Role of EspZ in Enteropathogenic Escherichia coli Virulence

Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhea, particularly in developing countries. EPEC belongs to the attaching and effacing (A/E) family of pathogens. All A/E pathogens harbor a type III secretion system (T3SS) that delivers virulence proteins directly into host epithelial cells. These proteins mediate diverse structural and functional alterations that likely facilitate pathogenesis. We recently demonstrated that EspZ, a secreted protein unique to A/E pathogens, is a critical virulence factor and that mutant strains lacking espZ are impaired for pathogenesis in both mouse and rabbit models of infection. EspZ prevents premature death of cultured intestinal epithelial cells by inhibiting intrinsic apoptosis. We hypothesized that EspZ promotes cell survival by engaging host proteins. Yeast two-hybrid studies identified the mitochondrial fission protein, hFis1, as a putative EspZ interactor. Co-immunoprecipitation studies confirmed EspZ-hFis1 interaction, and hFis1 was shown to be re-distributed in infected cells. These observations are consistent with the established role of hFis1 in intestinal cell survival pathways. The goal of my studies is to validate hFis1-EspZ interactions in epithelial cell cyto-protection and, eventually, to establish the significance of this pathway in EPEC virulence.Release after 31-Dec-2025Originally set to release after 13-Dec-2018; contacted by Graduate College 29-Nov-2018 to extend embargo through 30-Dec-2020. Kimberly Contacted again 13-Jan-2021 to extend embargo through 31-Dec-2025 Laure

Clostridium scindens exacerbates experimental necrotizing enterocolitis via upregulation of the apical sodium-dependent bile acid transporter

Supplemental Figure S1: https://doi.org/10.25422/azu.data.24208272Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in premature infants. Evidence indicates that bile acid homeostasis is disrupted during NEC: ileal bile acid levels are elevated in animals with experimental NEC, as is expression of the apical sodium-dependent bile acid transporter (Asbt). In addition, bile acids, which are synthesized in the liver, are extensively modified by the gut microbiome, including via the conversion of primary bile acids to more cytotoxic secondary forms. We hypothesized that the addition of bile acid-modifying bacteria would increase susceptibility to NEC in a neonatal rat model of the disease. The secondary bile acid-producing species Clostridium scindens exacerbated both incidence and severity of NEC. C. scindens upregulated the bile acid transporter Asbt and increased levels of intraenterocyte bile acids. Treatment with C. scindens also altered bile acid profiles and increased hydrophobicity of the ileal intracellular bile acid pool. The ability of C. scindens to enhance NEC requires bile acids, as pharmacological sequestration of ileal bile acids protects animals from developing disease. These findings indicate that bile acid-modifying bacteria can contribute to NEC pathology and provide additional evidence for the role of bile acids in the pathophysiology of experimental NEC.NEW & NOTEWORTHY Necrotizing enterocolitis (NEC), a life-threatening gastrointestinal emergency in premature infants, is characterized by dysregulation of bile acid homeostasis. We demonstrate that administering the secondary bile acid-producing bacterium Clostridium scindens enhances NEC in a neonatal rat model of the disease. C. scindens-enhanced NEC is dependent on bile acids and driven by upregulation of the ileal bile acid transporter Asbt. This is the first report of bile acid-modifying bacteria exacerbating experimental NEC pathology.NIH R01DK117652 NIH P30CA02307412 month embargo; first published 7 November 2023This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

An Engineered Synthetic Biologic Protects Against Clostridium difficile Infection

Morbidity and mortality attributed to Clostridium difficile infection (CDI) have increased over the past 20 years. Currently, antibiotics are the only US FDA-approved treatment for primary C. difficile infection, and these are, ironically, associated with disease relapse and the threat of burgeoning drug resistance. We previously showed that non-toxin virulence factors play key roles in CDI, and that colonization factors are critical for disease. Specifically, a C. difficile adhesin, Surface Layer Protein A (SlpA) is a major contributor to host cell attachment. In this work, we engineered Syn-LAB 2.0 and Syn-LAB 2.1, two synthetic biologic agents derived from lactic acid bacteria, to stably and constitutively express a host-cell binding fragment of the C. difficile adhesin SlpA on their cell-surface. Both agents harbor conditional suicide plasmids expressing a codonoptimized chimera of the lactic acid bacterium's cell-wall anchoring surface-protein domain, fused to the conserved, highly adherent, host-cell-binding domain of C. difficile SlpA. Both agents also incorporate engineered biocontrol, obviating the need for any antibiotic selection. Syn-LAB 2.0 and Syn-LAB 2.1 possess positive biophysical and in vivo properties compared with their parental antecedents in that they robustly and constitutively display the SlpA chimera on their cell surface, potentiate human intestinal epithelial barrier function in vitro, are safe, tolerable and palatable to Golden Syrian hamsters and neonatal piglets at high daily doses, and are detectable in animal feces within 24 h of dosing, confirming robust colonization. In combination, the engineered strains also delay (in fixed doses) or prevent (when continuously administered) death of infected hamsters upon challenge with high doses of virulent C. difficile. Finally, fixeddose Syn-LAB ameliorates diarrhea in a non-lethal model of neonatal piglet enteritis. Taken together, our findings suggest that the two synthetic biologics may be effectively employed as non-antibiotic interventions for CDI.National Institutes of Health [AI121590]; US Department of Veterans Affairs [1I01BX001183-01]; USDA CSREES Hatch Program [ARZT-570410-A-02-139]; Asset Development Award from Tech Launch ArizonaOpen access journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]