Glycosylation of pilin and nonpilin protein constructs by Pseudomonas aeruginosa 1244

Abstract

PilO is an oligosaccharyl transferase (OTase) that catalyzes the O-glycosylation of Pseudomonas aeruginosa 1244 pilin by adding a single O-antigen repeating unit to the β carbon of the C-terminal residue (a serine). While PilO has an absolute requirement for Ser/Thr at this position, it is unclear if this enzyme must recognize other pilin features. To test this, pilin constructs containing peptide extensions terminating with serine were tested for the ability to support glycosylation. It was found that a 15-residue peptide, which had been modeled on the C-proximal region of strain 1244 pilin, served as a PilO substrate when it was expressed on either group II or group III pilins. In addition, adding a 3-residue extension culminating in serine to the C terminus of a group III pilin supported PilO activity. A protein fusion composed of strain 1244 pilin linked at its C terminus with Escherichia coli alkaline phosphatase (which, in turn, contained the above-mentioned 15 amino acids at its C terminus) was glycosylated by PilO. E. coli alkaline phosphatase lacking the pilin membrane anchor and containing the 15-residue peptide was also glycosylated by PilO. Addition of the 3-residue extension did not allow glycosylation of either of these constructs. Site-directed mutagenesis of strain 1244 pilin residues of the C-proximal region common to the group I proteins showed that this structure was not required for glycosylation. These experiments indicate that pilin common sequence is not required for glycosylation and show that nonpilin protein can be engineered to be a PilO substrate.

FIG. 1.

Comparison of the P. aeruginosa 683 PilA primary structure with pilin group I, II, and III representatives. Sequence identity is indicated by gray highlighting. The alignment of the three representative pilin groups is based on the common sequence of the N-proximal region and the disulfide loop region. Cysteines forming the disulfide loop are in white with a black background. Identity between PA14 and 683 pilin residues is indicated by a colon. The GenBank accession numbers for strains 1244, PA103, and PA14 are CAA58768.1, P08015.1, and ABJ13792.1, respectively.

FIG. 2.

Features of the fusions employed in this study. (A) Parent and modified pilins; (B) pilin-alkaline phosphatase constructs (modified and unmodified); (C) parent and modified alkaline phosphatase. The cartoons show the relative sizes and orientations of construct components (gray boxes, PilA; white boxes, PhoA) as well as the modification site (black boxes). The protein product name is listed in the first column of each panel. The plasmid containing the gene expressing this protein is in the second. The third column shows essential features of the protein construct. Here, the C-terminal additions are in white with a black background. The PilA-PhoA junctions are shown in black with a gray background.

FIG. 3.

Western blot of natural and modified PilA of Pseudomonas aeruginosa PA103 in the presence and absence of PilO. Antibodies used were monoclonal antibody 2.97, which is specific for strain P. aeruginosa PA103 pilin (A); monoclonal antibody 11.14, which is specific for the O antigen of P. aeruginosa IATS serotype O7 (B); monoclonal antibody O11, which is specific for the O antigen of P. aeruginosa IATS serotype O11 (C); and monoclonal antibody 6.45, which is specific for the disulfide loop region of strain 1244 pilin (D). The plasmids of panels A and B were expressed in P. aeruginosa 1244N3. The plasmids of panels C and D were expressed in P. aeruginosa PA103wzy_PaO11::aacC1. Glycosylated and nonglycosylated 1244 pilin was produced from pPAC46 and pPAC24, respectively.

FIG. 4.

Western blot of natural and modified P. aeruginosa 683 PilA in the presence or absence of PilO. The antibodies used were a polyclonal preparation raised against pure 683 pilin (A) or a monoclonal antibody against IATS serotype O11 (B). All plasmids were expressed in P. aeruginosa PA103wzy_PaO11::aacC1. Strain 683 pilin and strain PA103 pilin were supplied as purified proteins. pPAC24 contains a functional pilA gene but no pilO.

FIG. 5.

Western blot of natural and modified PilA-PhoA fusions in the presence of PilO. The antibodies used were a monoclonal antibody specific for E. coli PhoA (A); monoclonal antibody 5.44, which was specific for a P. aeruginosa 1244 pilin epitope (B); and monoclonal antibody O11, which was specific for P. aeruginosa IATS serotype O11 (C). All plasmids were expressed in P. aeruginosa PA103wzy_PaO11::aacC1 containing pUCP26PilO. See the legend to Fig. 2 for construct details.

FIG. 6.

Western blot of natural and modified E. coli PhoA produced in the presence of PilO. The antibodies used were a monoclonal antibody specific for E. coli PhoA (A) and monoclonal antibody O11, which was specific for P. aeruginosa IATS serotype O11 (B). All plasmids were expressed in P. aeruginosa PA103wzy_PaO11::aacC1 containing pUCP26PilO. See the legend to Fig. 2 for construct details.

FIG. 7.

Glycosylation of mutant P. aeruginosa 1244 pilins. Cloned pilA from this organism was subjected to site-directed mutagenesis, resulting in clones which produced the pilins seen. The primary antibody employed was monoclonal antibody 5.44. Pilin produced from pPAC46 was the glycosylated control, while pilin from pS148A, which has been shown to be nonglycosylated (9), was the negative control.