A selective antibiotic for Lyme disease.

Lyme disease is on the rise. Caused by a spirochete Borreliella burgdorferi, it affects an estimated 500,000 people in the United States alone. The antibiotics currently used to treat Lyme disease are broad spectrum, damage the microbiome, and select for resistance in non-target bacteria. We therefore sought to identify a compound acting selectively against B. burgdorferi. A screen of soil micro-organisms revealed a compound highly selective against spirochetes, including B. burgdorferi. Unexpectedly, this compound was determined to be hygromycin A, a known antimicrobial produced by Streptomyces hygroscopicus. Hygromycin A targets the ribosomes and is taken up by B. burgdorferi, explaining its selectivity. Hygromycin A cleared the B. burgdorferi infection in mice, including animals that ingested the compound in a bait, and was less disruptive to the fecal microbiome than clinically relevant antibiotics. This selective antibiotic holds the promise of providing a better therapeutic for Lyme disease and eradicating it in the environment.

A synthetic antibiotic class overcoming bacterial multidrug resistance.

The dearth of new medicines effective against antibiotic-resistant bacteria presents a growing global public health concern1. For more than five decades, the search for new antibiotics has relied heavily on the chemical modification of natural products (semisynthesis), a method ill-equipped to combat rapidly evolving resistance threats. Semisynthetic modifications are typically of limited scope within polyfunctional antibiotics, usually increase molecular weight, and seldom permit modifications of the underlying scaffold. When properly designed, fully synthetic routes can easily address these shortcomings2. Here we report the structure-guided design and component-based synthesis of a rigid oxepanoproline scaffold which, when linked to the aminooctose residue of clindamycin, produces an antibiotic of exceptional potency and spectrum of activity, which we name iboxamycin. Iboxamycin is effective against ESKAPE pathogens including strains expressing Erm and Cfr ribosomal RNA methyltransferase enzymes, products of genes that confer resistance to all clinically relevant antibiotics targeting the large ribosomal subunit, namely macrolides, lincosamides, phenicols, oxazolidinones, pleuromutilins and streptogramins. X-ray crystallographic studies of iboxamycin in complex with the native bacterial ribosome, as well as with the Erm-methylated ribosome, uncover the structural basis for this enhanced activity, including a displacement of the [Formula: see text] nucleotide upon antibiotic binding. Iboxamycin is orally bioavailable, safe and effective in treating both Gram-positive and Gram-negative bacterial infections in mice, attesting to the capacity for chemical synthesis to provide new antibiotics in an era of increasing resistance.

Retapamulin-Assisted Ribosome Profiling Reveals the Alternative Bacterial Proteome.

The use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding the cellular proteome. Although several such examples have been serendipitously found in bacteria, genome-wide mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin-enhanced Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the genes, but strikingly, it also revealed putative internal start sites in a number of Escherichia coli genes. Experiments demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose synthesis is initiated at internal in-frame and out-of-frame start sites, can be functionally important and contribute to the "alternative" bacterial proteome. The internal start sites may also play regulatory roles in gene expression.

Macrolones target bacterial ribosomes and DNA gyrase and can evade resistance mechanisms.

Growing resistance toward ribosome-targeting macrolide antibiotics has limited their clinical utility and urged the search for superior compounds. Macrolones are synthetic macrolide derivatives with a quinolone side chain, structurally similar to DNA topoisomerase-targeting fluoroquinolones. While macrolones show enhanced activity, their modes of action have remained unknown. Here, we present the first structures of ribosome-bound macrolones, showing that the macrolide part occupies the macrolide-binding site in the ribosomal exit tunnel, whereas the quinolone moiety establishes new interactions with the tunnel. Macrolones efficiently inhibit both the ribosome and DNA topoisomerase in vitro. However, in the cell, they target either the ribosome or DNA gyrase or concurrently both of them. In contrast to macrolide or fluoroquinolone antibiotics alone, dual-targeting macrolones are less prone to select resistant bacteria carrying target-site mutations or to activate inducible macrolide resistance genes. Furthermore, because some macrolones engage Erm-modified ribosomes, they retain activity even against strains with constitutive erm resistance genes.

Functional domains of a ribosome arresting peptide are affected by surrounding nonconserved residues.

Expression of the Escherichia coli tnaCAB operon, responsible for L-tryptophan (L-Trp) transport and catabolism, is regulated by L-Trp-directed translation arrest and the ribosome arresting peptide TnaC. The function of TnaC relies on conserved residues distributed throughout the peptide, which are involved in forming an L-Trp binding site at the ribosome exit tunnel and inhibiting the ribosome function. We aimed to understand whether nonconserved amino acids surrounding these critical conserved residues play a functional role in TnaC-mediated ribosome arrest. We have isolated two intragenic suppressor mutations that restore arrest function of TnaC mutants; one of these mutations is located near the L-Trp binding site, while the other mutation is located near the ribosome active site. We used reporter gene fusions to show that both suppressor mutations have similar effects on TnaC mutants at the conserved residues involved in forming a free L-Trp binding site. However, they diverge in suppressing loss-of-function mutations in a conserved TnaC residue at the ribosome active site. With ribosome toeprinting assays, we determined that both suppressor mutations generate TnaC peptides, which are highly sensitive to L-Trp. Puromycin-challenge assays with isolated arrested ribosomes indicate that both TnaC suppressor mutants are resistant to peptidyl-tRNA cleavage by puromycin in the presence of L-Trp; however, they differ in their resistance to puromycin in the absence of L-Trp. We propose that the TnaC peptide two functionally distinct segments, a sensor domain and a stalling domain, and that the functional versatility of these domains is fine-tuned by the nature of their surrounding nonconserved residues.

Inhibition of translation termination by the antimicrobial peptide Drosocin.

The proline-rich antimicrobial peptide (PrAMP) Drosocin (Dro) from fruit flies shows sequence similarity to other PrAMPs that bind to the ribosome and inhibit protein synthesis by varying mechanisms. The target and mechanism of action of Dro, however, remain unknown. Here we show that Dro arrests ribosomes at stop codons, probably sequestering class 1 release factors associated with the ribosome. This mode of action is comparable to that of apidaecin (Api) from honeybees, making Dro the second member of the type II PrAMP class. Nonetheless, analysis of a comprehensive library of endogenously expressed Dro mutants shows that the interactions of Dro and Api with the target are markedly distinct. While only a few C-terminal amino acids of Api are critical for binding, the interaction of Dro with the ribosome relies on multiple amino acid residues distributed throughout the PrAMP. Single-residue substitutions can substantially enhance the on-target activity of Dro.

Programmed Ribosomal Frameshifting Generates a Copper Transporter and a Copper Chaperone from the Same Gene.

Metal efflux pumps maintain ion homeostasis in the cell. The functions of the transporters are often supported by chaperone proteins, which scavenge the metal ions from the cytoplasm. Although the copper ion transporter CopA has been known in Escherichia coli, no gene for its chaperone had been identified. We show that the CopA chaperone is expressed in E. coli from the same gene that encodes the transporter. Some ribosomes translating copA undergo programmed frameshifting, terminate translation in the -1 frame, and generate the 70 aa-long polypeptide CopA(Z), which helps cells survive toxic copper concentrations. The high efficiency of frameshifting is achieved by the combined stimulatory action of a "slippery" sequence, an mRNA pseudoknot, and the CopA nascent chain. Similar mRNA elements are not only found in the copA genes of other bacteria but are also present in ATP7B, the human homolog of copA, and direct ribosomal frameshifting in vivo.

The context of the ribosome binding site in mRNAs defines specificity of action of kasugamycin, an inhibitor of translation initiation.

Kasugamycin (KSG) is an aminoglycoside antibiotic widely used in agriculture and exhibits considerable medical potential. Previous studies suggested that KSG interferes with translation by blocking binding of canonical messenger RNA (mRNA) and initiator transfer tRNA (tRNA) to the small ribosomal subunit, thereby preventing initiation of protein synthesis. Here, by using genome-wide approaches, we show that KSG can interfere with translation even after the formation of the 70S initiation complex on mRNA, as the extent of KSG-mediated translation inhibition correlates with increased occupancy of start codons by 70S ribosomes. Even at saturating concentrations, KSG does not completely abolish translation, allowing for continuing expression of some Escherichia coli proteins. Differential action of KSG significantly depends on the nature of the mRNA residue immediately preceding the start codon, with guanine in this position being the most conducive to inhibition by the drug. In addition, the activity of KSG is attenuated by translational coupling as genes whose start codons overlap with the coding regions or the stop codons of the upstream cistrons tend to be less susceptible to drug-mediated inhibition. Altogether, our findings reveal KSG as an example of a small ribosomal subunit-targeting antibiotic with a well-pronounced context specificity of action.

Context-based sensing of orthosomycin antibiotics by the translating ribosome.

Orthosomycin antibiotics inhibit protein synthesis by binding to the large ribosomal subunit in the tRNA accommodation corridor, which is traversed by incoming aminoacyl-tRNAs. Structural and biochemical studies suggested that orthosomycins block accommodation of any aminoacyl-tRNAs in the ribosomal A-site. However, the mode of action of orthosomycins in vivo remained unknown. Here, by carrying out genome-wide analysis of antibiotic action in bacterial cells, we discovered that orthosomycins primarily inhibit the ribosomes engaged in translation of specific amino acid sequences. Our results reveal that the predominant sites of orthosomycin-induced translation arrest are defined by the nature of the incoming aminoacyl-tRNA and likely by the identity of the two C-terminal amino acid residues of the nascent protein. We show that nature exploits this antibiotic-sensing mechanism for directing programmed ribosome stalling within the regulatory open reading frame, which may control expression of an orthosomycin-resistance gene in a variety of bacterial species.

Charting the sequence-activity landscape of peptide inhibitors of translation termination.

Apidaecin (Api), an unmodified 18-amino-acid-long proline-rich antibacterial peptide produced by bees, has been recently described as a specific inhibitor of translation termination. It invades the nascent peptide exit tunnel of the postrelease ribosome and traps the release factors preventing their recycling. Api binds in the exit tunnel in an extended conformation that matches the placement of a nascent polypeptide and establishes multiple contacts with ribosomal RNA (rRNA) and ribosomal proteins. Which of these interactions are critical for Api's activity is unknown. We addressed this problem by analyzing the activity of all possible single-amino-acid substitutions of the Api variants synthesized in the bacterial cell. By conditionally expressing the engineered api gene, we generated Api directly in the bacterial cytosol, thereby bypassing the need for importing the peptide from the medium. The endogenously expressed Api, as well as its N-terminally truncated mutants, retained the antibacterial properties and the mechanism of action of the native peptide. Taking advantage of the Api expression system and next-generation sequencing, we mapped in one experiment all the single-amino-acid substitutions that preserve or alleviate the on-target activity of the Api mutants. Analysis of the inactivating mutations made it possible to define the pharmacophore of Api involved in critical interactions with the ribosome, transfer RNA (tRNA), and release factors. We also identified the Api segment that tolerates a variety of amino acid substitutions; alterations in this segment could be used to improve the pharmacological properties of the antibacterial peptide.

A method for tritiation of iboxamycin permits measurement of its ribosomal binding.

Hydrogen-tritium exchange is widely employed for radioisotopic labeling of molecules of biological interest but typically involves the metal-promoted exchange of sp2-hybridized carbon-hydrogen bonds, a strategy that is not directly applicable to the antibiotic iboxamycin, which possesses no such bonds. We show that ruthenium-induced 2'-epimerization of 2'-epi-iboxamycin in HTO (200 mCi) of low specific activity (10 Ci/g, 180 mCi/mmol) at 80 °C for 18 h affords after purification tritium-labeled iboxamycin (3.55 µCi) with a specific activity of 53 mCi/mmol. Iboxamycin displayed an apparent inhibition constant (Ki, app) of 41 ± 30 nM towards Escherichia coli ribosomes, binding approximately 70-fold more tightly than the antibiotic clindamycin (Ki, app = 2.7 ± 1.1 µM).

Nascent peptide in the ribosome exit tunnel affects functional properties of the A-site of the peptidyl transferase center.

The ability to monitor the nascent peptide structure and to respond functionally to specific nascent peptide sequences is a fundamental property of the ribosome. An extreme manifestation of such response is nascent peptide-dependent ribosome stalling, involved in the regulation of gene expression. The molecular mechanisms of programmed translation arrest are unclear. By analyzing ribosome stalling at the regulatory cistron of the antibiotic resistance gene ermA, we uncovered a carefully orchestrated cooperation between the ribosomal exit tunnel and the A-site of the peptidyl transferase center (PTC) in halting translation. The presence of an inducing antibiotic and a specific nascent peptide in the exit tunnel abrogate the ability of the PTC to catalyze peptide bond formation with a particular subset of amino acids. The extent of the conferred A-site selectivity is modulated by the C-terminal segment of the nascent peptide, where the third-from-last residue plays a critical role.

Co-produced natural ketolides methymycin and pikromycin inhibit bacterial growth by preventing synthesis of a limited number of proteins.

Antibiotics methymycin (MTM) and pikromycin (PKM), co-produced by Streptomyces venezuelae, represent minimalist macrolide protein synthesis inhibitors. Unlike other macrolides, which carry several side chains, a single desosamine sugar is attached to the macrolactone ring of MTM and PKM. In addition, the macrolactone scaffold of MTM is smaller than in other macrolides. The unusual structure of MTM and PKM and their simultaneous secretion by S. venezuelae bring about the possibility that two compounds would bind to distinct ribosomal sites. However, by combining genetic, biochemical and crystallographic studies, we demonstrate that MTM and PKM inhibit translation by binding to overlapping sites in the ribosomal exit tunnel. Strikingly, while MTM and PKM readily arrest the growth of bacteria, ∼40% of cellular proteins continue to be synthesized even at saturating concentrations of the drugs. Gel electrophoretic analysis shows that compared to other ribosomal antibiotics, MTM and PKM prevent synthesis of a smaller number of cellular polypeptides illustrating a unique mode of action of these antibiotics.

Context-specific inhibition of translation by ribosomal antibiotics targeting the peptidyl transferase center.

The first broad-spectrum antibiotic chloramphenicol and one of the newest clinically important antibacterials, linezolid, inhibit protein synthesis by targeting the peptidyl transferase center of the bacterial ribosome. Because antibiotic binding should prevent the placement of aminoacyl-tRNA in the catalytic site, it is commonly assumed that these drugs are universal inhibitors of peptidyl transfer and should readily block the formation of every peptide bond. However, our in vitro experiments showed that chloramphenicol and linezolid stall ribosomes at specific mRNA locations. Treatment of bacterial cells with high concentrations of these antibiotics leads to preferential arrest of translation at defined sites, resulting in redistribution of the ribosomes on mRNA. Antibiotic-mediated inhibition of protein synthesis is most efficient when the nascent peptide in the ribosome carries an alanine residue and, to a lesser extent, serine or threonine in its penultimate position. In contrast, the inhibitory action of the drugs is counteracted by glycine when it is either at the nascent-chain C terminus or at the incoming aminoacyl-tRNA. The context-specific action of chloramphenicol illuminates the operation of the mechanism of inducible resistance that relies on programmed drug-induced translation arrest. In addition, our findings expose the functional interplay between the nascent chain and the peptidyl transferase center.

Nascent peptide assists the ribosome in recognizing chemically distinct small molecules.

Regulation of gene expression in response to the changing environment is critical for cell survival. For instance, binding of macrolide antibiotics to the ribosome promotes translation arrest at the leader open reading frames ermCL and ermBL, which is necessary for inducing the antibiotic resistance genes ermC and ermB. Cladinose-containing macrolides such as erythromycin (ERY), but not ketolides such as telithromycin (TEL), arrest translation of ermCL, whereas either ERY or TEL stall ermBL translation. How the ribosome distinguishes between chemically similar small molecules is unknown. We show that single amino acid changes in the leader peptide switch the specificity of recognition of distinct molecules, triggering gene activation in response to ERY alone, to TEL alone or to both antibiotics or preventing stalling altogether. Thus, the ribosomal response to chemical signals can be modulated by minute changes in the nascent peptide, suggesting that protein sequences could have been optimized for rendering translation sensitive to environmental cues.

The Berkeleylactones, Antibiotic Macrolides from Fungal Coculture.

A carefully timed coculture fermentation of Penicillium fuscum and P. camembertii/clavigerum yielded eight new 16-membered-ring macrolides, berkeleylactones A-H (1, 4, 6-9, 12, 13), as well as the known antibiotic macrolide A26771B (5), patulin, and citrinin. There was no evidence of the production of the berkeleylactones or A26771B (5) by either fungus when grown as axenic cultures. The structures were deduced from analyses of spectral data, and the absolute configurations of compounds 1 and 9 were determined by single-crystal X-ray crystallography. Berkeleylactone A (1) exhibited the most potent antimicrobial activity of the macrolide series, with low micromolar activity (MIC = 1-2 µg/mL) against four MRSA strains, as well as Bacillus anthracis, Streptococcus pyogenes, Candida albicans, and Candida glabrata. Mode of action studies have shown that, unlike other macrolide antibiotics, berkeleylactone A (1) does not inhibit protein synthesis nor target the ribosome, which suggests a novel mode of action for its antibiotic activity.

Macrolide antibiotics allosterically predispose the ribosome for translation arrest.

Translation arrest directed by nascent peptides and small cofactors controls expression of important bacterial and eukaryotic genes, including antibiotic resistance genes, activated by binding of macrolide drugs to the ribosome. Previous studies suggested that specific interactions between the nascent peptide and the antibiotic in the ribosomal exit tunnel play a central role in triggering ribosome stalling. However, here we show that macrolides arrest translation of the truncated ErmDL regulatory peptide when the nascent chain is only three amino acids and therefore is too short to be juxtaposed with the antibiotic. Biochemical probing and molecular dynamics simulations of erythromycin-bound ribosomes showed that the antibiotic in the tunnel allosterically alters the properties of the catalytic center, thereby predisposing the ribosome for halting translation of specific sequences. Our findings offer a new view on the role of small cofactors in the mechanism of translation arrest and reveal an allosteric link between the tunnel and the catalytic center of the ribosome.

Interactions of the TnaC nascent peptide with rRNA in the exit tunnel enable the ribosome to respond to free tryptophan.

A transcriptional attenuation mechanism regulates expression of the bacterial tnaCAB operon. This mechanism requires ribosomal arrest induced by the regulatory nascent TnaC peptide in response to free L-tryptophan (L-Trp). In this study we demonstrate, using genetic and biochemical analyses, that in Escherichia coli, TnaC residue I19 and 23S rRNA nucleotide A2058 are essential for the ribosome's ability to sense free L-Trp. We show that the mutational change A2058U in 23S rRNA reduces the concentration dependence of L-Trp-mediated tna operon induction, whereas the TnaC I19L change suppresses this phenotype, restoring the sensitivity of the translating A2058U mutant ribosome to free L-Trp. These findings suggest that interactions between TnaC residue I19 and 23S rRNA nucleotide A2058 contribute to the creation of a regulatory L-Trp binding site within the translating ribosome.