Browsing by Author "Estes, Mary K."

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    A novel system to culture human intestinal organoids under physiological oxygen content to study microbial-host interaction
    (Public Library of Science, 2024) Fofanova, Tatiana Y.; Karandikar, Umesh C.; Auchtung, Jennifer M.; Wilson, Reid L.; Valentin, Antonio J.; Britton, Robert A.; Grande-Allen, K. Jane; Estes, Mary K.; Hoffman, Kristi; Ramani, Sashirekha; Stewart, Christopher J.; Petrosino, Joseph F.
    Mechanistic investigation of host-microbe interactions in the human gut are hindered by difficulty of co-culturing microbes with intestinal epithelial cells. On one hand the gut bacteria are a mix of facultative, aerotolerant or obligate anaerobes, while the intestinal epithelium requires oxygen for growth and function. Thus, a coculture system that can recreate these contrasting oxygen requirements is critical step towards our understanding microbial-host interactions in the human gut. Here, we demonstrate Intestinal Organoid Physoxic Coculture (IOPC) system, a simple and cost-effective method for coculturing anaerobic intestinal bacteria with human intestinal organoids (HIOs). Using commensal anaerobes with varying degrees of oxygen tolerance, such as nano-aerobe Bacteroides thetaiotaomicron and strict anaerobe Blautia sp., we demonstrate that IOPC can successfully support 24–48 hours HIO-microbe coculture. The IOPC recapitulates the contrasting oxygen conditions across the intestinal epithelium seen in vivo. The IOPC cultured HIOs showed increased barrier integrity, and induced expression of immunomodulatory genes. A transcriptomic analysis suggests that HIOs from different donors show differences in the magnitude of their response to coculture with anaerobic bacteria. Thus, the IOPC system provides a robust coculture setup for investigating host-microbe interactions in complex, patient-derived intestinal tissues, that can facilitate the study of mechanisms underlying the role of the microbiome in health and disease.
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    Enteroaggregative E. coli Adherence to Human Heparan Sulfate Proteoglycans Drives Segment and Host Specific Responses to Infection
    (Public Library of Science, 2020) Rajan, Anubama; Robertson, Matthew J.; Carter, Hannah E.; Poole, Nina M.; Clark, Justin R.; Green, Sabrina I.; Criss, Zachary K.; Zhao, Boyang; Karandikar, Umesh; Xing, Yikun; Margalef-Català, Mar; Jain, Nikhil; Wilson, Reid L.; Bai, Fan; Hyser, Joseph M.; Petrosino, Joseph; Shroyer, Noah F.; Blutt, Sarah E.; Coarfa, Cristian; Song, Xuezheng; Prasad, BV Venkataram; Amieva, Manuel R.; Grande-Allen, Jane; Estes, Mary K.; Okhuysen, Pablo C.; Maresso, Anthony W.
    Enteroaggregative Escherichia coli (EAEC) is a significant cause of acute and chronic diarrhea, foodborne outbreaks, infections of the immunocompromised, and growth stunting in children in developing nations. There is no vaccine and resistance to antibiotics is rising. Unlike related E. coli pathotypes that are often associated with acute bouts of infection, EAEC is associated with persistent diarrhea and subclinical long-term colonization. Several secreted virulence factors have been associated with EAEC pathogenesis and linked to disease in humans, less certain are the molecular drivers of adherence to the intestinal mucosa. We previously established human intestinal enteroids (HIEs) as a model system to study host-EAEC interactions and aggregative adherence fimbriae A (AafA) as a major driver of EAEC adherence to HIEs. Here, we report a large-scale assessment of the host response to EAEC adherence from all four segments of the intestine across at least three donor lines for five E. coli pathotypes. The data demonstrate that the host response in the duodenum is driven largely by the infecting pathotype, whereas the response in the colon diverges in a patient-specific manner. Major pathways altered in gene expression in each of the four enteroid segments differed dramatically, with responses observed for inflammation, apoptosis and an overwhelming response to different mucin genes. In particular, EAEC both associated with large mucus droplets and specific mucins at the epithelial surface, binding that was ameliorated when mucins were removed, a process dependent on AafA. Pan-screening for glycans for binding to purified AafA identified the human ligand as heparan sulfate proteoglycans (HSPGs). Removal of HSPG abrogated EAEC association with HIEs. These results may mean that the human intestine responds remarkably different to distinct pathobionts that is dependent on the both the individual and intestinal segment in question, and uncover a major role for surface heparan sulfate proteoglycans as tropism-driving factor in adherence and/or colonization.