Team:YAU-China/Fragmentation mode
Lysis Mechanism
Since the extracellular polysaccharide hydrolase PslG and PelA used to destroy the biofilm are intracellular enzymes, they can’t be effectively released to play an extracellular role. We designed two pathways to lyse engineered bacteria to release both enzymes out of the cell.
The first is a passive approach:
Pf4 filamentous phage is a mild phage that specifically recognizes Pseudomonas aeruginosa. During the long-term evolution of Pseudomonas aeruginosa, a correlation has been developed between Pseudomonas aeruginosa and Pf4 phage. Pseudomonas aeruginosa produces a large number of Pf4 phage in the formation of biofilms. Pf4 phage plays a key role in the development and structural integrity of biofilms. We knocked out the entire Pf4 phage gene cluster in the Pseudomonas aeruginosa genome to enhance the sensitivity of the engineered bacteria to Pf4. When the engineered bacteria contacts the biofilm, the Pf4 phage in biofilm infects and lyses the engineered bacteria, and the PelA and PslG hydrolases are released to hydrolyze exopolysaccharide, thus destroying biofilm.
We collected Pf4 phages from planktonic cultures of PAO1Δpf4r (Pf4r functions as a repressor for Pf4 phage production), 10 μl of the serially diluted Pf4 phage solution was applied to PAO1 and PAO1Δpf4 lawns, respectively. The plaques were visualized after 8 h of incubation. As expected, deletion of pf4 increased Pf4 sensitivity 10000 folds compared to the PAO1 wild-type strain (Fig. 3).
The second active way:
In order to expand the scope of application of engineering bacteria, we added an active lysis method on the basis of passive lysis. We selected the PA2069 promoter mainly regulated by the autoinducer synthase RhlI and the transcriptional regulator RhlR in Pseudomonas aeruginosa (Fig. 4. A). RhlI produces the autoinducer N-butyryl-L-homoserine lactone (BHL). When the bacteria grow to a stable phase, BHL and RhlR protein form a complex to activate the PA2069 promoter to express the lysozyme-activating protein PrtN, which induces engineering strain lysis (Fig.4. B), and releases the two enzymes (PelA and PslG) accumulated intracellularly into the environment.
It can be seen that the growth of Pseudomonas aeruginosa strains with PrtN overexpression was significantly degraded after 24 hours of bacterial culture.
The two cleavage methods work together on the engineered bacteria constructed by our team, which can effectively solve the problem that the extracellular polysaccharide hydrolase can’t be secreted extracellularly. Moreover, the two cleavage mechanisms can be used as part of the safety mechanism of our experimental design to solve the problem of engineered bacteria escaping.
References:
[1] Matsui H, Sano Y, Ishihara H, Shinomiya T. Regulation of pyocin genes in Pseudomonas aeruginosa by positive (prtN) and negative (prtR) regulatory genes[J]. Journal of Bacteriology, 1993, 175(5):1257-1263.
[2] Mukherjee S, Moustafa D A, Stergioula V, Smith C D, Goldberg J B, Bassler B L. The PqsE and RhlR proteins are an autoinducer synthase–receptor pair that control virulence and biofilm development in Pseudomonas aeruginosa[J]. PNAS, 2018, 115(40): E9411-E9418.
[3] Li Y M, Liu X X, Tang K H, Wang P X, Zeng Z S, Guo Y X, Wang X X. Excisionase in Pf filamentous prophage controls lysis‐lysogeny decision‐making in Pseudomonas aeruginosa[J]. Molecular Microbiology , 2019, 111(2): 495–513.