Mechanisms of action of aminoglycoside antibiotics in eucaryotic protein synthesis

Abstract

Tetrahymena thermophila is a eucaryotic organism that is highly susceptible to growth inhibition by aminoglycoside antibiotics. Concentrations of paromomycin, gentamicin G418, and hygromycin B at 22, 10, and 17 ,uM, respectively, inhibited growth by 50%. A combination of in vitro and in vivo methods was used to determine the mechanisms of action of these aminoglycoside antibiotics on protein synthesis in T. thermophila. Analysis of polysome profiles from paromomycin-and gentamicin G418-treated cells showed clear, progressive depletions of polysomes concomitant with an inhibition of in vivo [14C]lysine incorporation. In vitro, paromomycin and gentamicin G418, which are disubstituted 2-deoxystreptamine-containing molecules, were not very effective inhibitors of either the translocation of peptidyl-tRNA or the elongation of nascent polypeptide chains on polysomes. In contrast, we found that the translocation of phe-tRNA on polyuridylate programmed ribosomes was susceptible to inhibition by paromomycin. We conclude that the primary inhibitory action of paromomycin and gentamicin G418 was at (i) an early stage of elongation after initiation, (ii) the initiation stage of translation, or (iii) a stage of translation before initiation. Hygromycin B, which is a monosubstituted 2-deoxystreptamine-containing aminoglycoside, potently inhibited the elongation of nascent chains during the translation of polysomes. In addition, the in vitro translation of polysomes from two hygromycin B-resistant mutants was resistant to the inhibition of elongation caused by hygromycin B.Aminoglycoside antibiotics are well-established inhibitors of growth, stimulators of misreading, and inhibitors of protein synthesis in procaryotes and eucaryotes (18). Vazquez and co-workers have studied the in vitro activities of many aminoglycoside antibiotics in eucaryotic model systems derived from yeasts and mammalian cells (20 and references therein). Of the aminoglycoside antibiotics tested, those active in inhibiting protein synthesis were usually found to inhibit the polypeptide chain elongation phase (20). Model systems to study the interaction of antibiotics with the ribosome at separate stages of the elongation cycle have proven to be powerful tools in the elucidation of antibiotic action on ribosomes. Furthermore, these studies have greatly enhanced the understanding of the structure-function relationships of ribosomes (8,15).Eucaryotic protein synthesis has not, however, received as much attention as have procaryotic systems, and the majority of current concepts about the structure of eucaryotic ribosomes have been derived by analogy with the extensive information available from Escherichia coli ribosomes. Model systems derived from different eucaryotic sources do not have uniform responses to a particular aminoglycoside, as is exemplified by the failure of paromomycin (PM) to inhibit the cytoplasmic protein synthesis of yeasts (14), mammalian cells (21), and wheat germ (22). In contrast, cytoplasmic protein synthesis in the lower eucaryote Tet...

Multiple Antibiotic Resistance in Arabidopsis Is Conferred by Mutations in a Chloroplast-Localized Transport Protein

“…These findings agree with our data; however, Aufsatz et al report that resistance is kanamycin specific and does not carry over to gentamicin or hygromycin. We did not see hygromycin resistance in any of our mar1 mutants, which was expected because hygromycin has effects against eukaryotic ribosomes and therefore acts in the cytoplasm of the plant cell (Eustice and Wilhelm, 1984). However, we do show that mar1 mutants are multiply resistant to several aminoglycosides, including gentamicin (Fig.…”

“…S1 and S2). These compounds, while structurally similar to other aminoglycosides, are distinct in that they inhibit both prokaryotic and eukaryotic protein synthesis (Eustice and Wilhelm, 1984). No resistance was found to antibiotics of other classes, including spectinomycin (an aminocyclitol), tetracycline, chloramphenicol, and lincomycin (Fig.…”

Antiviral Activity of Geneticin against Bovine Viral Diarrhoea Virus

“…In spite of the literature that demonstrated aminoglycosides inhibited protein synthesis [17,18], the selective inhibition of viral cytopathology by only G418 suggested that the antiviral activity of this aminoglycoside is different from that commonly seen with inhibitors of viral translation and replication. To assess the effect of G418 on the translation of viral proteins, we used viral NS3 protein as a marker of BVDV translation at 24 h post-infection with an MOI of 10.…”

“…Moreover, G418 did not inhibit viral RNA synthesis, suggesting that it did not affect viral polymerase (NS5B) formation and activity. These data suggest that G418, although being an inhibitor of protein synthesis [17,18], could possibly inhibit virus-induced cytopathology without blocking translation or replication. In addition, because virus entry precedes translation, it is highly unlikely that G418 blocks viral mechanisms before translation and replication.…”

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“…5 GUS expression at two stage 5a GUS foci in callus on resting medium (after 6-days co-cultivation); 5b GUS expression in leaf tissues of green shoots developed on RSM medium compared with non-transformed control leaf tissue from seedling All these data were resulted from three replicates with 25 calli per replication; data are presented after arcsine transformations as avg. means ± standard error followed by the same letter are not significantly different from each other (LSD > 0.05) Wilhelm 1984). When the treatment 9 was used in the selection media against transformed rice calli for 8 weeks, it resulted in survival of only the transgenic calli.…”