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The ability of P. aeruginosa to cause serious infections is mediated by a variety of virulence factors. One of these, the somatic pilus, promotes colonization by adhesion and dissemination by twitching motility. Pili are thin protein filaments which extend from the cell's pole. They are composed of protein monomers called pilins which have molecular weights of approximately 15-16,000 and are arranged in a helical fashion in the pilus. We have demonstrated that each of the pilin monomers of P. aeruginosa 1244 is glycosylated with a single saccharide, and that this glycan is the lipopolysaccharide repeating unit of this organism. This structure is O-linked through the b -carbon of the carboxy-terminal (serine) residue, and is surface-exposed as determined by immunogold electron microscopy using a glycan-specific monoclonal antibody. The presence of the glycan strongly influences pilus hydrophobicity. Competition studies between a mutant unable to glycosylate pilin and wildtype strains using a mouse respiratory model revealed that the organism producing the pilin glycan significantly outgrew the strain that failed to produce glycosylated pili, suggesting a role for this modification in pathogenicity.
Studies using P. aeruginosa 1244 mutants defective in synthesis of the O-antigen indicated that the O-antigen biosynthetic pathway is the source of the pilin glycan. Pilin glycosylation resulting from expression of heterologous O-antigen gene clusters in strain 1244 supported these results, and, more importantly, showed that the pilin glycosylation machinery had extremely low glycan substrate specificity. Recent experiments using truncated O-antigen precursors have shown that this substrate permissiveness is due to the ability of the pilin glycosylation system to only recognize common features of the reducing end sugar of the O-antigen repeating unit. As with the glycan, the pilin substrate specificity is low. For example, glycosylation of 1244 pilin does not require a specific sequence motif, but rather needs only a carboxy- terminal serine (or threonine) and a charge-compatible pilus surface.
We have previously described a gene ( pilO ) that is required for pilin glycosylation. Recent research from our lab has shown that PilO shares a sequence motif with Wzy and WaaL, enzymes that also utilize the O-antigen repeating unit as substrate but show little overall sequence similarity with the pilin glycosylating enzyme. Mutagenesis experiments showed that the C-proximal portion of PilO is required for catalysis. The enzyme is located in the cytoplasmic membrane with putative catalytic segments in the periplasm. These results indicated that pilin glycosylation occurs in this chamber after the carrier lipid-bound O-antigen repeating unit had been transported from the cytoplasm.
Horzempa, J., Held, T.K., Cross, A.S., Furst, F., Qutyan, M., Neely, A.N., and Castric, P. 2008. Immunization with Pseudomonas aeruginosa 1244 pilin provides O-antigen-specific protection. Clin. Vacc. Immunol. 15: in press
Qutyan, M., Paliotti, M., and Castric, P. 2007. PilO of Pseudomonas aeruginosa 1244: subcellular location and domain assignment. Molec. Microbiol. 66:1444-1458.
Horzempa, J., Dean, C.R., Goldberg, JB, Castric, P. 2006. Pseudomonas aeruginosa 1244 Pilin Glycosylation: Glycan Substrate Recognition. J. Bacteriol. 188:4244-52.
Horzempa, J., Comer, J.E., Davis, S.A., Castric, P. 2006. Glycosylation substrate specificity of Pseudomonas aeruginosa 1244 pilin. J. Biol. Chem. 281:1128-36.
Smedley, J.G. 3rd, Jewell, E., Roguskie, J., Horzempa, J., Syboldt, A, Stolz, D.B., Castric, P. 2005. Influence of pilin glycosylation on Pseudomonas aeruginosa 1244 pilus function. Infect. Immun. 73:7922-31.
DiGiandomenico, A., Matewish, M.J., Bisaillon, A., Stehle, J., Lam, J.S., Castric, P. 2002. Glycosylation of Pseudomonas aeruginosa 1244 Pilin: Glycan Substrate Specificity. Molec. Microbiol. 46:519-130.
Comer, J.E., Marshall, M.A., Blanch, V.J., Deal, C.D., Castric, P. 2002. Identification of the Pseudomonas aeruginosa 1244 Pilin Glycosylation Site. Infect. Immun. 70:2837-2845.
Castric, P.A., Cassels, F.J., Carlson, R.W. 2001. Structural Characterization of the Pseudomonas aeruginosa 1244 Pilin Glycan. J. Biol. Chem. 276:26479-26485.
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Email: castric@duq.edu |
