Liposomal Delivery of Newly Identified Prophage Lysins in a Pseudomonas aeruginosa Model

Abstract

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium that presents resistance to several antibiotics, thus, representing a major threat to human and animal health. Phage-derived products, namely lysins, or peptidoglycan-hydrolyzing enzymes, can be an effective weapon against antibiotic-resistant bacteria. Whereas in Gram-positive bacteria, lysis from without is facilitated by the exposed peptidoglycan layer, this is not possible in the outer membrane-protected peptidoglycan of Gram-negative bacteria. Here, we suggest the encapsulation of lysins in liposomes as a delivery system against Gram-negative bacteria, using the model of P. aeruginosa. Bioinformatic analysis allowed for the identification of 38 distinct complete prophages within 66 P. aeruginosa genomes (16 of which newly sequenced) and led to the identification of 19 lysins of diverse sequence and function, 5 of which proceeded to wet lab analysis. The four purifiable lysins showed hydrolytic activity against Gram-positive bacterial lawns and, on zymogram assays, constituted of autoclaved P. aeruginosa cells. Additionally, lysins Pa7 and Pa119 combined with an outer membrane permeabilizer showed activity against P. aeruginosa cells. These two lysins were successfully encapsulated in DPPC:DOPE:CHEMS (molar ratio 4:4:2) liposomes with an average encapsulation efficiency of 33.33% and 32.30%, respectively. The application of the encapsulated lysins to the model P. aeruginosa led to a reduction in cell viability and resulted in cell lysis as observed in MTT cell viability assays and electron microscopy. In sum, we report here that prophages may be important sources of new enzybiotics, with prophage lysins showing high diversity and activity. In addition, these enzybiotics following their incorporation in liposomes were able to potentiate their antibacterial effect against the Gram-negative bacteria P. aeruginosa, used as the model.

Phage Lytic Protein CHAPSH3b Encapsulated in Niosomes and Gelatine Films

“…Indeed, the cationic surfactant CTAB is an ammonium salt with known antimi-crobial activity that provokes cell lysis [39]. Similarly to our findings, empty liposomes (DMPC:DOPE:CHEMS, molar ratio 4:4:2), designed to encapsulate lysins Pa7 and Pa119, also exhibited a lytic effect against Pseudomonas aeruginosa cultures, likely due to membrane destabilization [40]. Despite the impact of CTAB on cell viability, it is important to note that the highest antimicrobial efficacy was observed when testing endolysin-loaded niosomes.…”

New Antimicrobial Strategies to Treat Multi-Drug Resistant Infections Caused by Gram-Negatives in Cystic Fibrosis

“…Lipid coating is functional also for non-antibiotic entities, such as prophage lysins [192]; from a set of 19 proteins identified from 38 prophages within different P. aeruginosa genomes, lysins Pa7 and Pa119 were found to be biologically active against P. aeruginosa cells once encapsulated in dipalmitoylphosphatidylcholine:dioleoylphosphatidylethanolamine:cholesteryl hemisuccinate (DPPC:DOPE:CHEMS) liposomes. Free lysins showed a bactericidal effect at 25 µg/mL, while their encapsulated counterparts were already active at 6.25 µg/mL; interestingly, the time needed for the treatment to be effective seemed to be dependent on lysin release and accumulation at the peptidoglycan site.…”