(515) Identification of a key gene in Pseudomonas aeruginosa regulating neutrophilic inflammatory signals in infected airways

Background: Pseudomonas aeruginosa is an opportunistic bacterial pathogen that commonly infects the lungs of people with cystic fibrosis. Airway infection with P. aeruginosa triggers neutrophil-mediated inflammation, which can severely damage the airway, driving morbidity and mortality.

Methods: The P. aeruginosa (strain PA14) non-redundant transposon insertion mutant library was screened to identify genes involved in bacterial-induced neutrophil migration. To determine the effect of bacterial mutations on the induction of neutrophil trans-epithelial migration, lung epithelial cells grown on the apical side of transwell filters were infected with bacterial mutants. After infection, neutrophils were added to the basolateral side of the transwell and the number of neutrophils that migrated to the apical side was quantified. Production of leukotriene B₄ (LTB₄), an eicosanoid that drives neutrophil chemotaxis, was measured by enzyme-linked immunoassay. Motility, adhesion, and neutrophil chemotaxis assays were performed on the mutants identified.

Results: Mutant PA14 23790::MAR2xT7 with a transposon insertion in the P. aeruginosa leuB gene showed a significant reduction in neutrophil transmigration compared to the wild-type. The neutrophil transmigration defect in the leuB transposon insertion mutant was not rescued by increasing dose of infection. An in-frame deletion mutant in the leuB gene (PA14DleuB) showed a similar reduction in neutrophil trans-epithelial migration and also a reduction in LTB₄ production compared to wild-type. Interestingly, when exogenous leucine, the biosynthetic product of the leuABCD operon was supplemented during transmigration assays, neutrophil migration and LTB₄ production were restored. PA14DleuB displayed normal levels of motility and adhesion but showed decreased chemotaxis towards neutrophils as compared to wild-type PA14.

Conclusions: These findings suggest that leuB plays an important role in the neutrophil response to P. aeruginosa infection in the airway, perhaps as a component of a signal peptide that induces neutrophil chemotaxis. Future research is necessary to further elucidate the role of leuB in driving airway inflammation in cystic fibrosis and to identify targets for therapeutics to mitigate airway damage.

Acknowledgements: This work was supported by the Cystic Fibrosis Foundation (YONKER18Q0, YONKER20A0-KB) and NIH/NHLBI (5K08HL143183).

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