铜绿假单胞菌DNA甲基转移酶调控基因表达和毒力表型的机制研究

大部分原核生物缺乏组蛋白和核小体结构，其表观遗传控制主要依赖 DNA 甲基化，由 DNA甲基转移酶 (DNA MTase) 介导。近年来，多项研究表明，DNA 甲基化在调节细菌生理生化活动上具有重要作用。铜绿假单胞菌 (Pseudomonas aeruginosa) 是一种常见的革兰氏阴性条件致病菌，能够引起难以治愈的急性和慢性感染。同时，铜绿假单胞菌也是病原微生物领域的模式细菌，其致病性表型的调控机制 (例如群体感应、生物被膜、分泌系统) 在近几十年来被广泛研究。然而，目前对于铜绿假单胞菌 DNA 甲基化等表观遗传因素探究较少。本项目以两株铜绿假单胞菌临床菌株 TBCF10839 和 LYSZa7 为模型，通过测序、生物信息学分析、生物化学和分子生物学等技术，研究DNA甲基转移酶对细菌转录调控及重要毒力表型的影响。首先，我们通过检测质粒转化效率验证了 LYSZa7 菌株中两个 DNA 甲基转移酶 PaeZa7HsdMS 和 PaeZa7Mod 所在的 R-M 系统的功能，使用单分子实时测序技术和液相色谱串联质谱验证了 DNA 甲基转移酶的 DNA 甲基化功能，并利用 AlphaFold2 对 DNA 甲基转移酶的结构进行了预测。接下来，我们对菌株中几个 DNA 甲基转移酶对转录调控和重要表型的影响进行了研究。课题组前期实验发现，DNA 甲基转移酶 M.TBCFORFCP (CP) 参与调控活性氮氧化合物抵抗一氧化氮还原酶 (NOR) 家族基因的表达，并影响 NO 稳态及对巨噬细胞吞噬的抵抗作用。本研究进一步通过构建定点突变株，证明了距离预测的转录因子 Dnr 和 RpoN 结合位点较近的甲基化基序的甲基化状态会影响 nosR 基因的转录水平。此外，铜绿假单胞菌 LYSZa7 中 DNA 甲基转移酶 PaeZa7Mod 的缺失降低了铜绿假单胞菌 LYSZa7 Ⅲ 型分泌系统 (T3SS) 中 exsA，pscQ 和 pcrD 等基因的表达、增加铁载体合成相关的 pvdA，pchE 和 pchF 等基因的表达。细胞毒力实验和大蜡螟幼虫感染模型表明，DNA甲基转移酶 PaeZa7Mod 的缺失显著降低细菌毒力。差异甲基化基因的富集分析结果表示，甲基转移酶 PaeZa7Mod 对应的基序在 T3SS 相关基因和铁载体合成相关基因中富集，而转录水平的变化可能是由 LYSZa7 基因组相应区域甲基化状态的缺失所导致。铜绿假单胞菌 LYSZa7 中 DNA 甲基转移酶 PaeZa7HsdMS 的缺失增加了铁载体的合成，从而增加细菌摄铁能力。综上，本研究为 DNA 甲基化调控铜绿假单胞菌生理生化活动提供了实验证据，为进一步探究 DNA 甲基转移酶调控细菌表型奠定了基础。

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