Glycosylation of b-Type Flagellin of Pseudomonas aeruginosa: Structural and Genetic Basis

Department of Medicine/Infectious Diseases, P.O. Box 100277, JHMHC, University of Florida, Gainesville, FL 32610, USA.
Journal of Bacteriology (Impact Factor: 2.81). 07/2006; 188(12):4395-403. DOI: 10.1128/JB.01642-05
Source: PubMed


The flagellin of Pseudomonas aeruginosa can be classified into two major types-a-type or b-type-which can be distinguished on the basis of molecular weight and reactivity with type-specific antisera. Flagellin from the a-type strain PAK was shown to be glycosylated with a heterogeneous O-linked glycan attached to Thr189 and Ser260. Here we show that b-type flagellin from strain PAO1 is also posttranslationally modified with an excess mass of up to 700 Da, which cannot be explained through phosphorylation. Two serine residues at positions 191 and 195 were found to be modified. Each site had a deoxyhexose to which is linked a unique modification of 209 Da containing a phosphate moiety. In comparison to strain PAK, which has an extensive flagellar glycosylation island of 14 genes in its genome, the equivalent locus in PAO1 comprises of only four genes. PCR analysis and sequence information suggested that there are few or no polymorphisms among the islands of the b-type strains. Mutations were made in each of the genes, PA1088 to PA1091, and the flagellin from these isogenic mutants was examined by mass spectrometry to determine whether they were involved in posttranslational modification of the type-b flagellin. While mutation of PA1088, PA1089, and PA1090 genes altered the composition of the flagellin glycan, only unmodified flagellin was produced by the PA1091 mutant strain. There were no changes in motility or lipopolysaccharide banding in the mutants, implying a role that is limited to glycosylation.

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Available from: Amrisha Verma, Sep 20, 2014
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    • "Of the known flagellin modification structures only some P. aeruginosa strains produce a structure composed of a sugar plus a phosphate and a methylated amino acid, similar to the C. difficile type A flagellin modification (Verma et al., 2006). Interestingly, as with C. difficile, the P. aeruginosa flagellins are also subject to strain-specific variation of the PTMs, some being modified as the C. difficile type A strain such as strain PA01 and others, such as the strain PAK being modified with a larger glycan composed of a number of monosaccharide moieties (Verma et al., 2006). "
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    ABSTRACT: Clostridium difficile is a prominent nosocomial pathogen, proliferating and causing enteric disease in individuals with a compromised gut microflora. We characterised the post-translational modification of flagellin in C. difficile 630. The structure of the modification was solved by nuclear magnetic resonance and shown to contain an N-acetylglucosamine substituted with a phosphorylated N-methyl-L-threonine. A reverse genetics approach investigated the function of the putative four-gene modification locus. All mutants were found to have truncated glycan structures by LC-MS/MS, taking into account bioinformatic analysis, we propose that the open reading frame CD0241 encodes a kinase involved in the transfer of the phosphate to the threonine, the CD0242 protein catalyses the addition of the phosphothreonine to the N-acetylglucosamine moiety and CD0243 transfers the methyl group to the threonine. Some mutations affected motility and caused cells to aggregate to each other and abiotic surfaces. Altering the structure of the flagellin modification impacted on colonisation and disease recurrence in a murine model of infection, showing that alterations in the surface architecture of C. difficile vegetative cells can play a significant role in disease. We show that motility is not a requirement for colonisation, but that colonisation was compromised when the glycan structure was incomplete.
    Full-text · Article · Aug 2014 · Molecular Microbiology
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    • "O-linked glycosylation of the flagellins has been described for an increasing number of bacteria in both Gram-negative and Gram-positive systems. Gram-negative examples include pathogens such as Aeromonas (Tabei et al., 2009), Pseudomonas (Verma et al., 2006), Campylobacter (Thibault et al., 2001), and Helicobacter (Josenhans et al., 2002), with Gram-positive pathogen examples including Clostridium (Twine et al., 2008) and Listeria (Schirm et al., 2004). Furthermore, the flagellar glycosylation process appears to be essential for the formation and function of an intact flagellar filament, as mutation of genes involved in the biosynthesis of the core glycan results in aflagellated cells (Josenhans et al., 2002; McNally et al., 2006; Canals et al., 2007; Tabei et al., 2009). "
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    ABSTRACT: Bacterial swimming is mediated by rotation of a filament that is assembled via polymerisation 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 polymerisation competent glycosylated form.
    Full-text · Article · Feb 2014 · Molecular Microbiology
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    • "Pseudomonas aeruginosa PAK FliC 2 11 residues (pentose, hexose, deoxyhexose, hexuronic) attached via L-rhamnose IL-8 stimulation, virulence Schirm et al. (2004a) Pseudomonas aeruginosa PAO1 FliC 2 356 Da (209 Da and rhamnose phosphate) N/D Verma et al. (2006), Lindhout et al. (2009) Pseudomonas syringae pv. tabaci FliC 6 β-D-Quip4N(3-hydroxy-1-oxobutyl)2Me-(133)-α- L-Rhap-(132)-α-L-Rhap Host specificity Taguchi et al. (2006), Takeuchi et al. (2007) Gram-positive Clostridium botulinum Langeland (group I) "
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