Raustad, N. | bioRxiv Science

bioRxiv2024

A phosphorylation signal activates genome-wide transcriptional control by BfmR, the global regulator of Acinetobacter resistance and virulence.

The nosocomial pathogen Acinetobacter baumannii is a major threat to human health. The sensor kinase-response regulator system, BfmS-BfmR, is essential to multidrug resistance and virulence in the bacterium and represents a potential antimicrobial target. Important questions remain about how the system controls resistance and pathogenesis. Although BfmR knockout alters expression of >1000 genes, its direct regulon is undefined. Moreover, how phosphorylation controls the regulator is unclear. Here, we address these problems by combining mutagenesis, ChIP-seq, and in vitro phosphorylation to study the functions of phospho-BfmR. We show that phosphorylation is required for BfmR-mediated gene regulation, antibiotic resistance, and sepsis development in vivo. Consistent with activating the protein, phosphorylation induces dimerization and target DNA affinity. Integrated analysis of genome-wide binding and transcriptional profiles of BfmR led to additional key findings: (1) Phosphorylation dramatically expands the number of genomic sites BfmR binds; (2) DNA recognition involves a direct repeat motif widespread across promoters; (3) BfmR directly regulates 303 genes as activator (eg, capsule, peptidoglycan, and outer membrane biogenesis) or repressor (pilus biogenesis); (4) BfmR controls several non-coding sRNAs. These studies reveal the centrality of a phosphorylation signal in driving A. baumannii disease and disentangle the extensive pathogenic gene-regulatory network under its control.

microbiology↗

bioRxiv2022

Genome-wide phage susceptibility analysis in Acinetobacter baumannii reveals capsule modulation as a central determinant of phage defense

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 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. Author SummaryAntibiotic-resistant infections with Acinetobacter baumannii are 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-resistant A. baumannii infections, and A. baumannii-specific phage often target the capsule. Here, we use high-throughput genetics to analyze how A. baumannii defends 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 in A. baumannii. This work thus uncovers a connection between phage pressure and the evolution of virulence in A. baumannii, and it identifies control mechanisms that may be leveraged for improving future phage-based antimicrobial therapies.

microbiology↗