Competitive Fitness of Essential Gene Knockdowns Reveals a Broad-Spectrum Antibacterial Inhibitor of the Cell Division Protein FtsZ.
To streamline the elucidation of antibacterial compounds' mechanism of action, comprehensive high-throughput assays interrogating multiple putative targets are necessary. However, current chemogenomic approaches for antibiotic target identification have not fully utilized the multiplexing potential of next-generation sequencing. Here, we used Illumina sequencing of transposon insertions to track the competitive fitness of a Burkholderia cenocepacia library containing essential gene knockdowns. Using this method, we characterized a novel benzothiadiazole derivative, 10126109 (C109), with antibacterial activity against B. cenocepacia, for which whole-genome sequencing of low-frequency spontaneous drug-resistant mutants had failed to identify the drug target. By combining the identification of hypersusceptible mutants and morphology screening, we show that C109 targets cell division. Furthermore, fluorescence microscopy of bacteria harboring green fluorescent protein (GFP) cell division protein fusions revealed that C109 prevents divisome formation by altering the localization of the essential cell division protein FtsZ. In agreement with this, C109 inhibited both the GTPase and polymerization activities of purified B. cenocepacia FtsZ. C109 displayed antibacterial activity against Gram-positive and Gram-negative cystic fibrosis pathogens, including Mycobacterium abscessus C109 effectively cleared B. cenocepacia infection in the Caenorhabditis elegans model and exhibited additive interactions with clinically relevant antibiotics. Hence, C109 is an enticing candidate for further drug development.
['Hogan|Andrew M|AM|', 'Scoffone|Viola C|VC|', 'Makarov|Vadim|V|', 'Gislason|April S|AS|', 'Tesfu|Haben|H|', 'Stietz|Maria S|MS|', 'Brassinga|Ann Karen C|AKC|', 'Domaratzki|Michael|M|', 'Li|Xuan|X|', 'Azzalin|Alberto|A|', 'Biggiogera|Marco|M|', 'Riabova|Olga|O|', 'Monakhova|Natalia|N|', 'Chiarelli|Laurent R|LR|0000-0003-0348-9764', 'Riccardi|Giovanna|G|', 'Buroni|Silvia|S|', 'Cardona|Silvia T|ST|0000-0002-0629-8886']
D000818:Animals D000900:Anti-Bacterial Agents D001426:Bacterial Proteins D019121:Burkholderia Infections D057508:Burkholderia cenocepacia D017173:Caenorhabditis elegans D003550:Cystic Fibrosis D003598:Cytoskeletal Proteins D004353:Drug Evaluation, Preclinical D055785:Gene Knockdown Techniques D020043:Genes, Essential D049452:Green Fluorescent Proteins D059014:High-Throughput Nucleotide Sequencing D006801:Humans D008826:Microbial Sensitivity Tests D009154:Mutation
essential genes mechanisms of action Burkholderia drug targets FtsZ Tn-seq fluorescent image analysis