Multifaceted Mechanism of Amicoumacin A Inhibition of Bacterial Translation

Abstract

Amicoumacin A (Ami) halts bacterial growth by inhibiting the ribosome during translation. The Ami binding site locates in the vicinity of the E-site codon of mRNA. However, Ami does not clash with mRNA, rather stabilizes it, which is relatively unusual and implies a unique way of translation inhibition. In this work, we performed a kinetic and thermodynamic investigation of Ami influence on the main steps of polypeptide synthesis. We show that Ami reduces the rate of the functional canonical 70S initiation complex (IC) formation by 30-fold. Additionally, our results indicate that Ami promotes the formation of erroneous 30S ICs; however, IF3 prevents them from progressing towards translation initiation. During early elongation steps, Ami does not compromise EF-Tu-dependent A-site binding or peptide bond formation. On the other hand, Ami reduces the rate of peptidyl-tRNA movement from the A to the P site and significantly decreases the amount of the ribosomes capable of polypeptide synthesis. Our data indicate that Ami progressively decreases the activity of translating ribosomes that may appear to be the main inhibitory mechanism of Ami. Indeed, the use of EF-G mutants that confer resistance to Ami (G542V, G581A, or ins544V) leads to a complete restoration of the ribosome functionality. It is possible that the changes in translocation induced by EF-G mutants compensate for the activity loss caused by Ami.

Differential Contribution of Protein Factors and 70S Ribosome to Elongation

“…To compare the contribution of homologous and heterologous elongation factors EF-Tu, we decided to monitor a relatively late event of the A-site binding-tRNA accommodation-that would allow us to detect potential changes caused by heterologous EF-Tu during accommodation or preceding steps. The aa-tRNA accommodation in the A site changes the fluorescence intensity of the proflavin label (Prf) located at the elbow region of the initiator tRNA(Prf20) in the P site of the ribosome [23,24]. The interaction of both ternary complexes EF-Tu•GTP•Phe-tRNA Phe with the ribosome resulted in a fluorescence intensity decrease (Figure 1a).…”

“…To study kinetics of A-site binding, initiation complex (0.05 µM) containing fMet-tRNA fMet (Prf20) (initiator tRNA labelled with proflavine at position 20 in the elbow region of tRNA) [23,24] was rapidly mixed with increasing concentration of ternary complex EF-Tu•GTP•Phe-tRNA Phe (0.1 µM, 0.2 µM, 0.4 µM, 0.8 µM, 1.2 µM, 2 µM) containing mesophilic or thermophilic EF-Tu. The reaction was monitored at 20 and 37 • C.…”

“…[24]; hygromycin B, 20 µM; viomycin, 200 µM; spectinomycin, 900 µM; streptomycin, 20 µM[31]; kirromycin, 150 µM[57].…”

Antimicrobial Bacillus: Metabolites and Their Mode of Action

“…In particular, amicoumacin A (36) ( Figure 10 ), produced by B. pumilus BN-103, inhibits B. subtilis 1779 with an MIC = 20.0 µg/mL. Further studies have shown that amicoumacin A inhibits the protein synthesis of methicillin-resistant Staphylococcus aureus by stabilizing the mRNA at the terminal E site on the ribosome during protein synthesis [ 107 ]. This disruption results in the perturbation of the membrane, leading to energy dissipation and eventual cell death [ 107 , 108 ].…”

“…Further studies have shown that amicoumacin A inhibits the protein synthesis of methicillin-resistant Staphylococcus aureus by stabilizing the mRNA at the terminal E site on the ribosome during protein synthesis [ 107 ]. This disruption results in the perturbation of the membrane, leading to energy dissipation and eventual cell death [ 107 , 108 ]. Prumycin (37) ( Figure 10 ), isolated from a culture broth of B. amyloliquefaciens SD-32, exerts bactericidal and fungicidal effects, such as on S. sclerotiorum, with an MIC value of 1.56 µg/mL [ 109 , 110 , 111 ].…”