The IBD candidate gene, PTPN2, Restricts Epithelial and Macrophage Invasion of a Novel Reporter Adherent‐Invasive Escherichia coli
Authors: Chatterjee Pritha , Spalinger Marianne , Gries Casey , Shawki Ali , Borneman James , McCole Declan 
Published: April 2020

BackgroundDysbiosis of the gut microbiota and expansion of pathobionts such as adherent‐invasive Escherichia coli (AIEC) are associated with the pathogenesis of Inflammatory Bowel Disease (IBD). Mice deficient in expression of the IBD gene candidate, Ptpn2, exhibit pronounced expansion of a novel murine adherent‐invasive Escherichia coli (mAIEC). The aim of this study was to generate a fluorescent mAIEC to study host susceptibility to mAIEC invasion, and to identify how loss of PTPN2 expression in host cells modifies mAIEC invasiveness.MethodsmAIEC was isolated from the distal colon of a Ptpn2 knockout (KO) mouse. mAIEC was made chemically competent and then transformed with a plasmid expressing mCherry fluorescent protein and a chloramphenicol resistance cassette. For bacterial adherence and invasion studies, parental Caco‐2bbe cells were infected with mAIEC‐mCherry at multiplicity of infection (MOI) of 10 for 3 hours. Caco‐2bbe intestinal epithelial cells (IEC), stably expressing either scrambled shRNA or shRNA against Ptpn2 were infected with mAIEC‐mCherry (MOI=10) for 5 hours. Peritoneal macrophages from Ptpn2 wildtype, heterozygous and KO mice were infected with mAIEC‐mCherry for 5 hours.ResultsWe previously demonstrated that mAIEC can adhere to and invade IECs. We next validated that mAIEC‐mCherry adhered to (0.94 ±0.61 bacteria/cell compared to control E. coli K12; 0.14±0.03 bacteria/cell, n=4, P=0.0039) IECs. mAIEC‐mCherry had comparable adherence (n=4, P=0.35) to IECs as control (unlabeled) mAIEC. Bacterial invasion of mAIEC‐mCherry invasion of IECs was visually confirmed by fluorescence microscopy. To better understand if the loss of expression of PTPN2, which drives increased luminal abundance of mAIEC, also affected mAIEC pathogenicity, we next tested if Ptpn2 deficiency in host cells altered mAIEC‐mCherry invasion. We observed by immunofluorescence that Ptpn2 knockdown (KD) IECs were more susceptible to mAIEC‐mCherry invasion compared to control IECs (n=3). We also confirmed that macrophages from Ptpn2‐KO mice were more susceptible to mAIEC‐mCherry invasion compared to wildtype and Ptpn2‐heterozygous mouse macrophages (n=5, wildtype vs. KO, P=0.0044; KO vs. heterozygous, P=−0.8665). Ptpn2‐heterozygous macrophages were also more susceptible to invasion than wildtype, indicating a gene‐dosing effect of macrophage PTPN2 loss on bacterial invasion (wildtype vs. heterozygous, P=0.0117). These data identify that loss of PTPN2 expression increases susceptibility to mAIEC invasion.ConclusionHere, we validated a newly generated fluorescently tagged mAIEC to study interactions between this novel mouse AIEC with host cells. We further demonstrated that Ptpn2 deficiency promotes mAIEC of intestinal epithelial cells and macrophages. These data suggest that IBD patients with loss‐of‐function mutations in PTPN2 may have increased susceptibility to colonization by disease‐relevant AIEC.Support or Funding InformationNIH 2R01DK091281 (DFM)