Lincosamide antibiotics stimulate dissociation of peptidyl-tRNA from ribosomes

Abstract

At nonpermissive temperatures the peptidyl-tRNA hydrolase ofpth(Ts) bacterial mutants is inactivated, and cells accumulate peptidyl-tRNA and die. Doses of erythromycin, lincomycin, or clindamycin that inhibited the growth of antibiotic-hypersensitive DB-11 pth+ cells accelerated the killing of DB-11 pth(Ts) cells at nonpermissive temperatures. Erythromycin and lincomycin also stimulated the accumulation of peptidyltRNA. Lincomycin and clindamycin stimulated peptidyl-tRNA dissociation from ribosomes.Lincosamide antibiotics inhibit protein synthesis by binding to procaryotic ribosomes. This binding was reported to block peptide bond formation and/or peptidyl-tRNA translocation from the A to the P site of the ribosome (8,18,22). Formerly, erythromycin and other macrolide antibiotics were also believed to have this mechanism (3,14). Since some mutations can confer resistance simultaneously to macrolides and lincosamides, it is possible that both classes inhibit protein synthesis as macrolides are known to do, by stimulating peptidyl-tRNA dissociation from ribosomes (16).Peptidyl-tRNA dissociates from ribosomes normally during protein synthesis (11,12). The effects are usually masked by peptidyl-tRNA hydrolase, an enzyme that catalyzes hydrolysis of intact peptide from dissociated peptidyl-tRNA. Cells with the pth(Ts) mutation grow normally at 30°C, but their hydrolase is inactivated if the temperature is raised to .40'C (2, 17), leading to peptidyl-tRNA accumulation, protein synthesis inhibition, and ultimately, cell death (13). If lincosamide antibiotics stimulate peptidyl-tRNA dissociation in a pth(Ts) mutant at high temperatures, they should impair cell survival and lead to a higher rate of peptidyltRNA accumulation.Cell survival. The Escherichia coli DB-11 met mutant was obtained from B. Weisblum, University of Wisconsin. DB-llpth(Ts) was isolated from a DB-11 met trp mutant by cotransducingpth(Ts) with trp+ by using P1 bacteriophage from AA7852 or from DB-11 by cotransducingpth(Ts) with Tetr (0.5 min apart [1]) from TnlO (Tetr) transposon-containing strain TGM8031. Peptidyl-tRNA hydrolase activity in DB-llpth(Ts) was demonstrated to be temperature sensitive (data not shown).Bacteria were grown to saturation overnight at 30°C in LP8 medium (61 mM Na2HPO4, 9.6 mM KH2PO4, 43 mM NaCl, 5.6 mM D-glucose, 5 g of Difco yeast extract per liter, 10 g of Difco tryptone per liter, adjusted to pH 8.0 with NaOH). The method was essentially that described previously (13). The fraction surviving was determined by dividing the density of CFU present after various times at the inactivation temperature by the density at time zero.For comparison with previous results (16), we report the effects of chloramphenicol and erythromycin, as well as lincosamide antibiotics. At elevated temperatures, growth of wildtypepth+ cells was arrested by the doses used (Fig. 1) Mutant pth(Ts) cells showed the expected loss of colonyforming ability at the high temperature because of accumulation of peptidyl-tRNA in the absence of peptidyl-tRNA hydrol...

Characterization of peptidyl-tRNA hydrolase encoded by open reading frame Rv1014c of Mycobacterium tuberculosis H37Rv

“…Macrolide action results in high fall-off rates of peptidyl-tRNA from the ribosome, leading to peptidyl-tRNA accumulation (Menninger and Coleman, 1993). Accumulation of peptidyl-tRNA in the cytoplasm is also caused by the action of lincosamide antibiotics, and has been suggested to be toxic for bacteria (Menninger and Otto, 1982).…”

Inhibition of Translation and Cell Growth by Minigene Expression

“…An essential enzyme, PTH, is responsible for peptidyl-tRNA recycling (23); PTH depletion results in cell death, most probably because of starvation for tRNA Lys sequestered in the form of peptidyl-tRNA (17). Several factors have been identified that may affect the rate of peptidyl-tRNA drop-off, including effect of antibiotics (26)(27)(28), codon-anticodon mispairing (4,11), and presence of consecutive rare codons at the beginning of the genes (9). Involvement of ribosomal termination (16) and initiation factors (19) in peptidyl-tRNA dissociation has been demonstrated.…”

Cumulative Effects of Several Nonenzymatic Mechanisms on the Resistance of Pseudomonas aeruginosa to Aminoglycosides

“…Therefore, Pth plays a crucial role in the recycling of essential tRNA molecules such as tRNA Lys sequestered as peptidyl-tRNAs (12), especially when the translation process aborts, for instance, because of the action of antibiotics targeting the ribosome (28). RT-PCR experiments showed that the transcript levels of pth were not abolished in mutant FE57 but just slightly reduced compared to that of PAO1 (ratio of 0.34 as a mean value of two distinct experiments), probably because of a polar effect of Tn5-associated promoters (4).…”