1Department of Medicine, Division of Gastroenterology, University of California-San Diego School of Medicine, San Diego, California 92103-8414; and 2Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
Submitted 26 September 2003 ; accepted in final form 31 May 2004
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ABSTRACT |
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chloride secretion; Salmonella typhimurium; epidermal growth factor
Salmonella, a gram-negative bacterium that can cause gastroenteritis and enteric fever, is a significant cause of diarrheal disease worldwide. All Salmonella serotypes share the ability to invade the host by inducing their own uptake into intestinal epithelial cells. The uptake into these nonphagocytic cells is facilitated by virulence proteins delivered into the host cell cytoplasm by specialized type III protein secretion systems (TTSS) (11). The genes coding for subunits of TTSS are present in a number of distantly related gram-negative pathogens such as Yersinia, Shigella, Salmonella, and Pseudomonas. In Salmonella, two separate TTSS are encoded in genetic loci called Salmonella pathogenicity islands (SPIs). Whereas the SPI-2 TTSS is essential for the systemic part of the infection, the SPI-1 TTSS contains the genes required for invasion, manipulating cellular signal transduction pathways and the subsequent triggering of the epithelial inflammatory response, which culminate in diarrhea (25). However, effectors secreted by the SPI-1 TTSS are not necessarily encoded within the borders of SPI-1, and several are found elsewhere on the chromosome.
SigD (also called SopB), which is encoded in SPI-1, is one such effector protein secreted from Salmonella and has been characterized as an inositol phosphate phosphatase. It is capable of hydrolyzing the intracellular messenger inositol 1,3,4,5,6-P5 to inositol 1,4,5,6-P4 (21a). Previous studies (13) have shown that infection of T84 human intestinal epithelial cells with Salmonella, but not other invasive bacteria, leads to an increase in intracellular inositol 1,4,5,6-tetrakisphosphate (IP4). Furthermore, this inositol 1,4,5,6-P4 isoform of IP4 indirectly increases Cl secretion by counteracting the inhibitory effect of epidermal growth factor (EGF) on basolateral K+ efflux (3), thus supporting increased Cl secretion. The effect of EGF on secretion is mediated via the EGF receptor (EGFr) and consequent activation of the downstream effector phosphatidylinositol 3-kinase (PI3K). In turn, this enzyme activates phosphorylation of phosphatidylinositol 3,4-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3) (30). In addition to its effect on IP4, SigD has been shown to countermand PIP3 signaling by hydrolyzing it to PIP2 (19).
Salmonella mutants lacking sigD retain a normal ability to invade epithelial cells in vitro (30) but are attenuated in their ability to induce fluid secretion and neutrophil accumulation in a bovine ligated ileal loop model infected with Salmonella dublin (21a). We hypothesized that the attenuated diarrheal phenotype of sigD mutants might result from their inability to countermand signals that normally limit Cl secretion. Thus, by acting as an inositol phosphate phosphatase, we explored the possibility that SigD might reverse the inhibitory effect of EGF on Ca2+-dependent Cl secretion. In the present study, we therefore sought to define cell physiological correlates of the known biochemical properties of this particular bacterial effector protein.
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MATERIALS AND METHODS |
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Cell culture and infection.
The colonic epithelial cell line, T84, was cultured routinely in DMEM/Ham's F-12 medium (1:1) (JRH, Lenexa, KS) supplemented with 5% newborn calf serum (vol/vol) (HyClone, Logan, UT). For Ussing chamber/voltage clamp studies, 5 x 105 cells were seeded onto 12-mm Millicell-HA Transwells (Millipore, Bedford, MA). For experiments involving immunoprecipitation and Western blotting,
106 cells were seeded onto 30-mm Millicell-HA Transwells. Cells seeded onto Millicell filters were cultured for 1015 days before use. Under these conditions, T84 cells develop a polarized phenotype characteristic of epithelial cells in vivo (12). The cell monolayers were infected apically for the times indicated with either wild-type Salmonella typhimurium SL1344, an isogenic mutant strain containing a deletion in sigD, or the sigD mutant complemented with sigD provided on a plasmid (19). Bacteria were maintained using LB broth or LB agar plates. Before infection, bacteria were grown overnight without shaking in LB broth supplemented with 300 mM NaCl. After an optical density (at 600 nm) of
0.4 was reached, bacteria were washed twice in PBS and resuspended in Ringer solution before infection.
Colony-forming units and IL-8 determination. T84 monolayers were grown to confluence and infected apically with bacteria at a multiplicity of infection (MOI) of 25 for 1 h to allow bacterial entry to occur. Extracellular bacteria were then removed by washing the monolayers, and gentamicin (50 µg/ml) was added to kill any remaining extracellular bacteria. Numbers of intracellular bacteria were measured by counting colony-forming units after lysing epithelial cells in 50% PBS and 0.1% Triton X-100 and plating serial dilutions of the lysate on LB agar plates for overnight culture.
IL-8 secretion was determined using T84 intestinal epithelial cells grown to confluence on permeable supports and infected with bacteria for 1 h as described above, followed by incubation of the cells for 8 h. Supernatants were harvested, and IL-8 was measured by ELISA as described previously (14).
Immunoprecipitation and Western blotting. T84 cell monolayers were washed three times in Ringer solution and allowed to equilibrate for 30 min at 37°C. Cells were then infected (±inhibitors) as appropriate. Incubations were terminated by washing twice with ice-cold PBS. Ice-cold lysis buffer was added (consisting of 1% Triton X-100, 1 mM NaVO4, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml antipain, 1 mM NaF, 1 mM EDTA, and 100 µg/ml phenylmethylsulfonyl fluoride in PBS), and the cells were incubated at 4°C for 30 min. Cells were then scraped into microcentrifuge tubes and centrifuged at 10,000 rpm for 10 min, and the pellet was discarded. An aliquot was removed from each sample to determine protein content, and samples were adjusted so that they contained equal amounts of protein. For immunoprecipitation studies, monoclonal EGFr antibody (5 µg) was added to each sample and allowed to incubate, with shaking, for 60 min at 4°C, followed by addition of 40 µl protein A sepharose, and an additional incubation for 60 min at 4°C. After centrifugation, pellets were washed once with ice-cold lysis buffer, twice with ice-cold PBS, and then resuspended in gel loading buffer (50 mM Tris, pH 6.8, 2% SDS, 100 mM dithiothreitol, 0.2% bromophenol blue, 20% glycerol). The samples were boiled for 5 min, and proteins were separated by SDS-PAGE. Separated proteins were transferred onto polyvinylidene difluoride membrane (DuPont NEN) overnight at 4°C. The membrane was washed in 1% blocking buffer for 30 min, followed by incubation of the membrane with an appropriate dilution of primary antibody in 1% blocking buffer for 60 min. This was followed by washing three times in TBS with 1% Tween. After washes, a horseradish peroxidase-conjugated secondary antibody was added to the membrane in 1% blocking buffer and allowed to incubate for an additional 30 min. This was followed by a further washing three times in TBS with 1% Tween. Immunoreactive proteins were detected by an enhanced chemiluminescense detection kit and exposure of the membrane to X-ray film. Quantitation of protein phosphorylation was performed by densitometry using National Institutes of Health Image software.
Measurement of ion transport across T84 cells. T84 cells were grown as monolayers on permeable filter supports as described above. After 1015 days in culture, monolayers were infected (or treated appropriately as controls), mounted in Ussing chambers (aperture = 0.6 cm2), voltage-clamped to zero potential difference, and monitored for changes in short-circuit current, which in this cell line are wholly reflective of active chloride secretion (12). Short-circuit current measurements were carried out in Ringer solution containing (in mM) 140 Na+, 5.2 K+, 1.2 Ca2+, 0.8 Mg2+, 119.8 Cl, 25 HCO3, 2.4 H2PO4, and 10 glucose. Results were normalized and expressed as µA/cm2.
Measurement of transepithelial resistance across T84 epithelial monolayers. Monolayers of T84 cells were grown on permeable filter supports, as described above, and infected or treated appropriately as controls. Transepithelial resistance (TER) was monitored at various times thereafter with a voltohmeter.
Immunohistochemical analysis of T84 epithelial monolayers. For immunohistochemical analysis, infected and control epithelial monolayers were fixed in 5% neutral buffered formalin and blocked (1% BSA, 0.2% blocking reagent, 0.3% Triton X-100). Sections were incubated respectively with antibodies, subsequently washed three times in PBS, and incubated with rabbit anti-goat IgG Cy3-, goat anti-rabbit IgG Cy5-conjugated secondary antibody, or phalloidin-FITC. Images were captured with a DeltaVision Restoration microscope system (Applied Precision, Issaquah, WA) attached to an inverted, wide-field fluorescent microscope (Nikon TE-200). Optical sections were acquired in 0.2-µm steps in the z-axis, and the resolution of x40 images was enhanced using DeltaVision's deconvolution process and the algorithm by Agard et al. (1). Images were saved, processed, and analyzed using the DeltaVision software package softWoRx (version 2.50). Quantification of fluorescence was accomplished using only the linear range of the digital camera.
Statistical analysis. All data are expressed as means ± SE for a series of n experiments. Student's t-tests were used to compare paired data. One-way analysis of variance with a Student-Neuman-Keul posttest was used when three or more groups of data were compared. P values <0.05 were considered to be statistically significant.
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RESULTS |
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Immunolabeling of tight junctional proteins shows a more distinct temporary loss of actin structure and loss of colocalization of ZO-1 and occludin in monolayers infected with wild-type vs. sigD mutant S. typhimurium. To explore how the early differences in the effects of wild-type and sigD infection on TER occur, a role for SigD in inducing changes in tight junctional protein structures was investigated using protein immunolabeling followed by deconvolution microscopy. Costaining for F-actin (green) and ZO-1 (red) (Fig. 8) showed that, in monolayers infected with wild-type bacteria for 20 min (Fig. 8A), the apical actin structure was temporarily lost in a large percentage of the cells with locally condensed accumulation, whereas the loss was more limited after infection with sigD Salmonella (arrows). Interestingly, in both wild-type and sigD mutant-infected monolayers, an elongation/distortion of cell shape was observed at this time point of infection (arrows). Furthermore, after 40 min of infection, the distortion of the cells declined, and the actin structure appeared to reorganize. Actin, however, was still locally accumulated mainly in cells infected with wild-type bacteria where this recovery also appeared only partial.
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DISCUSSION |
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In this study, deletion of SigD had no influence on the ability of Salmonella to invade T84 cells, as also found previously (30). SigD has been shown to participate in the bacteria-induced host membrane ruffling thought to be utilized in Salmonella invasion via its role as a phosphatase (28). However, as shown by Zhou et al. (30), the role of SigD in bacterial entry may be redundant with the roles of two other effector proteins, SopE and SopE2. Hence, if only one of these effector proteins is missing, the bacteria can still enter host cells with high efficiency. Our findings on invasion support this interpretation. Similarly, we also showed that the induction of IL-8 by wild-type and sigD mutant Salmonella was similar, suggesting that other early cellular anti-infectious responses to both wild-type and mutant Salmonella might be comparable as well.
Wild-type and sigD S. typhimurium initiated similar initial signaling responses in T84 cells, notably EGFr activation. Investigators at one of our laboratories have recently shown that certain effects of Salmonella on epithelial signaling require only bacterial attachment rather than invasion (5). Thus activation of EGFr induced by both wild-type and sigD Salmonella may occur before any actual invasion. Moreover, although activation of the EGFr probably does not participate in Salmonella invasion (20), prompt phosphorylation of this basolaterally located receptor (24) after apical infection of a polarized monolayer is striking and deserving of further investigation (5). Studies on epithelial signaling in response to Salmonella infection, especially relating to the role of specific effector proteins, are still in relative infancy, but this rapidly moving field of investigation should ultimately shed light on pathophysiological mechanisms.
Although Salmonella induces activation of EGFr, the pattern and downstream effects differ from the mode of EGFr activation by EGF itself. This together with the SigD-induced actions on ion transport pathways induced by EGF is intriguing. Although we do not yet have a definite answer, our laboratorys prior work with signaling through the EGFr leads us to believe that Salmonella may induce divergent dimerization of the EGFr than EGF itself. The EGFr (ErbB1) is part of the ErbB family, which consists of, thus far, four receptors (ErbB14) (6, 7, 26). On activation, and dependent on agonist, these receptors form dimers. Data suggest that EGF will induce dimerization of ErbB1 and ErbB2 (18), whereas, for instance, CCh will induce homorization between two ErbB1s (18), and the downstream effects of these activations differ accordingly. Without certainty, we speculate that Salmonella will form dimerization between ErbB receptors too. The actual EGFr activation does not, however, seem to interfere with the phosphatase activity induced by Salmonella through SigD. Thus, although Salmonella does activate the EGFr, this activation may induce different actions than what is observed with EGF.
Although wild-type and sigD mutant Salmonella had similar properties with respect to invasion, initial host cell signaling, and IL-8 production, their effects on epithelial secretory responses were divergent. Thus wild-type Salmonella modified secretory responses and associated signaling, whereas sigD mutant Salmonella did not. First, calcium-dependent Cl secretion was decreased in wild-type but not sigD Salmonella-infected cell monolayers, indicating a role for SigD in controlling secretory responses. Indeed, several inositol phosphates and phosphatidylinositol phosphates have been shown to interfere with cellular secretory pathways (3, 13, 17). This suggests that the ability of SigD to function as an inositol phosphate phosphatase renders the protein as a key mediator of Salmonella-induced alterations in epithelial secretory responses. Second, we observed that, although wild-type Salmonella with intact phosphatase activity was capable of abrogating the inhibitory effect of EGF on chloride secretion, the sigD mutant was not. This finding strongly supports our initial hypothesis, which held that because SigD acts as inositol phosphate phosphatase and converts Ins(1,3,4,5,6)P5 to Ins(3,4,5,6)P4 (21a), it can thereby antagonize inhibitory actions of PIP3 on potassium efflux (3, 9, 17). Likewise, the ability of SigD to convert PIP3 to PIP2 (19, 21a) would also be predicted to abrogate the inhibitory effect of EGF on chloride secretion, which is known to depend on the enzyme responsible for PIP3 production, PI3K (29). Thus SigD induces diverse effects on chloride secretory responses, including inhibition of agonist-induced Cl secretion, while at the same time antagonizing normal cellular inhibitory pathways that would otherwise limit secretion by themselves, perhaps via IP4 production and/or PIP3 metabolism (Fig. 10A). On the other hand, it is unlikely that wild-type Salmonella abrogates responses to EGF by virtue of prior activation of the receptor for this ligand, because this effect was induced equally by the wild-type bacteria and the sigD mutant.
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SigD has been shown to mediate bacterial entry by stimulating membrane ruffling and actin cytoskeleton rearrangements (15). Although a loss in actin structure was also seen in sigD mutant-infected monolayers, it was less extensive in these early time points, and this may contribute to differing effects of the bacterial strains on the initial drop in TER. Other studies have shown that SigD induces effects on the actin-organizing small GTP proteins, Cdc42, whereas another effector protein, SopE, induces effects on both Cdc42 and Rac (15). Thus a possible explanation for the delayed reduction in TER and the reduced effect on actin structure in epithelial monolayers infected with the sigD mutant could be the lack of an early SigD-induced effect on Cdc42, although eventually this can be compensated for by SopE. This, together with the PKC-dependent early TER reduction we observed, may link the bacterial effector protein-induced changes in actin structure and the pathophysiological response of reduced TER. SigD is also known to play a role in bacterial-induced membrane ruffling (28), perhaps contributing to epithelial dysfunction even before invasion. Furthermore, the reduction in colocalization of occludin and ZO-1 was considerably less remarkable in sigD mutant- than in wild type-infected monolayers, and a relation between PKC activation, altered occludin phosphorylation, and increased permeability have been suggested (10). Thus, although these data do not prove conclusively that SigD directly impairs colocalization of occludin and ZO-1 rather than influencing this association downstream from an effect on the actin cytoskeleton, the faster reduction in TER observed in wild-type vs. sigD mutant-infected monolayers may be attributable to the deranged localization of these tight junctional proteins. Therefore, the early reduction in TER produced by SigD is dependent on 1) a possible direct action on actin filaments, 2) a disruption of the colocalization of occludin and ZO-1, and 3) possible mechanical rupture of tight junctions as reflected by the altered contour of individual cells in infected monolayers. On the other hand, the ability of wild-type Salmonella to activate EGFr is unlikely to be involved in reducing TER, because this effect was evoked in an equivalent manner by the SigD mutant. We have also shown that an EGFr kinase inhibitor fails to prevent Salmonella-induced resistance changes (5).
In summary, we have presented evidence that specific cell physiological parameters of epithelial monolayers infected with S. typhimurium are affected by the bacterial effector protein SigD. The effects of SigD extend to both the regulation of Cl secretory responses as well as the time course of changes in epithelial resistance. SigD may interfere with intestinal epithelial secretion via several mechanisms. First, its function as an inositol phosphate phosphatase may antagonize inhibitory pathways for epithelial secretion (Fig. 10A). Second, it apparently is required, at least initially, through a PKC-dependent mechanism, to alter the integrity of epithelial tight junctions through its actions on actin structure and ocludin/ZO-1 colocalization (Fig. 10B). The reversal of EGF-induced inhibition of Cl secretion by the wild-type but not the sigD-deficient mutant also supports the hypothesis that Salmonella likely increases Cl secretion in vivo by counteracting negative regulatory epithelial signaling pathways. Attenuation of signaling pathways that normally limit Cl secretion may account, at least in part, for the diarrhea that so often accompanies Salmonella infection.
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GRANTS |
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ACKNOWLEDGMENTS |
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This work was presented, in part, at the 2001 Annual Meeting of Experimental Biology and has been published in abstract form (FASEB J 15: 656.5, 2001).
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FOOTNOTES |
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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.
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