Bojsen Rasmus, Regenberg Birgitte, Gresham David, Folkesson Anders
National Veterinary Institute, Technical University of Denmark, Frederiksberg, Denmark.
Department of Biology, University of Copenhagen, Copenhagen, Denmark.
Sci Rep. 2016 Feb 23;6:21874. doi: 10.1038/srep21874.
Fungal infections are an increasing clinical problem. Decreased treatment effectiveness is associated with biofilm formation and drug recalcitrance is thought to be biofilm specific. However, no systematic investigations have tested whether resistance mechanisms are shared between biofilm and planktonic populations. We performed multiplexed barcode sequencing (Bar-seq) screening of a pooled collection of gene-deletion mutants cultivated as biofilm and planktonic cells. Screening for resistance to the ergosterol-targeting fungicide amphotericin B (AmB) revealed that the two growth modes had significant overlap in AmB-persistent mutants. Mutants defective in sterol metabolism, ribosome biosynthesis, and the TORC1 and Ras pathways showed increased persistence when treated with AmB. The ras1, ras2 and tor1 mutants had a high-persister phenotype similar to wild-type biofilm and planktonic cells exposed to the TORC1 pathway inhibitor rapamycin. Inhibition of TORC1 with rapamycin also increased the proportion of persisters in Candida albicans and Candida glabrata. We propose that decreased TORC1-mediated induction of ribosome biosynthesis via Ras can lead to formation of AmB-persister cells regardless of whether the cells are in planktonic or biofilm growth mode. Identification of common pathways leading to growth mode-independent persister formation is important for developing novel strategies for treating fungal infections.
真菌感染正成为一个日益严重的临床问题。治疗效果的降低与生物膜形成有关，并且药物顽固性被认为具有生物膜特异性。然而，尚无系统性研究测试生物膜群体和浮游群体之间的耐药机制是否相同。我们对作为生物膜和浮游细胞培养的基因缺失突变体混合文库进行了多重条形码测序（Bar-seq）筛选。对靶向麦角固醇的抗真菌剂两性霉素B（AmB）的耐药性筛选显示，两种生长模式在对AmB有耐受性的突变体中存在显著重叠。在麦角固醇代谢、核糖体生物合成以及TORC1和Ras途径中存在缺陷的突变体在用AmB处理时表现出更高的耐受性。ras1、ras2和tor1突变体具有高耐受性表型，类似于暴露于TORC1途径抑制剂雷帕霉素的野生型生物膜和浮游细胞。用雷帕霉素抑制TORC1也增加了白色念珠菌和光滑念珠菌中耐受性细胞的比例。我们提出，无论细胞处于浮游还是生物膜生长模式，通过Ras降低TORC1介导的核糖体生物合成诱导都可导致AmB耐受性细胞的形成。识别导致与生长模式无关的耐受性细胞形成的共同途径对于开发治疗真菌感染的新策略很重要。
