Efficient Translocation of EspC into Epithelial Cells Depends on Enteropathogenic Escherichia coli and Host Cell Contact
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感染与免疫杂志 2006年第4期
Department of Cell Biology, Centro de Investigacion y de Estudios Avanzados (Cinvestav-Zacatenco), Ap. Postal 14-740, 07000 Mexico, DF, Mexico
ABSTRACT
EspC is an autotransporter protein secreted by enteropathogenic Escherichia coli (EPEC). The pathogenic role of EspC in EPEC infection is unknown. We have shown that the purified EspC produces enterotoxicity and cytotoxicity; for the latter effect, EspC must be internalized. However, the internalization mechanism is unknown. Here we show that azithromycin (an inhibitor of pinocytosis), but not drugs affecting caveole-, clathrin-, or receptor-mediated endocytosis, inhibited purified EspC internalization and cytoskeletal disruption, suggesting that purified EspC is internalized by pinocytosis. Furthermore, unlike in cholera toxin, we were unable to detect a receptor on epithelial cells by pretreatment at 4°C. Upon EspC entry, it is delivered directly into the cell cytosol, as shown by the fact that drugs that inhibit intracellular trafficking had no effect on cytoskeletal disruption. All these data suggest that purified EspC internalization is not a physiological internalization mechanism; hence, we explored EspC internalization during the infection of epithelial cells by EPEC. Like other EPEC virulence factors, EspC secretion is stimulated by EPEC when it is grown in cell culture medium and enhanced by the presence of epithelial cells. Physiologically secreted EspC was efficiently internalized during EPEC and host cell interaction. Additionally, the lack of EspC internalization caused by using an isogenic mutant prevented the cytopathic effect caused by EPEC. These data suggest that EPEC uses an efficient mechanism to internalize milieu-secreted EspC into epithelial cells; once inside the cells, EspC is able to induce the cytopathic effect caused by EPEC.
INTRODUCTION
Enteropathogenic Escherichia coli (EPEC) infection is a leading cause of infantile diarrhea that can be severe and lethal (15) in developing countries. The hallmark of EPEC infection is a histopathological lesion formed at the mucosal intestinal surface that resembles a pedestal-like structure known as the attaching and effacing (A/E) lesion (18, 25). The genes responsible for the A/E phenotype are located in a 35.6-kb pathogenicity island termed LEE (locus of enterocyte effacement) (22, 23). The LEE contains diverse genes encoding secreted proteins for the type III secretion system (TTSS) that are termed EPEC-secreted proteins (Esp). EPEC directly injects virulence factors into the target cell through its TTSS (6, 16). In this way, the bacterial proteins are translocated to the cytoplasm, where they interact with host components and alter signaling pathways, resulting in disease (29). However, the pathophysiology of EPEC-induced diarrhea is not yet fully understood.
A second pathogenicity island of EPEC that encodes EspC, an autotransporter protein, has been identified; unlike proteins secreted by the TTSS, EspC secretion is mediated by the type V secretion system (24, 38). EspC shows the three classical domains (signal sequence, passenger domain, and translocation unit) of autotransporter proteins that were first described for the immunoglobulin A (IgA) protease of Neisseria gonorrhoeae (33, 38). EspC also has a conserved serine protease motif similar to that of the IgA protease but does not cleave IgA like several other members of the autotransporter family of proteins. In fact, EspC belongs to the subfamily of serine protease autotransporters of Enterobacteriaceae, which includes Tsh, SepA, Pic, EspP, Sat, and Pet; none of them cleaves IgA. Additionally, a deletion mutant in espC by allelic exchange has been shown to be indistinguishable from its isogenic parent for adherence, invasion, actin rearrangement, and Tir phosphorylation, events that are crucial for A/E lesion formation (38).
Recently, we showed that the purified EspC has enterotoxic and cytotoxic activities on rat jejunum preparations mounted in Ussing chambers and on cultured epithelial cells, respectively (24, 30). Our group has also found that EspC causes cytotoxic effects, including cytoskeletal damage that depends on EspC internalization and on its functional serine protease motif (30). These activities are similar to those induced by the Pet toxin, a homolog autotransporter to EspC (70% similarity) that is secreted by enteroaggregative E. coli. Pet is internalized by epithelial cells and undergoes retrograde transport, leading to the cytotoxic effects (3, 31). Like Pet, EspC must be internalized into the cell to cause cytoskeletal damage. However, EspC (120 μg/ml) reached the cell cytosol after 6 h of incubation, whereas Pet (38 μg/ml) entry occurs after 15 to 30 min of incubation. After 8 h, EspC is heterogeneously distributed inside the cells, whereas Pet is located perinuclearly after only 1 h (30). These findings suggest that the purified EspC relies on different mechanisms of internalization and intracellular trafficking than those that are used by the Pet toxin. In this work, we demonstrate that purified EspC internalization is mediated by pinocytosis, an unspecific endocytic pathway for entering the cell. The pinocytosis of purified EspC by epithelial cells required 8 h of incubation and a high dose of protein. However, in EPEC-infected HEp-2 cells, EspC internalization occurred after 30 min of infection, suggesting that it is the physiological mechanism of internalization of this autotransporter protein. Additionally, during EPEC infection, EspC internalization increased cytoskeletal damage, whereas in espC isogenic mutant-treated cells, the actin stress fibers were preserved.
MATERIALS AND METHODS
Bacterial strains. EPEC prototype E2348/69 (O127:H6) has been described previously (19), MAS111 is an E2348/69 derivative with an espC deletion, and MAS120 is a JPN15/pCVD450 derivative with an espC deletion (38), kindly given by Brett Finlay. JML174 is a minimum espC clone expressing HB101; arabinose (0.2% wt/vol) added to the culture medium stimulates EspC supernatant secretion (24) since espC minimal fragment is cloned in pBAD30 and grown in Luria-Bertani (LB) broth supplemented with 0.2% glycerol and 0.2% glucose (repression) or 0.2% arabinose (induction). MAS111(pJLM174) was constructed in this study based on a standard protocol (35), which is an EPECespC strain complemented with pJLM174, a plasmid encoding the espC gene. All strains were routinely grown in LB broth or minimum essential medium (MEM) (without supplements) aerobically at 37°C. When needed, cultures were supplemented with arabinose (0.2%), ampicillin (100 μg/ml), or tetracycline (15 μg/ml). EPEC cultures were activated for 3 h as previously described (34), indicating bacteria from culture in logarithmic phase which were grown in tissue culture medium.
Purification of EspC recombinant protein. HB101(pJLM174) was grown overnight in LB plus arabinose (0.2% wt/vol) and ampicillin (100 μg/ml) at 37°C under shaking. The supernatants were obtained by centrifugation at 7,000 x g for 15 min, filter sterilized through 0.22-μm-diameter filters (Corning, Inc., Cambridge, MA), and concentrated 100-fold in an Ultrafree centrifugal filter device with a 100-kDa cutoff (Millipore, Bedford, MA), producing a homogeneous purified EspC (30). Recombinant proteins were filter sterilized again, aliquoted, and quantified by the Bradford method (1). Untreated cells (similar conditions but without EspC) and cells treated with supernatants from HB101(pJLM174), which was grown in the presence of glucose (repression of EspC expression) instead of arabinose and concentrated as mentioned above, produced the same results (30); therefore, we used untreated cells.
Tissue culture cells. The human epithelial cell line HEp-2 (ATCC CCL23) was cultured in MEM supplemented with 10% fetal calf serum (HyClone, Logan, UT), 1% nonessential amino acids, 5 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells were normally harvested with 10 mM EDTA and 0.25% trypsin (GIBCO BRL, Grand Island, NY) in phosphate-buffered saline (PBS) (pH 7.4), resuspended in the appropriate volume of supplemented MEM, and incubated at 37°C in a humidified atmosphere of 5% CO2.
HEp-2 cell cultures, drug treatments, and EspC toxicity. HEp-2 cells were seeded onto eight-well LabTek slides (VWR, Bridgeport, NJ) at a density of 4 x 104 cells/well and incubated for 24 h. Cells were washed with PBS (pH 7.4) and incubated at 37°C in MEM (without supplements) or with chlorpromazine (25 μM), filipine (2 μg/ml), monodansyl cadaverine (200 μM/ml), NH4Cl (30 mM), monensin (10 μM), or brefeldin A (30 mM) for 30 min or with azithromycin (50 μg/ml) for 3 h. Another set of HEp-2 cells were treated by incubation at 4°C for 30 min. Drug-treated and control HEp-2 cells were incubated with EspC (120 μg/ml) diluted in MEM (without supplements) for 8 or 10 h. HEp-2 cells pretreated at 4°C were incubated with EspC (120 μg/ml) or cholera toxin (CTx) (0.5 μg/ml) (Sigma Aldrich, Inc., St. Louis, MO) and diluted in precold medium during 6 h at 4°C. After the incubation time, the medium was aspirated and cells were washed twice with PBS and processed by means of the methods described below.
(i) Giemsa staining. Cells were fixed with 70% methanol and stained with 10% Giemsa (Sigma). Slides were analyzed at a x40 magnification by standard bright field light microscopy. Toxic activity was scored by using a modified scale from a previous work (32).
(ii) Immunostaining. Cells were fixed with 2% formalin-PBS, washed, permeabilized by adding 0.1% Triton X-100 in PBS, and stained with 0.05 μg/ml of tetramethyl rhodamine isothiocyanate-phalloidin and with a rabbit anti-EspC polyclonal antibody as previously described (30) or a rabbit anti-CT polyclonal antibody (Sigma Aldrich, Inc.), followed by an anti-rabbit fluorescein-labeled antibody. Slides were mounted on Gelvatol, covered with glass coverslips, and examined under a Leica TCS SP2 confocal microscope.
Analysis of EspC secretion by EPEC. EspC secretion was analyzed in the supernatant of EPEC cultures in LB medium and MEM as well as in supernatants of EPEC-infected HEp-2 cells.
(i) Secretion of EspC into EPEC supernatant. Bacterial strains were grown aerobically in 2 ml of LB broth at 37°C for 16 h under shaking. Cultures were diluted 1:50 in MEM or LB broth and incubated at 37°C in a shaking incubator until reaching an optical density at 600 nm (OD600) of 0.3 (2 h). Bacterial cultures were pelleted by centrifugation at 16,000 x g for 10 min, and supernatants were passed through a 0.22-μm filter (Millipore Co., Bedford, MA), concentrated by the addition of trichloroacetic acid (10% vol/vol), and then incubated on ice for 1 h. Secreted proteins were pelleted by centrifugation at 23,000 x g for 20 min. The pellets were resuspended in equal volumes of SDS-PAGE loading buffer and analyzed by SDS-PAGE and Western blotting. Briefly, precipitated proteins were transferred to nitrocellulose membranes, blocked in PBS-Tween 20 (0.05% vol/vol) and nonfat dry milk (5% wt/vol), and probed with rabbit anti-EspC polyclonal antibodies. Bound antibody was detected with horseradish peroxidase-conjugated secondary anti-rabbit antibody and enhanced chemiluminescence reagents (Amersham, Naperville, IL).
(ii) Secretion of EspC into EPEC-infected HEp-2 cell supernatants. HEp-2 cells were seeded at a density of 1.25 x 106 cells in 60-mm petri dishes (Corning, Inc.) and incubated at 37°C during 24 h. When indicated, cell cultures were fixed with 2% formalin-PBS, extensively washed with PBS (pH 7.4), and incubated in MEM for 30 min before infection. HEp-2 cells, fixed HEp-2 cells, or mock-treated cells in petri dishes were infected with activated EPEC cultures (multiplicity of infection [MOI], 10) during 2 h. After this time, supernatants containing bacteria and secreted proteins were obtained, separated by centrifugation at 16,000 x g for 10 min, filter sterilized through 0.22-μm-diameter filters (Corning, Inc.), trichloracetic acid (TCA) concentrated, and analyzed by Western blotting (as described above) or by enzyme-linked immunosorbent assay (ELISA). Briefly, the ELISA analysis was performed as follows: the supernatants were serially diluted in carbonate/bicarbonate buffer and used to coat 96-well ELISA plates (Corning, Inc.) at 4°C overnight. Plates were washed with PBS-Tween (0.05%) and blocked with bovine serum albumin (1%) for 1 h at 37°C. Then, plates were washed, incubated with a rabbit anti-EspC polyclonal antibody (1:500) for 1 h at 37°C, and detected with a horseradish peroxidase-conjugated anti-rabbit antibody (1:3,000) for 1 h at 37°C. The color reaction was developed with o-phenylenediamine-H2O2 (Sigma Aldrich, Inc.) and stopped with sulfuric acid (2 N). The final OD was measured at 490 nm.
Confocal microscopy. HEp-2 cells were seeded onto eight-well LabTek slides (VWR, Bridgeport, NJ) at a density of 4 x 104 cells/well. Before infection with activated EPEC cultures, cells were washed three times with warm PBS (pH 7.4) and incubated at 37°C in MEM (without supplements) during 30 min. Infections were performed at the indicated time in the presence of D-mannose (1%) (Research Organics, Inc. Cleveland, OH). Infected HEp-2 cells were washed with PBS, fixed with 2% formalin-BS, permeabilized or not permeabilized with Triton X-100 (0.1%), immunostained, and analyzed through confocal microscopy as described above.
Cell fractionation. HEp-2 cells grown in 60-mm petri dishes were infected with activated cultures (MOI, 10) of either EPEC wild-type, isogenic EspC mutant (espC), or complemented strain (espC+espC) during the indicated times. EPEC-infected HEp-2 cells were incubated in the presence of D-mannose (1%) and the appropriate antibiotic. Cells were delicately washed three times with ice-cold PBS (pH 7.4) and scraped into a buffer consisting of Tris-HCl (0.25 M) (pH 7.5), phenylmethylsulfonyl fluoride (50 μg/ml), aprotinin (0.5 μg/ml), and EDTA (0.5 μM). Then, cells were lysed by three repeating freeze-thaw cycles (5 min incubation in a dry ice-ethanol bath and 3 min incubation in a thermoblock at 37°C) (39). Cells were scraped into ice-cold PBS. The cell lysates were ultracentrifuged at 100,000 x g for 1 h at 4°C, and the supernatant fraction containing soluble cytoplasmic proteins was obtained. Pellets containing HEp-2 cell membranes, adherent bacteria, nuclei, and cytoskeletal proteins were washed with cold PBS and resuspended in PBS. Protein concentrations were estimated by the Bradford method (1) using bovine serum albumin as standard. Equivalent volumes were boiled for 7 min, analyzed by SDS-PAGE, and electrotransferred to nitrocellulose membranes for Western blot analyses, essentially as described above. The identity of cellular fractions was confirmed with a mouse monoclonal antiactin antibody (a gift of Manuel Hernández) for cytosolic proteins and a rabbit anti-pan cadherin polyclonal antibody (Zymed laboratories, Inc.) for the membrane-insoluble fraction.
RESULTS
Internalization of EspC does not involve classical endocytosis or intracellular trafficking. Unlike Pet, a homolog autotransporter, EspC does not undergo endocytosis by conventional mechanisms. As shown in Fig. 1B, EspC caused a cytotoxic effect, characterized by cell rounding and detachment, in HEp-2 cells incubated with purified recombinant EspC (120 μg/ml) for 8 h as previously reported (30). Drugs used to inhibit endocytosis by caveole-coated vesicles (Fig. 1C), clathrin-coated vesicles (Fig. 1E), or receptor-mediated endocytosis (Fig. 1G) were unable to abolish the cytotoxic effect of EspC on HEp-2 cells. Additionally, EspC-treated cells were fixed and permeabilized for immunostaining with anti-EspC antibodies to visualize EspC internalization by confocal microscopy. None of the drugs mentioned was able to block EspC internalization into epithelial cells (data not shown). These data indicate that EspC is not internalized by endocytosis. Additionally, drugs were used to inhibit vesicular trafficking, such as that of NH4Cl, monensin or brefeldin A. EspC was able to cause the cytotoxic effect even though the endosomes were alkalinized by NH4Cl (Fig. 1I), the H+ ATPase was blocked by monensin (Fig. 1J), or the Golgi apparatus was disrupted by brefeldin A (Fig. 1K). As with the other drugs, EspC internalization was verified through confocal microscopy, yielding the same results, i.e., no inhibition of EspC internalization (data not shown). These results indicate that EspC does not undergo intracellular trafficking.
Uptake of purified EspC occurs by pinocytosis, and no receptor is required. Recently, it has been shown that azithromycin, a lysomotropic antibiotic, impairs fluid-phase pinocytosis in cultured fibroblasts (41). To investigate whether EspC is internalized by pinocytosis, HEp-2 cells were preincubated with azithromycin (50 μg/ml) and then treated with EspC (120 μg/ml) for 10 h. Actin cytoskeleton was stained with rhodamine-phalloidin and EspC was localized by immunofluorescence using anti-EspC antibodies; slides were analyzed through confocal microscopy. Figure 2A displays cells treated with only azithromycin as a control, showing the integrity of the F-actin cytoskeleton. When EspC entered the cells, they suffered the cytotoxic effects characterized by loss of stress fibers, cell rounding, and detachment (Fig. 2B). These EspC-caused effects were prevented by pretreatment with azithromycin, as shown by the fact that these cells were seen as untreated cells and EspC was unable to enter the cells, avoiding the F-actin cytoskeleton disruption (Fig. 2C).
EspC internalization is abolished by an inhibitor of pinocytosis (A to C) and no receptor on the cell surface is required (D to L). HEp-2 cells were directly treated with EspC (120 μg/ml) for 10 h (B) or pretreated (3 h) with 50 μg/ml of azithromycin (C). HEp-2 cells treated with only azithromycin were used as a negative control (A). Cells were fixed, permeabilized, and stained with rhodamine-phalloidin and a rabbit anti-EspC polyclonal antibody followed by secondary fluorescein-labeled anti-rabbit IgG antibody. Slides were observed through confocal microscopy. To detect a possible receptor for EspC, HEp-2 cells were pretreated at 4°C for 30 min and then treated with 0.5 μg/ml CTx or 120 μg/ml of EspC for 6 h at 4°C. Cells were fixed and permeabilized; actin cytoskeleton was stained with rhodamine-phalloidin (D and G), and the toxins were stained with rabbit anti-CTx (E) or anti-EspC (H) polyclonal antibodies followed by secondary fluorescein-labeled anti-rabbit IgG antibody. Slides were observed through confocal microscopy. Panels F and I are the merge of D to E and G to H, respectively. Untreated cells were incubated at 4°C for 6 h (J). Panels K and L are top sections from the panels E and H, respectively. The arrow in panel F shows CTx detection.
The above data suggest that the purified EspC does not require a cell receptor. To search for this possibility, HEp-2 cells were preincubated at 4°C and then treated with EspC (120 μg/ml) or CTx (0.5 μg/ml) and kept at 4°C. At this temperature, many of the cell functions are stopped and, indeed, the CTx-treated cells were observed as rounding cells, with no preservation of the cytoskeleton (Fig. 2D). However, this effect is not caused by CTx (Fig. 2J) since its localization, determined by immunostaining with anti-CTx (Fig. 2E), was not intracellular but on the cell surface as detected in a middle section (Fig. 2F) and confirmed in a top section (Fig. 2K), suggesting that CTx binds to its receptor, the gangloside GM1. On the other hand, EspC-treated cells were also observed as rounding cells (induced by the temperature) (Fig. 2G) but, contrary to CTx, EspC was not immunolocalized on the cell surface with anti-EspC antibodies as detected in a middle section (Fig. 2H and I) and confirmed in a top section (Fig. 2L), suggesting that EspC has no receptor on the cell surface.
Secretion of EspC by EPEC is regulated by the culture medium and cell contact. Since the internalization process and the cytotoxic effects of EspC require a high dose of protein and an unusual mechanism of internalization, we decided to investigate how EspC is secreted and internalized during an EPEC infection. To search for the efficiency of EspC secretion by EPEC, the wild-type strain, an isogenic mutant in EspC, and its complementation with the espC gene as well as anti-EspC antibodies were tested for EspC secretion and antibody specificity. Concentrated supernatants from overnight culture of these strains were analyzed by immunoblot using rabbit anti-EspC polyclonal antibodies. The EspC minimal clone HB101(pJLM174) was able to secrete around 5 μg/ml of EspC into the medium, whereas the wild-type EPEC strain E2348/69 secreted a calculated concentration of 40-fold less EspC, specifically detected by the anti-EspC polyclonal antibody (Fig. 3A). As expected, the isogenic espC mutant (MAS111-espC) was unable to secrete EspC into the medium, thereby being undetectable by the anti-EspC antibody (Fig. 3A). The anti-EspC antibodies were also unable to detect EspC in concentrated supernatants from EPEC JPN15 mutated in espC and transcomplemented with pCVD450 (Fig. 3A), a perA-encoded plasmid, to increase EPEC protein secretion, whereas these antibodies detected EspC in concentrated supernatants from the MAS111 strain transcomplemented with the plasmid pJLM174 at a similar concentration as that secreted by the minimal clone HB101(pJLM174) (Fig. 3A).
Since EspC expression is regulated by Ler (9), a regulator of genes into the LEE pathogenecity island, some of which are overexpressed in MEM cell culture medium and upon cell contact, we looked for overexpression of EspC in MEM and during EPEC and epithelial cell interaction. Overnight EPEC culture in LB broth was diluted in either serum-free MEM tissue culture medium or LB broth and incubated until it reached an OD600 of 0.3 (about 2 h). Filtered, TCA-precipitated supernatants were analyzed by immunoblotting by using the rabbit anti-EspC polyclonal antibody. EPEC secreted three times more EspC protein into the bacteria grown in MEM than into those grown in LB broth (Fig. 3B). EspC secretion was higher in precipitated supernatants (2-h cultures) from bacteria infecting epithelial cells than in supernatants from mock infection without cells (Fig. 3C). Moreover, when the epithelial cells were fixed and then inoculated with EPEC, the secretion of EspC into the medium was similar to that in supernatants from mock infection without cells (Fig. 3C). ELISA analysis of EspC secretion in supernatants from EPEC, under these three conditions, showed that EPEC secreted about 55% more EspC in supernatants from EPEC infecting live cells and about 25% less in supernatants of bacteria interacting with fixed cells relative to mock infection without cells (Fig. 3D). These data suggest that secretion of EspC by EPEC, similarly to other proteins secreted by TTSS and regulated by Ler, is increased in the MEM culture medium and by the interaction with the host cells.
Intracellular internalization of EspC is enhanced during EPEC-epithelial cell interaction. Since EspC is hypersecreted during the interaction of EPEC and epithelial cells, we investigated the efficiency of EspC internalization under this condition. HEp-2 cells were incubated with EPEC at various times, and EspC internalization was detected by immunofluorescence and analyzed through confocal microscopy. After 30 min of infection, the merge of the green and red channels showed that when the actin cytoskeleton is preserved, an incipient and localized EspC internalization is observed (Fig. 4A); it is better visualized through the merge of the Nomarsky interference contrast and the green channel (Fig. 4C). Additionally, the Nomarsky interference contrast allowed us to detect that this localized EspC internalization occurred underneath some bacteria that were attached to epithelial cells (Fig. 4B). All these events were more obvious after 1 h of infection, revealing preserved cytoskeleton (Fig. 4D) with localized EspC internalization (Fig. 4D and F) occurring underneath bacteria attached to epithelial cells (Fig. 4E). Even though EspC internalization into HEp-2 cells was analyzed through confocal microscopy and revealed as middle cuts, similar experiments were performed in which cells were not permeabilized to confirm that EspC was not on the cell surface but inside the cells. In these cells infected with EPEC for 1 h, it was possible to observe bacteria attached to epithelial cells by means of the Nomarsky interference contrast (Fig. 4H) but, in the merge of this optic Nomarsky with the green channel, EspC was not observed (Fig. 4I) because the anti-EspC antibodies were unable to enter the cells to mark EspC.
Interestingly, EspC was efficiently translocated by EPEC into the cell upon pedestal formation. HEp-2 cells were infected with EPEC for 1.5 h and 3 h to detect pedestal formation. Fluorescent actin staining and immunolocalization of EspC were performed, and the cells were analyzed through confocal microscopy. HEp-2 cells that were treated for 1.5 h with EPEC showed some pedestal formations without evident cytoskeletal damage (Fig. 5A). Interestingly, over the pedestal formations, EspC was mainly secreted and internalized into the cells and was detected as dense green spots by the anti-EspC antibodies (Fig. 5A). After 3 h, when the pedestals were completely established, EspC was distributed homogeneously inside the cells and the cytoskeleton was incipiently damaged, showing loss of the stress fibers and cell contraction (Fig. 5B). As expected, after 3 h, the isogeneic espC mutant MAS111 was able to form pedestals but unable to secrete and internalize EspC into the cells, and the cytoskeleton was better preserved than that in cells treated with the wild-type strain (Fig. 5C). Whereas the espC mutant complemented with the pJLM174 plasmid was able to form pedestals and to secrete and internalize EspC over the pedestals, the EspC was distributed inside the cells after 3 h (Fig. 5D).
Remarkably, EspC secretion seemed to be coupled to its internalization, as shown by the fact that we were unable to detect EspC binding to the epithelial cell membrane by immunoblotting using anti-EspC antibodies to probe the membrane fraction of EPEC or espC+espC-infected epithelial cells during different times (Fig. 6A). Uninfected cells and cells treated with EPEC(espC) were used as negative controls. As a control, the presence of membrane proteins was evidenced by detecting a transmembranal protein, cadherin, by using anticadherin antibodies (Fig. 6A). However, in all the cytoplasmic fractions of cells that were treated with bacteria producing EspC (i.e., EPEC or espC+espC-infected epithelial cells), it was possible to detect EspC (Fig. 6B). EspC translocation was a time-dependent event that correlated with data obtained by confocal microscopy; at 1 h, a weak signal was detected by the anti-EspC antibodies, which was more evident after 2 h, and a strong recognition was detected after 6 h of incubation (Fig. 6B). Whereas, in EPEC(espC)-treated cells, the anti-EspC antibodies were unable to detect EspC in the cytoplasmic fraction, as expected, the complemented bacteria (espC+espC) were able to translocate EspC into epithelial cells at concentrations similar to those reached by the wild type after 6 h of infection (Fig. 6B).
These data suggest that EPEC is able to translocate an autotransporter protein which was previously secreted to the milieu into epithelial cells. Furthermore, EspC protein inside the cells is able to cause the cytopathic effect produced by EPEC on epithelial cells.
DISCUSSION
EPEC strains are an extremely common cause of diarrhea in young children throughout the developing world. However, EPEC-induced diarrhea occurs through an unclear mechanism. Even if the decreased absorption due to pedestal formation might be a possible explanation, the role of microvilli effacement and pedestal formation in the disease remains undefined (7). The secretion of chloride has been shown in EPEC infection, a common effect of bacterial enterotoxins, but no enterotoxin has been implicated (4). Recently, EspC, an autotransporter protein that has no participation in signal transduction and pedestal formation (38), has been shown to have enterotoxic activity (24). We have shown that EspC also produces cytotoxic effects on epithelial cells, and under light microscopy, the cell damage seems to be similar to that previously reported for Pet (30). However, the uptake and trafficking mechanisms appear to be different from those used by Pet (30). Here we show that Pet internalization is different from EspC internalization. The purified EspC protein is internalized by pinocytosis, and no receptor is required, suggesting that this process of internalization by using purified EspC is not a physiological event. However, during EPEC infection, EspC is efficiently internalized into eukaryotic cells.
Unlike other toxins (27, 26, 36, 40), which mainly use a cell receptor, EspC does not have a receptor on the epithelial cell. This was shown by the fact that we were unable to find EspC on the cell surface when the cell functions were blocked by chilling the cells at 4°C using Ctx as a positive control. Furthermore, in EPEC-infected epithelial cells, it was possible to find EspC in the supernatants and inside the cells but not on the cell membrane or in the membrane fraction. Interestingly, for the case of Pet, we were able to find Pet on the cell surface by chilling the cells at 4°C (Navarro-Garcia et al., submitted for publication). The lack of a cell receptor for EspC suggests that EspC uptake is not a receptor-mediated endocytosis. Indeed, drugs affecting endocytosis by caveole-coated vesicles, clathrin-coated vesicles, or receptor-mediated endocytosis, such as filipin, monodansyl cadaverine, or chlorpromazine, respectively, were unable to abolish the cytotoxic effect of EspC on HEp-2 cells. Again, these results were different from those obtained for its homolog Pet, which undergoes endocytosis by only clathrin-coated pits (Navarro-Garcia et al., submitted). All these results suggest that EspC does not undergo intracellular trafficking; to assess this possibility, the translocation of EspC from endosomes to cytosol was tested by alkalinizing the endosomes with NH4Cl or by blocking the H+ ATPase. Unlike diphtheria, anthrax, botulinum, and tetanus toxins (37) but similar to Pet (31), EspC cytotoxicity is not affected by these inhibitors, indicating that this protein is not translocated from endosomes to the cytosol. Furthermore, Pet is transported from endosomes to the Golgi apparatus (31; Navarro-Garcia et al., submitted). However, EspC does not seem to be transported to the Golgi apparatus because brefeldin A was unable to inhibit EspC cytotoxicity. Additionally, we were unable to detect EspC in the Golgi apparatus through confocal microscopy (data no shown).
In an attempt to find an inhibitor for EspC internalization, we used azithromycin, an antibiotic that was recently used to inhibit the fluid-phase pinocytosis in fibroblasts (41); azithromycin was able to inhibit EspC internalization, suggesting that EspC is internalized by fluid-phase pinocytosis. Pinocytosis plays a central role in cell physiology by allowing a number of cellular events, such as (i) bulk uptake of extracellular solutes, (ii) receptor-mediated uptake of ligands, (iii) transepithelial transport of macromolecules, and (iv) a large influx of membranes for the recycling of constituents inserted at the cell surface by exocytosis. Interestingly, the purified EspC internalization appears to be accomplished through the bulk uptake mechanism for extracellular solutes; since EspC, unlike Pet, is found in large amounts and irregularly distributed inside the cells (30), a high dose is needed for its internalization and no receptor is involved, However, the question of how EspC is delivered into the cytosol from vesicles is not answered since various and different drugs affecting protein trafficking did not inhibit EspC cytosol translocation. Together, these data suggest that the purified EspC internalization into epithelial cells is not a physiological event and that perhaps other bacterial factors are involved in this process.
EspC is a protein secreted by the type V secretion system (38), which is one of the most abundant and the first to be secreted by EPEC (17). Recently, it was shown that EspC is a non-LEE encoded protein that is encoded in its own pathogenicity island (24). However, the LEE-encoded regulator (Ler), an important regulator of the expression of genes implicated in forming the A/E lesion, strongly activates (31-fold) the espC promoter and increases the levels of EspC that are secreted from EPEC as measured by the promoter activity with lacZ fusion in the E. coli K-12 strain (9). Here we found that EPEC secretes more EspC protein (threefold) in MEM medium than in LB medium, and this increase is augmented twofold when EPEC is infecting epithelial cells, as measured by densitometry and ELISA, suggesting the complexity and multifactorial mechanism implicated in the expression of EPEC virulence factors encoded within and outside the LEE. Furthermore, Gauthier et al. (11) found that an agent (called compound 1) decreased the amount of secreted proteins not by interfering with the TTSS apparatus, but by affecting the amount of type III secretion-associated products (Tir, EspB, EscJ, and EscC) and of the non-LEE encoded virulence protein (EspC).
Even though EspC secretion is highly increased during EPEC and epithelial cell interaction, it is impossible to reach a 120-μg/ml concentration in the medium culture of EPEC-infected cells, which is the concentration used in experiments with purified EspC. Actually, we were unable to quantify it by the Bradford methodology and it was detected by only the ELISA and densitometry. However, considering the amount of EspC secreted by the type V secretion system during EPEC infection (ca. 0.625 μg/ml), EPEC was able to efficiently internalize EspC into epithelial cells (after 30 min). EspC uptake by the cells is a dose-dependent event, reaching amounts comparable to those of actin after 6 h and becoming saturated after this time. Moreover, EspC internalization occurs just beneath the infecting bacteria and simultaneously to the pedestal formation. Once the pedestal is formed, EspC internalization is not focalized over the pedestal but is distributed along the cytosol. Thus, this EspC homogenous distribution inside the cells is different than that which is observed when purified EspC is internalized to the cells (30). As expected, EspC internalization was not detected when epithelial cells were infected with EPEC(espC), but it was detected when the espC mutant was complemented with the pJLM174 plasmid.
EspC internalization correlated with the cytopathic effect caused by EPEC, which is clearly observed through confocal microscopy using the wild type and the complemented strain but not with the espC isogenic mutant. This effect was observed in a previous study (30) and here by using purified EspC. Further experiments are in progress to clearly associate the cytopathic effect with EspC.
Additional studies are also needed to explore which EPEC factors are involved in the efficient internalization of EspC. We can visualize at least two possibilities; given the dependence of the identified non-LEE effectors on the LEE TTSS for delivery, such as EspG (8, 21) and Cif (20) for EPEC, EspFU (2, 10, 42) and NleA (12) for enterohemorrhagic E. coli, and EspI (28) and seven non-LEE effectors that have recently been identified in the mouse-specific A/E pathogen (5), we would expect that EspC delivery is also related to the TTSS. However, it is clear by this and other studies that EspC is secreted to the extracellular medium by the type V secretion system, complicating how TTSS is getting EspC from the milieu since TTSS allows protein translocation from bacterial cytoplasm to eukaryotic cytoplasm. Another possibility is that EPEC might increase pinocytosis at the contact site with the cells as has been reported for Salmonella and Shigella. SopB plays a key role in assembling this Salmonella-containing vacuole by inducing profuse macropinocytosis events to accompany bacterial uptake (13), whereas for Shigella flexneri, the cytoskeletal rearrangements induced by the Shigella effector proteins result in the bacterium being internalized by epithelial cells within a macropinocytic vacuole (14). In both cases, a TTSS is involved; however, both events implicate macropinocytosis of the whole bacterium. Further studies in our laboratory are being addressed to search for these two possibilities. Notwithstanding, here we show that EspC is an important non-LEE virulence factor that, in nonphysiological conditions (i.e., purified protein), is not efficiently internalized, because no receptor is involved in its uptake, no intracellular trafficking exists, and no explanation for its translocation from endosomes or Golgi apparatus to the cytosol can be given. On the other hand, the physiologically secreted EspC by EPEC, which is enhanced in tissue culture media and by cell contact, is efficiently internalized during EPEC and epithelial cell interaction. This latter event is deemed to be important for the cytopathic effect produced by EPEC.
ACKNOWLEDGMENTS
This work was supported by a grant from Consejo Nacional de Ciencia y Tecnología (Mexico) (CONACYT, 44660-M) to F.N.-G.
We thank Rocio Huerta and Adrian Canizalez for their technical help.
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ABSTRACT
EspC is an autotransporter protein secreted by enteropathogenic Escherichia coli (EPEC). The pathogenic role of EspC in EPEC infection is unknown. We have shown that the purified EspC produces enterotoxicity and cytotoxicity; for the latter effect, EspC must be internalized. However, the internalization mechanism is unknown. Here we show that azithromycin (an inhibitor of pinocytosis), but not drugs affecting caveole-, clathrin-, or receptor-mediated endocytosis, inhibited purified EspC internalization and cytoskeletal disruption, suggesting that purified EspC is internalized by pinocytosis. Furthermore, unlike in cholera toxin, we were unable to detect a receptor on epithelial cells by pretreatment at 4°C. Upon EspC entry, it is delivered directly into the cell cytosol, as shown by the fact that drugs that inhibit intracellular trafficking had no effect on cytoskeletal disruption. All these data suggest that purified EspC internalization is not a physiological internalization mechanism; hence, we explored EspC internalization during the infection of epithelial cells by EPEC. Like other EPEC virulence factors, EspC secretion is stimulated by EPEC when it is grown in cell culture medium and enhanced by the presence of epithelial cells. Physiologically secreted EspC was efficiently internalized during EPEC and host cell interaction. Additionally, the lack of EspC internalization caused by using an isogenic mutant prevented the cytopathic effect caused by EPEC. These data suggest that EPEC uses an efficient mechanism to internalize milieu-secreted EspC into epithelial cells; once inside the cells, EspC is able to induce the cytopathic effect caused by EPEC.
INTRODUCTION
Enteropathogenic Escherichia coli (EPEC) infection is a leading cause of infantile diarrhea that can be severe and lethal (15) in developing countries. The hallmark of EPEC infection is a histopathological lesion formed at the mucosal intestinal surface that resembles a pedestal-like structure known as the attaching and effacing (A/E) lesion (18, 25). The genes responsible for the A/E phenotype are located in a 35.6-kb pathogenicity island termed LEE (locus of enterocyte effacement) (22, 23). The LEE contains diverse genes encoding secreted proteins for the type III secretion system (TTSS) that are termed EPEC-secreted proteins (Esp). EPEC directly injects virulence factors into the target cell through its TTSS (6, 16). In this way, the bacterial proteins are translocated to the cytoplasm, where they interact with host components and alter signaling pathways, resulting in disease (29). However, the pathophysiology of EPEC-induced diarrhea is not yet fully understood.
A second pathogenicity island of EPEC that encodes EspC, an autotransporter protein, has been identified; unlike proteins secreted by the TTSS, EspC secretion is mediated by the type V secretion system (24, 38). EspC shows the three classical domains (signal sequence, passenger domain, and translocation unit) of autotransporter proteins that were first described for the immunoglobulin A (IgA) protease of Neisseria gonorrhoeae (33, 38). EspC also has a conserved serine protease motif similar to that of the IgA protease but does not cleave IgA like several other members of the autotransporter family of proteins. In fact, EspC belongs to the subfamily of serine protease autotransporters of Enterobacteriaceae, which includes Tsh, SepA, Pic, EspP, Sat, and Pet; none of them cleaves IgA. Additionally, a deletion mutant in espC by allelic exchange has been shown to be indistinguishable from its isogenic parent for adherence, invasion, actin rearrangement, and Tir phosphorylation, events that are crucial for A/E lesion formation (38).
Recently, we showed that the purified EspC has enterotoxic and cytotoxic activities on rat jejunum preparations mounted in Ussing chambers and on cultured epithelial cells, respectively (24, 30). Our group has also found that EspC causes cytotoxic effects, including cytoskeletal damage that depends on EspC internalization and on its functional serine protease motif (30). These activities are similar to those induced by the Pet toxin, a homolog autotransporter to EspC (70% similarity) that is secreted by enteroaggregative E. coli. Pet is internalized by epithelial cells and undergoes retrograde transport, leading to the cytotoxic effects (3, 31). Like Pet, EspC must be internalized into the cell to cause cytoskeletal damage. However, EspC (120 μg/ml) reached the cell cytosol after 6 h of incubation, whereas Pet (38 μg/ml) entry occurs after 15 to 30 min of incubation. After 8 h, EspC is heterogeneously distributed inside the cells, whereas Pet is located perinuclearly after only 1 h (30). These findings suggest that the purified EspC relies on different mechanisms of internalization and intracellular trafficking than those that are used by the Pet toxin. In this work, we demonstrate that purified EspC internalization is mediated by pinocytosis, an unspecific endocytic pathway for entering the cell. The pinocytosis of purified EspC by epithelial cells required 8 h of incubation and a high dose of protein. However, in EPEC-infected HEp-2 cells, EspC internalization occurred after 30 min of infection, suggesting that it is the physiological mechanism of internalization of this autotransporter protein. Additionally, during EPEC infection, EspC internalization increased cytoskeletal damage, whereas in espC isogenic mutant-treated cells, the actin stress fibers were preserved.
MATERIALS AND METHODS
Bacterial strains. EPEC prototype E2348/69 (O127:H6) has been described previously (19), MAS111 is an E2348/69 derivative with an espC deletion, and MAS120 is a JPN15/pCVD450 derivative with an espC deletion (38), kindly given by Brett Finlay. JML174 is a minimum espC clone expressing HB101; arabinose (0.2% wt/vol) added to the culture medium stimulates EspC supernatant secretion (24) since espC minimal fragment is cloned in pBAD30 and grown in Luria-Bertani (LB) broth supplemented with 0.2% glycerol and 0.2% glucose (repression) or 0.2% arabinose (induction). MAS111(pJLM174) was constructed in this study based on a standard protocol (35), which is an EPECespC strain complemented with pJLM174, a plasmid encoding the espC gene. All strains were routinely grown in LB broth or minimum essential medium (MEM) (without supplements) aerobically at 37°C. When needed, cultures were supplemented with arabinose (0.2%), ampicillin (100 μg/ml), or tetracycline (15 μg/ml). EPEC cultures were activated for 3 h as previously described (34), indicating bacteria from culture in logarithmic phase which were grown in tissue culture medium.
Purification of EspC recombinant protein. HB101(pJLM174) was grown overnight in LB plus arabinose (0.2% wt/vol) and ampicillin (100 μg/ml) at 37°C under shaking. The supernatants were obtained by centrifugation at 7,000 x g for 15 min, filter sterilized through 0.22-μm-diameter filters (Corning, Inc., Cambridge, MA), and concentrated 100-fold in an Ultrafree centrifugal filter device with a 100-kDa cutoff (Millipore, Bedford, MA), producing a homogeneous purified EspC (30). Recombinant proteins were filter sterilized again, aliquoted, and quantified by the Bradford method (1). Untreated cells (similar conditions but without EspC) and cells treated with supernatants from HB101(pJLM174), which was grown in the presence of glucose (repression of EspC expression) instead of arabinose and concentrated as mentioned above, produced the same results (30); therefore, we used untreated cells.
Tissue culture cells. The human epithelial cell line HEp-2 (ATCC CCL23) was cultured in MEM supplemented with 10% fetal calf serum (HyClone, Logan, UT), 1% nonessential amino acids, 5 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells were normally harvested with 10 mM EDTA and 0.25% trypsin (GIBCO BRL, Grand Island, NY) in phosphate-buffered saline (PBS) (pH 7.4), resuspended in the appropriate volume of supplemented MEM, and incubated at 37°C in a humidified atmosphere of 5% CO2.
HEp-2 cell cultures, drug treatments, and EspC toxicity. HEp-2 cells were seeded onto eight-well LabTek slides (VWR, Bridgeport, NJ) at a density of 4 x 104 cells/well and incubated for 24 h. Cells were washed with PBS (pH 7.4) and incubated at 37°C in MEM (without supplements) or with chlorpromazine (25 μM), filipine (2 μg/ml), monodansyl cadaverine (200 μM/ml), NH4Cl (30 mM), monensin (10 μM), or brefeldin A (30 mM) for 30 min or with azithromycin (50 μg/ml) for 3 h. Another set of HEp-2 cells were treated by incubation at 4°C for 30 min. Drug-treated and control HEp-2 cells were incubated with EspC (120 μg/ml) diluted in MEM (without supplements) for 8 or 10 h. HEp-2 cells pretreated at 4°C were incubated with EspC (120 μg/ml) or cholera toxin (CTx) (0.5 μg/ml) (Sigma Aldrich, Inc., St. Louis, MO) and diluted in precold medium during 6 h at 4°C. After the incubation time, the medium was aspirated and cells were washed twice with PBS and processed by means of the methods described below.
(i) Giemsa staining. Cells were fixed with 70% methanol and stained with 10% Giemsa (Sigma). Slides were analyzed at a x40 magnification by standard bright field light microscopy. Toxic activity was scored by using a modified scale from a previous work (32).
(ii) Immunostaining. Cells were fixed with 2% formalin-PBS, washed, permeabilized by adding 0.1% Triton X-100 in PBS, and stained with 0.05 μg/ml of tetramethyl rhodamine isothiocyanate-phalloidin and with a rabbit anti-EspC polyclonal antibody as previously described (30) or a rabbit anti-CT polyclonal antibody (Sigma Aldrich, Inc.), followed by an anti-rabbit fluorescein-labeled antibody. Slides were mounted on Gelvatol, covered with glass coverslips, and examined under a Leica TCS SP2 confocal microscope.
Analysis of EspC secretion by EPEC. EspC secretion was analyzed in the supernatant of EPEC cultures in LB medium and MEM as well as in supernatants of EPEC-infected HEp-2 cells.
(i) Secretion of EspC into EPEC supernatant. Bacterial strains were grown aerobically in 2 ml of LB broth at 37°C for 16 h under shaking. Cultures were diluted 1:50 in MEM or LB broth and incubated at 37°C in a shaking incubator until reaching an optical density at 600 nm (OD600) of 0.3 (2 h). Bacterial cultures were pelleted by centrifugation at 16,000 x g for 10 min, and supernatants were passed through a 0.22-μm filter (Millipore Co., Bedford, MA), concentrated by the addition of trichloroacetic acid (10% vol/vol), and then incubated on ice for 1 h. Secreted proteins were pelleted by centrifugation at 23,000 x g for 20 min. The pellets were resuspended in equal volumes of SDS-PAGE loading buffer and analyzed by SDS-PAGE and Western blotting. Briefly, precipitated proteins were transferred to nitrocellulose membranes, blocked in PBS-Tween 20 (0.05% vol/vol) and nonfat dry milk (5% wt/vol), and probed with rabbit anti-EspC polyclonal antibodies. Bound antibody was detected with horseradish peroxidase-conjugated secondary anti-rabbit antibody and enhanced chemiluminescence reagents (Amersham, Naperville, IL).
(ii) Secretion of EspC into EPEC-infected HEp-2 cell supernatants. HEp-2 cells were seeded at a density of 1.25 x 106 cells in 60-mm petri dishes (Corning, Inc.) and incubated at 37°C during 24 h. When indicated, cell cultures were fixed with 2% formalin-PBS, extensively washed with PBS (pH 7.4), and incubated in MEM for 30 min before infection. HEp-2 cells, fixed HEp-2 cells, or mock-treated cells in petri dishes were infected with activated EPEC cultures (multiplicity of infection [MOI], 10) during 2 h. After this time, supernatants containing bacteria and secreted proteins were obtained, separated by centrifugation at 16,000 x g for 10 min, filter sterilized through 0.22-μm-diameter filters (Corning, Inc.), trichloracetic acid (TCA) concentrated, and analyzed by Western blotting (as described above) or by enzyme-linked immunosorbent assay (ELISA). Briefly, the ELISA analysis was performed as follows: the supernatants were serially diluted in carbonate/bicarbonate buffer and used to coat 96-well ELISA plates (Corning, Inc.) at 4°C overnight. Plates were washed with PBS-Tween (0.05%) and blocked with bovine serum albumin (1%) for 1 h at 37°C. Then, plates were washed, incubated with a rabbit anti-EspC polyclonal antibody (1:500) for 1 h at 37°C, and detected with a horseradish peroxidase-conjugated anti-rabbit antibody (1:3,000) for 1 h at 37°C. The color reaction was developed with o-phenylenediamine-H2O2 (Sigma Aldrich, Inc.) and stopped with sulfuric acid (2 N). The final OD was measured at 490 nm.
Confocal microscopy. HEp-2 cells were seeded onto eight-well LabTek slides (VWR, Bridgeport, NJ) at a density of 4 x 104 cells/well. Before infection with activated EPEC cultures, cells were washed three times with warm PBS (pH 7.4) and incubated at 37°C in MEM (without supplements) during 30 min. Infections were performed at the indicated time in the presence of D-mannose (1%) (Research Organics, Inc. Cleveland, OH). Infected HEp-2 cells were washed with PBS, fixed with 2% formalin-BS, permeabilized or not permeabilized with Triton X-100 (0.1%), immunostained, and analyzed through confocal microscopy as described above.
Cell fractionation. HEp-2 cells grown in 60-mm petri dishes were infected with activated cultures (MOI, 10) of either EPEC wild-type, isogenic EspC mutant (espC), or complemented strain (espC+espC) during the indicated times. EPEC-infected HEp-2 cells were incubated in the presence of D-mannose (1%) and the appropriate antibiotic. Cells were delicately washed three times with ice-cold PBS (pH 7.4) and scraped into a buffer consisting of Tris-HCl (0.25 M) (pH 7.5), phenylmethylsulfonyl fluoride (50 μg/ml), aprotinin (0.5 μg/ml), and EDTA (0.5 μM). Then, cells were lysed by three repeating freeze-thaw cycles (5 min incubation in a dry ice-ethanol bath and 3 min incubation in a thermoblock at 37°C) (39). Cells were scraped into ice-cold PBS. The cell lysates were ultracentrifuged at 100,000 x g for 1 h at 4°C, and the supernatant fraction containing soluble cytoplasmic proteins was obtained. Pellets containing HEp-2 cell membranes, adherent bacteria, nuclei, and cytoskeletal proteins were washed with cold PBS and resuspended in PBS. Protein concentrations were estimated by the Bradford method (1) using bovine serum albumin as standard. Equivalent volumes were boiled for 7 min, analyzed by SDS-PAGE, and electrotransferred to nitrocellulose membranes for Western blot analyses, essentially as described above. The identity of cellular fractions was confirmed with a mouse monoclonal antiactin antibody (a gift of Manuel Hernández) for cytosolic proteins and a rabbit anti-pan cadherin polyclonal antibody (Zymed laboratories, Inc.) for the membrane-insoluble fraction.
RESULTS
Internalization of EspC does not involve classical endocytosis or intracellular trafficking. Unlike Pet, a homolog autotransporter, EspC does not undergo endocytosis by conventional mechanisms. As shown in Fig. 1B, EspC caused a cytotoxic effect, characterized by cell rounding and detachment, in HEp-2 cells incubated with purified recombinant EspC (120 μg/ml) for 8 h as previously reported (30). Drugs used to inhibit endocytosis by caveole-coated vesicles (Fig. 1C), clathrin-coated vesicles (Fig. 1E), or receptor-mediated endocytosis (Fig. 1G) were unable to abolish the cytotoxic effect of EspC on HEp-2 cells. Additionally, EspC-treated cells were fixed and permeabilized for immunostaining with anti-EspC antibodies to visualize EspC internalization by confocal microscopy. None of the drugs mentioned was able to block EspC internalization into epithelial cells (data not shown). These data indicate that EspC is not internalized by endocytosis. Additionally, drugs were used to inhibit vesicular trafficking, such as that of NH4Cl, monensin or brefeldin A. EspC was able to cause the cytotoxic effect even though the endosomes were alkalinized by NH4Cl (Fig. 1I), the H+ ATPase was blocked by monensin (Fig. 1J), or the Golgi apparatus was disrupted by brefeldin A (Fig. 1K). As with the other drugs, EspC internalization was verified through confocal microscopy, yielding the same results, i.e., no inhibition of EspC internalization (data not shown). These results indicate that EspC does not undergo intracellular trafficking.
Uptake of purified EspC occurs by pinocytosis, and no receptor is required. Recently, it has been shown that azithromycin, a lysomotropic antibiotic, impairs fluid-phase pinocytosis in cultured fibroblasts (41). To investigate whether EspC is internalized by pinocytosis, HEp-2 cells were preincubated with azithromycin (50 μg/ml) and then treated with EspC (120 μg/ml) for 10 h. Actin cytoskeleton was stained with rhodamine-phalloidin and EspC was localized by immunofluorescence using anti-EspC antibodies; slides were analyzed through confocal microscopy. Figure 2A displays cells treated with only azithromycin as a control, showing the integrity of the F-actin cytoskeleton. When EspC entered the cells, they suffered the cytotoxic effects characterized by loss of stress fibers, cell rounding, and detachment (Fig. 2B). These EspC-caused effects were prevented by pretreatment with azithromycin, as shown by the fact that these cells were seen as untreated cells and EspC was unable to enter the cells, avoiding the F-actin cytoskeleton disruption (Fig. 2C).
EspC internalization is abolished by an inhibitor of pinocytosis (A to C) and no receptor on the cell surface is required (D to L). HEp-2 cells were directly treated with EspC (120 μg/ml) for 10 h (B) or pretreated (3 h) with 50 μg/ml of azithromycin (C). HEp-2 cells treated with only azithromycin were used as a negative control (A). Cells were fixed, permeabilized, and stained with rhodamine-phalloidin and a rabbit anti-EspC polyclonal antibody followed by secondary fluorescein-labeled anti-rabbit IgG antibody. Slides were observed through confocal microscopy. To detect a possible receptor for EspC, HEp-2 cells were pretreated at 4°C for 30 min and then treated with 0.5 μg/ml CTx or 120 μg/ml of EspC for 6 h at 4°C. Cells were fixed and permeabilized; actin cytoskeleton was stained with rhodamine-phalloidin (D and G), and the toxins were stained with rabbit anti-CTx (E) or anti-EspC (H) polyclonal antibodies followed by secondary fluorescein-labeled anti-rabbit IgG antibody. Slides were observed through confocal microscopy. Panels F and I are the merge of D to E and G to H, respectively. Untreated cells were incubated at 4°C for 6 h (J). Panels K and L are top sections from the panels E and H, respectively. The arrow in panel F shows CTx detection.
The above data suggest that the purified EspC does not require a cell receptor. To search for this possibility, HEp-2 cells were preincubated at 4°C and then treated with EspC (120 μg/ml) or CTx (0.5 μg/ml) and kept at 4°C. At this temperature, many of the cell functions are stopped and, indeed, the CTx-treated cells were observed as rounding cells, with no preservation of the cytoskeleton (Fig. 2D). However, this effect is not caused by CTx (Fig. 2J) since its localization, determined by immunostaining with anti-CTx (Fig. 2E), was not intracellular but on the cell surface as detected in a middle section (Fig. 2F) and confirmed in a top section (Fig. 2K), suggesting that CTx binds to its receptor, the gangloside GM1. On the other hand, EspC-treated cells were also observed as rounding cells (induced by the temperature) (Fig. 2G) but, contrary to CTx, EspC was not immunolocalized on the cell surface with anti-EspC antibodies as detected in a middle section (Fig. 2H and I) and confirmed in a top section (Fig. 2L), suggesting that EspC has no receptor on the cell surface.
Secretion of EspC by EPEC is regulated by the culture medium and cell contact. Since the internalization process and the cytotoxic effects of EspC require a high dose of protein and an unusual mechanism of internalization, we decided to investigate how EspC is secreted and internalized during an EPEC infection. To search for the efficiency of EspC secretion by EPEC, the wild-type strain, an isogenic mutant in EspC, and its complementation with the espC gene as well as anti-EspC antibodies were tested for EspC secretion and antibody specificity. Concentrated supernatants from overnight culture of these strains were analyzed by immunoblot using rabbit anti-EspC polyclonal antibodies. The EspC minimal clone HB101(pJLM174) was able to secrete around 5 μg/ml of EspC into the medium, whereas the wild-type EPEC strain E2348/69 secreted a calculated concentration of 40-fold less EspC, specifically detected by the anti-EspC polyclonal antibody (Fig. 3A). As expected, the isogenic espC mutant (MAS111-espC) was unable to secrete EspC into the medium, thereby being undetectable by the anti-EspC antibody (Fig. 3A). The anti-EspC antibodies were also unable to detect EspC in concentrated supernatants from EPEC JPN15 mutated in espC and transcomplemented with pCVD450 (Fig. 3A), a perA-encoded plasmid, to increase EPEC protein secretion, whereas these antibodies detected EspC in concentrated supernatants from the MAS111 strain transcomplemented with the plasmid pJLM174 at a similar concentration as that secreted by the minimal clone HB101(pJLM174) (Fig. 3A).
Since EspC expression is regulated by Ler (9), a regulator of genes into the LEE pathogenecity island, some of which are overexpressed in MEM cell culture medium and upon cell contact, we looked for overexpression of EspC in MEM and during EPEC and epithelial cell interaction. Overnight EPEC culture in LB broth was diluted in either serum-free MEM tissue culture medium or LB broth and incubated until it reached an OD600 of 0.3 (about 2 h). Filtered, TCA-precipitated supernatants were analyzed by immunoblotting by using the rabbit anti-EspC polyclonal antibody. EPEC secreted three times more EspC protein into the bacteria grown in MEM than into those grown in LB broth (Fig. 3B). EspC secretion was higher in precipitated supernatants (2-h cultures) from bacteria infecting epithelial cells than in supernatants from mock infection without cells (Fig. 3C). Moreover, when the epithelial cells were fixed and then inoculated with EPEC, the secretion of EspC into the medium was similar to that in supernatants from mock infection without cells (Fig. 3C). ELISA analysis of EspC secretion in supernatants from EPEC, under these three conditions, showed that EPEC secreted about 55% more EspC in supernatants from EPEC infecting live cells and about 25% less in supernatants of bacteria interacting with fixed cells relative to mock infection without cells (Fig. 3D). These data suggest that secretion of EspC by EPEC, similarly to other proteins secreted by TTSS and regulated by Ler, is increased in the MEM culture medium and by the interaction with the host cells.
Intracellular internalization of EspC is enhanced during EPEC-epithelial cell interaction. Since EspC is hypersecreted during the interaction of EPEC and epithelial cells, we investigated the efficiency of EspC internalization under this condition. HEp-2 cells were incubated with EPEC at various times, and EspC internalization was detected by immunofluorescence and analyzed through confocal microscopy. After 30 min of infection, the merge of the green and red channels showed that when the actin cytoskeleton is preserved, an incipient and localized EspC internalization is observed (Fig. 4A); it is better visualized through the merge of the Nomarsky interference contrast and the green channel (Fig. 4C). Additionally, the Nomarsky interference contrast allowed us to detect that this localized EspC internalization occurred underneath some bacteria that were attached to epithelial cells (Fig. 4B). All these events were more obvious after 1 h of infection, revealing preserved cytoskeleton (Fig. 4D) with localized EspC internalization (Fig. 4D and F) occurring underneath bacteria attached to epithelial cells (Fig. 4E). Even though EspC internalization into HEp-2 cells was analyzed through confocal microscopy and revealed as middle cuts, similar experiments were performed in which cells were not permeabilized to confirm that EspC was not on the cell surface but inside the cells. In these cells infected with EPEC for 1 h, it was possible to observe bacteria attached to epithelial cells by means of the Nomarsky interference contrast (Fig. 4H) but, in the merge of this optic Nomarsky with the green channel, EspC was not observed (Fig. 4I) because the anti-EspC antibodies were unable to enter the cells to mark EspC.
Interestingly, EspC was efficiently translocated by EPEC into the cell upon pedestal formation. HEp-2 cells were infected with EPEC for 1.5 h and 3 h to detect pedestal formation. Fluorescent actin staining and immunolocalization of EspC were performed, and the cells were analyzed through confocal microscopy. HEp-2 cells that were treated for 1.5 h with EPEC showed some pedestal formations without evident cytoskeletal damage (Fig. 5A). Interestingly, over the pedestal formations, EspC was mainly secreted and internalized into the cells and was detected as dense green spots by the anti-EspC antibodies (Fig. 5A). After 3 h, when the pedestals were completely established, EspC was distributed homogeneously inside the cells and the cytoskeleton was incipiently damaged, showing loss of the stress fibers and cell contraction (Fig. 5B). As expected, after 3 h, the isogeneic espC mutant MAS111 was able to form pedestals but unable to secrete and internalize EspC into the cells, and the cytoskeleton was better preserved than that in cells treated with the wild-type strain (Fig. 5C). Whereas the espC mutant complemented with the pJLM174 plasmid was able to form pedestals and to secrete and internalize EspC over the pedestals, the EspC was distributed inside the cells after 3 h (Fig. 5D).
Remarkably, EspC secretion seemed to be coupled to its internalization, as shown by the fact that we were unable to detect EspC binding to the epithelial cell membrane by immunoblotting using anti-EspC antibodies to probe the membrane fraction of EPEC or espC+espC-infected epithelial cells during different times (Fig. 6A). Uninfected cells and cells treated with EPEC(espC) were used as negative controls. As a control, the presence of membrane proteins was evidenced by detecting a transmembranal protein, cadherin, by using anticadherin antibodies (Fig. 6A). However, in all the cytoplasmic fractions of cells that were treated with bacteria producing EspC (i.e., EPEC or espC+espC-infected epithelial cells), it was possible to detect EspC (Fig. 6B). EspC translocation was a time-dependent event that correlated with data obtained by confocal microscopy; at 1 h, a weak signal was detected by the anti-EspC antibodies, which was more evident after 2 h, and a strong recognition was detected after 6 h of incubation (Fig. 6B). Whereas, in EPEC(espC)-treated cells, the anti-EspC antibodies were unable to detect EspC in the cytoplasmic fraction, as expected, the complemented bacteria (espC+espC) were able to translocate EspC into epithelial cells at concentrations similar to those reached by the wild type after 6 h of infection (Fig. 6B).
These data suggest that EPEC is able to translocate an autotransporter protein which was previously secreted to the milieu into epithelial cells. Furthermore, EspC protein inside the cells is able to cause the cytopathic effect produced by EPEC on epithelial cells.
DISCUSSION
EPEC strains are an extremely common cause of diarrhea in young children throughout the developing world. However, EPEC-induced diarrhea occurs through an unclear mechanism. Even if the decreased absorption due to pedestal formation might be a possible explanation, the role of microvilli effacement and pedestal formation in the disease remains undefined (7). The secretion of chloride has been shown in EPEC infection, a common effect of bacterial enterotoxins, but no enterotoxin has been implicated (4). Recently, EspC, an autotransporter protein that has no participation in signal transduction and pedestal formation (38), has been shown to have enterotoxic activity (24). We have shown that EspC also produces cytotoxic effects on epithelial cells, and under light microscopy, the cell damage seems to be similar to that previously reported for Pet (30). However, the uptake and trafficking mechanisms appear to be different from those used by Pet (30). Here we show that Pet internalization is different from EspC internalization. The purified EspC protein is internalized by pinocytosis, and no receptor is required, suggesting that this process of internalization by using purified EspC is not a physiological event. However, during EPEC infection, EspC is efficiently internalized into eukaryotic cells.
Unlike other toxins (27, 26, 36, 40), which mainly use a cell receptor, EspC does not have a receptor on the epithelial cell. This was shown by the fact that we were unable to find EspC on the cell surface when the cell functions were blocked by chilling the cells at 4°C using Ctx as a positive control. Furthermore, in EPEC-infected epithelial cells, it was possible to find EspC in the supernatants and inside the cells but not on the cell membrane or in the membrane fraction. Interestingly, for the case of Pet, we were able to find Pet on the cell surface by chilling the cells at 4°C (Navarro-Garcia et al., submitted for publication). The lack of a cell receptor for EspC suggests that EspC uptake is not a receptor-mediated endocytosis. Indeed, drugs affecting endocytosis by caveole-coated vesicles, clathrin-coated vesicles, or receptor-mediated endocytosis, such as filipin, monodansyl cadaverine, or chlorpromazine, respectively, were unable to abolish the cytotoxic effect of EspC on HEp-2 cells. Again, these results were different from those obtained for its homolog Pet, which undergoes endocytosis by only clathrin-coated pits (Navarro-Garcia et al., submitted). All these results suggest that EspC does not undergo intracellular trafficking; to assess this possibility, the translocation of EspC from endosomes to cytosol was tested by alkalinizing the endosomes with NH4Cl or by blocking the H+ ATPase. Unlike diphtheria, anthrax, botulinum, and tetanus toxins (37) but similar to Pet (31), EspC cytotoxicity is not affected by these inhibitors, indicating that this protein is not translocated from endosomes to the cytosol. Furthermore, Pet is transported from endosomes to the Golgi apparatus (31; Navarro-Garcia et al., submitted). However, EspC does not seem to be transported to the Golgi apparatus because brefeldin A was unable to inhibit EspC cytotoxicity. Additionally, we were unable to detect EspC in the Golgi apparatus through confocal microscopy (data no shown).
In an attempt to find an inhibitor for EspC internalization, we used azithromycin, an antibiotic that was recently used to inhibit the fluid-phase pinocytosis in fibroblasts (41); azithromycin was able to inhibit EspC internalization, suggesting that EspC is internalized by fluid-phase pinocytosis. Pinocytosis plays a central role in cell physiology by allowing a number of cellular events, such as (i) bulk uptake of extracellular solutes, (ii) receptor-mediated uptake of ligands, (iii) transepithelial transport of macromolecules, and (iv) a large influx of membranes for the recycling of constituents inserted at the cell surface by exocytosis. Interestingly, the purified EspC internalization appears to be accomplished through the bulk uptake mechanism for extracellular solutes; since EspC, unlike Pet, is found in large amounts and irregularly distributed inside the cells (30), a high dose is needed for its internalization and no receptor is involved, However, the question of how EspC is delivered into the cytosol from vesicles is not answered since various and different drugs affecting protein trafficking did not inhibit EspC cytosol translocation. Together, these data suggest that the purified EspC internalization into epithelial cells is not a physiological event and that perhaps other bacterial factors are involved in this process.
EspC is a protein secreted by the type V secretion system (38), which is one of the most abundant and the first to be secreted by EPEC (17). Recently, it was shown that EspC is a non-LEE encoded protein that is encoded in its own pathogenicity island (24). However, the LEE-encoded regulator (Ler), an important regulator of the expression of genes implicated in forming the A/E lesion, strongly activates (31-fold) the espC promoter and increases the levels of EspC that are secreted from EPEC as measured by the promoter activity with lacZ fusion in the E. coli K-12 strain (9). Here we found that EPEC secretes more EspC protein (threefold) in MEM medium than in LB medium, and this increase is augmented twofold when EPEC is infecting epithelial cells, as measured by densitometry and ELISA, suggesting the complexity and multifactorial mechanism implicated in the expression of EPEC virulence factors encoded within and outside the LEE. Furthermore, Gauthier et al. (11) found that an agent (called compound 1) decreased the amount of secreted proteins not by interfering with the TTSS apparatus, but by affecting the amount of type III secretion-associated products (Tir, EspB, EscJ, and EscC) and of the non-LEE encoded virulence protein (EspC).
Even though EspC secretion is highly increased during EPEC and epithelial cell interaction, it is impossible to reach a 120-μg/ml concentration in the medium culture of EPEC-infected cells, which is the concentration used in experiments with purified EspC. Actually, we were unable to quantify it by the Bradford methodology and it was detected by only the ELISA and densitometry. However, considering the amount of EspC secreted by the type V secretion system during EPEC infection (ca. 0.625 μg/ml), EPEC was able to efficiently internalize EspC into epithelial cells (after 30 min). EspC uptake by the cells is a dose-dependent event, reaching amounts comparable to those of actin after 6 h and becoming saturated after this time. Moreover, EspC internalization occurs just beneath the infecting bacteria and simultaneously to the pedestal formation. Once the pedestal is formed, EspC internalization is not focalized over the pedestal but is distributed along the cytosol. Thus, this EspC homogenous distribution inside the cells is different than that which is observed when purified EspC is internalized to the cells (30). As expected, EspC internalization was not detected when epithelial cells were infected with EPEC(espC), but it was detected when the espC mutant was complemented with the pJLM174 plasmid.
EspC internalization correlated with the cytopathic effect caused by EPEC, which is clearly observed through confocal microscopy using the wild type and the complemented strain but not with the espC isogenic mutant. This effect was observed in a previous study (30) and here by using purified EspC. Further experiments are in progress to clearly associate the cytopathic effect with EspC.
Additional studies are also needed to explore which EPEC factors are involved in the efficient internalization of EspC. We can visualize at least two possibilities; given the dependence of the identified non-LEE effectors on the LEE TTSS for delivery, such as EspG (8, 21) and Cif (20) for EPEC, EspFU (2, 10, 42) and NleA (12) for enterohemorrhagic E. coli, and EspI (28) and seven non-LEE effectors that have recently been identified in the mouse-specific A/E pathogen (5), we would expect that EspC delivery is also related to the TTSS. However, it is clear by this and other studies that EspC is secreted to the extracellular medium by the type V secretion system, complicating how TTSS is getting EspC from the milieu since TTSS allows protein translocation from bacterial cytoplasm to eukaryotic cytoplasm. Another possibility is that EPEC might increase pinocytosis at the contact site with the cells as has been reported for Salmonella and Shigella. SopB plays a key role in assembling this Salmonella-containing vacuole by inducing profuse macropinocytosis events to accompany bacterial uptake (13), whereas for Shigella flexneri, the cytoskeletal rearrangements induced by the Shigella effector proteins result in the bacterium being internalized by epithelial cells within a macropinocytic vacuole (14). In both cases, a TTSS is involved; however, both events implicate macropinocytosis of the whole bacterium. Further studies in our laboratory are being addressed to search for these two possibilities. Notwithstanding, here we show that EspC is an important non-LEE virulence factor that, in nonphysiological conditions (i.e., purified protein), is not efficiently internalized, because no receptor is involved in its uptake, no intracellular trafficking exists, and no explanation for its translocation from endosomes or Golgi apparatus to the cytosol can be given. On the other hand, the physiologically secreted EspC by EPEC, which is enhanced in tissue culture media and by cell contact, is efficiently internalized during EPEC and epithelial cell interaction. This latter event is deemed to be important for the cytopathic effect produced by EPEC.
ACKNOWLEDGMENTS
This work was supported by a grant from Consejo Nacional de Ciencia y Tecnología (Mexico) (CONACYT, 44660-M) to F.N.-G.
We thank Rocio Huerta and Adrian Canizalez for their technical help.
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