Search bioRxiv⌕ Search

Biology subjects

Neisewander, I.

Publications and source records attributed to Neisewander, I..

2 recordsLinked to original sources
bioRxiv2024

Massively parallel combination screen reveals small molecule sensitization of antibiotic-resistant Gram-negative ESKAPE pathogens

Antibiotic resistance, especially in multidrug-resistant ESKAPE pathogens, remains a worldwide problem. Combination antimicrobial therapies may be an important strategy to overcome resistance and broaden the spectrum of existing antibiotics. However, this strategy is limited by the ability to efficiently screen large combinatorial chemical spaces. Here, we deployed a high-throughput combinatorial screening platform, DropArray, to evaluate the interactions of over 30,000 compounds with up to 22 antibiotics and 6 strains of Gram-negative ESKAPE pathogens, totaling to over 1.3 million unique strain-antibiotic-compound combinations. In this dataset, compounds more frequently exhibited synergy with known antibiotics than single-agent activity. We identified a compound, P2-56, and developed a more potent analog, P2-56-3, which potentiated rifampin (RIF) activity against Acinetobacter baumannii and Klebsiella pneumoniae. Using phenotypic assays, we showed P2-56-3 disrupts the outer membrane of A. baumannii. To identify pathways involved in the mechanism of synergy between P2-56-3 and RIF, we performed genetic screens in A. baumannii. CRISPRi-induced partial depletion of lipooligosaccharide transport genes (lptA-D, lptFG) resulted in hypersensitivity to P2-56-3/RIF treatment, demonstrating the genetic dependency of P2-56-3 activity and RIF sensitization on lpt genes in A. baumannii. Consistent with outer membrane homeostasis being an important determinant of P2-56-3/RIF tolerance, knockout of maintenance of lipid asymmetry complex genes and overexpression of certain resistance-nodulation-division efflux pumps - a phenotype associated with multidrug-resistance - resulted in hypersensitivity to P2-56-3. These findings demonstrate the immense scale of phenotypic antibiotic combination screens using DropArray and the potential for such approaches to discover new small molecule synergies against multidrug-resistant ESKAPE strains. Significance StatementThere is an unmet need for new antibiotic therapies effective against the multidrug-resistant, Gram-negative ESKAPE pathogens. Combination therapies have the potential to overcome resistance and broaden the spectrum of existing antibiotics. In this study, we use DropArray, a massively parallel combinatorial screening tool, to assay more than 1.3 million combinations of small molecules against the Gram-negative ESKAPE pathogens, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. We discovered a synthetic small molecule potentiator, P2-56, of the antibiotic rifampin effective in A. baumannii and K. pneumoniae. We generated P2-56-3, a more potent derivative of P2-56, and found that it likely potentiates rifampin by compromising the outer membrane integrity. Our study demonstrates a high-throughput strategy for identifying antibiotic potentiators against multidrug-resistant bacteria.

microbiology↗
bioRxiv2024

Identification of essential genes that support fitness of Acinetobacter baumannii efflux pump overproducers in the presence of fluoroquinolone

Elevated expression of Resistance-Nodulation-Cell Division (RND) drug transporters is commonly observed in clinical isolates of Acinetobacter baumannii, a nosocomial pathogen associated with multidrug-resistant (MDR) infections. We describe here a CRISPRi platform directed toward identifying essential gene hypomorphs that preferentially change resistance to the fluoroquinolone antibiotic ciprofloxacin in RND pump overproducers. An sgRNA library including single and double nucleotide mutations directed against essential genes of A. baumannii was constructed and introduced into multiple strain backgrounds, allowing strain-specific, titratable knockdown efficiencies to be analyzed. Other than NusG depletions, there were few candidates in the absence of drug treatment that showed lowered fitness specifically in strains overexpressing the RND efflux pumps AdeAB, AdeIJK, or AdeFGH. In the presence of ciprofloxacin, the hypomorphs that caused hypersensitivity were predicted to result in outer membrane dysfunction, with the AdeFGH overproducer appearing particularly sensitive to such disruptions. Most notably, depletion of the predicted monovalent cation-proton antiporter component PhaF compromised efflux pump function, as depletion of the antiporter resulted in increased ciprofloxacin accumulation in strains overproducing AdeFGH and disrupted cytosolic pH. On the other hand, depletions of translation-associated proteins and components of the proton-pumping ATP synthase conferred fitness benefits in the presence of the drug in at least two pump-overproducing strains. Therefore, pump overproduction exacerbated stress caused by defective outer membrane integrity, while the activity of at least one efflux pump overproducer required the function of an antiporter that maintains cytosolic pH homeostasis. ImportanceAcinetobacter baumannii clinical isolates are increasingly multidrug-resistant, leaving patients with few effective treatment options. Many of these isolates are fluoroquinolone-resistant due to drug target mutations in the two major type II topoisomerases, as well as mutations that activate RND efflux pump expression. This work identifies essential gene products that support the fitness of efflux pump hyperexpressers during treatment with the fluoroquinolone antibiotic ciprofloxacin, most of which are involved in OM biogenesis. These findings suggest new strategies for combination therapy with currently available fluoroquinolones to sensitize and combat high-level resistant strains.

microbiology↗