Biology subjects

Bentley, W. E.

Publications and source records attributed to Bentley, W. E..

7 recordsLinked to original sources

bioRxiv2025

Genetically-programmed Hypervesiculation of Lactiplantibacillus plantarum Increases Production of Bacterial Extracellular Vesicles with Therapeutic Efficacy in a Preclinical Inflammatory Bowel Disease Model

Inflammatory bowel diseases (IBD) affect over 6 million people globally and current treatments achieve only 10-20% rates of durable disease remission. Bacterial extracellular vesicles (BEVs) from probiotic lactic acid bacteria (LAB) are a promising novel therapeutic with mechanisms holding potential to drive increased rates of durable disease remission, including immunomodulation and intestinal epithelial tissue repair. However, translation of these cell-secreted nanovesicles is limited by long standing biomanufacturing hurdles, especially low production yields due to low biogenesis rates from cells. Here, our goal was to identify a candidate probiotic LAB that produces BEVs effective in a preclinical mouse model of IBD, and then genetically engineer the LAB for at least 10-fold increased production yields of BEVs, thereby passing a critical production threshold. We identified Lactiplantibacillus plantarum as a candidate LAB producing BEVs effective in treating acute dextran sulfate sodium (DSS)-induced murine colitis, and with greater efficacy than BEVs from probiotic Escherichia coli Nissle 1917. We then genetically engineered a hypervesiculating L. plantarum strain by inducible expression of a peptidoglycan-modifying enzyme, resulting in a 66-fold increase in BEV productivity. Finally, we confirmed hypervesiculating L. plantarum BEVs were therapeutically effective in the acute DSS mouse model of colitis and found these BEVs were superior in reducing mucosal tissue damage compared to live L. plantarum cells. These findings demonstrate that BEVs from genetically engineered hypervesiculating strain of L. plantarum are a promising preclinical therapeutic candidate for IBD that overcomes historical biomanufacturing limitations of BEV therapeutics.

bioengineering↗

bioRxiv2024

Yersinia pseudotuberculosis growth arrest during type-III secretion system expression is associated with altered ribosomal protein expression and decreased gentamicin susceptibility

It has been long appreciated that expression of the Yersinia type-III secretion system (T3SS) in culture is associated with growth arrest. Here we sought to understand whether this impacts expression of ribosomal protein genes, which were among the most highly abundant transcripts in exponential phase Yersinia pseudotuberculosis based on RNA-seq analysis. To visualize changes in ribosomal protein expression, we generated a fluorescent transcriptional reporter with the promoter upstream of rpsJ/S10 fused to a destabilized gfp variant. We confirmed reporter expression significantly increases in exponential phase and decreases as cells transition to stationary phase. We then utilized a mouse model of systemic Y. pseudotuberculosis infection to compare T3SS and S10 reporter expression during clustered bacterial growth in the spleen, and found that cells expressing high levels of the T3SS had decreased S10 levels, while cells with lower T3SS expression retained higher S10 expression. In bacteriological media, growth inhibition with T3SS induction and a reduction in S10 expression were observed in subsets of cells, while cells with high expression of both T3SS and S10 were also observed. Loss of T3SS genes resulted in rescued growth and heightened S10 expression. To understand if clustered growth impacted bacterial gene expression, we utilized droplet-based microfluidics to encapsulate bacteria in spherical agarose droplets, and also observed growth inhibition with high expression of T3SS and reduced S10 levels that better mirrored phenotypes observed in the mouse spleen. Finally, we show that T3SS expression is sufficient to promote tolerance to the ribosome-targeting antibiotic, gentamicin. Collectively, these data indicate that the growth arrest associated with T3SS induction leads to decreased expression of ribosomal protein genes, and this results in reduced antibiotic susceptibility. Author SummarySlow-growing bacterial cells have reduced antibiotic susceptibility, rendering them very difficult to eliminate during antibiotic treatment. However, for many key virulence factors (bacterial factors required to promote infection), it remains unclear whether expression is sufficient to slow bacterial growth and impact antibiotic susceptibility. Using Yersinia pseudotuberculosis, we found ribosomal protein expression fluctuated based on growth rate, and we generated a fluorescent reporter construct to detect altered ribosomal protein expression within individual bacterial cells. We then asked if expression of a key virulence factor in Yersinia, the type-III secretion system (T3SS), is sufficient to lower ribosomal protein expression, since it has been well established that T3SS induction results in growth arrest. We found high levels of T3SS expression promotes slowed growth and antibiotic tolerance, and bacterial cells that survive treatment with a ribosome-targeting antibiotic, gentamicin, have heightened levels of T3SS and lower levels of S10 expression.

microbiology↗

bioRxiv2023

Detecting Features of Protein Structure through their Mediator-Accessible Redox Activities

Protein function relies on sequence, folding, and post-translational modification, and molecular measurements are commonly used to reveal these structural features. Here, we report an alternative approach that represents these molecular features as readily measurable electronic patterns and validate this experimental approach by detecting structural perturbations commonly encountered during protein biomanufacturing. Specifically, we studied a monoclonal antibody standard (NISTmAb) and focused on the electronic detection of variants that have undergone interchain-disulfide bond reduction and methionine oxidation. Electronic detection of these structural perturbations is based on mediated electrochemical probing (MEP) that discerns patterns associated with the antibodys mediator-accessible redox activity. We demonstrate that MEP can rapidly (within minutes) and quantitatively transduce the proteins structural features into robust electronic signals that can enable bioprocess monitoring and control. More broadly, the ability to transduce information of a proteins molecular structure into a more convenient electronic domain offers fundamentally new opportunities to apply the power of microelectronics and real-time data analytics to chemical and biological analysis.

bioengineering↗

bioRxiv2023

Redox Active Plant Phenolic, Acetosyringone, for Electrogenetic Signaling

Redox is a unique, programmable modality capable of bridging communication between biology and electronics. Previous studies have shown that the E. coli redox-responsive OxyRS regulon can be re-wired to accept electrochemically generated hydrogen peroxide (H2O2) as an inducer of gene expression. Here we report that the redox-active phenolic plant signaling molecule acetosyringone (AS) can also induce gene expression from the OxyRS regulon. AS must be oxidized, however, as the reduced state present under normal conditions cannot induce gene expression. Thus, AS serves as a "pro-signaling molecule" that can be activated by its oxidation - in our case by application of oxidizing potential to an electrode. We show that the OxyRS regulon is not induced electrochemically if the imposed electrode potential is in the mid-physiological range. Electronically sliding the applied potential to either oxidative or reductive extremes induces this regulon but through different mechanisms: reduction of O2 to form H2O2 or oxidation of AS. Fundamentally, this work reinforces the emerging concept that redox signaling depends more on molecular activities than molecular structure. From an applications perspective, the creation of an electronically programmed "pro-signal" dramatically expands the toolbox for electronic control of biological responses in microbes, including in complex environments, cell-based materials, and biomanufacturing.

bioengineering↗

bioRxiv2023

Redox-enabled Electronic Interrogation and Feedback Control of Hierarchical and Networked Biological Systems

We enable microelectronic devices to interrogate biologys molecular communication, perform computations, and in real time control biological systems, including at several hierarchical levels: proteins, cells, and cell consortia. A key driver is establishing electronic access to and from biologys native redox networks. First, redox-mediated electro-biofabrication facilitates facile assembly of biological components onto microelectronic systems, then electrode-actuated redox allows digital programming of enzyme activity, and further, redox-mediated electrogenetics facilitates closed-loop electronic control of cellular function. Specifically, we show algorithm-based feedback control of enzyme activity, cellular genetic circuits (via eCRISPR) and cell consortia behavior, all enabled by electronic data transfer. We further demonstrate electronic switching of cell-cell quorum sensing communication from one autoinducer network to another, creating an electronically controlled "bilingual" cell. We suggest these methodologies will not only help us to better understand biological systems, but design and control those currently unimagined.

synthetic biology↗

bioRxiv2023

High performance anion exchange chromatography purification of probiotic bacterial extracellular vesicles enhances purity and anti-inflammatory efficacy

Bacterial extracellular vesicles (BEVs), including outer membrane vesicles (OMVs), have emerged as a promising new class of vaccines and therapeutics to treat cancer and inflammatory diseases, among other applications. However, clinical translation of BEVs is hindered by a current lack of scalable and efficient purification methods. Here, we address downstream BEV biomanufacturing limitations by developing a method for orthogonal size- and charge-based BEV enrichment using tangential flow filtration (TFF) in tandem with high performance anion exchange chromatography (HPAEC). The data show that size-based separation co-isolated protein contaminants, whereas size-based TFF with charged-based HPAEC dramatically improved purity of BEVs produced by probiotic Gram-negative Escherichia coli and Gram-positive lactic acid bacteria (LAB). E. coli BEV purity was quantified using established biochemical markers while improved LAB BEV purity was assessed via observed potentiation of anti-inflammatory bioactivity. Overall, this work establishes orthogonal TFF + HPAEC as a scalable and efficient method for BEV purification that holds promise for future large-scale biomanufacturing of therapeutic BEV products.

bioengineering↗

bioRxiv2022

Electrogenetic signaling and information propagation for controlling microbial consortia via programmed lysis

To probe signal propagation and genetic actuation in microbial consortia, we have coopted the components of both redox and quorum sensing (QS) signaling into a communication network for guiding composition by "programming" cell lysis. Here, we use an electrode to generate hydrogen peroxide as a redox cue that determines consortia composition. The oxidative stress regulon of Escherichia coli, OxyR, is employed to receive and transform this signal into a QS signal that coordinates the lysis of a subpopulation of cells. We examine a suite of information transfer modalities including "monoculture" and "transmitter-receiver" models, as well as a series of genetic circuits that introduce time-delays for altering information relay, thereby expanding design space. A simple mathematical model aids in developing communication schemes that accommodate the transient nature of redox signals and the "collective" attributes of QS signals. We suggest this platform methodology will be useful in understanding and controlling synthetic microbial consortia for a variety of applications, including biomanufacturing and biocontainment.

synthetic biology↗