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

Abstract

Pseudomonas aeruginosa routinely causes chronic lung infection in individuals with muco-obstructive airway diseases (MADs). In MADs, P. aeruginosa forms antibiotic-tolerant biofilm-like aggregates within hyperconcentrated airway mucus. While the contribution of mucin hyper-concentration to antibiotic tolerance and bacterial aggregation has been described, less is known about the bacterial factors involved. We previously found that P. aeruginosa populations isolated from people with MADs exhibited significant variability in antibiotic tolerance. This variability is not explained by antibiotic resistance or the mucus environment, suggesting bacterial-driven mechanisms play a crucial role in treatment outcomes. Here, we investigated the contribution of flagellar motility to aggregate formation and tolerance by manipulating motility behaviors. Similar to prior studies, we found that loss of flagellar motility resulted in increased aggregation and tolerance to various antibiotics. We identified novel differential roles of the MotAB and MotCD stators, which power flagellar rotation, in antimicrobial tolerance and aggregate formation. In addition, we found that control of fliC expression was important for aggregate formation and antibiotic tolerance. Constitutive expression of fliC allowed P. aeruginosa to overcome entropic forces of mucin, antagonizing aggregate formation and increasing antibiotic efficacy. Lastly, we demonstrate that neutrophil elastase, an abundant antimicrobial protease in chronic lung infection, promotes antibiotic treatment failure by impairing flagellar motility leading to antibiotic-tolerant aggregate formation. These results underscore the crucial role of flagellar motility in aggregate formation and antibiotic tolerance, enhancing our understanding of how P. aeruginosa adapts to the MADs lung environment.

Importance: Antibiotic treatment failure of Pseudomonas aeruginosa infection is a key driver of mortality in muco-obstructive airway diseases (MADs). The bacterial mechanisms that contribute to antibiotic tolerance in MADS infection are poorly understood. We investigated the impact of swimming motility behaviors on P. aeruginosa antibiotic tolerance in the context of the diseased mucus environment. Loss of flagellar motility, a common adaptation in chronic lung infection, drives antibiotic tolerance by promoting aggregate formation under physiologically relevant mucin concentrations. We uncovered novel roles of the flagellar stators in motility and mucus aggregate formation. Furthermore, neutrophil elastase, an abundant host-derived antimicrobial protease, promotes antibiotic tolerance and aggregation by impairing flagellar motility. These results further our understanding of the formation of antibiotic-tolerant aggregates within the MADs airway, revealing potential new targets to improve antibiotic treatment of chronic P. aeruginosa airway infection.