环二鸟苷酸和群体感应系统在兔子脓胸模型中铜绿假单胞菌生物膜形成中的作用

Whether bacteria can form biofilms within the pleural space of pleural empyema or not remains unclear，and warrants further investigation. Our previous woks：1) have constructed some Pseudomonas aeruginosa (PA) strains with fluorescence-based reporter for gauging the intracellular second messenger -- C-di-GMP levels，2) have demonstrated both quorum sensing (QS) systems and C-di-GMP can control the formation of PA-biofilms (PA-Bfs), 3）have observed that PA can form PA-Bf-alike construction in the fluid space of pleural empyema, and 4) have found that PA can undergo adaptive evolution in human airway during pulmonary infection. We hypothesize that both quorum sensing systems and C-di-GMP can regulate PA to form suspended PA-Bfs in the pleural fluid, and form sessile PA-Bfs on the visceral and parietal pleura in the pleural empyema. . Based on the previous studies, PA strains equipped with fluorescence-based reporter for gauging C-di-GMP levels and PA mutants with deficient in QS systems are applied in our current study, respectively. We use these strains to construct diverse models in vitro an in vivo, respectively. We utilize different techniques, such as PNA-based fluorescence in situ hybridization, PCR, flow cytometry, fluorescence microscope/ confocal laser scanning microscope, and whole-genome sequencing, etc. . Thus, we (1) construct in vitro aerobic and anaerobic models, and explore the effects of quorum sensing system and C-di-GMP on the characteristics of various PA strains, such as the cellular morphology, the extracellular virulence factors (pyocyanin, LasA protease, LasB elastase, and rhamnolipids), various motilities (swimming, swarming, and twitching), the antibiotics susceptibility, the expression of CdrA gene, the ability of PA-Bfs formation, etc.; (2)set up rabbit anaerobic pleural empyema models, and explore the effects of quorum sensing system and C-di-GMP signal pathway on the lifestyles of PA strains (the antibiotic susceptibility, the PA-Bfs formation ability, the cellular morphology, and the extracellular virulence factors as mentioned above), the characteristics of pleural fluid (the total leukocyte count; the indexes of pH, PaO2 and PaCO2, respectively; the concentrations of protein and LDH, respectively; and the levels of TNF-α,IL-1,IL-6 and IL-8,respectively), and the severity of pleural adhesion and empyema, respectively; (3) build rabbit pleural empyema models with intrapleural tissue plasminogen activator and DNase therapy, and explore the effects of intrapleural therapy on the formation/dispersion of PA-Bfs, elucidate the associations between the quorum sensing system and C-di-GMP, PA-Bfs, and drugs;(4) longitudinally collect the isolates during rabbit pleural infection, perform comparative genomics analyses，detect the nucleotide differences, and elucidate the associations between QS systems, C-di-GMP, and the adaptive evolution of PA in pleural empyema. In summary, we try to find out the roles of C-di-GMP and QS systems in the formation of PA-Bf in New Zealand rabbit pleural empyema.

目前尚无细菌在脓胸中形成生物膜的相关研究。我们以前的工作发现，细胞交流工具群体感应系统(QS)和第二信使环二鸟苷酸(GMP)能调控铜绿假单胞菌株(PA)形成生物膜（Bf），PA在脓胸胸腔里形成Bf样结构，及PA在肺部发生适应性进化过程。我们假设QS和GMP调控PA在脓胸的胸膜上和胸液中形成Bf。本研究在原基础上，使用分子克隆构建菌株，运用PNA-FISH、共聚焦激光显微镜、流式、PCR、全基因组测序等方法，1）探讨体外、兔脓胸模型中，QS和GMP对PA（药敏、毒力因子、CdrA基因、运动、Bf形成）、胸液（免疫指标、细胞凋亡、脓胸程度）和胸膜黏连的影响；2）探讨胸腔注入的药物（DNase+tPA）能否通过干预QS系统和GMP通路而影响Bf的形成，及其它上述指标；3）探讨脓胸厌氧环境下QS系统和GMP通路与PA进化过程的关系。综上，多方面阐述QS和GMP通路在脓胸Bf形成中的作用。

背景：识别致病病原体对于诊断和治疗脓胸至关重要。铜绿假单胞菌是引起脓胸和化脓性关节炎的常见病原菌，而铜绿假单胞菌的感染常与生物膜的形成有关。.目的：首先，我们想确定引起脓胸的病原菌；其次，我们想明确铜绿假单胞菌是否引起胸膜腔和关节腔内生物膜的形成。最后，我们研究了c-di-GMP对铜绿假单胞菌脓胸模型中生物膜形成的影响。.方法：我们通过Illumina测序平台对45名脓胸患者的胸腔积液进行宏基因组快照，以评估其分类学和抗生素耐药组结构。此外，分别成功建立了PAO1、PAO1ΔwspF和PAO1/plac-yhjH诱导的化脓性关节炎和胸膜脓胸兔模型。.结果：（1）宏基因组学结果显示，胸腔积液中微生物群的变化一般是分层的，而不是连续的。平衡良好的微生物共生状态的存在可能对病原体定植和药物摄入有不同的反应；（2）扫描电镜显示PAO1菌株分别被胸膜腔和关节腔内自产的细胞外基质包围。PNA-FISH也证实了纤维沉积物中的生物膜。PNA-FISH分析显示，PAO1ΔwspF组的红色荧光大小大于 PAO1和 PAO1/plac-yhjH 组。.贡献：（1）这项宏基因组学研究提供了对脓胸病理生物学的更深入理解，并表明潜在的耐药基因可能会阻碍脓胸的抗菌治疗; (2)这是第一个分别在脓胸和化脓性关节炎动物模型中报告铜绿假单胞菌形成生物膜的研究。此外，c-di-GMP信号分子在胸膜腔和关节腔的生物膜形成中发挥了重要作用。

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