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

Abstract

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.

FIG. 1.

(A) Deduced amino acid sequence of PAK flagellin showing the location of the predicted TLR5 binding site (boxed). The numbering of the amino acids in the flagellin molecule has been done after exclusion of the first methionine. (B) The locations of mutated amino acids in FliC-expressing strains. The putative TLR5 binding regions (residues 88 to 97) are in italics.

FIG. 2.

(A) Immunoblot of bacterial samples from complemented PAKΔC strains showing the expression of FliC with anti-FliC antiserum. Left margin shows molecular size markers (in kDa). (B) 10% Coomassie-stained SDS-PAGE gels demonstrating the purities of flagellin preparations from the wild type and different mutant strains. Left margin shows molecular size markers (kDa). The minor contaminating bands seen come from the BL21 background and do not contribute towards IL-8 signaling, as confirmed by doing the assay with an equivalent amount of total protein [4.0 × 10⁻¹⁰ M flagellin] purified from BL21 cells through a nickel column (data not shown).

FIG. 3.

Electron micrographs showing the expression of flagella in wild-type PAKC, motile mutant PAKCM3 (L88A), and nonmotile mutant PAKCM2 (Q89A, R92E). (A) Wild-type (WT) strain PAKC demonstrates a fully formed, normal, wavy flagellum. (B) Mutant PAKCM3 demonstrates a fully formed, normal flagellum similar to that of the wild type. (C and D) EM shows the presence of a deformed and fragile flagellum on PAKCM2. Many broken pieces of fragile flagella were seen floating in the medium as depicted in panel C and at higher magnification in panel E.

FIG. 4.

Dose-response curve. A549 cells stimulated with different concentrations of flagellin purified from pETC demonstrated a significantly higher IL-8 production at 4.0 × 10⁻¹⁰ M flagellin concentration compared to those at the other concentrations used (P < 0.001). Similarly, the purified flagella from the PAK wild-type strain and the PAO1 wild-type strain produced maximum IL-8 responses at a concentration of 4.0 × 10⁻¹⁰ M in comparison to those at the other concentrations used (P < 0.001). The means ± SD were determined from at least three independent experiments.

FIG. 5.

IL-8 responses of A549 cells incubated with flagellins purified from motile and nonmotile/partially motile site-specific mutants. A549 cells stimulated with flagellin at a concentration of 4.0 × 10⁻¹⁰ M purified from the motile and nonmotile/partially motile TLR5 binding site mutants show significant (P < 0.001) reductions in IL-8 levels compared to that of the wild type (WT). IL-8 response produced by flagellin purified from a motile non-TLR5 binding site mutant at 4.0 × 10⁻¹⁰ M concentration was not significantly different from that produced by wild-type flagellin. Data shown are means ± SD of four different experiments.

FIG. 6.

IL-8 responses of A549 cells stimulated with flagellin purified from pETC, pETCM3, and flagellin mixed in equimolar quantities of pETC and pETCM3. Cells incubated with the mutant flagellin [4.0 × 10⁻¹⁰ M] from pETCM3 demonstrated a significant (P < 0.001) decrease in IL-8 levels compared to cells incubated with flagellin from pETC at the same concentration. On the other hand, the cells stimulated with a mixed flagellin sample (pETC+pETCM3) showed a significant (P < 0.001) increase in IL-8 response in comparison to cells incubated with flagellin at the 4.0 × 10⁻¹⁰ M concentration from pETCM3 alone. Data shown are means ± SD of four different experiments.

FIG. 7.

IL-8 responses of A549 cells incubated with a 4.0 × 10⁻¹⁰ M flagellar preparation purified from PAK wild type (PAKWT), PAO1 wild type (PAO1WT), PAKrfbc, and PAO1rfbc. A549 cells incubated with the flagellar preparation from glycosylation-defective mutant strains PAKrfbc and PAO1rfbc showed a significant (P < 0.001) reduction in IL-8 response compared to those of their respective wild-type strains. Data shown are means ± SD of four different experiments.