Liaison, mode d'action et synergie de la virginiamycine S : un inhibiteur de la synthèse protéique bactérienne
(1992)
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- Authors
- Supervisors
- Cocito, Carlo
- Abstract
- The Streptogramin family of antibiotics, which include the virginiamycin, is composed of two groups of chemically unrelated compounds: type A; such as virginiamycin M (VM), and type B, such as virginiamycin S (VS). Both types of Streptogramins are simultaneously produced by Streptomyces. Each single type of compound reversibly inhibits growth of gram positive bacteria, whereas their synergistic action on protein synthesis induces a bactericidal effect. <BR> The present work was focused on virginiamycine S. Its aim was explore the binding of this antibiotic to ribosome and its autonomous and synergistic (with VM) action. This research was divided in four distinct parts, each delineated in one of the following paragraphs. <BR> A cell-free polypeptide synthesis system, based on the translation of several two-base random copolymers, was developed to study the mode of action of virginiamycine S on the translation elongation step. Under these conditions, VS-induced inhibitory action increased with the polymerization reaction rate and the peptide size. The slowing down of synthesis due to virginiamycine S was related to a decreased efficiency of peptide bond formation. In addition, VS was able to stimulate a premature release of peptidyl-tRNA. This premature release was dependent on the nature of the C-terminal amino acid residue of the growing peptidyl chain. All these effects might be due to the local conformational change induced by binding of the antibiotic to ribosome. This change would result in altered interaction between peptidyl-t-RNA and the donor site of peptidyltransférase. Moreover, contrary to VM, VS was able to act on translating ribosomes. Using the same approach, it was shown that erythromycin (M14 MAcrolide) exerts a similar mode of action. Location of the VS binding site into the bacterial ribosomal three-dimensional structure was investigated by identifying its constitutive ribosomal proteins. Therefore, ribosome affinitu labelling was performed with a VS derivative (N-hydroxysuccinimide ester, VSE) able to covalently link with amino groups of proteins. Identification of affinity-labeled proteins, using the VSE derivative, was performed by an immunoenzymometric procedure. A aspecific linkage of VSE with a restricted region of the large ribosomal subunit was observed. The fact that more than 80% of the label was associated with both L18 and L22 proteins argues for the contribution of both these proteins to the VS binding site and, indirectly, to the peptidyltransférase domain of bacterial ribosomes. No modification of the labelling pattern was observed when the coupling reaction was performed in presence of virginiamycine M, which is otherwise known to induce a conformational change to the VS binding site. In view of the literature data, giving direct or indirect information on the location of L18 and L22 into the three-dimensional structure of ribosome, these results indicate that that VS binding site is situated at the base of the central protuberance of the 50S subunit, the purative location of peptidyltransférase domain. <BR> Understanding of the molecular interactions between VS and ribosomes was further expanded here to the role played by a bivalent cation in VS binding. This part of the study was based on the increase in intensity of the intrinsic fluorescence of VS when chelated to alkaline earth cations or bound to ribosomes. Fluorescence lifetime measurements, equilibrium titrations and stopped-flow spectrofluorometry allowed to observe VS•MG²+ dissociation in the presence and absence of ribosome. Our results strongly suggest that the interaction between VS and ribosome is partly provided by a salt bridge between suitable acceptor atoms of the ribosome and the 3-OH-picolinyl residue of VS. <BR> The study of VS inhibitory action was completed by an analysis of the in vivo synergistic action mechanism observed in the presence of VS and VM. Synergistic effect on bacterial growth was shown to require the simultaneous presence of both components to the active ribosomal population. VM concentration constituted the limiting factor of the synergic reaction. The decrease in bacterial viability was related to both protein synthesis inhibition and antibiotic-ribosome binding of type A Streptogramins of the ribosomes. It also clearly excludes a catalytic mode of VM promoted effect in ribosome affinity for VS? On the other hand, VS, which did not directly affect the association constant of VM to ribosomes, promotes the binding of VM by inducing the premature release stimulation of peptidyl-tRNA. Accordingly, a model was elaborated founding the synergistic mechanism on the sudden inhibition of polysomes. Therefore, the functional ribosomal population would rapidly decrease, leading to a dramatic loss of cellular viability
- Affiliations
Citations
Nyssen, E. (1992). Liaison, mode d’action et synergie de la virginiamycine S : un inhibiteur de la synthèse protéique bactérienne. https://hdl.handle.net/2078.5/111172