ORF4333催化铜绿假单胞菌甲基化调控生物被膜形成的作用与机制

Bacteria genome could be methylated under the catalysis of methyltransferase, which plays an important role in phenotypic regulation of bacterial biofilms, drug resistance, and pathogenicity. However, the catalytic specificity and epigenetic regulation mechanism of most of the bacterial methyltransferases are poorly understood. Our previous study isolated a clinical strain of Pseudomonas aeruginosa, named as PA1. Bioinformatic analysis revealed that ORF4333 of PA1 is a putative m6A methyltransferase, and single-molecule real-time sequencing confirmed that the PA1 genome contains m6A-modified motifs, and biofilm formation of the PA1 orf4333 gene-knockout strain was down-regulated; meanwhie, the m6A-modified motifs were all lost when orf4333 was knocked-out. Through transcriptome sequencing and quantitative PCR experiments, we found that the biofilm related gene fecA was significantly down-regulated, and the fecA promoter region contains m6A-modified motifs, while fecA was positively regulated by fecI. Thus we speculated that ORF4333 can catalyze the methylation of m6A motifs, and affect the binding of fecI to the promoter region of fecA, so as to participate in the regulation of biofilm formation. To verify the hypothesis, this project intends to further characterize the ORF4333 methyltransferase activity and target specificity in vitro and in vivo; explore the effect of ORF4333 on the functions of FecI and FecA in the regulation of biofilm formation; verify the binding site of FecI at the promoter region of fecA gene and reveal the mechanism of ORF4333 catalyzed methylation through FecI and FecA to regulate the biofilm formation. This project will provide a theoretical basis for understanding of the Pseudomonas aeruginosa life mechanism and control of the related infectious disease.

细菌编码的特定甲基转移酶通过甲基化修饰，在生物被膜、耐药及致病性等表型调控中发挥重要作用。然而，目前对多数细菌甲基转移酶催化特异性和表观遗传调控机制不清。我们前期从临床分离到一株铜绿假单胞菌，预测其编码推定甲基转移酶ORF4333，单分子测序证实其基因组含m6A修饰基序，且推定基因敲除株生物被膜下调，同时丢失所有m6A修饰基序。进一步实验发现敲除株生物被膜相关基因fecA显著下调，且其启动子区含m6A修饰基序，而fecA受fecI正向调控。推测ORF4333通过甲基化修饰，影响fecI与fecA启动子区结合，参与生物被膜调控。因此，本项目拟进一步验证ORF4333甲基转移酶活性与特异性；探讨其调控生物被膜的作用；验证FecI在fecA基因启动子区结合位点，并揭示ORF4333催化的甲基化通过FecI与FecA调控生物被膜形成的机制，为理解铜绿假单胞菌生命机理及控制相关感染疾病提供理论依据。

DNA甲基化修饰是生物体表观遗传学的重要基础之一，在基因表达调控、生物拮抗中发挥重要作用。细菌编码的特定甲基转移酶通过甲基化修饰，在生物被膜、耐药及致病性等表型调控中发挥重要作用。然而，目前对多数细菌甲基转移酶催化特异性和表观遗传调控机制不清。我们前期从临床分离到一株铜绿假单胞菌PA1，通过使用单分子-实时（SMRT）测序技术对PA1的基因组进行了重测序及注释分析，预测其编码推定Ⅰ型甲基转移酶ORF4333。甲基化组分析表明，其基因组含m6A修饰基序。构建ORF4333表达基因敲除株PA1Δhsd后，SMRT测序发现PA1Δhsd丢失所有m6A修饰基序。通过构建ORF4333修饰特异性报告质粒、甲基转移酶体外催化试验、回补实验，结合核苷酸LC-MS及单分子测序分析，进一步验证了ORF4333甲基转移酶活性与修饰特异性，鉴定了PA1的Ⅰ型甲基转移酶ORF4333的识别基序为CYYA(N)6CTTC和GAAG(N)6TRRG。药敏实验及动物攻毒实验结果表明，hsd位点的缺失并未显著影响PA1的耐药性及毒力，但导致其生物被膜形成能力的显著削弱。转录组比较分析发现与野生型PA1相比，I型R-M系统缺失突变体PA1Δhsd有270个差异表达基因（DEGs），其中95个基因表达上调，175个基因表达下调。DEGs显著富集于与核糖体、柠檬酸循环及碳代谢相关途径。与野生株相比，敲除株PA1Δhsd生物被膜相关基因fecA显著下调，且其启动子区含m6A修饰基序，而fecA受FecI正向调控。推测ORF4333通过甲基化修饰，影响fecI与fecA启动子区结合，参与生物被膜调控。表达、纯化FecI蛋白，通过PCR获得fecA基因调控区DNA探针，再通过体外催化实验构建甲基化的调控区DNA探针，然而，无论DNA探针甲基化与否，凝胶阻滞实验（EMSA）均未检测到探针与FecI的结合。因此，我们围绕ORF4333甲基转移酶调控的其他表型以及与噬菌体的互作进行了探讨与拓展研究，分离并鉴定了新型铜绿假单胞菌裂解性噬菌体TC6，建立了PA11样噬菌体新属；鉴定了噬菌体裂解酶LysP108的抗菌活性及噬菌体尾丝蛋白Gp50的耐药菌快速检测应用。本课题研究结果为理解铜绿假单胞菌表观遗传机理及控制耐药菌感染疾病提供新的线索和依据。