Search

Streptogramin antibiotics consist of two types of molecules, group A and group B. The group B molecule quinupristin (RP 57669) and the group A molecule dalfopristin (RP 54476) constitute the first water-soluble semisynthetic streptogramin, quinupristin/dalfopristin (RP 59500). When group B molecules bind to 50S subunits or to tightly coupled ribosomes, there is an increase in their fluorescence intensity, which is proportional to the concentration of the antibiotic-ribosome complex formed. We found here that the background fluorescence of unbound quinupristin is 10-fold lower than that of unbound virginiamycin S, a natural group B molecule often used experimentally. The association constants were found (i) to be similar for the binding of the two group B molecules to tightly coupled 70S ribosomes in the absence of the group A molecules (quinupristin: 3.5 x 10(7) M(-1); virginiamycin S: 2.8 x 10(7) M(-1)) and (ii) to similarly increase about 20-fold in the presence of the corresponding group A molecule (quinupristin + dalfopristin: 69 x 10(7) M(-1); virginiamycin S + virginiamycin M: 60 x 10(7) M(-1)). Similar results were obtained with 50S ribosomal subunits. Additionally, we provide evidence that the failure of the group B molecules to inhibit poly(Phe) synthesis is due to the displacement of the group B molecule during poly(Phe) polymerization on the ribosome, indicating that the artificial poly(Phe) peptide competes with the binding of the group B molecule.

The streptogramins and related antibiotics (the lincosamides and macrolides) (MLS) are important inhibitors of bacterial protein synthesis. The key reaction in this process is the formation of a peptide bond between the growing peptide chain (peptidyl-tRNA) linked to the P-site of the 50S ribosome and aminoacyl-tRNA linked to the A site. This reaction is catalysed by the peptidyl transferase catalytic centre of the 50S ribosome. Type A and B streptogramins in particular have been shown to block this reaction through the inhibition of substrate attachment to the A and P sites and inhibition of peptide chain elongation. Synergy between type A and B components results from conformational changes imposed upon the peptidyl transferase centre by type A compounds and by inhibition of both early and late stages of protein synthesis. The conformational change increases ribosomal affinity for type B streptogramins. Microbial resistance to the MLSB antibiotics is largely attributable to mutations of rRNA bases, producing conformational changes in the peptidyl transferase centre. This can result in resistance to a single inhibitor or to a group of antibiotics (MLSB). The activity of type A streptogramin is retained thus explaining the improved inhibitory action of the combined streptogramins against macrolide and lincosamide-resistant strains. However, the development of resistance to the streptogramins may be less of a problem because of the synergic effect of type A and B compounds which has also been demonstrated in strains resistant to MLSB i.e., high level resistance to the combined streptogramins is only likely when type A streptogramin resistance determinants are present along with type B streptogramin resistance determinants.

Protein synthesis is catalysed by ribosomes and cytoplasmic factors. Bacterial ribosomes (70S) are made up of 2 subunits (50S and 30S) containing ribosomal RNA (rRNA) and ribosomal proteins: the 30S binds messenger RNA and begins the ribosomal cycle (initiation), whereas 50S binds transfer RNA (tRNA) derivatives and controls elongation. The key reaction, peptide bond formation, is promoted by the catalytic centre of 50S (the peptidyl transferase centre), and the growing peptide chain (peptidyl-tRNA) attached at the donor P site undergoes peptide linkage with an aminoacyl-tRNA at the acceptor A site. This reaction is inhibited by several antibiotics, the best known being chloramphenicol, and the macrolide-lincosamide-streptogramin (MLS) group. These inhibitors have a reversible action, except for streptogramins that are composed of A and B components, which are bacteriostatic alone, but bactericidal when combined. The peptidyl transferase centre has been identified at the 50S surface, and the binding sites of inhibitors have been mapped within this domain: some of these sites overlap (e.g. those of macrolides, and type B streptogramins, which compete for binding to ribosomes). Chloramphenicol blocks the catalytic portion, and A streptogramins the substrate sites of the peptidyl transferase centre. Macrolides and type B streptogramins interfere with the formation of long polypeptides and cause a premature detachment of incomplete peptide chains. The synergism between types A and B streptogramins is due to induction by type A streptogramins of an increased ribosome affinity for type B streptogramins. Microbial resistance to antibiotics mainly involves inactivation of inhibitors and modification of targets (mutations of ribosomal proteins or rRNA genes). Alterations of rRNA bases can induce resistance to a single inhibitor or to a group of antibiotics (e.g. MLSB). The impact of resistance in chemotherapy is less important for streptogramins than for other inhibitors, because the synergistic effect of A and B streptogramins also applies to strains resistant to the MLSB group. It is proposed that mutations and modifications of rRNA bases induce conformational ribosomal changes that prevent antibiotics binding to the target. Conformational changes are also triggered by type A streptogramins: they are responsible for their synergism with type B streptogramins.

Previous findings suggest the location of the central loop of domain V of 23S rRNA within the peptidyltransferase domain of ribosomes. This enzymatic activity is inhibited by some antibiotics, including type A (virginiamycin M or VM) and type B (virginiamycin S or VS) synergimycins, antibiotics endowed with a synergistic action in vivo. In the present work, the ability of VM and VS to modify the accessibility of 23S rRNA bases within ribosomes to chemical reagents has been explored. VM afforded a protection of rRNA bases A2037, A2042, G2049 and C2050. Moreover, when ribosomes were incubated with the two virginiamycin components, the base A2062, which was protected by VS alone, became accessible to dimethyl sulphate (DMS). Modified reactivity to chemical reagents of different rRNA bases located either in the central loop of domain V or in its proximity furnishes experimental evidence for conformational ribosome alterations induced by VM binding.

Synergism of streptogramins A (virginiamycin M, VM) and B (virginiamycin S, VS), peptidyltransferase inhibitors, was explored in EM4/pLC7-21 (wild type) and EM4/pERY (VS-resistant). These bacterial strains contained multicopy plasmids carrying an rrnH operon with wild type (pLC7-21) or mutated (A2058----U transversion) 23 S rRNA gene. Ribosomes with wild type and mutated rRNA were both present in EM4/pERY. The latter particles did not bind VS; in the presence of VM, however, high affinity VS binding occurred. As shown previously, VS protected against chemical reagents certain bases in domain V rRNA and VM in the stems flanking this loop. Differences between wild type and mutant ribosomes were observed: A2058, A2059, A2062, and G2505, protected by VS and ERY in EM4/pLC7-21, were unshielded in EM4/pERY. A2062 was shielded by VM in EM4/pERY, not in EM4/pLC7-21, and G2505 of mutant ribosomes became protected by VS when VM was simultaneously present. Induction by VM of a high affinity VS binding site in VS-sensitive and -resistant ribosomes indicates A2058 mutation to entail a conformational change of this site, which is counteracted by VM fixation. Accessibility of A2062 to chemical reagents (unlike behavior of EM4/pERY and EM4/pLC7-21 in the presence of VM) implies different conformations for wild type and mutant ribosomes.

The affinity of ribosomes for VS (virginiamycin S, a type B synergimycin) is known to be increased by VM (virginiamycin M, a type A synergimycin). Erythromycin, a macrolide, displaces ribosome-bound VS in the absence of VM, but is ineffective in its presence. In the present work, the ability of spiramycin and tylosin (macrolide subgroups) derivatives to displace ribosome-bound VS, in the presence and in the absence of VM, has been explored. All macrolides with in-vitro activity displaced ribosome-bound VS: the displacement curves produced by tylosin and spiramycin derivatives virtually overlapped. When VM was added to these systems, displaced VS became readily attached to ribosomes in the case of erythromycin, did not bind appreciably within 20 min incubation in the presence of tylosin, and underwent a slow binding in the case of an N-substituted tylosin. The 16-membered macrolides (leucomycin, spiramycin and tylosin subgroups) can therefore be distinguished from the 14-membered macrolides (erythromycin subgroup) by the antagonistic effect displayed toward VM.