Pseudomonas aeruginosa Evasion of Phagocytosis Is Mediated by Loss of Swimming Motility and Is Independent of Flagellum Expression

Abstract

Pseudomonas aeruginosa is a pathogenic Gram-negative bacterium that causes severe opportunistic infections in immunocompromised individuals; in particular, severity of infection with P. aeruginosa positively correlates with poor prognosis in cystic fibrosis (CF) patients. Establishment of chronic infection by this pathogen is associated with downregulation of flagellar expression and of other genes that regulate P. aeruginosa motility. The current paradigm is that loss of flagellar expression enables immune evasion by the bacteria due to loss of engagement by phagocytic receptors that recognize flagellar components and loss of immune activation through flagellin-mediated Toll-like receptor (TLR) signaling. In this work, we employ bacterial and mammalian genetic approaches to demonstrate that loss of motility, not the loss of the flagellum per se, is the critical factor in the development of resistance to phagocytosis by P. aeruginosa. We demonstrate that isogenic P. aeruginosa mutants deficient in flagellar function, but retaining an intact flagellum, are highly resistant to phagocytosis by both murine and human phagocytic cells at levels comparable to those of flagellum-deficient mutants. Furthermore, we show that loss of MyD88 signaling in murine phagocytes does not recapitulate the phagocytic deficit observed for either flagellum-deficient or motility-deficient P. aeruginosa mutants. Our data demonstrate that loss of bacterial motility confers a dramatic resistance to phagocytosis that is independent of both flagellar expression and TLR signaling. These findings provide an explanation for the well-documented observation of nonmotility in clinical P. aeruginosa isolates and for how this phenotype confers upon the bacteria an advantage in the context of immune evasion.Pseudomonas aeruginosa is an opportunistic Gram-negative bacterial pathogen that causes severe infections in immunocompromised patients and in the pulmonary compartment of patients suffering from cystic fibrosis (CF) (13,14). In CF patients, disease severity is positively correlated with colonization by P. aeruginosa and the establishment of chronic infection. As part of the colonization process, the bacteria undergo a number of genetic changes that assist in their ability to survive in the mammalian host and to evade detection and clearance by the immune system (9, 21). One such change that has been phenotypically characterized for P. aeruginosa is loss of flagellar motility (12,17). Furthermore, the loss of flagellar gene expression and motility function is associated with increased bacterial burdens and increased disease severity in CF patients (12,17). While downregulation of flagellar expression has been inferred to confer a survival advantage on P. aeruginosa once it colonizes the host by evasion of both phagocytic receptors and TLR5-driven inflammatory signaling, the exact contribution of flagellum downregulation with respect to successful immune evasion is unclear (5, 17, 18).Nonopsonic phagocytosis of P. aeruginosa by murine and human mac...

Pseudomonas aeruginosaflagellar motility activates the phagocyte PI3K/Akt pathway to induce phagocytic engulfment

“…With regard to the contribution of bacterial motility to the recognition and clearance of P. aeruginosa, we recently identified that bacterial flagellar motility is a pattern-recognition signal for phagocytic engulfment by innate immune cells (1,19). Bacteria which have lost flagellar motility, either through loss of the flagellar structural protein FliC or the loss of the stator complexes in the flagellar motor, are ϳ100-fold more resistant to phagocytosis (1,19) by the cells critical for clearance of P. aeruginosa: neutrophils and macrophages (17).…”

“…Bacteria which have lost flagellar motility, either through loss of the flagellar structural protein FliC or the loss of the stator complexes in the flagellar motor, are ϳ100-fold more resistant to phagocytosis (1,19) by the cells critical for clearance of P. aeruginosa: neutrophils and macrophages (17). The P. aeruginosa stators are two partially redundant complexes, MotAB and MotCD, which are not necessary for flagellar assembly, but are required for flagellar rotation and swimming motility (5,33).…”

“…Multiple reports have demonstrated that flagellar function is a critical virulence factor for the establishment, persistence, and inflammatory profile of P. aeruginosa infection (1,2,19,28). P. aeruginosa swims via a single, polar, monotrichous flagellum, which it rotates by proton motive force (33).…”

“…The vast majority of patients with the genetic disorder cystic fibrosis (CF) exhibit chronic pulmonary infection with P. aeruginosa, leading to chronic and fatal inflammation and pneumonia (22,26). For both acute and chronic infections, bacterial flagellar swimming motility is a key determinant of P. aeruginosa colonization of the host (1,2,20,23,24).…”

Bacterial host interactions in cystic fibrosis

“…In addition to biofilm formation providing resistance to phagocytosis, additional strategies are also used to overcome this host response mechanism. P. aeruginosa can evade phagocytosis by becoming non-motile [37]. Similarly, Staphylococcus aureus forms polysaccharide encased cells under oxygen limited conditions which confer resistance to neutrophil killing [35].…”