Genome-wide phage susceptibility analysis inAcinetobacter baumanniireveals capsule modulation strategies that determine phage infectivity

Abstract

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.

A phosphorylation signal activates genome-wide transcriptional control by BfmR, the global regulator ofAcinetobacterresistance and virulence

“…An additional cell envelope pathway directly activated by BfmR is periplasmic protein folding, which included two disulfide bond generation enzymes (DsbB and DsbC) (62) and the periplasmic chaperone SurA (63). Upregulation of this pathway could in theory contribute to how A. baumannii responds to and alleviates a major stress, defective periplasmic disulfide formation, shown previously to be a signal detected by BfmRS (9). On the other hand, a group of cell envelope proteins was directly repressed by BfmR.…”

“…An additional cell envelope pathway directly activated by BfmR is periplasmic protein folding, which included two disulfide bond generation enzymes (DsbB and DsbC) (63), the chaperone SurA, and a DegPfamily protease (ACX60_RS04935) (64). Upregulation of this pathway could contribute to how A. baumannii counteracts a major stress, defective periplasmic disulfide formation, shown previously to be a signal for BfmRS (9), and suggests the system may respond to other forms of protein misfolding in the envelope analogous to E. coli Cpx (65). In tandem, a separate group of cell envelope proteins was directly repressed by BfmR.…”

“…BfmRS was first identified as a regulator of biofilm formation (8). The system has since been shown to regulate a number of additional features relevant to nosocomial infection, including capsule production (9,10), resistance to serum killing (6,7), fitness and virulence in animal models (6,11,12), antibiotic resistance and tolerance (6,7,13), desiccation tolerance (14,15), and motility (16). For many of these phenotypes, knockouts of BfmR (response regulator) and BfmS (membrane sensor kinase) have opposing effects.…”

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 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 OD600 of 0.5.…”