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bioRxiv · 10.1101/2024.10.25.620240

Flagellar motility and the mucus environment influence aggregation mediated antibiotic tolerance of Pseudomonas aeruginosa in chronic lung infection

Higgs, M. G.·Greenwald, M. A.·Roca, C.·Macdonald, J. K.·Sidders, A. E.·Conlon, B.·Wolfgang, M. C.
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Abstract

Pseudomonas aeruginosa frequently causes chronic lung infection in individuals with muco-obstructive airway diseases (MADs). Chronic P. aeruginosa infections are difficult to treat, primarily owing to antibiotic treatment failure, which is often observed in the absence of antimicrobial resistance. In MADs, P. aeruginosa forms biofilm-like aggregates within the luminal mucus. While the contribution of mucin hyperconcentration towards antibiotic tolerance has been described, the mechanism for mucin driven antibiotic tolerance and the influence of aggregates have not been fully elucidated. In this study, we investigated the contribution of flagellar motility towards aggregate formation as it relates to the diseased mucus environment. We found that loss of flagellar motility resulted in increased P. aeruginosa aggregation and tolerance to multiple classes of antibiotics. Further, we observed differential roles in antimicrobial tolerance of the motAB and motCD stators, which power the flagellum. Additionally, we found that control of fliC expression was important for aggregate formation and antibiotic tolerance as a strain constitutively expressing fliC was unable to form aggregates and was highly susceptible to treatment. Lastly, we demonstrate that neutrophil elastase, an abundant immune mediator and biomarker of chronic lung infection, promotes aggregation and antibiotic tolerance by impairing flagellar motility. Collectively, these results highlight the key role of flagellar motility in aggregate formation and antibiotic tolerance and deepens our understanding of how the MADs lung environment promotes antibiotic tolerance of P. aeruginosa. IMPORTANCEAntibiotic recalcitrance of chronic Pseudomonas aeruginosa infections in muco-obstructive airway diseases is a primary driver of mortality. Mechanisms that drive antibiotic tolerance are poorly understood. We investigated motility phenotypes related to P. aeruginosa adaptation and antibiotic tolerance in the diseased mucus environment. Loss of flagellar motility drives antibiotic tolerance by promoting aggregate formation. Regulation of flagellar motility appears to be a key step in aggregate formation as the inability to turn off flagellin expression resulted in poor aggregate formation and increased antibiotic susceptibility. These results deepen our understanding of the formation of antibiotic tolerant aggregates within the MADs airway and opens novel avenues and targets for treatment of chronic P. aeruginosa infections.

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Higgs, M. G., Greenwald, M. A., Roca, C., Macdonald, J. K., Sidders, A. E., Conlon, B., Wolfgang, M. C.. 2024-10-25. Flagellar motility and the mucus environment influence aggregation mediated antibiotic tolerance of Pseudomonas aeruginosa in chronic lung infection. https://doi.org/10.1101/2024.10.25.620240

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