Development of a biological containment strategy for recombinant Lactobacillus reuteri

历史上抗生素在农业和医学上的过度使用导致了细菌抗生素的增加 耐药性是21世纪世纪医疗保健的最大威胁之一。到目前为止，还没有任何战略可以 在不使用抗生素的情况下从复杂的微生物群落中根除病原体。工程益生菌 根除病原体提供了巨大的潜力。然而，为了确保病人的安全， 需要采取适当的策略，当 会出现并发症。在满足这一需求之前，临床应用微生物疗法杀死病原体 细菌将是不现实的。我们的长期目标是开发益生菌罗伊氏乳杆菌（Lr）， 靶向病原体的治疗递送载体。本申请的总体目标是开发一种高效的 生物防护平台我们的中心假设是，我们可以利用噬菌体（即噬菌体）， 通过CRISPR递送至重组Lr实现生物遏制。我们的假设是 基于我们的初步数据，证明与这项工作相关的噬菌体可以在体内感染Lr。 此外，我们实验室发表的工作证明了CRISPR-Cas在Lr中的功能。的理由 拟议的研究是，一旦我们开发了Lr的机器，我们的平台将是一个步进 Stone用于研究LR介导的抗菌治疗剂递送的R 01应用。我们计划完成 通过完成两个具体目标实现总体目标。在目标#1中，我们将开发重组人免疫球蛋白， 通过CRISPR-Cas对Lr的菌株特异性杀伤。Lr基因将被改造为编码CRISPR阵列， 工程公司将实施减少“逃跑者”的战略。在目标#2中，我们将优化噬菌体介导的 在胃肠道转运过程中杀死重组Lr，我们将评估我们的生物制剂的功能， 体内的密封系统。我们将确定在胃肠道中杀死Lr所需的最佳噬菌体浓度。 完成后，我们希望我们的工作将产生积极的影响，因为我们的杀死开关可以适应任何 细菌与裂解噬菌体，并带来了应用重组细菌治疗疾病更接近一步， 诊所重要的是，我们的生物遏制战略预计将是高度选择性的，因此不会造成 对微生物群的干扰。

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.