Identification of two distinct types of flagellar cap proteins, FliD, in Pseudomonas aeruginosa

Abstract

Binding of Pseudomonas aeruginosa strain PAK to mucin has been shown to be mediated by the flagellar cap protein, product of the fliD gene. Since the flagellar cap is very likely an exposed structure, the FliD polypeptide should be recognized by the host immune system, analogous to the recognition of dominant epitopes located in the exposed parts of the flagellin polypeptide within the assembled flagellum. In P. aeruginosa, a number of distinct flagellin variants are made, and these variable sequences presumably allow the newly infected P. aeruginosa to escape recognition by the antibody induced during a previous infection. Since similar mechanisms may direct the selection of FliD variants, we examined the extent of sequence heterogeneity among various FliD sequences among a selected group of P. aeruginosa. The results of PCR and nucleotide sequencing of the fliD region of eight different P. aeruginosa strains (laboratory strains PAK, PAO1, and PA103; clinical strains 1244, CS2, and CS32; cystic fibrosis strains CS29 and MDR) suggested that there were two distinct types of FliD in P. aeruginosa, which we named A type and B type. The results of Western blotting using the polyclonal antibodies raised against the purified FliD of A type (PAK) or B type (PAO1) further confirmed the existence of two distinct antigenic types of FliD proteins, with no cross-reactivity between the two serotypes. Further Western immunoblot analysis of the same strains using polyclonal FliC antibody showed that the strains with A-type FliD possessed a-type FliC and those with B-type FliD had b-type FliC. Similar Western blot analyses of 50 more P. aeruginosa strains obtained from varied sources revealed that all strains contained either A-type or B-type FliD, suggesting the existence of only two types of FliD in P. aeruginosa and indicating that fliC and fliD were coinherited. This limited diversity of FliC and FliD serotypes seems to be a unique feature of flagellar proteins. A chromosomal mutant having an insertion in the fliD gene of P. aeruginosa PAO1 was constructed. The motility defect of this mutant and a previously constructed PAK fliD mutant was better complemented with the fliD gene of the homologous types.

FIG. 1

Schematic representation of the fliDST regions of E. coli and P. aeruginosa strains PAK and PAO1. Locations and orientations of various primers used in PCR are shown. The deletion of ca. 300 bp in P. aeruginosa PAO1 is represented by the open triangle. The unique SmaI site used to insert the Gm^r cassette in the PAO1 fliD gene and BamHI cleavage sites are marked.

FIG. 2

Alignment of deduced amino acid sequences of the PAK (upper row) and PAO1 (lower row) fliD genes, using the GAP program of the Genetics and Computer Group package.

FIG. 3

Western blot analysis of different P. aeruginosa strains using specific polyclonal antibodies against A- and B-type FliD and a-type FliC proteins. Whole cell extracts of P. aeruginosa strains in panel A were probed with the PAK (A-type) FliD antibodies. The identical samples in panels B and C were probed with the PAO1 (B-type) FliD antibodies and a-type FliC antibodies, respectively.

FIG. 4

Soft L-agar (0.3%) plates showing the motility phenotypes of PAK and its derivatives (A) and of PAO1 and its derivatives (B). (A) 1, wild-type PAK; 2, A-type fliD mutant PAK-D; 3, PAK-D containing vector control pET15BVP; 4, PAK-D complemented with pET15BVP_a (A-type fliD); 5, PAK-D containing pET15BVPD_b (B-type fliD). (B) 1, wild-type PAO1; 2, B-type fliD mutant PAO-D; 3, PAO-D containing vector control pET15BVP; 4, PAO-D complemented with pET15BVPD_a (A-type fliD); 5, PAK-D containing pET15BVPD_b (B-type fliD).

FIG. 5

Phylogenetic analysis of the FliD sequences. The tree was constructed using P. aeruginosa (Pa) sequences from this work and additional sequences retrieved from GenBank: Bsu, Bacillus subtilis (U56901); Hly1 and Hly2, Helicobacter pylori (accession no. U82981) and H. pylori J99 (accession no. AE001500); Eco, E. coli K-12 MG1655 (accession no. AE000285); Tpa, Treponema pallidum (accession no. AE001257); Bbu, Borrelia burgdorferi (accession no. U66699); PaPAK, P. aeruginosa strain PAK (accession no. L81176); Vpa, Vibrio parahaemolyticus (accession no. U52957); and Sty, Salmonella enterica serovar Typhimurium (accession no. M33541). Bootstrap values represent 100 replications as indicated at branch points.