Interactions between Enterohemorrhagic Escherichia coli (EHEC) and Gut Commensals at the Interface of Human Colonoids

Martins, Fernando Henrique; Rajan, Anubama; Carter, Hannah; Baniasadi, Hamid; Maresso, Anthony W.; Sperandio, Vanessa

doi:10.1128/mbio.01321-22

Cited by 11 publications

(3 citation statements)

References 48 publications

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“…In summary, refined patient-derived organoid model systems are effective tools for understanding the interplay between oxygen level, microbiota, and innate immune signals in health and disease. IEOs are also valuable models for, e.g., colon cancer ( 94 ) and studies of enteric infections ( 95 , 96 ). However, the relatively hypoxic gut environment influencing barrier function and inflammatory tone is not appropriately modeled by standard atmospheric oxygen culture conditions.…”

Section: Discussionmentioning

confidence: 99%

Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids

et al. 2023

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BackgroundThe epithelium in the colonic mucosa is implicated in the pathophysiology of various diseases, including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial organoids from the colon (colonoids) can be used for disease modeling and personalized drug screening. Colonoids are usually cultured at 18-21% oxygen without accounting for the physiological hypoxia in the colonic epithelium (3% to <1% oxygen). We hypothesize that recapitulating the in vivo physiological oxygen environment (i.e., physioxia) will enhance the translational value of colonoids as pre-clinical models. Here we evaluate whether human colonoids can be established and cultured in physioxia and compare growth, differentiation, and immunological responses at 2% and 20% oxygen.MethodsGrowth from single cells to differentiated colonoids was monitored by brightfield images and evaluated with a linear mixed model. Cell composition was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing (scRNA-seq). Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data.ResultsColonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. No differences in expression of cell markers for cells with proliferation potential (KI67 positive), goblet cells (MUC2 positive), absorptive cells (MUC2 negative, CK20 positive) and enteroendocrine cells (CGA positive) were found between colonoids cultured in 2% and 20% oxygen. However, the scRNA-seq analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CXCL2, CXCL5, CXCL10, CXCL12, CX3CL1 and CCL25, and NGAL upon TNF + poly(I:C) treatment, but there appeared to be a tendency towards lower pro-inflammatory response in 2% oxygen. Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks.ConclusionsOur results suggest that colonoids studies can and should be performed in physioxia when the resemblance to in vivo conditions is important.

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Section: Discussionmentioning

confidence: 99%

Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids

et al. 2023

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“…Enterohemorrhagic Escherichia coli (EHEC) is a human foodborne pathogen that colonizes the colon and causes outbreaks of bloody diarrhea and hemolytic uremic syndrome worldwide ( 1 ). The formation of attaching and effacing (A/E) intestinal lesions is one of the major features of EHEC virulence and pathogenesis, which is dependent on the secretion of proteins by the type III secretion system (T3SS) ( 2 ).…”

Section: Introductionmentioning

confidence: 99%

Enterohemorrhagic Escherichia coli effector EspF triggers oxidative DNA lesions in intestinal epithelial cells

Fang,

Fu,

et al. 2024

Infect Immun

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Attaching/effacing (A/E) pathogens induce DNA damage and colorectal cancer by injecting effector proteins into host cells via the type III secretion system (T3SS). EspF is one of the T3SS-dependent effector proteins exclusive to A/E pathogens, which include enterohemorrhagic Escherichia coli . The role of EspF in the induction of double-strand breaks (DSBs) and the phosphorylation of the repair protein SMC1 has been demonstrated previously. However, the process of damage accumulation and DSB formation has remained enigmatic, and the damage response is not well understood. Here, we first showed a compensatory increase in the mismatch repair proteins MutS homolog 2 (MSH2) and MSH6, as well as poly(ADP-ribose) polymerase 1, followed by a dramatic decrease, threatening cell survival in the presence of EspF. Flow cytometry revealed that EspF arrested the cell cycle at the G2/M phase to facilitate DNA repair. Subsequently, 8-oxoguanine (8-oxoG) lesions, a marker of oxidative damage, were assayed by ELISA and immunofluorescence, which revealed the accumulation of 8-oxoG from the cytosol to the nucleus. Furthermore, the status of single-stranded DNA (ssDNA) and DSBs was confirmed. We observed that EspF accelerated the course of DNA lesions, including 8-oxoG and unrepaired ssDNA, which were converted into DSBs; this was accompanied by the phosphorylation of replication protein A 32 in repair-defective cells. Collectively, these findings reveal that EspF triggers various types of oxidative DNA lesions with impairment of the DNA damage response and may result in genomic instability and cell death, offering novel insight into the tumorigenic potential of EspF. IMPORTANCE Oxidative DNA lesions play causative roles in colitis-associated colon cancer. Accumulating evidence shows strong links between attaching/effacing (A/E) pathogens and colorectal cancer (CRC). EspF is one of many effector proteins exclusive to A/E pathogens with defined roles in the induction of oxidative stress, double-strand breaks (DSBs), and repair dysregulation. Here, we found that EspF promotes reactive oxygen species generation and 8-oxoguanine (8-oxoG) lesions when the repair system is activated, contributing to sustained cell survival. However, infected cells exposed to EspF presented 8-oxoG, which results in DSBs and ssDNA accumulation when the cell cycle is arrested at the G2/M phase and the repair system is defective or saturated by DNA lesions. In addition, we found that EspF could intensify the accumulation of nuclear DNA lesions through oxidative and replication stress. Overall, our work highlights the involvement of EspF in DNA lesions and DNA damage response, providing a novel avenue by which A/E pathogens may contribute to CRC.

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“…This process has been implicated in the pathogenesis of IBD and CRC ( Vona et al, 2021 ; Pan et al, 2022 ). On the other hand, the reduction in ROS is facilitated by the influence of gut bacteria-generated beneficial metabolites, specifically short-chain fatty acids (SCFAs), which are considered metabolic byproducts produced by certain bacterial species ( Martins et al, 2022 ; Dey and Ray Chaudhuri, 2023 ). These SCFAs can serve as an energy source for other bacterial species through a phenomenon referred to as cross-feeding ( Evans et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

The role of gut microbiota in intestinal disease: from an oxidative stress perspective

Sun,

Wang,

et al. 2024

Front. Microbiol.

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Recent studies have indicated that gut microbiota-mediated oxidative stress is significantly associated with intestinal diseases such as colorectal cancer, ulcerative colitis, and Crohn’s disease. The level of reactive oxygen species (ROS) has been reported to increase when the gut microbiota is dysregulated, especially when several gut bacterial metabolites are present. Although healthy gut microbiota plays a vital role in defending against excessive oxidative stress, intestinal disease is significantly influenced by excessive ROS, and this process is controlled by gut microbiota-mediated immunological responses, DNA damage, and intestinal inflammation. In this review, we discuss the relationship between gut microbiota and intestinal disease from an oxidative stress perspective. In addition, we also provide a summary of the most recent therapeutic approaches for preventing or treating intestinal diseases by modifying gut microbiota.

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Interactions between Enterohemorrhagic Escherichia coli (EHEC) and Gut Commensals at the Interface of Human Colonoids

Abstract: Enterohemorrhagic E. coli (EHEC) is a natural human pathogen that poorly colonizes mice. Hence, the use of murine models to understand features of EHEC infection is a challenge.

Cited by 11 publications

References 48 publications

Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids

Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids

Enterohemorrhagic Escherichia coli effector EspF triggers oxidative DNA lesions in intestinal epithelial cells

The role of gut microbiota in intestinal disease: from an oxidative stress perspective

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