and fixed with 2% paraformaldehyde in PBS before being photographed.
significant effect on the viability of any of the bacterial species used in this study. The effect of this drug on the viability of bacteria in the intracellular environment was tested by first infecting HeLa cells with HB101/pVM101 for 60 min. The cells were then washed and incubated for 2 h in medium containing 100 ,ug of gentamicin per ml and with or without 1 ,uM staurosporine. Staurosporine did not have any effect on the viability of intracellular E. coli. The inhibitory effect of staurosporine on PK is only very slowly reversed after the drug is removed. We used this property to exclude the possibility that staurosporine inhibited invasion by affecting some bacterial function. HeLa cells were pretreated for 60 min with 1 ,uM staurosporine, washed several times to remove the drug, and infected with HB101/pVM101. This staurosporine pretreatment still reduced the invasion rate by 98%. Microscopic studies of the effect of staurosporine on the binding of HB101/pVM101 to HeLa cells demonstrated that staurosporine does not inhibit the binding of this bacterium to the epithelial cell surface (Fig. 2), indicating that this drug acts by preventing internalization of the bacteria rather than by blocking their binding to the host cell surface. To examine the effect of staurosporine on internalization, we performed an experiment in which the initial binding was uncoupled from the subsequent internalization event. Binding of HB101/pVM101 to HeLa cells without staurosporine was accomplished by incubation at 4°C for 60 min. (At this temperature, HB101/pVM101 binds to the epithelial cell surface but is not internalized [21].) The cells were washed five times with cold medium to remove the unbound bacteria. Medium with or without 1 ,uM staurosporine was added,
and the temperature was shifted to 37°C to allow internalization of the bound bacteria. Internalization of HB1O1/ pVM101 was reduced by 1,000-fold in the staurosporinesupplemented cultures. The effect of staurosporine on the shape of HeLa cells (Fig. 2) and on the cell cytoskeleton (16) might suggest that it inhibits internalization by paralyzing nonspecifically the phagocytic capability of these cells. However, our results indicated that this is not the case, since HeLa cells treated with 1 puM staurosporine still internalized S. typhimurium (which enters cells by an actin-requiring process similar to phagocytosis [11]) as efficiently as nontreated cells. Moreover, 1 ,uM staurosporine did not have any effect on the efficiency of nonspecific uptake of E. coli HB101 by the murine macrophagelike J774 cell line. All the above-described results indicate that staurosporine does not affect bacterial binding but prevents specifically the internalization promoted by invasin. Staurosporine is a potent inhibitor of different PK classes, including PKC, cAMP-dependent PK, and some TPKs. Of these various enzymes, TPK and PKC are the most likely candidates to participate in transducing the internalization signal that is generated upon binding of invasin to its P, integrin receptor. Both enzymes (PKC and TPKs such as pp6O-src) are localized in focal contacts associated with integrins (22, 23). Moreover, integrins and proteins which are believed to be involved in anchoring integrins to actin filaments, such as vinculin and talin, have been shown to be potential substrates for both enzymes (2, 23, 25, 30). Therefore, we attempted to identify which of these kinase activities is needed for invasion promoted by invasin.
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A 2 18.3
00.5
D
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-
71.8 4-
4X.2 FIG. 3. Effect of PK inhibitors on TPA-induced phosphorylation of p80. HeLa cells were labeled with 32Pi and pretreated with different PK inhibitors. PKC activity was then induced by the addition of TPA to a concentration of 0.1 ,uM and, after 5 min of induction, the reaction was stopped and the cells were extracted.
Electrophoresis was performed with an 8% SDS-polyacrylamide gel. The location of the major PKC substrate, p80, is indicated by the arrowhead, and the locations of other proteins whose phosphorylation was affected by the inhibitors are indicated by arrows. The extracts that were analyzed were as follows: lane A, cells induced with TPA and treated with 300 ,uM H7; lane B, cells induced with TPA and treated with 0.35 ,uM calphostin C; lane C, cells induced with TPA and treated with 0.5 ,uM staurosporine; lane D, cells induced with TPA; and lane E, cells that were not induced with TPA. Numbers at left are in kilodaltons.
Role of PKC in the internalization of HB101/pVM101. To examine whether PKC activity is needed for invasion promoted by invasin, we tested the effect of inhibitors which are known to have a higher specificity for PKC than for TPK (3, 33). Calphostin C (at a final concentration of 0.35 jiM) and H7 and H9 (both at a final concentration of 300 jiM) did not block the invasion of HeLa cells by HB101/pVM101 or S.
typhimunium.
These drugs were toxic for HeLa cells at
concentrations higher than those used, as determined by trypan blue staining of the treated cells. The effect of these inhibitors on PKC activity in intact cells was examined by analyzing the effect of the inhibitors on the phosphorylation of the major PKC substrate, p80 (29, 32). The cells were preincubated for 1 h with PKC inhibitors (0.5 jiM staurosporine, 0.35 FM calphostin C, or 300 jiM H7). PKC was then induced for 5 min by the addition of TPA to a final concentration of 0.1 jiM. The cells were lysed, and the cells extracts were analyzed by SDS-polyacrylamide gel electrophoresis. While staurosporine strongly inhibited TPA-induced p80 phosphorylation, H7 and calphostin C had a less inhibitory effect on p80 phosphorylation (Fig. 3), confirming other studies that showed that calphostin C and H7 are not as potent PKC inhibitors as staurosporine (the 50% inhibitory concentrations for PKC are as follows:
staurosporine, 0.003 jiM; calphostin C, 0.05 jiM; H7, 6 jiM; and H9, 15 ,uM [3, 33]). Interestingly, calphostin C and H7 inhibited the phosphorylation of proteins other than p80, whose phosphorylation was only moderately induced by TPA (Fig. 3). These results suggest that H7 and calphostin C
are not suitable for examining the correlation between PKC activity and other processes in intact cells. An alternate approach was used to determine whether PKC activity is correlated with invasion efficiency. PKC is activated in vivo by the short-living second messenger diacylglycerol. Similarly, PKC can be activated, as shown earlier, by phorbol esters, such as TPA, which act as stable analogs of diacylglycerol. However, long-term exposure to TPA down-regulates the enzyme activity by sensitizing the enzyme to proteolytic degradation and by specifically inhibiting its transcription by an unknown mechanism (6, 36). We hypothesized that if PKC is needed for invasin-mediated uptake of E. coli, it would be possible to block this uptake by using TPA to down-regulate PKC. For determination of the TPA concentration and incubation time needed to down-regulate PKC in HeLa cells, PKC from untreated cells and from cells which had been incubated with TPA for 24, 48, and 72 h was partially purified and its activity was assayed. We found that 24 h of incubation with 0.1 ,uM TPA was sufficient to reduce the PKC activity to its minimum level, which was 5.8% of the activity in nontreated cells (Fig. 4A and B). This 24-h pretreatment also reduced invasion by HB101/pVM101 to 17.7% ± 10.9% (an average of five different experiments) of the invasion of nontreated cells (Fig. 4C). To examine whether TPA inhibited invasion by a mechanism consistent with down-regulation of PKC, we tested the invasion of cells which had been pretreated with 0.1 ,uM TPA for 30 min (under these conditions, PKC is activated rather than down-regulated). In contrast to 24 h of pretreatment, 30 min of pretreatment did not inhibit invasion (Fig. 4C). The possibility that TPA inhibited invasion by affecting bacterial viability, reducing the binding of the bacteria to the host cells, or paralyzing the phagocytic machinery was also examined. Incubation for 2 h with concentrations of TPA as high as 1 ,uM did not have any effect on bacterial viability. To examine whether this drug affected the viability of the intracellular bacteria, we infected HeLa cells with HB1O1/ pVM101 for 90 min and then washed and incubated them in medium containing 100 ,ug of gentamicin per ml to kill extracellular bacteria. After 2 h of incubation, this medium was replaced by a medium containing 10 jig of gentamicin per ml and 0.1 jiM TPA, and the cells were incubated for an additional 24 h before being lysed to determine the number of viable bacteria inside them. This 24-h TPA treatment did not affect the viability of the intracellular E. coli. We also determined whether TPA had any effect on the binding of the bacteria to the HeLa cells or on the phagocytic capability of these cells, since TPA, like staurosporine, induces actin filament rearrangement and cell rounding (31) (Fig. 2). We used S. typhimurium to test the capability of TPA-treated cells to internalize bacteria. As seen with staurosporine, down-regulation of PKC with TPA did not inhibit the uptake of S. typhimurium. In addition, TPA treatment did not reduce the binding of HB101/pVM101 to HeLa cells (Fig. 2). These results demonstrate that TPA treatment inhibits the internalization of HB101/pVM101 by HeLa cells. Although TPA treatment inhibited invasin-mediated bacterial uptake by HeLa cells, staurosporine was a much more efficient inhibitor (Fig. 1 and 4C). Thus, to correlate PKC activity and invasion efficiency, we determined the residual PKC activity in staurosporine-treated HeLa cells and compared it with the residual PKC activity in HeLa cells treated for 24 h with TPA. The PKC specific activity in cells treated for 1 h with 1 jiM staurosporine was 6.1% of the specific activity in nontreated cells. While both TPA and staurospo-
BLOCKAGE OF BACTERIAL UPTAKE BY TPK INHIBITORS
VOL. 60, 1992
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FIG. 5. Effect of the TPK inhibitors genistein and tyrphostinAG34 on invasion promoted by invasin (0) and on invasion by S. typhimurium (O) of HeLa cells. (A) Genistein was added 15 min before infection. (B) Tyrphostin-AG34 was added 1 h before infection.
T 10
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FIG. 4. Effect of TPA on PKC activity and efficiency of bacterial invasion. PKC was partially purified from HeLa cells which had been left untreated (A) or pretreated for 24 h with 0.1 p.M TPA (B). Fractionation of the cell extracts with a MonoQ column and assays of PKC activity were carried out as described elsewhere (6). Histone Hi phosphorylation activity in the presence (-) or absence (E) of added Ca2 diolein, and phosphatidylserine is shown. (C) To determine invasion levels, we preincubated HeLa cells with 0.1 P.M TPA for 30 min or 24 h before infecting them with HB101/pVM101 and compared invasion levels with those for untreated cells.
rine treatments reduced PKC activity to similar extents (5.8 and 6.1%, respectively), staurosporine blocked invasion much more efficiently than TPA, suggesting that the inhibition of PKC is not the major reason for the inhibition of invasion by staurosporine. Internalization of HB101/pVM101 by HeLa cells can be blocked by specific TPK inhibitors. Since staurosporine also
inhibits some TPK activities, we examined the effect of two specific TPK inhibitors, tyrphostin-AG34 and genistein, on invasin-mediated bacterial uptake. These two drugs inhibit TPK by different mechanisms: tyrphostin-AG34 competes with the tyrosine residue binding to the kinase, and genistein inhibits the binding of ATP to the enzyme (1, 35). However, both drugs are highly specific TPK inhibitors (1, 14, 35). Both genistein and tryphostin-AG34 strongly inhibited the invasion of HeLa cells by HB101/pVM101 (98.5 and 93.2% inhibition, respectively), but they did not have a similar effect on invasion by S. typhimurium (Fig. 5A and B). We also examined, as described for staurosporine, the possibility that these drugs affect the invasion assay not by blocking internalization but by some other mechanism. The exposure of extracellular HB101/pVM101 or S. typhimurium for 3 h to either 250 ,uM genistein or 1 mM tyrphostin-AG34 in MEM did not affect bacterial viability. Genistein at 250 ,uM also did not affect the viability of intracellular HB101/pVM101 after 5 h of incubation. Microscopic examination revealed that these drugs do not reduce the efficiency of binding of the bacteria to the cells. Moreover, neither drug affects the morphology of the HeLa
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1000
100
c
10 0
co(U
.-1
.01 | 0
, 10
20
30
40
time (min) after genistein removal
FIG. 6. Invasion of HeLa cells by HB101/pVM101 after genistein removal. The bacteria adhered to but did not invade HeLa cells when the medium was supplemented with 250 ,uM genistein. The rate of internalization after removal of the drug was determined as described in the text and compared with that for untreated cells (100% invasion).
cells, suggesting that they do not
cause
major cytoskeleton
rearrangement.
The inhibition of TPK by genistein is known to be highly reversible (1, 17). We predicted that if genistein blocked invasion by inhibiting TPK activity, then the inhibition of invasion should also be reversible. To examine this possibility, we infected HeLa cells in medium containing 250 ,uM genistein with HB101/pVM101 for 90 min to allow binding. At this point, the cells were washed five times with PBS to remove the drug and the unbound bacteria, and prewarmed MEM was added instead to allow bacterial internalization. To monitor the internalization rate, we killed extracellular bacteria at different times after genistein removal by the addition of gentamicin to a final concentration of 100 ,ug/ml and incubation for an additional 90 min. The number of internalized bacteria was determined and compared with the invasion efficiency for nontreated cells and for cells from which genistein was not removed. The bound bacteria were internalized rapidly after genistein removal (Fig. 6). Less than 5 min after genistein removal, there was more than a 100-fold increase in the number of intracellular bacteria, and 20 min after genistein removal, the number of intracellular bacteria was the same as in nontreated cells (Fig. 6). These results suggest that the internalization of HB101/pVM101 may be dependent on some genistein-sensitive TPK activity in the host cell. DISCUSSION In this study, we used the PK inhibitor staurosporine to demonstrate a correlation between the efficiency of invasion promoted by the Yersinia inv gene product, invasin, and host cell PK activity. Further analysis with the specific TPK inhibitors genistein and tyrphostin-AG34 indicated that TPK activity in host cells may be essential for invasin-mediated bacterial internalization. Staurosporine and long-term TPA treatments reduced PKC activity to similar extents (6.1 and 5.8%, respectively), but the staurosporine treatment inhibited bacterial uptake about 90-fold more than the TPA treatment. Therefore, it seems that the major mechanism of the effect of staurospo-
rine on bacterial internalization is the inhibition of a PK other than PKC, possibly TPK inhibition. The PKC down-regulation experiment also suggested that PKC plays some role in the process of bacterial uptake by epithelial cells. However, the inhibitory effect of TPA on invasion may also result from some secondary effect of long-term PKC activation and down-regulation or, alternatively, TPA may interact directly with different unknown targets to inhibit invasion. The inhibition of invasion of several invasive bacteria after 1 h of TPA treatment has been described (8). However, under the conditions used by Ewanowich et al. (8), PKC was probably activated rather than down-regulated, and different bacterial strains were used. Nevertheless, analyses of the TPA effects in both studies may be interpreted in several different ways. Therefore, it is not yet clear whether PKC activity is needed for invasin-promoted bacterial uptake. The potential involvement of TPK in regulating the function of the invasin receptor integrin and of focal contact has been suggested by several studies (5, 23). However, in normal epithelial cells integrin has not been shown to be functionally affected by TPK activity. In this study, we showed that integrin-mediated bacterial internalization can be blocked by TPK inhibitors. However, it is not yet clear whether binding of the bacteria to integrin induces TPK activity to trigger internalization or, alternatively, whether some protein that is involved in this internalization must be phosphorylated to function. We are currently trying to examine these possibilities, but we have not yet detected the induction of specific tyrosine protein phosphorylation by invasin. Further investigation of how the binding of invasin to integrin induces internalization might provide new insights into how integrins interact with the cytoskeleton, how these interactions are regulated, and how they mediate signal transduction. Interestingly, one of the Yersinia virulence genes (the Y. enterocolitica gene yop-51 or its homologous gene in Y. pseudotuberculosis, yopH) encodes a specific tyrosine phosphatase (15). It is not clear how this enzyme contributes to bacterial virulence or whether it has anything to do with the correlation of invasin-mediated invasion with tyrosine phosphorylation. It will be interesting to determine whether the expression of the tyrosine phosphatase by intracellular yersiniae inhibits further infection of host cells by extracellular yersiniae. The results presented in this study substantiate previous observations that the mechanism of S. typhimunium invasion is different from that of Yersinia species. In a previous study, we reported that anti-integrin antibodies that inhibit invasinmediated invasion do not affect S. typhimurium invasion (13). We have found that, in contrast to S. typhimunum, Y. enterocolitica and HB101/pVM101 do not induce any major cytoskeleton rearrangement when they invade (13). Furthermore, the different profiles of the sensitivities of the two invasion systems to PK inhibitors indicate that S. typhimurium and Yersinia species are internalized by different pathways. Further studies of signaling between different invasive bacteria and their host cells will contribute to an understanding of these virulence factors. They may also shed new light on the dynamics of the cytoskeleton-membrane interaction and some aspects of endocytosis. In addition, these studies may lead to the identification and development of new chemotherapeutic agents that block host cell invasion by pathogens.
VOL. 60, 1992
BLOCKAGE OF BACTERIAL UPTAKE BY TPK INHIBITORS ACKNOWLEDGMENTS
We thank E. Lim and H. D. Fong for technical assistance. We also thank J. Bliska, V. Miller, and S. Falkow for plasmid pVM101 and A. Levitzki for tyrphostin-AG34. This work was supported by an operating grant to B.B.F. from the Medical Research Council of Canada and the NCE as part of the Canadian Bacterial Diseases Network Research Program. I.R. is a recipient of a long-term fellowship from the European Molecular Biology Organization. 1.
2.
3. 4.
5. 6.
7. 8.
9.
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11. 12. 13. 14.
15. 16.
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