Abstract 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. Subject terms: Cellular microbiology, Malnutrition, Bacterial toxins __________________________________________________________________ Enterotoxigenic Escherichia coli infections have been linked to non-diarrheal sequelae however, the reasons for this are unclear. Here, the authors present an additional role of heat-labile toxin in disrupting the structure and function of intestinal epithelial cells. Introduction 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^[52]1. Enterotoxigenic E. coli (ETEC), initially discovered as a cause of severe, cholera-like illness^[53]2, are one of the most common pathogens associated with moderate-severe diarrhea among children under the age of 5 years^[54]3,[55]4, and are perennially the most common cause of diarrhea in travelers^[56]5 to endemic regions where these organisms are thought to account for hundreds of millions of cases of diarrheal illness each year^[57]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^[58]7,[59]8, and malnutrition^[60]9,[61]10, adding significantly to the morbidity as well as deaths from diarrhea and other infections^[62]11. The risk of stunting multiplies with each episode of diarrheal illness in children under the age of two years^[63]12, a period during which children residing in impoverished areas commonly sustain multiple ETEC infections^[64]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^[65]13–[66]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^[67]16,[68]17, nutrient malabsorption, and wasting. The basic molecular mechanisms underpinning acute watery diarrhea caused by ETEC are well-established^[69]18. ETEC produces heat-labile (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 ADP-ribosylation 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^[70]19. In its central role as a second messenger, cAMP governs a diverse array of cellular processes^[71]20 and modulates the transcription of multiple genes through a number of cAMP-responsive transcriptional activators and repressors^[72]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^[73]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 cAMP-response elements (CRE) in the promoter regions of target genes^[74]21–[75]23. Notably, cholera toxin (CT), LT, and dibutyryl-cyclic AMP all induce hypersecretion and impact the architecture of gastrointestinal epithelia in rodent small intestine^[76]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^[77]25,[78]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 associated LT-producing ETEC with undernutrition^[79]27, suggesting that heat-labile 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. Results Heat-labile toxin markedly alters the transcriptomes ofintestinal 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. [80]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^[81]28,[82]29, and cAMP signaling is known to be aberrant in some transformed cells^[83]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. [84]1b). Notably, however, we found substantial statistically significant overlap in genes significantly modulated in Caco-2 cells and enteroids (Supplementary Table [85]3 and Supplementary Dataset [86]1) with the transcription of hundreds of genes significantly up- or down-regulated in both groups. Gene ontology enrichment analysis, as well as ontology-independent investigation of genes (CompBio, Supplementary Fig. [87]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 [88]2). Fig. 1. Heat-labile toxin modulates expression of multiple genes in intestinal epithelia. [89]Fig. 1 [90]Open in a new tab Model at left depicts the E. coli heat-labile toxin^[91]92 based on PDB structure [92]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 LT-treated 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]93. 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^[94]30 (Fig. [95]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^[96]31; (2) ezrin, myo1a^[97]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^[98]30 that are stabilized by a tripartite complex of MYO7B, ANKS4B^[99]33, and USH1C^[100]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. [101]2b). Similarly, RT-PCR confirmed decreased expression of multiple genes involved in microvilli biogenesis, including VIL1 encoding villin (Fig. [102]3a), and we were able to demonstrate that production of villin was depressed in polarized small intestinal enteroids (Fig. [103]3b and Supplementary Fig. [104]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. [105]3c). Fig. 2. Heat-labile toxin modulates multiple genes involved in microvillus assembly. [106]Fig. 2 [107]Open in a new tab a Diagram at the top (adapted from ref. [108]30) depicts molecules involved in key elements of microvillus development. b Heatmaps of RNA-seq data obtained following treatment of Caco-2 intestinal cells (left) with mLT (n = 2 biologically independent samples) or LT (n = 2) relative to untreated cells (n = 2); and ileal enteroids (right) treated with LT (n = 3) relative to control untreated cells (n = 3). Comparisons were made with DESeq2^[109]93. Bars indicate absolute log[2]fold change values + SE. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 10^−4, and *****p ≤ 10^−5. Fig. 3. Heat-labile toxin impairs the effective formation of small intestinal microvilli. [110]Fig. 3 [111]Open in a new tab a Quantitative RT-PCR data from n = 9 biologically independent samples for selected microvillus genes comparing untreated (−) and LT-treated (+) ileal epithelial cells (enteroid line 235D). b Villin production is suppressed in small intestinal enteroids following treatment (t + 18 h) with LT (100 µg/ml). Shown are representative confocal images obtained showing membrane (CellMask, blue), nuclei (white), villin (gold), and merged image. The graph at right shows apical villin geometric mean fluorescence intensity data relative to the corresponding cytoplasmic signal. Each symbol (n = 12) represents a unique region of interest. For a, b ****p < 0.0001, ***p < 0.001 by Mann–Whitney two-tailed testing. c TEM images of small intestinal microvilli following treatment LT (right) compared to control untreated cells (left). The graph at right shows the length of microvilli when enteroids (n = 2 biologically independent samples) are treated before (pre) and after (post) differentiation on polarized ileal cells ****<0.0001 by ANOVA (Kruskal–Wallis, nonparametric testing). 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^[112]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. [113]4a). These included transporters for Zinc, known both to be deficient in children with enteropathy^[114]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)^[115]36 by differentiated intestinal epithelial cells lining the surface of intestinal villi of the proximal small intestine^[116]37,[117]38 (Fig. [118]4b) was repressed as was the production of the corresponding protein (supplementary figure [119]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^[120]39–[121]41 (Fig. [122]4c and Supplementary Fig. [123]3b). Finally, we found that thiamine transport was significantly depressed following exposure to LT (Fig. [124]4d) providing additional evidence that ETEC can impair transport of critical nutrients. Fig. 4. Heat-labile toxin alters the transcription of multiple brush border SLC genes. [125]Fig. 4 [126]Open in a new tab a Heatmap indicating key SLC genes modulated by heat-labile toxin (LT) compared to enzymatically inactive E112K LT mutant (mLT), or untreated (ø) Caco-2 cells (left) and human small intestinal (ileal) enteroids (Hu235D, right). Bars indicate absolute log[2] fold change values + SE. Comparisons were made with DESeq2^[127]93. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 10^−4, *****p ≤ 10^−5. Real-time qRT-PCR confirming LT-mediated modulation of genes in ileal (Hu235D) enteroids encoding b the major thiamine transporter SLC19A3 and c the SP1 cis-regulatory element. Data reflect two independent experiments with two replicates each. d Uptake of [^3H]-thiamine by Hu235D cells is impaired following LT treatment. Data presented in b–d are from two independent experiments with n = 3 replicates each. (*<0.05, **<0.01 by Mann–Whitney two-tailed comparisons). 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. [128]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. [129]5b, c). In mice challenged with a wild-type ETEC isolate that makes ST and LT, but not an LT/ST-toxin-negative mutant (jf4763, Supplementary Table [130]1), we observed significant alteration in the architecture of the intestinal brush border with significant shortening and disorganization of the microvilli (Fig. [131]5d, e and Supplementary Fig. [132]4a–d) reminiscent of the earlier ultrastructural studies of patients with tropical sprue^[133]17 and V. cholerae infections^[134]25. Similarly, we found that in mice challenged with a strain containing an isogenic mutation in eltA (jf571, Supplementary Table [135]1) encoding the LT A subunit, which still makes heat-stable toxins, the microvillus architecture was preserved (Fig. [136]5f, g), suggesting that LT is the principal toxin underlying the enteropathic changes to the enterocyte surfaces. Fig. 5. ETEC disrupts in vivo formation of small intestinal microvilli. [137]Fig. 5 [138]Open in a new tab Timeline at the top depicts the challenge with ETEC or control nontoxigenic isolate or sham (PBS) challenge. a Quantitative PCR results for genes involved in brush border development in small intestinal samples obtained from infant mice (n = 9/group) 7 days after challenge with toxigenic ETEC (jf876), nontoxigenic ETEC (jf4763, LT^−/ST^−) PBS controls. Comparisons between data represent ANOVA, Kruskal–Wallis testing where ***p ≤ 0.001, **p ≤ 0.01, and *p ≤ 0.05. b Immunofluorescence images of small intestinal sections showing villin expression (blue), and nuclei (white). c Mean villin fluorescence intensity normalized per enterocyte (n = 4 mice/group) ****p ≤ 0.0001 by Mann–Whitney (two-tailed) nonparametric comparisons. d Representative transmission electron microscopy (TEM) images of the small intestinal brush border from mice challenged with toxin-negative (∆, left) and toxigenic ETEC (wt, right). e Microvillus length ****p ≤ 0.0001 by Mann–Whitney (two-tailed) nonparametric comparisons. Data represent geometric mean length from n = 5 mice per group in three independent experiments. f TEM images from mice challenged with jf570 (eltA::Km^R), sham PBS controls, or mice challenged with wild-type ETEC. g Length of microvilli (dashed horizontal lines represent geometric means). ****p < 0.0001 by Kruskal–Wallis. 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. [139]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^[140]8,[141]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. [142]5B). Finally, we found that the growth kinetics of mice repeatedly challenged with wild-type ETEC [143]H10407 was significantly retarded relative to those challenged with the isogenic LT-mutant jf876 (Supplementary Fig. [144]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. [145]6a), consistent with increased levels of antibodies in the stomachs of infant mice (Fig. [146]6b). Notably, we found that maternal vaccination with LT completely abrogated changes to the microvilli (Fig. [147]6c, d) further substantiating the importance of LT in driving changes to the epithelial architecture. Fig. 6. Maternal vaccination with LT mitigates microvillus disruption inneonatal mice. [148]Fig. 6 [149]Open in a new tab Timeline depicts vaccination and challenge (top): maternal intranasal (i.n.) vaccinations with 10 µg LT/immunization (yellow arrows on days 0, 14, 28) and neonatal challenge at 3 days of age (green arrow) followed by sacrifice and tissue collection at 7 days post-infection (gray arrow). a Kinetic ELISA data of from triplicate samples of breast milk anti-LT (IgA, and IgG in n = 2 immunized dams and 1 un-immunized control). *<0.05 by Mann–Whitney two-tailed nonparametric testing. b Anti-LT antibodies in the gastric contents of neonatal mice at day 53. ****p < 0.0001 by Mann–Whitney two-tailed comparisons. c Representative transmission electron microscopy images of brush border microvilli from unvaccinated mice challenged with wild-type ETEC (left), vaccinated mice challenged with wild-type ETEC, and vaccinated un-challenged controls. d Microvillus lengths (based on image analysis of n = 5 mice group) ****<0.0001 Kruskal–Wallis comparisons. 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^[150]23. Therefore, we performed transcription factor target enrichment analysis^[151]42,[152]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 [153]4 and Supplementary Dataset [154]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^[155]44, and to be involved in intestinal epithelial repair^[156]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^[157]46,[158]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^[159]48. Of note, the transcription of both paralogs was found to be significantly depressed following exposure of intestinal epithelia to LT (Fig. [160]7a, b), as were levels of HNF4γ in nuclear fractions from toxin-treated cells (Fig. [161]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. [162]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. [163]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. [164]6A, B), leading us to speculate that LT-mediated phosphorylation of HNF4 by PKA, interrupts this critical transcription module (Supplementary Fig. [165]6C–E). Fig. 7. Enterotoxigenic E. coli heat-labile toxin impairs production of HNF4 nuclear receptors. [166]Fig. 7 [167]Open in a new tab a Heatmap demonstrating the impact of LT on transcription of paralogous transcription factors HNF4α and HNF4γ in Caco-2 cells (left) and ileal enteroids, (Hu235D, right) ø untreated, mLT mutant LT. b qRT-PCR (TaqMan) data confirming decreased transcription of HNF4 transcription factors following treatment of enteroids with LT (n = 9 biologically independent samples). ****<0.0001 by Mann–Whitney two-tailed comparisons. c HNF4γ is decreased in nuclear fractions obtained from small intestinal enteroids following treatment with LT. Shown in the HNF4γ immunoblot are samples from four independent experiments, with the graph below-representing quantitation of signal intensity normalized to the lamin B1 nuclear protein (p = 0.0017, paired t-test, one-tailed). Bars indicate mean ± 95% confidence intervals. d HNF4 transcription Gaussia luciferase reporter assay showing a decrease in signal following treatment of TR104-transfected Caco-2 cells with LT. 2 experimental replicates (n = 15 samples total) ***p < 0.001 Wilcoxon matched pairs, one-tailed). e Confocal microscopy of representative Ileal sections from sham-challenged (PBS) left, and ETEC-infected mice (right). Immunofluorescence intensity of HNF4γ signal in sections from n = 5 control mice, and n = 6 ETEC-challenged mice. Membranes (yellow) were strained with CellMask orange (Thermo Fisher [168]C10045), nuclei (blue) were stained with DAPI, and HNFγ immunostaining was represented in white. Each symbol represents a microscopic region of interest. Bars represent geographic means (p < 0.0001, Mann–Whitney two-tailed comparisons). 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^[169]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^[170]50,[171]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^[172]25 and tropical sprue^[173]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^[174]52 is not presently clear. Similarly, while tropical sprue remains a leading cause of malabsorption in regions where infectious diarrhea is prevalent^[175]53–[176]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^[177]56 repeatedly assailed by diarrhea while residing in endemic regions^[178]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^[179]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^[180]35,[181]59,[182]60, the possible contribution of zinc deficiency to enteropathic changes^[183]61, and the salutary effects of zinc in the treatment of children with diarrhea^[184]62,[185]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^[186]3, and follow-on studies of children enrolled in these studies have also linked infections with ETEC encoding heat-stable toxin to growth faltering^[187]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^[188]64. While LT-producing ETEC have been specifically linked to malnutrition among children in Bangladesh^[189]27, and our in vitro and animal studies point to the potential importance of LT, additional effort will be needed to correlate toxin-induced 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^[190]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^[191]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^[192]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^[193]66. cAMP second messaging also engages multiple signaling pathways converging at CREB^[194]67, and PKA can phosphorylate and modulate the activity of multiple transcription factors to act either as transcriptional activators or repressors, including SP1^[195]68. Notably, putative binding sites for HNF4, a cAMP-modulated transcription factor^[196]48 known to regulate genes needed for the formation of microvilli^[197]46,[198]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^[199]69. HNF4 activates the transcription of SMAD4, and in turn, SMAD4 activates the transcription of HNF4^[200]70. Both transcription factors then engage genes needed for the effective differentiation of stem cells to mature enterocytes^[201]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^[202]71 and are involved in complex signaling networks implicated in cell growth, differentiation, and morphogenesis^[203]72,[204]73. cAMP can also exert potent epigenetic influences on transcription. CREB-binding protein (CBP) possesses intrinsic histone acetyl-transferase (HAT) activity^[205]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^[206]75. Increased intestinal permeability is a recognized hallmark of enteropathy in young children in LMICs^[207]76. Given the known negative impacts of cholera toxin on epithelial barrier function^[208]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