Chemical probing for examining the structure of modified RNAs and ligand binding to RNA.

Among different RNA modifications, the helix 69 (H69) region of the bacterial ribosomal RNA (rRNA) contains three pseudouridines (Ψs). H69 is functionally important due to its location in the heart of the ribosome. Several structural and functional studies have shown the importance of Ψ modifications in influencing the H69 conformation as well as maintaining key interactions in the ribosome during protein synthesis. Therefore, a need exists to understand the influence of modified nucleosides on conformational dynamics of the ribosome under solution conditions that mimic the cellular environment. In this review on chemical probing, we provide detailed protocols for the use of dimethyl sulfate (DMS) to examine H69 conformational states and the influence of Ψ modifications under varying solution conditions in the context of both ribosomal subunits and full ribosomes. The use of DMS footprinting to study the binding of aminoglycosides to the H69 region of bacterial rRNA as a potential antibiotic target will also be discussed. As highlighted in this work, DMS probing and footprinting are versatile techniques that can be used to gain important insight into RNA local structure and RNA-ligand interactions, respectively.

Antibiotics Targeting the 30S Ribosomal Subunit: A Lesson from Nature to Find and Develop New Drugs.

The use of antibiotics has revolutionized medicine, greatly improving our capacity to save millions of lives from otherwise deadly bacterial infections. Unfortunately, the health-associated benefits provided by antibiotics have been counteracted by bacteria developing or acquiring resistance mechanisms. The negative impact to public health is now considered of high risk due to the rapid spreading of multi-resistant strains. More than 60 % of clinically relevant antibiotics of natural origin target the ribosome, the supramolecular enzyme which translates the genetic information into proteins. Although many of these antibiotics bind the small ribosomal subunit, only a few are reported to inhibit the initiation of protein synthesis, with none reaching commercial availability. Counterintuitively, translation initiation is the most divergent phase of protein synthesis between prokaryotes and eukaryotes, a fact which is a solid premise for the successful identification of drugs with reduced probability of undesired effects to the host. Such a paradox is one of its kind and deserves special attention. In this review, we explore the inhibitors that bind the 30S ribosomal subunit focusing on both the compounds with proved effects on the translation initiation step and the underreported translation initiation inhibitors. In addition, we explore recent screening tests and approaches to discover new drugs targeting translation.

Aminomethyl Spectinomycins as Therapeutics for Drug-Resistant Gonorrhea and Chlamydia Coinfections.

Bacterial sexually transmitted infections are widespread and common, with Neisseria gonorrhoeae (gonorrhea) and Chlamydia trachomatis (chlamydia) being the two most frequent causes. If left untreated, both infections can cause pelvic inflammatory disease, infertility, ectopic pregnancy, and other sequelae. The recommended treatment for gonorrhea is ceftriaxone plus azithromycin (to empirically treat chlamydial coinfections). Antibiotic resistance to all existing therapies has developed in gonorrheal infections. The need for new antibiotics is great, but the pipeline for new drugs is alarmingly small. The aminomethyl spectinomycins, a new class of semisynthetic analogs of the antibiotic spectinomycin, were developed on the basis of a computational analysis of the spectinomycin binding site of the bacterial 30S ribosome and structure-guided synthesis. The compounds display particular potency against common respiratory tract pathogens as well as the sexually transmitted pathogens that cause gonorrhea and chlamydia. Here, we demonstrate the in vitro potencies of several compounds of this class against both bacterial species; the compounds displayed increased potencies against N. gonorrhoeae compared to that of spectinomycin and, significantly, demonstrated activity against C. trachomatis that is not observed with spectinomycin. Efficacies of the compounds were compared to those of spectinomycin and gentamicin in a murine model of infection caused by ceftriaxone/azithromycin-resistant N. gonorrhoeae; the aminomethyl spectinomycins significantly reduced the colonization load and were as potent as the comparator compounds. In summary, data produced by this study support aminomethyl spectinomycins as a promising replacement for spectinomycin and antibiotics such as ceftriaxone for treating drug-resistant gonorrhea, with the added benefit of treating chlamydial coinfections.

Ribosomal binding and antibacterial activity of ethylene glycol-bridged apidaecin Api137 and oncocin Onc112 conjugates.

Recent surveillance data on antimicrobial resistance predict the beginning of the post-antibiotic era with pan-resistant bacteria even overcoming polymyxin as the last available treatment option. Thus, new substances using novel modes of antimicrobial action are urgently needed to reduce this health threat. Antimicrobial peptides are part of the innate immune system of most vertebrates and invertebrates and accepted as valid substances for antibiotic drug development efforts. Especially, short proline-rich antimicrobial peptides (PrAMP) of insect origin have been optimized for activity against Gram-negative strains. They inhibit protein expression in bacteria by blocking the 70S ribosome exit tunnel (oncocin-type) or the assembly of the 50S subunit (apidaecin-type binding). Thus, apidaecin analog Api137 and oncocin analog Onc112 supposedly bind to different nearby or possibly partially overlapping binding sites. Here, we synthesized Api137/Onc112-conjugates bridged by ethylene glycol spacers of different length to probe synergistic activities and binding modes. Indeed, the antimicrobial activities against Escherichia coli and Pseudomonas aeruginosa improved for some constructs, although the conjugates did not bind better to the 70S ribosome of E. coli than Api137 and Onc112 using 5(6)-carboxyfluorescein-labelled Api137 and Onc112 in a competitive fluorescence polarization assay. In conclusion, Api137/Onc112-conjugates showed increased antimicrobial activities against P. aeruginosa and PrAMP-susceptible and -resistant E. coli most likely because of improved membrane interactions, whereas the interaction to the 70S ribosome was most likely not improved relying still on the independent apidaecin- and oncocin-type binding modes. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Chemically related 4,5-linked aminoglycoside antibiotics drive subunit rotation in opposite directions.

Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. Here we show that aminoglycosides chemically related to neomycin-paromomycin, ribostamycin and neamine-each bind to sites within h44 and H69 to perturb bridge B2 and affect subunit rotation. Neomycin and paromomycin, which only differ by their ring-I 6'-polar group, drive subunit rotation in opposite directions. This suggests that their distinct actions hinge on the 6'-substituent and the drug's net positive charge. By solving the crystal structure of the paromomycin-ribosome complex, we observe specific contacts between the apical tip of H69 and the 6'-hydroxyl on paromomycin from within the drug's canonical h44-binding site. These results indicate that aminoglycoside actions must be framed in the context of bridge B2 and their regulation of subunit rotation.

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.

Bactericidal effect of sulbactam against Acinetobacter baumannii ATCC 19606 studied by 2D-DIGE and mass spectrometry.

Acinetobacter baumannii has been associated with several severe hospital-acquired infections such as ventilator-associated pneumonia and meningitis. Sulbactam, a ß-lactamase inhibitor, is usually combined with ß-lactam antibiotics to treat infections. It has been found that sulbactam alone may be used to treat infections caused by A. baumannii, although the mechanism of the bactericidal effect remains unknown. In this study, proteomics was used to analyse protein intensity changes and to identify the proteins of A. baumannii following sulbactam treatment. In total, 54 proteins were found to exhibit significant changes in intensity. Proteins with reduced intensity included ATP-binding cassette (ABC) transporters as well as 30S and 50S ribosomal subunit proteins. These proteins are essential for nutrient import and protein synthesis and are vital for bacterial survival. The amplified proteins included glutamine synthetase, malic enzyme, RNA polymerase subunit α, and the molecular chaperones DnaK and GroEL, which function in metabolism, DNA and protein synthesis, and repair machinery. These amplified proteins were increased to rescue bacteria, however they could not overcome the effects of the reduced proteins and the bacteria were killed. This is the first report that the reduction of ABC transporters and 30S and 50S ribosomal subunit proteins plays an important role in the bactericidal effect of sulbactam against A. baumannii.

A structural basis for streptomycin-induced misreading of the genetic code.

During protein synthesis, the ribosome selects aminoacyl-transfer RNAs with anticodons matching the messenger RNA codon present in the A site of the small ribosomal subunit. The aminoglycoside antibiotic streptomycin disrupts decoding by binding close to the site of codon recognition. Here we use X-ray crystallography to define the impact of streptomycin on the decoding site of the Thermus thermophilus 30S ribosomal subunit in complexes with cognate or near-cognate anticodon stem-loop analogues and messenger RNA. Our crystal structures display a significant local distortion of 16S ribosomal RNA induced by streptomycin, including the crucial bases A1492 and A1493 that participate directly in codon recognition. Consistent with kinetic data, we observe that streptomycin stabilizes the near-cognate anticodon stem-loop analogue complex, while destabilizing the cognate anticodon stem-loop analogue complex. These data reveal how streptomycin disrupts the recognition of cognate anticodon stem-loop analogues and yet improves recognition of a near-cognate anticodon stem-loop analogue.

The antibiotic Furvina® targets the P-site of 30S ribosomal subunits and inhibits translation initiation displaying start codon bias.

Furvina®, also denominated G1 (MW 297), is a synthetic nitrovinylfuran [2-bromo-5-(2-bromo-2-nitrovinyl)-furan] antibiotic with a broad antimicrobial spectrum. An ointment (Dermofural®) containing G1 as the only active principle is currently marketed in Cuba and successfully used to treat dermatological infections. Here we describe the molecular target and mechanism of action of G1 in bacteria and demonstrate that in vivo G1 preferentially inhibits protein synthesis over RNA, DNA and cell wall synthesis. Furthermore, we demonstrate that G1 targets the small ribosomal subunit, binds at or near the P-decoding site and inhibits its function interfering with the ribosomal binding of fMet-tRNA during 30S initiation complex (IC) formation ultimately inhibiting translation. Notably, this G1 inhibition displays a bias for the nature (purine vs. pyrimidine) of the 3'-base of the codon, occurring efficiently only when the mRNA directing 30S IC formation and translation contains the canonical AUG initiation triplet or the rarely found AUA triplet, but hardly occurs when the mRNA start codon is either one of the non-canonical triplets AUU or AUC. This codon discrimination by G1 is reminiscent, though of opposite type of that displayed by IF3 in its fidelity function, and remarkably does not occur in the absence of this factor.

Allosteric control of the ribosome by small-molecule antibiotics.

Protein synthesis is targeted by numerous, chemically distinct antibiotics that bind and inhibit key functional centers of the ribosome. Using single-molecule imaging and X-ray crystallography, we show that the aminoglycoside neomycin blocks aminoacyl-transfer RNA (aa-tRNA) selection and translocation as well as ribosome recycling by binding to helix 69 (H69) of 23S ribosomal RNA within the large subunit of the Escherichia coli ribosome. There, neomycin prevents the remodeling of intersubunit bridges that normally accompanies the process of subunit rotation to stabilize a partially rotated ribosome configuration in which peptidyl (P)-site tRNA is constrained in a previously unidentified hybrid position. Direct measurements show that this neomycin-stabilized intermediate is incompatible with the translation factor binding that is required for distinct protein synthesis reactions. These findings reveal the functional importance of reversible intersubunit rotation to the translation mechanism and shed new light on the allosteric control of ribosome functions by small-molecule antibiotics.

Following the intersubunit conformation of the ribosome during translation in real time.

We report the direct observation of conformational rearrangements of the ribosome during multiple rounds of elongation. Using single-molecule fluorescence resonance energy transfer, we monitored the intersubunit conformation of the ribosome in real time as it proceeds from codon to codon. During each elongation cycle, the ribosome unlocks upon peptide bond formation, then reverts to the locked state upon translocation onto the next codon. Our data reveal both the specific and cumulative effects of antibiotics on individual steps of translation and uncover the processivity of the ribosome as it elongates. Our approach interrogates the precise molecular events occurring at each codon of the mRNA within the full context of ongoing translation.

Aminoglycoside association pathways with the 30S ribosomal subunit.

Many aminoglycoside antibiotics target bacterial ribosomes and alter their proper functioning as translational machinery leading to bacterial death. To better understand their several inhibitory mechanisms we applied Brownian dynamics and investigated the kinetics and association of paromomycin, an aminoglycoside representative, with the entire 30S ribosomal subunit. We determined that aminoglycoside specific binding at the ribosomal aminoacyl-tRNA site (A-site) begins with antibiotic diffusion toward any point on the 30S subunit and is followed by exploration of the 30S surface. Surprisingly, there is no direct electrostatic steering of the antibiotic to the A-site. Furthermore, we discovered two possible entrances to the A-site around which the mobility of paromomycin is high. The antibiotic also visits binding sites for other drugs targeting the 30S subunit. We found that paromomycin interacts with different sites located along the helix 44 of 16S rRNA, which might explain the recent experimental findings that paromomycin's other inhibitory role arises from overstabilizing the ribosomal 70S complex. In addition, our simulations revealed an alternate binding cleft in the 30S subunit that may be important for paromomycin's inhibitory effect on translocation. The diffusion limited rate of association was estimated of the order of 10(9) (M.s)(-1) with no dependence on the ionic strength of the solution; the physical origins of this result are explained.

Three methods to assay inhibitors of ribosomal subunit assembly.

The inhibition of bacterial ribosomal subunit formation is a novel target for translational inhibitors. Inhibition of subunit biogenesis has been shown to be equivalent to the inhibition of protein biosynthesis for many antibiotics. This chapter describes three methods for examining the inhibition of subunit formation in growing bacterial cells. The first method permits the determination of the IC50 value for inhibition of assembly and protein synthesis. The second is a pulse and chase labeling procedure to measure the kinetics of subunit formation. The third procedure allows an examination of ribosome reformation after antibiotic removal as a part of the post-antibiotic effect. Together these procedures give a description of the relative inhibitory effects of an antibiotic on translation and subunit formation.

Interaction between RsgA and the ribosome.

RsgA is a unique GTP hydrolytic protein that is widely found in bacteria and plants, and is activated by the small subunit of the ribosome. Disruption of the gene for RsgA from the genome affects the growth of cells, the subunit association of the ribosome in cells and maturation of 16S ribosomal RNA. Here, we investigated the interaction between EscherichiacoliRsgA and the ribosome. Several antibiotics bound to the decoding center of the small subunit inhibited the ribosome-dependent GTPase activity of RsgA, suggesting that RsgA binds to the decoding center. Chemical footprinting was also performed to further investigate the interaction.