evernimicin and avilamycin

The ribosome is one of the major targets for therapeutic antibiotics; however, the rise in multidrug resistance is a growing threat to the utility of our current arsenal. The orthosomycin antibiotics evernimicin (EVN) and avilamycin (AVI) target the ribosome and do not display cross-resistance with any other classes of antibiotics, suggesting that they bind to a unique site on the ribosome and may therefore represent an avenue for development of new antimicrobial agents. Here we present cryo-EM structures of EVN and AVI in complex with the Escherichia coli ribosome at 3.6- to 3.9-Å resolution. The structures reveal that EVN and AVI bind to a single site on the large subunit that is distinct from other known antibiotic binding sites on the ribosome. Both antibiotics adopt an extended conformation spanning the minor grooves of helices 89 and 91 of the 23S rRNA and interacting with arginine residues of ribosomal protein L16. This binding site overlaps with the elbow region of A-site bound tRNA. Consistent with this finding, single-molecule FRET (smFRET) experiments show that both antibiotics interfere with late steps in the accommodation process, wherein aminoacyl-tRNA enters the peptidyltransferase center of the large ribosomal subunit. These data provide a structural and mechanistic rationale for how these antibiotics inhibit the elongation phase of protein synthesis.

Topics: Amino Acid Sequence; Aminoglycosides; Anti-Bacterial Agents; Binding Sites; Cryoelectron Microscopy; Escherichia coli; Molecular Sequence Data; Molecular Structure; Oligosaccharides; Peptide Chain Elongation, Translational; Ribosome Subunits, Large, Bacterial; Single Molecule Imaging

Two structurally unique ribosomal antibiotics belonging to the orthosomycin family, avilamycin and evernimicin, possess activity against Enterococci, Staphylococci, and Streptococci, and other Gram-positive bacteria. Here, we describe the high-resolution crystal structures of the eubacterial large ribosomal subunit in complex with them. Their extended binding sites span the A-tRNA entrance corridor, thus inhibiting protein biosynthesis by blocking the binding site of the A-tRNA elbow, a mechanism not shared with other known antibiotics. Along with using the ribosomal components that bind and discriminate the A-tRNA-namely, ribosomal RNA (rRNA) helices H89, H91, and ribosomal proteins (rProtein) uL16-these structures revealed novel interactions with domain 2 of the CTC protein, a feature typical to various Gram-positive bacteria. Furthermore, analysis of these structures explained how single nucleotide mutations and methylations in helices H89 and H91 confer resistance to orthosomycins and revealed the sequence variations in 23S rRNA nucleotides alongside the difference in the lengths of the eukaryotic and prokaryotic α1 helix of protein uL16 that play a key role in the selectivity of those drugs. The accurate interpretation of the crystal structures that could be performed beyond that recently reported in cryo-EM models provide structural insights that may be useful for the design of novel pathogen-specific antibiotics, and for improving the potency of orthosomycins. Because both drugs are extensively metabolized in vivo, their environmental toxicity is very low, thus placing them at the frontline of drugs with reduced ecological hazards.

Topics: Aminoglycosides; Anti-Bacterial Agents; Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Drug Resistance, Bacterial; Gram-Positive Bacteria; Microbial Sensitivity Tests; Models, Molecular; Mutation; Nucleic Acid Conformation; Oligosaccharides; Protein Biosynthesis; Ribosomal Proteins; Ribosomes; RNA, Ribosomal; RNA, Ribosomal, 23S; RNA, Transfer; Sequence Alignment; Species Specificity

Orthosomycins are oligosaccharide antibiotics that include avilamycin, everninomicin, and hygromycin B and are hallmarked by a rigidifying interglycosidic spirocyclic ortho-δ-lactone (orthoester) linkage between at least one pair of carbohydrates. A subset of orthosomycins additionally contain a carbohydrate capped by a methylenedioxy bridge. The orthoester linkage is necessary for antibiotic activity but rarely observed in natural products. Orthoester linkage and methylenedioxy bridge biosynthesis require similar oxidative cyclizations adjacent to a sugar ring. We have identified a conserved group of nonheme iron, α-ketoglutarate-dependent oxygenases likely responsible for this chemistry. High-resolution crystal structures of the EvdO1 and EvdO2 oxygenases of everninomicin biosynthesis, the AviO1 oxygenase of avilamycin biosynthesis, and HygX of hygromycin B biosynthesis show how these enzymes accommodate large substrates, a challenge that requires a variation in metal coordination in HygX. Excitingly, the ternary complex of HygX with cosubstrate α-ketoglutarate and putative product hygromycin B identified an orientation of one glycosidic linkage of hygromycin B consistent with metal-catalyzed hydrogen atom abstraction from substrate. These structural results are complemented by gene disruption of the oxygenases evdO1 and evdMO1 from the everninomicin biosynthetic cluster, which demonstrate that functional oxygenase activity is critical for antibiotic production. Our data therefore support a role for these enzymes in the production of key features of the orthosomycin antibiotics.

Topics: Aminoglycosides; Anti-Bacterial Agents; Catalytic Domain; Crystallography, X-Ray; Cyclization; Hydrogen; Hygromycin B; Metals; Micromonospora; Multigene Family; Oligosaccharides; Open Reading Frames; Oxidation-Reduction; Oxygen; Oxygenases; Phylogeny; Protein Binding; Protein Structure, Secondary; Reproducibility of Results; Streptomyces

Topics: Aminoglycosides; Animals; Clinical Trials, Phase III as Topic; Drug Resistance, Bacterial; Gas Chromatography-Mass Spectrometry; Humans; Molecular Conformation; Molecular Structure; Oligosaccharides

Six high-level evernimicin-resistant Enterococcus faecium isolates were identified among 304 avilamycin-resistant E. faecium isolates from animals and 404 stool samples from humans with diarrhea. All four animal isolates, and one of the human isolates, were able to transfer resistance to a susceptible E. faecium strain. The resulting transconjugants all tested positive for the presence of emtA, a gene encoding a methyltransferase previously linked with high-level evernimicin resistance. The four transconjugants derived from animal isolates all carried the same plasmid, while a differently sized plasmid was found in the isolate from humans. This study demonstrated a low incidence of high-level evernimicin resistance mediated by the emtA gene in different E. faecium isolates of animal and human origin.

Topics: Aminoglycosides; Animal Feed; Animals; Anti-Bacterial Agents; Cattle; Chickens; Conjugation, Genetic; Denmark; Diarrhea; Drug Resistance; Enterococcus faecium; Humans; Methyltransferases; Oligosaccharides; Reverse Transcriptase Polymerase Chain Reaction; Swine

Fragments (414 bp) of the gene-encoding ribosomal protein L16 from Enterococcus faecium and Enterococcus faecalis that were resistant and susceptible to the oligosaccharide antibiotics avilamycin and evernimicin (SCH 27899) were sequenced and compared. The susceptible E. faecalis and E. faecium isolates had sequences that were similar to those of the type strains. All resistant E. faecalis isolates contained the same base pair variation [CGT (Arg-56) --> CAT (His-56)]. The same variation and two additional variations [ATC (Ile-52) --> ACC (Thr-52) and ATC (Ile-52) --> AGC (Ser-52)] were found in the resistant E. faecium isolates. This study indicated that resistance to the oligosaccharides in enterococci is associated with variations in the ribosomal protein L16.

Topics: Aminoglycosides; Anti-Bacterial Agents; Bacterial Proteins; Base Sequence; DNA, Bacterial; Enterococcus; Genetic Variation; Microbial Sensitivity Tests; Molecular Sequence Data; Oligosaccharides; Ribosomal Proteins

The emergence of multiresistant bacteria has increased the need for new antibiotics or modifications of older antibiotics. One promising agent might be the everninomicin SCH 27899, an oligosaccharide antibiotic recently developed by Schering Plough. However, another oligosaccharide, avilamycin, that is structurally very similar has been used as a growth promoter for food animals in the EU for several years, and a very frequent occurrence of resistance to avilamycin has been found among Enterococcus faecium isolates from broilers in Denmark. This study was conducted to investigate whether the resistance to avilamycin was associated with decreased susceptibility to everninomicin. From broilers, a total of 31 avilamycin susceptible and 55 avilamycin resistant (MIC >16 microg/mL) E. faecium isolates were selected. From pigs, 21 avilamycin-susceptible and eight avilamycin-resistant Enterococcus faecalis and 50 avilamycin-susceptible and two avilamycin-resistant E. faecium isolates were selected. All isolates were tested for susceptibility to everninomicin by E-test. The avilamycin-susceptible enterococci isolates had MICs to everninomicin from 0.064 to 0.75 microg/mL (MIC50 = 0.38 microg/mL) and the avilamycin-resistant isolates had MICs from 1.5 to 16 microg/mL (MIC50 = 3 microg/mL). Complete agreement between decreased susceptibility to avilamycin and everninomicin was found. This study showed that the use of avilamycin as a growth promoter for broilers and pigs has created a reservoir of E. faecium and E. faecalis isolates with decreased susceptibility to everninomicin among food animals already before this antibiotic have been finally developed for human use.

Topics: Aminoglycosides; Animal Feed; Animals; Anti-Bacterial Agents; Chickens; Drug Resistance, Microbial; Drug Utilization; Enterococcus; Gram-Positive Bacterial Infections; Growth Substances; Humans; Microbial Sensitivity Tests; Oligosaccharides; Swine