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: 3.19). 07/2006; 188(12):4395-403. DOI: 10.1128/JB.01642-05
Source: PubMed

ABSTRACT 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: We have previously shown that the pro-inflammatory cytokine TNF (tumour necrosis factor) could drive sLex (sialyl-Lewisx) biosynthesis through the up-regulation of the BX transcript isoform of ST3GAL4 sialyltransferase gene in lung epithelial cells and human bronchial mucosa. Here, we show that the TNF-induced up-regulation of ST3GAL4 BX transcript is mediated by the mitogen and stress activated protein kinases MSK1/2 through Erk (extracellular signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) pathways, and increases sLex expression on high molecular weight glycoproteins in the inflamed airway epithelium. We also show that the TNF-induced sLex expression increases the adhesion of Pseudomonas aeruginosa PAO1 and PAK strains to lung epithelial cells in a FliD-dependent manner. These results suggest that Erk and p38 MAPK, and the downstream kinase MSK1/2 should be considered as potential targets to hamper inflammation, bronchial mucins glycosylation changes and P. aeruginosa binding in the lung of patients suffering from lung diseases such as chronic bronchitis or cystic fibrosis.
    Biochemical Journal 10/2013; · 4.65 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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.
    Molecular Microbiology 02/2014; · 5.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Protein glycosylation had been considered as an eccentricity of a few bacteria. However, through advances in analytical methods and genome sequencing, it is now established that bacteria possess both N-linked and O-linked glycosylation pathways. Both glycosylation pathways can modify multiple proteins, flagellins from Archaea and Eubacteria being one of these. Flagella O-glycosylation has been demonstrated in many polar flagellins from Gram-negative bacteria and in only the Gram-positive genera Clostridium and Listeria. Furthermore, O-glycosylation has also been demonstrated in a limited number of lateral flagellins. In this work, we revised the current advances in flagellar glycosylation from Gram-negative bacteria, focusing on the structural diversity of glycans, the O-linked pathway and the biological function of flagella glycosylation.
    International Journal of Molecular Sciences 01/2014; 15(2):2840-57. · 2.46 Impact Factor

Full-text (2 Sources)

Available from
Sep 20, 2014