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

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.