KKL-35 Exhibits Potent Antibiotic Activity against Legionella Species Independently of trans -Translation Inhibition

Abstract

ABSTRACTtrans-Translation is a ribosome-rescue system that is ubiquitous in bacteria. Small molecules defining a new family of oxadiazole compounds that inhibit trans-translation have been found to have broad-spectrum antibiotic activity. We sought to determine the activity of KKL-35, a potent member of the oxadiazole family, against the human pathogen Legionella pneumophila and other related species that can also cause Legionnaires' disease (LD). Consistent with the essential nature of trans-translation in L. pneumophila, KKL-35 inhibited the growth of all tested strains at submicromolar concentrations. KKL-35 was also active against other LD-causing Legionella species. KKL-35 remained equally active against L. pneumophila mutants that have evolved resistance to macrolides. KKL-35 inhibited the multiplication of L. pneumophila in human macrophages at several stages of infection. No resistant mutants could be obtained, even during extended and chronic exposure. Surprisingly, KKL-35 was not synergistic with other ribosome-targeting antibiotics and did not induce the filamentation phenotype observed in cells defective for trans-translation. Importantly, KKL-35 remained active against L. pneumophila mutants expressing an alternate ribosome-rescue system and lacking transfer-messenger RNA, the essential component of trans-translation. These results indicate that the antibiotic activity of KKL-35 is not related to the specific inhibition of trans-translation and its mode of action remains to be identified. In conclusion, KKL-35 is an effective antibacterial agent against the intracellular pathogen L. pneumophila with no detectable resistance development. However, further studies are needed to better understand its mechanism of action and to assess further the potential of oxadiazoles in treatment.

Ribosome Rescue Pathways in Bacteria

“…In 2013, members of a class of small molecules, the so-called KKLs, showed broad-spectrum antibiotic activity and were proposed to inhibit trans -translation in vitro and in vivo ( Ramadoss et al, 2013 ). However, it was challenged that trans -translation is the only or even major target of KKLs ( Mace et al, 2017 ; Brunel et al, 2018 ; Tresse et al, 2019 ) and the subject is matter of an ongoing discussion ( Aron et al, 2020 ; Guyomar et al, 2020 ). This has not however deterred attempts to develop more sensitive and selective high-throughput screening assays ( Guyomar et al, 2020 ; Thépaut et al, 2020 ) but only time will tell whether they will lead to discovery of the elusive molecules that specifically target and inhibit bacterial ribosome rescue systems.…”

A chemical-genetic map of the pathways controlling drug potency in Mycobacterium tuberculosis

“…In light of our findings, we suggest designating rv1473 as oprA (oxazolidinone phenicol resistance A). Finally, these results show that trans-translation may be a target for synergistic drug combinations (Brunel et al, 2018), which could be important in increasing the potency and decreasing toxicity of oxazolidinones. In addition to guiding rational development of synergistic drug combinations, our results illustrate the power of combining chemical-genetics with comparative genomics to discover new mechanisms of acquired drug resistance.…”

“…In light of our findings, we suggest designating rv1473 as oprA ( o xazolidinone p henicol r esistance A ). Finally, these results show that trans-translation may be a target for synergistic drug combinations (Brunel et al, 2018), which could be important in increasing the potency and decreasing toxicity of oxazolidinones.…”

Pyrazinamide/Pyrazinoic Acid Resistance in Mycobacterium Tuberculosis: Recent Findings and Implications for Improving the Treatment of Tuberculosis

“…Inhibitors of trans-translation have been identified and at least one (KKL-35) was shown to be active against MTB under both aerobic and anoxic conditions [3], although others have questioned KKL-35's mode of action [10].…”