c-di-GMP的磷酸二酯酶PA4781在抗菌肽Merecidin抑制铜绿假单胞菌生物被膜中的作用

The role of c-di-GMP phosphodiesterase PA4781 in the inhibition of Pseudomonas aeruginosa biofilm by the antimicrobial peptide merecidin

摘要:

【背景】抗菌肽merecidin可抑制临床菌株铜绿假单胞菌PA03生物被膜。PA4781基因是课题组通过生物信息学分析筛选出的差异表达基因，PA4781作为细菌第二信使分子环二鸟苷酸(cyclic diguanylate，c-di-GMP)的磷酸二酯酶具有降解c-di-GMP的作用，其在抗菌肽merecidin抑制生物被膜中的作用机制尚不清楚。【目的】研究细菌第二信使分子c-di-GMP的磷酸二酯酶PA4781基因在抗菌肽merecidin抑制铜绿假单胞菌生物被膜中的作用。【方法】利用单碱基突变技术敲除PA4781基因，sanger测序方法检测敲除的正确性。采用结晶紫染色法观察PA03菌株、PA4781过表达菌株、PA4781敲除菌株24 h生物被膜生长情况，以及在抗菌肽merecidin 24、48、72 μmol/L作用下各菌株生物被膜的生长情况。采用对羟基联苯溶液显色法检测在抗菌肽Merecidin 48、72 μmol/L作用下，PA03菌株、PA4781过表达菌株、PA4781敲除菌株生物被膜藻酸盐的变化情况。【结果】sanger测序结果显示，用pnCasPA- BEC系统成功实现了靶点位置的单碱基突变，提前终止了PA4781的转录；结晶紫染色结果显示，培养24 h时，在24 μmol/L抗菌肽Merecidin作用下PA03菌株、PA4781过表达菌株、PA4781敲除菌株生物被膜形成情况无显著性差异(p>0.05)，在抗菌肽Merecidin 48、72 μmol/L处理下，过表达株与正常株和敲除株有显著性差异(p<0.05)，生物被膜明显减少，敲除株生物被膜厚度高于PA03组(p<0.05)。随着抗菌肽Merecidin浓度升高各组藻酸盐含量下降，其中过表达菌株在抗菌肽Merecidin作用下藻酸盐生成量抑制率最高，可达65%。【结论】抗菌肽Merecidin能够促进细菌第二信使分子磷酸二酯酶PA4781的表达，为抗菌肽Merecidin抑制铜绿假单胞菌生物被膜的作用机制可能通过细菌第二信使分子这一信号途径提供新的研究思路。

Abstract:

[Background] The antimicrobial peptide merecidin can inhibit the clinical strain Pseudomonas aeruginosa PA03 biofilm. The PA4781 gene is a differentially expressed gene selected by bioinformatics analysis. As a phosphodiesterase, PA4781 has function to degrade c-di-GMP, which is a bacterial second messenger molecule. PA4781 plays a role in inhibiting biofilm in the antimicrobial peptide merecidin, while the mechanism of action is still unclear. [Objective] To study the role of the phosphodiesterase PA4781 gene, which degrades the bacterial second messenger molecule c-di-GMP, in the inhibition of Pseudomonas aeruginosa biofilm by the antimicrobial peptide merecidin. [Methods] The PA4781 gene was knocked out by approach of base editing and the sanger sequencing method was used to detect the correctness of knockout. Crystal violet staining was used to observe the growth of biofilm in PA03 strain, PA4781 overexpressing strain, PA4781 knockout strain for 24 hours, and the development of biofilm of each strain under the action of antimicrobial peptide mericidin 24, 48, 72 μmol/L. Dihydroxybiphenyl solution chromogenic method was used to detect alginate production under interference from antibacterial peptide mericidin 48, 72 μmol/L to the PA03 strain, PA4781 overexpressing strain and PA4781 knockout strain. Alginate is an exopolysaccharide polymer composed of mannituronic acid and guloruronic acid which produced by various bacteria. It is an important component of Pseudomonas aeruginosa biofilm. [Results] The results of sanger sequencing showed that the pnCasPA-BEC system successfully realized the single-base mutation at target position and terminated the transcription of PA4781 in advance. The results of crystal violet staining showed that under the treatment of 24 μmol/L antimicrobial peptide merecidin, there was no significant difference in the formation of biofilm between the three groups (p>0.05). Under the treatment of 48 μmol/L and 72 μmol/L antimicrobial peptide merecidin, there was a significant difference between the overexpression group with the normal group with the knockout group (P<0.05), the biofilm was significantly reduced, and the biofilm thickness of the knockout group was higher than that of the PA03 group (P<0.05). With the increase of the concentration of the antimicrobial peptide mericidin, the alginate content of each group decreased, and the overexpression strain had the highest inhibition rate of alginate production under the action of the antimicrobial peptide merecidin, which reached to 65%. [Conclusion] Antibacterial peptide merecidin can promote the expression of bacterial second messenger molecule c-di-GMP phosphodiesterase PA4781, which may provide a new research idea for the mechanism of antibacterial peptide merecidin inhibiting Pseudomonas aeruginomonas biofilm through the signaling pathway of bacterial second messenger molecule.