Antibiotic that inhibits trans-translation blocks binding of EF-Tu to tmRNA but not to tRNA.

IMPORTANCE: Elongation factor thermo-unstable (EF-Tu) is a universally conserved translation factor that mediates productive interactions between tRNAs and the ribosome. In bacteria, EF-Tu also delivers transfer-messenger RNA (tmRNA)-SmpB to the ribosome during trans-translation. We report the first small molecule, KKL-55, that specifically inhibits EF-Tu activity in trans-translation without affecting its activity in normal translation. KKL-55 has broad-spectrum antibiotic activity, suggesting that compounds targeted to the tmRNA-binding interface of EF-Tu could be developed into new antibiotics to treat drug-resistant infections.

Physiology of trans-translation deficiency in Bacillus subtilis - a comparative proteomics study.

trans-Translation is the most effective ribosome rescue system known in bacteria. While it is essential in some bacteria, Bacillus subtilis possesses two additional alternative ribosome rescue mechanisms that require the proteins BrfA or RqcH. To investigate the physiology of trans-translation deficiency in the model organism B. subtilis, we compared the proteomes of B. subtilis 168 and a ΔssrA mutant in the mid-log phase using gel-free label-free quantitative proteomics. In chemically defined medium, the growth rate of the ssrA deletion mutant was 20% lower than that of B. subtilis 168. An 35 S-methionine incorporation assay demonstrated that protein synthesis rates were also lower in the ΔssrA strain. Alternative rescue factors were not detected. Among the 34 proteins overrepresented in the mutant strain were eight chemotaxis proteins. Indeed, both on LB agar and minimal medium the ΔssrA strain showed an altered motility and chemotaxis phenotype. Despite the lower growth rate, in the mutant proteome ribosomal proteins were more abundant while proteins related to amino acid biosynthesis were less abundant than in the parental strain. This overrepresentation of ribosomal proteins coupled with a lower protein synthesis rate and down-regulation of precursor supply reflects the slow ribosome recycling in the trans-translation-deficient mutant.

Druggable differences: Targeting mechanistic differences between trans-translation and translation for selective antibiotic action.

Bacteria use trans-translation to rescue stalled ribosomes and target incomplete proteins for proteolysis. Despite similarities between tRNAs and transfer-messenger RNA (tmRNA), the key molecule for trans-translation, new structural and biochemical data show important differences between translation and trans-translation at most steps of the pathways. tmRNA and its binding partner, SmpB, bind in the A site of the ribosome but do not trigger the same movements of nucleotides in the rRNA that are required for codon recognition by tRNA. tmRNA-SmpB moves from the A site to the P site of the ribosome without subunit rotation to generate hybrid states, and moves from the P site to a site outside the ribosome instead of to the E site. During catalysis, transpeptidation to tmRNA appears to require the ribosomal protein bL27, which is dispensable for translation, suggesting that this protein may be conserved in bacteria due to trans-translation. These differences provide insights into the fundamental nature of trans-translation, and provide targets for new antibiotics that may have decrease cross-reactivity with eukaryotic ribosomes.

Ribosome collisions: New ways to initiate ribosome rescue.

When ribosomes collide while translating the same mRNA, a MutS-like protein can pry apart the leading ribosome and a nuclease can cut the mRNA between the collided ribosomes to initiate ribosome rescue and recycling.

trans-Translation inhibitors bind to a novel site on the ribosome and clear Neisseria gonorrhoeae in vivo.

Bacterial ribosome rescue pathways that remove ribosomes stalled on mRNAs during translation have been proposed as novel antibiotic targets because they are essential in bacteria and are not conserved in humans. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans-translation, the main ribosome rescue pathway in bacteria. Here, we report optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles, producing MBX-4132, which clears multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Single particle cryogenic-EM studies of non-stop ribosomes show that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein bL27, suggesting a novel mechanism for specific inhibition of trans-translation by these molecules. These results show that trans-translation is a viable therapeutic target and reveal a new conformation within the bacterial ribosome that may be critical for ribosome rescue pathways.

Pathogen-specific antimicrobials engineered de novo through membrane-protein biomimicry.

Precision antimicrobials aim to kill pathogens without damaging commensal bacteria in the host, and thereby cure disease without antibiotic-associated dysbiosis. Here we report the de novo design of a synthetic host defence peptide that targets a specific pathogen by mimicking key molecular features of the pathogen's channel-forming membrane proteins. By exploiting physical and structural vulnerabilities within the pathogen's cellular envelope, we designed a peptide sequence that undergoes instructed tryptophan-zippered assembly within the mycolic acid-rich outer membrane of Mycobacterium tuberculosis to specifically kill the pathogen without collateral toxicity towards lung commensal bacteria or host tissue. These mycomembrane-templated assemblies elicit rapid mycobactericidal activity and enhance the potency of antibiotics by improving their otherwise poor diffusion across the rigid M. tuberculosis envelope with respect to agents that exploit transmembrane protein channels for antimycobacterial activity. This biomimetic strategy may aid the design of other narrow-spectrum antimicrobial peptides.

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

New antibiotics are urgently needed to address the mounting resistance challenge. In early drug discovery, one of the bottlenecks is the elucidation of targets and mechanisms. To accelerate antibiotic research, we provide a proteomic approach for the rapid classification of compounds into those with precedented and unprecedented modes of action. We established a proteomic response library of Bacillus subtilis covering 91 antibiotics and comparator compounds, and a mathematical approach was developed to aid data analysis. Comparison of proteomic responses (CoPR) allows the rapid identification of antibiotics with dual mechanisms of action as shown for atypical tetracyclines. It also aids in generating hypotheses on mechanisms of action as presented for salvarsan (arsphenamine) and the antirheumatic agent auranofin, which is under consideration for repurposing. Proteomic profiling also provides insights into the impact of antibiotics on bacterial physiology through analysis of marker proteins indicative of the impairment of cellular processes and structures. As demonstrated for trans-translation, a promising target not yet exploited clinically, proteomic profiling supports chemical biology approaches to investigating bacterial physiology.

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

Staphylococcus aureus is a leading cause of infection in the United States, and due to the rapid development of resistance, new antibiotics are constantly needed. trans-Translation is a particularly promising antibiotic target because it is conserved in many bacterial species, is critical for bacterial survival, and is unique among prokaryotes. We have investigated the potential of KKL-40, a small-molecule inhibitor of trans-translation, and find that it inhibits both methicillin-susceptible and methicillin-resistant strains of S. aureus KKL-40 is also effective against Gram-positive pathogens, including a vancomycin-resistant strain of Enterococcus faecalis, Bacillus subtilis, and Streptococcus pyogenes, although its performance with Gram-negative pathogens is mixed. KKL-40 synergistically interacts with the human antimicrobial peptide LL-37, a member of the cathelicidin family, to inhibit S. aureus but not other antibiotics tested, including daptomycin, kanamycin, or erythromycin. KKL-40 is not cytotoxic to HeLa cells at concentrations that are 100-fold higher than the effective MIC. We also find that S. aureus develops minimal resistance to KKL-40 even after multiday passage at sublethal concentrations. Therefore, trans-translation inhibitors could be a particularly promising drug target against S. aureus, not only because of their ability to inhibit bacterial growth but also because of their potential to simultaneously render S. aureus more susceptible to host antimicrobial peptides.

Bioresponsive peptide-polysaccharide nanogels - A versatile delivery system to augment the utility of bioactive cargo.

We report the design, synthesis and efficacy of a new class of gel-like nano-carrier, or 'nanogel', prepared via templated electrostatic assembly of anionic hyaluronic acid (HA) polysaccharides with the cationic peptide amphiphile poly-L-lysine (PLL). Small molecules and proteins present during nanogel assembly become directly encapsulated within the carrier and are precisely released by tuning the nanogel HA:PLL ratio to control particle swelling. Remarkably, nanogels exhibit versatile and complimentary mechanisms of cargo delivery depending on the biologic context. For example, in mammalian cells, nanogels are rapidly internalized and escape the endosome to both deliver membrane-impermeable protein cargo into the cytoplasm and improve chemotherapeutic potency in drug resistant cancer cells. In bacteria, nanogels permeabilize microbial membranes to sensitize bacterial pathogens to the action of a loaded antibiotic. Thus, peptide nanogels represent a versatile, readily scalable and bio-responsive carrier capable of augmenting and enhancing the utility of a broad range of biomolecular cargoes.

A New Mechanism for Ribosome Rescue Can Recruit RF1 or RF2 to Nonstop Ribosomes.

Bacterial ribosomes frequently translate to the 3' end of an mRNA without terminating at an in-frame stop codon. In all bacteria studied to date, these "nonstop" ribosomes are rescued using trans-translation. Genes required for trans-translation are essential in some species, but other species can survive without trans-translation because they express an alternative ribosome rescue factor, ArfA or ArfB. Francisella tularensis cells lacking trans-translation are viable, but F. tularensis does not encode ArfA or ArfB. Transposon mutagenesis followed by deep sequencing (Tn-seq) identified a new alternative ribosome rescue factor, now named ArfT. arfT can be deleted in wild-type (wt) cells but not in cells that lack trans-translation activity. Overexpression of ArfT suppresses the slow-growth phenotype in cells lacking trans-translation and counteracts growth arrest caused by trans-translation inhibitors, indicating that ArfT rescues nonstop ribosomes in vivo Ribosome rescue assays in vitro show that ArfT promotes hydrolysis of peptidyl-tRNA on nonstop ribosomes in conjunction with F. tularensis release factors. Unlike ArfA, which requires RF2 for activity, ArfT can function with either RF1 or RF2. Overall, these results indicate that ArfT is a new alternative ribosome rescue factor with a distinct mechanism from ArfA and ArfB.IMPORTANCEFrancisella tularensis is a highly infectious intracellular pathogen that kills more than half of infected humans if left untreated. F. tularensis has also been classified as a potential bioterrorism agent with a great risk for deliberate misuse. Recently, compounds that inhibit ribosome rescue have been shown to have antibiotic activity against F. tularensis and other important pathogens. Like all bacteria that have been studied, F. tularensis uses trans-translation as the main pathway to rescue stalled ribosomes. However, unlike most bacteria, F. tularensis can survive without any of the known factors for ribosome rescue. Our work identified a F. tularensis protein, ArfT, that rescues stalled ribosomes in the absence of trans-translation using a new mechanism. These results indicate that ribosome rescue activity is essential in F. tularensis and suggest that ribosome rescue activity might be essential in all bacteria.

Ribosome Rescue Inhibitors Kill Actively Growing and Nonreplicating Persister Mycobacterium tuberculosis Cells.

The emergence of Mycobacterium tuberculosis (MTB) strains that are resistant to most or all available antibiotics has created a severe problem for treating tuberculosis and has spurred a quest for new antibiotic targets. Here, we demonstrate that trans-translation is essential for growth of MTB and is a viable target for development of antituberculosis drugs. We also show that an inhibitor of trans-translation, KKL-35, is bactericidal against MTB under both aerobic and anoxic conditions. Biochemical experiments show that this compound targets helix 89 of the 23S rRNA. In silico molecular docking predicts a binding pocket for KKL-35 adjacent to the peptidyl-transfer center in a region not targeted by conventional antibiotics. Computational solvent mapping suggests that this pocket is a druggable hot spot for small molecule binding. Collectively, our findings reveal a new target for antituberculosis drug development and provide critical insight on the mechanism of antibacterial action for KKL-35 and related 1,3,4-oxadiazole benzamides.

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

Bacillus anthracis, 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 B. anthracis spores makes it necessary to identify chemical agents that target and kill spores. Here, we demonstrate that a tetrazole-based trans-translation inhibitor, KKL-55, is bactericidal against vegetative cells of B. anthracis in culture. Using a fluorescent analog, we show that this class of compounds colocalizes with developing endospores and bind purified spores in vitro 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 B. anthracis spores are killed early in the germination process. At ≥2× MIC, KKL-55 protected macrophages from death after infection with B. anthracis 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 B. anthracis spores and suggest that there is an early requirement for trans-translation in germinating spores.

Anti-tubercular Activity of Pyrazinamide is Independent of trans-Translation and RpsA.

Pyrazinamide (PZA) is a first line anti-tubercular drug for which the mechanism of action remains unresolved. Recently, it was proposed that the active form of PZA, pyrazinoic acid (POA), disrupts the ribosome rescue process of trans-translation in Mycobacterium tuberculosis. This model suggested that POA binds within the carboxy-terminal domain of ribosomal protein S1 (RpsA) and inhibits trans-translation leading to accumulation of stalled ribosomes. Here, we demonstrate that M. tuberculosis RpsA interacts with single stranded RNA, but not with POA. Further, we show that an rpsA polymorphism previously identified in a PZA resistant strain does not confer PZA resistance when reconstructed in a laboratory strain. Finally, by utilizing an in vitro trans-translation assay with purified M. tuberculosis ribosomes we find that an interfering oligonucleotide can inhibit trans-translation, yet POA does not inhibit trans-translation. Based on these findings, we conclude that the action of PZA is entirely independent of RpsA and trans-translation in M. tuberculosis.

Teaching broader impacts of science with undergraduate research.

Science plays an important role in most aspects of society, and scientists face ethical decisions as a routine part of their work, but science education frequently omits or segregates content related to ethics and broader impacts of science. Undergraduate research experiences have the potential to bridge traditional divides in education and provide a holistic view of science. In practice, these experiences can be inconsistent and may not provide the optimal learning environment. We developed a course that combines seminar and independent research elements to support student learning during undergraduate research, makes ethical and societal impacts of science clear by relating them to the students' own research projects, and develops students' ethical decision-making skills. Here, we describe the course and provide resources for developing a similar course.

Human Cells Require Non-stop Ribosome Rescue Activity in Mitochondria.

Bacteria use trans-translation and the alternative rescue factors ArfA (P36675) and ArfB (Q9A8Y3) to hydrolyze peptidyl-tRNA on ribosomes that stall near the 3' end of an mRNA during protein synthesis. The eukaryotic protein ICT1 (Q14197) is homologous to ArfB. In vitro ribosome rescue assays of human ICT1 and Caulobacter crescentus ArfB showed that these proteins have the same activity and substrate specificity. Both ArfB and ICT1 hydrolyze peptidyl-tRNA on nonstop ribosomes or ribosomes stalled with ≤6 nucleotides extending past the A site, but are unable to hydrolyze peptidyl-tRNA when the mRNA extends ≥14 nucleotides past the A site. ICT1 provided sufficient ribosome rescue activity to support viability in C. crescentus cells that lacked both trans-translation and ArfB. Likewise, expression of ArfB protected human cells from death when ICT1 was silenced with siRNA. These data indicate that ArfB and ICT1 are functionally interchangeable, and demonstrate that ICT1 is a ribosome rescue factor. Because ICT1 is essential in human cells, these results suggest that ribosome rescue activity in mitochondria is required in humans.

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

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.

Mechanisms of ribosome rescue in bacteria.

Ribosomes that stall during translation need to be rescued to ensure that the protein synthesis capacity of the cell is maintained. Stalling arises when ribosomes become trapped at the 3' end of an mRNA, which occurs when a codon is unavailable, as this leads to the arrest of elongation or termination. In addition, various factors can induce ribosome stalling in the middle of an mRNA, including the presence of specific amino acid sequence motifs in the nascent polypeptide. Almost all bacteria use a mechanism known as trans-translation to rescue stalled ribosomes, and some species also have other rescue mechanisms that are mediated either by the alternative ribosome-rescue factor A (ArfA) or ArfB. In this Review, I summarize the recent studies that have demonstrated the conditions that trigger ribosome stalling, the pathways that bacteria use to rescue stalled ribosomes and the physiological effects of these processes.

Release of nonstop ribosomes is essential.

UNLABELLED: Bacterial ribosomes frequently translate to the 3' end of an mRNA without terminating at a stop codon. Almost all bacteria use the transfer-messenger RNA (tmRNA)-based trans-translation pathway to release these "nonstop" ribosomes and maintain protein synthesis capacity. trans-translation is essential in some species, but in others, such as Caulobacter crescentus, trans-translation can be inactivated. To determine why trans-translation is dispensable in C. crescentus, a Tn-seq screen was used to identify genes that specifically alter growth in cells lacking ssrA, the gene encoding tmRNA. One of these genes, CC1214, was essential in ΔssrA cells. Purified CC1214 protein could release nonstop ribosomes in vitro. CC1214 is a homolog of the Escherichia coli ArfB protein, and using the CC1214 sequence, ArfB homologs were identified in the majority of bacterial phyla. Most species in which ssrA has been deleted contain an ArfB homolog, suggesting that release of nonstop ribosomes may be essential in most or all bacteria. IMPORTANCE: Genes that are conserved across large phylogenetic distances are expected to confer a selective advantage. The genes required for trans-translation, ssrA and smpB, have been found in >99% of sequenced bacterial genomes, suggesting that they are broadly important. However, these genes can be deleted in some species without loss of viability. The identification and characterization of C. crescentus ArfB reveals why trans-translation is not essential in C. crescentus and suggests that many other bacteria are likely to use ArfB to survive when trans-translation is compromised.

Cell-based assay to identify inhibitors of the Hfq-sRNA regulatory pathway.

Noncoding small RNAs (sRNAs) act in conjunction with the RNA chaperone Hfq to regulate gene expression in bacteria. Because Hfq is required for virulence in several bacterial pathogens, the Hfq-sRNA system is an attractive target for antibiotic development. A reporter strain in which the expression of yellow fluorescent protein (YFP) is controlled by Hfq-sRNA was engineered to identify inhibitors of this system. A reporter that is targeted by Hfq in conjunction with the RybB sRNA was used in a genetic screen to identify inhibitors from a library of cyclic peptides produced in Escherichia coli using split-intein circular ligation of peptides and proteins (SICLOPPS), an intein-based technology. One cyclic peptide identified in this screen, RI20, inhibited Hfq-mediated repression of gene expression in conjunction with both RybB and an unrelated sRNA, MicF. Gel mobility shift assays showed that RI20 inhibited binding of Hfq to RybB and MicF with similar Ki values. These data suggest that RI20 inhibits Hfq activity by blocking interactions with sRNAs and provide a paradigm for inhibiting virulence genes in Gram-negative pathogens.