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J. Biol. Chem., Vol. 277, Issue 52, 50716-50724, December 27, 2002

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Escherichia coli K1 Internalization via Caveolae Requires Caveolin-1 and Protein Kinase C Interaction in Human Brain Microvascular Endothelial Cells^*

Sunil K. Sukumaran, Michael J. Quon^§, and Nemani V. Prasadarao^¶

From the  Division of Infectious Diseases, Children's Hospital, and the Keck School of Medicine, University of Southern California, Los Angeles, California 90027 and the ^§ Diabetes Unit, Laboratory of Clinical Investigation, NCCAM, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, August 29, 2002, and in revised form, October 3, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The morbidity and mortality associated with Escherichia coli K1 meningitis during the neonatal period have remained significant over the last decade and are once again on the rise. Transcytosis of brain microvascular endothelial cells (BMEC) by E. coli within an endosome to avoid lysosomal fusion is crucial for dissemination into the central nervous system. Central to E. coli internalization of BMEC is the expression of OmpA (outer membrane protein A), which interacts with its receptor for the actin reorganization that leads to invasion. However, nothing is known about the nature of the signaling events for the formation of endosomes containing E. coli K1. We show here that E. coli K1 infection of human BMEC (HBMEC) results in activation of caveolin-1 for bacterial uptake via caveolae. The interaction of caveolin-1 with phosphorylated protein kinase C (PKC) at the E. coli attachment site is critical for the invasion of HBMEC. Optical sectioning of confocal images of infected HBMEC indicates continuing association of caveolin-1 with E. coli during transcytosis. Overexpression of a dominant-negative form of caveolin-1 containing mutations in the scaffolding domain blocked the interaction of phospho-PKC with caveolin-1 and the E. coli invasion of HBMEC, but not actin cytoskeleton rearrangement or the phosphorylation of PKC. The interaction of caveolin-1 with phospho-PKC was completely abrogated in HBMEC overexpressing dominant-negative forms of either focal adhesion kinase or PKC. Treatment of HBMEC with a cell-permeable peptide that represents the scaffolding domain, which was coupled to an antennapedia motif of a Drosophila transcription factor significantly blocked the interaction of caveolin-1 with phospho-PKC and E. coli invasion. These results show that E. coli K1 internalizes HBMEC via caveolae and that the scaffolding domain of caveolin-1 plays a significant role in the formation of endosomes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A broad range of pathogens have the capacity to induce their own uptake by host cells via classical endocytosis (1, 2). Because the endocytic pathway mediated by caveolae appears to avoid fusion of lysosomes, pathogens often utilize this pathway to survive within the host cells. Caveolae are indentations in the plasma membrane thought to be involved in transcytosis, signal transduction, and uptake of membrane components and extracellular ligands. Although it is known that caveolae have the capacity to pinch off as endocytic vesicles and can internalize a variety of ligands, the process seems to be selective under normal culture conditions. Caveolae contain a distinct group of molecules, including cholesterol, a 22-kDa protein, caveolin-1 and various glycolipids, and glycosylphosphatidylinositol-anchored molecules (3-8). Furthermore, receptors for various growth factors and hormones, including epidermal growth factor, platelet-derived growth factor, and insulin, have been localized in caveolae (9-12). Caveolin-1 is assumed to take a hairpin-loop conformation in the lipid bilayer, thereby exposing both the N and C termini to the cytoplasmic surface (13). A stretch of amino acids referred to as the "scaffolding domain" within caveolin-1 interacts with many signaling proteins (3-5, 13). Although the mechanism of caveola formation for transcytosis of small molecules has been well established, it is not clearly known how bacterial pathogens induce signaling events that lead to caveola formation during invasion of host cells.

One of the crucial events in Escherichia coli meningitis is the traversal of E. coli across the blood-brain barrier (BBB).¹ During this process, E. coli cells seek refuge in a safe compartment in human brain microvascular endothelial cells (HBMEC), which form a lining of the BBB. The BBB exhibits selective permeability mainly for transport of macromolecules, liquids, and nutrients between the blood and the brain. However, E. coli could exploit the mechanisms of transport of macromolecules to promote their entry. Using HBMEC culture as an in vitro model of the BBB, we have demonstrated that E. coli invasion is a complex, multifactorial process that involves important virulence factors like S-fimbriae, OmpA, IbeA, and IbeB (14-18). However, our studies so far have suggested that OmpA interaction with endothelial cells via a gp96-like receptor on HBMEC is critical for invasion (19). OmpA-mediated E. coli invasion of HBMEC induces the phosphorylation of FAK and its interaction with phosphatidylinositol 3-kinase (20, 21). We have further shown that the invading E. coli cells induce phosphorylation of PKC in a phosphatidylinositol 3-kinase-dependent manner and that PKC is recruited to the plasma membrane, where it interacts with its substrate, MARCKS (myristoylated alanine-rich C kinase substrate) (22). These signaling events lead to the accumulation of actin beneath the E. coli entry site, which is required for the generation of lamellipodia (23). These protrusions of HBMEC enwrap the E. coli cell, which is progressively drawn into a compartment in the host cell. However, the nature of the compartment in which the E. coli cell resides in order to cross the cell is not known.

The interaction of phospho-PKC with caveolin-1 has been shown to be crucial for the formation of caveolae, which contain a conserved consensus phosphorylation site for activated PKC (24-26). Thus, the increased PKC activity that we observed during E. coli invasion could be directed toward initiation of the interaction with caveolin-1, which may play a key role in regulating the internalization of E. coli. Previous studies have shown that pathogens like E. coli expressing FimH antigen and Chlamydia trachomatis prefer caveola-dependent endocytosis to invade eukaryotic cells and actively recruit caveolin to the sites of bacterial entry (1, 2, 27). In addition, the internalization of SV40 via caveolae has been shown to trigger actin rearrangements and dynamin recruitment to the sites of entry (28-32). These events appear to depend on the presence of cholesterol and on the activation of tyrosine kinases that phosphorylate proteins in caveolae. However, these studies used only general tyrosine kinase inhibitors and thus did not reveal the nature of specific kinases involved in caveola formation.

This report describes, for the first time, the role of PKC in initiating caveolin-1-mediated uptake of E. coli into HBMEC via caveolae. During E. coli invasion, the activated PKC migrated to the cell membrane and interacted with caveolin-1, which co-localized with condensed actin beneath the bacterial entry site. Interference of the PKC interaction with caveolin-1, either by overexpression of a dominant-negative mutant form of caveolin-1 or by introduction of a peptide that represents the scaffolding domain into HBMEC, significantly reduced the invasion. In addition, we also demonstrate the association of caveolin-1 with internalized E. coli by confocal microscopy.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacteria-- A rifampin-resistant mutant of E. coli K1 strain RS218 (serotype O18:K1:H7), E44, has been isolated from the cerebrospinal fluid of a neonate with meningitis and invades HBMEC in a cell culture model. E91 is a noninvasive derivative of E44 in which the ompA gene was disrupted (16). The bacteria were grown in brain heart infusion broth with appropriate antibiotics as necessary. All bacterial media were purchased from Difco.

Materials-- Monoclonal antibodies to phospho-PKC were purchased from Cell Signaling Technology Inc. (Beverly, MA). Antibodies to PKC (polyclonal) and caveolin-1 were obtained from Transduction Laboratories (Lexington, KY). Fluorescein isothiocyanate-conjugated secondary antibodies and rhodamine-phalloidin were obtained from Molecular Probes, Inc. (Eugene, OR). Cy3-conjugated secondary antibody was from Rockland Immunochemicals (Gilbertsville, PA). Monoclonal anti-actin antibody was obtained from Oncogene Research Products (Boston, MA). Normal goat serum and Vectashield mounting medium with 4',6-diamidino-2-phenylindole were obtained from Vector Laboratories, Inc. (Burlingame, CA). The PepTag nonradioactive PKC assay was purchased from Promega (Madison, WI). SuperSignal chemiluminescence reagent was obtained from Pierce. All other chemicals were obtained from Sigma.

Expression Plasmids and Antennapedia (AP) Peptides-- Wild-type caveolin-1 (a HindIII/BamHI fragment (~600 bp) containing cDNA for Myc-tagged canine caveolin-1) was blunt-ended and ligated in the sense orientation into the HpaI site of pCIS2. Mutant caveolin-1 in which F92A and V94A point mutations were introduced was cloned into the pCIS2 vector. The generation of these plasmids was described in detail previously (33). A peptide corresponding to the putative scaffolding domain of caveolin-1 (Cav, DGIWKASFTTFTVTKYWFYR, amino acids 82-101) or the scrambled control peptide (Cav-X, WGIDKAFFTTSTVTYKWFRY) was synthesized as a fusion peptide with the C terminus of the AP internalization sequence (RQIKIWFQNRRMKWKK) (34). The peptides were purified and analyzed by reverse-phased high pressure liquid chromatography. The synthetic peptides (20 µg each) were labeled using a fluoresceinamine labeling kit (Panvera, Madison, WI). The fluoresceinated peptides were purified by a Sephadex G-15 column.

HBMEC Culture Maintenance and Transfections-- HBMEC were isolated and cultured as described previously (14). HBMEC cultures were maintained in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 10% NuSerum, 2 mM glutamine, 1 mM sodium pyruvate, 100 µg/ml streptomycin, 100 units/ml penicillin, essential amino acids, and vitamins. HBMEC were transfected with mammalian expression vectors using LipofectAMINE. Briefly, DNA/LipofectAMINE in RPMI 1640 medium was added to 30-40% confluent HBMEC monolayers. After 6 h of incubation at 37 °C, the cells were washed with RPMI 1640 medium, and complete medium was added. The next day, the medium was replaced with medium containing G418, and the cells were maintained in the same medium until they were confluent. For introduction of AP-Cav and AP-Cav-X peptides into HBMEC, the peptides were dissolved in sterile water, added to the culture medium, and incubated with the cells for 6 h. The cells were washed before performing invasion assays.

E. coli Invasion Assays-- Confluent HBMEC in 24-well plates were incubated with 1 × 10⁷ E. coli cells in experimental medium (1:1 mixture of Ham's F-12 and Medium 199 containing 5% heat-inactivated fetal bovine serum) for 90 min at 37 °C. The monolayers were washed three times with RPMI 1640 medium and further incubated in experimental medium containing gentamycin (100 µg/ml) for 1 h to kill extracellular bacteria. The monolayers were washed again and lysed with 0.5% Triton X-100. The intracellular bacteria were enumerated by plating on sheep blood-agar plates. In duplicate experiments, the total cell-associated bacteria were determined as described for invasion, except that the gentamycin step was omitted. In some experiments, HBMEC were pretreated with various inhibitors for 30 min prior to the addition of bacteria. The effects of these inhibitors on HBMEC were assessed by the trypan blue exclusion method, and the effects on bacterial viability were tested by colony plate counting (16).

Preparation of Cytosolic and Membrane Fractions of HBMEC-- For detection of PKC activity, confluent monolayers of HBMEC grown on collagen-coated dishes (60-mm diameter) were washed with RPMI 1640 medium, and E. coli cells suspended in experimental medium were added. Following stimulation for varying periods of time (0, 5, 10, 15, and 30 min), the cells were rinsed twice in ice-cold phosphate-buffered saline and placed on ice. The cells in each 60-mm dish were harvested by scraping on ice into 2 ml of cell homogenization buffer consisting of 20 mM Tris (pH 7.5), 0.25 M sucrose, 10 mM EGTA, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 2 mM dithiothreitol. The cells were subjected to mild sonication, and the cell lysates were used for the PepTag assay as described below. To obtain the membrane and cytosolic fractions, the cell lysates in the above buffer were initially centrifuged at 5000 × g to remove the debris, followed by centrifugation at 100,000 × g at 4 °C for 45 min. The supernatant from this step was designated as the cytosolic fraction, and the pellet was designated as the membrane fraction. The procedures for immunoprecipitation, Western blotting, and scanning of the bands were described previously (22).

PepTag Assay for Nonradioactive Detection of PKC Activity-- The PepTag assay utilizes a brightly colored, fluorescent peptide substrate that is highly specific to PKC (Promega). Phosphorylation by PKC changes the net charge of the substrate from +1 to 1, thereby allowing the phosphorylated and non-phosphorylated versions of the substrate to be separated on an agarose gel (0.8%). The phosphorylated species migrates toward the positive electrode, whereas the non-phosphorylated substrate migrates toward the negative electrode. The phosphorylated peptide in the band can then be visualized under UV light. For PKC assay, HBMEC total lysates or membrane proteins (10-25 µg/10 µl) were incubated with PKC reaction mixture (25 µl) according to the manufacturer's protocol at 30 °C for 30 min. The reactions were stopped by placing the tubes in a boiling water bath. After adding 80% glycerol (1 µl), the samples were loaded onto an 0.8% agarose gel in 50 mM Tris-HCl (pH 8.0) and separated in the same buffer at 100 V for 15 min, and the bands were visualized under UV light and photographed.

Immunofluorescence Staining-- HBMEC were grown in eight-well chamber slides coated with collagen and infected with E. coli as described above. The monolayers were then washed with phosphate-buffered saline and fixed in 2% paraformaldehyde for 15 min at room temperature. Subsequently, the monolayers were incubated with 5% normal goat serum in phosphate-buffered saline containing 1% Triton X-100 for 30 min and further incubated with primary antibody in the same mixture for 1 h at room temperature. The cells were then washed with phosphate-buffered saline and incubated with secondary antibodies conjugated to the fluorochromes Cy3 and fluorescein isothiocyanate, respectively, or rhodamine-phalloidin for 30 min at room temperature. The cells were washed again; the chambers were removed; and the slides were mounted in Vectashield anti-fade solution containing 4',6-diamidino-2-phenylindole. Cells were viewed under a Leica DMRA microscope with Plan-apochromat ×40/1.25 NA and ×63/1.40 NA oil immersion objective lenses. Images were acquired with a SkyVision-2/VDS digital CCD (12-bit, 1280 × 1024 pixels) camera in unbinned or 2 × 2-binned models using EasyFISH software, saved as 16-bit monochrome, and merged as 24-bit RGB TIFF images (Applied Spectral Imaging Inc., Carlsbad, CA). Optical sectioning was carried out using a Leica TCS SP confocal laser scanning microscope, and images were analyzed using MetaMorph Version 5.0 software (Universal Imaging Corp., Downingtown, PA). The images were assembled and labeled by Adobe Photoshop Version 6.0.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inhibitors of Caveola Formation Significantly Reduce E. coli Invasion of HBMEC-- Our previous studies showed that PKC activated in E. coli invasion is critical for actin accumulation at the site of E. coli entry (23). Thus, we speculated that PKC might interact with caveolin-1, one of the molecules that has a PKC activation site and is a constituent of caveolae. Thus, to examine the role of caveolae, E. coli invasion assays were performed following treatment of HBMEC with various concentrations of filipin, which is reported to cause disassembly of caveolae and enclosed receptors found in caveolae (35, 36). Filipin was found to be effective in blocking the invasion of OmpA⁺ E. coli (E44) in a dose-dependent manner with a 50% inhibitory concentration of 2 µM and 80% inhibition at 4 µM ((1.12 ± 0.03) × 10⁴ cfu/well for untreated HBMEC versus (0.2 ± 0.05) × 10⁴ cfu/well for 4 µM filipin; p < 0.001) (Fig. 1A). However, the total cell-associated bacteria (represented as binding) did not differ between untreated and filipin-treated cells, indicating that the inhibition was not due to inefficient binding of E. coli to HBMEC. In contrast, OmpA E. coli (E91), which did not show significant invasion in untreated cells, also showed no inhibition of either total cell-associated bacteria or background invasion in filipin-treated HBMEC. Similarly, pretreatment of HBMEC with another inhibitor, cyclodextrin, an agent that inhibits caveola formation by sequestering plasma membrane cholesterol by inducing its efflux, also showed significant inhibitory effect on E. coli invasion. A dose of 2 mM exerted 40% inhibition, whereas a dose of 4 mM exerted a 75% inhibitory effect on invasion of HBMEC ((1.0 ± 0.3) × 10⁴ cfu/well for untreated HBMEC versus (0.21 ± 0.10) × 10⁴ cfu/well for cyclodextrin-treated HBMEC; p < 0.001) (Fig. 1B) without significant differences in the total cell-associated bacteria. These results suggest that caveolae may play a role in E. coli invasion.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Inhibition of E. coli invasion of HBMEC by filipin and cyclodextrin. Various concentrations of either filipin or cyclodextrin were incubated with confluent monolayers of HBMEC before performing the E. coli invasion assays. Similarly, the total extracellular bacteria were determined as described under "Experimental Procedures." OmpA E. coli (E91) was used as a negative control with maximum concentrations of the inhibitors. The results are expressed as relative invasion or binding, taking OmpA+ E. coli values as 100%. The error bars represent the means ± S.D. of three individual experiments carried out in triplicate.

Phosphorylated PKC Binds to Caveolin-1 in E. coli Invasion-- Having determined the importance of caveola formation in bacterial invasion, our next effort was to identify the activation of caveolin-1 during bacterial invasion. Because caveolin-1 activation requires interaction with phosphorylated PKC, immunoprecipitation studies were performed using anti-PKC antibody from total cell lysates of HBMEC infected with either E44 or E91. Western blot analysis of the immune complexes using anti-phospho-PKC and anti-caveolin-1 antibodies indicated an association of caveolin-1 with phospho-PKC only in HBMEC infected with E44 (invasive strain) and not in the cells infected with E91 (noninvasive strain) (Fig. 2A). The association of caveolin-1 with phospho-PKC was observed within 5 min post-infection with E44 and peaked at 15 min, followed by a decline at 30 min. Densitometric analysis of caveolin-1 bands indicated a 3-fold increase in the association with phospho-PKC when infected with E44 (Fig. 2B). In contrast, the noninvasive E. coli strain, despite containing similar levels of PKC as revealed by blotting, showed neither phosphorylated species of PKC nor caveolin-1. To rule out the possibility that the absence of caveolin-1 in E91 cell lysates is not due to low levels of caveolin-1, the total lysates of HBMEC infected with E44 and E91 were also immunoblotted with anti-caveolin-1 antibody. The immunoblot showed equal quantities of caveolin-1 in cell lysates. These results suggest that phosphorylated PKC interacts with caveolin-1 in E. coli invasion of HBMEC.

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Association of caveolin-1 with phospho-PKC. A, total cell lysates (200 µg of protein) of HBMEC infected with either E44 or E91 for varying periods of time were immunoprecipitated (IP) with anti-PKC antibody, and the immune complexes were separated by 10% SDS-PAGE. The proteins were then transferred to a nitrocellulose membrane; the upper portion of the blot (above 40 kDa) was immunoblotted with anti-phospho-PKC, and the lower portion with anti-caveolin-1 antibody. The upper portion was stripped with Restore Western Stripping buffer and reprobed with anti-PKC antibody to verify equality of loading in all the lanes. In a separate experiment, total cell lysates (20 µg) were subjected to Western blotting (WB) with anti-caveolin-1 antibody. B, the blots in A were scanned on a densitometer and plotted as the areas of the individual bands. C and D, total cell lysates of FAK- or FRNK-transfected HBMEC or wild-type PKC (PKC-WT)- or PKC/CAT-KR-transfected HBMEC, respectively, infected with E44 were subjected to immunoprecipitation with anti-PKC antibody, followed by immunoblotting with the same antibody or anti-caveolin-1 antibody. The blot used for anti-PKC antibody was stripped and reprobed with anti-phospho-PKC antibody. In addition, the cell lysates were also subjected to immunoblotting with anti-caveolin-1 antibody.

Our previous studies showed that FAK activation is important for PKC phosphorylation (22); thus, we next examined whether blocking FAK activation would inhibit caveolin-1 association with phospho-PKC. Wild-type FAK- and FAK-related non-kinase (FRNK)-transfected HBMEC were infected with E44, and cell lysates were analyzed for caveolin-1 activation. FRNK has been shown to negatively regulate the function of FAK (37). Our previous studies showed that overexpression of FRNK in HBMEC blocks E. coli cell-induced activation of FAK and subsequent actin rearrangements (20). As shown in Fig. 2C, immunoprecipitation with anti-PKC antibody pulled down significant quantities of PKC in both cell lysates. However, we observed significant phosphorylation of PKC only in wild-type FAK-transfected HBMEC, and the pattern was similar to that previously reported for non-transfected and infected HBMEC (22). When probed with anti-caveolin-1 antibody, the immune complexes showed profound association with caveolin-1 in these cells. However, in FRNK-transfected HBMEC, neither phospho-PKC nor the corresponding association of caveolin-1 was observed, suggesting that the interaction of PKC with caveolin-1 is downstream of FAK activation.

To confirm this sequence of events, we further examined the association of caveolin-1 with phospho-PKC in cell lysates of HBMEC overexpressing a dominant-negative mutant form of PKC. PKC/CAT-KR (a mutant in which the catalytic subunit of PKC has been mutated)- and wild-type PKC-transfected HBMEC were infected with E44 for various time points, and the cell lysates were analyzed. As expected, wild-type PKC-transfected HBMEC showed significant phosphorylation of PKC between 10 and 15 min post-infection, whereas PKC/CAT-KR-transfected cells showed no activation at all (Fig. 2D). When stripped and reprobed with anti-caveolin-1 antibody, the blot showed association of caveolin-1 similar to that in non-transfected HBMEC, whereas there were no observable levels of associated caveolin-1 in PKC/CAT-KR-transfected HBMEC, suggesting that an intact and active PKC is necessary for the association of caveolin-1. Lack of either phospho-PKC or caveolin-1 was not due to unequal loading of the proteins, as the blot of cell lysates with anti-caveolin-1 antibody showed equal amounts of the protein. Taken together, these results suggest that E. coli cell-induced activation of caveolin-1 may be required for the formation of caveolae and is downstream of FAK and PKC.

Localization of Caveolin-1 at the E. coli Entry Site with Actin and Phospho-PKC-- Because E. coli invasion of HBMEC appears to be mediated by caveolae, we next examined the distribution of caveolin-1 by immunocytochemistry. Our previous studies showed that invading E. coli cells induce actin accumulation at the E. coli attachment site (23); thus, HBMEC infected with either E44 or E91 were fixed, permeabilized, and stained for both actin and caveolin-1. We observed several groups of bacteria attached to HBMEC, although the actin accumulation was observed only beneath select groups (Fig. 3, A and B). We have previously shown that only E. coli cells that are entering the cell elicit actin condensation, whereas those merely attached to the surface do not (23). Furthermore, the co-localization of caveolin-1 with actin at the bacterial entry site was also observed (Fig. 3, C and D). In contrast, such a pattern was not present in E91-infected cells (Fig. 3, E-H), as would be expected, because E91 does not activate the signals necessary for either actin accumulation or caveolin-1 activation. Because immunoprecipitation studies revealed that phospho-PKC interacted with caveolin-1, we also stained the infected HBMEC with anti-phospho-PKC and anti-caveolin-1 antibodies. Strong accumulation of caveolin-1 beneath the E. coli entry site was observed (Fig. 3J), with caveolin-1 co-localization with phospho-PKC in E44-infected cells (Fig. 3, K and L), but not in E91-infected cells (Fig. 3, M-P). Taken together, these results suggest that activated PKC is probably recruited to the plasma membrane to interact with caveolin-1 to initiate the formation of caveolae and pulls the bacterium into the cytoplasm utilizing the force of the actin network.

View larger version (86K): [in this window] [in a new window]   Fig. 3.   Localization of caveolin-1 and phospho-PKC in HBMEC infected with OmpA+ E. coli. Confluent monolayers of HBMEC infected with either E44 (A-D and I-L) or E91 (E-H and M-P) were fixed and stained with rhodamine-phalloidin (B and F) and anti-caveolin-1 antibody (C and G). Similarly, in a separate experiment, the monolayers were stained with both anti-caveolin-1 (J and N) and anti-phospho-PKC (K and O) antibodies. Both green and red fluorescence was visualized using a dual-filter mode (D, H, L, and P). Arrows indicate the positions of bacteria, caveolin, actin, and phospho-PKC.

Transcellular Association of Caveolin-1 with E. coli in HBMEC-- Transmission electron microscopy of E. coli invasion of HBMEC revealed that the bacterium resides in an endosome and crosses HBMEC with no signs of multiplication (23). In addition, we did not observe any lysosomal association with endosomes, indicating that E. coli cells somehow avoid lysosomal killing. The results described herein suggest that the endosomal compartment might contain caveolin-1, which was previously shown to be responsible for avoiding lysosomal fusion (13). Thus, to confirm the presence of caveolin-1 during the transcellular process, we utilized the confocal laser microscopy Z section technique. HBMEC monolayers were infected with E44 for 30 min, fixed, permeabilized, and stained for caveolin-1 with anti-caveolin-1 antibody followed by fluorescein isothiocyanate-conjugated secondary antibody. E. coli cells were labeled with either anti-K1 (capsular polysaccharide) or anti-S-fimbria antibody followed by Cy3-conjugated secondary antibody. We obtained ~45 Z sections of 0.3 µm thickness from the top to the bottom of the cell; however, we have presented only five representative sections for each label. As shown in Fig. 4, several bacteria attached to HBMEC near the surface, and the corresponding caveolin-1 section revealed the distribution of caveolin-1 throughout the cells. A clear condensation of caveolin-1 around or beneath the bacteria in several places was observed (Fig. 4, A and F). Further sectioning showed that more bacteria were invading the cells especially at the center of monolayer. Interestingly, in this section, the density of caveolin-1 was slightly reduced throughout the cell, but more clear association was observed with the bacteria (Fig. 4, B and G). The sections that represent the middle of the cell (Fig. 4, D and I) showed a group of bacteria with significant association of caveolin-1. Other bacteria that showed accumulation of caveolin-1 around them at the top of the cell showed decreased association of caveolin-1, suggesting that these bacteria were still in the process of invasion at the plasma membrane. However, the bacteria at the center appeared to have already entered the cell. This group of bacteria continued to associate with caveolin-1 until reaching the bottom of the cell (Fig. 4, E-J). We also stained HBMEC infected with OmpA E. coli in a similar fashion, but did not observe any bacteria invading the cells and correspondingly no association of caveolin-1 (data not shown).

View larger version (80K): [in this window] [in a new window]   Fig. 4.   Association of caveolin-1 with OmpA+ E. coli during the transcellular process. Confluent monolayers of HBMEC were infected with E44 for 30 min, fixed, and stained with anti-caveolin-1 antibody followed by fluorescein isothiocyanate-coupled secondary antibody (A-E). The bacteria were stained with anti-K1 capsular polysaccharide antibody and then with Cy3-labeled secondary antibody (F-J). Multiple optical sections of 0.3-µm thickness of each label were accumulated, but only five representative sections are presented. Arrows indicate the position of the bacteria and the corresponding accumulation of caveolin-1.

In addition, an optical section obtained from the middle of the cell infected with OmpA⁺ E. coli from a different experiment was analyzed for the co-localization of caveolin-1 with OmpA⁺ E. coli using MetaMorph imaging software. As shown in Fig. 5A, the optical section contained several bacteria inside the cell. The corresponding caveolin-1 staining showed accumulation of caveolin-1 around the bacteria, although discontinuously, but not in other areas where there were no bacteria (Fig. 5B). Overlay of these two images clearly showed that OmpA⁺ E. coli and caveolin-1 were co-localized (Fig. 5C). Orthogonal sections of this optical slice also showed that E. coli (red) and caveolin-1 (green) were co-localized (yellow) when viewed from the area where several bacteria had invaded (Fig. 5D). Moreover, the fluorescence intensities of caveolin-1 and OmpA⁺ E. coli were calculated from this optical section and converted into a line scan. The area of the section that was taken for these calculations is indicated with a blue line. In agreement with our above results that caveolin-1 accumulated at the bacterial entry site, we observed significant association of caveolin-1 (green) with E. coli (red) (peaks at positions 150-175, 210-225, and 250-260). These results strongly suggest that caveolin-1 accumulates around the invading E. coli cells and is probably present within the endosomal membranes that are formed via caveolae.

View larger version (141K): [in this window] [in a new window]   Fig. 5.   Scanning of fluorescence intensities of E. coli and caveolin-1 from an optical section of OmpA+ E. coli cell-infected HBMEC. Confluent monolayers of HBMEC infected with E44 were stained for caveolin-1 with anti-caveolin-1 antibody, and bacteria were stained with anti-K1 capsular polysaccharide antibody followed by fluorescent secondary antibody as described under "Results." Optical sections were accumulated, and one section was analyzed for fluorescence scanning. Magnified images of bacteria stained in red (A), caveolin-1 stained in green (B), and an overlay of both A and B (C) are shown. The orthogonal projections of the optical section were viewed from XZ and YZ angles at a point where the two red lines intersect (D). The area of the section used for scanning is indicated by a blue line. The fluorescence intensities of red (E. coli) and green (caveolin-1) were plotted as a line scan (E) using MetaMorph Version 5.0 software.

Overexpression of a Dominant-negative Form of Caveolin-1 in HBMEC Blocks E. coli Invasion-- Because caveolin-1 was found to be associated with phospho-PKC, we decided to analyze the importance of this association in E. coli invasion. Phospho-PKC is reported to bind to a specific region of caveolin-1 known as the scaffolding domain. Thus, HBMEC were transfected with a mutant form of caveolin-1 in which two amino acids responsible for phospho-PKC interaction in the scaffolding domain (Cav/HBMEC) were mutated. We also transfected HBMEC with wild-type caveolin-1 (Cav⁺/HBMEC) and pcDNA3 as positive and negative controls, respectively. Although efforts were initially directed toward stable transfection, we were unsuccessful in obtaining stable colonies. Thus, "ephémeré (French for short-lived) transfections" were done at 30-40% confluence of HBMEC and continued in the presence of G418 until cultures were 90-100% confluent. For each experiment, a portion of the transfected cells were stained with anti-Myc antibody to assess the efficiency of transfection, and we found that 50-60% of the cells significantly expressed the Myc-tagged proteins (data not shown). Expression of mutant proteins was also verified by Western blot analysis for the expression of caveolin proteins. Total cell lysates of HBMEC and pcDNA3/HBMEC showed a band reactive to anti-caveolin-1 antibody, whereas Cav⁺/HBMEC and Cav/HBMEC showed two bands (Fig. 6A). The upper band was the Myc-tagged caveolin-1, which migrated slightly slower than that of native caveolin-1. When reprobed with anti-Myc antibody, the same blot revealed one band in both Cav⁺/HBMEC and Cav/HBMEC, suggesting that the caveolin-1 plasmid-transfected HBMEC express significant amounts of Myc-tagged caveolin-1. The equality of loading of total proteins in each lane was examined by blotting the cell lysates with anti-actin antibody. Invasion assays were then performed using these mutants. The results showed that E. coli invasion of Cav/HBMEC was inhibited by >70% compared with either non-transfected or pcDNA3-transfected HBMEC ((1.2 ± 0.2) × 10⁴ cfu/well for normal HBMEC and (1.1 ± 0.3) × 10⁴ cfu/well for pcDNA3/HBMEC versus (0.35 ± 0.1) × 10⁴ cfu/well for Cav/HBMEC) (Fig. 6B). Interestingly, the E. coli invasion of Cav⁺/HBMEC was observed to be 20% more compared with normal HBMEC ((1.5 ± 0.3) × 10⁴ cfu/well for Cav⁺/HBMEC). However, no differences in the extent of binding of E. coli to these cells were observed. OmpA E. coli was also used in these invasion assays and showed no significant differences in the levels of background invasion compared with pcDNA3/HBMEC. These results suggest a definite role played by caveolin-1 in mediating E. coli entry into HBMEC.

View larger version (38K): [in this window] [in a new window]   Fig. 6.   Inhibition of E. coli invasion and association of caveolin-1 with phospho-PKC in HBMEC overexpressing a dominant-negative form of caveolin-1. A, total cell lysates (20 µg of protein) of HBMEC, pcDNA3/HBMEC (p/HBMEC), Cav+/HBMEC, and Cav/HBMEC were blotted with anti-caveolin-1, anti-Myc, or anti-actin antibody. The arrow indicates Myc-tagged caveolin-1. B, confluent monolayers of various HBMEC were used for OmpA+ E. coli invasion assays. In simultaneous experiments, the total cell-associated bacteria were also determined. The results are expressed as either relative binding or invasion, taking HBMEC values as 100%. The error bars represent the means ± S.D. of four separate experiments performed in triplicate. C, total cell lysates (20 µg of protein) of Cav+/HBMEC and Cav/HBMEC infected with E44 for varying periods of time were subjected to the PepTag assay for PKC activity. D, ~200 µg of total cell lysate proteins of Cav+/HBMEC and Cav/HBMEC infected with E44 were immunoprecipitated (IP) with anti-phospho-PKC antibody. The immune complexes were subjected to Western blotting (WB) with anti-phospho-PKC and anti-caveolin-1 antibodies. In addition, the total cell lysates (20 µg) were also subjected to immunoblotting with anti-caveolin-1 antibody.

To demonstrate that the decrease in the E. coli invasion of Cav/HBMEC is due to the inability of phospho-PKC to interact with caveolin-1, we first examined the activation of PKC in these cells by a nonradioactive PepTag assay. Total cell lysates of both Cav⁺/HBMEC and Cav/HBMEC showed significant activation of PKC between 10 and 15 min post-infection with E44 (Fig. 6C), suggesting that overexpression of mutant caveolin-1 did not affect E. coli cell-induced PKC activation. Next, we examined whether PKC interacts with caveolin-1 by immunoprecipitation. Concomitant with the PepTag assay results, we observed significant amounts of phospho-PKC in both Cav⁺/HBMEC and Cav/HBMEC (Fig. 6D). However, no association of caveolin-1 was found in Cav/HBMEC, whereas only Cav⁺/HBMEC showed considerable amounts of caveolin-1 coprecipitated with phospho-PKC. When subjected to Western blotting with anti-caveolin-1 antibody, the total cell lysates of the transfected HBMEC indicated the presence of equal quantities of caveolin-1. This indicates that the absence of PKC interaction with caveolin-1 might be responsible for the inhibition of E. coli invasion of HBMEC. In addition, this interaction could be crucial for the formation of caveolae.

Absence of Phospho-PKC Interaction with Caveolin-1 at the Plasma Membrane in Cav/HBMEC-- Our previous studies showed that PKC activated by invading E. coli cells translocates to the plasma membrane for further signaling events (22). One such event could be its interaction with caveolin-1, which is important for the formation of caveolae. Thus, we also examined whether the inhibition of E. coli invasion in Cav/HBMEC is due to the inability of phospho-PKC to interact with caveolin-1 at the plasma membrane. Membrane fractions of both Cav⁺/HBMEC and Cav/HBMEC infected with E44 were prepared and assessed for the activation of PKC by PepTag assay. As expected, both membrane fractions showed peak activation of PKC at 15 min post-infection (Fig. 7A). The Cav⁺/HBMEC membrane fractions showed activation even at 5 min post-infection. The membrane fractions were then immunoprecipitated with anti-phospho-PKC antibody, followed by immunoblotting with the same antibody and anti-caveolin-1 antibody. We observed that a greater amount of phospho-PKC had been recruited to the membrane fraction in Cav⁺/HBMEC, with a corresponding increased interaction with caveolin-1 (Fig. 7B). Cav/HBMEC showed normal levels of phospho-PKC compared with non-transfected and infected HBMEC, but showed no association of caveolin-1. These results suggest that despite the translocation of activated PKC to the plasma membrane, its inability to interact with caveolin-1 blocks E. coli invasion of Cav/HBMEC.

View larger version (49K): [in this window] [in a new window]   Fig. 7.   Absence of caveolin-1 and phospho-PKC interaction in HBMEC membrane fractions. Membrane fractions of Cav+/HBMEC and Cav/HBMEC were prepared and subjected to either PepTag assay (A) or immunoprecipitation (IP) with anti-phospho-PKC antibody, followed by Western blotting (WB) with anti-phospho-PKC and anti-caveolin-1 antibodies (B).

Immunofluorescence studies on Cav⁺/HBMEC infected with OmpA⁺ E. coli showed strong co-localization of caveolin at actin condensation sites (Fig. 8, A-D). In these cells, caveolin recruitment was much greater at the bacterial entry site compared with non-transfected HBMEC. Similarly, the density of PKC recruitment was also much greater than in control cells, indicating that overexpression of caveolin may increase the interaction with PKC, thus increasing the formation of more caveolae necessary for invasion. This could be the reason for slightly increased E. coli invasion of Cav⁺/HBMEC. In contrast, in Cav/HBMEC, although there was significant phospho-PKC and actin accumulation beneath the bacteria, there was no detectable accumulation of caveolin-1 (Fig. 8, I-P). Thus, it is clear that an intact scaffolding domain contributes to the association of caveolin-1 with PKC, thereby facilitating bacterial invasion.

View larger version (101K): [in this window] [in a new window]   Fig. 8.   Overexpression of a dominant-negative form of caveolin-1 blocks the accumulation of caveolin-1 at the E. coli entry site. Confluent monolayers of Cav+/HBMEC (A-H) and Cav/HBMEC (I-P) were infected with E44 for 15 min, fixed, and stained with rhodamine-phalloidin (B and J) or anti-caveolin-1 antibody (C, F, and N). The anti-caveolin-1 antibody reactivity was assessed with fluorescein isothiocyanate-labeled secondary antibody. Some monolayers were stained with anti-phospho-PKC antibody (G and O) followed by Cy3-labeled secondary antibody. The red and green fluorescence was observed in a dual-filter mode (D, H, L, and P). The bacteria were visualized under transmitted light with a blue filter (A, E, I, and M). Arrows indicate the position of the bacteria and accumulation of actin, caveolin-1, and phospho-PKC.

Cell-permeable Scaffolding Peptide of Caveolin-1 Blocks E. coli Invasion-- Because the binding of PKC to the scaffolding peptide of caveolin-1 has been found to be crucial to the internalization of caveolae, we used a peptide that represents the caveolin-1 scaffolding domain (amino acid residues 82-101) and a scrambled peptide (control) to examine their effect on E. coli invasion. These peptides were synthesized with a 16-amino acid portion of the AP homeodomain (RQIKIWFQNRRMKWKK), a Drosophila transcription factor, at the N-terminal side of both the scaffolding peptide (AP-Cav) and the scrambled peptide (AP-Cav-X). The AP protein facilitates homogeneous uptake of peptides or oligonucleotides into cultured mammalian cells through a non-endocytic and non-degradative pathway (34). To analyze the efficiency of entry of the peptides into the cell, the peptides were labeled with fluoresceinamine and then added to the cells. These fluoresceinated peptides entered 60-70% of the cells at a 2 µM concentration within 6 h of incubation and distributed throughout the cell (Fig. 9, A and B). HBMEC pretreated with AP-Cav showed significant inhibition of E. coli invasion compared with HBMEC pretreated with AP-Cav-X in a dose-dependent manner ((0.4 ± 0.2) × 10⁴ cfu/well for AP-Cav versus (1.1 ± 0.3) × 10⁴ cfu/well for AP-Cav-X at 4 µM; p < 0.01) (Fig. 9C). In contrast, the total cell-associated bacteria did not differ significantly between AP-Cav- and AP-Cav-X-treated HBMEC, suggesting that lack of invasion is not due to the inability of bacteria to bind to the cells. Consistent with the ability of AP-Cav to inhibit the E. coli invasion, immunoprecipitation of the cell lysates of HBMEC treated with the scaffolding peptide with anti-phospho-PKC antibody resulted in the inhibition of caveolin-1 association with phospho-PKC (Fig. 9D). In contrast, AP-Cav-X-pretreated HBMEC showed an association of caveolin-1 with phospho-PKC similar to that in untreated HBMEC. Lack of caveolin-1 association with phospho-PKC is not due to the absence of caveolin-1 in these fractions, as the total cell lysates showed equal quantities of caveolin-1 when immunoblotted with anti-caveolin-1 antibody. In addition, we also examined the association of actin and phospho-PKC by immunocytochemistry. The results were similar to those obtained for Cav⁺/HBMEC and Cav/HBMEC (similar to Fig. 8), in which we observed accumulation of actin and phospho-PKC at the sites of E. coli entry, but not co-localization of caveolin-1 in AP-Cav-pretreated HBMEC. This confirms our hypothesis that E. coli cells use caveolae as a mode of entry into HBMEC, thereby probably ensuring their survival by avoiding fusion with lysosomes.

View larger version (50K): [in this window] [in a new window]   Fig. 9.   Inhibition of E. coli invasion of HBMEC by a cell-permeable peptide that represents the scaffolding domain. Fluoresceinated AP-Cav and AP-Cav-X peptides (4 µM) were incubated with confluent monolayers of HBMEC for 6 h, washed, and viewed under transmitted light with a blue filter (A) or fluorescence (B). Only AP-Cav peptide-treated cells are shown. Confluent monolayers of HBMEC were treated with various concentrations of either AP-Cav or AP-Cav-X peptide as described under "Experimental Procedures," washed, and used for E. coli invasion assays (C). In duplicate experiments, the total cell-associated bacteria (represented as binding) were determined. The peptide-treated and E44-infected HBMEC lysates were subjected to immunoprecipitation (IP) studies with anti-phospho-PKC antibody. The immune complexes were probed with either anti-phospho-PKC or anti-caveolin-1 antibody. In addition, total cell lysates were also subjected to Western blotting (WB) with anti-caveolin-1 antibody to verify the presence of caveolin-1 (D).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

E. coli invasion of HBMEC occurs by a zipper-like mechanism in which the host cell plasma membrane enwraps the invading bacteria and becomes an endosome (23). This mechanism requires E. coli cell-induced HBMEC cytoskeletal rearrangements for the accumulation of actin at the site of bacterial entry. Transcytosis of E. coli in the endosome occurs within 30 min post-infection without multiplication. In addition, there is no evidence of bacterial killing inside the endosome, which could be due to the ability of bacteria within the endosome to avoid the fusion of lysosomes. The nature of this endosome is not known to date. A few studies have attempted to establish the role of caveolae in the entry of pathogens such as E. coli expressing FimH and Chlamydia (1, 2, 27). However, the molecular events leading to the formation of caveolae for any pathogenic microorganism have not been described thus far.

Our findings underscore the critical nature of caveola formation in E. coli invasion of HBMEC via interaction of caveolin-1 with phospho-PKC. In addition, these data indicate that the integrity of cholesterol-enriched microdomains that are normally present in HBMEC must be necessary for invasion. This may explain why the drugs filipin and cyclodextrin, which are known to specifically disrupt raft microdomains, inhibit most E. coli entry. Because our previous studies showed that E. coli OmpA interacts with a 95-kDa gp96 like molecule for invasion of HBMEC (19), it is possible that the OmpA receptor might be enriched in caveolae during the invasion process. In agreement with this concept, we observed clustering of the OmpA receptor beneath the E. coli entry site by immunocytochemistry by 15 min post-infection. Receptors for insulin have been shown to cluster within caveolae and to interact with caveolin-1 to differentially modulate post-receptor signaling (33). Thus, it is reasonable to speculate that OmpA interacts with its receptor, gp96, which in turn interacts with caveolin-1 and clusters within the caveolae for further signaling necessary for E. coli invasion. However, caveolae are small relative to E. coli to be transported across the cell; therefore, it would be possible that several OmpA molecules on E. coli induce multiple raft domains after interaction with HBMEC receptors and that these are subsequently united to surround the bacterium.

Our studies show that E. coli invasion of HBMEC induces activation of PKC by three folds in an OmpA-dependent manner (22). Activated PKC then translocates to the plasma membrane for further signaling events. A major finding of this study is that the activated PKC interacts with caveolin-1, a specific marker of caveolae. We also found by immunocytochemistry that PKC activated by invading E. coli cells co-localizes with caveolin-1. In addition, these two molecules are also present at the actin condensation sites beneath E. coli attachment, reflecting possible signaling complex formation around the caveolae for efficient transcytosis of E. coli. However, the role of caveolin-1 in further signaling events is not clear at this point. Interestingly, overexpression of dominant-negative forms of either FAK (FRNK) or PKC (PKC/CAT-KR) inhibited the association of phospho-PKC with caveolin-1, indicating that both FAK and PKC may be upstream of caveolin-1 interaction. Thus, targeting of phospho-PKC to caveolae could be an important event in E. coli invasion. Several studies have previously shown that PKC regulates the membrane invaginations and is enriched in caveolae, and our results are in agreement with these observations (4, 25).

We further demonstrated that phospho-PKC interacts at the scaffolding domain of caveolin-1, as overexpression of a dominant-negative form of caveolin-1 in which the amino acids in the scaffolding domain have been mutated significantly blocked E. coli invasion of HBMEC. Consistent with these findings, introduction of a cell-permeable peptide that represents the caveolin-1 scaffolding domain resulted in significant inhibition of E. coli invasion. In contrast, a scrambled peptide showed no such inhibitory activity. Bucci et al. (34) have successfully used this peptide both in vitro and in vivo for selective inhibition of acetylcholine-induced vasodilation and nitric oxide production. The uptake of this peptide by cells was rapid and independent of membrane fluidity and is a useful tool for studying the scaffolding domain-mediated signaling pathways. Indeed, in vitro biochemical studies have indicated that the caveolin-1 scaffolding domain regulates the activity of PKC (3, 4, 6). In addition, overexpression of a dominant-negative form of caveolin-1 in HBMEC prevented accumulation of caveolin-1 at the bacterial entry site despite the recruitment of phospho-PKC to the membrane. These results are in agreement with the immunoprecipitation studies in which phospho-PKC present in the membrane fractions of Cav/HBMEC showed no association with caveolin-1. These observations suggest that phospho-PKC recruitment to the E. coli entry site does not require its interaction with caveolin-1, but is absolutely necessary for efficient E. coli invasion. Similarly, we observed actin accumulation at the bacterial attachment site despite absence of invasion. Because our previous studies showed that PKC activation is important for actin accumulation beneath the bacteria (22), PKC might play two differential roles in E. coli invasion: one in actin rearrangements and the other in caveolin-1 activation and effective caveola internalization. These results are in sharp contrast to those observed for SV40 internalization via caveolae, in which either actin condensation or tyrosine phosphorylation occurs only after SV40-induced caveola formation (31). As a control, we also used wild-type caveolin-1-transfected HBMEC in E. coli invasion assays, which showed a slight increase in invasion. Previous studies by other investigators have shown that overexpression of caveolin-1 induces the formation of caveolae in several cell types (3, 5, 13). Thus, the increase in E. coli invasion could be due to nonspecific trapping of bacteria in caveolae. Alternatively, overexpression of caveolin-1 increases the turnover of receptors to the cell surface, and these receptors are subsequently responsible for internalization of more E. coli.

The survival of E. coli in the endosome depends on the ability of the intracellular parasite to resist endosomal acidification and lysosomal fusion. Our studies show that during transcytosis, E. coli cells remain enclosed in the endosome containing caveolin-1, suggesting that caveola-mediated entry may play an important role in preventing lysosomal fusion. The caveola-mediated SV40 and C. trachomatis entry pathways transport the microorganisms to the endoplasmic reticulum, a normal target for endocytic cargo, rather than the endosomal/lysosomal compartment (30-32). However, caveolin-1 may not be the sole molecule responsible for the avoidance of lysosomal fusion, as shown for Chlamydia psittaci, in which early gene expression by the bacterium in the endosome is important (38). Despite poor understanding of the mechanisms that lead to lysosomal avoidance, the continuing association of caveolin with E. coli in caveola-mediated entry may have important implications in the crossing of the BBB.

In summary, we have shown that E. coli internalization of HBMEC occurs via caveolae. The scaffolding domain of caveolin-1 interacts with PKC at the plasma membrane beneath the E. coli entry site. Because invasion by E. coli depends on OmpA interaction with its receptor, a gp96-like protein, identification of the role of caveolin-1 in bringing these receptor molecules into caveolae furthers our understanding of the mechanism of invasion.

	ACKNOWLEDGEMENTS

We thank Soman Abraham for initial encouragement and Philippe Sansonetti and Barbara Driscoll for critical reading of the manuscript. We also thank K. S. Kim and M. F. Stins for providing HBMEC, Salam Khan (University of Wurtzburg, Wurtzburg, Germany) for providing anti-SfaA antibody, and George McNamara (Children's Hospital Research Institute Image Core Facility, Los Angeles) for assistance with confocal imaging.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant AI40567 (to N. V. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Div. of Infectious Diseases, MS 51, Children's Hospital, 4650 Sunset Blvd., Los Angeles, CA 90027. Tel.: 323-669-5465; Fax: 323-660-2661; E-mail: pnemani@chla.usc.edu.

Published, JBC Papers in Press, October 16, 2002, DOI 10.1074/jbc.M208830200

	ABBREVIATIONS

The abbreviations used are: BBB, blood-brain barrier; HBMEC, human brain microvascular endothelial cell(s); FAK, focal adhesion kinase; PKC, protein kinase C; AP, antennapedia; cfu, colony-forming units; FRNK, FAK-related non-kinase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES