Tetrazole-based trans-translation inhibitors kill Bacillus anthracis spores to protect host cells

Bacillus anthracis, the causative agent of anthrax, remains a significant threat to humans, including potential use in bioterrorism and biowarfare. The capacity to engineer strains with increased pathogenicity coupled with the ease of disseminating lethal doses of B. anthracis spores makes it necessary to identify chemical agents that target and kill spores. Here, we demonstrate that a tetrazole-based trans-translation inhibitor, KKL-55, is bactericidal against vegetative cells of B. anthracis in culture. Using a fluorescent analog, we show that this class of compounds co-localizes with developing endospores and bind purified spores in vitro. KKL-55 was effective against spores at concentrations close to its MIC for vegetative cells. Spore germination was inhibited at 1.2x MIC, and spores were killed at 2x MIC. In contrast, ciprofloxacin killed germinants at concentrations close to its MIC but did not prevent germination even at 32x MIC. Because toxins are released by germinants, macrophages infected by B. anthracis spores are killed early in the germination process. At ≥2x MIC, KKL-55 protected macrophages from death after infection with B. anthracis spores. Ciprofloxacin required concentrations of ≥8x MIC to exhibit a similar effect. Taken together, these data indicate that KKL-55 and related tetrazoles are good lead candidates for therapeutics targeting B. anthracis spores and suggest that there is an early requirement for trans-translation in germinating spores.