Search for: All records

Multidrug-resistant bacteria necessitate innovative antibacterial strategies. Bacteriophages (phages) offer a promising alternative; however, bacterial immune defenses limit their effectiveness. Small-molecule inhibitors of these defenses may facilitate mechanistic studies and serve as adjuvants to enhance phage therapy. Here, we identify inhibitors targeting the bacterial cyclic oligonucleotide-based anti-phage signaling system (CBASS) effector nuclease Cap5. Cap5 is hypothesized to degrade genomic DNA in virally infected cells, leading to cell death through abortive infection. Guided by the crystal structure of the Cap5 SAVED domain bound to its activating ligand, we performed structure-guided virtual screening to identify candidate inhibitors. Biochemical assays revealed ∼16% of the top docking hits inhibited Cap5. Cellular assays revealed one compound could enter E. coli cells and inhibit Cap5 activity. Our integrated approach—combining structure-based virtual screening with biochemical validation—provides a framework for discovering small-molecule inhibitors of bacterial immune defenses to advance adjunctive therapies and deepen our understanding of phage-bacteria interactions. 

ABSTRACT Multidrug-resistant (MDR) bacteria pose a significant public health challenge, underscoring the urgent need for innovative antibacterial strategies. Bacteriophages (phages), viruses that specifically target bacteria, offer a promising alternative; however, bacterial immune defenses often limit their effectiveness. Developing small-molecule inhibitors of these defenses can facilitate mechanistic studies and serve as adjuvants to enhance phage therapy. Here, we identify novel inhibitors targeting the bacterial cyclic oligonucleotide-based anti-phage signaling system (CBASS) effector Cap5. Cap5 is an HNH endonuclease activated by a cyclic nucleotide to degrade genomic DNA in virally infected cells, leading to cell death through abortive infection. Guided by the crystal structure of the Cap5 SAVED domain bound to its activating ligand, we performed structure-guided virtual screening to identify candidate inhibitors. Biochemical assays revealed that approximately 16% of the top docking hits exhibited inhibitory activity. Further cellular assays demonstrated that one potent compound could enterE. colicells and inhibit Cap5 activity. Our integrated approach—combining structure-based virtual screening with biochemical validation—provides a robust framework for discovering small-molecule inhibitors of bacterial immune defenses to advance adjunctive therapies and deepen our understanding of phage-bacteria interactions. 

The rise of antibiotic resistance motivates a revived interest in phage therapy. However, bacteria possess dozens of anti-phage immune systems that confer resistance to therapeutic phages. Chemical inhibitors of these anti-phage immune systems could be employed as adjuvants to overcome resistance in phage-based therapies. Here, we report a class of chemical inhibitors that selectively inhibit type II Thoeris anti-phage immune systems from diverse bacteria—including antibiotic-resistant pathogens, thereby sensitizing phage-resistant bacteria to phages. These inhibitors block the biosynthesis of a histidine-ADPR intracellular “alarm” signal by ThsB, thereby preventing ThsA from arresting phage replication. Chemical inhibition of the Thoeris defense improves the efficacy of a model phage therapy against a phage-resistant clinical isolate of P. aeruginosa in a mouse infection, suggesting a therapeutic potential. These findings demonstrate that the selective inhibition of anti-phage defense systems can improve the efficacy of therapeutic phages, suggesting a strategy to circumvent phage-therapy resistance. 

Many antiviral immune systems have recently been discovered in bacteria. The mechanisms of several are obscure, as are their individual significance for antiphage defense. To shed light on the mechanism and significance of the two-component type I Thoeris antiphage immune system, we leveraged high-throughput phenotypic screening to identify three small molecule inhibitors. The inhibitors target the ThsA NADase component, inhibiting its 3ʹ-cADPR-activated filamentation. The temporal control afforded by the small-molecule inhibitors allowed us to answer an outstanding question in antiviral immunity—is persistent immunity required to repress phage titers, or do immune systems become unnecessary after eradicating infectious phages? We found that Thoeris immunity must be maintained, as chemical inhibition enabled repressed phages to revive and overtake the bacterial population. Furthermore, due to the cooperative nature of antiviral immunity, we found that Thoeris must be inhibited in only 10% of the bacteria to cause phage-induced lysis of the entire population. 

Abstract The rise of antibiotic resistance motivates a revived interest in phage therapy. However, bacteria possess dozens of anti-bacteriophage immune systems that confer resistance to therapeutic phages. Chemical inhibitors of these anti-phage immune systems could be employed as adjuvants to overcome resistance in phage-based therapies. Here, we report that anti-phage systems can be selectively inhibited by small molecules, thereby sensitizing phage-resistant bacteria to phages. We discovered a class of chemical inhibitors that inhibit the type II Thoeris anti-phage immune system. These inhibitors block the biosynthesis of a histidine-ADPR intracellular ‘alarm’ signal by ThsB and prevent ThsA from arresting phage replication. These inhibitors promiscuously inhibit type II Thoeris systems from diverse bacteria—including antibiotic-resistant pathogens. Chemical inhibition of the Thoeris defense improved the efficacy of a model phage therapy against a phage-resistant strain ofP. aeruginosain a mouse infection, suggesting a therapeutic potential. Furthermore, these inhibitors may be employed as chemical tools to dissect the importance of the Thoeris system for phage defense in natural microbial communities. 

Abstract To overtake competitors, microbes produce and secrete secondary metabolites that kill neighboring cells and sequester nutrients. This natural product-mediated competition likely evolved in complex microbial communities that included viral pathogens. From this ecological context, we hypothesized that microbes secrete metabolites that “weaponize” natural pathogens (i.e., bacteriophages) to lyse their competitors. Indeed, we discovered a bacterial secondary metabolite that sensitizes other bacteria to phage infection. We found that this metabolite provides the producer (aStreptomycessp.) with a fitness advantage over its competitor (Bacillus subtilis) by promoting phage infection. The phage-promoting metabolite, coelichelin, sensitizedB. subtilisto a wide panel of lytic phages, and it did so by preventing the early stages of sporulation through iron sequestration. Beyond coelichelin, other natural products may provide phage-mediated competitive advantages to their producers—either by inhibiting sporulation or through yet-unknown mechanisms.