The Myxobacterial Antibiotic Myxovalargin: Biosynthesis, Structural Revision, Total Synthesis, and Molecular Characterization of Ribosomal Inhibition

Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent

Combinations of Macrolide Resistance Determinants in Field Isolates of Mannheimia haemolytica and Pasteurella multocida▿

Respiratory tract infections in cattle are commonly associated with the bacterial pathogens Mannheimia haemolytica and Pasteurella multocida. These infections can generally be successfully treated in the field with one of several groups of antibiotics, including macrolides. A few recent isolates of these species exhibit resistance to veterinary macrolides with phenotypes that fall into three distinct classes. The first class has type I macrolide, lincosamide, and streptogramin B antibiotic resistance and, consistent with this, the 23S rRNA nucleotide A2058 is monomethylated by the enzyme product of the erm(42) gene. The second class shows no lincosamide resistance and lacks erm(42) and concomitant 23S rRNA methylation. Sequencing of the genome of a representative strain from this class, P. multocida 3361, revealed macrolide efflux and phosphotransferase genes [respectively termed msr(E) and mph(E)] that are arranged in tandem and presumably expressed from the same promoter. The third class exhibits the most marked drug phenotype, with high resistance to all of the macrolides tested, and possesses all three resistance determinants. The combinations of erm(42), msr(E), and mph(E) are chromosomally encoded and intermingled with other exogenous genes, many of which appear to have been transferred from other members of the Pasteurellaceae. The presence of some of the exogenous genes explains recent reports of resistance to additional drug classes. We have expressed recombinant versions of the erm(42), msr(E), and mph(E) genes within an isogenic Escherichia coli background to assess their individually contributions to resistance. Our findings indicate what types of compounds might have driven the selection for these resistance determinants

Inhibition of protein synthesis on the ribosome by tildipirosin compared with other veterinary macrolides

Tildipirosin is a 16-membered-ring macrolide developed to treat bacterial pathogens, including Mannheimia haemolytica and Pasteurella multocida, that cause respiratory tract infections in cattle and swine. Here we evaluated the efficacy of tildipirosin at inhibiting protein synthesis on the ribosome (50% inhibitory concentration [IC(50)], 0.23 ± 0.01 μM) and compared it with the established veterinary macrolides tylosin, tilmicosin, and tulathromycin. Mutation and methylation at key rRNA nucleotides revealed differences in the interactions of these macrolides within their common ribosomal binding site

Visualizing the 16-Membered Ring Macrolides Tildipirosin and Tilmicosin Bound to Their Ribosomal Site

The veterinary antibiotic tildipirosin (20,23-dipiperidinyl-mycaminosyl-tylonolide, Zuprevo) was developed recently to treat bovine and swine respiratory tract infections caused by bacterial pathogens such as <i>Pasteurella multocida.</i> Tildipirosin is a derivative of the naturally occurring compound tylosin. Here, we define drug–target interactions by combining chemical footprinting with structure modeling and show that tildipirosin, tylosin, and an earlier tylosin derivative, tilmicosin (20-dimethylpiperidinyl-mycaminosyl-tylonolide, Micotil), bind to the same macrolide site within the large subunit of <i>P. multocida</i> and <i>Escherichia coli</i> ribosomes. The drugs nevertheless differ in how they occupy this site. Interactions of the two piperidine components, which are unique to tildipirosin, distinguish this drug from tylosin and tilmicosin. The 23-piperidine of tildipirosin contacts ribosomal residues on the tunnel wall while its 20-piperidine is oriented into the tunnel lumen and is positioned to interfere with the growing nascent peptide

Visualizing the 16-Membered Ring Macrolides Tildipirosin and Tilmicosin Bound to Their Ribosomal Site

The veterinary antibiotic tildipirosin (20,23-dipiperidinyl-mycaminosyl-tylonolide, Zuprevo) was developed recently to treat bovine and swine respiratory tract infections caused by bacterial pathogens such as <i>Pasteurella multocida.</i> Tildipirosin is a derivative of the naturally occurring compound tylosin. Here, we define drug–target interactions by combining chemical footprinting with structure modeling and show that tildipirosin, tylosin, and an earlier tylosin derivative, tilmicosin (20-dimethylpiperidinyl-mycaminosyl-tylonolide, Micotil), bind to the same macrolide site within the large subunit of <i>P. multocida</i> and <i>Escherichia coli</i> ribosomes. The drugs nevertheless differ in how they occupy this site. Interactions of the two piperidine components, which are unique to tildipirosin, distinguish this drug from tylosin and tilmicosin. The 23-piperidine of tildipirosin contacts ribosomal residues on the tunnel wall while its 20-piperidine is oriented into the tunnel lumen and is positioned to interfere with the growing nascent peptide

Visualizing the 16-Membered Ring Macrolides Tildipirosin and Tilmicosin Bound to Their Ribosomal Site

The veterinary antibiotic tildipirosin (20,23-dipiperidinyl-mycaminosyl-tylonolide, Zuprevo) was developed recently to treat bovine and swine respiratory tract infections caused by bacterial pathogens such as <i>Pasteurella multocida.</i> Tildipirosin is a derivative of the naturally occurring compound tylosin. Here, we define drug–target interactions by combining chemical footprinting with structure modeling and show that tildipirosin, tylosin, and an earlier tylosin derivative, tilmicosin (20-dimethylpiperidinyl-mycaminosyl-tylonolide, Micotil), bind to the same macrolide site within the large subunit of <i>P. multocida</i> and <i>Escherichia coli</i> ribosomes. The drugs nevertheless differ in how they occupy this site. Interactions of the two piperidine components, which are unique to tildipirosin, distinguish this drug from tylosin and tilmicosin. The 23-piperidine of tildipirosin contacts ribosomal residues on the tunnel wall while its 20-piperidine is oriented into the tunnel lumen and is positioned to interfere with the growing nascent peptide