Multidrug resistant [MDR] pathogens represent a global health threat and a challenge for modern medicine; and, as bacterial resistance to new antibiotics is now outpacing the antibiotic development effort, it is critical to develop new effective antimicrobial alternatives. The antimicrobial resistance crisis is bringing new interests worldwide to develop phage-based therapies. Over the last decade, efforts in the U.S. to produce phage therapeutics targeting different bacterial pathogens have shown promising results, including successful treatments of life-threatening infections in human patients. Safety of phage therapy is still a concern in the U.S.; however FDA has highlighted requirements for phage preparation: they need to be safe, pure, potent, exclusively lytic, non-transducing, and lacking undesirable genes (antibiotic resistance, virulence factors) and bacterial endotoxins. If bacteriophages for therapy have historically been isolated from natural environments, recent progresses in phage genetics and genome engineering have proven successful to generate synthetic, strictly lytic derivatives targeting pathogens. The development of synthetic phages against MDR pathogens would require pipelines to accelerate our knowledge on newly discovered phages and their potential for synthetic biology. Critical insights into their biology, i.e. genome structure, phage replication cycle, genetic content (essential genes versus dispensable [antibiotic resistance and virulence genes]), interaction with the target host, are a prerequisite. Here, we propose a platform to (i) mine the genomes of MDR pathogens, a gold mine to identify dormant lysogenic phages directly from within their natural host; and (ii) develop high-throughput pipelines to quickly gain knowledge on the phage biology to guide our efforts to engineer synthetic phages as therapeutics. We will use the Group A Streptococcus (GAS), a ?Concerning Threat? on the 2019 CDC ?18 MDR pathogens? Watch List, as our model. We showed that Tn-seq could identify functional lysogenic phages from cryptic ones in GAS genomes, and mutations to reboot dormant prophages into their lytic cycle.
In Aim 1, we will produce the critical knowledge to guide decision on what phages to select for therapeutic potential using synthetic biology: we will experimentally assess phage genome organization, phage replication/transduction mechanisms, host range and cell surface receptor(s).
In Aim 2, we will implement a design-build-test-learn cycle pipeline to optimize the synthetic biology effort, i.e. deletion of undesirable genes and addition of ?payload? genes, to enhance their potential as therapy phages. Finally, we will use in vivo model of wound infection to test the efficacy of the synthetic phages we generated. Our overarching goal is to develop the tools and experience to apply our synthetic biology phage-engineering platform to other MDR pathogens.
Preliminary Tn-seq analyses looking at Group A Streptococcus (GAS) essential genes identified lethal mutations found within prophages present in the bacterium chromosome: these correspond to genes for lytic cycle repressors; and our results show that it is possible to use Tn-seq to reboot dormant prophages from its bacterium host. The primary goal of this R21 is to develop a synthetic biology platform driven by Tn-seq results to engineer ?synthetic-lytic? phages with the goal to further characterize their biology using high throughput analyses, and to develop and produce safe, efficacious, optimized phages for therapy of GAS wound infections; and ultimately to apply this platform to treat multidrug-resistant infections.
