Intestinal organoids model human responses to infection by commensal and Shiga toxin producing Escherichia coli

Infection with Shiga toxin (Stx) producing Escherichia coli O157:H7 can cause the potentially fatal complication hemolytic uremic syndrome, and currently only supportive therapy is available. Lack of suitable animal models has hindered study of this disease. Induced human intestinal organoids (iHIOs), generated by in vitro differentiation of pluripotent stem cells, represent differentiated human intestinal tissue. We show that iHIOs with addition of human neutrophils can model E. coli intestinal infection and innate cellular responses. Commensal and O157:H7 introduced into the iHIO lumen replicated rapidly achieving high numbers. Commensal E. coli did not cause damage, and were completely contained within the lumen, suggesting defenses, such as mucus production, can constrain non-pathogenic strains. Some O157:H7 initially co-localized with cellular actin. Loss of actin and epithelial integrity was observed after 4 hours. O157:H7 grew as filaments, consistent with activation of the bacterial SOS stress response. SOS is induced by reactive oxygen species (ROS), and O157:H7 infection increased ROS production. Transcriptional profiling (RNAseq) demonstrated that both commensal and O157:H7 upregulated genes associated with gastrointestinal maturation, while infection with O157:H7 upregulated inflammatory responses, including interleukin 8 (IL-8). IL-8 is associated with neutrophil recruitment, and infection with O157:H7 resulted in recruitment of human neutrophils into the iHIO tissue

Molecular Defects of Vitamin B6 Metabolism Associated with Neonatal Epileptic Encephalopathy

Neonatal epileptic encephalopathy (NEE) is a seizure disorder that occurs within hours from birth and arises from central nervous system (CNS) dysfunctions of various origins, including metabolic or inflammatory conditions, abnormalities of brain structure and cerebrovascular diseases. In some rare circumstances, NEE is refractory to conventional antiepileptic drugs (AEDs) but responds very well to treatment with vitamin B6 in the form of either pyridoxine (PN) or pyridoxal 5’-phosphate (PLP). Vitamin B6-dependent NEE derives either from a deficiency of PLP, from inborn errors in enzymes, such as pyridoxine 5’-phosphate oxidase (PNPOx) and pyridoxal kinase (PL kinase) involved in the PLP salvage pathway or from inherited mutations of enzymes, such as -aminoadipic semialdehyde dehydrogenase (also known as antiquitin) involved in other metabolic pathways, which lead to the accumulation of intermediates that react with PLP, reducing its availability. Clinical phenotypes observed in vitamin B6-dependent NEE patients may include fetal distress, hypoglycemia, acidosis, anemia, and asphyxia. The health state of untreated patients may undergo progressive deterioration, which can lead to death within weeks. Surviving children are usually mentally retarded and are dependent on vitamin B6 to control the disease. Several known cases of B-dependent NEE, however do not or only mildly manifest some of the above clinical features, and are characterized by mild to moderate developmental delay. This chapter will review the molecular mechanism of how in-born errors in PNPOx or antiquitin affect PLP levels in the cell and lead to NEE. We will also review important clinical and general features associated with PLP dependent NEE, and provide some directions for clinicians to diagnose and treat or manage the diseas

Shiga Toxin Binding to Glycolipids and Glycans

Background: Immunologically distinct forms of Shiga toxin (Stx1 and Stx2) display different potencies and disease outcomes, likely due to differences in host cell binding. The glycolipid globotriaosylceramide (Gb3) has been reported to be the receptor for both toxins. While there is considerable data to suggest that Gb3 can bind Stx1, binding of Stx2 to Gb3 is variable. Methodology: We used isothermal titration calorimetry (ITC) and enzyme-linked immunosorbent assay (ELISA) to examine binding of Stx1 and Stx2 to various glycans, glycosphingolipids, and glycosphingolipid mixtures in the presence or absence of membrane components, phosphatidylcholine, and cholesterol. We have also assessed the ability of glycolipids mixtures to neutralize Stx-mediated inhibition of protein synthesis in Vero kidney cells. Results: By ITC, Stx1 bound both Pk (the trisaccharide on Gb3) and P (the tetrasaccharide on globotetraosylceramide, Gb4), while Stx2 did not bind to either glycan. Binding to neutral glycolipids individually and in combination was assessed by ELISA. Stx1 bound to glycolipids Gb3 and Gb4, and Gb3 mixed with other neural glycolipids, while Stx2 only bound to Gb3 mixtures. In the presence of phosphatidylcholine and cholesterol, both Stx1 and Stx2 bound well to Gb3 or Gb4 alone or mixed with other neutral glycolipids. Pre-incubation with Gb3 in the presence of phosphatidylcholine and cholesterol neutralized Stx1, but not Stx2 toxicity to Vero cells

Glycolipid binding of Stx B-subunits.

<p>Serial dilutions of Stx B-subunits were titrated against immobilized glycolipids to obtain the dose response curves. <b>A. Gb3, B. Gb3+PC+Ch, C. Gb4, D. Gb4+PC+Ch</b>. B-subunits were incubated with methanol-coated wells as negative controls. Binding was assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101173#pone-0101173-g002" target="_blank">Figure 2</a>. The RFU signal is the mean of at least three independent experiments and error bars indicate SD. Symbols represent experimental data, while lines represent the fitted model for that data analyzed with Prism5 (GraphPad software, La Jolla, CA).</p

Binding of Stx holotoxins to glycolipid mixtures in absence of PC and Ch.

<p>Binding was assessed by ELISA at 37°C using serial dilutions of Stx variants. <b>A. Gb3; B. Gb3+Gal-Cer; C. Gb3+Glc-Cer; D. Gb3+Lac-Cer; E. Gb4; F. Gb4+Gal-Cer; Gb4+Glc-Cer; Gb4+Lac-Cer.</b> Mixtures of glycolipids were prepared in methanol in the ratio of 1∶1 of the two glycolipids. Total concentration of 200 ng glycolipid was added per well. As negative control toxins were incubated with plate sham-coated with methanol. In all experiments, background RFU values obtained in methanol were subtracted from each value. The RFU signal is the mean of three independent experiments and error bars indicate standard deviation (SD).</p

Sources of B-subunit plasmids used in this study.

<p>Sources of B-subunit plasmids used in this study.</p

Binding of holotoxin and B-subunits to Lyso-Gb3.

<p>ELISA was used to study binding of serial dilutions of <b>A. Stx holotoxins</b> and <b>B. B-subunits</b>. Binding was assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101173#pone-0101173-g002" target="_blank">Figure 2</a>. The RFU signal is the mean of three independent experiments and error bars indicate SD.</p

EC₅₀ values (in), glycolipid binding dissociation constants for Stx holotoxin and B-subunits (N.D.: Not determined due to insignificant binding).

<p>EC₅₀ values (in), glycolipid binding dissociation constants for Stx holotoxin and B-subunits (N.D.: Not determined due to insignificant binding).</p

Glycolipids used in this study.

<p>Glycolipids used in this study.</p

Sources of Stx-producing strains used in this study.

<p>Sources of Stx-producing strains used in this study.</p