F1hA, a component of the flagellum assembly apparatus of Pseudomonas aeruginosa, plays a role in internalization by corneal epithelial cells

Pseudomonas aeruginosa invades various epithelial cell types in vitro and i n vivo. The P. aeruginosa genome possesses a gene (flhA) which encodes a pr otein that is believed to be part of the export apparatus for flagellum ass embly and which is homologous to invA of Salmonella spp. Because invA is re quired for invasion of Salmonella spp., a role for flhA in P. aeruginosa in vasion was explored using cultured rabbit corneal epithelial cells. An flhA mutant of P. aeruginosa strain PAO1 was constructed and was shown to be no nmotile. Complementation with flhA in trans restored motility. Corneal cell s were infected for 3 h with the wild type (PAO1), the flhA mutant, the flh A mutant complemented with flhA in trans, an flhA mutant containing the pla smid vector control, or an fliC mutant (nonmotile mutant control). Invasion was quantified by amikacin exclusion assays. Both the flhA and the fliC mu tants invaded at a lower level than the wild-type strain did, suggesting th at both fliC and flhA played roles in invasion. However, loss of motility w as not sufficient to explain the reduced invasion by flhA mutants, since ce ntrifugation of bacteria onto cells did not restore invasion to wild-type l evels. Unexpectedly, the flhA mutant adhered significantly better to cornea l epithelial cells than wild-type bacteria or the fliC mutant did. The perc entage of adherent bacteria that invaded was reduced by similar to 80% for the flhA mutant and similar to 50% for the fliC mutant, showing that only p art of the role of flhA in invasion involves fliC. Invasion was restored by complementing the flhA mutant with flhA in trans but not by the plasmid ve ctor control. Intracellular survival assays, in which intracellular bacteri a were enumerated after continued incubation in the presence of antibiotics , showed that although flhA and fliC mutants had a reduced capacity for epi thelial cell entry, they were not defective in their ability to survive wit hin those cells after entry. These results suggest that the flagellum assem bly type III secretion system plays a role in P. aeruginosa invasion of epi thelial cells. Since the flhA mutants were not defective in their ability t o adhere to corneal epithelial cells, to retain viability at the cell surfa ce, or to survive inside epithelial cells after entry, the role of flhA in invasion of epithelial cells is likely to occur during the process of bacte rial internalization.