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Abstract Interactions between epithelial and immune cells with the gut microbiota have wide-ranging effects on many aspects of human health. Therefore, there is value in developing in vitro models capable of performing highly controlled studies of such interactions. However, several critical factors that enable long term homeostasis between bacterial and mammalian cultures have yet to be established. In this study, we explored a model consisting of epithelial and immune cells, as well as four different bacterial species (Bacteroides fragilisKLE1958,Escherichia coliMG1655,Lactobacillus rhamnosusKLE2101, orRuminococcus gnavusKLE1940), over a 50 hour culture period. Interestingly, both obligate and facultative anaerobes grew to similar extents in aerobic culture environments during the co-culture period, likely due to measured microaerobic oxygen levels near the apical surface of the epithelia. It was demonstrated that bacteria elicited reactive oxygen species (ROS) production, and that the resulting oxidative damage heavily contributed to observed epithelial barrier damage in these static cultures. Introduction of a ROS scavenger significantly mitigated oxidative damage, improving cell monolayer integrity and reducing lipid peroxidation, although not to control (bacteria-free culture) levels. These results indicate that monitoring and mitigating ROS accumulation and oxidative damage can enable longer term bacteria-intestinal epithelial cultures, while also highlighting the significance of additional factors that impact homeostasis in mammalian cell-bacteria systems.
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Transcriptional programming in a Bacteroides consortium
Abstract Bacteroidesspecies are prominent members of the human gut microbiota. The prevalence and stability ofBacteroidesin humans make them ideal candidates to engineer as programmable living therapeutics. Here we report a biotic decision-making technology in a community ofBacteroides(consortium transcriptional programming) with genetic circuit compression. Circuit compression requires systematic pairing of engineered transcription factors with cognate regulatable promoters. In turn, we demonstrate the compression workflow by designing, building, and testing all fundamental two-input logic gates dependent on the inputs isopropyl-β-D-1-thiogalactopyranoside and D-ribose. We then deploy complete sets of logical operations in five human donorBacteroides, with which we demonstrate sequential gain-of-function control in co-culture. Finally, we couple transcriptional programs with CRISPR interference to achieve loss-of-function regulation of endogenous genes—demonstrating complex control over community composition in co-culture. This work provides a powerful toolkit to program gene expression inBacteroidesfor the development of bespoke therapeutic bacteria.
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- PAR ID:
- 10368716
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 13
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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