A quadruple knockout of LasIR and rhlIR of Pseudomonas aeruginosa PAO1 that retains wild-type twitching motility has equivalent infectivity and persistence to PAO1 in a mouse model of lung infection

It has been widely reported that quorum-sensing incapable strains of Pseudomonas aeruginosa are less virulent than wildtype strains. However, quorum sensing mutants of P. aeruginosa have been shown to develop other spontaneous mutations under prolonged culture conditions, and one of the phenotypes of P. aeruginosa that is frequently affected by this phenomenon is type IV pili-dependent motility, referred to as twitching motility. As twitching motility has been reported to be important for adhesion and colonisation, we aimed to generate a quorum-sensing knockout for which the heritage was recorded and the virulence factor production in areas unrelated to quorum sensing was known to be intact. We created alas IRrhlIR quadruple knockout in PAO1 using a published technique that allows for the deletion of antibiotic resistancecartridges following mutagenesis, to create an unmarked QS knockout of PAO1, thereby avoiding the need for use ofantibiotics in culturing, which can have subtle effects on bacterial phenotype. We phenotyped this mutant demonstratingthat it produced reduced levels of protease and elastase, barely detectable levels of pyoverdin and undetectable levels ofthe quorum sensing signal molecules N-3-oxododecanoly-L-homoserine lactone and N-butyryl homoserine lactone, butretained full twitching motility. We then used a mouse model of acute lung infection with P. aeruginosa to demonstrate that the lasIRrhlIR knockout strain showed equal persistence to wild type parental PAO1, induced equal or greater neutrophilin filtration to the lungs, and induced similar levels of expression of inflammatory cytokines in the lungs and similar antibody responses, both in terms of magnitude and isotype. Our results suggest, in contrast to previous reports, that lack of quorum sensing alone does not significantly affect the immunogenicity, infectiveness and persistence of P. aeruginosa in a mouse model of acute lung infection.