Phage extracellular polysaccharide depolymerases to combat Pseudomonas aeruginosa biofilms

项目总结/摘要 需氧革兰氏阴性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。铜绿假单胞菌具有广泛的毒力 这些因素不仅会导致广泛的组织损伤，还会干扰免疫防御。期间 慢性感染，如可存在于囊性纤维化患者的肺中或伤口感染中， 的铜绿假单胞菌经历了特征性的进化适应，包括从嗜酸性到生物膜的转变 forms.生物膜是一种结构化的多细胞生物群落， 在细菌耐药性进化和感染抗生素耐受性中起重要作用。2017年，多药- 据估计，耐药铜绿假单胞菌在住院患者中造成32，600例感染和2，700例死亡 只有在美国，每年增加约7.67亿美元的医疗费用。新的抗菌和 因此，迫切需要抗生物膜剂和方法来更好地控制或彻底治愈P。 铜绿感染。噬菌体与细菌共同进化了30亿年， 并且已经出现了特定的抗菌机制，赋予它们杀死细菌的独特优势。作为 抗生物膜剂，这些病毒特别可以编码胞外多糖解聚酶（EPD）， 它们用来降解对生物膜产生和毒性很重要的细菌聚合物。噬菌体EPD可以 切割与细胞表面（LPS或荚膜）相关的细菌胞外多糖 多糖）或构成生物膜基质，允许病毒体隧穿进入细菌生物膜至细菌细胞 表面。这些噬菌体酶在底物特异性方面表现出很大的多样性，并且可以通过噬菌体抗体来鉴定。 噬菌体噬菌斑周围出现不断增加的晕圈，后者是酶促作用的结果。 甚至在没有噬菌体感染的情况下发生的细菌胞外多糖的降解。知识 然而，噬菌体EPD，特别是铜绿假单胞菌的EPD，目前有些有限。只有相对较少的EPD 已经表征了靶向铜绿假单胞菌菌株的基因。此外，还不清楚EPD在多大程度上 噬菌体感染过程需要活性，或者噬菌体的数量和密度如何可能变化 细菌胞外多糖影响这一过程。我们对它的结构知之甚少， 抗铜绿假单胞菌EPD的底物结合和切割结构域。我们在拟议项目中的目标 因此，鉴定和表征多种多样的抗铜绿假单胞菌EPD， 确定它们对不同Pa生物膜和毒力的影响。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，因此使它们成为 对于广泛的应用非常有吸引力，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.