Phage extracellular polysaccharide depolymerases to combat Pseudomonas aeruginosa biofilms

项目摘要/摘要 需氧革兰氏阴性杆菌铜绿假单胞菌是最重要和最危险的细菌之一 医院环境中的微生物。它能够适应并在许多生态位中茁壮成长，从 水和土壤环境对植物和动物组织的影响。铜绿假单胞菌具有多种毒力 这些因素不仅会造成广泛的组织损伤，还会干扰免疫防御。在.期间 慢性感染，可出现在囊性纤维化患者的肺部或伤口感染人群中 铜绿假单胞菌经历了特有的进化适应，包括从浮游到生物膜的转变 表格。生物膜是嵌入在自己产生的聚合物基质和Play中的结构化多细胞群落 在细菌耐药性进化和感染耐药中的重要作用。2017年，多种药物- 据估计，耐药铜绿假单胞菌导致32,600名住院患者感染，2,700人死亡 仅在美国，每年增加的医疗费用约为7.67亿美元。新型抗菌剂和 因此，迫切需要抗生物被膜剂和方法来更好地控制或彻底治愈P. 铜绿假单胞菌感染。噬菌体已经与细菌共同进化了30亿年，而且效率很高 特定的抗菌机制已经出现，赋予了它们杀菌的独特优势。AS 抗生物被膜剂，这些病毒尤其能编码胞外多糖解聚酶(Epd)。 它们用来降解对生物膜生产和毒力至关重要的细菌聚合物。噬菌体EPD可以 切断与细胞表面(脂多糖或胶囊)相关的细菌胞外多糖 或组成生物膜基质，允许病毒粒子通过隧道进入细菌生物膜到达细菌细胞 表面。这些噬菌体酶在底物专一性上表现出很大的多样性，可以通过 在噬菌体斑块周围出现一个不断增加的晕圈，后者是酶促反应的结果 细菌胞外多糖的降解，即使在没有噬菌体感染的情况下也会发生。了解以下内容 然而，噬菌体EPD，特别是铜绿假单胞菌，目前在某种程度上是有限的。只有相对较少的环保署 已经确定了以铜绿假单胞菌为靶标的基因。此外，环保署在多大程度上仍不得而知。 噬菌体感染过程所需的活性，或噬菌体数量和密度的可能变化 细菌胞外多糖会影响这一过程。人们对这种结构也知之甚少， 底物结合和抗P。铜绿假单胞菌EPD。我们在拟议项目中的目标 因此鉴定和表征的是多重、多样、抗P。铜绿假单胞菌EPD和 它们对不同的PA生物被膜和毒力的影响的测定。重组噬菌体的应用 人造蛋白质提供了绕过抗生素治疗障碍的绝佳机会，从而使它们 非常适合广泛的应用，特别是在医疗和工业环境中。

Project Summary/Abstract The aerobic Gram-negative bacterium Pseudomonas aeruginosa is one of the most important and dangerous microbes inhabiting the hospital environment. It is able to adapt to and thrive in many ecological niches, from water and soil environments to plant and animal tissues. P. aeruginosa possesses a wide array of virulence factors that not only cause extensive tissue damage but also interfere with the immune defenses. During chronic infections, as can be present in the lungs of cystic fibrosis patients or in wound infections, populations of P. aeruginosa undergo characteristic evolutionary adaptations, including transition from planktonic to biofilm forms. Biofilms are structured, multicellular communities embedded in self-produced polymer matrix and play important roles in bacterial antibiotic-resistance evolution and infection antibiotic tolerance. In 2017, multidrug- resistant P. aeruginosa caused an estimated 32,600 infections among hospitalized patients and 2,700 deaths only in the U.S., adding roughly $767 million annually to the cost of medical treatments. New antibacterial and anti-biofilm agents and approaches therefore are urgently needed to better control or outright cure P. aeruginosa infections. Bacteriophages have co-evolved with bacteria for three billion years and highly efficient and specific antibacterial mechanisms have emerged, granting them unique advantages to kill bacteria. As anti-biofilm agents, these viruses in particular can encode extracellular polysaccharide depolymerases (EPDs) that they use to degrade bacterial polymers important for biofilm production and virulence. Phage EPDs can cleave bacterial extracellular polysaccharides either associated with the cell surface (LPS or capsule polysaccharides) or making up biofilm matrix, allowing virions to tunnel into bacterial biofilms to bacterial cell surfaces. These phage enzymes display a great diversity in substrate specificity and can be identified by the appearance of a constantly increasing halo zone around the phage plaques, the latter as a result of enzymatic degradation of bacterial extracellular polysaccharides that occurs even without phage infection. Knowledge of phage EPDs, particularly of P. aeruginosa, is currently somewhat limited, however. Only relatively few EPD genes have been characterized that target P. aeruginosa strains. It further is unknown to what extent EPD activity is required for the phage infection process or how possible variations in the amount and density of the bacterial extracellular polysaccharides influence this process. Not much is known as well about the structure, substrate binding, and cleaving domains of anti-P. aeruginosa EPDs. Our goal in the proposed project therefore is the identification and characterization of multiple, diverse, anti-P. aeruginosa EPDs and determination of their impact on diverse Pa biofilms and virulence. The use of bacteriophage recombinantly manufactured proteins offers a great opportunity to bypass antibiotic therapy hurdles, therefore making them very attractive for a broad range of applications especially in medical and industrial settings.