Inhibitors of Ribosome Rescue Arrest Growth of Francisella tularensis at All Stages of Intracellular Replication

Francisella tularensis ssp. tularensis is a gram‐negative, facultative intracellular bacterium, which is classified as a Tier 1 Select Agent by the CDC due to its high infectivity and ease of propagation. Attempts to develop an effective vaccine have been unsuccessful, due in part to the organism's ability to suppress or bypass the host immune response early after infection. F. tularensis strains resistant to multiple antibiotics are a biowarfare threat. In the absence of an effective vaccine, new antibiotic targets and compounds are needed to ensure biodefense. Bacteria require at least one pathway to rescue ribosomes stalled at the end of mRNAs. The primary pathway for ribosome rescue is trans ‐translation, which is conserved in >99% of sequenced bacterial genomes. Some bacterial species also have backup systems, such as ArfA or ArfB, which can rescue ribosomes in the absence of sufficient trans ‐translation activity. Small molecule inhibitors of ribosome rescue have broad‐spectrum antimicrobial activity against bacteria grown in liquid culture. These compounds were tested against F. tularensis to determine if they can limit bacterial proliferation during infection of eukaryotic cells. The inhibitors KKL‐10 and KKL‐40 exhibited exceptional antimicrobial activity against both attenuated and fully virulent strains of F. tularensis in vitro and during ex vivo infection. Addition of KKL‐10 or KKL‐40 to macrophage or liver cells at any time after infection by F. tularensis prevented further bacterial proliferation. When macrophages were stimulated with the pro‐inflammatory cytokine interferon‐γ before being infected by F. tularensis , addition of KKL‐10 or KKL‐40 reduced intracellular bacteria by >99%, indicating that the combination of cytokine induced stress and a nonfunctional ribosome rescue pathway is fatal to F. tularensis . Neither KKL‐10 nor KKL‐40 were cytotoxic to eukaryotic cells in culture. These results demonstrate that ribosome rescue is required for F. tularensis growth at all stages of its infection cycle, and suggest that KKL‐10 and KKL‐40 will be good lead compounds for antibiotic development. Support or Funding Information This work was supported by NIH (AI077917 to Girish S. Kirimanjeswara and GM068720 to Kenneth C. Keiler) and CAS/PSU start up funds to Girish S. Kirimanjeswara. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.