Relative Roles of Flagellin and Swimming Motility in Corneal Infection by Pseudomonas Aeruginosa

Abstract

Abstract: : Purpose:The Pseudomonas aeruginosa flagellum is composed of flagellin protein and is required for swimming motility. The aim of this study was to distinguish the roles of flagellin and swimming motility in corneal disease pathogenesis caused by P. aeruginosa. Methods: Wild–type invasive P. aeruginosa strain PAK (flagellum positive, swimming motility positive) was compared to two isogenic mutants; 1) PAK motA–motB(ΔΔ), which lacks the stator proteins and ability to drive flagella motion from the proton motive force (flagellum positive, swimming motility negative), and 2) PAK fliC which lacks the ability to produce the flagellin protein (flagellum negative, swimming motility negative). These mutants were derived using chromosomal transposon deletion of specific motility alleles. Ability to invade corneal epithelial cells in vitro was compared using gentamicin survival assays. C57BL/6 mice and the corneal scratch model were used to compare in vivo virulence. Infections were graded on days 1,2,4,and 7 and colonization levels determined at 48 hours. Results: Lack of swimming motility caused a significantly reduced ability to invade corneal epithelial cells, irrespective of whether or not flagella were expressed (15–fold decrease, p = 0.002). Surprisingly, the presence of flagella on bacteria lacking swimming motility reduced epithelial cell invasion even further (5–fold, p = 0.0026). Flagellin expression also reduced disease severity in vivo (fliC mutant 3.2 ± 0.24 versus motAB mutant 1.2 ± 0.19, p = 0.002); while swimming motility had little impact on pathology (disease scores of 1.4 ± 0.2 for wild–type versus 1.2 ± 0.19 for motAB mutant, p = .33). There was no significant difference in colonization rates (CFU) between wild–type PAK and either of the swimming defective mutants (wild–type 6.16 ± 1.69 x 10⁶ , motAB mutant 7.82 ± 0.22 x 10⁶, fliC mutant 5.87± 3.13 x 10⁶, p = 0.1). Conclusions: The capacity to express flagellin hindered epithelial cell invasion in vitro, and it reduced disease severity without affecting bacterial colonization levels in vivo. Flagellin protein is known to activate innate immunity via TLR signaling; whether this relates at all to our findings is yet to be determined. Although swimming motility enhanced corneal epithelial cell invasion in vitro, it contributed little to disease pathogenesis in vivo in the corneal infection scratch model.

Keywords: Pseudomonas • cornea: epithelium • keratitis