Abstract
Macrolide antibiotics, such as erythromycin and josamycin, are natural polyketide products harboring 14- to 16-membered macrocyclic lactone rings to which various sugars are attached. These antibiotics are used extensively in the clinic because of their ability to inhibit bacterial protein synthesis. More recently, some macrolides have been shown to also possess anti-inflammatory and other therapeutic activities in mammalian cells. To better understand the targets and effects of this drug class in mammalian cells, we used a genome-wide shRNA screen in K562 cancer cells to identify genes that modulate cellular sensitivity to josamycin. Among the most sensitizing hits were proteins involved in mitochondrial translation and the mitochondrial unfolded protein response, glycolysis, and the mitogen-activated protein kinase signaling cascade. Further analysis revealed that cells treated with josamycin or other antibacterial agents exhibited impaired oxidative phosphorylation and metabolic shifts to glycolysis. Interestingly, we observed that knockdown of the mitogen-activated protein kinase kinase kinase 4 (MAP3K4) gene, which contributes to p38 mitogen-activated protein kinase signaling, sensitized cells only to josamycin but not to other antibacterial agents. There is a growing interest in better characterizing the therapeutic effects and toxicities of antibiotics in mammalian cells to guide new applications in both cellular and clinical studies. To our knowledge, this is the first report of an unbiased genome-wide screen to investigate the effects of a clinically used antibiotic on human cells.
Highlights
Macrolide antibiotics, such as erythromycin and josamycin, are natural polyketide products harboring 14- to 16-membered macrocyclic lactone rings to which various sugars are attached
We previously optimized a high-density shRNA library (ϳ25 shRNAs per protein-coding gene and ϳ10,000 negative controls) and validated its performance in pooled genetic screens [36, 37]. Using this shRNA library, we conducted a genomewide screen to identify gene knockdowns that greatly altered the sensitivity of the human K562 erythroleukemia cell line to josamycin
Because sensitivity to macrolide antibiotics varies based on drug and cell type [35, 38], josamycin was titrated into a K562 cell culture and found to reduce cell growth with an IC50 of 39 M (Fig. 1B)
Summary
Amita Gupta‡§1,2, Aye Ökesli-Armlovich§¶1,3, X David Morgensʈ, Michael C. Bassik§ʈ, and Chaitan Khosla‡§¶**4 From the Departments of ‡Chemical Engineering, ¶Chemistry, ʈGenetics, and **Biochemistry and §Stanford Chemistry, Engineering and Medicine for Human Health (ChEM-H), Stanford University, Stanford, California 94305
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