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.
Bacteriophages, viruses that kill bacteria, encode extracellular polysaccharide depolymerases (EPDs) which represent promising anti-Pseudomonas aeruginosa biofilm agents. Knowledge about those enzymes affecting P. aeruginosa biofilms is limited, however, and systematic efforts are needed toward both enhancing anti- biofilm activity and subsequent preclinical development. Here we propose the identification and preparation of multiple, diverse, new anti-P. aeruginosa recombinant EPDs, and testing them in purified form against a diversity of P. aeruginosa biofilms.
