胞外蛋白在铜绿假单胞菌生物被膜中的作用

Biofilms, defined as surface-associated communities of microorganisms, are prevalent in environmental and clinical settings. It is estimated that biofilms cost the nation billions of dollar annually due to damage to devices, product contamination, energy losses, and infections. Pseudomonas aeruginosa can readily form biofilms on a variety of surfaces and has become a model organism for biofilm research. Bacteria within biofilms are usually embedded within an extracellular polymeric substance (EPS) matrix, which confers a critical property of the biofilm resistance phenotype. The EPS matrix of P. aeruginosa biofilms is an ill-defined mix of polysaccharides, nucleic acids and proteins. Glycans as an extracellular matrix component of animal tissues can bind and regulate the activities of secreted proteins, which play a major role in chemical signaling between cells. It has been known that the EPS matrix of a biofilm contains proteins, but little is known about the roles of proteins as a biofilm matrix component and whether polysaccharides in the biofilm matrix have a similar regulation role as eukaryotic glycans. Leucine aminopeptidase is upregulated by quorum sensing system of P. aeruginosa. This protein is usually associated with extracellular membrane vesicles, which can serve as delivery vehicles to traffic the quorum sensing signaling molecular within a biofilm population and are also a component of biofilm matrix. Recently, we found that P. aeruginosa aminopeptidase (PaAP) was copurified with Psl exopolysaccharide,a key biofilm matrix component of P. aeruginos. Moreover, a Tn5 insertion mutation of PaAP showed an increased attachment in a microtiter dish biofilm assay. We hypothesize that PaAP has a relationship with Psl, can function as a matrix component in P. aeruginosa biofilm, and plays a critical role in biofilm development and persistence. Therefore, the objective of this project is to discover the roles of PaAP and associated mechanism(s) that are involved in P. aeruginosa biofilm development and persistence under a variety of environmental conditions. We will also explore other potential biofilm matrix proteins. An understanding of the mechanisms will lead to strategies to control biofilm-related complications in natural and clinical settings. The specific aims are to:..1..Determine the contributions of PaAP in biofilm development, resistance to antimicrobial agents, and pathogenesis...2..Define interactions and/or regulations among the three main biofilm matrix components, exopolysaccaride, eDNA, and proteins. ..3..Determine other potential biofilm matrix proteins involved in biofilm development and persistence.

生物被膜(biofilm)是微生物依附于某载体表面形成的微群落膜性集合体。生物被膜里的微生物被包埋在胞外多聚基质形成的网中，具有更强的适应外界环境的能力。胞外多糖，eDNA和蛋白是基质网的三个主要组份。至今胞外蛋白组份在生物被膜中的作用知之甚少。已知铜绿假单胞菌氨肽酶是分泌蛋白，其表达受群感效应系统调控。我们前期的工作表明，氨肽酶与基质网关键多糖Psl之间有某种关联，而且该酶的缺失影响生物被膜形成。本研究拟用现代分子生物学、生物化学与遗传分析相结合的方法，阐明氨肽酶类胞外蛋白在生物被膜形成与发育中的功能及作用机制；对生物被膜抗药性、抗逆性和环境适应性的贡献；解析基质网中各组份间的相互作用与调控；并筛选基质网中其它与生物被膜相关的胞外蛋白；进一步揭示生物被膜形成的分子机理及抗性的根源，为控制生物被膜带来的各种问题提供理论依据和解决办法，并为利用生物被膜中的胞外蛋白和酶类奠定基础。

细菌生物被膜中有许多胞外蛋白，特别是各种酶类，但它们在生物被膜中的作用尚不明晰。本研究从氨肽酶入手来探究胞外蛋白组份在生物被膜形成与发育中的作用；该组份与多糖，eDNA的关系；以及该组份对生物被膜抗药性、抗逆性和环境适应性的贡献。进一步揭示生物被膜形成的分子机理，为利用或控制及预防生物被膜带来的各种问题提供理论基础及其解决办法。氨肽酶，是一种从蛋白质或者小的寡肽 N-端去除氨基酸的酶。铜绿假单胞菌氨肽酶由基因位点PA2939 编码（PaAP），属于亮氨酸氨肽酶。该酶的活性在有弹性蛋白酶或其它蛋白酶存在时被激活。它是分泌型蛋白，在微氧的条件下表达量较高。四年来的研究揭示氨肽酶是生物被膜中不可或缺的重要组分，它除了通过分解胞外的小肽和蛋白为生物被膜细菌提供营养外，还调控另一种关键的生物被膜胞外基质，胞外多糖。具体的结果如下:1) 氨肽酶缺失导致胞外多糖合成增加，细菌的吸附能力增强，从而生物被膜生物量在早期迅速增长；2）在生物被膜发育后期，氨肽酶的缺少确导致大量死细菌产生，生物被膜提前分散瓦解以及Psl多糖丝状网的破坏；3）进一步的研究表明死菌分泌的PslG是导致Psl多糖降解以及生物被膜提前分散瓦解的主要原因；4）施氏假单胞菌中氨肽酶的缺失也导致该菌生物被膜的提前分散瓦解，而且在氮源缺乏时表现尤为明显。这暗示着氨肽酶在生物被膜中起到营养再利用的功能，该功能的缺乏导致大量死菌产生。这些结果不仅表明氨肽酶在生物被膜中功能的普遍性，同时提示氨肽酶可以作为针对铜绿假单胞菌感染的靶标蛋白。