Genome-wide phage susceptibility analysis in Acinetobacter baumannii reveals capsule modulation strategies that determine phage infectivity

Abstract

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.

Development of a sensor for disulfide bond formation in diverse bacteria

“…Considering that disulfide bonds are required for the stability of virulence factors, toxins, antibiotic resistance, and cell envelope biogenesis proteins, lack of disulfide bond formation in many gram-negative pathogens results in virulence attenuation as well as antibiotic and phage susceptibility (6,7,21,23,45). However, the pathway is not essential for aerobic laboratory growth (17), thus facilitating the development of methods to search for molecules that identify molecules on-target in pathogenic bacteria, also known as target-based and whole cell-based approaches (46).…”

Genetic synergy between Acinetobacter baumannii undecaprenyl phosphate biosynthesis and the Mla system impacts cell envelope and antimicrobial resistance

“…Samples were prepared similar to previous descriptions ( 80 , 81 ). Three milliliters of overnight cultures was diluted 1:100 in 5 mL fresh LB and grown to mid-log with an OD 600 of 0.5.…”

Genetic synergy inAcinetobacter baumanniiundecaprenyl biosynthesis and maintenance of lipid asymmetry impacts outer membrane and antimicrobial resistance

“…Samples were prepared similarly to previous descriptions ( 81 , 82 ). Three-mL overnight cultures were diluted 1:100 in 5 mL fresh LB and grown to mid-log with an OD 600 of 0.5.…”