Engineering a Cortisol Sensing Enteric Probiotic.

Chronic stress can lead to prolonged adrenal gland secretion of cortisol, resulting in human ailments such as anxiety, post-traumatic stress disorder, metabolic syndrome, diabetes, immunosuppression, and cardiomyopathy. Real time monitoring of chronic increases in cortisol and intervening therapies to minimize the physiological effects of stress would be beneficial to prevent these endocrine related illnesses. Gut microbiota have shown the ability to secrete, respond, and even regulate endocrine hormones. One such microbe, Clostridium scindens, responds transcriptionally to cortisol. We engineered these cortisol responsive genetic elements from C. scindens into an enteric probiotic, E. coli Nissle 1917, to drive the expression of a fluorescent reporter allowing for the designing, testing, and building of a robust and physiologically relevant novel cortisol probiotic sensor. This smart probiotic was further engineered to be more sensitive and to respond to elevated cortisol by expressing tryptophan decarboxylase, thereby bestowing the ability to generate tryptamine and serotonin. Here we show that upon cortisol treatment the smart probiotic produces measurable amounts of tryptamine. Accumulated levels of these neuromodulators should improve mood, anxiety, and depression and drive down cortisol levels. Importantly, this work can serve as a model for the engineering of a sense-and-respond probiotic to modulate the gut-brain axis.

In Vitro Fermentation Evaluation of Engineered Sense and Respond Probiotics in Polymicrobial Communities.

Synthetic biology provides a means of engineering tailored functions into probiotic bacteria. Of particular interest is introducing microbial sense and response functions; however, techniques for testing in physiologically relevant environments, such as those for the intended use, are still lacking. Typically, engineered probiotics are developed and tested in monoculture or in simplified cocultures still within ideal environments. In vitro fermentation models using simplified microbial communities now allow us to simulate engineered organism behavior, specifically organism persistence and intended functionality, within more physiologically relevant, tailored microbial communities. Here, probiotic bacteria Escherichia coli Nissle and Lactobacillus plantarum engineered with sense and response functionalities were evaluated for the ability to persist and function without adverse impact on commensal bacteria within simplified polymicrobial communities with increasing metabolic competition that simulate gut microbe community dynamics. Probiotic abundance and plasmid stability, measured by viability qPCR, decreased for engineered E. coli Nissle relative to monocultures as metabolic competition increased; functional output was not affected. For engineered L. plantarum, abundance and plasmid stability were not adversely impacted; however, functional output was decreased universally as metabolic competition was introduced. For both organisms, adverse effects on select commensals were not evident. Testing engineered probiotics in more physiologically relevant in vitro test beds can provide critical knowledge for circuit design feedback and functional validation prior to the transition to more costly and time-consuming higher-fidelity testing in animal or human studies.

Evaluation of Human Performance Aiding Live Synthetically Engineered Bacteria in a Gut-on-a-Chip.

Synbiotics are a new class of live therapeutics employing engineered genetic circuits. The rapid adoption of genetic editing tools has catalyzed the expansion of possible synbiotics, exceeding traditional testing paradigms in terms of both throughput and model complexity. Herein, we present a simplistic gut-chip model using common Caco2 and HT-29 cell lines to establish a dynamic human screening platform for a cortisol sensing tryptamine producing synbiotic for cognitive performance sustainment. The synbiotic, SYN, was engineered from the common probiotic E. coli Nissle 1917 strain. It had the ability to sense cortisol at physiological concentrations, resulting in the activation of a genetic circuit that produces tryptophan decarboxylase and converts bioavailable tryptophan to tryptamine. SYN was successfully cultivated within the gut-chip showing log-phase growth comparable to the wild-type strain. Tryptophan metabolism occurred quickly in the gut compartment when exposed to 5 µM cortisol, resulting in the complete conversion of bioavailable tryptophan into tryptamine. The flux of tryptophan and tryptamine from the gut to the vascular compartment of the chip was delayed by 12 h, as indicated by the detectable tryptamine in the vascular compartment. The gut-chip provided a stable environment to characterize the sensitivity of the cortisol sensor and dynamic range by altering cortisol and tryptophan dosimetry. Collectively, the human gut-chip provided human relevant apparent permeability to assess tryptophan and tryptamine metabolism, production, and transport, enabled host analyses of cellular viability and pro-inflammatory cytokine secretion, and succeeded in providing an efficacy test of a novel synbiotic. Organ-on-a-chip technology holds promise in aiding traditional therapeutic pipelines to more rapidly down select high potential compounds that reduce the failure rate and accelerate the opportunity for clinical intervention.