H3K18乙酰化修饰调控白念珠菌唑类药物耐受机制的研究

Candida is one of the important pathogens of infectious fungus diseases. It has been found that drug resistance of C.albicans could lead to treatment failure. In our previous work, we identified lysine 18 as critical acetylation sites on Histone H3 of C.albicans, found inhibition of deacetylase activity can reduce C.albicans' tolerance to azoles and deacetylation of H3K18 is associated with increasing C.albicans' tolerance to azoles. But the mechanism of H3K18ac regulates C.albicans' sensitivity to azoles has not been reported. We try to build acetylation and deacetylation simulation of H3K18 mutant strains(K18R and K18Q) of C.albicans, study the drug tolerance and related gene/protein expression by drug sensitivity experiment, QPCR and WB/PRM. We also try to seek H3K18ac targeting genes by ChIP-Seq. This is first study to find the molecular mechanisms of the interaction between H3K18ac and azole drug tolerance of C.albicans, screening the new therapeutic targets of C.albicans for future drug development.

念珠菌是真菌感染性疾病重要的致病菌之一，临床上发现治疗白念珠菌系统感染的几种一线药物均存在不同程度的耐药现象，严重影响治疗效果。课题组首次发现白念珠菌组蛋白H3蛋白第18位赖氨酸存在乙酰化修饰（H3K18ac），发现抑制去乙酰化酶活性降低白念珠菌对唑类药物的耐受，并通过体外诱导耐药浮游菌株和构建生物膜模型，发现H3蛋白K18位点乙酰化修饰水平下调（去乙酰化修饰）与白念珠菌对唑类药物耐受增强相关，但H3K18ac调控白念珠菌对唑类药物敏感性的机制尚未见报道。课题组拟通过对构建的白念珠菌H3K18去乙酰化模拟突变株和乙酰化模拟突变株开展药物敏感性、QPCR和WB/PRM实验，研究其对药物耐受和相关基因/蛋白表达的影响，同时应用ChIP-Seq寻找H3K18ac靶向调控基因，拟从表观遗传学角度率先深入研究H3K18ac影响白念珠菌对唑类药物耐受的分子机制，筛选白念珠菌新的治疗靶点。

念珠菌是真菌感染性疾病重要的致病菌之一，临床上发现治疗白念珠菌系统感染的几种一线药物均存在不同程度的耐药现象，严重影响治疗效果。本项目从构建白念珠菌生物膜模型和诱导耐药菌株入手，通过激光共聚焦、蛋白质组学、WB、qPCR等方法观察组蛋白H3K18乙酰化修饰在白念珠菌不同状态或药物耐受下的表达情况，尝试构建H3K18位点突变菌株和去乙酰化酶突变菌株，通过ChIP-Seq寻找H3K18乙酰化修饰靶向调控的下游基因和通路，同时向上游探索组蛋白去乙酰化酶对白念珠菌表观修饰的影响。研究发现：①通过构建白念珠菌生物膜模型，发现组蛋白H3表达上调，但是H3蛋白上第18位赖氨酸乙酰化修饰（H3K18ac）下调，指出H3K18ac可能与白念珠菌生物膜形成相关；②通过诱导白念珠菌氟康唑耐药菌株，发现浮游状态下的菌株，随着氟康唑耐受溶度增加，H3K18乙酰化修饰水平下调（去乙酰化增强）；③通过ChIP-Seq明确了H3K18乙酰化修饰靶向调控的下游基因和通路，发现三个与生物膜形成相关的基因Bcr1，Tec1，Ndt80在72h表达量上调最高，同样mRNA表达上调的还有PCK1，MDH1，ACC1，NRP1，SUR2，ZPR1，SPR28，CAALFM_CR04620CA，但GST2基因表达下调，其中PCK1，MDH1在TCA cycle通路中发挥作用，说明H3K18乙酰化修饰直接调控生物膜相关基因和糖代谢等重要通路；④证实去乙酰化酶Sir2抑制剂AK-1可以调控白念珠菌乙酰化修饰的表达。本研究有助于筛选白念珠菌新的治疗靶点，为后续临床治疗和药物开发做铺垫。