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Enterotoxigenic Escherichia coli heat-labile toxin drives enteropathic changes in small intestinal epithelia

Enterotoxigenic E. coli (ETEC) produce heat-labile (LT) and/or heat-stable (ST) enterotoxins, and commonly cause diarrhea in resource-poor regions. ETEC have been linked repeatedly to sequelae in children including enteropathy, malnutrition, and growth impairment. Although cellular actions of ETEC enterotoxins leading to diarrhea are well-established, their contributions to sequelae remain unclear. LT increases cellular cAMP to activate protein kinase A (PKA) that phosphorylates ion channels driving intestinal export of salt and water resulting in diarrhea. As PKA also modulates transcription of many genes, we interrogated transcriptional profiles of LT-treated intestinal epithelia. Here we show that LT significantly alters intestinal epithelial gene expression directing biogenesis of the brush border, the major site for nutrient absorption, suppresses transcription factors HNF4 and SMAD4 critical to enterocyte differentiation, and profoundly disrupts microvillus architecture and essential nutrient transport. In addition, ETEC-challenged neonatal mice exhibit substantial brush border derangement that is prevented by maternal vaccination with LT. Finally, mice repeatedly challenged with toxigenic ETEC exhibit impaired growth recapitulating the multiplicative impact of recurring ETEC infections in children. These findings highlight impacts of ETEC enterotoxins beyond acute diarrheal illness and may inform approaches to prevent major sequelae of these common infections including malnutrition that impact millions of children.

Infectious diarrhea remains a leading cause of death and morbidity among young children in low-middle-income countries where access to clean water and sanitation remains in short supply 1 . Enterotoxigenic E. coli (ETEC), initially discovered as a cause of severe, cholera-like illness 2 , are one of the most common pathogens associated with moderate-severe diarrhea among children under the age of 5 years 3,4 , and are perennially the most common cause of diarrhea in travelers 5 to endemic regions where these organisms are thought to account for hundreds of millions of cases of diarrheal illness each year 6 .
Importantly, ETEC infections have been linked to non-diarrheal sequelae, including "environmental enteric dysfunction (EED)," a condition characterized by impaired nutrient absorption, impaired growth 7,8 , and malnutrition 9,10 , adding significantly to the morbidity as well as deaths from diarrhea and other infections 11 . The risk of stunting multiplies with each episode of diarrheal illness in children under the age of two years 12 , a period during which children residing in impoverished areas commonly sustain multiple ETEC infections 8 . However, the molecular pathogenesis underlying the intestinal changes associated with EED, and the contribution of individual pathogens, including ETEC, remain poorly understood.
Similarly, toxin-producing E. coli have also been repeatedly identified in patients with tropical sprue [13][14][15] , a condition classically described in adults residing for extended periods of time in areas where ETEC diarrheal disease is common. Like EED, tropical sprue is associated with changes to the small intestinal villous architecture, including ultrastructural alteration of the epithelial brush border formed by the microvilli 16,17 , nutrient malabsorption, and wasting.
The basic molecular mechanisms underpinning acute watery diarrhea caused by ETEC are well-established 18 . ETEC produces heatlabile (LT) and/or heat-stable (ST) enterotoxins that activate the production of cAMP and cGMP second messengers, respectively, leading to activation of cellular kinases that in turn modulate the activity of sodium and chloride channels in the apical membrane of intestinal epithelial cells to promote net efflux of salt and water into the intestinal lumen resulting in watery diarrhea.
LT and cholera toxin (CT) share~85% amino acid identity, and both toxins exert their major effects on the cell through the ADPribosylation of the alpha subunit of Gs (Gsα), a stimulatory intracellular guanine nucleotide-binding protein. Inhibition of Gsα GTPase activity leads to constitutive activation of adenylate cyclase and increased production of intracellular cAMP 19 .
In its central role as a second messenger, cAMP governs a diverse array of cellular processes 20 and modulates the transcription of multiple genes through a number of cAMP-responsive transcriptional activators and repressors 21 . cAMP activates protein kinase A (PKA), a heterotetramer, by liberating its two regulatory subunits from the catalytic subunits, which are then free to phosphorylate a wide variety of cytoplasmic and nuclear protein substrates 22 . PKA largely regulates transcription by phosphorylation of transcription factors, including the cyclic AMP response element binding protein (CREB) and the cAMP-response element modulator (CREM), which bind cAMPresponse elements (CRE) in the promoter regions of target genes [21][22][23] .
Notably, cholera toxin (CT), LT, and dibutyryl-cyclic AMP all induce hypersecretion and impact the architecture of gastrointestinal epithelia in rodent small intestine 24 , while small intestinal biopsies of patients with acute cholera exhibit marked changes in the ultrastructure of the intestinal brush border, the major absorptive surface in the small intestine 25,26 , including shortening and disruption of the microvilli. Consistent with these observations, studies of young children less than two years of age in Bangladesh have specifically asso-ciated LT-producing ETEC with undernutrition 27 , suggesting that heatlabile toxin may exert effects on intestinal mucosa that extend beyond acute diarrheal illness.
Here we demonstrate that in addition to the canonical effects of LT on the cellular export of salt and water into the intestinal lumen, this toxin impacts multiple genes involved in the formation of microvilli, resulting in marked alteration of the architecture of the intestinal brush border, the major site of nutrient absorption in the small intestine. These effects are compounded by the alteration of solute transporters within the brush border epithelia, potentially disrupting the absorption of multiple essential nutrients.

Heat-labile toxin markedly alters the transcriptomes of intestinal epithelial cells
Commensurate with the importance of cAMP as a second messenger, we found that compared to either untreated controls or cells treated with a catalytically inactive (E211K) mutant of LT, wild-type LT holotoxin substantially modulated transcription of many genes in intestinal epithelial cells. In RNA-seq studies of polarized Caco-2 intestinal epithelial cells, we found that 3832 genes were significantly (p ≤ 10 −5 ) upregulated and 3687 downregulated in response to LT, while the inactive toxin failed to induce significant changes in the transcriptome (Fig. 1a). However, Caco-2 cells are derived from distant metastases of a colon cancer tumor in which transcriptomes would likely be altered relative to untransformed intestinal epithelia 28,29 , and cAMP signaling is known to be aberrant in some transformed cells 23 . Therefore, to examine a more physiologically relevant target, we next examined the impact of LT on differentiated small intestinal enteroids. Here, we found that far fewer genes were differentially expressed (≤10 −5 ), with 746 significantly upregulated, and 561 downregulated in response to intoxication with LT (Fig. 1b). Notably, however, we found substantial statistically significant overlap in genes significantly modulated in Caco-2 cells and enteroids (Supplementary Table 3   Model at left depicts the E. coli heat-labile toxin 92 based on PDB structure 1LTS with the A1 subunit in blue, the A2 region in yellow, and pentameric B subunit in green. The E211K mutation of mLT is in the active site of the A1 subunit. a Scatterplot of RNAseq data right depicts differential expression profiles of Caco-2 cells following exposure to a heat-labile toxin (n = 2) relative to untreated cells (n = 2) and cells treated with the biologically inactive mLT (n = 2). (Because expression profiles of untreated and mLT-treated cells were virtually identical, their combined expression profiles totaling n = 4 replicates are compared here to LTtreated cells). b RNA-seq data from polarized small intestinal ileal enteroids treated with LT (n = 3) compared to control untreated (n = 3) cells. Differentially expressed genes were identified by DESeq2 93 .
as well as ontology-independent investigation of genes (CompBio, Supplementary Fig. 1) modulated by the toxin, highlighted multiple cellular components associated with both the development and function of the absorptive surface of the small intestine (Supplementary Dataset 2).

Heat-labile toxin impairs the development of small intestinal microvilli
Small intestinal enterocytes are each covered with hundreds of microvilli, complex structures comprised of a central core of actin filaments within protrusions of the plasma membrane. Collectively, the luminal surface of the intestine formed by these microvilli, known as the brush border, represents the major absorptive surface of the gastrointestinal tract. Three major classes of proteins are required for the biogenesis of microvilli 30 (Fig. 2a). These include (1) proteins such as villin, epsin, plastin, and EPS8 that bundle parallel clusters of actin filaments; BAIP2L1 (IRTKS) responsible for recruiting the EPS bundling protein to the tips of microvilli 31 ; (2) ezrin, myo1a 32 , myo6 that link the actin cytoskeleton with the plasma membrane; and (3) protocadherin molecules CDHR2 and CDHR5 engaged in extracellular heterotypic complexes between the tips of the microvilli 30 that are stabilized by a tripartite complex of MYO7B, ANKS4B 33 , and USH1C 33 . Interrogation of transcriptional profiles indicated that the transcription of each of these classes of genes was significantly altered following exposure to wild-type heat-labile toxin (Fig. 2b). Similarly, RT-PCR confirmed decreased expression of multiple genes involved in microvilli biogenesis, including VIL1 encoding villin (Fig. 3a), and we were able to demonstrate that production of villin was depressed in polarized small intestinal enteroids ( Fig. 3b and Supplementary Fig. 2). In addition, TEM images of polarized small intestinal enteroids exposed to heat-labile toxin demonstrated significantly shortened and disorganized microvillus structures on the apical surface of enterocytes (Fig. 3c).
Heat-labile toxin modulates the transcription of multiple brush border nutrient transport genes The human solute carrier (SLC) gene superfamily is comprised of more than 50 gene families thought to encode more than 300 functional transporters 34 . Many of the SLC proteins are enriched in the small intestinal brush border, where they transport critical nutrients, including amino acids, oligopeptides, sugars, and vitamins. We found that transcription of many SLC genes was altered in Caco-2 cells as well as small intestinal enteroids (Fig. 4a). These included transporters for Zinc, known both to be deficient in children with enteropathy 35 , and a micronutrient critical for intestinal homeostasis. Likewise, transcription of SLC19A3 encoding the principal SLC responsible for uptake of the water-soluble B vitamin thiamine (vitamin B1) 36 by differentiated intestinal epithelial cells lining the surface of intestinal villi of the proximal small intestine 37,38 (Fig. 4b) was repressed as was the production of the corresponding protein (supplementary figure 3a). Moreover, we found that LT treatment of human small intestinal organoids also interfered with the transcription of the cis-regulatory element specificity protein 1 (SP1) previously shown to govern the transcription of SLC19A3 39-41 ( Fig. 4c and Supplementary Fig. 3b). Finally, we found that thiamine transport was significantly depressed following exposure to LT (Fig. 4d) providing additional evidence that ETEC can impair transport of critical nutrients.  LT-producing ETEC disrupts the absorptive architecture of the small intestine To further study the potential impact of ETEC toxins on intestinal architecture, we performed challenge studies in infant mice. Compared to sham-challenged (PBS) controls, or mice challenged with a toxin-deficient strain of ETEC, we again noted down-regulation of genes involved in F-actin bundling, membrane cross-linking, and intermicrovillus adhesion complex formation, all required for intestinal microvilli (Fig. 5a) biogenesis. Likewise, on examination of small intestinal villi we found that the production of villin in enterocyte brush borders was substantially decreased relative to sham-challenged controls (Fig. 5b, c). In mice challenged with a wild-type ETEC isolate that makes ST and LT, but not an LT/ST-toxinnegative mutant (jf4763, Supplementary Table 1), we observed significant alteration in the architecture of the intestinal brush border with significant shortening and disorganization of the microvilli (Fig. 5d, e and Supplementary Fig. 4a-d) reminiscent of the earlier ultrastructural studies of patients with tropical sprue 17 and V. cholerae infections 25 . Similarly, we found that in mice challenged with a strain containing an isogenic mutation in eltA (jf571, Supplementary Table 1) encoding the LT A subunit, which still makes heat-stable toxins, the microvillus architecture was preserved ( Fig. 5f, g), suggesting that LT is the principal toxin underlying the enteropathic changes to the enterocyte surfaces.
Despite the dramatic toxin-dependent changes to the absorptive surface of the intestine, the early growth kinetics of suckling mice challenged a single time with either wild-type bacteria or a heat-labile toxin deletion mutant were surprisingly similar ( Supplementary  Fig. 5A) and paralleled those of sham-challenged controls. However, enteropathy in young children is thought to reflect damage elicited by repeated infections. Children in endemic regions typically suffer multiple ETEC infections before their second birthday, and the risk of enteropathic sequelae increases multiplicatively per episode 8,12 . Therefore, to assess the contribution of repeated ETEC infections to growth impairment, we compared the growth kinetics of suckling mice challenged a single time to those repeatedly infected with wild-type toxigenic ETEC. These studies demonstrated a clear impact of repeated infection on growth ( Supplementary Fig. 5B). Finally, we found that the growth kinetics of mice repeatedly challenged with wild-type ETEC H10407 was significantly retarded relative to those challenged with the isogenic LT-mutant jf876 ( Supplementary Fig. 5C). Therefore, repeated infections in this model appear to recapitulate impacts observed following repeated ETEC infection in children, and our data suggest that these features are at least in part driven by LT.

Maternal vaccination with LT prevents brush border disruption
To address whether enteropathic changes to the small intestine can be prevented by vaccination, and to further define the role of LT, we vaccinated mouse dams with heat-labile toxin and examined the brush border ultrastructure in suckling mice. Vaccinated dams but not sham vaccinated controls expressed significant levels of IgA and IgG in breast milk (Fig. 6a), consistent with increased levels of antibodies in the stomachs of infant mice (Fig. 6b). Notably, we found that maternal vaccination with LT completely abrogated changes to the microvilli ( Fig. 6c, d) further substantiating the importance of LT in driving changes to the epithelial architecture.

LT modulates key transcription factors that govern enterocyte development
Despite the marked alteration in transcription mediated by LT, the majority of genes critical for brush border development lacked conserved CRE sites 23 . Therefore, we performed transcription factor target enrichment analysis 42,43 to identify potential transcription factors responsible for differential regulation of genes significantly (P < 0.05) upregulated or downregulated by LT in both Caco-2 and enteroid RNA-seq datasets (Supplementary Table 4 and Supplementary Dataset 1). The upregulated genes were most significantly (p = 4.2 × −10 −3 ) linked to transcription factor targets of AP-1 encoded by the c-jun gene, previously shown to be regulated by cAMP 44 , and to be involved in intestinal epithelial repair 45 . Notably, however, downregulated genes were most significantly enriched in targets for the HNF4α transcription factor or its intestine-specific paralog HNF4γ (P = 1.9 × −10 −4 ) that were recently shown to regulate multiple genes required for brush border development 46,47 . Importantly, PKA has also been shown to phosphorylate HNF4 α at a consensus recognition site within the DNA binding domain, shared with HNF4 γ, inhibiting transcription 48 . Of note, the transcription of both paralogs was found to be significantly depressed following exposure of intestinal epithelia to LT (Fig. 7a, b), as were levels of HNF4γ in nuclear fractions from toxin-treated cells (Fig. 7c), suggesting that activation of cAMP can interfere with transcription mediated by HNF4. To further examine the impact of LT on HNF4-mediated transcription, we introduced a transcriptional reporter plasmid containing six tandem copies of the HNF4 transcriptional response element (5′-CAAAGGTCA-3′) linked to a human codon-optimized Gaussia princeps luciferase into Caco-2 cells. These assays demonstrated that HNF4-mediated transcription was dramatically reduced in cells treated with LT (Fig. 7d). Similarly, we found that relative to nuclei of intestinal epithelial cells in ileal segments from sham-challenged control mice, those from ETEC-challenged mice exhibited significantly less HNFγ (Fig. 7e) further supporting a role for ETEC in modulating the production of this important transcription factor. Chen et al described a "feed-forward regulatory module" essential to enterocyte differentiation in which HNF4 and SMAD4 transcription factors reciprocally activate each other's transcription. As would be predicted from this model, we found that transcription of SMAD4 was also impaired by LT ( Supplementary Fig. 6A, B), leading us to speculate that LT-mediated phosphorylation of HNF4 by PKA, interrupts this critical transcription module ( Supplementary  Fig. 6C-E).
Collectively, the current studies demonstrate that in addition to their known canonical effects on ion transport that culminate in watery diarrhea, ETEC toxins can drive appreciable derangement of enterocyte architecture and function by interfering with key pathways in intestinal epithelia that govern the formation of mature enterocytes capable of effective nutrient absorption. These findings have important implications for our understanding and prevention of enteropathic conditions linked to ETEC.

Discussion
Understanding the molecular events that lead to sequelae of undernutrition and growth faltering following ETEC infections may be key to the effective design of prevention strategies, including vaccines 49 . Although the molecular mechanisms involved in the fluid and ion fluxes into the intestinal lumen leading to diarrhea are firmly established, only recently has evidence emerged to suggest that ETEC toxins may incite previously unappreciated changes in small intestinal epithelia 50,51 . The present studies, initiated to identify additional effects of LT, were prompted by an appreciation that cyclic nucleotides, particularly cAMP, govern a multitude of cellular pathways, potentially resulting in collateral impacts that extend beyond the acute episodes of diarrhea. Consistent with this model, we found that exposure of intestinal cells to heat-labile toxin altered the transcription of hundreds of genes. A central theme highlighted in the analysis of these transcriptional alterations is that LT affects major classes of genes involved in the biogenesis of microvilli and the function of the intestinal brush border, the major site of nutrient uptake in the small intestine, potentially offering a direct molecular link to sequelae of malnutrition and impaired growth in children. The potential clinical relevance of the observations reported here is highlighted by remarkably similar ultrastructural alteration of intestinal epithelial cells seen in small intestinal biopsies of patients with acute cholera 25 and tropical sprue 17 . In both entities, the brush border was noted to be abnormal, with shortened, irregular microvilli. Importantly, however, despite the structural and functional similarity between LT and CT, clinical cholera, unlike ETEC infections, has not been linked to enteropathy or attendant sequelae. Whether this relates to the repetitive nature of ETEC infections compared to the durable protective immunity that follows a single V. cholerae infection 52 is not presently clear. Similarly, while tropical sprue remains a leading cause of malabsorption in regions where infectious diarrhea is prevalent [53][54][55] , the most debilitating forms of this illness have not typically followed isolated cases of traveler's diarrhea, but occur in resident populations or expatriates 56 repeatedly assailed by diarrhea while residing in endemic regions 57 . Likewise, our data also highlight the potential importance of repeated infections on the development of sequelae.
The negative impacts of LT on brush border architecture, with a commensurate reduction in surface area available for nutrient absorption, are compounded by the alteration of multiple SLC genes that encode transporters critical for the uptake of essential vitamins and other molecules. Importantly, small intestinal biopsies obtained from Zambian children with enteropathy and refractory stunting exhibited similar changes in SLC gene expression profiles 58 . The decreased transcription of molecules required for intestinal zinc uptake, in cells treated with LT is particularly intriguing given the known aberrations in zinc absorption in children with enteropathy 35,59,60 , the possible contribution of zinc deficiency to enteropathic changes 61 , and the salutary effects of zinc in the treatment of children with diarrhea 62,63 .
Further study will be needed to precisely delineate the role of ETEC LT and ST enterotoxins, alone and in combination, in driving enteropathic changes to the intestine and sequelae. ST-producing ETEC were most strongly associated with moderate to severe diarrhea in GEMS 3 , and follow-on studies of children enrolled in these studies have also linked infections with ETEC encoding heat-stable toxin to growth faltering 7 . However, our earlier analysis of a global collection of more than 1100 ETEC isolates, including those collected in GEMS, demonstrated that slightly more than half of all ST-encoding strains also encoded LT, and that roughly one-third of the isolates overall encoded LT alone, ST-LT, or ST only 64 . While LT-producing ETEC have been specifically linked to malnutrition among children in Bangladesh 27 , and our in vitro and animal studies point to the potential importance of LT, additional effort will be needed to correlate toxininduced morphologic and functional perturbation of the intestinal brush border with outcomes in children. Importantly, the long-term morbidity associated with ETEC infections does not appear to correlate with the severity of diarrhea as both mild illness 4 , and perhaps asymptomatic colonization may lead to growth faltering.
Further refinement of animal models that can faithfully recapitulate features of enteropathy are also needed. Indeed, conventional mice lack genes that could be required to reproduce the full effects of ETEC. For instance, each of the carcinoembryonic antigen cell adhesion molecules (CEACAMs) that are substantially upregulated on human small intestinal epithelia in response to LT, and which we have recently shown to play a critical role in ETEC interactions with human small intestine 50 , are absent in mice.
While the precise mechanism underlying LT-mediated modulation of genes required for microvillus biogenesis and absorptive function of the brush border is presently unclear, stimulation of adenylate cyclase invokes many cAMP-responsive nuclear factors that may serve either to activate or repress transcription. Genes implicated in the development of microvilli are mostly devoid of consensus palindromic (TGACGTCA) or "half" (CGTCA) cAMP-response element (CRE) sites within their promoter regions for direct modulation by CREB 65 , which typically is involved as a transcriptional activator, and both CREB and CREM can yield several alternatively spliced variants that may act as either activators or repressors 66 . cAMP second messaging also engages multiple signaling pathways converging at CREB 67 , and PKA can phosphorylate and modulate the activity of multiple transcription factors to act either as transcriptional activators or repressors, including SP1 68 . Notably, putative binding sites for HNF4, a cAMP-modulated transcription factor 48 known to regulate genes needed for the formation of microvilli 46,47 , were significantly enriched in the promotors of genes downregulated by LT. Both HNF4α and HNF4γ possess canonical PKA recognition sites within their DNA binding motifs, and PKA phosphorylation of these sites interrupts transcription 69 . HNF4 activates the transcription of SMAD4, and in turn, SMAD4 activates the transcription of HNF4 70 . Both transcription factors then engage genes needed for the effective differentiation of stem cells to mature enterocytes 70 . Modulation of the activity of this transcription factor by LT would therefore be predicted to have a marked impact on pathways critical to intestinal epithelial homeostasis.
We should also note that increases in cellular cAMP can impact multiple cellular pathways independent of PKA. Included among these are pathways governed by a more recently discovered family of cellular cAMP-binding molecules, exchange proteins activated by cAMP (EPACs). EPACs appear to play critical roles as guanine exchange factors that regulate GTPase proteins 71 and are involved in complex signaling networks implicated in cell growth, differentiation, and morphogenesis 72,73 .
cAMP can also exert potent epigenetic influences on transcription. CREB-binding protein (CBP) possesses intrinsic histone acetyltransferase (HAT) activity 74 , and can therefore modulate chromatin remodeling, enhancing access to transcription factors. In addition, cAMP messaging through PKA leads to phosphorylation-dependent activation of the histone demethylase enzyme PHF2 to promote the transcription of multiple genes that can impact the transition from stem cells to epithelial cells 75 .
Increased intestinal permeability is a recognized hallmark of enteropathy in young children in LMICs 76 . Given the known negative impacts of cholera toxin on epithelial barrier function 77 , and its structural and functional similarity to a heat-labile toxin, LT may exert additional enteropathic effects beyond the impact on brush border biogenesis described here.
Altogether it seems likely that multiple pathways governed by increases in cellular cAMP may underlie the morphologic and functional disruption of the brush border epithelial observed in our studies. Nevertheless, the data presented here provide compelling evidence that the heat-labile toxin of ETEC ultimately impacts multiple genes required for the biogenesis and function of the brush border, the major site of nutrient absorption in the human small intestine. The findings may have significant implications for our understanding of sequelae linked to ETEC, including environmental enteropathy in young children, and tropical sprue in adults. The increased acknowledgement of long-term morbidity linked to ETEC and an improved understanding of the role of ETEC enterotoxins as drivers of this morbidity may also strengthen the case for vaccines 49 specifically engineered to prevent both acute illness and sequelae.

Methods
The studies reported here comply with all relevant ethical regulations and the study protocols have been approved by the IACUC, Institutional Biosafety, and Institutional Review Boards at Washington University in Saint Louis School of Medicine. To induce differentiation and polarization of enteroids, cells were washed once to remove Matrigel, followed by trypsinization and centrifugation at 1100 × g for 5 min. Cells were then resuspended in 1:1 CM and primary medium with Y-27632 and SB431541 as described above. Cells were then plated on semiporous filters (Transwells®, 6.5 mm insert, 24 well plate, 0.4-μm polyester membrane, Corning Incorporated, Kennebunk, ME, USA) that had been previously coated with Collagen IV (Millipore Sigma). Transwell® inserts were rinsed with DMEM/F12 with Hepes, 10% FBS, L-glutamine, Penicillin, and streptomycin. Cells were allowed to grow to confluency in 50% conditioned media (CM) and then changed to a differentiation medium (5% CM in primary medium + ROCK inhibitor) for 48 h. Differentiated cells were used for toxin treatment.

Propagation of Caco-2 cells
Caco-2 intestinal epithelial cells were obtained from ATCC (ATCC HTB-37) and cultured at 37°C, 5% CO 2 , in Eagle's MEM supplemented with 20% of fetal bovine serum (FBS). To generate polarized monolayers, 1 × 10 5 cells were seeded onto polystyrene membrane filters (0.4 μM, 6.5 mm diameter insert, Transwell, Corning) and grew for at least a week prior to toxin treatment. Media was replaced with fresh media every two days.

Toxin treatment
Polarized differentiated cells were treated with a heat-labile toxin (LT) in a differentiation medium (100 ng/mL) in a volume of 700 µL at the basolateral side of the Transwell insert and 100 µL volume on the apical aspect of the monolayer. Treated cells were incubated at 37°C for 18 h and then fixed for transmission electron microscopy or fluorescence microscopy, or lysed for RNA extraction (GE Healthcare, Buckinghamshire, UK). LT and mutant LT (E211K, mLT) were kindly provided by Dr. John D. Clements, Tulane University, New Orleans, Louisianna, USA.

RNA extraction and cDNA synthesis
RNA was extracted using the Illustra RNAspin Mini RNA extraction kit (GE Healthcare, Buckinghamshire, UK). Three biological replicates per treatment (untreated and LT treated) were submitted to the Genome Technology Access Center (GTAC) at Washington University in St. Louis School of Medicine for RNA-seq library preparation. An Agilent Bioanalyzer was used to determine the integrity of RNA samples. cDNA was generated using Superscript TM Vilo TM cDNA synthesis kit (Invitrogen by Thermo Fisher) after normalizing RNA concentrations.

RNA-seq library preparation
Library preparation was performed with 10 ng of total RNA, integrity was determined using an Agilent bioanalyzer. ds-cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Clontech) per the manufacturer's protocol. cDNA was fragmented using a Covaris E220 sonicator using peak incident power 18, duty factor 20%, cycles/burst 50, and time 120 s. cDNA was blunt-ended, had an A base added to the 3′ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique index tags. Fragments were sequenced on an Illumina HiSeq-2500 using single reads extending 50 bases.

RNA-seq data processing and analysis
RNA-seq reads were aligned to the Ensembl top-level human genome assembly with STAR version 2.7.3a. Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:feature-Count version 1.54.1. Read counts were used as input for DESeq2 differential gene expression analysis (version 1.24.0) 78 with default settings, and a minimum P value significance threshold of 10 −5 (after false discovery rate [FDR 79 ] correction for the number of tests). Fragments per kilobase per million reads mapped (FPKM) values for relative gene expression were calculated from DESeq2-normalized read counts, and Z-scores were calculated per gene using the average and standard deviations of FPKM values across samples. Log 2 fold changes were identified from differential expression output. Pathway enrichment analysis for KEGG 80 and Gene Ontology (GO) 81 pathways among gene sets of interest was performed using the over-representation analysis tool provided on the WebGestalt 42 web server (version 2019). Heatmap and bar graph visualization was performed with Microsoft Excel.
Transcript counts were produced by Sailfish version 0.6.3. Sequencing performance was assessed for the total number of aligned reads, the total number of uniquely aligned reads, genes and transcripts detected, ribosomal fraction known junction saturation, and read distribution over known gene models with RSeQC version 2.3.
All gene-level and transcript counts were then imported into the R/Bioconductor package EdgeR and TMM normalization size factors were calculated to adjust for samples for differences in library size. Ribosomal features, as well as any feature not expressed in at least the smallest condition size minus one sample were excluded from further analysis and TMM size factors were recalculated to created effective TMM size factors. The TMM size factors and the matrix of counts were then imported into R/Bioconductor package Limma and weighted likelihoods based on the observed mean-variance relationship of every gene/transcript and sample were then calculated for all samples with the voomWithQualityWeights function. The performance of the samples was assessed with a spearman correlation matrix and multidimensional scaling plots. Gene/transcript performance was assessed with plots of the residual standard deviation of every gene to their average log count with a robustly fitted trend line of the residuals. Generalized linear models were then created to test for gene/transcript level differential expression. Differentially expressed genes and transcripts were then filtered for FDR-adjusted p values less than or equal to 0.05.
The biological interpretation of the large set of features found in the Limma results were then elucidated for global transcriptomic changes in known Gene Ontology (GO) and KEGG terms with the R/ Bioconductor packages GAGE and Pathview. Briefly, GAGE measures for perturbations in GO or KEGG terms based on changes in observed log 2 fold changes for the genes within that term versus the background log 2 fold changes observed across features not contained in the respective term as reported by Limma. For GO terms with an adjusted statistical significance of FDR ≤0.05, heatmaps were automatically generated for each respective term to show how genes co-vary or coexpress across the term in relation to a given biological process or molecular function. In the case of KEGG-curated signaling and metabolism pathways, Pathview was used to generate annotated pathway maps of any perturbed pathway with an unadjusted statistical significance of p value ≤0.05. Genes significantly modulated in both Caco-2 cells and enteroids were subjected to further ontology-free analysis via CompBio v1.4 (PercayAI, Inc., www.percayai.com/compbio) 82 to identify unifying biological themes in sets of genes differentially expressed between pairwise comparisons of different groups.

Quantitative real-time PCR
Quantitative real-time PCR was performed using a QuantStudio 3 realtime detection system (Applied Biosystems). Fast SYBR Green master mix (Applied Biosystems/Thermo Fisher) was used for qPCR analysis. Disassociation curve analysis was performed to assess the specificity of amplification for each sample, and PCR product size was verified by agarose gel electrophoresis. Percent expression was normalized by GAPDH and analyzed using the comparative threshold cycle (Ct) method. Amplification of SLC19A3 transcripts from small intestinal enteroids was performed as recently described 83 with relative gene expression normalized to β actin. HNF4a, HNFg, and SMAD4 gene expression was determined by TaqMan (Thermo Fisher) using validated primer and probe sets. Primers and TaqMan probes used in this study are listed in Supplemental Table 2.

Transcriptional reporter assays
To assess the impact of LT on HNF4-mediated transcription, pTR104 (GeneCopoeia) carrying six copies of the HNF4 transcriptional response element upstream of a secreted Gaussia luciferase (Gluc) reporter gene was transfected (FuGENE, Promega) into confluent Caco-2 cells. Media (EMEM, ATCC) were exchanged 24 h after transfection, and after an additional 24 h, baseline samples of media were removed from each well and stored at −80°C. Wells were then treated with a heat-labile toxin (100 ng/ml) overnight (16 h). At the end of treatment cell culture supernatants media were transferred to blackwalled microplates (Greiner 655086) and processed as directed (Secrete-Pair Luminescence, GeneCopoeia, Inc. Rockville, MD), followed by luminescence detection (Synergy H1, BioTek). Data were expressed as Relative Light Units (RLU) before and after treatment with LT.
Following treatment with overnight treatment with LT, cells were resuspended in 80 µl of EMEM media and mixed with an equal volume of ONEGLO EX Reagent (Nano-Glo® Dual-Luciferase® Reporter Assay System, Promega) for 20 min on an orbital shaker at 300 rpm, then read on the luminometer as above.
Villin. Differentiated LT-treated and control enteroid monolayers were lysed using NE-PER TM nuclear and cytoplasmic extraction reagents (Thermo Scientific). Cell membrane pellets were solubilized in PBS containing 1% Triton X-100 supplemented with a protease inhibitor cocktail (Pierce TM protease inhibitor mini, Thermo Scientific). Equal amounts of total protein were loaded on a 4-20% gradient SDS-PAGE gel (Mini Protean TGX, Bio-Rad), then blotted onto nitrocellulose membranes and probed with anti-villin mouse monoclonal antibody (1:1000; Cat# SC-66022; Santa Cruz Biotechnology) followed by detection with HRP-conjugated anti-mouse secondary antibody (1:1000; Cat# 7076 S; Cell Signaling Technology). Blots were developed with Clarity Western ECL substrate (Bio-Rad) and imaged with a c600 imaging system (Azure Biosystems). Relative Villin expression signals were then analyzed with respect to corresponding Coomassiestained gel using ImageJ analysis software.

Thiamin uptake
Initial rates (30 min; at 37°C) of carrier-mediated thiamin uptake were examined in LT-treated (100 ng/ml; overnight) and untreated control differentiated small intestinal enteroid monolayers incubated in Krebs-Ringer buffer (pH 7.4) containing [ 3 H]-thiamin (15 nM). Enteroid monolayers were then washed with ice-cold Krebs-Ringer buffer, followed by lysis with NaOH and neutralization with 10 N HCl. The radioactive content was counted using a liquid scintillation counter as described previously 83 . Uptake of thiamin by its respective and distinct carrier-mediated mechanism was determined by subtracting uptake of [ 3 H]-thiamin in the presence of a high pharmacological concentration (1 mM) of unlabeled thiamin from uptake in their absence; all uptake data points were calculated relative to total protein content (in milligrams) of the different preparations and presented as a percentage relative to simultaneously performed controls.

Confocal microscopy
Cell monolayers were fixed with 2% paraformaldehyde for 30 min at 37°C and then washed 3× with PBS prior to blocking with 1% BSA in PBS for 1 h at room temperature. Villin expression was detected using 1:100 dilution of anti-villin antibodies raised in mice (Santa Cruz Biotechnology) for 1 h at room temperature, followed by additional 1 h incubation with 1:200 dilution of fluorescence-tagged goat anti-mouse IgG Alexa Fluor 488 (Thermo Fisher) (Supplemental Table 5). Cell membranes were stained with 1:2000 dilution of CellMask orange (Invitrogen) and nuclei were counterstained with DAPI (1:1000) and mounted on glass slides using Prolong Gold antifade reagent (Invitrogen). Images were captured and analyzed on a Nikon C2 confocal microscope equipped with NIS-Elements AR 5.11.01 software (Nikon).

Electron microscopy
For ultrastructural analyses, in vitro-grown differentiated polarized monolayers of human ileal enteroid samples, as well as mouse intestinal biopsy samples, were fixed in 2% paraformaldehyde/2.5% glutaraldehyde (Ted Pella, Inc., Redding, CA) in 100 mM sodium cacodylate buffer, pH 7.2 for 2 h at room temperature and then overnight at 4°C. Samples were washed in sodium cacodylate buffer and postfixed in 2% osmium tetroxide (Ted Pella, Inc) 1 h at room temperature. Samples were then rinsed in dH 2 0, dehydrated in a graded series of ethanol, and embedded in Eponate 12 resin (Ted Pella, Inc.). Sections of 95 nm were cut with a Leica Ultracut UCT ultramicrotome (Leica Microsystems Inc., Bannockburn, IL), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA Inc., Peabody, MA) equipped with an AMT 8 megapixel digital camera and AMT Image Capture Engine V602 software (Advanced Microscopy Techniques, Woburn, MA). Images were analyzed using ImageJ software for microvilli length and structures.

Identification of potential transcription factor target sites
The CREB targets database 65 (http://natural.salk.edu/CREB/) was used to identify potential categorical CRE full (TGACGTCA) or half (TGACG/ CGTCA) sites within the promotor regions of genes modulated by the heat-labile toxin. Following previously described protocol 23 , 5 kilobase upstream sequences from each human gene were retrieved from Ensembl 86 and were searched for the CREB-binding sites TGACGTCA and TGACGTAA (and their reverse complements), based on sequences retrieved from the TRANSFAC 87 database (matrix ID: V$CREB_01). CREB sequence matches were identified in the upstream sequences of 1,302 genes. WebGestalt 42 was used for overall functional enrichment of potential transcription factor binding sequences upstream from genes that were significantly (p < 0.05) up-and down-regulated in both Caco-2 cells and enteroids.

Suckling mouse challenge studies
Studies in mice were performed under protocol 20-0438 approved by the Washington University in Saint Louis Institutional Animal Care and Use Committee. In neonatal mouse challenge studies, 3-day-old CD-1 mice (male and female) were inoculated with wild-type ETEC (H10407), isogenic toxin deletion mutants, or sterile PBS (sham negative controls). Inocula were prepared from frozen glycerol stocks maintained at −80°C grown overnight (~16 h) in 2 ml Luria Bertani (LB) broth in a shaking incubator (at 37°C, 225 rpm). Cultures were then diluted 1:100 in 20 ml of fresh LB, grown for an additional 2 h, and bacteria were harvested by centrifugation (6000 rpm, 5 min at 4°C). Pellets were then washed once in ice-cold sterile PBS, then resuspended to a density of~1 × 10 5 colony forming units (CFU) per 20 µl inoculum. CFU in each inoculum were determined by plating serial dilutions LB-agar plates. Mice were then inoculated with ETEC directly into the stomach through the abdominal wall using a 31 g insulin needle. Following infection, mice were marked with tattoo ink to define groups and returned to the cage. Seven days post-infection, mice were sacrificed, and the ileal tissue collected was fixed for microscopy or preserved for RNA extraction.

Bacterial strain construction
The strains used in these studies are described in Supplementary  Table 1. Strain jf4763 is devoid of all enterotoxins and was constructed from a previously engineered ST-negative mutant of H10407, jf2848 88 . Briefly, jf2848 was first transformed with pKD46, a lambda red recombinase helper plasmid with selection on ampicillin. Jf2848(pKD46) was then transformed with a PCR amplicon generated with primers _to replace the eltAB genes encoding LT with a spectinomycin resistance cassette resulting in jf4763. The strain was subsequently validated by PCR and by testing supernatants in GM1 ganglioside ELISA to confirm the lack of LT production.

Vaccination and immunologic assessment
Adult female mouse dams were vaccinated intranasally three times at 2-week intervals with 10 µg LT holotoxin in PBS, with the last dose administered~2 weeks prior to anticipated delivery. Control mice were mock vaccinated with PBS. Fecal pellets collected from vaccinated dams were used to assess mucosal responses to LT. To collect breast milk, the dam was separated from the litter~2 h prior to milking. Oxytocin (Sigma O3251), 0.002 IU/g, was injected IP to stimulate milk production, followed by collection via pipette tip. Anti-LT immune responses in maternal fecal extracts and breast milk, and stomach contents and sera of neonates (male and female) were assessed by GM1 ganglioside 89 kinetic ELISA 90 for anti-LT IgA and IgG, using plates coated with GM1 (Sigma G7641), as previously described 91 .

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
RNAseq data were deposited at the Sequence Read Archive (SRA) on the NCBI website https://www.ncbi.nlm.nih.gov/sra under BioProject accession number PRJNA875141. Primary source data and original images are available on Figshare as detailed in Supplementary Table 6 of the supplementary information.