Mitochondrial ribosome as the target for the macrolide antibiotic clarithromycin in the helminth Echinococcus multilocularis

Abstract

The mitochondrial rRNA of the tapeworm species Echinococcus multilocularis carries an adenine at sequence position 2058 (numbering according to that for Escherichia coli) of the large-subunit rRNA (lsrRNA), while the nucleus-encoded rRNA, as determined in this study, is characterized by 2058G. This indicates a dichotomy in the drug susceptibilities of ribosomes: cytoplasmic ribosomes are predicted to be resistant to macrolide antibiotics, while mitochondrial ribosomes lack the most common chromosomal resistance determinant, lsrRNA 2058G. Upon incubation with the macrolide clarithromycin, the formation of vesicles from metacestode tissue was reduced in a dose-dependent manner. Electron microscopy revealed distinct morphological alterations both of the mitochondria and of the vesicle wall (e.g., loss of microtriches) in drug-treated vesicles. Adult worms lost their motility and displayed morphological changes (shortening and constriction of proglottids and the presence of vacuoles) upon incubation with clarithromycin. Our findings demonstrate that macrolides have distinct in vitro effects on E. multilocularis, endorsing the use of sequence-based in silico approaches for exploitation of available ribosomal drugs as anthelmintic agents.

FIG. 1.

Effects of drugs on vesicle formation of E. multilocularis after 14 days of in vitro cultivation. 1, control, no drug; 2, 100 μg/ml clarithromycin; 3, 33 μg/ml clarithromycin; 4, 10 μg/ml clarithromycin; 5, hygromycin (100 μg/ml); 6, mebendazole (10 μg/ml). The mean values of the numbers of vesicles per well and the standard deviations are shown; experiments were done in quadruplicate and were repeated three times (twice with the two lower concentrations of clarithromycin). The results for all treatments were statistically significantly lower (P < 0.01, t test based on log + 1 transformed data) than those for the control treatment.

FIG. 2.

Effects of drugs on adult worms in vitro after 2 days. (A) Control (no drugs); (B) clarithromycin at 100 μg/ml.

FIG. 3.

Micrographs of E. multilocularis metacestode tissue in semithin sections (A and C) and ultrathin sections (B and D) of controls (no drugs) and after in vitro incubation with clarithromycin (100 μg ml⁻¹) for 14 days. The vesicles of the controls (A and B) are characterized by a thick laminated layer (LL) which is lined with several layers of cells (germinal layer [GL]) and by the presence of a distinct distal tegument (TE) and numerous microtriches (MT) protruding into the acellular laminated layer. Note the mitochondria with distinct cristae (arrows, panel B). After incubation with clarithromycin (C and D), the vesicles were smaller and had laminated layers of various thicknesses (C). The cells lining the laminated layers were thin and often detached. They contained condensed mitochondria and mitochondria with indistinct cristae (D, arrowheads). Microtriches and the tegument were largely lacking (D). Bars, 50 μm (A and C) or 1 μm (B and D).