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.

James J Lazenby,Margaret A Cooley,Jennelle Kyd,Phoebe E Griffin,Cynthia B Whitchurch

doi:10.1371/journal.pone.0060973

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.

Highlights

The role of quorum sensing (QS) and biofilm formation in Pseudomonas aeruginosa biology is clear, and previous reports have suggested that knocking out QS systems in P. aeruginosa results in decreased virulence in animal models of infection [1,2,3,4,5] and has impacts on bacterial growth rate in culture
P. aeruginosa and E. coli were cultured in LB-Lennox broth (LB) or cation-adjusted Mueller Hinton broth (CAMHB) or on LB solidified with 1.5% agar (LBA)
After verification of the PAO1DrhlIR mutant phenotypes, PAO1DlasIR and PAO1DlasIRrhlIR deletion mutants were created. These mutants were assayed to verify that they had phenotypes consistent with published observations and that they had not acquired secondary mutations that result in defective twitching motility

Summary

Introduction

The role of quorum sensing (QS) and biofilm formation in Pseudomonas aeruginosa biology is clear, and previous reports have suggested that knocking out QS systems in P. aeruginosa results in decreased virulence in animal models of infection [1,2,3,4,5] and has impacts on bacterial growth rate in culture. It has been shown that, unlike the PAO1 parental strain, a PAO1 mutant with defective pili is unable to bind to respiratory epithelial cells and cannot induce production of the proinflammatory and neutrophil-recruiting chemokine interleukin (IL)-8 [16] It has been reported in many studies that type-IV pili are integral for adherence to and colonization of mucosal surfaces (reviewed in Hahn et al [17]). It is important that twitching motility remains intact in any QS knockout P. aeruginosa used for in vivo infection studies assessing the role of QS in bacterial infectivity and persistence

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