Sequence Polymorphism in the Glycosylation Island and Flagellins of Pseudomonas aeruginosa

Abstract

A genomic island consisting of 14 open reading frames, orfA to orfN was previously identified in Pseudomonas aeruginosa strain PAK and shown to be essential for glycosylation of flagellin. DNA microarray hybridization analysis of a number of P. aeruginosa strains from diverse origins showed that this island is polymorphic. PCR and sequence analysis confirmed that many P. aeruginosa strains carry an abbreviated version of the island (short island) in which orfD, -E and -H are polymorphic and orfI, -J, -K, -L, and -M are absent. To ascertain whether there was a relationship between the inheritance of the short island and specific flagellin sequence variants, complete or partial nucleotide sequences of flagellin genes from 24 a-type P. aeruginosa strains were determined. Two distinct flagellin subtypes, designated A1 and A2, were apparent. Strains with the complete 14-gene island (long island) were almost exclusively of the A1 type, whereas strains carrying the short island were associated with both A1-and A2-type flagellins. These findings indicate that P. aeruginosa possesses a relatively low number of distinct flagellin types and probably has the capacity to further diversify this antigenic surface protein by glycosylation.Pseudomonas aeruginosa is a motile opportunistic Gramnegative bacterium that possesses a single polar flagellum. The flagellum of this organism is structurally similar to those of other Gram-negative bacteria and contains a relatively complex basal body and hook structure attached to a filament primarily consisting of the assembled flagellin subunit protein.Although the role of flagellar motility in virulence of many pathogenic microorganisms including P. aeruginosa has been well established (9,18,19), the recent discovery that flagellin is one of the most potent stimuli of the host innate immune response gives added significance to the expression of flagella in the context of infection. Flagellin-based signaling in a variety of cells is mediated by Toll-like receptor 5 (11), and in P. aeruginosa, it has been shown to be more potent than Pseudomonas lipopolysaccharide (17).The flagellin proteins of P. aeruginosa can be classified as a-type or b-type, based on their reactions with specific polyclonal antibodies and their molecular weights (1, 2, 16). The a-type flagellins are reported to be heterologous and have variable molecular masses ranging from 45 to 52 kDa, whereas b-type flagellins are essentially conserved in sequence and have an almost invariant molecular mass of 53 kDa. However, between the two types, the N-and C-terminal domains are conserved while the central domain of the a-type is said to be hypervariable, leading to antigenic or serological variations (21, 29). The a-type flagellins have been further classified into several subtypes based on their H-antigenic components (2), nucleotide sequence variations (23), or restriction fragment length polymorphisms (20).Totten and Lory first noted anomalous migration and heterogeneity of the a-type flagellins during sodium dodecyl sulfate...

Comparative phylogenomics of the food-borne pathogen Campylobacter jejuni reveals genetic markers predictive of infection source

“…The modifications increase the hydrophilicity of flagellin and often influence the cells' immunogenicity and their interaction with eukaryotic cells (22,23). The biological significance of the glycosylation island in C. jejuni remains to be determined, but different glycoforms appear to be expressed in different hosts or environments and may provide them with a specific survival advantage (25). Variant flagellin glycoforms have been shown to be expressed in different hosts or environments in the opportunistic pathogen P. aeruginosa that may provide the pathogen with a specific survival advantage (25).…”

“…The biological significance of the glycosylation island in C. jejuni remains to be determined, but different glycoforms appear to be expressed in different hosts or environments and may provide them with a specific survival advantage (25). Variant flagellin glycoforms have been shown to be expressed in different hosts or environments in the opportunistic pathogen P. aeruginosa that may provide the pathogen with a specific survival advantage (25). Recently, flagellin glycosylation in the plant pathogen Pseudomonas syringae has been shown to be involved in determining plant host specificity (26).…”

Flagellar glycosylation – a new component of the motility repertoire?

“…Genetic studies have revealed considerable polymorphism in the glycosylation islands of a-type strains (Arora et al, 2004), indicative of considerable diversity in the composition of the respective flagellar glycans. The identification of a glycosylation island containing four genes, two of which display homology to genes within the PAK glycosylation island, from the genome of strain PAO, suggests that the flagellin from b-type strains may also be glycosylated.…”

“…Additionally, the rhamnose moiety can be further modified through sequential extension, as was shown in the case of PAK, with a heterogeneous glycan capped with a conserved trisaccharide. In contrast to other species, where flagellar filament assembly is inhibited when the glycosylation process is interrupted, lack of glycosylation through mutation of the rhamnose-specific glycosyltransferase gene orfN from the flagellar glycosylation island had no effect on flagellar assembly or subsequent motility in P. aeruginosa.Genetic studies have revealed considerable polymorphism in the glycosylation islands of a-type strains (Arora et al, 2004), indicative of considerable diversity in the composition of the respective flagellar glycans. The identification of a glycosylation island containing four genes, two of which display homology to genes within the PAK glycosylation island, from the genome of strain PAO, suggests that the flagellin from b-type strains may also be glycosylated.…”

Superchannel of Bacteria: Biological Significance and New Horizons

“…12) Furthermore, part of the central domain (approximately 100 amino acid residues) of p5 and of p6 is missing in comparison with the structures of Escherichia coli and Salmonella typhimurium flagellins, resulting in the formation of shaft-type flagella with no propeller. Since certain pseudomonades that form a polar flagellum produce flagellins lacking part of the central domain, 15) the deletions observed in p5 and p6 are irrelevant to the fact that strain A1 has no flagella.…”