Roles of specific amino acids in the N terminus of Pseudomonas aeruginosa flagellin and of flagellin glycosylation in the innate immune response.

The Toll-like receptor 5 (TLR5) binding site has been predicted to be in the N terminus of the flagellin molecule. In order to better define the interaction between the N-terminal amino acids of Pseudomonas aeruginosa flagellin and TLR5, site-specific mutations were generated between residues 88 and 97 of P. aeruginosa PAK flagellin as well as outside of this region. The mutant flagellins were expressed in Escherichia coli BL21(plysS), purified by affinity chromatography, and passed through a polymyxin B column to remove contaminating lipopolysaccharide (LPS). Their ability to stimulate interleukin-8 (IL-8) release from A549 cells was examined. The cloned mutated genes were used to complement a PAK fliC mutant in order to test for effects on motility and on IL-8 release by purified flagellar preparations. All the mutations, single or double, in the predicted TLR5 binding region reduced IL-8 signaling to less than 95% of the wild-type flagellin levels, but the single mutation outside the binding region had no effect. Changes made at two amino acid sites resulted in loss/reduction of motility; however, changes made at single sites, i.e., Q83A, L88A, R90A, M91A, L94A, and Q97A, had no effect on motility. The mutated genes encoding two of the motile but poorly signaling flagellins had no compensatory mutations to allow motility. Thus, while it is speculated that pathogen-associated molecular patterns (PAMPs) have evolved in locations that are essential to maintain function, it appears that there is tolerance for at least single amino acid changes in the PAMP of P. aeruginosa flagellin. The purpose of flagellin glycosylation in P. aeruginosa is unknown. In order to examine its role, if any, in signaling an inflammatory response, we used whole flagella from the motile chromosomal mutant strains PAKrfbC and PAO1rfbC, which are defective in flagellin glycosylation. IL-8 release from A549 cells stimulated with nonglycosylated flagellar preparations (having less then 1 picogram of LPS/mug) was significantly reduced compared to their respective wild-type flagellar preparations, indicating a role of flagellar glycosylation in the proinflammatory action of Pseudomonas flagellin. The basis of the latter activity is unknown, since the glycosylation sites are found in the D3 domain of flagellins and the TLR5 binding site is located in the D1 domain. Thus, P. aeruginosa flagellin has evolved additional flagellar signaling mechanisms over that described for Salmonella flagellin.