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

Abstract

trans-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 transfermessenger 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 transtranslation 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. KEYWORDS Legionella, trans-translationL egionella pneumophila is a ubiquitous freshwater bacterium that infects a wide spectrum of environmental protozoans. Human-made systems, such as sanitary water networks and air-cooling towers, can disseminate contaminated water through aerosolization. The breathing of microscopic droplets contaminated with L. pneumophila can lead to infection of alveolar macrophages and development of a lifethreatening pneumonia called Legionnaires' disease (LD) or legionellosis. LD remains an important cause of both morbidity and mortality in Europe, with over 6,900 cases being reported in 2014 (1). Guidelines for the management of LD recommend the use of macrolides (with a preference for azithromycin) or fluoroquinolones (levofloxacin or moxifloxacin) to treat the infection (2, 3). Despite a rapid diagnosis and the correct administration of antibiotics, the death rate among those with LD is over 10% (4). L. Citation Brunel R, Descours G, Durieux I, Doublet P, Jarraud S, Charpentier X. 2018. KKL-35 exhibits potent antibiotic activity against Legionella species independently of transtranslation inhibition. Antimicrob Agents

A Small-Molecule Inhibitor of trans -Translation Synergistically Interacts with Cathelicidin Antimicrobial Peptides To Impair Survival of Staphylococcus aureus

“…Finally, generation of resistance against KKL-40 is limited, even in S. aureus, a pathogen notorious for its ability to acquire antibiotic resistance (1,2). These results are consistent with those seen in L. pneumophila, E. coli, and S. flexneri, where no resistance to KKL-35 was generated (8,9). Developing resistance to KKL-40 may be more difficult because mutations that prevent binding to the ribosome are lethal, because mutations would be required in all rRNA genes, or because there is a second, unidentified target for KKL-40.…”

“…In addition to S. aureus, we find that KKL-40 is effective against several other Gram-positive pathogens, including a vancomycin-resistant strain of Enterococcus faecalis, Bacillus subtilis, and Streptococcus pyogenes. Although activity has been seen against Gramnegative species, including Shigella flexneri, F. tularensis, and L. pneumophila (8,9), as well as H. influenzae, Y. pestis, B. mallei, and B. pseudomallei in this study, we saw little to no activity against E. coli, P. aeruginosa, K. pneumoniae, or A. baumannii. However, KKL-40 potently inhibited the growth of two mutant E. coli strains that have increased permeability, and coincubation of KKL-40 with polymyxin B, which disrupts the outer membrane of Gram-negative bacteria (22), lowered the effective MIC of KKL-40 in a synergistic manner.…”

“…A family of oxadiazole benzamides, including KKL-35 and KKL-40 ( Fig. 1), was shown to have potent antibiotic activity against Bacillus anthracis, Mycobacterium tuberculosis, Francisella tularensis, Legionella pneumophila, and ⌬tolC strains of Escherichia coli (7)(8)(9)(10). Biochemical and cross-linking data indicated that KKL-35 and KKL-40 bind to a site on 23S rRNA that is not bound by other drugs, and this binding inhibits trans-translation but not translation (7,8).…”

Safe and easy in vitro evaluation of tmRNA-SmpB-mediated trans-translation from ESKAPE pathogenic bacteria

“…To date, trans-translation has not been yet exploited for clinical use. In the search for inhibitors specific to the process, initial assays led to the discovery of 1,3,4 oxadiazole molecules (Ramadoss et al, 2013), but their specificity for trans-translation in vivo is still in question (Macé et al, 2017;Brunel et al, 2018). It has been suggested that trans-translation is inhibited by pyrazinamide (PZA), a first-line anti-tuberculosis drug (Shi et al, 2011), but it was finally recently shown the action of PZA is entirely independent of trans-translation in M. tuberculosis (Dillon et al, 2017).…”