Competitive fitness of essential gene knockdowns reveals a broad-spectrum antibacterial inhibitor of the cell division protein FtsZ [Mechanisms of Action]

To streamline 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 identification of hypersusceptible mutants and morphology screening, we show that C109 targets cell division. Furthermore, fluorescence microscopy of bacteria harboring 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.

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