Peter Castric

The role of pilin glycosylation in Pseudomonas aeruginosa pathogenesis

Pseudomonas aeruginosa is a consummate opportunistic pathogen that is commonly associated with hospital-acquired infections. This organism is a particularly serious problem because of its natural and acquired antibiotic resistance and its arsenal of potent virulence factors. The type IVa pili of P. aeruginosa are thin protein filaments that extend from the cell's pole. They mediate adhesion to host tissue and facilitate a mode of surface-associated motility called twitching. Both of these traits are important to survival of this organism its soil/water environment and both are crucial to its ability to act as a pathogen.

The pilus fiber is made up of a monomeric subunit called pilin. We have demonstrated that each of the pilin monomers of P. aeruginosa 1244 is glycosylated with a single oligosaccharide, and that this glycan is the lipopolysaccharide repeating unit of this organism. This structure is O-linked through the -carbon of the carboxy-terminal (serine) residue, and is surface-exposed on the pilus fiber.

Work in our lab indicated that the presence of the pilin glycan influences virulence. Competition studies between a mutant unable to glycosylate pilin and a wild type strain using a mouse respiratory model revealed that the organism producing the pilin glycan significantly outgrew the strain that failed to produce glycosylated pili. This suggested a role for pilin glycosylation in pathogenicity. A research goal of our laboratory is the determination of the specific role of pilus glycosylation in pathogenicity.

Work in our lab also focuses on vaccine design. In spite of extensive research, there is currently no effective vaccine against P. aeruginosa infections. The ability of this organism to attach heterologous O-antigen repeating units to pilins suggests the possibility of constructing a vaccine that could provide both pilin and O-antigen specific protection. Both of these structures are strongly immunogenic and have previously been considered as vaccine candidates.

Selected Publications (student authors from my lab are cited in italic)

Qutyan, M., Henkel, M., Horzempa, J., Quinn, M., and P. Castric. 2010. Glycosylation of Pilin and Nonpilin Protein Constructs by Pseudomonas aeruginosa 1244. J. Bacteriol. 192:5972-5981. PMID: 20833803

PilO is an oligosaccharyl transferase (OTase) that catalyzes the O-glycosylation of Pseudomonas aeruginosa group I pilins. This enzyme attaches a single O-antigen repeating unit to the β-carbon of the C-terminal residue (a serine). The present work was carried out to explore the specificity of PilO for its protein substrate. It was found that adding a fifteen residue peptide (terminating in serine) to the C-terminus of a group II pilin (which does not normally support glycosylation) allowed this protein to serve as substrate. Likewise, modification of a group III pilin (which is not normally glycosylated and which is structurally dissimilar from the other two pilin groups) with the C-terminal attachment of this peptide or the residues AAS allowed glycosylation. A protein fusion composed of a group I pilin linked at its C-terminus with Escherichia coli alkaline phosphatase which contained the above mentioned fifteen residues at its C-end also served as a PilO substrate. In addition, modified E. coli alkaline phosphatase in the absence of attached pilin could be glycosylated. This work, along with previous studies (Horzempa 06) provides evidence that PilO has a very simple, but very specific, protein substrate requirement and that engineered nonpilin protein could be glycosylated by this enzyme. These studies suggest that a variety of proteins can be modified to act as PilO substrate and that these finding have specific application in vaccine design.

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Horzempa, J., Held, T.K., Cross, A.S., Furst, D., Qutyan, M., Neeley, A.N., and P. Castric. 2008. Immunization with a Pseudomonas aeruginosa 1244 pilin provides O-antigen-specific protection. Clin. Vacc. Immunol. 15:590-597. PMID: 18272666 PDF

The O-antigen is both a major structural outer-membrane component and the dominant epitope of most Gram-negative bacteria. Pseudomonas aeruginosa 1244 produces a Type IV pilus and covalently links an O-antigen repeating unit to each pilin monomer. Here we show that immunization of mice with pure pilin from strain 1244, using either the mouse respiratory or the thermal injury model, resulted in protection from challenge with a pilus-null O-antigen-producing 1244 mutant. These results provide evidence that the pilin glycan stimulates a protective response that targets the O-antigen, suggesting that this system could be utilized as the basis for developing a variety of protective anti-Gram-negative bioconjugate vaccines.

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Qutyan, M., Paliotti, M., and P. Castric. 2007. PilO of Pseudomonas aeruginosa 1244: subcellular location and domain assignment. Mol. Microbiol. 66:1444-1468. PMID: 18005110

PilO of Pseudomonas aeruginosa 1244 catalyzes the attachment of an O-antigen repeating unit to the β-carbon of the pilin carboxy-terminal residue, a serine. The present study was conducted to locate the catalytic region of this enzyme and to establish the cellular location of the pilin glycosylation reaction. While PilO was not detectable by protein stain in extracts of P. aeruginosa or Escherichia coli, it was found that a MalE/PilO fusion protein was produced in significant amounts. This fusion complemented a P. aeruginosa 1244 mutant containing a pilO deletion and targeted to the cytoplasmic membrane of Escherichia coli. Wzy and WaaL, enzymes that also utilize the O-antigen repeating unit as substrate, share significant sequence similarity with PilO in a putative periplasmic loop region. PilO constructs in which portions of this common sequence were deleted or replaced with histidine residues lacked glycosylating activity, indicating that this region is important in pilin glycosylation. Deletions of segments downstream from the common region also prevented enzyme activity. Topology studies showed that the two PilO regions associated with enzyme activity were located in the periplasm. These results establish the catalytic site region and provide evidence that pilin glycosylation occurs in the periplasmic space of this organism.

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Horzempa, J., Dean, C.R., Goldberg, J.B., and P. Castric. 2006. Pseudomonas aeruginosa 1244 pilin glycosylation: glycan substrate recognition. J. Bacteriol. 188:4244-4252. PMID: 16740931

The pilin of Pseudomonas aeruginosa 1244 is glycosylated with an oligosaccharide that is structurally identical to the O-antigen repeating unit of this organism. Concordantly, the metabolic source of the pilin glycan is the O-antigen biosynthetic pathway. The present study was conducted to investigate glycan substrate recognition in the 1244 pilin glycosylation reaction. Comparative structural analysis of O-subunits that had been previously shown to be compatible with the 1244 glycosylation machinery revealed similarities among sugars at the presumed reducing termini of these oligosaccharides. We therefore hypothesized that the glycosylation substrate was within the sugar at the reducing end of the glycan precursor. Since much is known of PA103 O-antigen genetics, and because the sugar at the reducing termini of the O7 (strain 1244) and O11 (strain PA103) are identical (β-N-acetyl fucosamine), we utilized PA103 and strains that express lipopolysaccharide (LPS) with a truncated O-antigen subunit to test our hypothesis. LPS from a strain mutated in the wbjE gene produced an incomplete O-subunit, consisting only of the monosaccharide at the reducing end (β-D-N-acetyl fucosamine). Expression of pilAO₁₂₄₄ in PA103 wbjE::aacC1 followed by Western blotting of extracts of these cells indicated that pilin produced had been modified by the addition of material consistent with a single N-acetyl fucosamine. This was confirmed by analyzing endopeptidase-treated pilin by mass spectrometry. Collectively, these data suggest the pilin glycosylation substrate recognition features lie within the reducing-end moiety of the O-repeat, and structures of the remaining sugars are irrelevant.

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Horzempa, J., Comer, J.E., Davis, S., and P. Castric. 2006. Glycosylation substrate specificity of Pseudomonas aeruginosa 1244 pilin. J. Biol. Chem. 281:1128-1136. PMID: 16286455

The β-carbon of the Pseudomonas aeruginosa 1244 pilin C-terminal Ser is a site of glycosylation. Previous work has shown that the glycan originates in the O-antigen biosynthetic pathway and that glycosylation has extremely low glycan substrate specificity. The present study was conducted to determine the pilin structures necessary for glycosylation. It was found that while Thr could be tolerated at the pilin C-terminus, the blocking of the Ser carboxyl group with the addition of an Ala prevented glycosylation. Pilin from strain PA103 was not glycosylated by P. aeruginosa 1244, even when the C-terminal residue was converted to Ser. Substituting the disulfide loop region of strain PA103 pilin with that of strain 1244 allowed glycosylation to take place. Neither conversion of 1244 pilin disulfide loop Cys residues to Ala nor the deletion of segments of this structure prevented glycosylation. It was noted that the PA103 pilin disulfide loop environment was electronegative, while that of strain 1244 pilin had an overall positive charge. Insertion of a positive charge into the PA103 pilin disulfide loop of a mutant containing Ser at the C-terminus allowed glycosylation to take place. Extending the "tail" region of the PA103 mutant pilin containing Ser at its terminus resulted in robust glycosylation. These results suggest that the terminal Ser is the major pilin glycosylation recognition feature, and that this residue cannot be substituted at its carboxyl group. While no other specific recognition features are present, the pilin surface must be compatible with the reaction apparatus for glycosylation to occur.

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Smedley, J., III, Jewell, E., Roguskie, J., Horzempa, J., Syboldt, A., Beer Stolz, D. and P. Castric. 2005. Influence of pilin glycosylation on Pseudomonas aeruginosa 1244 pilus function. Infect. Immun. 73:7922-7931. PMID:16299283

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of nosocomial pneumonia. Among its virulence factors, the type IV pili of P. aeruginosa strain 1244 contain a covalently linked, three-sugar glycan of previously unknown significance. The work described in this paper was carried out to determine the influence of the P. aeruginosa 1244 pilin glycan on pilus function, as well as a possible role in pathogenesis. To accomplish this, a deletion was introduced into the pilO gene of this organism. The isogenic knockout strain produced, 1244G7, was unable to glycosylate pilin, but could produce pili normal in appearance and quantity. In addition, this strain could carry out twitching motility, was sensitive to pilus-specific bacteriophages, and could form a normal biofilm. Analysis of whole cells and isolated pili from wild type P. aeruginosa strain 1244 by transmission electron microscopy with a glycan-specific immunogold label showed that this saccharide was distributed evenly over the fiber surface. The presence of the pilin glycan reduced the hydrophobicity of purified pili, as well as whole cells. With regard to pathogenicity, P. aeruginosa strains producing glycosylated pili were commonly found among clinical isolates, and particularly among those strains isolated from sputum. Competition index analysis using a mouse respiratory model comparing strain 1244 and 1244G7 indicated that the presence of the pilin glycan allowed for significantly greater survival in the lung environment. These results collectively suggest that the pilin glycan is a significant virulence factor and may aid in the establishment of infection.

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DiGiandomenico, A., Matewish, M.J., Bisaillon, A., Stehle, J., Lam, J.S., and P. Castric. 2002. Glycosylation of Pseudomonas aeruginosa 1244 Pilin: Glycan Substrate Specificity. Molec. Microbiol. 46:519-130. PMID: 12406226

The structural similarity between the pilin glycan and the O-antigen of Pseudomonas aeruginosa 1244 suggested that they have a common metabolic origin. Mutants of this organism lacking functional wbpM or wbpL genes synthesized no O-antigen and produced only non-glycosylated pilin. Complementation with plasmids containing functional wbpM or wbpL genes fully restored the ability to produce both O-antigen and glycosylated pilin. Expression of a cosmid clone containing the O-antigen biosynthetic gene cluster from P. aeruginosa PA103 (LPS serotype O11) in P. aeruginosa 1244 (LPS serotype O7) resulted in the production of strain 1244 pili that contained both O7 and O11 antigens. The presence of the O11 repeating unit was confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Expression of the O-antigen biosynthesis cluster from Escherichia coli O157:H7 in strain 1244 resulted in the production of pilin that contained both the endogenous Pseudomonas as well as the Escherichia O157 O-antigens. A role for pilO in the glycosylation of pilin in P. aeruginosa is evident as the cloned pilAO operon produced glycosylated strain 1244 pilin in eight heterologous P. aeruginosa strains. Removal of the pilO gene resulted in the production of unmodified strain 1244 pilin. These results show that the pilin glycan of P. aeruginosa 1244 is a product of the O-antigen biosynthetic pathway. In addition, the structural diversity of the O-antigens used by the 1244 pilin glycosylation apparatus indicates that the glycan substrate specificity of this reaction is extremely low.

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Comer, J.E., Marshall, M.A., Blanch, V.J., Deal, C.D., and P. Castric. 2002. Identification of the Pseudomonas aeruginosa 1244 Pilin Glycosylation Site. Infect. Immun. 70:2837-2845. PMID: 12010970

Previous work (P. Castric, F. J. Cassels, and R. W. Carlson, J. Biol. Chem. 276:26479-26485, 2001) has shown the Pseudomonas aeruginosa 1244 pilin glycan to be covalently bound to a serine residue. N-terminal sequencing of pilin fragments produced from endopeptidase treatment and identified by reaction with a glycan-specific monoclonal antibody indicated that the glycan was present between residue 75 and the pilin carboxy terminus. Further sequencing of these peptides revealed that serine residues 75, 81, 84, 105, 106, and 108 were not modified. Conversion of serine 148, but not serine 118, to alanine by site-directed mutagenesis, resulted in loss of the ability to carry out pilin glycosylation when tested in an in vivo system. These results showed the pilin glycan to be attached to residue 148, the carboxy-terminal amino acid. The carboxy-proximal portion of the pilin disulfide loop, which is adjacent to the pilin glycan, was found to be a major linear B-cell epitope, as determined by peptide epitope mapping analysis. Immunization of mice with pure pili produced antibodies that recognized the pilin glycan. These sera also reacted with P. aeruginosa 1244 lipopolysaccharide as measured by Western blotting and enzyme-linked immunosorbent assay.

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Castric, P.A., Cassels, F.J., and R.W. Carlson. 2001. Structural Characterization of the Pseudomonas aeruginosa 1244 Pilin Glycan. J. Biol. Chem. 276:26479-26485. PMID: 11342554

An antigenic similarity between lipopolysaccharide (LPS) and glycosylated pilin of Pseudomonas aeruginosa 1244 was noted. We purified a glycan-containing molecule from proteolytically digested pili and showed it to be composed of three sugars and serine. This glycan competed with pure pili and LPS for reaction with an LPS-specific monoclonal antibody, which also inhibited twitching motility by P. aeruginosa bearing glycosylated pili. One-dimensional NMR analysis of the glycan indicated the sugars to be 5N-β-OHC(4)7NfmPse, Xyl, and FucNAc. The complete proton assignments of these sugars as well as the serine residue were determined by COSY and TOCSY. Electrospray ionization mass spectrometry (MS) determined the mass of this molecule to be 771.5. The ROESY NMR spectrum, tandem MS/MS analysis, and methylation analysis provided information on linkage and the sequence of oligosaccharide components. These data indicated that the molecule had the following structure: α-5N β OHC(4)7NFmPse-(2→4)-β-Xyl-(1→3)-β-FucNAc-(1→3)-β-Ser.

The work in the references cited here was supported by the National Institute of Allergy and Infectious Disease.