PMC

Flagellin is shown in orange, TLR5 in white. The LLR9 loop of TLR5 is shown as a thick blue band. Positions 90 and 93 in flagellin are represented using Van der Waals surfaces. (A) Top and side views of hTLR5 with wild-type SaTy flagellin. (B) Top and side views of mTLR5 with wild-type SaTy flagellin. (C) Top view of hTLR5 with flagellin mutant R90N E93R. (D) Top view of mTLR5 with flagellin mutant R90N E93R.

(a) Schematic overview of experimental setup to determine flagellin leakage during filament assembly. The black elements represent the quantified fractions. (b) Coomassie-staining of total extracellular flagellin (filaments attached to cell bodies and flagellin secreted into culture supernatant), polymerized flagellin (filaments attached to cell bodies), detached and secreted flagellin (FliC detected in culture supernatant), detached flagellin (filaments detached from cell bodies as collected by ultracentrifugation of cleared culture supernatant) and secreted flagellin (FliC monomers detected in the culture supernatant after ultracentrifugation) of wild-type (WT) and ∆flgK mutant strains. Relative extracellular flagellin levels in the wild-type were 1.000 ± 0.175 for total extracellular flagellin, 0.756 ± 0.046 for polymerized flagellin, 0.239 ± 0.037 for detached and secreted flagellin, 0.057 ± 0.036 for detached flagellin and 0.085 ± 0.021 for secreted flagellin (mean ± s.d., n = 4).
DOI: http://dx.doi.org/10.7554/eLife.23136.004

(A) Glycosylation status of the flagellin protein from PAKorfN mutant and the complemented strains assessed by Western blots. The wild-type PAK flagellin migrates as a diffuse band indicating glycosylation of flagellin (lane 2). The flagellin from the PAKorfN mutant migrates much faster, suggesting loss of glycosylation (lane 3). Introduction of the complete orfN gene on a plasmid (pMMB194H) complements the glycosylation defect of the PAKorfN mutant (lane 5), whereas the vector control (pMMB66) remains nonglycosylated (lane 4). Size markers (lane 1) are in kDa. (B) Glycosylation status of the flagellins from PAKorfA mutant and the complemented strains assessed by Western blots. The wild-type PAK flagellin migrates as a diffuse band indicating glycosylation of flagellin (lane 2). The flagellin from the PAKorfA mutant migrates faster than the wild-type flagellin, suggesting loss of glycosylation (lane 3). Introduction of the complete orfA gene on a plasmid (pSP329Gm10EH) restores flagellin glycosylation in the PAKorfA mutant (lane 5) but not in mutants carrying the vector control (pSP329Gm) (lane 4). Lane 1 contains the size markers. (C) Effect of chemical deglycosylation on flagellin mobility during SDS/PAGE. Lane 1, PAK flagellin; lane 2, deglycosylated PAK flagellin; lane 3, PAKorfA mutant flagellin; lane 4, PAO1 flagellin; lane 5, deglycosylated PAO1 flagellin; lane 6, horseradish peroxidase; lane 7, deglycosylated horseradish peroxidase; and lane 8, molecular mass markers in kDa.

Western blot of TFMS-treated and untreated flagellin proteins with human anti-a flagellin MAb. Lane 1, 170018 flagellin; lane 2, TFMS-treated 170018 flagellin; lane 3, 5933 flagellin; lane 4, TFMS-treated 5933 flagellin; lane 5, 5939 flagellin; and lane 6, TFMS-treated 5939 flagellin.