Genome-wide phage susceptibility analysis inAcinetobacter baumanniireveals capsule modulation strategies that determine phage infectivity
Authors: Bai Jinna , Raustad Nicole , Denoncourt Jason , van Opijnen Tim , Geisinger Edward 
Published: October 2022

Phage have gained renewed interest as an adjunctive treatment for life-threatening infections with the resistant nosocomial pathogenAcinetobacter baumannii. Our understanding of howA. baumanniidefends 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 inA. baumanniiusing Tn-seq. These studies focused on the lytic phage Loki, which targetsAcinetobacterby 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 providesA. baumanniiwith 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 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 alterA. baumanniivirulence, is also a major determinant of susceptibility to phage.

Author Summary

Antibiotic-resistant infections withAcinetobacter baumanniiare a major problem in critical care units and have increased in frequency during the COVID-19 pandemic. The virulence of these infections depends on a polysaccharide capsule surrounding the bacterium. Phage, or viruses that kill bacteria, represent a promising alternative therapy against highly antibiotic-resistantA. baumanniiinfections, andA. baumannii-specific phage often target the capsule. Here, we use high-throughput genetics to analyze howA. baumanniidefends against phage and identify ways to potentiate their killing activity. We found that stressing the bacteria in ways that cause augmented production of capsule also causes hyper-susceptibility to phage. By contrast, turning off the stress response, or mutating the capsule structure, causes complete phage resistance. Altering another surface structure, lipooligosaccharide, or an intracellular protease also enhances phage attack. Modulating the amounts or makeup of capsular polysaccharide is known to influence virulence inA. baumannii. This work thus uncovers a connection between phage pressure and the evolution of virulence inA. baumannii, and it identifies control mechanisms that may be leveraged for improving future phage-based antimicrobial therapies.