Bacteria can be genetically engineered to kill specific pathogens or inhibit their virulence. We previously developed a synthetic genetic system that allows a laboratory strain of Escherichia coli to ...sense and kill Pseudomonas aeruginosa in vitro. Here, we generate a modified version of the system, including a gene encoding an anti-biofilm enzyme, and use the probiotic strain Escherichia coli Nissle 1917 as host. The engineered probiotic shows in vivo prophylactic and therapeutic activity against P. aeruginosa during gut infection in two animal models (Caenorhabditis elegans and mice). These findings support the further development of engineered microorganisms with potential prophylactic and therapeutic activities against gut infections.
Abstract
Clostridioides difficile
infection (CDI) results in significant morbidity and mortality in hospitalised patients. The pathogenesis of CDI is intrinsically related to the ability of
C. ...difficile
to shuffle between active vegetative cells and dormant endospores through the processes of germination and sporulation. Here, we hypothesise that dysregulation of microbiome-mediated bile salt metabolism contributes to CDI and that its alleviation can limit the pathogenesis of CDI. We engineer a genetic circuit harbouring a genetically encoded sensor, amplifier and actuator in probiotics to restore intestinal bile salt metabolism in response to antibiotic-induced microbiome dysbiosis. We demonstrate that the engineered probiotics limited the germination of endospores and the growth of vegetative cells of
C. difficile
in vitro and further significantly reduced CDI in model mice, as evidenced by a 100% survival rate and improved clinical outcomes. Our work presents an antimicrobial strategy that harnesses the host-pathogen microenvironment as the intervention target to limit the pathogenesis of infection.
The skin microbiome plays a central role in inflammatory skin disorders such as atopic dermatitis (AD). In AD patients, an imbalance between pathogenic Staphylococcus aureus (S. aureus) and resident ...skin symbionts creates a state of dysbiosis which induces immune dysregulation and impairs skin barrier function. There are now exciting new prospects for microbiome‐based interventions for AD prevention. In the hopes of achieving sustained control and management of disease in AD patients, current emerging biotherapeutic strategies aim to harness the skin microbiome associated with health by restoring a more diverse symbiotic skin microbiome, while selectively removing pathogenic S. aureus. Examples of such strategies are demonstrated in skin microbiome transplants, phage‐derived anti‐S. aureus endolysins, monoclonal antibodies, and quorum sensing (QS) inhibitors. However, further understanding of the skin microbiome and its role in AD pathogenesis is still needed to understand how these biotherapeutics alter the dynamics of the microbiome community; to optimize patient selection, drug delivery, and treatment duration; overcome rapid recolonization upon treatment cessation; and improve efficacy to allow these therapeutic options to eventually reach routine clinical practice.
Novel approaches to modify the host skin bacteria (microbiome) provide exciting avenues for prevention and treatment of inflammatory skin diseases like atopic dermatitis. This review summarizes the emerging bio‐engineering strategies which aim to harness the skin microbiome associated with health to restore a healthy skin bacterial communities, while selectively removing harmful skin bacteria.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The proteasome inhibitor MG132 had been shown to prevent galactose induction of the S. cerevisiae GAL1 gene, demonstrating that ubiquitin proteasome-dependent degradation of transcription factors ...plays an important role in the regulation of gene expression. The deletion of the gene encoding the F-box protein Mdm30 had been reported to stabilize the transcriptional activator Gal4 under inducing conditions and to lead to defects in galactose utilization, suggesting that recycling of Gal4 is required for its function. Subsequently, however, it was argued that Gal4 remains stably bound to the enhancer under inducing conditions, suggesting that proteolytic turnover of Gal4 might not be required for its function. We have performed an alanine-scanning mutagenesis of ubiquitin and isolated a galactose utilization-defective ubiquitin mutant. We have used it for an unbiased suppressor screen and identified the inhibitor Gal80 as a suppressor of the transcriptional defects of the ubiquitin mutant, indicating that the protein degradation of the inhibitor Gal80, and not of the activator Gal4, is required for galactose induction of the GAL genes. We also show that in the absence of Gal80, Mdm30 is not required for Gal4 function, strongly supporting this hypothesis. Furthermore, we have found that Mediator controls the galactose-induced protein degradation of Gal80, which places Mediator genetically upstream of the activator Gal4. Mediator had originally been isolated by its ability to respond to transcriptional activators, and here we have discovered a leading role for Mediator in the process of transcription. The protein kinase Snf1 senses the inducing conditions and transduces the signal to Mediator, which initiates the degradation of the inhibitor Gal80 with the help of the E3 ubiquitin ligase SCF(Mdm30). The ability of Mediator to control the protein degradation of transcriptional inhibitors indicates that Mediator is actually able to direct its own recruitment to gene promoters.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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IJS, KILJ, NUK, PNG, UL, UM
Abstract The discovery of antimicrobial drugs and their subsequent use has offered an effective treatment option for bacterial infections, reducing morbidity and mortality over the past 60 years. ...However, the indiscriminate use of antimicrobials in the clinical, community and agricultural settings has resulted in selection for multidrug-resistant bacteria, which has led to the prediction of possible re-entrance to the pre-antibiotic era. The situation is further exacerbated by significantly reduced antimicrobial drug discovery efforts by large pharmaceutical companies, resulting in a steady decline in the number of new antimicrobial agents brought to the market in the past several decades. Consequently, there is a pressing need for new antimicrobial therapies that can be readily designed and implemented. Recently, it has become clear that the administration of broad-spectrum antibiotics can lead to collateral damage to the human commensal microbiota, which plays several key roles in host health. Advances in genetic engineering have opened the possibility of reprogramming commensal bacteria that are in symbiotic existence throughout the human body to implement antimicrobial drugs with high versatility and efficacy against pathogenic bacteria. In this review, we discuss recent advances and potentialities of engineered bacteria in providing a novel antimicrobial strategy against antibiotic resistance.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPUK, ZRSKP
Antimicrobial resistance is an emerging phenomenon that undermines current antimicrobial therapies and leads to higher economic and healthcare costs. Constant development of novel antimicrobial ...strategies are needed to counter this rising challenge. The human microbiome presents an avenue for reconstituted next generation probiotics to provide in situ treatment and prevention against pathogenic infections. Herein, probiotic Escherichia coli Nissile 1917 strain is engineered through synthetic biology principles into novel antimicrobial therapeutics capable of targeting pathogenic infections of the human gastrointestinal tract.In this doctoral research thesis, four projects were completed under two themes - Tools Development and Antimicrobial Strategies, towards the aim of developing non-antibiotic-based probiotics engineering approaches against gastrointestinal infections. Specifically, strategies were devised to target Pseudomonas aeruginosa and Clostridium difficile infections, two common opportunistic nosocomial infections that occur in the human gastrointestinal tracts.A probiotic expression system with self-selecting plasmid capability was developed through strain engineering (Chapter 3). This system was further engineered and tested for the treatment of P. aeruginosa infection in Caenorhabditis elegans animal model (Chapter 4). Next, the probiotic was equipped with an engineered biosensor for the detection of dysbiosis in the lower gastrointestinal tract (Chapter 5). A novel prophylactic antimicrobial strategy against C. difficile infection was then developed through utilisation of this dysbiosis biosensor (Chapter 6).Toolkit available for synthetic biology engineering of probiotics was expanded through these projects. The probiotic expression system and dysbiosis biosensor were optimised for functioning in the lower gastrointestinal tract. Antimicrobial strategies that utilised the tools for targeting P. aeruginosa and C. difficile were validated in vivo and in vitro respectively. These results provide proof-of-concept for employing engineered probiotics as antimicrobial strategies for the treatment and prevention of gastrointestinal infections. They can be further developed for clinical application as alternatives to antibioticbased therapies, and thereby providing potential solutions to the issue of emerging antimicrobial resistance.
The impact of advanced age on the optimal duration of dual antiplatelet therapy (DAPT) in patients with acute coronary syndrome (ACS) undergoing percutaneous coronary revascularization (PCI) is still ...greatly debated. Therefore, the aim of the present sub-analysis of the REDUCE trial was to assess the impact of age on the comparison between a short 3 months vs standard 12 months DAPT in ACS patients treated with the COMBO Dual Stent Therapy.
The REDUCE trial is a prospective, multicenter, investigator-initiated study that randomized ACS patients undergoing PCI with the COMBO drug eluting stent to either 3 or 12 months of DAPT. The study population was divided according to age (<or ≥ 75 years). Primary study endpoint was a composite of all-cause mortality, myocardial infarction, definite/probable stent thrombosis (ST), stroke, target-vessel revascularization (TVR) and bleeding (BARC II, III, V) at 12 months. Secondary endpoints were cardiovascular mortality and the individual components of the primary endpoint within 24 months.
From June 2014 to May 2016, 1496 patients were included in the study, of whom 205 (13.7%) ≥75 years of age. Among them, 50.7% of the elderly and 50.2% of younger patients were assigned to the 3-month DAPT treatment. Baseline characteristics were well matched between the two arms, except for a higher rate of males (p=0.02) and a reduced number of lesions on the right coronary artery (p=0.02) in elderly patients treated for the short DAPT duration. Median follow-up was 682.5 days IQR:667-731. At 12 months, no difference in the primary endpoint was observed according to DAPT duration in both patients aged ≥75 years (22.1% vs 18.8%, HR 95%CI = 1.6 0.73–3.5, p=0.24) and younger ones (9.7% vs 10.9%, HR 95%CI = 0.85 0.59–1.27, p=0.44; p INT = 0.15). Results were confirmed after correction for baseline differences among the elderly (adjusted HR 95%CI = 1.7 0.75–3.9, p=0.21). Comparable rates of survival, thrombotic (MI, stent thrombosis, TVR, stroke) and bleeding events were observed with the two DAPT strategies, with no impact of age.
The present study shows that among ACS patients randomized in the REDUCE trial, a 3-month DAPT strategy was comparable to a standard 12-month DAPT at a 2-year follow-up for both ischemic and bleeding endpoints, in elderly and younger patients. Thus, despite presenting the limitations of a subgroup analysis, our study strengthens the feasibility of a shorter DAPT duration even among high-risk subsets of ACS patients.
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•Advanced age enhances the bleeding risk associated with dual antiplatelet therapy.•The optimal duration of dual antiplatelet therapy in elderly patients with acute coronary syndromes (ACS) is debated.•The REDUCE trial compared 3 vs 12 months DAPT in ACS patients treated with the COMBO Dual Stent Therapy.•We provide a sub-analysis of the REDUCE trial according to age.•No age-related difference was observed in the primary composite or individual thrombotic and bleeding endpoints at 12 months.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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