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

Abstract

It has been widely reported that quorum-sensing incapable strains of Pseudomonas aeruginosa are less virulent than wild type 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 a lasIRrhlIR quadruple knockout in PAO1 using a published technique that allows for the deletion of antibiotic resistance cartridges following mutagenesis, to create an unmarked QS knockout of PAO1, thereby avoiding the need for use of antibiotics in culturing, which can have subtle effects on bacterial phenotype. We phenotyped this mutant demonstrating that it produced reduced levels of protease and elastase, barely detectable levels of pyoverdin and undetectable levels of the quorum sensing signal molecules N-3-oxododecanoly-L-homoserine lactone and N-butyryl homoserine lactone, but retained 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 neutrophil infiltration 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.

A quorum-sensing inhibitor blocks Pseudomonas aeruginosa virulence and biofilm formation

“…Apparently, that phenotype is high virulence. Consistent with our results and interpretation, the quadruple ΔlasI, ΔlasR, ΔrhlI, ΔrhlR mutant remains virulent in a mouse lung infection model (29). Lastly, we note that the double lasR, rhlR null mutant is not hypervirulent in the nematode assay.…”

Insights into host–pathogen interactions from state‐of‐the‐art animal models of respiratory Pseudomonas aeruginosa infections

“…In contrast, a quadruple mutant that affects quorum sensing by deletion of genes responsible for synthesizing the signaling molecules or their receptors ( lasIRrhlIR ) did not show a phenotype in the acute lung infection model. This is consistent with the idea that quorum sensing is only required in the chronic phase of the infection and confirms the idea that the phenotype of the bfmS deletion is not due to the impact on quorum sensing.…”

Links between Anr and Quorum Sensing in Pseudomonas aeruginosa Biofilms

“…We believe that the relationship between Anr and HHQ in quorum sensing mutants may functionally compensate for effects of losing LasR, and this could help explain the ability for P. aeruginosa lasR mutants to thrive in infections. A recent study demonstrated equal infectivity between WT PAO1 and a lasRI rhlRI quadruple mutant in a mouse lung model, suggesting that homoserine lactone signaling in general may be dispensable for infection in this context (85). Future studies will be aimed at measuring the role for Anr regulation of HHQ production in infections.…”