Wagner Mark C, Molnar Elizabeth E, Molitoris Bruce A, Goebl Mark G
Department of Medicine, Division of Nephrology, and the Indiana Center for Biological Microscopy, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122, USA.
Antimicrob Agents Chemother. 2006 Feb;50(2):587-95. doi: 10.1128/AAC.50.2.587-595.2006.
Gentamicin continues to be a primary antibiotic against gram-negative infections. Unfortunately, associated nephro- and ototoxicity limit its use. Our previous mammalian studies showed that gentamicin is trafficked to the endoplasmic reticulum in a retrograde manner and subsequently released into the cytosol. To better dissect the mechanism through which gentamicin induces toxicity, we have chosen to study its toxicity using the simple eukaryote Saccharomyces cerevisiae. A recent screen of the yeast deletion library identified multiple gentamicin-sensitive strains, many of which participate in intracellular trafficking. Our approach was to evaluate gentamicin sensitivity under logarithmic growth conditions. By quantifying growth inhibition in the presence of gentamicin, we determined that several of the sensitive strains were part of the Golgi-associated retrograde protein (GARP) and homotypic fusion and vacuole protein sorting (HOPS) complexes. Further evaluation of their other components showed that the deletion of any GARP member resulted in gentamicin-hypersensitive strains, while the deletion of other HOPS members resulted in less gentamicin sensitivity. Other genes whose deletion resulted in gentamicin hypersensitivity included ZUO1, SAC1, and NHX1. Finally, we utilized a Texas Red gentamicin conjugate to characterize gentamicin uptake and localization in both gentamicin-sensitive and -insensitive strains. These studies were consistent with our mammalian studies, suggesting that gentamicin toxicity in yeast results from alterations to intracellular trafficking pathways. The identification of genes whose absence results in gentamicin toxicity will help target specific pathways and mechanisms that contribute to gentamicin toxicity.
庆大霉素仍然是治疗革兰氏阴性菌感染的主要抗生素。不幸的是，其相关的肾毒性和耳毒性限制了它的使用。我们之前在哺乳动物身上进行的研究表明，庆大霉素以逆行方式转运至内质网，随后释放到细胞质中。为了更好地剖析庆大霉素诱导毒性的机制，我们选择使用简单的真核生物酿酒酵母来研究其毒性。最近对酵母缺失文库的筛选鉴定出多个对庆大霉素敏感的菌株，其中许多参与细胞内转运。我们的方法是在对数生长条件下评估庆大霉素敏感性。通过量化庆大霉素存在时的生长抑制情况，我们确定其中几个敏感菌株是高尔基体相关逆行蛋白（GARP）和同型融合与液泡蛋白分选（HOPS）复合体的一部分。对它们其他成分的进一步评估表明，任何GARP成员的缺失都会导致对庆大霉素超敏的菌株，而其他HOPS成员的缺失则导致对庆大霉素的敏感性降低。其他基因的缺失导致对庆大霉素超敏，这些基因包括ZUO1、SAC1和NHX1。最后，我们利用德克萨斯红-庆大霉素偶联物来表征庆大霉素在庆大霉素敏感和不敏感菌株中的摄取和定位。这些研究与我们在哺乳动物身上进行的研究一致，表明酵母中庆大霉素的毒性是由细胞内转运途径的改变引起的。鉴定出缺失后会导致庆大霉素毒性的基因将有助于靶向导致庆大霉素毒性的特定途径和机制。
