FlhA, a Component of the Flagellum Assembly Apparatus of Pseudomonas aeruginosa , Plays a Role in Internalization by Corneal Epithelial Cells

Abstract

Pseudomonas aeruginosa invades various epithelial cell types in vitro and in vivo. The P. aeruginosa genome possesses a gene (flhA) which encodes a protein that is believed to be part of the export apparatus for flagellum assembly and which is homologous to invA of Salmonella spp. Because invA is required for invasion of Salmonella spp., a role for flhA in P. aeruginosa invasion was explored using cultured rabbit corneal epithelial cells. An flhA mutant of P. aeruginosa strain PAO1 was constructed and was shown to be nonmotile. Complementation with flhA in trans restored motility. Corneal cells were infected for 3 h with the wild type (PAO1), the flhA mutant, the flhA mutant complemented with flhA in trans, an flhA mutant containing the plasmid vector control, or an fliC mutant (nonmotile mutant control). Invasion was quantified by amikacin exclusion assays. Both the flhA and the fliC mutants invaded at a lower level than the wild-type strain did, suggesting that both fliC and flhA played roles in invasion. However, loss of motility was not sufficient to explain the reduced invasion by flhA mutants, since centrifugation of bacteria onto cells did not restore invasion to wild-type levels. Unexpectedly, the flhA mutant adhered significantly better to corneal epithelial cells than wild-type bacteria or the fliC mutant did. The percentage of adherent bacteria that invaded was reduced by ϳ80% for the flhA mutant and ϳ50% for the fliC mutant, showing that only part 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 vector control. Intracellular survival assays, in which intracellular bacteria were enumerated after continued incubation in the presence of antibiotics, showed that although flhA and fliC mutants had a reduced capacity for epithelial cell entry, they were not defective in their ability to survive within those cells after entry. These results suggest that the flagellum assembly type III secretion system plays a role in P. aeruginosa invasion of epithelial cells. Since the flhA mutants were not defective in their ability to adhere to corneal epithelial cells, to retain viability at the cell surface, 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 bacterial internalization.Pseudomonas aeruginosa is an opportunistic gram-negative pathogen that can cause sight-threatening corneal infection in contact lens wearers and life-threatening infections among neutropenic patients, individuals with severe burns, and those with cystic fibrosis (3, 21). Although P. aeruginosa is traditionally thought to be an extracellular pathogen, evidence is accumulating to implicate corneal epithelial cell invasion in the pathogenesis of corneal disease caused by this bacterium (9, 10). Blocking the cystic fibrosis transmembrane conductance regulator, which has been shown to be a corneal cell factor involved in P. aeruginosa invasion, reduces corneal pa...

Genome-wide Expression Analyses of Campylobacter jejuni NCTC11168 Reveals Coordinate Regulation of Motility and Virulence by flhA

“…Additional flagellar biosynthetic and modification genes regulated by these alternate sigma factors in C. jejuni include the 28 -regulated flaG (22,23) and the 54 -regulated flgD, flgE, and Cj1293 (8,(15)(16)(17)24). In some organisms, including H. pylori, flgM homologues provide negative feedback on 28 (25). In contrast, flgM does not appear to be a strong repressor of 28 -regulated genes in C. jejuni (21).…”

“…In C. jejuni and Helicobacter pylori, flgR (an NtrC homologue) is required to activate class II flagellar genes with 54 promoters (14,20,21). As in the enterobacteriaceae, the major flagellin, flaA, belongs to class III and is controlled with a 28 promoter in both C. jejuni and H. pylori, and in both of these organisms, the minor flagellin, flaB, is controlled by 54 . Additional flagellar biosynthetic and modification genes regulated by these alternate sigma factors in C. jejuni include the 28 -regulated flaG (22,23) and the 54 -regulated flgD, flgE, and Cj1293 (8,(15)(16)(17)24).…”

“…As in the enterobacteriaceae, the major flagellin, flaA, belongs to class III and is controlled with a 28 promoter in both C. jejuni and H. pylori, and in both of these organisms, the minor flagellin, flaB, is controlled by 54 . Additional flagellar biosynthetic and modification genes regulated by these alternate sigma factors in C. jejuni include the 28 -regulated flaG (22,23) and the 54 -regulated flgD, flgE, and Cj1293 (8,(15)(16)(17)24). In some organisms, including H. pylori, flgM homologues provide negative feedback on 28 (25).…”

“…In peritrichous organisms, such as those belonging to the family of Enterobacteriaceae, flhC and flhD are the master regulators of the flagellar cascade, acting on class II promoters. Class II genes encode proteins forming part of the hook and basal body complex, the flagellar export complex, and 28 . Class III genes encode flagellin proteins required for extension of the flagellar filament.…”

EprS, an autotransporter serine protease, plays an important role in various pathogenic phenotypes of Pseudomonas aeruginosa

“…Indeed, deletion of elastase has been demonstrated to reduce the invasiveness of P. aeruginosa into epithelial cells (Cowell et al, 2003). Moreover, a reduction of flagellum-mediated motilities leads to a lowering of the invasion efficiency in P. aeruginosa (Fleiszig et al, 2001). Therefore, these observations support the idea that a decreased production of elastase and a reduction of flagellum-mediated motilities, which are phenotypic changes represented by the eprS mutant, would be involved in a reduction of the invasiveness of P. aeruginosa.…”