Engineered Probiotic for the Inhibition of Salmonella via Tetrathionate-Induced Production of Microcin H47

Abstract

Complications arising from antibiotic-resistant bacteria are becoming one of the key issues in modern medicine. Members of drug-resistant Enterobacteriaceae spp. include opportunistic pathogens (e.g., Salmonella spp.) that are among the leading causes of morbidity and mortality worldwide. Overgrowth of these bacteria is considered a hallmark of intestinal dysbiosis. Microcins (small antimicrobial peptides) produced by some gut commensals can potentially cure these conditions by inhibiting these pathogens and have been proposed as a viable alternative to antibiotic treatment. In this proof-of-concept work, we leverage this idea to develop a genetically engineered prototype probiotic to inhibit Salmonella spp. upon exposure to tetrathionate, a molecule produced in the inflamed gut during the course of Salmonella infection. We developed a plasmid-based system capable of conferring the ability to detect and utilize tetrathionate, while at the same time producing microcin H47. We transferred this plasmid-based system to Escherichia coli and demonstrated the ability of the engineered strain to inhibit growth of Salmonella in anaerobic conditions while in the presence of tetrathionate, with no detectable inhibition in the absence of tetrathionate. In direct competition assays between the engineered E. coli and Salmonella, the engineered E. coli had a considerable increase in fitness advantage in the presence of 1 mM tetrathionate as compared to the absence of tetrathionate. In this work, we have demonstrated the ability to engineer a strain of E. coli capable of using an environmental signal indicative of intestinal inflammation as an inducing molecule, resulting in production of a microcin capable of inhibiting the organism responsible for the inflammation.

Developing a new class of engineered live bacterial therapeutics to treat human diseases

“…In some instances, it may be preferable to activate effector functions under more specific conditions, rather than constitutively throughout the gastrointestinal tract. For example, coupling gene expression to biosensors of reactive oxygen and nitrogen species for the treatment of inflammatory bowel disease may help deliver effectors specifically where activity is beneficial 7,28 . Regulating effector expression in response to bacterial quorum sensing molecules 29 , pH 30 , specific carbon sources [31][32][33] , temperature 34 , or combinations of these signals may allow for exquisitely tuned effector functions in various intestinal microenvironments 7 .…”

“…demonstrated that the EcN-based gastrointestinal delivery of anti-biofilm enzyme, dispersin B (DspB), resulted in a reduction of pre-colonized P. aeruginosa abundance in both nematode and murine models 29 . Other groups have reported the successful production of antimicrobial peptides from EcN that are effective for significantly decreasing murine colonization by Enterococcal species 36 or Salmonella typhimurium 28 . In the face of increasingly prevalent antibiotic-resistant pathogens, novel EcN-based antimicrobials offer promise for the treatment of infections caused by organisms recalcitrant to traditional approaches.…”

Adaptive Strategies of the Candidate Probiotic E. coli Nissle in the Mammalian Gut

“…Probiotics are live microorganisms that provide health benefits when consumed, including enhancement of gut epithelial barrier function, blocking of pathogen binding, and vitamin synthesis (Hill et al, 2014). There has been increasing success in expanding the therapeutic scope and efficacy of probiotics through genetic engineering, with prior work demonstrating preclinical efficacy against infectious and metabolic diseases (Durrer et al, 2017;Hwang et al, 2017;Isabella et al, 2018;Palmer et al, 2018). Engineered probiotics are exciting platforms for in situ drug synthesis and delivery, provided they maintain appropriate abundance and activity at their target site.…”

“…We focus on the probiotic Escherichia coli Nissle 1917 (EcN), an E. coli clade B2 commensal ( Figure S1). EcN has a long history of use in humans (Westendorf et al, 2005), has demonstrated efficacy against inflammatory bowel disease (Scaldaferri et al, 2016), and has been gaining increased attention as a chassis for engineered function (Hwang et al, 2017;Isabella et al, 2018;Kurtz et al, 2019;Palmer et al, 2018). We expected several factors to drive adaptation of EcN in the mouse gut.…”

Engineering probiotics as living diagnostics and therapeutics for improving human health

“…As an innovative and alternative measure to treat infectious diseases, probiotic therapy contributes to reduction of the development of multi-resistant bacteria. It has been proposed that probiotics can inhibit pathogens by different mechanisms, including secretion of antibacterial chemicals, stimulation and modulation of the immune responses, competition of nutrition and specific adhesion sites, and inhibition of toxic protein expression in gastrointestinal pathogens [59][60][61][62]. According to the mechanistic basis, various probiotics have been developed, and most of them demonstrated great specificity and efficacy for the inhibition of pathogens.…”