Pseudomonas evades immune recognition of flagellin in both mammals and plants

Abstract

The building blocks of bacterial flagella, flagellin monomers, are potent stimulators of host innate immune systems. Recognition of flagellin monomers occurs by flagellin-specific pattern-recognition receptors, such as Toll-like receptor 5 (TLR5) in mammals and flagellin-sensitive 2 (FLS2) in plants. Activation of these immune systems via flagellin leads eventually to elimination of the bacterium from the host. In order to prevent immune activation and thus favor survival in the host, bacteria secrete many proteins that hamper such recognition. In our search for Toll like receptor (TLR) antagonists, we screened bacterial supernatants and identified alkaline protease (AprA) of Pseudomonas aeruginosa as a TLR5 signaling inhibitor as evidenced by a marked reduction in IL-8 production and NF-κB activation. AprA effectively degrades the TLR5 ligand monomeric flagellin, while polymeric flagellin (involved in bacterial motility) and TLR5 itself resist degradation. The natural occurring alkaline protease inhibitor AprI of P. aeruginosa blocked flagellin degradation by AprA. P. aeruginosa aprA mutants induced an over 100-fold enhanced activation of TLR5 signaling, because they fail to degrade excess monomeric flagellin in their environment. Interestingly, AprA also prevents flagellin-mediated immune responses (such as growth inhibition and callose deposition) in Arabidopsis thaliana plants. This was due to decreased activation of the receptor FLS2 and clearly demonstrated by delayed stomatal closure with live bacteria in plants. Thus, by degrading the ligand for TLR5 and FLS2, P. aeruginosa escapes recognition by the innate immune systems of both mammals and plants.

Figure 1. Fractionated P. aeruginosa supernatant inhibits TLR5 activation.

HEK/TLR5 cells were transfected with a NF-κB reporter construct. Cells were incubated with 20-fold diluted elution fraction from a Q sepharose column (inhibitor) for 30 min and subsequently challenged with various concentrations recombinant flagellin of S. Typhimurium (□). Flagellin without inhibitor (▪) and LPS (•). (A) After 6 h the IL-8 concentration in the cell culture supernatant was measured by ELISA. (B) NF-κB activation was determined by measuring luciferase activity in a luminometer and expressed as fold increase of luciferase activity over stimulation with culture medium alone. The presented data are representative for the inhibition of TLR5 signaling that is typically observed with purifications of P. aeruginosa supernatant.

Figure 2. AprA prevents flagellin-induced IL-8 production by HEK/TLR5 cells.

(A) Schematic representation of the gene cluster of aprA, aprI, genes involved in secretion, aprD, aprE, and aprF and aprX on the genome of P. aeruginosa strain PAO1²⁶. (B) Different concentrations of recombinant flagellin from S. Typhimurium were treated with 1 µg/ml recombinant His-AprA for 30 min and subsequently added to HEK/TLR5 cells. After 6 h IL-8 was measured in the supernatant by ELISA. (C) His-AprA concentration-dependent inhibition of flagellin-induced HEK/TLR5 cell activation. HEK/TLR5 cells were treated with varying concentrations of AprA and challenged with 30 ng/ml flagellin of P. aeruginosa. (D) Recombinant flagellin of P. aeruginosa was incubated with buffer or 1 µg/ml His-AprA for 30 min and subsequently added to HEK/TLR5 cells for IL-8 release. (E) Recombinant flagellin of P. aeruginosa was incubated with 1 µg/ml recombinant His-AprA in the presence of PMB (10 µg/ml) for 30 min at 37°C, and subsequently added to human neutrophils. After 16 h IL-8 concentration was measured by ELISA. Results represent mean IL-8 concentration ± SEM from three independent experiments.

Figure 3. AprA of P. aeruginosa cleaves flagellin.

(A) HEK/TLR5 cells were treated for 30 min with AprA purified from P. aeruginosa culture supernatant and directly stimulated (□) or washed (○) before stimulation with varying concentrations flagellin from S. Typhimurium. As control cells were directly stimulated (•) or washed (▪) before addition of flagellin. After 6 h incubation, IL-8 was measured in the supernatant of HEK/TLR5 cells by ELISA. (B–F) Degradation of flagellin by recombinant AprA. Flagellin was mixed with 0, 0.01, 0.03, 0.1, 0.3, 1 and 3 µg/ml AprA for 60 min at 37°C in PBS and protein degradation was analyzed by SDS-PAGE and Coomassie staining. Cleavage of flagellin by AprA was compared for native monomeric (B) flagellin type B isolated from P. aeruginosa strain PAO25 (1 mg/ml), (C) flagellin type A isolated from clinical P. aeruginosa strain (150 µg/ml), (D) recombinant flagellin type B from P. aeruginosa (250 µg/ml) and (E) recombinant flagellin of S. Typhimurium (250 µg/ml). (F) Time-dependent degradation of flagellin by AprA. Flagellin (250 µg/ml) of S. Typhimurium was incubated with AprA for 0, 1, 3, 10, 30 and 60 min at 37°C in PBS.

Figure 4. Polymeric flagellin resists cleavage by AprA and AprI is an efficient endogenous inhibitor.

(A) Flagella isolated from PAO1 were treated for 20 min at 70°C to obtain monomeric flagellin. Untreated polymeric flagellin was compared with monomeric flagellin for susceptibility to AprA cleavage. Monomeric and polymeric flagellin was incubated with 0, 3, 1, 0.3, 0.1, 0.03 or 0.01 µg/ml AprA for 60 min at 37°C and analyzed by SDS-PAGE. (B and C) AprA (1 µg/ml) was incubated with 0, 0.03, 0.1, 0.3, 1, 3 or 10 µg/ml AprI and subsequently flagellin of (B) P. aeruginosa or (C) S. Typhimurium was added. Samples were analyzed by SDS-PAGE, untreated flagellin control is shown in the first lane followed by increasing AprI concentrations.

Figure 5. Culture supernatant of aprA mutant strains trigger TLR5.

(A) Dilutions of bacterial culture supernatants, collected from overnight grown wild-type (WT), and isogenic aprA mutant strains were used as flagellin source to stimulate HEK/TLR5 cells for IL-8 production. (B) 3 µg/ml recombinant AprA was added to the culture medium before inoculation with WT, aprA1 or aprA2 mutant strains. HEK/TLR5 cells were incubated with dilutions of bacterial culture supernatants and stimulated for 6 h. IL-8 production was measured by ELISA. All three data sets completely overlap in this graph. (C) Wild-type and mutant P. aeruginosa strains were grown in the presence of 10 µg/ml exogenous AprI and dilutions of the culture supernatants were added to HEK/TLR5 cells for IL-8 release. Data are expressed as mean IL-8 concentration ± SD from triplicates. (D) Human neutrophils were stimulated with dilutions of bacterial culture supernatants of wild-type and aprA2 mutant strain. PMB (10 µg/ml) was added prior to stimulation for 30 min at 37°C. After 16 h stimulation, IL-8 concentration in cell supernatant was determined. Results represent mean ± SEM of three independent experiments. (E) Culture supernatant of overnight grown wild-type and aprA mutant strains or recombinant AprA were analyzed for the presence of AprA by immunoblotting.

Figure 6. AprA prevents recognition of flagellin in Arabidopsis and prevents stomatal closure.

A. thaliana La-er seedlings were incubated with or without 500 nM flg22 or P. aeruginosa flagellin preincubated with 3 µg/ml AprA when indicated. (A). After treatment for 24 h, seedlings were stained for callose deposition by aniline blue and fluorescence was photographed under UV light. In the 3^rd row panels AprI was added before AprA treatment and in the bottom panels post AprA treatment (p.t.) of flagellin. (B) Examples of open (top), half open (middle) and closed (bottom) stomata, that were observed during the experiment. (C) Stomatal aperture on leaves of 5-week-old A. thaliana plants up to 40 minutes after treatment with P. aeruginosa PAO1 or isogenic aprA mutant strain (n = 108 to 224). Error bars indicate SEM. Asterisks indicate significant differences (Student's t-test; p<0.001) between WT and AprA mutant treated plants.

Figure 7. Proposed mechanism for AprA.

P. aeruginosa secretes AprA, which degrades free monomeric flagellin in the surrounding of the bacterium, whereas polymeric flagellin present in flagella is not affected. In this way flagellin is not recognized by TLR5 and FLS2, and thereby P. aeruginosa escapes activation of the innate immune system in both mammals and plants. AprA is secreted in one step over both membranes and is not present in its active form in the cytoplasm of the bacterium.