Cell Signaling by the Type IV Pili of Pathogenic Neisseria*

Neisseria gonorrhoeae andNeisseria meningitidis are Gram-negative bacterial pathogens that infect human mucosal epithelia. Type IV pilus-mediated adherence of these bacteria is a crucial early event for establishment of infection. In this work, we show that the type IV pili transduce a signal into the eucaryotic host cell. Purified adherent pili, but not pili from a low binding mutant, trigger an increase in the cytosolic free calcium ([Ca2+] i ) in target epithelial cells, a signal known to control many cellular responses. The [Ca2+] i increase was blocked by antibodies against CD46, a putative pilus receptor, suggesting a role for this protein in signal transduction. Pilus-mediated attachment was inhibited by depletion of host cell intracellular Ca2+ stores but not by removal of extracellular Ca2+. Further, kinase inhibition studies showed that pilus-mediated adherence is dependent on casein kinase II. In summary, these data reveal a novel function of the type IV pili, namely induction of signal transduction pathways in host cells.

Neisseria gonorrhoeae, the etiological agent of gonorrhea, and Neisseria meningitidis, which causes sepsis and/or meningitis, are two human-specific organisms. Bacterial adherence to epithelial cell surfaces plays an important role in the establishment of infection. Pili, or fimbriae, are assembled and expressed on the surface of many Gram-negative bacteria and have been shown to establish an important link in communication between the bacteria and the target cells.
Type IV pili of pathogenic Neisseria are essential during the initial stage of infection (1). Studies with human volunteers showed that non-piliated variants of N. gonorrhoeae are avirulent (2,3). The pilus consists of a major pilus subunit protein, PilE, and a minor pilus-associated protein, PilC. The adherence to epithelial cells is dependent on expression of PilC, and on sequence variation in PilE (4 -10). Most strains carry two pilC alleles, pilC1 and pilC2 (7,11). In N. gonorrhoeae MS11, mutants expressing either PilC1 or PilC2 adhere equally well to epithelial cells, whereas in N. meningitidis strain FAM20 or strain 8013, PilC1 ϩ , pilC2 Ϫ mutants, but not pilC1 Ϫ , PilC2 ϩ mutants, adhere well to cells (8,9,11,12). PilC has been suggested to be located at the tip of the pilus, and purified PilC inhibits adherence of both gonococci and meningococci (13).
However, PilC is also found in the bacterial membranes and is associated with the bacterial cell surface (11).
CD46 (membrane cofactor protein) acts as a eucaryotic receptor for gonococcal and meningococcal pili (14). CD46 is an abundant transmembrane glycoprotein involved in complement regulation on host cells and is expressed on virtually every human cell type except erythrocytes (15). Antibodies directed against CD46 as well as purified recombinant CD46 block binding of pathogenic Neisseria to target cells. Further, piliated, but not non-piliated, bacteria adhere to Chinese hamster ovary cells expressing human CD46 (14). It is likely that CD46, which is a human-specific protein, determines the host specificity of the pathogenic Neisseria species.
Colonization of epithelial cells by N. gonorrhoeae and N. meningitidis is followed by cellular invasion. The opacity proteins (Opa) are a family of invasion-associated outer membrane proteins that bind to CD66 (16 -19) and heparan sulfate proteoglycan receptors on human cells (20,21). The invasion of gonococci into HEC-1-B cells is enhanced by preincubation with fixed target cells, suggesting an induction of invasion-related functions upon contact with epithelial cells (22). It has also been shown that interaction between piliated and/or Opa expressing N. gonorrhoeae and epithelial cells leads to activation of nuclear factor-B, the activator protein 1, and production of inflammatory cytokines (23).
The mechanism behind bacterial signaling during adhesion and invasion has also been studied in enteropathogenic Escherichia coli (EPEC) 1 and species of Salmonella, Shigella, and Yersinia. In these systems, entry into nonphagocytic cells involves induction of host signal transduction mechanisms (24). The pathogenic Neisseria colonize the mucosal epithelia, invade the target cells, and disseminate into the blood stream. The [Ca 2ϩ ] in the extracellular space and in the blood are in the millimolar range. Within the eucaryotic cell, the [Ca 2ϩ ] i plays a central role in signal transduction. In a resting epithelial cell, the [Ca 2ϩ ] is around 100 nM. High storages of Ca 2ϩ are kept in the endoplasmic reticulum (ER) and are released upon signals and/or receptor activation.
To better understand the mechanism(s) involved in the induction of the host cell response to neisserial attachment, we examined the role of Ca 2ϩ signaling in the interaction of these bacteria with epithelial cells. We provide the first evidence showing that neisserial pili stimulate a Ca 2ϩ signal in host cells. Type IV pili from an adhesive strain, but not pili from a low binding pilC mutant, trigger mobilization of cytosolic free Ca 2ϩ in target epithelial cells. The [Ca 2ϩ ] i transient is associated with the pilus, which is then a novel Ca 2ϩ signaling factor.

EXPERIMENTAL PROCEDURES
Bacterial Strains-N. gonorrhoeae MS11 mk (P ϩ ) and MS11 mk (P Ϫ n), deleted in the 5Ј end of pilE, have been described (3). The MS11 mk strain sample used in our studies is designated MS11 mk (P ϩ )-u and is referred to in the text as MS11. Piliated (P ϩ ) and non-piliated (P Ϫ ) variants were distinguished by colony morphology under a binocular microscope. FAM20 mutant strains FAM20.1 and FAM20.2 with mutations in pilC1 and pilC2, respectively, have previously been described (11). The bacteria used did not express detectable levels of Opa, as detected by SDS-polyacrylamide gel electrophoresis of outer membrane preparations. Bacteria were grown on GCB-agar supplemented with Kellogg's complement (2) at 37°C in 5% CO 2 atmosphere and passaged every 18 -20 h.
Pili and Outer Membrane Preparations-Preparation of pili and outer membranes were performed as described previously (7). The pili preparations used (1 mg/ml) were crystallized and solubilized three times and contain less than 1% of minor proteins detected in Coomassie Blue-stained gels (shown in Ref. 7). For detection of Opa, outer membranes were heated to 100 or 37°C, subjected to 12% SDS-polyacrylamide gel electrophoresis, and stained with Coomassie Brilliant Blue.
Antibodies-Construction of recombinant CD46 has been described previously (14). The recombinant CD46, i.e. a maltose binding protein (MBP)-CD46 fusion protein, was purified over amylose columns and injected into a rabbit. The serum was extensively absorbed over MBPcoated CNBr-Sepharose columns (Amersham Pharmacia Biotech), to remove all MBP antibodies, and was finally purified over HiTrap protein A affinity columns (Pharmacia Biotech) to obtain IgG. The purified antiserum reacted strongly against CD46. The CD55 IgG 1 monoclonal antibody was purchased from Jackson ImmunoResarch Laboratories Inc. The monoclonal antibody GB24 IgG 1 was kindly provided by Dr. John Atkinson (Washington University, St. Louis). Antiserum against MS11 has been described previously (7). In the inhibition experiments, 50 g/ml of the antibodies was used.
Cell Lines and Growth Conditions-ME180 (ATCC HTB33), an epithelial-like human cell line from cervical carcinoma, was maintained in McCoy's 5A medium supplemented with 10% inactivated fetal bovine serum and 2 mM L-glutamine. Cell lines were maintained at 37°C, 5% CO 2 and occasionally grown in penicillin/streptomycin containing medium to prevent contaminations. All of the experiments were performed without fetal bovine serum, antibiotics, and L-glutamine. Media and growth supplements were purchased from Life Technologies, Inc. Cell culture materials were purchased from Costar.
Preparation of Defined Buffers-Preparation of Hepes-buffered saline (HBS) with defined free [Ca 2ϩ ] was constructed with the help of the MaxChelator software (45). The pH was set with NaOH to 7.4 at 20°C. Ionic strength of the buffers was calculated with the formula 0.5⌺C i Z i . The HBS contained 145 mM NaCl, 5 mM KCl, 1 mM Na 2 HPO 4 , 0.5 mM MgSO 4 , 10 mM Hepes, and 5 mM D-glucose. The buffer (P-HBS) used for permeabilized cells contained 110 mM KCl, 1 mM NaCl, 2 mM KH 2 PO 4 , 1 mM MgCl 2 , and 10 mM Hepes. The pH was set with KOH to 7.4 at 20°C. The solutions were sterile filtrated and stored in plastic bottles. To make a defined Ca 2ϩ buffer, an excess of CaCl 2 was added and buffered with EGTA down to the desired concentration of free Ca 2ϩ ions according to MaxChelator calculations.
Measurement of Intracellular Free Ca 2ϩ -The fluorescent Ca 2ϩ indicator fura-2 was used to measure [Ca 2ϩ ] i . ME180 cells, grown on poly-L-lysine (Sigma)-coated glass coverslips (Kebo), were incubated for 45 min at 37°C in McCoy's medium containing 0.5 mg/ml bovine serum albumin and 4 M fura-2 acetoxymethylester (AM) as described previously (25). The fluorometric microscopy system used has been described (26). Briefly, fura-2/AM loaded cells were placed in a 37°C perifusion chamber connected to an inverted epifluorescence microscope (Zeiss, Axiovert 35M). Upon assay, the ME180 cells were exposed to a perifusion of purified pili. The flow rate of 0.02 ml/min caused a delay of 10 s until the pili reached the cells. Pili were diluted 1:50 (final concentration of 20 g/ml) in HBS containing 1.5 mM free Ca 2ϩ . One single cell, isolated optically by the microscope, was analyzed by using a 100ϫ/1.3 NA oil-immersion objective. Each experiment was repeated at six independent occasions. The microscope was connected to a SPEX fluorolog-2 CM1T11I system for dual wavelength excitation fluorimetry. Upon binding to Ca 2ϩ , fura-2 shifts excitation maximum from 380 nm to 340 nm. The ratio between the fluorescence intensity at 340 and 380 nm (F340/380) gives a value of free Ca 2ϩ in the cytosol. Dissociation constant (K d ) of fura-2 was set to 225 nM. The background fluorescence was measured and subtracted before calculation of [Ca 2ϩ ] i . To compensate for variations in output light intensity from the two monochromators, the F340/380 values were corrected with both monochromators set at 360 nm. The [Ca 2ϩ ] i was calculated according to Grynkiewicz et al. (27).
Confocal Imaging-ME180 cells were grown on glass chamber slides (LAB-TEK). Upon assay, 80 M BAPTA/AM (Calbiochem) was added to the cells and incubated at 37°C for 30 min. The solution was removed, and fresh medium was added followed by an additional incubation of 15 min. MS11 P ϩ bacteria were allowed to bind for 60 min. Bacteria were detected with antiserum against MS11 diluted 1/100 and goat antirabbit IgG-fluorescein isothiocyanate diluted 1/500 (Sigma). We used the MultiProbe 2001 CLSM confocal laser scanning system (Molecular Dynamics) equipped with a diaphot 200 inverted microscope (Nikon). An excitation filter of 488 nm and the emission filter 510EFLP was used. The images were visualized by a 60ϫ/1.4 oil objective. The data were collected in a stack of 30 layers with a Z-stepsize of 1 m. Each image was then further processed by Photoshop 4.0 (Adobe Systems).
Permeabilization of Cells-For the permeabilization of cells, nonconfluent layers of ME180 cells were washed 3 ϫ 5 min in P-HBS. The cells were permeabilized with 0.5 g/ml digitonin in P-HBS for 15 min at 37°C (28). Trypan blue (0.01%) was added in a control well as an indicator of successful permeability. Binding assays were carried out in an ATP-generating system containing 10 mM phosphocreatine and 10 units/ml creatine phosphatase.
Binding Assays-The cells were grown in 24-well tissue culture plates for 2-3 days until each well contained about 10 5 cells. The monolayers were carefully washed three times in 500 l of HBS with 1.5 mM free Ca 2ϩ . Bacteria (10 8 /ml), grown for 18 -20 h, were suspended in HBS buffer. The bacterial suspension (50 l) was added to the cells, and binding was allowed for 60 min. The infected cell layers were washed 3 ϫ 5 min, treated with 1% saponin for 5 min, serially diluted, and spread onto GCB plates. The bacteria were grown at 37°C, 5% CO 2 overnight, and colony-forming units (cfu) were counted.
Thapsigargin (10 M, Calbiochem) or dantrolene (1, 5-(p-nitrophenyl) furfurylidene aminohydantoin, 0.5 M, Calbiochem) was preincubated with the cells for 30 min at 37°C prior to addition of bacteria. For adherence of bacteria in Ca 2ϩ free extracellular medium, the cells were first preincubated for 15 min in 5 mM EGTA. The binding assay was carried out in EGTA containing HBS. As a control, 0.01% trypan blue was added to one of the wells during chemical treatment or infection. Only 1% of the ME180 cells were permeable for trypan blue, which is not more than what is seen for uninfected or untreated cells.
Kinase Inhibition Assays-The used kinase inhibitors were staurosporine (Sigma), genistein (Sigma), BIMM I (bisindolylmaleimide I, Calbiochem), H-89 (Calbiochem), and DRB (5, 6-dichloro-1-␤-D-ribofuranosylbenzimidazole, Calbiochem). IC 50 values and used concentrations are shown in Table II. For the assay, nonconfluent layers of ME180 cells were preincubated with the inhibitor for 20 min at 37°C. The bacteria were then added to the cells and incubated for 60 min at 37°C, 5% CO 2 . The cells were washed, treated with saponin, and plated on GCB plates. Percentage of bacterial adherence is shown in Table II and was calculated as follows: 100 ϫ cfu per well/cfu per well for MS11 P ϩ in defined HBS with 1.5 mM free Ca 2ϩ .

Adherent Type IV Pili Trigger a Cytosolic Free Ca 2ϩ
Transient in Target Cells-The effects of highly purified pili of N. gonorrhoeae MS11 on Ca 2ϩ signaling in ME180 epithelial cells were examined using a spectrofluorometric instrument. The pili were introduced with a flow rate of 0.02 ml/min. After 6 min of perifusion, the pili (20 g/ml) induced a cytosolic free Ca 2ϩ ([Ca 2ϩ ] i ) increase from about 90 nM to 450 nM (Fig. 1A). The [Ca 2ϩ ] i responses were not elicited by control buffer (HBS) or by outer membrane preparations of MS11(P Ϫ n) (Fig. 1B).
Antibodies Directed Against CD46 Block the [Ca 2ϩ ] i Response-We have previously shown that antibodies directed against CD46 block pilus-mediated adherence of pathogenic Neisseria to ME180 cells (14). Consequently, we tested whether antibodies directed against CD46 could block the Ca 2ϩ signaling. As shown in Fig. 3, ME180 cells pretreated with polyclonal or monoclonal antibodies against CD46 did not respond with a [Ca 2ϩ ] i transient when exposed to pili. Pili still triggered a [Ca 2ϩ ] i transient in ME180 cells preincubated with monoclonal antibodies directed against CD55 (decay accelerating factor) ( Fig. 3C) or normal rabbit serum (data not shown). Adherence of piliated Neisseria to ME180 cells is not blocked by CD55 antibodies (data not shown). These data suggest that the Ca 2ϩ signal is transduced by the transmembrane cellular pilus receptor, CD46.
Ca 2ϩ Is Released from Intracellular Stores-The induction of [Ca 2ϩ ] i transients exclusively by pili from adherent Neisseria suggested that Ca 2ϩ signaling was necessary for adherence. Accordingly, the cells were treated in several ways to inhibit the [Ca 2ϩ ] i signals and then were exposed to bacteria in adherence assays. The [Ca 2ϩ ] i transients in mammalian cells can be mediated by release of Ca 2ϩ from intracellular stores and/or by open channels in the plasma membrane. Thapsigargin, an irreversible inhibitor of the ER Ca 2ϩ -ATPase (29), induces release of intracellular Ca 2ϩ , resulting in depletion of the ER stores. Preincubation of ME180 cells with thapsigargin for 30 min blocked attachment of MS11 P ϩ (Fig. 4). Further, ME180 monolayers were pretreated with dantrolene, a drug that prevents release of Ca 2ϩ from IP 3 -sensitive stores (30,31). As demonstrated in Fig. 4, the binding of MS11 P ϩ was reduced in dantrolene-treated cells. Spectrofluorometric [Ca 2ϩ ] i measurements showed that both thapsigargin and dantrolene block the pilus-mediated rises in [Ca 2ϩ ] i (data not shown). However, removal of external free Ca 2ϩ with 5 mM EGTA did not affect adherence of MS11 P ϩ to ME180 cells, indicating that Ca 2ϩ ions did not enter through channels in the plasma membrane (Fig. 4). To further address the requirement for intracellular calcium elevation in bacterial attachment, the ME180 cells were preincubated with the membrane-permeable calcium chelator BAPTA/AM. This agent is trapped inside cells after cleavage by cytosolic esterases. As shown in Fig. 5, A and B, chelation of the cytosolic free Ca 2ϩ with BAPTA reduced the adhesion of MS11 P ϩ to the host cells. Taken together, our data argue that Ca 2ϩ from intracellular stores is mobilized in response to binding of P ϩ bacteria.
The Cytosolic Free [Ca 2ϩ ] of the Host Cell Affects MS11 P ϩ Adherence-To examine the direct role of Ca 2ϩ in pilus-mediated adherence, we used permeabilized ME180 cells and buffers with defined free [Ca 2ϩ ]. Chemical permeabilization with digitonin creates pores of 8 -10 nm in the plasma membrane by complexing with membrane cholesterol and other unconjugated ␤-hydroxysterols (28). Fig. 5, C and D, show the adherence of MS11 P ϩ to permeabilized ME180 cells in P-HBS con-  taining 10 and 0 M Ca 2ϩ , respectively. The adherence of MS11 P ϩ is directly dependent on the [Ca 2ϩ ] in the buffer. Further, Fig. 6 shows that the free intracellular [Ca 2ϩ ] clearly influences the adherence of MS11 P ϩ to cells in a dose-dependent manner. At cytosolic [Ca 2ϩ ] of 200 nM, the binding was low; however, at 800 nM the bacterial adherence was close to that observed with intact cells. These data argue that efficient binding of piliated Neisseria to target epithelial cells requires an elevated cytosolic free [Ca 2ϩ ].
Neisserial Adhesion Is Dependent on Casein Kinase II-We employed kinase inhibitors of varying substrate specificity to gain further insight into the host cell signaling pathway. The effects of the various kinase inhibitors upon bacterial attachment is summarized in Table II. Staurosporine, genistein, or BIMM I did not block adherence of MS11 P ϩ to ME180 cells. Staurosporine, a broad range inhibitor, affects protein kinase A (PKA), protein kinase C (PKC), protein kinase G (PKG), Ca 2ϩ / calmodulin kinase (CaMK), and myosin light chain kinase (MLCK) (32). Genistein inhibits PKA, PKC, PKG, and tyrosine kinases (33). BIMM I inhibits PKC and PKA (34). In contrast, both H-89 (35) and DRB (36) reduced MS11 P ϩ adherence (Table II). H-89 blocked bacterial binding at concentrations known to inhibit casein kinase I (CK-I), casein kinase II (CK-II), CaMK, and MLCK. The possible involvement of CaMK and MLCK could be excluded, as staurosporine had no inhibitory effect. The highly specific CK-II inhibitor, DRB, clearly inhibited adherence of the bacteria, suggesting that CK-II takes part in the signal transduction event during pilus-dependent adherence. Finally, spectrofluorometric analysis showed that the pilus-induced Ca 2ϩ -peak occurred in ME180 cells pretreated with DRB (data not shown). Thus, the release of intracellular Ca 2ϩ may be followed by the action of CK-II.

DISCUSSION
In this study, we characterize signal transduction events that occur in human epithelial cells during pilus-dependent adherence of pathogenic Neisseria. We show that cytosolic Ca 2ϩ elevations occur in the host cells as they are exposed to pili and that the signal is most likely transduced by the pilus receptor, CD46. The increased Ca 2ϩ is due to release from intracellular stores since the depletion of intracellular Ca 2ϩ with thapsigargin or treatment with dantrolene inhibited adherence of the P ϩ bacteria to host cells. In addition, removal of extracellular Ca 2ϩ by EGTA did not affect the binding. Thus, the ability of the cells to release Ca 2ϩ from intracellular stores must play an essential role in pilus-mediated attachment.
Pilus-mediated adherence is a rapid event, resulting in bacteria that are firmly attached to the host cell within 5-10 min.
We suggest that the [Ca 2ϩ ] i transient induced by pili is needed as an initial step to establish a stable contact between the bacteria and the host cells. If the calcium-dependent signal is blocked, the bacteria will not form a secondary tight interaction with the host, and the interaction with the cells will be lost during the washing procedure. The Ca 2ϩ signal was detected in the epithelial cells after 6 min of perifusion. As the flow of pili into the chamber was 0.02 ml/min, there was a delay of at least 5 min until the concentration of pili in the chamber reached 20 g/ml. It is possible that the development of a successful signal transduction is concentration dependent and that the process may involve interactions with several domains of the pilus, or several pilus rods.
When using permeabilized cells, at least 800 nM Ca 2ϩ was needed to induce a strong pilus-mediated adherence. Therefore, it is likely that the pilus-induced Ca 2ϩ mobilization results in a local [Ca 2ϩ ] significantly higher than the 450 nM peak detected, which was representing the total concentration within the whole cell. The [Ca 2ϩ ] i transient caused by pili was blocked by antibodies directed against the putative neisserial pilus receptor, CD46. Because the pilus receptor CD46 is a transmembrane protein, it may transfer a signal across the host cell membrane. Such signals may prime the host cells for bacterial uptake. CD55 (also called decay accelerating factor) shares homologies with the putative repetitive domains of the CD46 protein (15). CD55 has no transmembrane domain or cytoplasmic tail. Preincubation of the host cells with antibodies against CD55 did not interfere with the pilus-induced Ca 2ϩ signal. The exact pilus component responsible for the signal remains to be determined. However, because a PilC1 mutant failed to trigger [Ca 2ϩ ] i release, it is likely that the bacteria are, directly or indirectly, dependent on PilC1 for signaling. Whether the signal is mediated by PilC, PilE, or another pilus protein remains to be determined.
Pathogenic bacteria have developed various mechanisms to interact with host tissue. Many bacteria that cause disease have the capacity to enter into and survive within eucaryotic cells. Most mechanisms for this involve subversion and exploitation of host cell functions. Entry into nonphagocytic cells involves in many cases triggering of host signal transduction mechanisms to accomplish a bacterial uptake, i.e. to induce rearrangements of the host cell cytoskeleton to stimulate protein synthesis or phosphorylation of host cell proteins. Salmonella typhimurium and EPEC are known to elevate [Ca 2ϩ ] i in target cells. Upon attachment to epithelial cells, EPEC induces a signal transduction cascade involving host cell IP 3 formation followed by [Ca 2ϩ ] i release from IP 3 -sensitive stores (37). However, the [Ca 2ϩ ] i of the cells was measured an hour or more after bacterial infection. Though extracellular Ca 2ϩ was removed, EPEC could still adhere to the cells, suggesting that the Ca 2ϩ was released from internal stores (38). Also S. typhimurium infection of cultured cells is accompanied by a marked increase in [Ca 2ϩ ] i (39,40). The Ca 2ϩ rise did no longer occur in strains carrying mutations in genes responsible for invasion, and chelators of intracellular Ca 2ϩ , but not extracellular Ca 2ϩ , block the entry of S. typhimurium into cultured epithelial cells (41). Also, Trypanosoma cruzi, an intracellular parasite that causes Chagas' disease in humans, produces a soluble factor that induces rapid and repetitive [Ca 2ϩ ] i transients in host cells (42).
Kinase inhibitors are widely used in all kinds of combinations to evaluate the pathway in which a signal is transduced. By using kinase inhibitors with overlapping specificities, we show that PKC, PKA, PKG, CaMK, and MLCK are most likely not involved in pilus-mediated adhesion of Neisseria to epithelial ME180 cells, as staurosporine, genistein, or BIMM I af-  a BIMM I, bisindolylmalcimide I; DRB, 5,6-dichloro-1-␤-D-ribofuranosyl-benzimidazole; tyr-P, broad range tyrosine kinases.
b The difference between this value and the control is not statistically significant. fected the adherence. However, H-89 and DRB with distinct substrate profiles significantly reduced binding at concentrations known to block CK-II. Sequence analysis shows a possible threonine phosphorylation site for CK-II at the cytoplasmic tail of CD46 (43). Thus, it is tempting to speculate that these sites might be phosphorylated by CK-II upon neisserial attachment. If so, the pilus-mediated mobilization of intracellular Ca 2ϩ is followed by the phosphorylation(s) event, as DRB did not inhibit the [Ca 2ϩ ] i transient.
Among other bacteria able to phosphorylate host cell proteins, S. typhimurium stimulates the epithelial growth factor receptor, initiating a signal transduction cascade resulting in the tyrosine phosphorylation and activation of the mitogenactivated protein kinase. In contrast to pathogenic Neisseria, the adherence of EPEC to host cells could be inhibited by staurosporine and genistein (44).
In summary, our data show that pili play a novel role as an inducer of signaling pathways in the target cells. The pilusmediated adherence is an initial event that involves interaction with CD46. Additionally, the pili transduce a signal into the host cell, involving [Ca 2ϩ ] i mobilization and CK-II activity. The detailed pathways in host cell signaling and the possible threonine phosphorylation of the CD46 cytoplasmic tail upon neisserial attachment is currently under investigation.