Differential effects of paromomycin on ribosomes of Leishmania mexicana and mammalian cells

Abstract

Paromomycin, an aminoglycoside antibiotic having low mammalian cell toxicity, is one of the drugs currently used in the chemotherapy of cutaneous and visceral leishmaniasis. In order to understand the mode of action of this antibiotic at the molecular level, we have investigated the effects of paromomycin on protein synthesis in Leishmania and its mammalian hosts. We were able to demonstrate that in vivo protein synthesis in the promastigote stage of the parasite and its proliferation rate are markedly inhibited by paromomycin while being only slightly affected by other aminoglycoside antibiotics, such as streptomycin and neomycin B. Furthermore, both in vitro polypeptide synthesis induced by poly(U) as mRNA and accuracy of translation are significantly decreased by paromomycin in cell-free systems containing ribosomal particles of Leishmania promastigotes. Conversely, when ribosomes from mammalian cells are used instead of the protozoan particles, polyphenylalanine synthesis is only barely reduced by the antibiotic and the translation misreading remains almost unaltered. Surface plasmon resonance analysis of the interaction between paromomycin and protozoan or mammalian cell ribosomal RNAs shows a strong binding of antibiotic to the parasite ribosomal decoding site and practically no interaction with the mammalian cell counterpart. Our results indicating differential effects of paromomycin on the translation processes of the Leishmania parasite and its mammalian hosts can explain the therapeutic efficiency of this antibiotic as an antileishmaniasis agent.

FIG. 1.

Inhibition of Leishmania mexicana growth by aminoglycoside antibiotics. Parasites were cultivated in SDM-79 medium in the absence of antibiotics (○) or with the addition of 0.4 mM streptomycin (•), 0.4 mM neomycin B (▴), or 0.2 mM (□) or 0.4 mM (▪) paromomycin. Parasite proliferation was followed for 72 h.

FIG. 2.

Effect of aminoglycoside antibiotics on Leishmania mexicana protein synthesis in vivo. Parasites at the exponential phase of growth in the absence or presence of antibiotics were labeled with radioactive methionine (Met) and treated as indicated in Materials and Methods. Cultures without antibiotics (○) or in the presence of 0.4 mM streptomycin (•), 0.4 mM neomycin B (▴), or 0.2 mM (□) or 0.4 mM (▪) paromomycin were used.

FIG. 3.

Effect of paromomycin on polyphenylalanine synthesis in cell-free systems. (A and B) S₃₀ cell fractions were obtained from the Crithidia fasciculata (•) or Leishmania mexicana (○) trypanosomatid parasites (A) or rat liver (B). (C) Polypeptide synthesis in reaction mixtures containing an S₁₅₀ supernatant fraction from Leishmania mexicana and purified ribosomes prepared from rat liver (▴) or Leishmania (▪) extracts in the presence of different antibiotic concentrations. Samples were incubated and treated as described in Materials and Methods. All values correspond to the average ± SD of three determinations.

FIG. 4.

Effect of paromomycin on translation misreading. (A) Reaction mixtures containing an S₁₅₀ supernatant fraction from Leishmania mexicana and purified ribosomal particles from trypanosomatid parasites (Crithidia fasciculata or Leishmania mexicana) or rat liver (mammalian cells system) were assayed in the absence and presence of 20 μM paromomycin. RL, Cf, and Lm, rat liver, Crithidia fasciculata, and Leishmania mexicana ribosomes, respectively. (B) The reaction mixtures contained an S₁₅₀ fraction from Leishmania mexicana and purified ribosomes from Leishmania (○) or rat liver (•). Assays were carried out at different paromomycin concentrations. Misreading frequencies shown are the average values ± SD for three experiments.

FIG. 5.

Surface plasmon resonance analysis (SPR) of the interaction of aminoglycoside antibiotics with L. mexicana rRNA. (A) SPR sensorgram of the interactions between paromomycin (8 to 0.06 mM) and immobilized L. mexicana rRNA (1,450 RU) after correction of nonspecific binding. (B) SPR sensorgram of the interaction of neomycin B (2 to 0.03 mM) and immobilized L. mexicana rRNA (1,560 RU) after correction of nonspecific binding. (C and D) Nonlinear analysis for the determination of the apparent K_D of the interaction between immobilized L. mexicana rRNA and aminoglycoside antibiotics, paromomycin, and neomycin B, respectively. (E) SPR sensorgram of the interactions between paromomycin (8 to 0.06 mM) and immobilized human rRNA (1,400 RU). (F) SPR sensorgram of the interactions between ampicillin (8 to 0.06 mM) and immobilized L. mexicana rRNA (1,510 RU). The sensorgrams are representative of at least three different experiments.