Flagellar glycosylation in Burkholderia pseudomallei and Burkholderia thailandensis

Abstract

Glycosylation of proteins is known to impart novel physical properties and biological roles to proteins from both eukaryotes and prokaryotes. In this study, gel-based glycoproteomics were used to identify glycoproteins of the potential biothreat agent Burkholderia pseudomallei and the closely related but nonpathogenic B. thailandensis. Top-down and bottom-up mass spectrometry (MS) analyses identified that the flagellin proteins of both species were posttranslationally modified by novel glycans. Analysis of proteins from two strains of each species demonstrated that B. pseudomallei flagellin proteins were modified with a glycan with a mass of 291 Da, while B. thailandensis flagellin protein was modified with related glycans with a mass of 300 or 342 Da. Structural characterization of the B. thailandensis carbohydrate moiety suggests that it is an acetylated hexuronic acid. In addition, we have identified through mutagenesis a gene from the lipopolysaccharide (LPS) O-antigen biosynthetic cluster which is involved in flagellar glycosylation, and inactivation of this gene eliminates flagellar glycosylation and motility in B. pseudomallei. This is the first report to conclusively demonstrate the presence of a carbohydrate covalently linked to a protein in B. pseudomallei and B. thailandensis, and it suggests new avenues to explore in order to examine the marked differences in virulence between these two species.

Fig. 1.

Identification of glycoproteins in B. pseudomallei and B. thailandensis. Protein extracts from B. pseudomallei K96243 (a) and B. thailandensis E264 (b) were separated by 2D electrophoresis and stained with ProQ Emerald 300. Indicated spots were identified by peptide mass fingerprinting following tryptic digestion. Marker sizes are indicated in kDa.

Fig. 2.

Electrospray mass spectrometry of intact flagellin proteins from B. thailandensis E264. The inset shows the reconstructed molecular mass profile showing a major peak at 40,174 Da and two minor peaks at 38,845 Da and 41,481 Da.

Fig. 3.

Mass spectrometry analyses of tryptic peptides from flagellin proteins of B. pseudomallei K96243. The nLC-MS/MS spectrum of the triply protonated T^185–213 glycopeptide ion of m/z 1145.6 from B. pseudomallei is shown. The spectrum is dominated in the low-molecular-m/z region by the carbohydrate oxonium ion (m/z 292.1) and what could be a fragmentation component (m/z 274.1). Loss of m/z 145²⁺ in the high-molecular-m/z region of the spectrum corresponds to loss of glycan of residue mass 291.1. From the observed mass of the glycopeptide, a mass excess of 582 Da was observed, corresponding to two glycans of 291 Da. Below the graph is the amino acid sequence of the B. pseudomallei K96243 FliC protein. The sequence of the T^185–213 glycopeptide is underlined. Sequences in bold indicate unmodified peptides observed in tryptic digests.

Fig. 4.

Mass spectrometry analyses of tryptic peptides from flagellin proteins of B. thailandensis E264. nLC-MS/MS spectrum of the triply protonated T^185–213 glycopeptide ion of m/z 1074.2 from B. thailandensis. The spectrum is dominated in the low-molecular-m/z region by the carbohydrate oxonium ion (m/z 343.1) and what could be fragmentation components (m/z 301.1 and 283.1). From the observed mass of the glycopeptide, a mass excess of 342 Da was observed, corresponding to a single glycan modification. Below the graph is the amino acid sequence of the B. thailandensis E264 FliC protein. The sequence of the T^185–211 glycopeptide is underlined. Sequences in bold indicate unmodified peptides observed in tryptic digests.

Fig. 5.

Electrospray mass spectrometry of B. pseudomallei K96243 and B. thailandensis E27 glycans. Second-generation product ion spectra of ions obtained from in-source dissociation of multiply charged flagellin ions. MS/MS spectra were acquired by increasing the radio frequency (RF) lens 1 voltage from 40 to 90 V, forming fragment ions in the orifice/skimmer region of the mass spectrometer and promoting the formation of labile ions from the intact flagellin MS/MS spectrum of B. pseudomallei K96243 m/z 292.1 (a) and B. thailandensis E264 m/z 343.1 (b). This yielded a series of predominant fragment ions which did not correspond to peptide type y and b ions. Plausible neutral losses of molecules such as acetyl groups and water are indicated.