Toxin-Free Host-Independent Synthesis of Bacteriophage of Gram-Negative Pathogens
Abstract
This proposal will address the Topic Area: Antimicrobial Resistance using synthetic biology tools to advance broader adoption of phage therapy as an alternative to small antibiotics. Background: Multidrug-resistant organisms (MDROs) pose one of the greatest emerging health threats to our military and civilian populations. Among the most challenging MDROs is Acinetobacter baumanii (AB), aka “Iraqibacter” because of its prevalence in battle-wound infections among Soldiers of the Iraq/Afghanistan campaigns. Phage therapy has the potential to cure and prevent bacterial infections that are otherwise resistant to the most commonly used antibiotics. Phages are viruses prevalent in the environment that prey upon bacteria, hijacking their host’s transcription and translation (TXTL) machinery to replicate. Unlike traditional antibiotics, therapeutic phage can specifically target and kill pathogenic strains of host bacteria while leaving “good” gut bacteria intact. Indeed, personalized phage therapy has been used to successfully treat systemic multidrug-resistant A. baumanii (MDRAB) infection in humans and wound infections in a murine model. Rationale: Despite positive outlook, wide-scale adoption of phage therapy faces several obstacles. Endotoxin contamination: Endotoxin is an immunogenic structural component of the outer cell-membrane of Gram-negative bacteria, such as A. baumanii. Crude phage of Gram-negative bacteria must be purified of endotoxin prior to in vivo application, as this contaminant can induce septic shock. Endotoxin cleanup is a time-consuming and inconsistent process, often dependent on individual phage characteristics. Narrow host range: Phage specificity for their hosts is advantageous for targeted antimicrobial activity, but introduces limitations to phage production. Without the appropriate host strain on hand, a particular phage cannot be produced. Cold-chain dependence: Shelf-stability of phage varies widely from strain to strain, making just-in-time production of personalized phage therapy followed by endotoxin cleanup the current paradigm. Innovation: The objective of this proposal is to overcome phage production bottlenecks by engineering universal endotoxin-free cell-free expression systems optimized for phage synthesis. After demonstrating proof of concept by producing MDRAB-phage, this platform can easily be adapted to synthesize infectious phage of any MDRO, regardless of host. This system will be shelf-stable to enable point-of-care production of phage. We expect to create a “just-add-water” kit for ready-to-use, personalized, endotoxin-free phage therapies. Benefits to Military, Department of Veterans Affairs (VA), and Civilian Patient Populations. Point-of-care phage production will enable cold-chain bypass, establishing a new paradigm for personalized phage therapy that cuts lead time from weeks to days. Using MDRAB-phage as a model system, this work will lead to the development of phage synthesis platforms that will improve access to phage therapy for any bacterial MDRO. Impact: Uncoupling phage production from bacterial culture will make phage therapy better than ever to fight MDROs. Combining rational design of CFES, phage engineering, and point-of-care production for personalized phage therapy will make it possible to treat any MDRO infection, anywhere. This platform will not only enable easier, more rapid access to characterized phage, but it will also provide a pipeline for novel engineered phage with new properties. Importantly, phage therapy presents a pathway to combat the drug-resistance crisis by providing targeted alternatives to traditional antibiotics.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Mar 10, 2021
- Source ID
- W81XWH2010071
Entities
People
- Lisa A Morici
Organizations
- Tulane University of Louisiana
- United States Army