Heat-Labile Enterotoxin Decreases Macrophage Phagocytosis of Enterotoxigenic Escherichia coli.

Enterotoxigenic E. coli (ETEC) are endemic in low-resource settings and cause robust secretory diarrheal disease in children less than five years of age. ETEC cause secretory diarrhea by producing the heat-stable (ST) and/or heat-labile (LT) enterotoxins. Recent studies have shown that ETEC can be carried asymptomatically in children and adults, but how ETEC subvert mucosal immunity to establish intestinal residency remains unclear. Macrophages are innate immune cells that can be exploited by enteric pathogens to evade mucosal immunity, so we interrogated the ability of ETEC and other E. coli pathovars to survive within macrophages. Using gentamicin protection assays, we show that ETEC H10407 is phagocytosed more readily than other ETEC and non-ETEC isolates. Furthermore, we demonstrate that ETEC H10407, at high bacterial burdens, causes nitrite accumulation in macrophages, which is indicative of a proinflammatory macrophage nitric oxide killing response. However, at low bacterial burdens, ETEC H10407 remains viable within macrophages for an extended period without nitrite accumulation. We demonstrate that LT, but not ST, intoxication decreases the number of ETEC phagocytosed by macrophages. Furthermore, we now show that macrophages exposed simultaneously to LPS and LT produce IL-33, which is a cytokine implicated in promoting macrophage alternative activation, iron recycling, and intestinal repair. Lastly, iron restriction using deferoxamine induces IL-33 receptor (IL-33R) expression and allows ETEC to escape macrophages. Altogether, these data demonstrate that LT provides ETEC with the ability to decrease the perceived ETEC burden and suppresses the initiation of inflammation. Furthermore, these data suggest that host IL-33/IL-33R signaling may augment pathways that promote iron restriction to facilitate ETEC escape from macrophages. These data could help explain novel mechanisms of immune subversion that may contribute to asymptomatic ETEC carriage.

Heat-Stable Enterotoxin Secretions Assessed via ICP-MS Reveal Iron-Mediated Regulation of Virulence in CFA/I- and CS6-Expressing ETEC Isolates.

Enterotoxigenic Escherichia coli (ETEC) are a significant cause of childhood diarrhea in low-resource settings. ETEC are defined by the production of heat-stable enterotoxin (ST) and/or heat-labile enterotoxin (LT), which alter intracellular cyclic nucleotide signaling and cause the secretion of water and electrolytes into the intestinal lumen. ETEC take cues from chemicals (e.g., glycans, bile salts, and solutes) that may be liberated following enterotoxin activity to recognize entrance into the host. ETEC then alter the expression of surface adhesins called colonization factors (CFs) to attach to the intestinal epithelium, proliferate, and cause disease. Here, we used an in vivo model of oral ST intoxication to determine its impact on luminal ion concentrations via ICP-MS. We also used functional assays, including Western blots, qPCR, and toxin activity assays, to assess the impact of luminal ion flux on CF and toxin expression. Finally, we assessed ETEC strains with CFs CFA/I or CS6 in a streptomycin mouse model of ETEC colonization. ST causes rapid and significant increases in luminal chloride but significant decreases in luminal magnesium and iron. We confirmed that increased sodium chloride suppresses CFA/I production in ETEC H10407 but does not affect CS6 production in ETEC 214-4. CFA/I production in ETEC H10407 is increased when magnesium becomes limiting, although it does not affect CS6 production in ETEC 214-4. Iron restriction via deferoxamine induces CFA/I expression in ETEC H10407 but not CS6 expression in ETEC 214-4. We demonstrate that ST production is suppressed via iron restriction in H10407, 214-4, and over 50 other ETEC clinical isolates. Lastly, we demonstrate that the iron restriction of mice using oral deferoxamine pre-treatment extends the duration of ETEC H10407 (CFA/I+) fecal shedding while accelerating ETEC 214-4 (CS6+) fecal shedding. Combined, these data suggest that enterotoxins modulate luminal ion flux to influence ETEC virulence including toxin and CF production.

Enterotoxigenic Escherichia coli Enterotoxins Regulate Epithelial to Immune Relay of IL-33 and IL-1Ra Cytokines.

Enterotoxigenic Escherichia coli (ETEC) remain a major cause of diarrheal mortality and morbidity in children in low-resource settings. Few studies have explored the consequences of simultaneous intoxication with heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT) despite the increased prevalence of wild ETEC isolates expressing both toxins. We therefore used a combination of tissue culture and murine models to explore the impact of simultaneous ST + LT intoxication on epithelial and myeloid cells. We report that LT induces sustained production of interleukin 33 (IL-33) and interleukin 1 receptor antagonist (IL-1Ra) in T84 intestinal epithelial cells via cAMP production and protein kinase A activation. We demonstrate that combined ST + LT intoxication hastens epithelial transcriptional responses induced more slowly by LT alone. ST- and LT-mediated luminal fluid accumulation in vivo correlates with significant increases in IL-33 and IL-1Ra in small intestinal mucosal scrapings. Additionally, IL-33 receptor (IL-33R)-deficient mice are significantly less susceptible to ST-mediated secretion than wildtype mice. In the immune compartment, IL-33 is sensed by myeloid cells, and LT suppresses IL-33-induced tumor necrosis factor α (TNF-α) secretion from macrophages and bone marrow-derived dendritic cells (BMDCs) but amplifies IL-33-mediated induction of IL-6 from BMDCs. In conclusion, our studies suggest that enterotoxin-induced IL-33 and IL-1Ra modulate intestinal inflammation and IL-1 receptor signaling in the intestinal mucosa in response to ETEC enterotoxins.

Elevated Extracellular cGMP Produced after Exposure to Enterotoxigenic Escherichia coli Heat-Stable Toxin Induces Epithelial IL-33 Release and Alters Intestinal Immunity.

Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries. Previous studies identified heat-stable enterotoxin (ST)-producing ETEC as a prevalent diarrheal pathogen in children younger than 5 years. While many studies have evaluated the interaction of ETEC heat-labile enterotoxin (LT) with host epithelium and immunity, few investigations have attempted similar studies with ST. To further understand ST pathogenesis, we examined the impact of ST on cGMP localization, epithelial cell cytokine production, and antibody development following immunization. In addition to robust intracellular cGMP in T84 cells in the presence of phosphodiesterase inhibitors (PDEis) that prevent the breakdown of cyclic nucleotides, we found that prolonged ST intoxication induced extracellular cGMP accumulation in the presence or absence of PDEis. Further, ST intoxication induced luminal cGMP in vivo in mice, suggesting that secreted cGMP may have other cellular functions. Using transcriptome sequencing (RNA-seq) and quantitative PCR (qPCR), we demonstrated that ST intoxication, or treatment with the clinically used ST mimic linaclotide, altered inflammatory cytokine gene expression, including the interleukin 1 (IL-1) family member IL-33, which could also be induced by cell-permeative 8-Br-cGMP. Finally, when present during immunization, ST suppressed induction of antibodies to specific antigens. In conclusion, our studies indicate that ST modulates epithelial cell physiology and the interplay between the epithelial and immune compartments.

Enterotoxigenic Escherichia coli Heat-Stable Toxin Increases the Rate of Zinc Release from Metallothionein and Is a Zinc- and Iron-Binding Peptide.

Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries. Previous studies have identified heat-stable enterotoxin (ST)-producing ETEC as one of the major diarrhea-causing pathogens in children younger than five years. In this study, we examined iron and zinc binding by both human and porcine ST variants and determined how host metallothionein could detoxify ST. We found that ST purified from ETEC culture supernatants eluted as a doublet during C18 reverse-phase chromatography. Leading edge fractions of the ST doublet were found to be devoid of iron, while trailing edge fractions of the ST doublet were found to contain measurable iron. Next, we found that purified ST could be reconstituted with iron under reducing and anaerobic conditions, and iron-bound ST attenuated the induction of cGMP in T84 epithelial cells. Moreover, we demonstrated that supernatants of ETEC 214-4 grown under increasing iron concentrations were only able to induce cGMP at iron concentrations greater than 5 µM. In vitro studies also demonstrated that ST binds zinc, and once bound, zinc removal from ST required denaturing conditions. Zinc-bound ST also failed to induce cGMP. We found that ST contributes disulfide bonds to the perceived oxidized glutathione pool, increases the rate of zinc release from metallothionein, and can be detoxified by metallothionein. Lastly, we showed ST induces transcriptional changes in genes previously shown to be regulated by deferoxamine. These studies demonstrate ST ETEC pathogenesis may be tied intimately to host mucosal metal status.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries, deployed military personnel, and travelers to regions of endemicity. The heat-stable toxin (ST) is a small nonimmunogenic secreted peptide with 3 disulfide bonds. It has been appreciated that dietary disulfides modulate intestinal redox potential and that ST could be detoxified using exogenous reductants. Using biochemical and spectroscopic approaches, we demonstrated that ST can separately bind iron and zinc under reducing conditions, thereby reducing ST toxicity. Moreover, we demonstrated that ST modulates the glutathione (GSH)/oxidized glutathione (GSSG) ratio and that ST should be considered a toxin oxidant. ST can be detoxified by oxidizing zinc-loaded metallothionine, causing free zinc to be released. These studies help lay a foundation to understand how diarrheal pathogens modulate intestinal redox potential and may impact how we design therapeutics and/or vaccines for the pathogens that produce them.