Megha Singhal

Role of SHP2 protein tyrosine phosphatase in SERT inhibition by enteropathogenic E. Coli (EPEC)

Enteropathogenic Escherichia coli (EPEC), one of the diarrheagenic E. coli pathotypes, is among the most important food-borne pathogens infecting children worldwide. Inhibition of serotonin transporter (SERT), which regulates extracellular availability of serotonin (5-HT), has been implicated previously in EPEC-associated diarrhea. EPEC was shown to inhibit SERT via activation of protein tyrosine phosphatase (PTPase), albeit the specific PTPase involved is not known. Current studies aimed to identify EPEC-activated PTPase and its role in SERT inhibition. Infection of Caco-2 monolayers with EPEC strain E2348/69 for 30 min increased the activity of Src-homology-2 domain containing PTPase (SHP2) but not SHP1 or PTPase 1B. Similarly, Western blot studies showed increased tyrosine phosphorylation of (p-tyrosine) SHP2, indicative of its activation. Concomitantly, EPEC infection decreased SERT p-tyrosine levels. This was associated with increased interaction of SHP2 with SERT, as evidenced by coimmunoprecipitation studies. To examine whether SHP2 directly influences SERT phosphorylation status or function, SHP2 cDNA plasmid constructs (wild type, constitutively active, or dominant negative) were overexpressed in Caco-2 cells by Amaxa electroporation. In the cells overexpressing constitutively active SHP2, SERT polypeptide showed complete loss of p-tyrosine. In addition, there was a decrease in SERT function, as measured by Na+Cl--sensitive [3H]5-HT uptake, and an increase in association of SERT with SHP2 in Caco-2 cells expressing constitutively active SHP2 compared with dominant-negative SHP2. Our data demonstrate that intestinal SERT is a target of SHP2 and reveal a novel mechanism by which a common food-borne pathogen uses cellular SHP2 to inhibit SERT.NEW & NOTEWORTHY The data presented in the current study reveal that intestinal serotonin transporter (SERT) is a target of the tyrosine phosphatase SHP2 and show a novel mechanism by which a common diarrheagenic pathogen, EPEC, activates cellular SHP2 to inhibit SERT function. These studies highlight host-pathogen interactions, which may be of therapeutic relevance in the management of diarrhea associated with enteric infections.

Serotonin is an endogenous regulator of intestinal CYP1A1 via AhR

Aryl hydrocarbon receptor (AhR) is a nuclear receptor that controls xenobiotic detoxification via induction of cytochrome P450 1A1 (CYP1A1) and regulates immune responses in the intestine. Metabolites of L-tryptophan activate AhR, which confers protection against intestinal inflammation. We tested the hypothesis that serotonin (5-HT) is an endogenous activator of AhR in intestinal epithelial cells. Treatment of Caco-2 monolayers with 5-HT induced CYP1A1 mRNA in a time- and concentration-dependent manner and also stimulated CYP1A1 activity. CYP1A1 induction by 5-HT was dependent upon uptake via serotonin transporter (SERT). Antagonism of AhR and knockdown of AhR and its binding partner aryl hydrocarbon receptor nuclear translocator (ARNT) attenuated CYP1A1 induction by 5-HT. Activation of AhR was evident by its nuclear translocation after 5-HT treatment and by induction of an AhR-responsive luciferase reporter. In vivo studies showed a dramatic decrease in CYP1A1 expression and other AhR target genes in SERT KO ileal mucosa by microarray analysis. These results suggest that intracellular accumulation of 5-HT via SERT induces CYP1A1 expression via AhR in intestinal epithelial cells, and SERT deficiency in vivo impairs activation of AhR. Our studies provide a novel link between the serotonergic and AhR pathways which has implications in xenobiotic metabolism and intestinal inflammation.

Infection with enteric pathogens Salmonella typhimurium and Citrobacter rodentium modulate TGF-beta/Smad signaling pathways in the intestine

ABSTRACT Salmonella and Citrobacter are gram negative, members of Enterobacteriaceae family that are important causative agents of diarrhea and intestinal inflammation. TGF-β1 is a pleiotropic multifunctional cytokine that has been implicated in modulating the severity of microbial infections. How these pathogens alter the TGF-β1 signaling pathways in the intestine is largely unknown. Streptomycin-pretreated C57BL/6J mouse model colonized with S. typhimurium for 8 hours (acute) and 4 days (chronic) infection and FVB/N mice infected with C. rodentium for 6 days were utilized. Results demonstrated an increase in TGF-β1 receptor I expression (p<0.05) in S. typhimurium infected mouse ileum at both acute and chronic post-infection vs control. This was associated with activation of Smad pathways as evidenced by increased phosphorylated (p)-Smad2 and p-Smad3 levels in the nucleus. The inhibitory Smad7 mRNA levels showed a significant up regulation during acute phase of Salmonella infection but no change at 4d post-infection. In contrast to Salmonella, infection with Citrobacter caused drastic downregulation of TGF receptor I and II concomitant with a decrease in levels of Smad 2, 3, 4 and 7 expression in the mouse colon. We speculate that increased TGF-β1 signaling in response to Salmonella may be a host compensatory response to promote mucosal healing; while C. rodentium decreases TGF-β1 signaling pathways to promote inflammation and contribute to disease pathogenesis. These findings increase our understanding of how enteric pathogens subvert specific aspects of the host-cellular pathways to cause disease.