Maf-dependent bacterial flagellin glycosylation occurs before chaperone binding and flagellar T3SS export

Abstract

Bacterial swimming is mediated by rotation of a filament that is assembled via polymerization of flagellin monomers after secretion via a dedicated flagellar Type III secretion system. Several bacteria decorate their flagellin with sialic acid related sugars that is essential for motility. Aeromonas caviae is a model organism for this process as it contains a genetically simple glycosylation system and decorates its flagellin with pseudaminic acid (Pse). The link between flagellin glycosylation and export has yet to be fully determined. We examined the role of glycosylation in the export and assembly process in a strain lacking Maf1, a protein involved in the transfer of Pse onto flagellin at the later stages of the glycosylation pathway. Immunoblotting, established that glycosylation is not required for flagellin export but is essential for filament assembly since non-glycosylated flagellin is still secreted. Maf1 interacts directly with its flagellin substrate in vivo, even in the absence of pseudaminic acid. Flagellin glycosylation in a flagellin chaperone mutant (flaJ) indicated that glycosylation occurs in the cytoplasm before chaperone binding and protein secretion. Preferential chaperone binding to glycosylated flagellin revealed its crucial role, indicating that this system has evolved to favour secretion of the polymerization competent glycosylated form.

Fig. 1

Maf1 is required for flagellin glycosylation.A. Western blot analysis of glycosylated (+pse) and unglycosylated (−pse) FlaA/B using α-FlaA/B(+pse) and α-FlaA/B(−pse) antibodies of whole-cell (WC) preparations and secreted fractions (SN) of A. caviae strains wild-type and maf1 mutant strains. A Coomassie-stained SDS-PAGE gel showing whole-cell preparations and secreted fractions is shown as a loading control. The samples were also probed with α-GroEL as a cell lysis control for secreted fractions and an additional loading control for whole-cell fractions.B. CID tandem mass spectra of the FlaB peptide ¹⁴⁶FQVGADANQTIGFSLSQAGGFSISGIAK¹⁷³. B(i) MS/MS of the triply charged ion m/z 1135.22 that elutes at 96.22 min from wild-type A. caviae. The ion corresponds to the peptide containing two Pse residues. B(ii) MS/MS of the quadruply charged ion m/z 851.66 that elutes at 96.25 min from wild-type A. caviae. The ion corresponds to the peptide containing two Pse residues. B(iii) MS/MS spectra from A. caviae maf1 mutant flagellin. The quadruply charged ion m/z 692.85 which elutes at 74.43 min corresponds to the unglycosylated peptide. S is the potentially modified serine residue.

Fig. 2

Flagellin directly interacts with Maf1 in vivo.A. Coomassie-stained SDS-PAGE gels (top panel) of co-purified Maf1-His and FlaA/B from A. caviae wild-type (far left); neuB-harbouring pBBR1MCS-maf1his (centre left); flaAB mutant harbouring pBBR1MCS-maf1his (centre right); and wild-type harbouring empty pBBR1MCS (far right); purified by nickel affinity chromatography. Western blot analysis of resulting purification fractions using α-His antibodies (middle panel) and α-FlaA/B(−pse) antibodies (lower panel). The load (L), flowthrough (FT) and wash (W) fractions were also analysed for the presence of Maf1-His and FlaA/B.B. Motility of A. caviae wild-type, maf1 mutant and maf1 mutant containing pBBR1MCS expressing maf1-his inoculated on 0.25% semisolid bacteriological agar.

Fig. 3

FlaJ is not required for flagellin glycosylation but is required for export.A. Motility of A. caviae wild-type, flaJ mutant, flaJ mutant harbouring pSRK flaA, and flaJ mutant harbouring pSRK_hisflaA inoculated on 0.25% semisolid bacteriological agar.B. Western blots of FlaA/B(+pse) using α-FlaA/B(+pse) antibodies of whole-cell preparations and secreted fractions of A. caviae strains (wild-type, wild-type + pSRK, wild-type + pSRK_hisflaA, maf1 mutant, flaJ mutant, flaJ mutant + pSRK, flaJ mutant + pSRK_flaA, and flaJ mutant + pSRK_hisflaA). Coomassie-stained SDS-PAGE gels showing whole-cell preparations and secreted fractions are given as a loading control. The samples were also probed with α-GroEL as a cell lysis control for secreted fractions and an additional loading control for whole-cell fractions.C. Western blots of FlaA/B(+pse) using α-FlaA/B(+pse) antibodies of whole-cell preparations and secreted fractions of A. caviae strains (wild-type, wild-type + pSRK, wild-type + pSRK_flaA-CBD), Coomassie-stained SDS-PAGE gels showing whole-cell preparations and secreted fractions are given as a loading control. The samples were also probed with α-GroEL as a cell lysis control for secreted fractions and an additional loading control for whole-cell fractions.D. Western blots of FlaA/B(+pse) using α-FlaA/B(+pse) antibodies of whole-cell (WC) preparations and secreted fractions (SN) of A. caviae wild-type and flaH mutant strains. A Coomassie-stained SDS-PAGE gel showing whole-cell preparations and secreted fractions is given as a loading control. The samples were also probed with α-GroEL as a cell lysis control for secreted fractions and an additional loading control for whole-cell fractions.

Fig. 4

FlaJ shows a stronger affinity for FlaA (+pse) than FlaA (−pse).A. Coomassie-stained SDS-PAGE gel and far Western blots of GST, GST-FlaJ, His-FlaJ, FlaA (−pse), FlaA-CBD (−pse), and FlaA (+pse) probed with purified GST (middle panel) or GST-FlaJ (lower panel) and α-GST antibodies.B. Coomassie-stained SDS-PAGE gel and representative far Western blot of FlaA (−pse), and FlaA (+pse) probed with purified GST-FlaJ and α-GST antibodies. Blots were performed in triplicate and the average signal density corresponding to deposited GST-FlaJ interacting with FlaA (−pse), and FlaA (+pse) was plotted as a % with the highest density band as 100%.

Fig. 5

Flagellin glycosylation occurs prior to chaperone binding.A. Coomassie-stained SDS-PAGE gel and Western blots of FlaA/B ‘handover’ event. Western blots of Maf1-His, GST-FlaJ and FlaA/B(+pse) using α-His, α-GST and α-FlaA/B(+pse) antibodies respectively, were analysed in the load (L) flowthrough (FT) and elution (E) purification fractions.B. Schematic showing overview of ‘handover’ event. The co-purified Maf1-His and FlaA/B complex was mixed with GST-FlaJ. The protein mixture was subjected to glutathione affinity chromatography. FlaA/B dissociates from His-Maf1 and binds GST-FlaJ, which subsequently binds the GST-resin. Maf1 is removed from the protein mixture in the flowthrough and during the wash stages. FlaA/B and GST-FlaJ are co-purified and released from the resin in the elution fraction.

Fig. 6

Model depicting the flagellin glycosylation and export pathway. Activated pseudaminic acid (CMP-Pse) either is transferred onto a sugar-antigen carrier lipid (ACL) by Lst to create an LPS O-antigen unit, which is subsequently transported across the cytoplasmic membrane by Lsg, or is transferred on to the central D2/3 domain of unglycosylated flagellin the cytoplasm in a Maf dependant manner. Glycosylated flagellin is then bound by the flagellin-specific chaperone FlaJ, which is dependent on presence of C-terminal chaperone-binding domain (CBD) and piloted to the basal body of the flagellar where it is exported via it's dedicated T3SS in an unfolded form. For polymerization, the flagellin is folded, exposing the central D2/3 domain and attached glycans along the surface of the filament. The cytoplasmic glycosylation process occurs independently of the presence of the flagellar cap FlaH and a functional flagellar filament.