Abstract
New therapies are necessary to combat increasingly antibiotic-resistant bacterial pathogens. We have developed a technology platform of computational, molecular biology, and microbiology tools which together enable on-demand production of phages that target virtually any given bacterial isolate. Two complementary computational tools that identify and precisely map prophages and other integrative genetic elements in bacterial genomes are used to identify prophage-laden bacteria that are close relatives of the target strain. Phage genomes are engineered to disable lysogeny, through use of long amplicon PCR and Gibson assembly. Finally, the engineered phage genomes are introduced into host bacteria for phage production. As an initial demonstration, we used this approach to produce a phage cocktail against the opportunistic pathogen Pseudomonas aeruginosa PAO1. Two prophage-laden P. aeruginosa strains closely related to PAO1 were identified, ATCC 39324 and ATCC 27853. Deep sequencing revealed that mitomycin C treatment of these strains induced seven phages that grow on P. aeruginosa PAO1. The most diverse five phages were engineered for nonlysogeny by deleting the integrase gene (int), which is readily identifiable and typically conveniently located at one end of the prophage. The Δint phages, individually and in cocktails, killed P. aeruginosa PAO1 in liquid culture as well as in a waxworm (Galleria mellonella) model of infection.IMPORTANCE The antibiotic resistance crisis has led to renewed interest in phage therapy as an alternative means of treating infection. However, conventional methods for isolating pathogen-specific phage are slow, labor-intensive, and frequently unsuccessful. We have demonstrated that computationally identified prophages carried by near-neighbor bacteria can serve as starting material for production of engineered phages that kill the target pathogen. Our approach and technology platform offer new opportunity for rapid development of phage therapies against most, if not all, bacterial pathogens, a foundational advance for use of phage in treating infectious disease.
Highlights
New therapies are necessary to combat increasingly antibiotic-resistant bacterial pathogens
The first step in our approach is to use two complementary computational tools (Islander and TIGER) to identify and precisely map the prophages present in genome sequences of bacteria closely related to the pathogen
Preemptive creation of prophage databases in this manner allows for rapid turnaround in phage therapy cases where time is limited—the physician can use the pathogen’s genome sequence, 16S rRNA gene sequence, or multilocus sequence typing (MLST) sequences to place it on a phylogenetic tree, to identify close relatives bearing large numbers of prophages
Summary
New therapies are necessary to combat increasingly antibiotic-resistant bacterial pathogens. We have developed two complementary computational tools, Islander [13] and TIGER [12], that identify and precisely map IGEs within bacterial (and archaeal) genome sequences This software reveals the bacterial host, complete sequence, and precise ends of each prophage. Our powerful computational prophage prediction software facilitates engineering with existing methodologies [3, 14, 15] This set of computational, molecular biology, and microbiology tools together constitute a technology platform for on-demand production of phage therapies against bacterial pathogens (Fig. 1). As an initial demonstration of this approach, we produced five engineered lysogeny-disabled phages that kill Pseudomonas aeruginosa PAO1 in liquid culture as well as in a waxworm model of infection. We foresee application of this platform to develop prepared or on-demand phage collections to target nearly any bacterial pathogen or to control undesirable components of environmental or clinical microbiomes
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