细菌生物被膜形成新因子及相应机制和耐受策略的解析

One of the most important features of a biofilm is the extracellular polymeric substance (EPS) that functions as a matrix, holding bacterial cells together. The EPS matrix and the biofilm-mode of growth are the causes of persistent bacterial infections and resistance to antibiotic treatment. Our goal is to discover novel factors/mechanisms that are required for Pseudomonas aeruginosa biofilm formation and persistence. The opportunistic pathogen P. aeruginosa causes a variety of infections in humans and is one of the most common bacteria to infect the cystic fibrosis (CF) lung. Once the lungs of CF patients are colonized by P. aeruginosa, the organism persists for years or decades and is rarely eradicated. We have recently reported that Psl polysaccharide (the product of polysaccharide synthesis locus) is a primary scaffolding matrix component in P. aeruginosa biofilm development. However, a portion of the psl bacteria is still able to attach on a surface and form a biofilm under static conditions or after extended growth with flow. In addition, most P. aeruginosa strains isolated from cystic fibrosis (CF) patient tend to loss of Psl and resistant to multiple drug. We hypothesize that factors in addition to Psl play a critical role in biofilm formation and persistence. The objective of this project is to discover novel Psl-independent factors and associated mechanism(s) that are involved in P. aeruginosa biofilm formation and persistence under a variety of environmental conditions. An understanding of the mechanisms will lead to strategies to control biofilm-related complications in clinical settings and improve the quality of life for patients who suffer from biofilm-related infections. The specific aims are to:. 1. Define novel factors and associated mechanism(s) that allow psl strains of P. aeruginosa to form biofilms. We will use complementary platform technologies (genetic, genomics, and transcriptomics) to generate profiles representing those factors that are important in biofim formation in the absence of Psl. We will also combine techniques that include real-time PCR, lectin and/or fluorescent dye staining coupled with confocal laser scanning microscopy (CLSM) to investigate whether the surface-attached psl bacteria contribute to the biofilm resistance/persistence phenotype in vitro.. 2. Determine whether the physiological heterogeneity of individual cell in planktonic culture of P. aeruginosa is one of reasons for the biofilm resistance/persistence. We will utilize novel techniques, Laser Tweezers Raman Spectroscopy (LTRS) combined with microfluidics to detect exopolysaccharides and separate individual bacterial cell that have significant different exopolysaccharides production and biofilm formation to further define their corresponding resistance/persistence mechanism.. 3. Determine the link between loss of exopolysaccharides production and increasing frequency of drug-resistant mutations. Which goes first?

生物被膜（Biofilm）是一种或多种微生物由胞外多聚基质相联合而形成的有结构的，类似组织的微生物集合体。铜绿假单胞菌的Psl和Pel胞外多糖在生物被膜形成及细菌群体结构维持中起关键作用，对生物被膜的耐药性也至关重要，它们能够作为一个结构屏障，阻挡抗生素药物进入生物被膜内。然而胞外多糖缺失菌株通过较长时间的培养仍可以形成有三维结构的生物被膜，这说明在不能有效合成胞外多糖时，细菌可以依赖其它机制形成生物被膜。一些初步的实验数据也暗示Psl胞外多糖产量高的菌与产量较低的菌很可能采用不同的耐药策略。基于此，本研究拟采用RNA-seq、TraDIS、激光拉曼与微流控相结合等先进技术，探究不依赖胞外多糖的生物被膜形成新因子与机制；阐明浮游细菌胞外多糖合成能力的个体差异与生物被膜形成能力的关系；揭示不产或低产Psl胞外多糖的菌株耐药的分子机理；挖掘新药物靶标，为开发清除生物被膜的有效方法奠定基础。

生物被膜（Biofilm）是一种或多种微生物由胞外多聚基质相联合而形成的有结构的，类似组织的微生物集合体。铜绿假单胞菌的Psl和Pel胞外多糖在生物被膜形成及细菌群体结构维持中起关键作用，对生物被膜的耐药性也至关重要，它们能够作为一个结构屏障，阻挡抗生素药物进入生物被膜内。已知铁可以作为一种信号分子调节生物被膜的形成与发育，然而其具体的调节机制尚不明确。本研究通过对非黏性铜绿假单胞菌野生型菌株PAO1及其Psl多糖缺失突变株WFPA800的转录组对比分析发现参与铁摄取和氧化应激反应的相关基因及基因簇均在Psl缺失突变株中大幅上调，提示Psl多糖的缺失造成了菌体的铁缺乏。研究进一步用实验验证了Psl多糖具有在体外及生物被膜体内环境中结合铁的能力，即Psl多糖可以作为蓄铁池为铜绿假单胞菌的菌细胞储存铁，维持胞周充足且适宜的铁浓度。而且，高铁信号通过抑制psl抑制子AmrZ的转录和鼠李糖脂的合成分别从转录和翻译后两个水平提高Psl多糖的产量，从而促进了生物被膜的形成，且这种调节机制亦适用于铜绿假单胞菌的黏性菌株。研究还发现，Psl是铜绿假单胞菌中唯一种对二价及三价铁离子都有结合能力的多糖，且与二价铁离子的结合能力更强。研究揭示了铜绿假单胞菌通过环境铁信号调控Psl多糖合成并利用Psl来结合和摄取环境铁的一种新的生存策略。此外，对施氏假单胞菌的研究揭示，通过大量分泌胞外多聚物形成生物被膜可以实现该植物根际微生物在有氧环境条件下的固氮作用。