Engineering probiotics to inhibit Clostridioides difficile infection by dynamic regulation of intestinal metabolism.

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.

Biotherapeutic Approaches in Atopic Dermatitis.

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.

Engineered probiotic Escherichia coli can eliminate and prevent Pseudomonas aeruginosa gut infection in animal models.

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.

Reprogrammable microbial cell-based therapeutics against antibiotic-resistant bacteria.

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.

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.

Mediator acts upstream of the transcriptional activator Gal4.

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.