Swimming Motility Mediates the Formation of Neutrophil Extracellular Traps Induced by Flagellated Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections often characterized by robust neutrophilic infiltration. Neutrophils provide the first line of defense against P. aeruginosa. Aside from their defense conferred by phagocytic activity, neutrophils also release neutrophil extracellular traps (NETs) to immobilize bacteria. Although NET formation is an important antimicrobial process, the details of its mechanism are largely unknown. The identity of the main components of P. aeruginosa responsible for triggering NET formation is unclear. In this study, our focus was to identify the main bacterial factors mediating NET formation and to gain insight into the underlying mechanism. We found that P. aeruginosa in its exponential growth phase promoted strong NET formation in human neutrophils while its NET-inducing ability dramatically decreased at later stages of bacterial growth. We identified the flagellum as the primary component of P. aeruginosa responsible for inducing NET extrusion as flagellum-deficient bacteria remained seriously impaired in triggering NET formation. Purified P. aeruginosa flagellin, the monomeric component of the flagellum, does not stimulate NET formation in human neutrophils. P. aeruginosa-induced NET formation is independent of the flagellum-sensing receptors TLR5 and NLRC4 in both human and mouse neutrophils. Interestingly, we found that flagellar motility, not flagellum binding to neutrophils per se, mediates NET release induced by flagellated bacteria. Immotile, flagellar motor-deficient bacterial strains producing paralyzed flagella did not induce NET formation. Forced contact between immotile P. aeruginosa and neutrophils restored their NET-inducing ability. Both the motAB and motCD genetic loci encoding flagellar motor genes contribute to maximal NET release; however the motCD genes play a more important role. Phagocytosis of P. aeruginosa and superoxide production by neutrophils were also largely dependent upon a functional flagellum. Taken together, the flagellum is herein presented for the first time as the main organelle of planktonic bacteria responsible for mediating NET release. Furthermore, flagellar motility, rather than binding of the flagellum to flagellum-sensing receptors on host cells, is required for P. aeruginosa to induce NET release.

Interactions between Neutrophils and Pseudomonas aeruginosa in Cystic Fibrosis

“…Phagocytes (macrophages) much more likely recognize and phagocytose flagellated P. aeruginosa than its aflagellated counterpart [103,204] (Figure 1). PMNs also phagocytose P. aeruginosa in a flagellum-dependent manner [205]. Impaired phagocytosis of flagellum-deficient P. aeruginosa is due to the loss of its motility, not flagellum expression [105].…”

“…It has been presumed that host-selective pressure inspires P. aeruginosa to lose its motility in chronic CF, especially in patients with poor clinical conditions [207,208]. The flagellum is also required for P. aeruginosa -induced superoxide formation and NET release in human PMNs [205]. Lack of flagellar motility, not flagellum expression per se, is responsible for impaired NET release induced by flagellated P. aeruginosa [205].…”

“…The flagellum is also required for P. aeruginosa -induced superoxide formation and NET release in human PMNs [205]. Lack of flagellar motility, not flagellum expression per se, is responsible for impaired NET release induced by flagellated P. aeruginosa [205]. This is consistent with previous data showing that early CF isolates of P. aeruginosa induce larger amounts of NETs and are more resistant against NET-mediated attacks than late isolates obtained from the same CF patients [128,170] (Figure 1).…”

“…ecDNA is a main component of P. aeruginosa aggregates, and ecDNA obtained from necrotic PMNs has been shown to support aggregate growth of P. aeruginosa [214]. NET formation can likely be the source of PMN DNA in CF airways, since P. aeruginosa elicits robust NET release in PMNs [122,128,170,205]. Pyocyanin, a redox pigment and toxin of P. aeruginosa , is present in chronic CF airways and promotes the biofilm-forming ability of ecDNA [64,221].…”

Neutrophil extracellular trap release driven by bacterial motility: Relevance to cystic fibrosis lung disease

“…A small population of flagellated P. aeruginosa could be found in CF airways while most P. aeruginosa are present in form of alginate-producing, elastase-negative bacteria. MucA mutations drive the mucoid, biofilm-forming phenotype, lasR mutations contribute to PMN recruitment 28 while outbreaks of flagellated bacteria from biofilms could be mainly responsible for PMN activation and NET release 14 . PMNs quickly and easily recognize motile, flagellated forms of P. aeruginosa and launch their robust effector mechanisms including NET release in response to them 14 .…”