The engineering of bacteria bearing azido-pseudaminic acid-modified flagella.

Catch a tiger by the tail: We have demonstrated that by feeding nonmotile mutant C. jejuni bacteria with a neutral azide-labelled pseudaminic acid precursor we can restore their ability to generate functional flagella. The presence of azido-pseudaminic acid on the surface of the flagella provides a bio-orthogonal chemical handle that can be used to modify the flagellar proteins.

Campylobacter jejuni glycosylation island important in cell charge, legionaminic acid biosynthesis, and colonization of chickens.

Previously, we identified five genes (Cj1321 to Cj1326, of which Cj1325 and Cj1326 are a single gene) in the O-linked flagellin glycosylation island that are highly prevalent in Campylobacter jejuni isolates from chickens. We report mutagenesis, functional, and structural data to confirm that this locus, and Cj1324 in particular, has a significant contributory role in the colonization of chickens by C. jejuni. A motile DeltaCj1324 mutant with intact flagella was considerably less hydrophobic and less able to autoagglutinate and form biofilms than the parent strain, 11168H, suggesting that the surface charge of flagella of Cj1324-deficient strains was altered. The physical and functional attributes of the parent were restored upon complementation. Structural analysis of flagellin protein purified from the DeltaCj1324 mutant revealed the absence of two legionaminic acid glycan modifications that were present in the parent strain, 11168H. These glycoform modifications were shown to be prevalent in chicken isolates and confirm that differences in the highly variable flagellin glycosylation locus can relate to the strain source. The discovery of molecular mechanisms influencing the persistence of C. jejuni in poultry aids the rational design of approaches to control this problematic pathogen in the food chain.

Identification of novel carbohydrate modifications on Campylobacter jejuni 11168 flagellin using metabolomics-based approaches.

It is well known that the flagellin of Campylobacter jejuni is extensively glycosylated by pseudaminic acid and the related acetamindino derivative, in addition to flagellin glycosylation being essential for motility and colonization of host cells. Recently, the use of metabolomics permitted the unequivocal characterization of unique flagellin modifications in Campylobacter, including novel legionaminic acid sugars in Campylobacter coli, which had been impossible to ascertain in earlier studies using proteomics-based approaches. To date, the precise identities of the flagellin glycosylation modifications have only been elucidated for C. jejuni 81-176 and C. coli VC167 and those present in the first genome-sequenced strain C. jejuni 11168 remain elusive due to lability and respective levels of individual glycan modifications. We report the characterization of the carbohydrate modifications on C. jejuni 11168 flagellin using metabolomics-based approaches. Detected as their corresponding CMP-linked precursors, structural information on the flagellin modifications was obtained using a combination of MS and NMR spectroscopy. In addition to the pseudaminic acid and legionaminic acid sugars known to be present on Campylobacter flagellin, two unusual 2,3-di-O-methylglyceric acid modifications of a nonulosonate sugar were identified. By performing a metabolomic analysis of selected isogenic mutants of genes from the flagellin glycosylation locus of this pathogen, these novel CMP-linked precursors were confirmed to be di-O-methylglyceric acid derivatives of pseudaminic acid and the related acetamidino sugar. This is the first comprehensive analysis of the flagellar modifications in C. jejuni 11168 and structural elucidation of di-O-methylglyceric acid derivatives of pseudaminic acid on Campylobacter flagellin.

Targeted metabolomics analysis of Campylobacter coli VC167 reveals legionaminic acid derivatives as novel flagellar glycans.

Glycosylation of Campylobacter flagellin is required for the biogenesis of a functional flagella filament. Recently, we used a targeted metabolomics approach using mass spectrometry and NMR to identify changes in the metabolic profile of wild type and mutants in the flagellar glycosylation locus, characterize novel metabolites, and assign function to genes to define the pseudaminic acid biosynthetic pathway in Campylobacter jejuni 81-176 (McNally, D. J., Hui, J. P., Aubry, A. J., Mui, K. K., Guerry, P., Brisson, J. R., Logan, S. M., and Soo, E. C. (2006) J. Biol. Chem. 281, 18489-18498). In this study, we use a similar approach to further define the glycome and metabolomic complement of nucleotide-activated sugars in Campylobacter coli VC167. Herein we demonstrate that, in addition to CMP-pseudaminic acid, C. coli VC167 also produces two structurally distinct nucleotide-activated nonulosonate sugars that were observed as negative ions at m/z 637 and m/z 651 (CMP-315 and CMP-329). Hydrophilic interaction liquid chromatography-mass spectrometry yielded suitable amounts of the pure sugar nucleotides for NMR spectroscopy using a cold probe. Structural analysis in conjunction with molecular modeling identified the sugar moieties as acetamidino and N-methylacetimidoyl derivatives of legionaminic acid (Leg5Am7Ac and Leg5AmNMe7Ac). Targeted metabolomic analyses of isogenic mutants established a role for the ptmA-F genes and defined two new ptm genes in this locus as legionaminic acid biosynthetic enzymes. This is the first report of legionaminic acid in Campylobacter sp. and the first report of legionaminic acid derivatives as modifications on a protein.

Functional characterization of the flagellar glycosylation locus in Campylobacter jejuni 81-176 using a focused metabolomics approach.

Bacterial genome sequencing has provided a wealth of genetic data. However, the definitive functional characterization of hypothetical open reading frames and novel biosynthetic genes remains challenging. This is particularly true for genes involved in protein glycosylation because the isolation of their glycan moieties is often problematic. We have developed a focused metabolomics approach to define the function of flagellin glycosylation genes in Campylobacter jejuni 81-176. A capillary electrophoresis-electrospray mass spectrometry and precursor ion scanning method was used to examine cell lysates of C. jejuni 81-176 for sugar nucleotides. Novel nucleotide-activated intermediates of the pseudaminic acid (Pse5NAc7NAc) pathway and its acetamidino derivative (PseAm) were found to accumulate within select isogenic mutants, and use of a hydrophilic interaction liquid chromatography-mass spectrometry method permitted large scale purifications of the intermediates. NMR with cryo probe (cold probe) technology was utilized to complete the structural characterization of microgram quantities of CMP-5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-L-glycero-alpha-L-manno-nonulosonic acid (CMP-Pse5NAc7Am), which is the first report of Pse modified at C7 with an acetamidino group in Campylobacter, and UDP-2,4-diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose, which is a bacillosamine derivative found in the N-linked proteinglycan. Using this focused metabolomics approach, pseB, pseC, pseF, pseI, and for the first time pseA, pseG, and pseH were found to be directly involved in either the biosynthesis of CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am. In contrast, it was shown that pseD, pseE, Cj1314c, Cj1315c, Cjb1301, Cj1334, Cj1341c, and Cj1342c have no role in the CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am pathways. These results demonstrate the usefulness of this approach for targeting compounds within the bacterial metabolome to assign function to genes, identify metabolic intermediates, and elucidate novel biosynthetic pathways.

Selective detection and identification of sugar nucleotides by CE-electrospray-MS and its application to bacterial metabolomics.

A novel method employing CE-ESMS and precursor ion scanning was developed for the selective detection of nucleotide-activated sugars. By using precursor ion scanning for fragment ions specific to the different nucleotide carriers, i.e., ions at m/z 322 for cytidine monophosphate, m/z 323, 385, and 403 for uridine diphosphate, m/z 362, 424, and 442, for guanosine diphosphate, and m/z 346, 408, and 426 for adenosine diphosphate, it was possible to selectively detect sugar nucleotides involved in the biosynthesis of glycoconjugates such as glycoproteins and lipopolysaccharides. Enhancement of sensitivity was achieved using N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) as a sample stacking buffer and provided detection limits between 0.2 and 3.8 pmol.mL(-)(1). The present CE-ESMS method provided linear dynamic ranges over the concentrations 0.2-164 nM (r(2) = 0.952-0.997) for different nucleotide sugar standards. The application of this method is demonstrated for the identification of intracellular pools of sugar nucleotides in wild type and isogenic mutants from the bacterial pathogen Campylobacter jejuni. By using product ion scanning (with and without front-end collision-induced dissociation), it was possible to determine the precise nature of unexpected sugar nucleotides involved in the biosynthesis of pseudaminic acid, a sialic acid-like sugar previously observed on the flagellin of some pathogenic bacteria.