Tetrazole-Based trans -Translation Inhibitors Kill Bacillus anthracis Spores To Protect Host Cells

Abstract

, 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 spores makes it necessary to identify chemical agents that target and kill spores. Here, we demonstrate that a tetrazole-based-translation inhibitor, KKL-55, is bactericidal against vegetative cells of in culture. Using a fluorescent analog, we show that this class of compounds colocalizes with developing endospores and bind purified spores KKL-55 was effective against spores at concentrations close to its MIC for vegetative cells. Spore germination was inhibited at 1.2× MIC, and spores were killed at 2× MIC. In contrast, ciprofloxacin killed germinants at concentrations close to its MIC but did not prevent germination even at 32× MIC. Because toxins are released by germinants, macrophages infected by spores are killed early in the germination process. At ≥2× MIC, KKL-55 protected macrophages from death after infection with spores. Ciprofloxacin required concentrations of ≥8× MIC to exhibit a similar effect. Taken together, these data indicate that KKL-55 and related tetrazoles are good lead candidates for therapeutics targeting spores and suggest that there is an early requirement for-translation in germinating spores.

KKL-35 exhibits potent antibiotic activity againstLegionellaspecies independently of trans-translation inhibition

“…KKL-35, along with other oxadiazoles (KKL-10 and KKL-40), were identified in a high-throughput screen for inhibitors of trans-translation activity in vitro and were initially found to display antibiotic activity against several species. Oxadiazoles have since shown potent antibiotic activity against additional pathogens such as Francisella tularensis, Bacillus anthracis or Mycobacterium tuberculosis (34,42,43). The antibiotic activity of oxadiazoles on these pathogens was assumed to derive from inhibition of trans-translation but was not demonstrated.…”

The Bewildering Antitubercular Action of Pyrazinamide

“…Following translation of this tag, the nascent peptide is released and targeted for proteolysis, and the ribosome disengages from tmRNA and is free to initiate translation of other available mRNA (109). This pathway is essential for viability of M. tuberculosis and many other bacterial pathogens and represents an outstanding novel target for drug discovery (114)(115)(116)(117). Indeed, structurally related families of oxadiazole and tetrazole-based compounds have recently been identified that inhibit trans-translation in a large number of bacterial species, including Gramnegative, Gram-positive, and mycobacterial species (114)(115)(116)(117).…”

Comparison of Proteomic Responses as Global Approach to Antibiotic Mechanism of Action Elucidation

“…Stalled ribosomes can quickly lead to the depletion of actively translating ribosomes by trapping other ribosomes in polysomes. We investigated the trans-translation inhibitors KKL-35 and KKL-40 (oxadiazoles), derivatives of which have been shown to crosslink to the 23S rRNA 53 , and KKL-55 54 (tetrazole), which likely has a different molecular target. The CoPR approach replaces labor-intense one-on-one comparisons with an expedient and reliable process that identifies the most closely matching responses.…”