Historical over-use of antibiotics in agriculture and medicine has led to an increase in bacterial antibiotic resistance, which is one of the largest threats in 21st century healthcare. As of today, there are no strategies to eradicate pathogens from a complex microbial community without the use of antibiotics. Engineered probiotics to eradicate pathogens offers great potential. However, to ensure safety to the patients, biological containment strategies need to be in-place that can rapidly shut down production of the recombinant molecule(s) when complications would occur. Until this need is met, clinical applications of microbial therapeutics to kill pathogenic bacteria will be unrealistic. Our long-term goal is to develop the probiotic Lactobacillus reuteri (Lr) as a therapeutic delivery vehicle to target pathogens. The overall objective of this application is to develop an efficient biological containment platform. Our central hypothesis is that we can exploit bacteriophages (i.e. phages) to achieve biological containment via CRISPR delivery to recombinant Lr. Our hypothesis has been formulated based on our preliminary data, demonstrating that the bacteriophages pertinent to this work can infect Lr in vivo. Also, published work from our laboratory demonstrated functionality of CRISPR-Cas in Lr. The rationale for the proposed research is, once we have developed the phages as Lr killing-machines, our platform will be a stepping stone for R01 applications studying Lr-mediated delivery of antimicrobial therapeutics. We plan to accomplish the overall objective by completing two specific aims.
In Aim #1 we will develop recombinant phages to achieve strain-specific killing of Lr via CRISPR-Cas. Lr phages will be engineered to encode CRISPR arrays targeting engineered Lr. Strategies will be implemented to reduce `escapers'.
In Aim #2 we will optimize phage-mediated killing of recombinant Lr during gastrointestinal transit, and we will assess functionality of our biological containment system in vivo. We will determine the optimized phage concentration needed kill Lr in the GI tract. Upon completion, we expect our work will have a positive impact because our kill switch can be adapted to any bacterium with a lytic phage, and brings the application of recombinant bacteria to treat disease a step closer to the clinic. Important, our biological containment strategy is expected to be highly selective, thus not causing perturbations to the microbiota.
Our proposal is relevant to human health as it pertains the development of a biological containment strategy for recombinant probiotic Lactobacillus reuteri. The project is relevant to NIH's mission because this work is criti- cally important to fulfill the promise to exploit tailored probiotics to treat disease in humans.
