Role of Flagella in Pathogenesis of Pseudomonas aeruginosa Pulmonary Infection

Pseudomonas aeruginosa strains are opportunistic pathogens associated with infections in immunocompromised hosts and patients with cystic fibrosis. Like many other mucosal pathogens, P. aeruginosa cells express flagella which provide motility and chemotaxis toward preferred substrates but also provide a ligand for clearance by phagocytic cells. We tested the role of flagella in the initial stages of respiratory tract infection by comparing the virulence of fliC mutants in a neonatal mouse model of pneumonia. In the absence of fliC, there was no mortality, compared with 30% mortality attributed to the parental strain PAK or 15% mortality associated with infection due to a pilA mutant PAK/NP (P < 0.0001). The fliC mutants caused pneumonia in only 25% of the mice inoculated, regardless of whether there was expression of the pilus, whereas the parental strain was associated with an 80% rate of pneumonia. Histopathological studies demonstrated that the fliC mutants caused very focal inflammation and that the organisms did not spread through the lungs as seen in infection due to either PAK or PAK/NP. Purified flagellin elicited an intense inflammatory response in the mouse lung. ¹²⁵I-labeled flagellin bound to the glycolipids GM1 and GD_1a and to asialoGM1 in an in vitro binding assay. However, flagellin-mediated binding to epithelial gangliosides was a relatively unusual event, as quantified by binding assays of wild-type or fliC mutant organisms to CHO Lec-2 cells with membrane-incorporated GM1. Fla⁺ organisms but not fliC mutants were efficiently taken up by murine macrophages. P. aeruginosa flagella are important in the establishment of respiratory tract infection and may act as a tether in initial interactions with epithelial membranes. This function is offset by the contribution of flagella to host clearance mechanisms facilitating phagocytic clearance and the role of flagellar genes in mucin binding and clearance.

Flagella are highly complex bacterial organelles which are unusually well conserved among diverse bacterial species. Over 50 genes are involved in the synthesis and function of flagella, suggesting that their preservation and role in chemotaxis and motility are important in the survival of many organisms (24). Flagella facilitate the acquisition of essential nutrients; thus, it seems likely that these organelles have a role in the virulence of pathogenic organisms. However, flagella are known to be highly immunogenic (16), and in certain settings Fla⁺ bacteria may be more readily cleared than Fla⁻ organisms (1).The contribution of flagella to the virulence of the opportunistic pathogen Pseudomonas aeruginosa has been examined in several animal models of infection (11, 19). Fla⁻ mutants were less invasive than motile strains in a mouse burn infection, and the administration of antiflagellum antibody had a significant protective effect (8). In a model of mucosal colonization, globally defective RpoN mutants (Pil⁻ Fla⁻) of P. aeruginosa were more deficient in their ability to colonize than to persist within the murine gastrointestinal tract (21). A rat model of Pseudomonas pneumonia was used to demonstrate the efficacy of human antiflagellum monoclonal antibody in attenuating pulmonary infection and reducing the spread of the organism within the rat lung (13). In these instrumented animals, large inocula of bacteria were instilled directly into the trachea. These studies do not directly address how flagella function in either the initial colonization of the upper respiratory tract or the subsequent infection of the lung by aspiration. Several flagellar genes, although not the flagellin structural gene fliC, have been shown to contribute to binding to respiratory mucin (2, 25). Attachment to mucin facilitates clearance from the respiratory tract via the normal mucociliary escalator. Flagella can function as ligands for macrophages and polymorphonuclear leukocytes (PMNs) which clear organisms from mucosal surfaces. In vivo selection of Fla⁻ mutants of P. aeruginosa has been demonstrated in human pulmonary infection in cystic fibrosis (CF) (14, 15). Although the first of sequential Pseudomonas isolates from a patient were motile and piliated, such as environmental strains of P. aeruginosa, genotypically identical isolates over the course of a chronic infection had an RpoN-like phenotype and failed to express functional flagella (15). There may be significant selective pressure for the emergence of Fla⁻ mutants which are less efficiently cleared by phagocytic cells (16, 29). Some respiratory pathogens such as Bordetella pertussis are nonmotile, and recent studies suggest that a bovine pathogen, Bordetella bronchoseptica, which can be motile, specifically turns off flagellar expression during the initial stages of infection (1).Since P. aeruginosa flagella are antigenically relatively homogeneous, falling into two large groups based on structural analysis and reactivity to antisera (28), they may provide a useful target for vaccine development. The ability of antiflagellum antibody to ameliorate infection in the rat lung suggests that flagella may provide a useful target for immune intervention and may be most effective as a strategy to block the initial infection of the respiratory tract by motile strains. However, the importance of normal flagellar function in the earliest stages of P. aeruginosa respiratory infection has not been established for genetically characterized strains. The purpose of this study was to examine (i) the virulence of wild-type and fliC mutant strains in the development of murine pneumonia and (ii) participation of flagella in stimulation of the subsequent immune response to the organism.