Identification of the flagellin glycosylation system in Burkholderia cenocepacia and the contribution of glycosylated flagellin to evasion of human innate immune responses

Abstract

Burkholderia cenocepacia is an opportunistic pathogen threatening patients with cystic fibrosis. Flagella are required for biofilm formation, as well as adhesion to and invasion of epithelial cells. Recognition of flagellin via the Toll-like receptor 5 (TLR5) contributes to exacerbate B. cenocepacia-induced lung epithelial inflammatory responses. In this study, we report that B. cenocepacia flagellin is glycosylated on at least 10 different sites with a single sugar, 4,6-dideoxy-4-(3-hydroxybutanoylamino)-D-glucose. We have identified key genes that are required for flagellin glycosylation, including a predicted glycosyltransferase gene that is linked to the flagellin biosynthesis cluster and a putative acetyltransferase gene located within the O-antigen lipopolysaccharide cluster. Another O-antigen cluster gene, rmlB, which is required for flagellin glycan and O-antigen biosynthesis, was essential for bacterial viability, uncovering a novel target against Burkholderia infections. Using glycosylated and nonglycosylated purified flagellin and a cell reporter system to assess TLR5-mediated responses, we also show that the presence of glycan in flagellin significantly impairs the inflammatory response of epithelial cells. We therefore suggest that flagellin glycosylation reduces recognition of flagellin by host TLR5, providing an evasive strategy to infecting bacteria.

Keywords: Bacteria; Carbohydrate Glycoprotein; Glycoprotein Biosynthesis; Lipopolysaccharide (LPS); Toll-like Receptor (TLR).

FIGURE 1.

SDS-PAGE and Western blot analyses of B. cenocepacia FliC. A, Coomassie-stained SDS-PAGE showing crude flagellar filaments (C), supernatant obtained after insoluble flagella were sedimented at 16,000 × g for 10 min (S), and purified flagellin after solubilization with 8 m urea and desalting (P). B, crude flagellar filaments from the B. cenocepacia parental strain (WT) and ΔBCAL0111 (Δ0111) were analyzed by Western blot with the AVIVA RFFL/ARP42986_P050 antibody. C, Coomassie Blue-stained SDS-PAGE of crude flagellar filaments from B. cenocepacia parental strain (WT) and ΔBCAL0111 (Δ0111) from the same preparation used in B. D, Coomassie Blue-stained SDS-PAGE of chemically deglycosylated (dgWT) and native (WT) flagellin. Arrows indicate the corresponding molecular masses of the protein standards in kDa.

FIGURE 2.

Mass spectra of purified flagellin preparations. A, B. cenocepacia flagellin. B, chemically deglycosylated flagellin. C, nonglycosylated flagellin purified from the ΔBCAL0111 mutant strain. D, flagellin purified from strain MH43 (ΔwbxD). Arrows indicate the difference of 231 m/z between ions.

FIGURE 3.

Structure of the B. cenocepacia FliC glycan d-Qui4N(3HOBut).

FIGURE 4.

GC/MS spectra after methanolysis and β-elimination of B. cenocepacia FliC glycan (K56-2) and control sample (O-antigen of P. stuartii O43). A, top two graphs correspond to an overview of entire spectra for P. stuartii O43 O-antigen and B. cenocepacia K56-2 FliC samples. Qui4N peaks at 13.8 and 14.4 (representing α- and β-configured derivatives) are indicated. Additional peaks detected in the O43 spectrum represent other sugars from the O-antigen (44). Additional peaks in the FliC spectrum represent derivatized amino acids released from the FliC protein during methanolysis. The lower two spectra show the characteristic fragmentation pattern of ions at 13.8 min (fragmentation pattern of ion at 14.4 min was identical). M corresponds to molecular weight of derivatized Qui4N (303 Da). B, top graph shows an overview of the GC spectrum of the glycan released from FliC during β-elimination. Inset shows the derivatized glycan (461 Da) with the characteristic fragmentation pattern of the sugar and 3-hydroxybutyric acid. Lower graph shows the MS/MS fragmentation spectrum of the ion at 24.23 min. Differences between fragment ions (Δ) correspond to CH₂CO (Δ42), CH₃CHO (Δ44), CH₃COO⁻ (Δ59), and CH₃COOH (Δ60).

FIGURE 5.

Gene organization of the fliC region (A) and the O-antigen cluster (B) in B. cenocepacia. Deletion mutants are indicated by thick bars. Vertical arrows indicate insertion sites of the rhamnose inducible pSC200 vector. Genes showed as striped arrows encode the predicted enzymes required for FliC glycosylation.

FIGURE 6.

Mass spectra of flagellin from various B. cenocepacia mutant strains. A, ΔBCAL3119–3131; B, MH1K pSC200/BCAL0111 grown in the presence of rhamnose; C, MH1K pSC200/BCAL0111 grown without rhamnose; D, ΔBCAL3123–3124; E, ΔBCAL3123–3124 pIN62/BCAL3123. Arrows indicate the Δmass of 231 Da.

FIGURE 7.

Silver-stained 14% SDS-PAGE of whole cell lysates of B. cenocepacia. Whole cell lysates from B. cenocepacia mutants were analyzed in silver-stained 14% SDS-PAGE. The strains used were as follows: MH1K (lane 1); ΔBCAL3119–3131 (lane 2); ΔBCAL3129 (lane 3); ΔBCAL0110 (lane 4); ΔBCAL0111 (lane 5); ΔBCAL3123–24 (lane 6); ΔBCAS0105 (lane 7); ΔBCAS0105 pGPΩTp/rmlD (lane 8); and MH1K pGPΩTp/rmlD (insertional mutant inactivating the last enzymatic step in dTDP-rhamnose biosynthesis; lane 9). Ladder-like bands (bracket) correspond to LPS-containing lipid A-core covalently linked to O-antigen polysaccharides of varying length. Single bands in the low molecular weight region (arrow) correspond to lipid A-core molecules without O-antigen.

FIGURE 8.

Gene organization in fliC clusters of other Burkholderia species. The identity to flmQ is indicated in parentheses. A, B. pseudomallei 668 (BURPS668; 49%), B. mallei NCTC 10247 (BMA10247; 49%), B. glumae (bglu_1g; 47%), B. xenovorans LB400 (Bxe_A; 49%), B. multivorans CGD2 (BURMUCGD2; 80%), B. vietnamiensis AU4i (L810; 89%). B, B. thailandensis E264, dotted line represents 11 genes inserted between the putative fliT and flmQ (BCAL0111) homologues. C, B. cepacia GG4. Genes showed as striped arrows represent BCAL0111 (flmQ) homologue, and aminotransferase represents a BCAL0110 homologue. GT, glycosyltransferase.

FIGURE 9.

Conditional lethal phenotypes of B. cenocepacia strains. Strains were cultured in LB supplemented with 0.5% (w/v) rhamnose (A) or without rhamnose (B). After initial growth for 4 h (arrow), cultures were diluted 1:100 in fresh medium and incubated for 18 h.

FIGURE 10.

Motility on soft LB agar plates (A) and biofilm formation (B) of B. cenocepacia strains. Data are representative of three independent experiments. Statistical analysis was performed by paired t test using two-tailed p values. Significant differences in comparison with B. cenocepacia parental strain (WT) as control are indicated by ** (p < 0.01) or *** (p < 0.005).

FIGURE 11.

Regulation of pro-inflammatory gene expression in THP1 cells by glycosylated and nonglycosylated forms of flagellin. THP1 cells were stimulated for 24 h in the absence (NT, nontreated) or presence of varying concentrations of fully glycosylated wild-type (WT) or nonglycosylated (Δ0111) forms of flagellin, purified from the B. cenocepacia parental or ΔBCAL0111 strains, respectively. Conditioned media were assayed for expression levels of IL-1β (A), TNF-α (B), and IL-6 (C). Data are representative of three independent experiments. Statistical analysis was performed by paired t test using two-tailed p values. Significant differences between samples from WT and Δ0111-treated cells are indicated by * (p < 0.05), ** (p < 0.01), or ***, (p < 0.001).

FIGURE 12.

Differential stimulation of TLR5 signaling by glycosylated and nonglycosylated forms of flagellin. A, HEK293 cells, stably expressing TLR5, were stimulated for 24 h in the absence (NT, nontreated) or presence of varying concentrations of fully glycosylated wild-type (WT) or nonglycosylated (Δ0111) forms of flagellin purified from the B. cenocepacia parental or ΔBCAL0111 strains, respectively. Conditioned medium was assayed for expression levels of IL-8. B, HEK293 cells, stably expressing TLR5, were transfected with a NFκB-regulated luciferase reporter gene and stimulated for 24 h as indicated above. Cell lysates were assayed for NFκB-regulated firefly luciferase activity, and fold induction levels of NFκB-regulated luciferase are expressed relative to nontreated (NT) cells. Data are representative of three independent experiments. Statistical analysis was performed by paired t test using two-tailed p values. Significant differences between samples from WT and Δ0111-treated cells are indicated by * (p < 0.05). HEK293 cells, stably expressing TLR5, were stimulated for indicated times with WT and Δ0111 flagellin (500 ng/ml). Cell lysates were immunoblotted for phosphorylated (p-) and total levels of p65 (C) and p38 (D), JNK and ERK MAPKs. β-Actin was used as a loading control.