Isolated Reporter Bacteria in Supramolecular Hydrogel Microwell Arrays

The combination of supramolecular hydrogels formed by low molecular weight gelator self-assembly via noncovalent interactions within a scaffold derived from polyethylene glycol (PEG) affords an interesting approach to immobilize fully functional, isolated reporter bacteria in novel microwell arrays. The PEG-based scaffold serves as a stabilizing element and provides physical support for the self-assembly of the C₂-phenyl-derived gelator on the micrometer scale. Supramolecular hydrogel microwell arrays with various shapes and sizes were used to isolate single or small numbers of Escherichia coli TOP10 pTetR-LasR-pLuxR-GFP. In the presence of the autoinducer <i>N</i>-(3-oxododecanoyl) homoserine lactone, the entrapped E. coli in the hydrogel microwell arrays showed an increased GFP expression. The shape and size of microwell arrays did not influence the fluorescence intensity and the projected size of the bacteria markedly, while the population density of seeded bacteria affected the number of bacteria expressing GFP per well. The hydrogel microwell arrays can be further used to investigate quorum sensing, reflecting communication in inter- and intraspecies bacterial communities for biology applications in the field of biosensors. In the future, these self-assembled hydrogel microwell arrays can also be used as a substrate to detect bacteria via secreted autoinducers

Reprogramming Probiotic <i>Lactobacillus reuteri</i> as a Biosensor for <i>Staphylococcus aureus</i> Derived AIP‑I Detection

Gram-positive <i>Staphylococcus aureus</i> infection that results in pneumonia, urinary tract infection, and in severe cases, sepsis, has recently been classified as a serious threat to public health. Rapid and cost-effective detection of these infections are costly and time-consuming. Here, we present probiotic lactic acid bacteria engineered to detect autoinducer peptide-I (AIP-I), a quorum sensing molecule produced by <i>Staphylococcus</i> sp. during pathogenesis. We achieved this by adapting the well-characterized <i>agr</i> quorum sensing (<i>agr</i>QS) from <i>Staphylococcus aureus</i> into <i>Lactobacillus reuteri</i>. The engineered biosensor is able to detect AIP-I levels in the nanomolar to micromolar range. We further investigated the function of the biosensor to detect real-time changes in AIP-I levels to understand the dynamics of <i>Staphylococcus aureus</i> under various strenuous conditions. The developed sensors would be useful for detection of <i>Staphylococcus</i> contamination in hospital settings and for high-throughput drug screening

Reprogramming Microbes to Be Pathogen-Seeking Killers

Recent examples of new genetic circuits that enable cells to acquire biosynthetic capabilities, such as specific pathogen killing, present an attractive therapeutic application of synthetic biology. Herein, we demonstrate a novel genetic circuit that reprograms Escherichia coli to specifically recognize, migrate toward, and eradicate both dispersed and biofilm-encased pathogenic Pseudomonas aeruginosa cells. The reprogrammed E. coli degraded the mature biofilm matrix and killed the latent cells encapsulated within by expressing and secreting the antimicrobial peptide microcin S and the nuclease DNaseI upon the detection of quorum sensing molecules naturally secreted by P. aeruginosa. Furthermore, the reprogrammed E. coli exhibited directed motility toward the pathogen through regulated expression of CheZ in response to the quorum sensing molecules. By integrating the pathogen-directed motility with the dual antimicrobial activity in E. coli, we achieved signifincantly improved killing activity against planktonic and mature biofilm cells due to target localization, thus creating an active pathogen seeking killer E. coli

An Engineered Probiotic Produces a Type III Interferon IFNL1 and Reduces Inflammations in <i>in vitro</i> Inflammatory Bowel Disease Models

The etiology of inflammatory bowel diseases (IBDs) frequently results in the uncontrolled inflammation of intestinal epithelial linings and the local environment. Here, we hypothesized that interferon-driven immunomodulation could promote anti-inflammatory effects. To test this hypothesis, we engineered probiotic Escherichia coli Nissle 1917 (EcN) to produce and secrete a type III interferon, interferon lambda 1 (IFNL1), in response to nitric oxide (NO), a well-known colorectal inflammation marker. We then validated the anti-inflammatory effects of the engineered EcN strains in two in vitro models: a Caco-2/Jurkat T cell coculture model and a scaffold-based 3D coculture IBD model that comprises intestinal epithelial cells, myofibroblasts, and T cells. The IFNL1-expressing EcN strains upregulated Foxp3 expression in T cells and thereafter reduced the production of pro-inflammatory cytokines such as IL-13 and -33, significantly ameliorating inflammation. The engineered strains also rescued the integrity of the inflamed epithelial cell monolayer, protecting epithelial barrier integrity even under inflammation. In the 3D coculture model, IFNL1-expressing EcN treatment enhanced the population of regulatory T cells and increased anti-inflammatory cytokine IL-10. Taken together, our study showed the anti-inflammatory effects of IFNL1-expressing probiotics in two in vitro IBD models, demonstrating their potential as live biotherapeutics for IBD immunotherapy

Hollow Fiber Membrane Decorated with Ag/MWNTs: Toward Effective Water Disinfection and Biofouling Control

The currently applied disinfection methods during water treatment provide effective solutions to kill pathogens, but also generate harmful byproducts, which are required to be treated with additional efforts. In this work, an alternative and safer water disinfection system consisting of silver nanoparticle/multiwalled carbon nanotubes (Ag/MWNTs) coated on a polyacrylonitrile (PAN) hollow fiber membrane, Ag/MWNTs/PAN, has been developed. Silver nanoparticles of controlled sizes were coated on polyethylene glycol-grafted MWNTs. Ag/MWNTs were then covalently coated on the external surface of a chemically modified PAN hollow fiber membrane to act as a disinfection barrier. A continuous filtration test using <i>E. coli</i> containing feedwater was conducted for the pristine PAN and Ag/MWNTs/PAN composite membranes. The Ag/MWNT coating significantly enhanced the antimicrobial activities and antifouling properties of the membrane against <i>E. coli</i>. Under the continuous filtration mode using <i>E. coli</i> feedwater, the relative flux drop over Ag/MWNTs/PAN was 6%, which was significantly lower than that over the pristine PAN (55%) at 20 h of filtration. The presence of the Ag/MWNT disinfection layer effectively inhibited the growth of bacteria in the filtration module and prevented the formation of biofilm on the surface of the membrane. Such distinctive antimicrobial properties of the composite membrane is attributed to the proper dispersion of silver nanoparticles on the external surface of the membrane, leading to direct contact with bacterium cells