Phage have gained renewed interest as an adjunctive treatment for life-threatening infections with the resistant nosocomial pathogen Acinetobacter baumannii. Our understanding of how A. baumannii defends against phage remains limited, although this information could lead to improved antimicrobial therapies. To address this problem, we identified genome-wide determinants of phage susceptibility in A. baumannii using Tn-seq. These studies focused on the lytic phage Loki, which targets Acinetobacter by unknown mechanisms. We identified 41 candidate loci that increase susceptibility to Loki when disrupted, and 10 that decrease susceptibility. Combined with spontaneous resistance mapping, our results support the model that Loki uses the K3 capsule as an essential receptor, and that capsule modulation provides A. baumannii with strategies to control vulnerability to phage. A key center of this control is transcriptional regulation of capsule synthesis and phage virulence by the global regulator BfmRS. Mutations hyperactivating BfmRS simultaneously increase capsule levels, Loki adsorption, Loki replication, and host killing, while BfmRS-inactivating mutations have the opposite effect, reducing capsule and blocking Loki infection. We identified novel BfmRS-activating mutations, including knockouts of a T2 RNase protein and the disulfide formation enzyme DsbA, that hypersensitize bacteria to phage challenge. We further found that mutation of a glycosyltransferase known to alter capsule structure and bacterial virulence can also cause complete phage resistance. Finally, additional factors including lipooligosaccharide and Lon protease act independently of capsule modulation to interfere with Loki infection. This work demonstrates that regulatory and structural modulation of capsule, known to alter A. baumannii virulence, is also a major determinant of susceptibility to phage.

Phage have gained renewed interest as an adjunctive treatment for life-threatening infections with the drug-resistant nosocomial pathogen Acinetobacter baumannii. Our understanding of the mechanisms used by A. baumannii to defend against phage remains limited, although this information could lead to improved antimicrobial therapies. To address this problem, we used Tn-seq to identify determinants of phage susceptibility in A. baumannii on a genome-wide scale. These studies focused on the lytic phage Loki, which uses unknown mechanisms to target Acinetobacter. We identified 41 candidate loci that increase susceptibility to Loki when disrupted, and 10 that decrease susceptibility. Combined with spontaneous resistance mapping, our results support the model that Loki uses the bacterial capsule as an essential receptor, and that modulation of capsule provides A. baumannii with key strategies to control vulnerability to phage. The center of this control is transcriptional capsule regulation by the two-component system BfmRS. Mutations hyperactivating BfmRS simultaneously increase capsule levels and Loki infectivity, while the opposite is true with BfmRS-inactivating mutations. We identified novel BfmRS-activating mutations, including knockouts of an RNase T2 homolog (RnaA) and the disulfide isomerase DsbA, that determine the outcome of a phage encounter. We further found that mutation of a glycosyltransferase (Gtr6) known to modify capsule structure and virulence in clinical strains can also confer complete phage resistance. Finally, additional factors including the outer core of lipooligosaccharide act independently of capsule modulation to interfere with Loki infection. This work demonstrates that regulatory and structural modulation of capsule, known to alter A. baumannii virulence, is also a major determinant of susceptibility to phage.