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Context-dependent activation kinetics elicited by soluble versus outer membrane 1
vesicle-associated heat-labile enterotoxin 2
Halima Chutkan1 and Meta J. Kuehn1,2* 3 4 5
Running title: Differential responses by soluble and vesicle-bound LT 6
7 8 Box 3711 9 Department of Molecular Genetics and Microbiology1 and Biochemistry2 10 Duke University Medical Center 11 Durham, NC 27710 12 13 *Corresponding author 14 email: [email protected] 15 919-684-254516
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.05336-11 IAI Accepts, published online ahead of print on 27 June 2011
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Abstract 17
Enterotoxigenic Escherichia coli (ETEC) is the leading cause of traveler’s and children’s 18
diarrhea worldwide. Among its virulence factors, ETEC produces heat-labile enterotoxin (LT). 19
Most secreted LT is associated with outer membrane vesicles that are rich in lipopolysaccharide. 20
The majority of prior studies have focused on soluble LT purified from ETEC periplasm. We 21
investigated the hypothesis that the extracellular vesicle context of toxin presentation might be 22
important in eliciting immune responses. We compared the polarized epithelial cell response to 23
apically applied soluble LT and LT-containing vesicles (LT+ vesicles) as well as controls using a 24
catalytically inactive mutant of LT and vesicles lacking LT. Although vesicle treatments with no 25
or catalytically inactive LT induced a modest amount of IL-6, samples containing catalytically 26
active LT elicited higher levels. A combination of soluble LT and LT-deficient vesicles induced 27
significantly higher IL-6 than either LT or LT+ vesicles alone. The responses to LT+ vesicles 28
were found to be independent of the canonical LT pathway because the inhibition of cyclic AMP 29
response element-binding protein (CREB) phosphorylation did not lead to a decrease in cytokine 30
gene expression levels. Furthermore, soluble LT caused earlier phosphorylation of CREB and the 31
activation of CRE compared with LT+ vesicles. Soluble LT also led to the activation of activator 32
protein 1, whereas LT+ vesicle IL-6 responses appeared to be mediated by NFκB. In summary, 33
the results demonstrate that soluble LT and vesicle-bound LT elicit similar cytokine responses 34
through different activation pathways.35
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Introduction 36
Enterotoxigenic Escherichia coli (ETEC) is the leading cause of traveler’s diarrhea (3), and 37
it has been estimated to cause approximately 10 million cases of traveler’s diarrhea worldwide 38
(45). ETEC is also the leading cause of morbidity and mortality due to diarrhea in children in 39
developing countries. A total of 280 million cases of diarrhea associated with ETEC were found 40
in children less than five years old in outpatient clinics in developing countries (50), and 41
mortality due to ETEC has been estimated at 170,000 deaths annually (33). Heat-labile 42
enterotoxin (LT) is a major virulence factor produced by ETEC and is known to contribute to the 43
disease (20). 44
LT is an AB5 toxin that is composed of a pentameric B subunit, which binds to host 45
receptors, and a catalytically active A subunit (12). The B pentameric ring binds to the 46
Galβ1,3GalNAcβ1(NeuAcα2,3),4Galβ1,4Glc ceramide (GM1) ganglioside on host cells, which 47
mediates internalization. Studies using soluble LT that was purified from the periplasm have led 48
to a detailed understanding of its complex trafficking pathway and activation inside mammalian 49
cells (12, 20, 39). Once internalized, LT is trafficked to the Golgi and the endoplasmic reticulum 50
(ER), where the A subunit is further processed. The modified A subunit then catalyzes ADP-51
ribosylation of the Gsα subunit in the adenylate cyclase pathway. This ribosylation leads to an 52
increase in cyclic adenosine monophosphate (cAMP) levels and an efflux of water and 53
electrolytes into the lumen of the intestine (12). LT activates protein kinase A (PKA), which 54
phosphorylates the cystic fibrosis transmembrane regulator (CFTR) and cAMP response element 55
(CRE)-binding protein (CREB) to transport Cl- into the intestinal lumen and induce gene 56
transcription (40). 57
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In contrast to the highly homologous cholera toxin (CT) produced by Vibrio cholerae, most 58
of the secreted LT is found associated with outer membrane vesicles (OMVs) (14, 18, 44). 59
OMVs are spherical structures secreted from all Gram-negative bacteria studied to date (26). 60
OMVs are enriched in outer membrane components, and the lumen of OMVs contains 61
periplasmic components. OMVs contain biologically active components and immunomodulatory 62
molecules (also known as pathogen-associated molecular patterns; PAMPs), such as LPS and 63
flagellin, that interact with and influence host cells (10). LT is found in the OMV lumen and, by 64
virtue of its ability to bind LPS, also bound to the surface of OMVs (17, 32). 65
Besides LT, other toxins from a variety of pathogenic bacteria have also been shown to be 66
enriched in OMVs, including cytolysin A (ClyA) from E. coli (49), leukotoxin A (LktA) from 67
Aggregatibacter actinomycetemcomitans (21), vacuolating toxin (VacA) from Helicobacter 68
pylori (11), and cytolethal distending toxin (CDT) from Campylobacter jejuni (29). Previous 69
studies have demonstrated differences in mammalian cell toxicity based on the presentation of 70
those toxins (e.g., in the context of OMVs versus soluble toxin). Wai et al. showed that 71
compared to equal amounts of ClyA purified from the periplasm, OMV-associated ClyA induced 72
higher cytotoxic activity in HeLa cells (49). The OMV context of ClyA presentation was shown 73
to facilitate the active oligomerized state of ClyA, leading to its higher activity (49). H. pylori 74
has been shown to secrete VacA in both a free soluble form and associated with OMVs (11). 75
Ricci et al. demonstrated that although OMV-associated VacA accounted for approximately 25% 76
of secreted toxin, the OMV-bound toxin showed lower vacuolating activity than soluble VacA 77
(37). However, as OMVs are complex entities, the OMV association of toxins is likely to affect 78
host cells beyond the differences in toxin potency. 79
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The gut is a unique environment that has developed tolerance to native microbiota. The 80
intestinal epithelium forms tight intracellular junctions and microvilli that inhibit the attachment 81
and invasion of intestinal organisms (1). Tight junctions also play a role in the polarization of 82
epithelial cells, resulting in distinct apical and basolateral surfaces (24). Commensal tolerance is 83
further maintained through a variety of mechanisms, including the subcellular localization of 84
Toll-like receptors (TLRs) and the inhibition of immune responses to commensal products, such 85
as LPS (1). Pathogenic bacteria subvert these defenses in a variety of ways, including epithelial 86
cell internalization and the elaboration of virulence factors, such as toxins (31). OMVs can also 87
penetrate the epithelial cell barrier. ETEC OMVs were found to enter cultured intestinal 88
epithelial cells via a specific LT-mediated pathway (23). 89
We hypothesized that, in addition to modulating its toxicity, the context of toxin 90
presentation is important in determining the host response. In particular, OMV-associated toxin 91
is likely to elicit a different inflammatory response than soluble toxin because of the presence of 92
LPS and other PAMPs. To investigate this theory, we compared the response of polarized human 93
intestinal epithelial cells to apically applied soluble LT, catalytically inactive LT (S63K), OMVs 94
containing either catalytically active LT (LT+ OMVs) or catalytically inactive LT (S63K 95
OMVs), and OMVs without LT (ΔLT OMVs). We found significant differences in the kinetics 96
of responses induced by soluble LT compared with OMV-bound LT. Our results show that 97
soluble LT and LT+ OMVs elicit different responses and act through different mechanisms. 98
Materials and Methods 99
Strains and growth conditions. The bacterial strains used in this study are listed in Table 1. 100
Strains were grown in CFA medium (1% casamino acids, 0.15% yeast extract, 0.005% MgSO4 101
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and 0.005% MnCl2) at 37°C with or without 100 µg/mL ampicillin. The LT S63K mutants were 102
constructed using the QuikChange mutagenesis kit (Qiagen) according to the manufacturer’s 103
instructions using the following primers: S63K sense, 5’-104
GACGGATATGTTTCCACTAAACTTAGTTTGAGAAGTGC-3’, and S63K anti-sense, 5’-105
GCACTTCTCAAACTAAGTTTAGTGGAAACATATCCGTC-3’. Transformations were 106
performed using a CaCl2 protocol, as previously described (17). To induce the expression of 107
plasmid-encoded wild-type and mutant LT, 100 µM isopropyl β-D-1-thiogalactopyranoside was 108
added to cultures. 109
Cell culture. The human intestinal epithelial T84 cell line (American Type Culture 110
Collection CCL-248) was maintained in a 1:1 ratio of Dulbecco’s modified Eagle’s medium and 111
Ham’s F12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; HyClone) and 112
1% penicillin–streptomycin amphotericin B (Gibco) at 37°C. Human embryonic kidney 293T 113
(HEK293T) cells (ATCC CRL-11268) were maintained in minimum essential medium 114
supplemented with 10% FBS at 37°C. For polarization assays, 4 x 105 T84 cells were seeded on 115
1.12-cm2 transwell inserts (Corning) and grown for 5-7 days. Tight junction formation was 116
measured using transepithelial electrical resistance (TEER) using a Millicell-ERS (Millipore), 117
and cells were used at TEERs ≥ 1,000 Ω·cm2. Cells were used between passages 65-73. 118
LT purification. LT was purified from strains containing wild-type LT or S63K LT as 119
described previously (32). The concentration of LT was quantified using the Bradford method, 120
with bovine serum albumin as the standard. The endotoxin concentration of LT was determined 121
using a limulus amebocyte lysate assay (Cambrex) and was found to be less than 1 EU/mL for 122
both wild-type LT and S63K LT in the concentrations used in the assays. 123
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OMV purification and standardization. OMVs were purified from strains expressing 124
wild-type LT or S63K LT or the ΔeltA strain as previously described (22). To quantify the 125
concentration of LT in LT+ OMVs, two-fold dilutions of LT were loaded on a 15% SDS-PAGE 126
gel with dilutions of LT+ OMVs. Proteins were transferred to a polyvinylidene fluoride 127
membrane (GE Healthcare), incubated with a LT-cross reactive rabbit polyclonal anti-CT 128
antibody (Sigma), incubated with a horseradish peroxidase-conjugated anti-rabbit antibody 129
(Sigma) and visualized using enhanced chemiluminescence (SuperSignal; Pierce). A standard 130
curve was calculated from the known concentrations of the soluble LT and used to calculate the 131
amount of LT in OMVs using densitometry values obtained using ImageJ software (National 132
Institutes of Health). S63K and ΔLT OMVs were standardized to LT+ OMVs by lipid content 133
using standard curves generated using the lipophilic dye FM4-64 (Molecular Probes). 134
Consequently, for example, in an experiment using “1 nM samples,” treatments were 1 nM 135
soluble LT (WT or S63K), OMV preparations containing 1 nM LT (LT+ OMVs and LT-136
supplemented ΔLT OMVs), and the equivalent amount of OMVs that did not contain wild-type 137
LT (S63K OMVs and ΔLT OMVs). 138
Signal pathway inhibitors. Inhibitors of PKA (adenosine 3′,5′-cyclic phosphorothioate-Rp; 139
Rp-camps), p38 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; 140
SB203580), JNK (anthra[1,9-cd]pyrazol-6(2H)-one, 1,9-pyrazoloanthrone; SP600125), MEK 141
(2′-amino-3′-methoxyflavone; PD98059) and NFκB (ammonium pyrrolidinedithiocarbamate; 142
PDTC) were purchased from Calbiochem. The CREB inhibitor H89 (N-[2-(p-143
bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride) was purchased from 144
Sigma-Aldrich. SB203580 (10 µM), SP600125 (10 µM), PD98059 (10 µM) and H89 (10 µM) 145
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were added bilaterally to polarized cells for 1 h, Rp-camps (100 µM) was added basolaterally for 146
1 h and PDTC (10 µM) was added bilaterally for 30 min before the addition of treatments. 147
RT-PCR. Samples standardized to either 1 nM or 200 pM LT or vehicle (PBS) were added 148
to the apical compartment of duplicate wells of polarized T84 cells for 6 h with or without 149
inhibitor pre-treatment for 1 h or 30 min. Total RNA was collected using the Qiagen RNeasy kit 150
according to the manufacturer’s instructions. The RNA was reverse-transcribed into cDNA using 151
oligo(dT) primers and SuperScript III (Invitrogen), according to the manufacturer’s instructions, 152
and the cDNA was used as the template in RT-PCR assays. RT-PCR was performed in a total 153
volume of 15 µL using iQ SYBR Green (Bio-Rad) and analyzed using an iCycler Real Time 154
Detection System (Bio-Rad). The gene-specific primers used for RT-PCR analysis were IL-6 155
sense, 5’-GACAGCCACTCACCTCTT -3’; IL-6 anti-sense, 5’-TGTTTTCTGCCAGTGCC-3’; 156
TNFα sense, 5’-CCCAGGCAGTCAGATCAT-3’; TNFα anti-sense, 5’-157
TCAGCTCCACGCCATT-3’; GAPDH sense, 5’-ACATCGCTCAGACACCAT-3’; and 158
GAPDH anti-sense, 5-‘GGGTCATTGATGGCAACA-3’. Results were analyzed using the 159
accompanying software, and gene expression was standardized to GAPDH levels. Results are 160
shown as the fold induction of the gene in relation to the corresponding mock treatments and 161
were measured using the 2-ΔΔCt method. 162
ELISA. Polarized T84 cells were treated apically with samples for 10 h, and both apical and 163
basal supernatants were collected and analyzed using IL-6 and TNFα ELISA kits (BD 164
Biosciences), according to the manufacturer’s instructions. 165
Flagellin immunoblotting. The samples used for all treatments and the vector (PBS) were 166
analyzed using 10% SDS-PAGE (BioRad) and transferred to a nylon membrane. The membrane 167
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was then blocked in Odyssey blocking buffer and incubated with rabbit anti-H7 (a flagellar 168
marker originally produced by Difco, a kind gift from Dr. Patrick Seed, Duke University). The 169
membrane was then incubated with a fluorescently conjugated anti-mouse antibody and imaged 170
using the Odyssey imaging system. The densitometry of the entire lane was determined using the 171
accompanying software. 172
Immunoblotting of nuclear extracts. Samples were added to polarized T84 cells in the 173
presence or absence of inhibitors for the indicated times, and nuclear protein was extracted as 174
previously described (42), with some modifications. Briefly, buffer A (10 mM HEPES, pH 8, 1.5 175
mM MgCl2, 10 mM KCl and 0.5 mM dithiothreitol (DTT)) supplemented with 0.4% Igepal was 176
directly added to cells in the transwell insert and incubated on ice for 15 min. Cells were then 177
collected, vortexed for 15 s, and centrifuged at 8,000 g for 2 min. The supernatant was discarded, 178
and the nuclear pellet was washed with buffer A. The nuclear pellet was then resuspended in 179
buffer B (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, 25% glycerol, 180
0.5 mM DTT and protease and phosphatase inhibitor cocktails (Sigma-Aldrich)) and incubated at 181
4°C for 2 h with shaking. The supernatant was then desalted using a desalting column (Thermo 182
Scientific) and analyzed using 10% SDS-PAGE. The proteins were transferred to a nitrocellulose 183
membrane and incubated with Odyssey blocking buffer before being incubated with mouse anti-184
TATA binding protein (TBP) (Abcam), mouse anti-pCREB (Millipore), rabbit anti-pMSK1 185
(S376) and rabbit anti-pMSK1 (T581) (Cell Signaling), and rabbit anti-pp65 S276 (Abcam). To 186
ensure that the nuclear preparations were free of cytosolic contamination, the membranes were 187
also incubated with mouse anti-β-tubulin. The β-tubulin monoclonal antibody developed by 188
Michael Klymkowsky was obtained from the Developmental Studies Hybridoma Bank 189
developed under the auspices of the NICHD and maintained by The University of Iowa, 190
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Department of Biology, Iowa City, IA 52242. The membranes were then incubated with the 191
appropriate fluorescently conjugated secondary antibodies and visualized using the Licor 192
Odyssey imaging system. No β-tubulin bands were detected in the nuclear extracts, indicating 193
that they were free from cytosolic contamination. The densitometry of the TBP and pCREB 194
bands was calculated using the accompanying Odyssey software. Results are presented as the 195
ratio of pCREB/TBP normalized to a mock value of 1 for each blot. 196
Luciferase assay. A total of 1 x 104 HEK293T cells were seeded into each well of a 96-well 197
plate 16 h before transfection. Cells were then transfected with the pCRE-Luc plasmid 198
(Stratagene) containing CRE fused to a firefly luciferase reporter gene and the pSV40-RL 199
plasmid (Promega) containing a Renilla luciferase reporter fused to a constitutive promoter using 200
Lipofectamine 2000 (Invitrogen) as previously described. Samples were added approximately 16 201
h post-transfection for the indicated time, and firefly luciferase and Renilla luciferase activities 202
were determined using the Dual-Glo Luciferase Assay (Promega), according to the 203
manufacturer’s instructions. The results are presented as the ratio of firefly luciferase activity to 204
Renilla luciferase activity. 205
Electrophoretic mobility shift assay. Nuclear extracts were prepared as described 206
previously. Extracts were then mixed with an equal volume of buffer C (20 mM HEPES, pH 7.9, 207
50 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, protease inhibitor cocktail and phosphatase inhibitor 208
cocktail), and the protein concentration was measured using the Bradford method. A total of 5 µg 209
of nuclear protein were analyzed using an electrophoretic mobility shift assay (EMSA) to 210
determine the activation of activator protein 1 (AP-1) according to the manufacturer’s 211
instructions (Gel Shift Assay System; Promega). Phosphorimages were scanned using the 212
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STORM 860 system (Molecular Dynamics) and analyzed using ImageQuant 5.2 software 213
(Molecular Dynamics). 214
Statistical analysis. Multiple comparisons were performed using a one-way analysis of 215
variance (ANOVA) followed by Tukey’s test. Comparisons between treated and untreated 216
samples were performed using Student’s t-test. All statistical analyses were performed using 217
GraphPad InStat (GraphPad Software, Inc.). Results are presented as means ± SEM, and all 218
experiments were performed in triplicate. 219
Results 220
Catalytically active LT-containing treatments induce higher expressions of IL-6 and TNFα 221
than other treatments 222
To investigate whether the context of LT affects the toxicity and host response to LT, we 223
treated polarized cultures of human T84 intestinal epithelial cells with either soluble LT or 224
equivalent concentrations of LT in the context of OMVs. Cytokine induction in response to LT 225
and OMVs, individually, has been widely studied using a variety of cultured mammalian cells (4, 226
5, 10, 41, 43). However, no studies on the pathways involved in cytokine responses have been 227
performed using the more physiologically relevant polarized epithelial cell model. As a result, 228
these experiments will also reveal host cell responses to strictly apical application of LT. 229
We designed a series of samples that would allow us to distinguish the effects of the OMVs, 230
the effects of active toxin, and the effects of the combination of OMVs with active toxin. The 231
samples were carefully standardized to allow comparative evaluations to be made. The amount 232
of LT in LT+ OMVs was first standardized to the amount of soluble LT. S63K OMVs and ΔLT 233
OMVs were then standardized to LT+ OMVs by their lipid content. Comparing equivalent 234
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treatments of LT+ OMVs and ΔLT OMVs was more appropriate than using pure LPS as an 235
endotoxin control because the presentation of LPS in OMVs is very different from preparations 236
of organically extracted, sonicated LPS. In addition, we combined soluble LT and ΔLT OMVs 237
(LT/ΔLT OMVs) in equivalent proportion to their concentrations in LT+ OMV preparations to 238
determine if the response induced by LT+ OMVs was merely additive or whether the native 239
context of toxin presentation played a role. 240
As shown in Fig. 1A, whereas all treatments induced TNFα expression compared with 241
mock, soluble LT, LT+ OMVs and the combination of LT/ΔLT OMVs induced higher gene 242
expression levels of TNFα than the other treatments. This induction appeared to be dependent on 243
the catalytic activity of LT because the catalytically inactive S63K LT treatments and the ΔLT 244
OMVs did not induce TNFα to the same level, even at the higher 1 nM dose (Fig. 1A). We noted 245
that the amount of TNFα induction was not significantly different between soluble and vesicle-246
associated LT. 247
As shown in Fig. 1B, the catalytically active LT-containing treatments induced higher gene 248
expression levels of IL-6 than the other treatments at both 1 nM and 200 pM. In addition, both 249
concentrations of LT induced equivalent amounts of IL-6, suggesting that the 200 pM treatment 250
already produced the maximal effect. Both ΔLT and S63K OMVs induced a modest amount of 251
IL-6, whereas S63K LT did not induce expression over mock. In contrast to TNFα, whose 252
expression was not significantly different between the LT-containing treatments, the 253
combination of LT and ΔLT OMVs led to significantly higher IL-6 levels than both soluble LT 254
and LT+ OMVs at both concentrations (ANOVA; p < 0.01 at 1 nM; p < 0.001 at 200 pM). This 255
induction was synergistic, as the level of induction was more than the combined results of LT 256
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and ΔLT OMVs. Notably, again, the amount of IL-6 gene induction was not significantly 257
different between soluble LT and LT+ OMVs at either concentration. 258
IL-6 ELISA results in Fig. 1C confirmed that the induction of gene expression also resulted 259
in an increase in protein concentration in the apical compartment. No IL-6 was found in the 260
basolateral supernatants, indicating the polarized secretion of this cytokine (data not shown). 261
TNFα protein expression was not found in either compartment of treated cell cultures, which 262
may be due to an extremely low, undetectable basal level of TNFα expression by these cells. 263
Overall, these results indicated that, independent of its context as a soluble molecule or as a 264
component of OMVS, equivalent amounts of catalytically active LT elicited substantial but 265
indistinguishable TNFα and IL-6 upregulation and IL-6 secretion in polarized epithelial cells. 266
We tested if levels of flagellin in the OMV preparations directly correlated with the 267
observed induction levels since flagellin is known to induce cytokine expression and to be 268
associated with OMVs (13, 34, 46). We analyzed the relative amounts of flagellin associated 269
with the OMV treatment preparations using immunoblotting. As shown in Fig. 1B, LT+ OMVs 270
and S63K OMVs had similar levels of flagellin and these were substantially higher than the 271
amount of flagellin present in ΔLT OMVs. Thus, although the flagellin present in OMVs might 272
contribute to the induction of TNFα and IL-6, the levels of flagellin did not correlate with the 273
differences in the levels of induction observed for the different OMV samples. 274
LT+ OMVs do not induce cytokine gene expression through CREB 275
To further examine the role of the catalytic activity of LT in inducing IL-6 and TNFα gene 276
expression, we used Rp-camps and H89 to examine if the induction occurs through PKA. As 277
mentioned earlier, LT acts by increasing cAMP levels, which activates PKA, leading to the 278
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phosphorylation of CREB. Rp-camps prevents the dissociation of the catalytic subunits of PKA 279
from their regulatory subunits, abolishing PKA activity, and H89 inhibits the phosphorylation of 280
CREB by PKA (30). As expected, Rp-camps significantly inhibited the induction of TNFα and 281
IL-6 in response to soluble LT, as determined 6 h post-treatment (Fig. 2A and B). However, Rp-282
camps inhibition of PKA activity notably did not have an effect on the responses induced by LT+ 283
OMVs at 6 h (Fig. 2A and B). The combination of LT/ΔLT OMVs showed an intermediate 284
response to inhibition by Rp-camps, since Rp-camps pretreatment significantly inhibited IL-6 285
gene induction but had no effect on TNFα gene induction. Pretreatment with the H89 inhibitor 286
led to a significant and more substantial reduction of IL-6 expression in response to soluble LT 287
and the combination of LT/ΔLT OMVs but showed no effect on IL-6 gene induction in response 288
to LT+ OMVs (Fig. 2B). However, whereas H89 showed similar effects as Rp-camps in TNFα 289
induction for most treatments, it led to a significant inhibition of TNFα expression in response to 290
LT+ OMVs (Fig 2A). In sum, it was notable that differences in the presentation of similar 291
concentrations of LT (LT+ OMVs compared with soluble LT) resulted in differences in the 292
kinetics of activation of PKA. 293
To examine the activation of CREB, immunoblots of nuclear extracts were performed to 294
determine the level of pCREB present in cells incubated with our samples and the blots 295
quantitatively analyzed by densitometry. Immunoblots showed that at 4 h, CREB was 296
phosphorylated in response to soluble LT and the combination of LT/ΔLT OMVs but not 297
phosphorylated in response to LT+ OMVs (Fig. 2C, upper panels). However, when assayed 2 h 298
later, CREB was phosphorylated in response to LT+ OMVs as well as soluble LT and the 299
combination of LT/ΔLT OMVs (Fig. 2C, lower panels). Densitometry analysis (Fig. 2D) showed 300
that pCREB was significantly increased (approximately 2-fold) in response to LT and LT/ΔLT 301
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OMVs compared to the other treatments at 4 h and increased to approximately 3.75-fold at 6 h. 302
LT+ OMV treatment led to a substantially higher CREB phosphorylation than mock at 6 h, 303
although this increase was not statistically significant. At both timepoints, both soluble LT and 304
the combination of LT/ΔLT OMVs induced significantly higher levels of pCREB than LT+ 305
OMVs. Therefore, treatment with LT in its native OMV-associated state appeared to activate 306
CREB more slowly compared with treatments containing soluble LT. 307
We next used an independent assay to further examine the differences in the kinetics of 308
cAMP-dependent activation. We compared the effects of soluble LT and OMV-bound LT on the 309
activation of CRE genetic elements using a dual luciferase reporter system. The CRE element is 310
activated in response to cAMP induction, therefore this assay provides a more sensitive detection 311
of the downstream effects of cAMP production. Because we found it technically unfeasible to 312
transfect T84 cells and maintain their ability to form a polarized monolayer, we used HEK293T 313
cells for these assays. HEK293T cells were transfected with a reporter plasmid containing the 314
CRE promoter fused to firefly luciferase as a reporter. To control for transfection efficiency, 315
cells were co-transfected with a plasmid that constitutively expressed Renilla luciferase. 316
The CRE reporter results were consistent with the kinetics of CREB activation (Fig 2E). 317
Although LT+ OMVs stimulated CRE activity approximately 5-fold at 4 h, this induction was not 318
significantly above mock-treated cells (p > 0.05). In comparison, after only 4 h, both soluble LT 319
and the combination of LT/ΔLT OMVs significantly induced CRE activity above mock 320
(approximately 15- and 22-fold higher than mock, respectively), and the response to the 321
combination of LT/ΔLT OMVs levels was significantly higher than that for the same amount of 322
soluble LT (p < 0.01). At 6 h, CRE activity was significantly induced by all of the treatments 323
containing active LT: soluble LT, LT+ OMVs, and the combination of LT/ΔLT OMVs 324
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(approximately 35-, 19-, and 28-fold, respectively). These results confirm that the LT present in 325
native LT+ OMVs causes the phosphorylation of CREB and activation of CRE, but that this is 326
delayed. However, as PKA pathway inhibition did not decrease LT+ OMV-mediated induction of 327
IL-6 and TNFα gene expression (Fig 2A and B), it should be noted that the activation of CREB 328
cannot account for the increased expression levels of these genes that is induced by LT+ OMVs. 329
The induction of TNFα, not IL-6, depends on ERK1/2 and p38 MAP kinases 330
We next investigated pathways other than via CREB activation through which LT+ OMVs 331
could induce the expression of TNFα and IL-6. MAP kinases, such as p38, ERK1/2 and JNK, 332
have long been known to play a role in the induction of cytokines in response to stimuli (2, 15, 333
19). Specific inhibitors of MEK1/2 and p38 were used to determine whether ERK1/2 and p38, 334
respectively, played a role in the induction of TNFα and IL-6 by LT and OMVs. 335
We found that p38 and ERK1/2 had similar roles in the induction of TNFα and that neither 336
played a role in IL-6 induction. MEK inhibition significantly reduced the induction of TNFα by 337
soluble LT, LT+ OMVs, and substantially reduced its induction by the combination of LT/ΔLT 338
OMVs (Fig. 3A). p38 inhibition also significantly reduced the induction of TNFα by soluble LT 339
and LT+ OMVs but, notably, did not affect the response to the combination of LT/ΔLT OMVs 340
(Fig. 3B). Neither inhibitor significantly reduced the induction of IL-6 by soluble LT, LT+ 341
OMVs, or ΔLT OMVs (Fig. 3C and 3D). Interestingly, p38 and ERK1/2 seemed to play, if 342
anything, opposite roles in the induction of IL-6 in response to the combination of LT/ΔLT 343
OMVs. Inhibition of ERK1/2 resulted in a significant decrease in IL-6, whereas p38 inhibition 344
significantly upregulated IL-6 expression (Fig 3C and D). Together, these data demonstrate that 345
both ERK1/2 and p38 play a role in the induction of TNFα in response to both LT and LT+ 346
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OMVs, but the induction of IL-6 in response to these treatments is independent of these MAP 347
kinases. 348
AP-1 is involved in the induction of TNFα and IL-6 in response to soluble LT but not LT+ 349
OMVs 350
We also determined the role of the JNK MAPK pathway in the induction of TNFα and IL-6 351
in response to soluble LT and LT+ OMVs. JNK pathway inhibition led to a significant decrease 352
in the level of expression of TNFα in response to soluble LT, but it did not significantly inhibit 353
TNFα induction for any of the other treatments (Fig. 4A). JNK pathway inhibition also led to a 354
significant decrease in the level of expression of IL-6 in response to soluble LT (Fig. 4B). But, in 355
contrast to TNFα, IL-6 expression levels induced in response to ΔLT OMVs and the combination 356
of LT/ΔLT OMVs were significantly reduced with inhibition of the JNK pathway. In addition, 357
we noted that JNK inhibition in cells treated with LT+ OMVs and the combination of LT/ΔLT 358
OMVs resulted in similar IL-6 levels (Fig. 54B), suggesting that JNK inhibition removes the 359
contribution of soluble LT to the induction of IL-6 by LT/ΔLT OMVs. It is not clear why the 360
same effect did not occur for TNFα induction by LT/ΔLT OMVs (Fig. 4A). Together, these 361
results show that the JNK pathway plays no role in LT+ OMV induction of either TNFα or IL-6 362
at 6 h and that this contrasts with the significant role JNK plays in their induction by soluble LT. 363
To confirm that soluble LT activates the JNK pathway, we performed an electrophoretic 364
mobility shift assay to determine the activation status of AP-1, a downstream effector of JNK. 365
Our results showed that AP-1 was induced in response to both soluble LT and the combination of 366
LT/ΔLT OMVs at 6 h (Fig. 4C), which is consistent with our RT-PCR results. Taken together, 367
our results show that AP-1 plays a role in the induction of IL-6 in response to soluble LT but not 368
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OMV-associated LT, providing further evidence that soluble LT and LT+ OMVs act through 369
different mechanisms to induce IL-6 gene expression. 370
LT and OMV treatments signal differentially through NFκB . 371
To examine the role of NFκB in the induction of TNFα and IL-6 by LT treatments, we 372
inhibited the activation of NFκB using PDTC, which prevents the binding of NFκB to DNA. 373
NFκB inhibition resulted in the downregulation of TNFα for all samples that contained 374
catalytically active LT (Fig. 5A). However, PDTC differentially affected IL-6 induction. NFκB 375
inhibition significantly decreased IL-6 induction by LT+ OMVs, ΔLT OMVs and S63K OMVs 376
to basal levels but NFκB inhibition did not reduce IL-6 gene induction in response to soluble LT 377
(Fig. 5B). Furthermore, we noted a PDTC-dependent decrease in the levels of IL-6 induced by 378
the combination of soluble LT/ΔLT OMVs to a level comparable to that of soluble LT. Thus, 379
NFκB inhibition appeared to act on the OMV contribution towards IL-6 induction but had no 380
effect on the contribution of soluble LT. These results further support our findings that soluble 381
LT and a comparable concentration of OMV-bound LT induce IL-6 through different pathways. 382
Discussion 383
Because ETEC is an important agent of disease worldwide, studies to elucidate the 384
mechanisms of its virulence factors, including LT, are important. Most studies on LT have been 385
performed on its soluble form in many different cell lines. However, recent studies have 386
demonstrated that LT has the propensity to bind LPS and thus become associated with secreted 387
OMVs (17, 18, 32). In addition, LT acts in the gut lumen, where tight barriers generated by the 388
epithelium allow only apical exposure of cells to toxin. Therefore, in this study, we determined 389
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the response of polarized intestinal epithelial cells to LT presented in soluble and insoluble 390
contexts. 391
We found significant differences in the mechanisms and kinetics of TNFα and IL-6 gene 392
induction elicited by soluble LT and LT+ OMVs. As summarized in Fig. 6, soluble LT elicited 393
IL-6 through two pathways, PKA and JNK. At early time points, by contrast, LT in the context 394
of LT+ OMVs only induced IL-6 through an independent pathway involving NFκB. Although 395
both OMV-bound and soluble LT elicited TNFα through some shared pathways, the PKA and 396
JNK pathways were unique to soluble LT. At later times, both LT and LT+ OMVs acted through 397
the PKA pathway (data not shown). 398
Whereas catalytically active LT-containing treatments (soluble LT, LT+ OMVs, and a 399
combination of LT/ΔLT OMVs) elicited substantially higher levels of TNFα and IL-6 expression 400
at 6 h compared with non-catalytically active treatments, this higher induction did not depend on 401
the activation of CREB. This result was unexpected. LT acts through the ADP-ribosylation of the 402
Gsα subunit of adenylate cyclase, which results in an increase in cAMP levels. cAMP then 403
activates PKA, which leads to Cl- efflux and the phosphorylation of CREB. As shown in Fig. 2, 404
LT+ OMVs showed delayed kinetics of CREB phosphorylation and CRE gene activity compared 405
to soluble LT and the combination of LT/ΔLT OMVs. Our data suggest that delayed kinetics 406
rather than decreased induction occurs for OMV-associated LT activation because at 9 h, LT+ 407
OMVs elicited significantly higher levels of IL-6 than soluble LT (p<0.001), and this increase 408
was significantly inhibited by Rp-camps (data not shown). Because PKA activation is also 409
responsible for phosphorylating the CFTR, resulting in Cl- and water efflux into the intestinal 410
lumen, our results also suggest that the onset of diarrhea may also be delayed in response to LT+ 411
OMVs compared to soluble toxin. 412
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There are many reasons why LT in its native OMV presentation might elicit different 413
responses than soluble LT. Multiple LT molecules are complexed with an OMV, thus fewer host 414
cells may become intoxicated by OMV-bound LT than soluble LT. By containing multiple LT 415
molecules, a few LT+ OMVs could elicit maximal CREB activation, whereas soluble LT would 416
elicit a more gradual, proportional response depending on how many individual LT molecules 417
were encountered by each cell. In addition, OMV internalization and trafficking could occur via 418
a different pathway than soluble LT, as supported by preliminary experiments (data not shown), 419
leading to differences in the efficiency of the intracellular processing of LT. The delayed kinetics 420
of activation could also suggest the inaccessibility of the toxin bound to OMVs. Removal of the 421
LT bound to LPS on the OMVs may be necessary to allow LT to progress through the canonical 422
retrograde trafficking pathway and may be inefficient inside the host cell. Further studies are 423
ongoing to elucidate the mechanistic basis for the observed differences. 424
H89, which inhibits CREB phosphorylation by PKA at low concentrations, was used as an 425
independent method to demonstrate the role of CREB in the induction of IL-6 and TNFα. H89 426
showed similar results as Rp-camps, which inhibits PKA activity, except that it led to a 427
significant decrease in TNFα induction in response to LT+ OMVs. However, this decrease may 428
not be dependent on CREB. In addition to its role as a CREB inhibitor, H89 has also been shown 429
to play a role in the inhibition of mitogen stress kinase 1 (MSK1) (16, 48), which phosphorylates 430
NFκB to induce NFκB-mediated gene transcription (48). To test whether this effect was relevant, 431
we tested but did not find any indication of MSK1 phosphorylation or NFκB phosphorylation in 432
any of our samples using immunoblotting (data not shown). This suggests that MSK1 does not 433
play a role in the induction of TNFα by LT+ OMVs. H89 has also been shown to inhibit protein 434
kinase D (PKD), which plays a role in Golgi-to-ER transport (27, 36). After binding to the host 435
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receptor ganglioside GM1, LT is internalized and transported to the Golgi and the ER, in which 436
the catalytic subunit is processed. H89 may interfere with this activation by inhibiting the 437
translocation of cargo from the Golgi to the ER, and this inhibition may also play a role in 438
inhibiting the TNFα response to LT+ OMVs. This possibility warrants further investigation. 439
This is the first study to show a role for AP-1 in the host response to LT. We discovered AP-440
1 only played a role in the induction of IL-6 by soluble LT and did not affect OMV responses, 441
even at later times. In addition, AP-1 was not activated in response to LT+ OMVs, even at later 442
time points. No previous studies have shown activation of AP-1 by LT, although IL-6 induction 443
in response to increased cAMP levels has been shown to be mediated, at least in part, by AP-1 444
(8). Dendorfer et al. also showed that cAMP induced IL-6 through mechanisms other than LPS, 445
and the IL-6 response to LPS was completely abrogated by mutations in NFκB-binding sites (8). 446
We found significant differences not only between soluble LT and LT+ OMVs but also among 447
overall OMV responses. A significant inhibition of IL-6 responses in response to NFκB 448
inhibition was only observed for treatments containing OMVs. In fact, in the combination of 449
LT/ΔLT OMVs, NFκB inhibition appeared to remove the OMV contribution to IL-6, resulting in 450
an IL-6 level similar to that induced by soluble LT alone. These results suggest an OMV 451
component is responsible for activating NFκB to induce IL-6. Although NFκB is an important 452
contributor to IL-6 induction, the IL-6 promoter contains multiple regulatory elements, including 453
binding sites for AP-1, NFκB, and NF-IL-6 (28). Additionally, previous studies have shown that 454
NFκB is not required for IL-6 induction and that this induction can be mediated by MAPKs (25, 455
35). Taken together, our results emphasize the different mechanisms through which soluble LT 456
and OMV treatments induce TNFα and IL-6 responses. 457
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OMVs were standardized according to lipid content, in consideration of the fact that LPS 458
would probably cause a dominant cytokine response. As a consequence of different protein:lipid 459
ratios in OMVs, however, this normalization process resulted in the protein concentrations of 460
LT+ OMVs and S63K OMVs being more than twice as high as the amount of protein in ΔLT 461
OMVs. This could have caused the differences in the levels of TNFα and IL-6 induction. 462
However, we propose that differences in OMV protein concentration were not a major factor in 463
the observed responses. First, despite similar levels of protein in LT+ OMVs and S63K OMVs, 464
the induction of cytokines in response to LT+ OMVs was significantly higher than S63K OMVs. 465
Second, S63K OMVs, with over two-fold higher protein levels, produced similar levels of TNFα 466
and IL-6 as ΔLT OMVs. Third, we used samples standardized to two concentrations of LT, 1 nM 467
and 200 pM. Although the amount of protein in the ΔLT OMVs at the 1 nM treatment was twice 468
that of LT+ OMVs at 200 pM, LT+ OMVs induced significantly more IL-6 than ΔLT OMVs. 469
Our results suggest that the response to the combination of LT/ΔLT OMVs is not merely 470
additive. IL-6 and TNFα responses induced by the combination of LT/ΔLT OMVs were not 471
equivalent to the combined responses of soluble LT and ΔOMVs. These results may be due to 472
the fact that the combination of LT/ΔLT OMVs actually consists of three distinct populations: 473
soluble LT, ΔLT OMVs, and LT bound to the surface of ΔLT OMVs. Although LT may bind to 474
the surface of ΔLT OMVs, this is not the native presentation because native LT+ OMVs also 475
contain LT in the lumen of the vesicle. The proportion of these populations, and therefore their 476
individual contribution, is difficult to determine. Nevertheless, it was valuable to use this 477
treatment as it allowed us to determine that the context of toxin presentation (i.e., in both the 478
lumen and on the surface of OMVs) was important in eliciting TNFα and IL-6 responses, which 479
were not merely a result of the presence of equivalent amounts of soluble LT and ΔLT OMVs. 480
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IL-6 and TNFα are pro-inflammatory cytokines that mediate acute inflammation. In addition 481
to their role as pro-inflammatory mediators, IL-6 and TNFα may also play protective roles in the 482
intestine, such as tissue repair after injury and protection from apoptosis (38, 47). Because ETEC 483
does not cause disease in mice, mouse models to study the in vivo effects of ETEC are not 484
available. In addition, little data have been published on the cytokine response of patients with 485
diarrhea caused by ETEC. However, other enteric pathogens have been shown to elicit TNFα 486
and IL-6 responses. Using in vivo studies, Dann et al. showed that IL-6 was produced in mouse 487
intestines in response to Citrobacter rodentium and that this induction was important in 488
preventing infection-induced apoptosis in the colonic epithelium (6). In clinical settings, both IL-489
6 and TNFα have been found in the stools of children who had diarrhea that was caused by 490
another enteric pathogen, Shigella dysenteriae (7). In addition, in children with enterocolitis, 491
serum IL-6 has been shown to be discriminative of bacterial etiology from viral etiology (51). 492
Although the mechanisms of IL-6 and TNFα induction in these studies have not been elucidated, 493
these reports show the relevance of IL-6 and TNFα induction by enteric pathogens. 494
In summary, we show that OMV-bound LT is not as effective at inducing CREB activation 495
at early times as soluble LT, however, LT+ OMVs can eventually induce similar amounts of 496
cytokine gene expression. These differences in CREB activation may be due to different 497
trafficking mechanisms within the cell. Whereas previous studies to determine the effects of LT 498
and vaccination strategies for ETEC have focused on soluble LT, our study emphasizes the 499
importance of studying virulence factors in their native context. 500
501
502
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Acknowledgments 503
The authors thank Dr. James Fleckenstein for strain H10407ΔeltA; Stefanie Hartman for 504
assistance with EMSAs; Dr. Ben Mudrak for cloning help and critical reading of the manuscript; 505
Dr. Patrick Seed for providing the anti-H7 antibody; and Dr. Hiroaki Matsunami, Kyla Selvig, 506
and Dan Rodriguez for piloting the CRE-Luciferase assay. This project was supported by grant 507
R01AI064464 from the National Institutes of Health. 508
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Figure Legends 650
Figure 1. Catalytically active LT-containing treatments elicit high levels of TNFα and IL-6 651
expression in polarized intestinal epithelial cells and induction is independent of flagellin. (A-B) 652
Polarized T84 cells were treated with 200 pM or 1 nM standardized amounts (see Methods) of 653
LT, LT+ OMVs, ΔLT OMVs, LT/ΔLT OMVs, S63K LT or S63K OMVs as indicated, and the 654
gene expression levels of (A) TNFα and (B) IL-6 were measured at 6 h using RT-PCR. (C) 655
Polarized T84 cells were incubated with samples standardized to 1 nM LT for 10 h, and IL-6 656
levels were measured in supernatants collected from the apical compartment using ELISA. (D) 657
Flagellin was detected in preparations of LT+ OMVs, ΔLT OMVs, LT/ΔLT OMVs and S63K 658
OMVs by immunoblotting with anti-FliC. The preparations were concentrated to allow for 659
detection of flagellin, but the concentrations remained proportional to those used in the 660
treatments described above. 661
662
Figure 2. TNFα and IL-6 induction in response to LT+ OMVs is independent of CREB 663
phosphorylation. (A-B) Polarized T84 cells were pretreated with 100 µM Rp-camps or 10 µM 664
H89 for 1 h and incubated with samples standardized to 200 pM LT. After 6 h, the levels of (A) 665
TNFα and (B) IL-6 (B) gene induction were evaluated using RT-PCR. Asterisks indicate 666
significant differences from the respective untreated sample. *p<0.05, **p<0.01, ***p<0.005. 667
(C) Nuclear extracts were prepared from polarized T84 cells incubated with samples 668
standardized to 1 nM LT for 4 h (upper panel) or 6 h (lower panel), and immunoblots were 669
performed to determine the phosphorylation status of CREB. TBP was used as a loading control. 670
The image shown is representative of three independent experiments. (D) Densitometry analysis 671
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of three independent immunoblots for each time point. Asterisks indicate significant differences 672
from the respective mock at the corresponding time. *p<0.05, **p<0.01, ***p<0.005. (E) 673
HEK293T cells were co-transfected with a firefly luciferase reporter fused to a CRE promoter 674
and a Renilla luciferase reporter fused to a constitutively active promoter. Transfected cells were 675
then incubated with samples standardized to 1 nM LT for 4 h and 6 h, and the firefly luciferase 676
activity was measured and normalized to Renilla luciferase values. Asterisks indicate significant 677
differences from the respective mock at the corresponding time. *p<0.05, **p<0.01, 678
***p<0.005. 679
680
Figure 3. MEK1/2 and p38 play similar roles in the induction of TNFα in response to both 681
soluble LT and LT+ OMVs but do not play a role in IL-6 induction. Polarized T84 cells were 682
pre-treated with for 1 h with inhibitors of MEK1/2 (10 µM PD98059) or p38 (10 µM SB203580) 683
before being incubated with samples standardized to 200 pM LT for 6 h. (A-B) TNFα levels 684
were measured using RT-PCR after pretreatment with inhibitors of (A) MEK1/2 and (B) p38. 685
(C-D) IL-6 levels were measured using RT-PCR after pretreatment with inhibitors of (C) 686
MEK1/2 and (D) p38. Asterisks indicate significant differences from the corresponding untreated 687
sample. *p<0.05, **p<0.01. 688
689
Figure 4. AP-1 plays a role in the induction of TNFα and IL-6 in response to soluble LT but not 690
LT+ OMVs. (A-B) Polarized T84 cells were pre-treated for 1 with a JNK inhibitor (10 µM 691
SP600125) followed by incubation with samples standardized to 200 pM LT for 6 h. The levels 692
of (A) TNFα and (B) IL-6 were measured using RT-PCR. Asterisks indicate significant 693
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differences from the corresponding untreated sample. *p<0.05, **p<0.01, ***p<0.005. (C) 694
Polarized T84 cells were incubated with samples standardized to 1 nM LT for 6 h, and nuclear 695
extracts were prepared. Nuclear protein (5 µg) was incubated with a radiolabelled 696
oligonucleotide that corresponded to the DNA-binding region of activated AP-1, and an EMSA 697
was performed to visualize binding. Densitometric values of the shifted DNA band are shown. 698
699
Figure 5. OMVs but not soluble LT induces IL-6 through NFκB. Polarized T84 cells were pre-700
treated for 30 min with an NFκB inhibitor (100 µM PDTC) followed by incubation with samples 701
standardized to 200 pM LT for 6 h. The levels of (A) TNFα and (B) IL-6 were measured using 702
RT-PCR. Asterisks indicate significant differences from the corresponding untreated sample. 703
*p<0.05, **p<0.01, ***p<0.005. 704
705
Figure 6. Overview of the different pathways through which soluble LT and LT+ OMVs elicit 706
TNFα and IL-6 responses in human intestinal epithelial cells. See discussion for details. 707
708
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Table 1. Strains used in this study. 709
Strain Description Relevant characteristics Reference
jf570 H10407 ΔeltA ETEC strain with polar insertion in
eltA (LT deficient)
(9)
MK1052 H10407 ΔeltA/pILT jf570 carrying inducible LT plasmid;
Ampr
(32)
MK1226 H10407 ΔeltA/pILT[S63K] jf570 carrying inducible S63K
mutant; Ampr
This work
MK741 DH5α degP::Tn5/pDsbA/pILT E. coli K12, degP knockout, carrying
a plasmid copy of dsbA and an
inducible LT plasmid; Kanr Cmr
Ampr
(32)
MK1250 DH5α
degP::Tn5/pDsbA/pILT[S63K]
E. coli K12, degP knockout, carrying
a plasmid copy of dsbA and an
inducible S63K plasmid; Kanr Cmr
Ampr
This work
710
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