Glycosylation of Pseudomonas aeruginosa strain Pa5196 type IV pilins with mycobacterium-like alpha-1,5-linked d-Araf oligosaccharides

Abstract

Pseudomonas aeruginosa is a gram-negative bacterium that uses polar type IV pili for adherence to various materials and for rapid colonization of surfaces via twitching motility. Within the P. aeruginosa species, five distinct alleles encoding variants of the structural subunit PilA varying in amino acid sequence, length, and presence of posttranslational modifications have been identified. In this work, a combination of mass spectrometry and nuclear magnetic resonance spectroscopy was used to identify a novel glycan modification on the pilins of the group IV strain Pa5196. Group IV pilins continued to be modified in a lipopolysaccharide (wbpM) mutant of Pa5196, showing that, unlike group I strains, the pilins of group IV are not modified with the O-antigen unit of the background strain. Instead, the pilin glycan was determined to be an unusual homo-oligomer of alpha-1,5-linked d-arabinofuranose (d-Araf). This sugar is uncommon in prokaryotes, occurring mainly in the cell wall arabinogalactan and lipoarabinomannan (LAM) polymers of mycobacteria, including Mycobacterium tuberculosis and Mycobacterium leprae. Antibodies raised against M. tuberculosis LAM specifically identified the glycosylated pilins from Pa5196, confirming that the glycan is antigenically, as well as chemically, identical to those of Mycobacterium. P. aeruginosa Pa5196, a rapidly growing strain of low virulence that expresses large amounts of glycosylated type IV pilins on its surface, represents a genetically tractable model system for elucidation of alternate pathways for biosynthesis of d-Araf and its polymerization into mycobacterium-like alpha-1,5-linked oligosaccharides.

FIG. 1.

Pa5196 pilins continue to be glycosylated in a wbpM mutant. A: Lipopolysaccharide samples were stained with silver (left) or detected via Western blotting using antibodies against serotype O11 (right). International Antigenic Typing Scheme strain O11 (IATS O11) and strain PA103 (both serotype O11) are positive controls; PAK (serotype O6) is a negative control. The wbpM mutant of Pa5196 lacks high-molecular-weight O antigen, as shown by silver staining and Western blotting, while the core region (at the bottom of the gel) is not affected. The band visible in all five lanes on the silver stain is residual proteinase K. B: Pilins from the indicated strains were detected with Coomassie (left) or with a fluorescent glycoprotein stain (right). M, molecular mass markers in kDa; G, glycoprotein controls; −, nonglycosylated (β-casein); +, glycosylated (RNase B). Lane 1, strain 1244 (group I, glycosylated); lane 2, PAK (group II, nonglycosylated); lane 3, Pa5196 (group IV); lane 4, Pa5196 wbpM mutant.

FIG. 2.

Analysis of the intact Pa5196 pilin by ESI-MS. The m/z value and charge state have been indicated for the most abundant ion in each of the multiply charged protein peak clusters. The reconstructed protein profile is shown in the inset. The interval between any two populations of proteins is 132 Da, equivalent to the mass of a single pentose residue.

FIG. 3.

Identification of pilin glycopeptides by MS/MS. A: LC-MS/MS spectrum of the triply protonated T^55-79 glycopeptide ion at m/z 1,080.8. The b and y fragment ions originate from fragmentation of the peptide backbone. Sequential neutral loss of pentose sugars from the doubly charged peptide ion is observed in the upper part of the MS/MS spectrum (indicated with a “P”). Oxonium ions corresponding to one and two pentoses were observed at m/z 133.1 and 265.1, respectively. This ion has six pentose residues and is the most abundant form of this glycopeptide. However, glycoforms ranging from three to seven pentoses were also observed, as is demonstrated in the expanded LC-MS spectrum of triply charged ions provided in the inset. B: LC-MS/MS spectrum of the triply protonated T^80-104 glycopeptide ion at m/z 1,133.1. This ion also contains six pentoses and is the most abundant form of this glycopeptide. MS/MS analysis was performed on many of the other glycoforms observed by LC-MS (inset shows triply charged ions). C: Map of Pa5196 pilin following trypsin and/or chymotrypsin digestion and nanoLC-MS/MS. The protein fragments identified by nanoLC-MS/MS (∼90% of the mature protein) are shown in regular font, the hydrophobic N-terminal fragment that could not be detected is in italics, and the two glycosylated tryptic peptides are underlined. The unmodified chymotryptic peptide ⁷³NATLVGKY⁸⁰ (boldface) overlaps both modified peptides. Two linked Cys residues are highlighted in gray, while the two Thr residues, which are the most likely sites of modification, are shown in reverse text. mF, methyl-Phe.

FIG. 4.

feCID-MS/MS analysis of the T^55-79 glycopeptide. The glycopeptides were fragmented by front-end-collision-induced dissociation (orifice voltage = 80 V) as they entered the mass spectrometer. MS/MS analysis was performed on the doubly protonated ion at m/z 1,291.1 corresponding to the T^55-79 peptide plus one pentose moiety. The observation of the pentose-modified y₁₇ and y₁₈ fragment ions at m/z 1,729.5 and 1,799.5 (+P) confirmed that the site of modification is Thr64 or Thr66.

FIG. 5.

1D ¹H spectrum (A) and a ¹H-¹³C HSQC spectrum (B) of the O-linked pilin glycan from P. aeruginosa. Labeled in the HSQC spectrum are the resonances from the two αAraf moieties that give rise to the most prominent glycan-associated peaks.

FIG. 6.

Recognition of P. aeruginosa 5196 pilins by antibodies to lipoarabinomannan. Sheared surface preparations from P. aeruginosa strains 1244 (group I), PAK (group II), and 5196 (group IV) were separated on 15% SDS-PAGE gels and stained with Coomassie or transferred to nitrocellulose and probed with either MAb CS-35 or polyclonal rabbit antisera, both raised against LAM purified from M. tuberculosis. A whole-cell lysate of M. smegmatis (M.smg) was included as a positive control. Masses of the molecular weight markers (in kDa) are indicated at the left.