基于RNA-Seq技术分析鼠源宿主防御肽对铜绿假单胞菌成熟生物被膜的清除作用

Eradication effect of Pseudomonas aeruginosa biofilms by mouse host defense peptide based on RNA-Seq technology

摘要:

[目的] 利用RNA-Seq技术探究修饰后的鼠源宿主防御肽CRAMP对铜绿假单胞菌PAO1成熟生物被膜的影响。[方法] 采用结晶紫法检测生物被膜量，激光共聚焦扫描显微镜（CLSM）观察生物被膜形态学变化；利用Illumina二代高通量测序平台，采用PE150测序策略分析了CRAMP修饰肽干预PAO1生物被膜与对照组在转录水平的基因表达差异；利用1，3-萘二酚方法测定了PAO1生物被膜中藻酸盐含量。[结果] CRAMP修饰肽能显著减少PAO1成熟生物被膜量，并且在0.98-62.50 μg/mL范围内呈一定浓度依赖性，CLSM显示CRAMP修饰肽能够显著减少细菌总荧光强度。转录组测序获得了12636700段干净读对，共鉴定出1582个差异基因，发现800个基因表达上调，782个基因表达下调。GO功能富集分析显示，1226个基因比对到GO功能分析数据库，在这些差异表达基因中，与分子功能、生物过程和细胞组成有关。KEGG通路富集分析显示，有603个表达差异显著基因比对到KEGG中的96条代谢途径，其中主要是各类氨基酸代谢途径、脂肪酸代谢途径、三羧酸循环、生物被膜调控系统等。分析发现CRAMP修饰肽可能作用于PAO1 c-di-GMP系统，增强细菌运动性和生物被膜分散，并且与其密度感应（quorum sensing，QS）系统和藻酸盐合成相关。最后经验证，CRAMP修饰肽显著减少了PAO1成熟生物被膜中藻酸盐的含量。[结论] CRAMP修饰肽对PAO1成熟生物被膜具有明显的清除作用，且能导致成熟生物被膜藻酸盐含量下降。通过转录组数据分析，可能是由于CRAMP修饰肽使PAO1 c-di-GMP水平下调导致的，具体机制还有待进一步探究。

关键词:铜绿假单胞菌;宿主防御肽;转录组学;c-di-GMP;密度感应;藻酸盐

Abstract:

[Objective] This study aimed to investigate the effect of modified mouse host defense peptide CRAMP on the mature biofilm of Pseudomonas aeruginosa PAO1 srtain based on RNA-Seq technology.[Methods] Crystal violet method was used to detect the biofilm biomass, and laser confocal scanning microscope (CLSM) was used to observe the morphological changes of biofilms. The gene expression differences at transcription level between biofilms treated with CRAMP and the non-peptide control were analyzed by the PE150 sequencing strategy using Illumina second-generation high-throughput sequencing platform. The content of alginate in PAO1 biofilms was determined by the 1,3-Dihydroxynaphthalene method.[Results] The biofilm biomass was significantly reduced by modified peptide CRAMP at the range of 0.98-62.50 μg/mL. CLSM showed that CRAMP could significantly reduce the total fluorescence intensity of biofilms. The results showed that 12636700 clean-read pairs were obtained by transcriptome sequencing. A total of 1582 differential genes were identified, including 800 genes that were up-regulated, and 782 genes were down-regulated. The GO functional enrichment analysis showed that 1226 genes were compared to the GO functional analysis database. Among these differentially expressed genes, they were related to molecular function, biological process, and cell composition. Enrichment analysis of the KEGG pathway showed that 603 differentially expressed genes were compared with 96 metabolic pathways in KEGG. There are various kinds of amino acid metabolism pathways, fatty acid metabolism pathway, tricarboxylic acid cycle, biofilm regulatory system, and so on. It was found that modified peptide CRAMP may act on PAO1 c-di-GMP system, enhance bacterial motility and biofilm dispersion, and be related to the quorum sensing (QS) system and alginate synthesis. Finally, it was verified that the modified peptide CRAMP significantly reduced the content of alginate in PAO1 biofilms.[Conclusion] The modified peptide CRAMP has an impair effect on PAO1 mature biofilms and can lead to the decrease of alginate content in mature biofilm. Transcriptomics analysis implied that CRAMP might exert its effectiveness by reducing the level of PAO1 c-di-GMP, and the mechanism remains to be further explored.

Key words:Pseudomonas aeruginosa;host defense peptide;transcriptomics;c-di-GMP;quorum sensing;alginate