Mortman, N. J. | Search bioRxiv

bioRxiv2020

Antibiotic hypersensitivity signatures identify targets for attack in the Acinetobacter baumannii cell envelope

Acinetobacter baumannii is an opportunistic pathogen that is a critical, high-priority target for new antibiotic development. Clearing of A. baumannii requires relatively high doses of antibiotics across the spectrum, primarily due to its protective cell envelope. Many of the proteins that support envelope integrity and modulate drug action are uncharacterized, largely because there is an absence of orthologs for several proteins that perform essential envelope-associated processes, impeding progress on this front. To identify targets that can synergize with current antibiotics, we performed an exhaustive analysis of A. baumannii mutants causing hypersensitivity to a multitude of antibiotic treatments. By examining mutants with antibiotic hypersensitivity profiles that parallel mutations in proteins of known function, we show that the function of poorly annotated proteins can be predicted and used to identify candidate missing link proteins in essential A. baumannii processes. Using this strategy, we uncovered multiple uncharacterized proteins with critical roles in cell division or cell elongation, and revealed that a predicted cell wall D,D-endopeptidase has an unappreciated function in lipooligosaccharide synthesis. Moreover, we provide a genetic strategy that uses hypersensitivity signatures to predict drug synergies, allowing the identification of {beta}-lactams that work cooperatively based on the cell wall assembly machineries that they preferentially target. These data reveal multiple pathways critical for envelope growth in A. baumannii that can be targeted in combination strategies for attacking the pathogen.

microbiology↗

bioRxiv2018

The landscape of intrinsic and evolved fluoroquinolone resistance in Acinetobacter baumannii includes suppression of drug-induced prophage replication

The emergence of fluoroquinolone resistance in nosocomial pathogens has restricted the clinical efficacy of this antibiotic class. In Acinetobacter baumannii, the majority of clinical isolates now show high-level resistance due to mutations in gyrA (DNA gyrase) and parC (Topo IV). To investigate the molecular basis for fluoroquinolone resistance, an exhaustive mutation analysis was performed in both drug sensitive and resistant strains to identify loci that alter the sensitivity of the organism to ciprofloxacin. To this end, parallel fitness tests of over 60,000 unique insertion mutations were performed in strains with various alleles in genes encoding the drug targets. The spectrum of mutations that altered drug sensitivity was found to be similar in the drug sensitive and double mutant gyrAparC background having resistance alleles in both genes. In contrast, introduction of a single gyrA resistance allele, resulting in preferential poisoning of Topo IV by ciprofloxacin, led to extreme alterations in the insertion mutation fitness landscape. The distinguishing feature of preferential Topo IV poisoning was induction of DNA synthesis in the region of two endogenous prophages, which appeared to occur in situ. Induction of the selective DNA synthesis in the gyrA background was also linked to enhanced activation of SOS response and heightened transcription of prophage genes relative to that observed in either the WT or gyrAparC double mutants. Therefore, the accumulation of mutations that result in the stepwise evolution of high ciprofloxacin resistance is tightly connected to suppression of hyperactivation of the SOS response and endogenous prophage DNA synthesis.\n\nImportanceFluoroquinolones have been extremely successful antibiotics. Their clinical efficacy derives from the ability to target multiple bacterial enzymes critical to DNA replication, the topoisomerases DNA gyrase and Topo IV. Unfortunately, mutations lowering drug affinity for both enzymes are now widespread, rendering these drugs ineffective for many pathogens. To undermine this form of resistance, we sought to understand how bacteria with target alterations differentially cope with fluoroquinolone exposures. We studied this problem in the nosocomial pathogen A. baumannii, which causes resistant, life-threating infections. Employing genome-wide approaches, we uncovered numerous pathways that could be exploited to lower fluoroquinolone resistance independently of target alteration. Remarkably, fluoroquinolone targeting of Topo IV in specific mutants caused dramatic prophage hyperinduction, a response that was muted in strains with DNA gyrase as the primary target. This work demonstrates that resistance evolution via target modification can profoundly modulate the antibiotic stress response, revealing potential resistance-associated liabilities.

microbiology↗