Methylation of Salmonella Typhimurium flagella promotes bacterial adhesion and host cell invasion

Abstract

The long external filament of bacterial flagella is composed of several thousand copies of a single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in Salmonella, which requires the methylase FliB. We show that both flagellins of Salmonella enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the surface hydrophobicity of flagellin, and enhances flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. Therefore, posttranslational methylation of flagellin facilitates adhesion of Salmonella Typhimurium to hydrophobic host cell surfaces, and contributes to efficient gut colonization and host infection.

Fig. 1. Structure of FljB and methylation pattern of the flagellins FliC and FljB.

a Cartoon representation of the structure of truncated FljB. The position of the N- and C-termini is shown at the end of the coiled-coil, and the extension of the domains D1, D2, and D3 is indicated below the structure. b Structural superposition of the individual domains. FljB domains are represented in blue, FliC in beige. The N- and C-termini of the structure are indicated, as well as the FljB numbering of the residues at the ends of the polypeptide segments defining the domains. c The structures of truncated FljB (blue) and truncated FliC (beige) have been superposed according to the D1 and D2 domains. The domain D3 shows a rotation of about 90° around an ideal axis starting from one end of the coiled-coil in D1 and passing through D2 (shown as dotted line). d Alignment of FljB and FliC and methylation pattern. Residues of domains D1, D2, and D3 have been aligned based on the structural superposition obtained with the EBI PDBeFold v2.59 server (51), either superposing the D1–D2 domains or the D3 domain. Residues of domain D0 have been aligned based on the sequence. The extent along the sequence of the domains is indicated above the alignment. Methylated lysine residues are indicated with stars above or below the alignment for FljB or FliC, respectively.

Fig. 2. Surface-exposed methylation of flagellin contributes to efficient colonization of the murine intestine.

a Schematic of a methylated flagellar filament and surface representation of the structure of FliC (top) and FljB (bottom). Methylation sites are highlighted in magenta and non-methylated lysines in black. b Streptomycin pre-treated C57BL/6 mice were infected with 10⁷ CFU of the FliC-expressing WT (fliC^ON) and isogenic ∆fliB mutant, each harboring a different antibiotic resistant cassette. The organ burden (small intestine, colon and cecum, respectively) was determined 2 days post-infection and used to calculate the competitive indices (CI). Each mouse is shown as an individual data point and significant differences were analyzed by a two-tailed Wilcoxon Signed Rank test. The bar graph represents the median of the data and asterisks indicate a significantly different phenotype to the value 1 (* = p < 0.05. WT vs. ∆fliB small intestine: p = 0.0135, WT vs. ∆fliB cecum: p = 0.010, WT vs. ∆fliB colon: p = 0.0384). n = 15 biologically independent animals. Source data are provided as a Source Data file.

Fig. 3. Effect of flagella methylation on swimming motility and flagellar assembly.

a Secreted flagellins from culture supernatants of Salmonella Typhimurium strains locked in expression of either FliC (fliC^ON) or FljB (fljB^ON). Secreted proteins were precipitated by addition of 10% TCA and fractionated according to their molecular weight by SDS-PAGE. Immunoblotting was performed using α-FliC/FljB antibodies (1:5,000). A representative immunoblot is shown. The experiment has been repeated three times with similar results. b Left: Histograms of the number of flagella per cell of the WT (fliC^ON) and an isogenic fliB mutant (fliC^ON ∆fliB). n = 79 bacteria for fliC^ON; n = 89 bacteria for fliC^ON ∆fliB. Average flagella numbers were calculated by Gaussian non-linear regression analysis. Right: Representative flagella immunostaining images. Flagellar filaments were immunostained using α-FliC primary (1:1000) and α-rabbit conjugated AlexaFluor 488 secondary antibodies (1:1000; green). DNA was stained with DAPI (blue). Scale bar = 5 µm. c Motility phenotypes of the WT and fliB mutants were analyzed in soft-agar plates containing 0.3% agar and quantified after 4 h incubation at 37 °C. Bottom: representative motility plate. Top: The diameters of the motility swarm were measured and normalized to the control strain. The bar graphs represent the mean of n = 10 biologically independent samples for the WT, ∆fliB, fljB^ON, fljB^ON ∆fliB, and ∆fliC∆fljB and n = 20 biologically independent samples for fliC^ON and fliC^ON ∆fliB. Replicates are shown as individual data points. Source data are provided as a Source Data file.

Fig. 4. Flagella methylation facilitates eukaryotic cell adhesion and invasion.

a Schematic illustration of Salmonella Typhimurium adhesion to eukaryotic epithelial cells, which is facilitated by methylated flagella. b Invasion of MODE-K murine epithelial cells by Salmonella Typhimurium expressing methylated or non-methylated flagella. Reported are relative invasion rates of MODE-K epithelial cells for various flagellin methylation mutants without (top: no spin) or with forced contact of the bacteria by centrifugation (bottom: +spin). The bar graphs represent the mean of the reported data. Top: n = 12 biologically independent samples for the WT, fliC^ON ∆fliB, fljB^ON, fljB^ON ∆fliB, ∆fliC ∆fljB, and ∆spi-1,2, n = 11 biologically independent samples for ∆fliB and n = 6 biologically independent samples for fliC^ON. Replicates are shown as individual data points and statistical significances were determined by a two-tailed Student’s t test (** = p < 0.01; *** = p < 0.001. WT vs. ∆fliB: p = 0.0010, fliC^ON vs. fliC^ON ∆fliB: p < 0.0001, fljB^ON vs. fljB^ON∆fliB: p = 0.0035). Bottom: n = 9 biologically independent samples for the WT, ∆fliB, fliC^ON∆fliB, fljB^ON, ∆fliC∆fljB and ∆spi-1,2, n = 3 biologically independent samples for fliC^ON and n = 6 biologically independent samples for fljB^ON∆fliB. Replicates are shown as individual data points and statistical significances were determined by a two-tailed Student’s t test (*** = p < 0.001. WT vs. ∆fliB: p = 0.0002, fliC^ON vs. fliC^ON∆fliB: p < 0.0001, fljB^ON vs. fljB^ON∆fliB: p = 0.0004). c Adhesion of Salmonella Typhimurium to MODE-K epithelial cells is reduced in the absence of flagella methylation. Adhesion was monitored using Salmonella Typhimurium strains deleted for spi-1 in order to prevent invasion of the eukaryotic host cells. The bar graphs represent the mean of the reported relative invasion rate data normalized to the inoculum. n = 18 biologically independent samples. Replicates are shown as individual data points and statistical significances were determined by a two-tailed Student’s t test (** = p < 0.01; *** = p < 0.001. WT vs. ∆fliB: p = 0.0069, fliC^ON vs. fliC^ON∆fliB: p = 0.0033, fljB^ON vs. fljB^ON∆fliB: p = 0.0003). Source data are provided as a Source Data file.

Fig. 5. Flagella methylation mediates adhesion to hydrophobic surfaces.

a Methylation increases hydrophobicity of the flagellar filament outer surface. Surface hydrophobicity distribution of the outer (top) and inner (bottom) surface of the FliC flagellar filament according to the Eisenberg scale (from green to white indicates increasing hydrophobicity) with methylation sites highlighted in magenta and non-methylated lysines in black. b Measured surface hydrophobicity (So) of methylated and non-methylated (∆fliB) flagellins using PRODAN on purified flagellar filaments. n = 6 independent experiments. Replicates are shown as individual data points and statistical significances were determined by a two-tailed Student’s t test (** = p < 0.01; *** = p < 0.001. FliC vs. FliC ∆fliB: p = 0.0020, FljB vs. FljB ∆fliB: p = 0.0009). c Adhesion of Salmonella Typhimurium to giant unilamellar vesicles (GUV) consisting of phosphatidylcholine (PC) from egg chicken is facilitated by the presence of methylated flagella. Top: schematic illustration of the adhesion of Salmonella to PC-GUVs, which is facilitated by methylated flagella. Bottom: Quantified adhesion of Salmonella mutants to PC-GUVs. WT, ∆fliB, fljB^ON∆fliB, fliC^ON∆fliB: n = 36; fljB^ON: n = 35; fliC^ON, ∆motAB ∆fliB, ∆flgK: n = 18; ∆flgK ∆fliB: n = 16; ∆motAB: n = 6 biologically independent samples. Replicates are shown as individual data points and statistical significances were determined by a two-tailed Student’s t test (* = p < 0.05; ** = p < 0.01; *** = p < 0.001; ns = not significant. WT vs. ∆fliB: p = 0.0437, fliC^ON vs. fliC^ON∆fliB: p = 0.0029, fljB^ON vs. fljB^ON∆fliB: p = 0.0009, ∆motAB vs. ∆motAB ∆fliB: p = 0.025). d Adhesion of Salmonella Typhimurium to GUVs consisting of phosphatidylglycerol (PG) is decreased by the presence of methylated flagella. Top: schematic illustration of the adhesion of Salmonella mutants to PG-GUVs. Bottom: Quantified adhesion of Salmonella mutants to PG-GUVs. The bar graphs represent the mean of the reported data. WT: n = 30; ∆fliB: n = 24; fliC^ON fliC^ON∆fliB, fljB^ON and fljB^ON∆fliB: n = 36 biologically independent samples. Replicates are shown as individual data points and statistical significances were determined by a two-tailed Student’s t test (** = p < 0.01; *** = p < 0.001. WT vs. ∆fliB: p = 0.0010, fliC^ON vs. fliC^ON∆fliB: p < 0.0001, fljB^ON vs. fljB^ON∆fliB: p < 0.0001). The bar graphs represent the mean of the reported data. Source data are provided as a Source Data file.