Attenuating the emergence of anti-fungal drug resistance by harnessing synthetic lethal interactions in a model organism

Drug resistance is a rapidly emerging concern, thus prompting the development of novel therapeutics or combinatorial therapy. Currently, combinatorial therapy targets are based on knowledge of drug mode of action and/or resistance mechanisms, constraining the number of target proteins. Unbiased genome-wide screens could reveal novel genetic components within interaction networks as potential targets in combination therapies. Testing this, in the context of antimicrobial resistance, we implemented an unbiased genome-wide screen, performed in Saccharomyces cerevisiae expressing a Candida glabrata PDR1+ gain-of-function allele. Gain-of-function mutations in this gene are the principal mediators of fluconazole resistance in this human fungal pathogen. Eighteen synthetically lethal S. cerevisiae genetic mutants were identified in cells expressing C. glabrata PDR1+. One mutant, lacking the histone acetyltransferase Gcn5, was investigated further. Deletion or drug-mediated inhibition of Gcn5 caused a lethal phenotype in C. glabrata cells expressing PDR1+ alleles. Moreover, deletion or drug-mediated inactivation of Gcn5, inhibited the emergence of fluconazole-resistant C. glabrata isolates in evolution experiments. Thus, taken together, the data generated in this study provides proof of concept that synthetically lethal genetic screens can identify novel candidate proteins that when therapeutically targeted could allow effective treatment of drug-resistant infections.

耐药性 (Drug resistance) 正快速成为日益凸显的重大公共卫生关切，由此推动了新型治疗手段或联合疗法 (combinatorial therapy) 的研发。当前，联合疗法的靶点选择多基于药物作用模式及/或耐药机制的既有认知，这限制了可靶向蛋白的数量。无偏倚全基因组筛选 (Unbiased genome-wide screens) 可揭示互作网络中的新型遗传组分，将其作为联合疗法的潜在靶点。为验证这一思路，本研究以抗菌药物耐药性 (antimicrobial resistance) 为研究背景，在表达光滑念珠菌 (Candida glabrata) PDR1+ 功能获得性等位基因 (gain-of-function allele) 的酿酒酵母 (Saccharomyces cerevisiae) 中开展了无偏倚全基因组筛选。该基因的功能获得性突变是这类人类致病真菌产生氟康唑 (fluconazole) 耐药性的主要介导因素。本研究在表达光滑念珠菌PDR1+的酿酒酵母细胞中，共鉴定出18株合成致死 (synthetically lethal) 酿酒酵母遗传突变体。其中一株缺失组蛋白乙酰转移酶 (histone acetyltransferase) Gcn5的突变体被进一步深入研究。敲除或药物介导的Gcn5抑制，会在表达PDR1+等位基因的光滑念珠菌细胞中引发致死表型 (lethal phenotype)。此外，敲除或药物介导的Gcn5失活，可抑制进化实验中氟康唑耐药光滑念珠菌分离株的产生。综上，本研究生成的数据证实了合成致死遗传筛选的概念可行性：该方法可鉴定出新型候选蛋白，对其进行靶向治疗即可有效应对耐药菌感染。