Cloning and comparison of fliC genes and identification of glycosylation in the flagellin of Pseudomonas aeruginosa a-type strains

Abstract

Pseudomonas aeruginosa a-type strains produce flagellin proteins which vary in molecular weight between strains. To compare the properties of a-type flagellins, the flagellin genes of several Pseudomonas aeruginosa a-type strains, as determined by interaction with specific anti-a monoclonal antibody, were cloned and sequenced. PCR amplification of the a-type flagellin gene fragments from five strains each yielded a 1.02-kb product, indicating that the gene size is not likely to be responsible for the observed molecular weight differences among the a-type strains. The flagellin amino acid sequences of several a-type strains (170,018, 5933, 5939, and PAK) were compared, and that of 170,018 was compared with that of PAO1, a b-type strain. The former comparisons revealed that a-type strains are similar in amino acid sequence, while the latter comparison revealed differences between 170,018 and PAO1. Posttranslational modification was explored for its contribution to the observed differences in molecular weight among the a-type strains. A biotin-hydrazide glycosylation assay was performed on the flagellins of three a-type strains (170,018, 5933, and 5939) and one b-type strain (M2), revealing a positive glycosylation reaction for strains 5933 and 5939 and a negative reaction for 170,018 and M2. Deglycosylation of the flagellin proteins with trifluoromethanesulfonic acid (TFMS) confirmed the glycosylation results. A molecular weight shift was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis for the TFMS-treated flagellins of 5933 and 5939. These results indicate that the molecular weight discrepancies observed for the a-type flagellins can be attributed, at least in part, to glycosylation of the protein. Anti-a flagellin monoclonal antibody reacted with the TFMS-treated flagellins, suggesting that the glycosyl groups are not a necessary component of the epitope for the human anti-a monoclonal antibody. Comparisons between a-type sequences and a b-type sequence (PAO1) will aid in delineation of the epitope for this monoclonal antibody.

FIG. 1

Comparison of a-type, 170018 (GenBank accession no. U76543), and b-type, PAO1 (GenBank accession no. U54775), flagellin amino acid sequences. Alignment was performed by the CLUSTAL method by using the DNA* computer program.

FIG. 2

Comparison of a-type flagellin amino acid sequences from P. aeruginosa PAK (41), 170018 (GenBank accession no. U76543), 5933 (GenBank accession no. AF003906), and 5939 (GenBank accession no. AF003905). Alignment was performed by the CLUSTAL method by using the DNA* computer program.

FIG. 3

(a) SDS-PAGE of P. aeruginosa flagellin proteins stained with Coomassie brilliant blue. Lane 1, protein standards; lane 2, 170018 flagellin (M_r, 45,000); lane 3, 5933 flagellin (M_r, 52,000); lane 4, 5939 flagellin (M_r, 41,000); lane 5, M2 (b-type) flagellin (M_r, 53,000); lane 6, transferrin; and lane 7, pepsin. (b) Western blot of flagellin proteins with human anti-a and anti-b MAbs. Anti-a MAb at a 1:1,000 dilution was added to anti-b MAb at a 1:1,000 dilution, and the mixture was added to the membrane. Lane 1, 170018 flagellin; lane 2, 5933 flagellin; lane 3, 5939 flagellin; lane 4, M2 flagellin; and lane 5, transferrin as a negative control. (c) Biotin-hydrazide glycosylation assay of the flagellin proteins of P. aeruginosa strains. Approximately 3 μg of each protein was separated by electrophoresis and transferred to a PVDF membrane. Meta-periodate (15 mM) in 50 mM sodium acetate buffer (pH 5.45) oxidized the sugars. Biotin-hydrazide (5 mM) was then added, followed by streptavidin-alkaline phosphatase. Lane 1, 170018 flagellin; lane 2, 5933 flagellin; lane 3, 5939 flagellin; lane 4, M2 flagellin; lane 5, glycoprotein transferrin as a positive control; and lane 6, pepsin as a negative control.

FIG. 4

SDS-PAGE of TFMS-treated and untreated flagellins stained with Coomassie brilliant blue. The deglycosylation assay revealed a molecular mass shift for the flagellins of strains 5933, 5939, and M2 after chemical removal of the glycosyl group. Lane 1, protein standards; lane 2, 170018 flagellin; lane 3, deglycosylated 170018 flagellin; lane 4, 5933 flagellin; lane 5, deglycosylated 5933 flagellin; lane 6, 5939 flagellin; lane 7, deglycosylated 5939 flagellin; lane 8, M2 flagellin; and lane 9, deglycosylated M2 flagellin.

FIG. 5

Western blot of TFMS-treated and untreated flagellin proteins with human anti-a flagellin MAb. Lane 1, 170018 flagellin; lane 2, TFMS-treated 170018 flagellin; lane 3, 5933 flagellin; lane 4, TFMS-treated 5933 flagellin; lane 5, 5939 flagellin; and lane 6, TFMS-treated 5939 flagellin.