INHIBITION OF CYTOPLASMIC AND ORGANELLAR PROTEIN-SYNTHESIS IN TOXOPLASMA-GONDII - IMPLICATIONS FOR THE TARGET OF MACROLIDE ANTIBIOTICS
INHIBITION OF CYTOPLASMIC AND ORGANELLAR PROTEIN-SYNTHESIS IN TOXOPLASMA-GONDII - IMPLICATIONS FOR THE TARGET OF MACROLIDE ANTIBIOTICS
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DOI：
10.1172/jci117665
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发表时间：
1995-01-01
影响因子：
15.9
通讯作者：
JOINER, KA
中科院分区：
文献类型：
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作者：
BECKERS, CJM;ROOS, DS;JOINER, KA
我们研究了蛋白合成抑制剂对弓形虫原虫活性的潜在靶点。尽管在细胞培养和体内实验中，纳米摩尔浓度的阿奇霉素和克林霉素都能阻止弓形虫的复制，但在细胞外或细胞内寄生虫中，用100 μ M阿奇霉素处理长达24小时，都没有观察到蛋白质标记的抑制作用。对二维凝胶上300个点的定量分析显示，100 μ M阿奇霉素没有选择性地消耗蛋白质。相反，环己亚胺以剂量依赖的方式抑制蛋白质合成。对弓形虫核糖体RNA大亚基编码基因肽基转移酶区域的核苷酸序列分析表明，弓形虫胞质核糖体与其他真核核糖体一样，对大环内酯类抗生素具有耐药性。将环己亚胺处理与二维凝胶分析相结合，揭示了一小部分寄生虫蛋白可能在线粒体核糖体上合成。100 μ M的四环素对这些蛋白的合成有抑制作用，而100 μ M的阿奇霉素和克林霉素对这些蛋白的合成没有抑制作用。据信来自弓形虫线粒体基因组的核糖体DNA序列可预测大环内酯/利可沙胺耐药性。弓形虫总DNA的PCR扩增鉴定出另一类原核型核糖体基因，类似于恶性疟原虫的质体样核糖体基因。这些基因编码的核糖体预计对林可沙胺/大环内酯类抗生素敏感，并可能作为弓形虫和相关寄生虫中阿奇霉素、克林霉素和其他蛋白质合成抑制剂的功能靶点。
We investigated potential targets for the activity of protein synthesis inhibitors against the protozoan parasite Toxoplasma gondii. Although nanomolar concentrations of azithromycin and clindamycin prevent replication of T. gondii in both cell culture and in vivo assays, no inhibition of protein labeling was observed in either extracellular or intracellular parasites treated with up to 100 mu M drug for up to 24 h. Quantitative analysis of > 300 individual spots on two-dimensional gels revealed no proteins selectively depleted by 100 mu M azithromycin. In contrast, cycloheximide inhibited protein synthesis in a dose-dependent manner. Nucleotide sequence analysis of the peptidyl transferase region from genes encoding the large subunit of the parasite's ribosomal RNA predict that the cytoplasmic ribosomes of T. gondii, like other eukaryotic ribosomes, should be resistant to macrolide antibiotics.Combining cycloheximide treatment with two-dimensional gel analysis revealed a small subset of parasite proteins likely to be synthesized on mitochondrial ribosomes. Synthesis of these proteins was inhibited by 100 mu M tetracycline, but not by 100 mu M azithromycin or clindamycin. Ribosomal DNA sequences believed to be derived from the T. gondii mitochondrial genome predict macrolide/lincosamide resistance. PCR amplification of total T. gondii DNA identified an additional class of prokaryotic-type ribosomal genes, similar to the plastid-like ribosomal genes of the Plasmodium falciparum. Ribosomes encoded by these genes are predicted to be sensitive to the lincosamide/macrolide class of antibiotics, and may serve as the functional target for azithromycin, clindamycin, and other protein synthesis inhibitors in Toxoplasma and related parasites.