Structural basis for the context-specific action of the classic peptidyl transferase inhibitor chloramphenicol.

Ribosome-targeting antibiotics serve as powerful antimicrobials and as tools for studying the ribosome, the catalytic peptidyl transferase center (PTC) of which is targeted by many drugs. The classic PTC-acting antibiotic chloramphenicol (CHL) and the newest clinically significant linezolid (LZD) were considered indiscriminate inhibitors of protein synthesis that cause ribosome stalling at every codon of every gene being translated. However, recent discoveries have shown that CHL and LZD preferentially arrest translation when the ribosome needs to polymerize particular amino acid sequences. The molecular mechanisms that underlie the context-specific action of ribosome inhibitors are unknown. Here we present high-resolution structures of ribosomal complexes, with or without CHL, carrying specific nascent peptides that support or negate the drug action. Our data suggest that the penultimate residue of the nascent peptide directly modulates antibiotic affinity to the ribosome by either establishing specific interactions with the drug or by obstructing its proper placement in the binding site.

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.

Effects of SDS on the activity and conformation of protein tyrosine phosphatase from thermus thermophilus HB27.

Deciphering the activity-conformation relationship of PTPase is of great interest to understand how PTPase activity is determined by its conformation. Here we studied the activity and conformational transitions of PTPase from thermus thermophilus HB27 in the presence of sodium dodecyl sulfate (SDS). Activity assays showed the inactivation of PTPase induced by SDS was in a concentration-dependent manner. Fluorescence and circular dichroism spectra suggested SDS induced significant conformational transitions of PTPase, which resulted in the inactivation of PTPase, and the changes of α-helical structure and tertiary structure of PTPase. Structural analysis revealed a number of hydrophobic and charged residues around the active sites of PTPase may be involved in the hydrophobic and ionic bonds interactions of PTPase and SDS, which are suggested to be the major driving force to result in PTPase inactivation and conformational transitions induced by SDS. Our results suggested the hydrophobic and charged residues around the active sites were essential for the activity and conformation of PTPase. Our study promotes a better understanding of the activity and conformation of PTPase.

Superoxide Dismutase and Pseudocatalase Increase Tolerance to Hg(II) in Thermus thermophilus HB27 by Maintaining the Reduced Bacillithiol Pool.

Mercury (Hg) is a widely distributed, toxic heavy metal with no known cellular role. Mercury toxicity has been linked to the production of reactive oxygen species (ROS), but Hg does not directly perform redox chemistry with oxygen. How exposure to the ionic form, Hg(II), generates ROS is unknown. Exposure of Thermus thermophilus to Hg(II) triggered ROS accumulation and increased transcription and activity of superoxide dismutase (Sod) and pseudocatalase (Pcat); however, Hg(II) inactivated Sod and Pcat. Strains lacking Sod or Pcat had increased oxidized bacillithiol (BSH) levels and were more sensitive to Hg(II) than the wild type. The ΔbshA Δsod and ΔbshA Δpcat double mutant strains were as sensitive to Hg(II) as the ΔbshA strain that lacks bacillithiol, suggesting that the increased sensitivity to Hg(II) in the Δsod and Δpcat mutant strains is due to a decrease of reduced BSH. Treatment of T. thermophilus with Hg(II) decreased aconitase activity and increased the intracellular concentration of free Fe, and these phenotypes were exacerbated in Δsod and Δpcat mutant strains. Treatment with Hg(II) also increased DNA damage. We conclude that sequestration of the redox buffering thiol BSH by Hg(II), in conjunction with direct inactivation of ROS-scavenging enzymes, impairs the ability of T. thermophilus to effectively metabolize ROS generated as a normal consequence of growth in aerobic environments.IMPORTANCEThermus thermophilus is a deep-branching thermophilic aerobe. It is a member of the Deinococcus-Thermus phylum that, together with the Aquificae, constitute the earliest branching aerobic bacterial lineages; therefore, this organism serves as a model for early diverged bacteria (R. K. Hartmann, J. Wolters, B. Kröger, S. Schultze, et al., Syst Appl Microbiol 11:243-249, 1989, https://doi.org/10.1016/S0723-2020(89)80020-7) whose natural heated habitat may contain mercury of geological origins (G. G. Geesey, T. Barkay, and S. King, Sci Total Environ 569-570:321-331, 2016, https://doi.org/10.1016/j.scitotenv.2016.06.080). T. thermophilus likely arose shortly after the oxidation of the biosphere 2.4 billion years ago. Studying T. thermophilus physiology provides clues about the origin and evolution of mechanisms for mercury and oxidative stress responses, the latter being critical for the survival and function of all extant aerobes.

High-resolution crystal structures of ribosome-bound chloramphenicol and erythromycin provide the ultimate basis for their competition.

The 70S ribosome is a major target for antibacterial drugs. Two of the classical antibiotics, chloramphenicol (CHL) and erythromycin (ERY), competitively bind to adjacent but separate sites on the bacterial ribosome: the catalytic peptidyl transferase center (PTC) and the nascent polypeptide exit tunnel (NPET), respectively. The previously reported competitive binding of CHL and ERY might be due either to a direct collision of the two drugs on the ribosome or due to a drug-induced allosteric effect. Because of the resolution limitations, the available structures of these antibiotics in complex with bacterial ribosomes do not allow us to discriminate between these two possible mechanisms. In this work, we have obtained two crystal structures of CHL and ERY in complex with the Thermus thermophilus 70S ribosome at a higher resolution (2.65 and 2.89 Å, respectively) allowing unambiguous placement of the drugs in the electron density maps. Our structures provide evidence of the direct collision of CHL and ERY on the ribosome, which rationalizes the observed competition between the two drugs.

Mode of Action of Kanglemycin A, an Ansamycin Natural Product that Is Active against Rifampicin-Resistant Mycobacterium tuberculosis.

Antibiotic-resistant bacterial pathogens pose an urgent healthcare threat, prompting a demand for new medicines. We report the mode of action of the natural ansamycin antibiotic kanglemycin A (KglA). KglA binds bacterial RNA polymerase at the rifampicin-binding pocket but maintains potency against RNA polymerases containing rifampicin-resistant mutations. KglA has antibiotic activity against rifampicin-resistant Gram-positive bacteria and multidrug-resistant Mycobacterium tuberculosis (MDR-M. tuberculosis). The X-ray crystal structures of KglA with the Escherichia coli RNA polymerase holoenzyme and Thermus thermophilus RNA polymerase-promoter complex reveal an altered-compared with rifampicin-conformation of KglA within the rifampicin-binding pocket. Unique deoxysugar and succinate ansa bridge substituents make additional contacts with a separate, hydrophobic pocket of RNA polymerase and preclude the formation of initial dinucleotides, respectively. Previous ansa-chain modifications in the rifamycin series have proven unsuccessful. Thus, KglA represents a key starting point for the development of a new class of ansa-chain derivatized ansamycins to tackle rifampicin resistance.

Drugging tRNA aminoacylation.

Inhibition of tRNA aminoacylation has proven to be an effective antimicrobial strategy, impeding an essential step of protein synthesis. Mupirocin, the well-known selective inhibitor of bacterial isoleucyl-tRNA synthetase, is one of three aminoacylation inhibitors now approved for human or animal use. However, design of novel aminoacylation inhibitors is complicated by the steadfast requirement to avoid off-target inhibition of protein synthesis in human cells. Here we review available data regarding known aminoacylation inhibitors as well as key amino-acid residues in aminoacyl-tRNA synthetases (aaRSs) and nucleotides in tRNA that determine the specificity and strength of the aaRS-tRNA interaction. Unlike most ligand-protein interactions, the aaRS-tRNA recognition interaction represents coevolution of both the tRNA and aaRS structures to conserve the specificity of aminoacylation. This property means that many determinants of tRNA recognition in pathogens have diverged from those of humans-a phenomenon that provides a valuable source of data for antimicrobial drug development.

Low-Molecular-Weight Thiols and Thioredoxins Are Important Players in Hg(II) Resistance in Thermus thermophilus HB27.

Mercury (Hg), one of the most toxic and widely distributed heavy metals, has a high affinity for thiol groups. Thiol groups reduce and sequester Hg. Therefore, low-molecular-weight (LMW) and protein thiols may be important cell components used in Hg resistance. To date, the role of low-molecular-weight thiols in Hg detoxification remains understudied. The mercury resistance (mer) operon of Thermus thermophilus suggests an evolutionary link between Hg(II) resistance and low-molecular-weight thiol metabolism. The mer operon encodes an enzyme involved in methionine biosynthesis, Oah. Challenge with Hg(II) resulted in increased expression of genes involved in the biosynthesis of multiple low-molecular-weight thiols (cysteine, homocysteine, and bacillithiol), as well as the thioredoxin system. Phenotypic analysis of gene replacement mutants indicated that Oah contributes to Hg resistance under sulfur-limiting conditions, and strains lacking bacillithiol and/or thioredoxins are more sensitive to Hg(II) than the wild type. Growth in the presence of either a thiol-oxidizing agent or a thiol-alkylating agent increased sensitivity to Hg(II). Furthermore, exposure to 3 µM Hg(II) consumed all intracellular reduced bacillithiol and cysteine. Database searches indicate that oah2 is present in all Thermus sp. mer operons. The presence of a thiol-related gene was also detected in some alphaproteobacterial mer operons, in which a glutathione reductase gene was present, supporting the role of thiols in Hg(II) detoxification. These results have led to a working model in which LMW thiols act as Hg(II)-buffering agents while Hg is reduced by MerA.IMPORTANCE The survival of microorganisms in the presence of toxic metals is central to life's sustainability. The affinity of thiol groups for toxic heavy metals drives microbe-metal interactions and modulates metal toxicity. Mercury detoxification (mer) genes likely originated early in microbial evolution in geothermal environments. Little is known about how mer systems interact with cellular thiol systems. Thermus spp. possess a simple mer operon in which a low-molecular-weight thiol biosynthesis gene is present, along with merR and merA In this study, we present experimental evidence for the role of thiol systems in mercury resistance. Our data suggest that, in T. thermophilus, thiolated compounds may function side by side with mer genes to detoxify mercury. Thus, thiol systems function in consort with mer-mediated resistance to mercury, suggesting exciting new questions for future research.

A Bright Future for Antibiotics?

Multidrug resistance is a global threat as the clinically available potent antibiotic drugs are becoming exceedingly scarce. For example, increasing drug resistance among gram-positive bacteria is responsible for approximately one-third of nosocomial infections. As ribosomes are a major target for these drugs, they may serve as suitable objects for novel development of next-generation antibiotics. Three-dimensional structures of ribosomal particles from Staphylococcus aureus obtained by X-ray crystallography have shed light on fine details of drug binding sites and have revealed unique structural motifs specific for this pathogenic strain, which may be used for the design of novel degradable pathogen-specific, and hence, environmentally friendly drugs.

Madumycin II inhibits peptide bond formation by forcing the peptidyl transferase center into an inactive state.

The emergence of multi-drug resistant bacteria is limiting the effectiveness of commonly used antibiotics, which spurs a renewed interest in revisiting older and poorly studied drugs. Streptogramins A is a class of protein synthesis inhibitors that target the peptidyl transferase center (PTC) on the large subunit of the ribosome. In this work, we have revealed the mode of action of the PTC inhibitor madumycin II, an alanine-containing streptogramin A antibiotic, in the context of a functional 70S ribosome containing tRNA substrates. Madumycin II inhibits the ribosome prior to the first cycle of peptide bond formation. It allows binding of the tRNAs to the ribosomal A and P sites, but prevents correct positioning of their CCA-ends into the PTC thus making peptide bond formation impossible. We also revealed a previously unseen drug-induced rearrangement of nucleotides U2506 and U2585 of the 23S rRNA resulting in the formation of the U2506•G2583 wobble pair that was attributed to a catalytically inactive state of the PTC. The structural and biochemical data reported here expand our knowledge on the fundamental mechanisms by which peptidyl transferase inhibitors modulate the catalytic activity of the ribosome.

Phenotypic Suppression of Streptomycin Resistance by Mutations in Multiple Components of the Translation Apparatus.

UNLABELLED: The bacterial ribosome and its associated translation factors are frequent targets of antibiotics, and antibiotic resistance mutations have been found in a number of these components. Such mutations can potentially interact with one another in unpredictable ways, including the phenotypic suppression of one mutation by another. These phenotypic interactions can provide evidence of long-range functional interactions throughout the ribosome and its functional complexes and potentially give insights into antibiotic resistance mechanisms. In this study, we used genetics and experimental evolution of the thermophilic bacterium Thermus thermophilus to examine the ability of mutations in various components of the protein synthesis apparatus to suppress the streptomycin resistance phenotypes of mutations in ribosomal protein S12, specifically those located distant from the streptomycin binding site. With genetic selections and strain constructions, we identified suppressor mutations in EF-Tu or in ribosomal protein L11. Using experimental evolution, we identified amino acid substitutions in EF-Tu or in ribosomal proteins S4, S5, L14, or L19, some of which were found to also relieve streptomycin resistance. The wide dispersal of these mutations is consistent with long-range functional interactions among components of the translational machinery and indicates that streptomycin resistance can result from the modulation of long-range conformational signals. IMPORTANCE: The thermophilic bacterium Thermus thermophilus has become a model system for high-resolution structural studies of macromolecular complexes, such as the ribosome, while its natural competence for transformation facilitates genetic approaches. Genetic studies of T. thermophilus ribosomes can take advantage of existing high-resolution crystallographic information to allow a structural interpretation of phenotypic interactions among mutations. Using a combination of genetic selections, strain constructions, and experimental evolution, we find that certain mutations in the translation apparatus can suppress the phenotype of certain antibiotic resistance mutations. Suppression of resistance can occur by mutations located distant in the ribosome or in a translation factor. These observations suggest the existence of long-range conformational signals in the translating ribosome, particularly during the decoding of mRNA.

The proline-rich antimicrobial peptide Onc112 inhibits translation by blocking and destabilizing the initiation complex.

The increasing prevalence of multidrug-resistant pathogenic bacteria is making current antibiotics obsolete. Proline-rich antimicrobial peptides (PrAMPs) display potent activity against Gram-negative bacteria and thus represent an avenue for antibiotic development. PrAMPs from the oncocin family interact with the ribosome to inhibit translation, but their mode of action has remained unclear. Here we have determined a structure of the Onc112 peptide in complex with the Thermus thermophilus 70S ribosome at a resolution of 3.1 Å by X-ray crystallography. The Onc112 peptide binds within the ribosomal exit tunnel and extends toward the peptidyl transferase center, where it overlaps with the binding site for an aminoacyl-tRNA. We show biochemically that the binding of Onc112 blocks and destabilizes the initiation complex, thus preventing entry into the elongation phase. Our findings provide a basis for the future development of this class of potent antimicrobial agents.

Ionic liquids for the concomitant use in extremophiles lysis and extremozymes extraction.

Ionic liquids have been successfully proposed to modify membrane permeability in cultures of a model extremophilic bacterium Thermus thermophilus HB27, which makes up the first time that aqueous solutions of these molten salts are applied in downstream stages of this kind of microorganisms. The presence of 1g/L of C10MIMCl entails a great solubilisation of cell biomass, thus allowing the release of intracellular and membrane-bound enzyme. The influence on the enzyme activity of two inorganic salts such as Na2CO3 and (NH4)2SO4, selected on the basis of their high salting out potential and biocompatibility with enzymes, respectively, was investigated. In parallel, their ability to trigger phase segregation was confirmed in the presence of the enzyme crude, leading to very high levels of enzyme extraction (96%). The validity of the strategy was confirmed by operating at bioreactor scale, and the main bioprocess parameters were obtained by modelling the experimental data.

Structural characterization of an alternative mode of tigecycline binding to the bacterial ribosome.

Although both tetracycline and tigecycline inhibit protein synthesis by sterically hindering the binding of tRNA to the ribosomal A site, tigecycline shows increased efficacy in both in vitro and in vivo activity assays and escapes the most common resistance mechanisms associated with the tetracycline class of antibiotics. These differences in activities are attributed to the tert-butyl-glycylamido side chain found in tigecycline. Our structural analysis by X-ray crystallography shows that tigecycline binds the bacterial 30S ribosomal subunit with its tail in an extended conformation and makes extensive interactions with the 16S rRNA nucleotide C1054. These interactions restrict the mobility of C1054 and contribute to the antimicrobial activity of tigecycline, including its resistance to the ribosomal protection proteins.

Structural analysis of base substitutions in Thermus thermophilus 16S rRNA conferring streptomycin resistance.

Streptomycin is a bactericidal antibiotic that induces translational errors. It binds to the 30S ribosomal subunit, interacting with ribosomal protein S12 and with 16S rRNA through contacts with the phosphodiester backbone. To explore the structural basis for streptomycin resistance, we determined the X-ray crystal structures of 30S ribosomal subunits from six streptomycin-resistant mutants of Thermus thermophilus both in the apo form and in complex with streptomycin. Base substitutions at highly conserved residues in the central pseudoknot of 16S rRNA produce novel hydrogen-bonding and base-stacking interactions. These rearrangements in secondary structure produce only minor adjustments in the three-dimensional fold of the pseudoknot. These results illustrate how antibiotic resistance can occur as a result of small changes in binding site conformation.

The antibiotics dityromycin and GE82832 bind protein S12 and block EF-G-catalyzed translocation.

The translocation of mRNA and tRNA through the ribosome is catalyzed by elongation factor G (EF-G), a universally conserved guanosine triphosphate hydrolase (GTPase). The mechanism by which the closely related decapeptide antibiotics dityromycin and GE82832 inhibit EF-G-catalyzed translocation is elucidated in this study. Using crystallographic and biochemical experiments, we demonstrate that these antibiotics bind to ribosomal protein S12 in solution alone as well as within the small ribosomal subunit, inducing long-range effects on the ribosomal head. The crystal structure of the antibiotic in complex with the 70S ribosome reveals that the binding involves conserved amino acid residues of S12 whose mutations result in in vitro and in vivo antibiotic resistance and loss of antibiotic binding. The data also suggest that GE82832/dityromycin inhibits EF-G-catalyzed translocation by disrupting a critical contact between EF-G and S12 that is required to stabilize the posttranslocational conformation of EF-G, thereby preventing the ribosome-EF-G complex from entering a conformation productive for translocation.

Antibiotic streptolydigin requires noncatalytic Mg2+ for binding to RNA polymerase.

Multisubunit RNA polymerase, an enzyme that accomplishes transcription in all living organisms, is a potent target for antibiotics. The antibiotic streptolydigin inhibits RNA polymerase by sequestering the active center in a catalytically inactive conformation. Here, we show that binding of streptolydigin to RNA polymerase strictly depends on a noncatalytic magnesium ion which is likely chelated by the aspartate of the bridge helix of the active center. Substitutions of this aspartate may explain different sensitivities of bacterial RNA polymerases to streptolydigin. These results provide the first evidence for the role of noncatalytic magnesium ions in the functioning of RNA polymerase and suggest new routes for the modification of existing and the design of new inhibitors of transcription.

Directed evolution for thermostabilization of a hygromycin B phosphotransferase from Streptomyces hygroscopicus.

To obtain a selection marker gene functional in a thermophilic bacterium, Thermus thermophilus, an in vivo-directed evolutionary strategy was conducted on a hygromycin B phosphotransferase gene (hyg) from Streptomyces hygroscopicus. The expression of wild-type hyg in T. thermophilus provided hygromycin B (HygB) resistance up to 60 °C. Through selection of mutants showing HygB resistance at higher temperatures, eight amino acid substitutions and the duplication of three amino acids were identified. A variant containing seven substitutions and the duplication (HYG10) showed HygB resistance at a highest temperature of 74 °C. Biochemical and biophysical analyses of recombinant HYG and HYG10 revealed that HYG10 was in fact thermostabilized. Modeling of the three-dimensional structure of HYG10 suggests the possible roles of the various substitutions and the duplication on thermostabilization, of which three substitutions and the duplication located at the enzyme surface suggested that these mutations made the enzyme more hydrophilic and provided increased stability in aqueous solution.

Stress fermentation strategies for the production of hyperthermostable superoxide dismutase from Thermus thermophilus HB27: effects of ions.

In this study, we explored how ammonium and metal ion stresses affected the production of recombinant hyperthermostable manganese superoxide dismutase (Mn-SOD). To improve Mn-SOD production, fed-batch culture in shake flasks and bioreactor fermentation were undertaken to examine the effects of [Formula: see text] and Mn(2+) feeding. Under the optimized feeding time and concentrations of [Formula: see text] and Mn(2+), the maximal SOD activity obtained from bioreactor fermentation reached some 480 U/ml, over 4 times higher than that in batch cultivation (113 U/ml), indicating a major enhancement of the concentration of Mn-SOD in the scale-up of hyperthermostable Mn-SOD production. In contrast, when the fed-batch culture with appropriate [Formula: see text] and Mn(2+) feeding was carried out in the same 5-L stirred tank bioreactor, a maximal SOD concentration of some 450 U/ml was obtained, again indicating substantial increase in SOD activity as a result of [Formula: see text] and Mn(2+) feeding. The isoelectric point (pI) of the sample was found to be 6.2. It was highly stable at 90 °C and circular dichroism measurements indicated a high α-helical content of 70 % as well, consistent with known SOD properties. This study indicates that [Formula: see text] and Mn(2+) play important roles in Mn-SOD expression. Stress fermentation strategies established in this study are useful for large-scale efficient production of hyperthermostable Mn-SOD and may also be valuable for the scale-up of other extremozymes.

Toxicity of indoxyl derivative accumulation in bacteria and its use as a new counterselection principle.

In this work we describe the conditional toxic effect of the expression of enzymes that cleave 5-bromo-4-chloro-3-indolyl (BCI) substrates and its use as a new counterselection principle useful for the generation of clean and unmarked mutations in the genomes of bacteria. The application of this principle was demonstrated in the thermophile Thermus thermophilus HB27 and in a mesophile for which currently no counterselection markers are available, Micrococcus luteus ATCC 27141. For T. thermophilus, the indigogenic substrate BCI-ß-glucoside was used in combination with the T. thermophilus ß-glucosidase gene (bgl). For M. luteus, a combination of BCI-ß-galactoside and the E. coli lacZ gene was implemented. We observed a strong growth-inhibiting effect when the strains were grown on agar plates containing the appropriate BCI substrates, the inhibition being proportional to the substrate concentration and the level of bgl/lacZ expression. The growth inhibition apparently depends on intracellular BCI substrate cleavage and accumulation of toxic indoxyl precipitates. The bgl and lacZ genes were used as counterselection markers for the rapid generation of scar-less chromosomal deletions in T. thermophilus HB27 (both in a Δbgl and in a wild type background) and in M. luteus ATCC 27141. In addition to Thermus and Micrococcus, sensitivity to BCI substrate cleavage was observed for other Gram-negative and Gram-positive species belonging to various bacterial phyla, including representatives of the genera Staphylococcus, Bacillus, Corynebacterium, Rhodococcus, Paracoccus and Xanthomonas. Thus, the toxicity of indoxyl derivative accumulation upon BCI substrate cleavage can be used for selection purposes in a broad range of microorganisms.