<i>Francisella tularensis</i> tmRNA system mutants are vulnerable to stress, avirulent in mice, and provide effective immune protection

Svetlanov, Anton; Puri, Neha; Mena, Patricio; Koller, Antonius; Karzai, A. Wali

doi:10.1111/j.1365-2958.2012.08093.x

Cited by 41 publications

(40 citation statements)

References 73 publications

(111 reference statements)

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“…In fact, trans-translation is essential in some species, including Shigella flexneri, Helicobacter pylori, Neisseria gonorrheae, and Mycoplasma species (14)(15)(16)(17). In addition, mutants lacking trans-translation activity are avirulent in Salmonella enterica, Yersinia pestis, Streptococcus pneumoniae, and Francisella tularensis (18)(19)(20)(21), so inhibitors of the pathway could be effective against a wide array of pathogens. We identified compounds that inhibit trans-translation using a high-throughput screen (HTS) and demonstrated that these compounds have antibiotic activity.…”

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confidence: 99%

Small molecule inhibitors of trans -translation have broad-spectrum antibiotic activity

Ramadoss

Alumasa

Cheng

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The trans-translation pathway for protein tagging and ribosome release plays a critical role for viability and virulence in a wide range of pathogens but is not found in animals. To explore the use of trans-translation as a target for antibiotic development, a highthroughput screen and secondary screening assays were used to identify small molecule inhibitors of the pathway. Compounds that inhibited protein tagging and proteolysis of tagged proteins were recovered from the screen. One of the most active compounds, KKL-35, inhibited the trans-translation tagging reaction with an IC 50 = 0.9 μM. KKL-35 and other compounds identified in the screen exhibited broad-spectrum antibiotic activity, validating trans-translation as a target for drug development. This unique target could play a key role in combating strains of pathogenic bacteria that are resistant to existing antibiotics.antibiotic target | tmRNA | non-stop translation T he increasing prevalence of antibiotic-resistant bacterial pathogens has spurred a search for new pathways that can be targeted for antibiotic development (1, 2). One pathway that has not been exploited is the trans-translation pathway, which resolves nonstop translation complexes. The components of trans-translation have been identified in every sequenced bacterial genome, and mutations in these components affect viability or virulence in a wide range of bacteria (3, 4), suggesting that inhibitors of trans-translation might be effective broad-spectrum antibiotics. In addition, the trans-translation pathway is not found in animals, so specific inhibitors are expected to have few side effects on the host.The purpose of trans-translation is to remove nonstop translation complexes, i.e., translation reactions in which the ribosome has reached the 3′ end of the mRNA without terminating at a stop codon (4-6). These complexes are prevalent in bacteria because bacterial ribosomes do not require any information from the 3′ end of the mRNA to initiate translation, and bacteria lack most of the mechanisms for mRNA proofreading found in eukaryotes (7). Because hydrolysis of peptidyl-tRNA by release factors requires a stop codon in the A site, normal translation termination cannot occur when the ribosome reads to the 3′ end of the mRNA and there is no in-frame stop codon. Transtranslation resolves nonstop translation complexes using a ribonucleoprotein complex containing transfer-messenger RNA (tmRNA) and the small protein SmpB (4-6). tmRNA-SmpB recognizes nonstop translation complexes and enters the ribosomal A site mimicking a tRNA (8). The nascent polypeptide is transferred to tmRNA, and a specialized reading frame within tmRNA is inserted into the mRNA channel (4-6). Translation resumes using this sequence as a message and terminates at a stop codon at the end of the reading frame, releasing the ribosome and a tagged protein (4-6). The tag sequence is recognized by proteases, and the tagged protein is rapidly degraded (9-12). The net reaction of trans-translation is the removal of all components o...

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confidence: 99%

Small molecule inhibitors of trans -translation have broad-spectrum antibiotic activity

Ramadoss

Alumasa

Cheng

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…We can use the method for monitoring of stress state with very good results. From a literature search, F. tularensis is quite resistant to oxidative stress and it can recovery from stress conditions more easily than the host organism 36 . IFN-was assessed in plasma samples (Table 5).…”

Section: Resultsmentioning

confidence: 99%

Organs of BALB/c mice can be injured in course of tularemia

Pavliš¹,

Kusakova²,

Novotný³

et al. 2014

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub

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Background. Francisella tularensis is a biological agent exploitable for bioterrorism and biological warfare purposes due to serious pathogenic progression and easy dissemination. Despite intensive research in the past, some adverse consequences remain unclear. One consequence of this pathogen is oxidative stress. Aims. The aim of this study was to undertake ex vivo assays for monitoring the disease in mice and increase our knowledge of the oxidative stress induced by tularemia. Methods. The mouse BALB/c model was chosen and the animals were infected by a dose 10 4 CFU of F. tularensis. After five days, the animals were euthanized. Blood immediately processed in plasma, spleen and liver were sampled from the cadavers. Oxidative stress markers, cytokines and histopathological were undertaken.Results. There was a significant link between oxidative stress and tularemia. Particularly elevated levels of malondialdehyde and decreased levels of low molecular weight antioxidants were found in the liver and spleen of tularemia-infected animals. The histopathological findings correlated well with the oxidative stress markers. The liver and spleen were proven to be significantly at risk from the disease and an association between stress and neutrophils in the affected organs was found. The histopathology excluded risk to other organs such as the kidney and or heart. Conclusions. Oxidative stress plays a significant role in tularemia infection in mice and this was confirmed by the histology.

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“…Svetlanov et al have shown that F. tularensis mutants lacking tmRNA or SmpB are viable but grow slowly, are more sensitive to stress, and have attenuated virulence (16). If these F. tularensis mutants with no trans-translation activity can survive, why is the growth of wild-type bacteria completely stopped by KKL-10 and KKL-40, which inhibit ribosome rescue?…”

Section: Discussionmentioning

confidence: 99%

“…This reaction also targets the nascent polypeptide and mRNA for degradation (10)(11)(12)(13). F. tularensis mutants lacking SmpB or tmRNA are attenuated for virulence in mouse models (16). Some bacteria, such as Escherichia coli, can survive without trans-translation because they contain ArfA, a protein that allows release factor 2 (RF-2) to terminate translation on ribosomes stalled at the 3= end of an mRNA (17)(18)(19).…”

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

Goralski

Dewan

Alumasa

et al. 2016

Antimicrob Agents Chemother

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c Bacteria require at least one pathway to rescue ribosomes stalled at the ends of mRNAs. The primary pathway for ribosome rescue is trans-translation, which is conserved in >99% of sequenced bacterial genomes. Some 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 the tier 1 select agent Francisella 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 macrophages or liver cells at any time after infection by F. tularensis prevented further bacterial proliferation. When macrophages were stimulated with the proinflammatory cytokine gamma interferon 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 was 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 are good lead compounds for antibiotic development.

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Francisella tularensis tmRNA system mutants are vulnerable to stress, avirulent in mice, and provide effective immune protection

Cited by 41 publications

References 73 publications

Small molecule inhibitors of trans -translation have broad-spectrum antibiotic activity

Small molecule inhibitors of trans -translation have broad-spectrum antibiotic activity

Organs of BALB/c mice can be injured in course of tularemia

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

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