Functional Characterization of the Flagellar Glycosylation Locus in Campylobacter jejuni 81–176 Using a Focused Metabolomics Approach

Abstract

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-␣-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-␣-D-glucopyranose, which is a bacillosamine derivative found in the N-linkedproteinglycan.Usingthisfocusedmetabolomicsapproach,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 CM-P-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.The availability of bacterial genomic sequences has provided unprecedented opportunity for comparative studies. The functional analysis of each respective genome is currently under way and often involves an integrative, multidisciplinary approach that combines bioinformatics, mutagenesis, proteomics, and microarray technologies. Most recently, metabolomics-based analyses, although limited in their number, are becoming established as an additional tool to elucidate gene function (1, 2). Metabolomics is the characterization of all low molecular weight compounds in a defined biological system and differs from classical metabolism studies by its greater breadth and speed of metabolite analysis. Although recent advances in mass spectrometry (MS), 2 such as the ultimate resolving capability of the Fourier-transform ion cyclotron resonance MS, and also in nuclear magnetic resonance spectroscopy (NMR) technologies, such as the development of higher magnetic field spectrometers and cryogenically cooled probes (cold probes), has greatly facilit...

A functional Campylobacter jejuni maf4 gene results in novel glycoforms on flagellin and altered autoagglutination behaviour

“…However, intracellular CMP-Pse5Am7Ac was detected, suggesting a role in glycosyl transfer to flagellin and not in monosaccharide biosynthesis. This is in contrast to a pseA mutant, which lacks intracellular CMPPse5Am7Ac, suggesting a defect in biosynthesis (McNally et al, 2006). Some of the maf genes including the (identical) maf1 and maf4 genes of strain 11168 contain a homopolymeric Gtract that is prone to slipped-strand mispairing, which may result in a shift of the ORF (Karlyshev et al, 2002).…”

“…Alternatively, the protein may have no role in the biosynthesis but may specifically transfer the novel Pse5Am7Ac derivative(s) to the flagellin protein. Also, insertional inactivation of the maf2 (pseD) gene does not interfere with the biosynthesis of pseudaminic acid precursors (McNally et al, 2006) but results in a specific lack of Pse5Am7Ac but not Pse5Ac7Ac glycans on flagellin , consistent with substrate-specific transfer of the glycans.…”

“…4a) indicates that the gene is not essential for motility of C. jejuni. This is surprising, as maf genes have been implicated as determinants of bacterial motility in strain 11168 (Karlyshev et al, 2002), although inactivation of maf2 and maf3 does not result in loss of motility in strain 81-176 McNally et al, 2006). The finding that insertional inactivation of maf4 still allowed flagella assembly enabled us to investigate the glycosylation status of the flagellin in the mutant strain.…”

“…Further characterization of flagellin using periodate treatment, specific lectins (Doig et al, 1996), and, at a later stage, state-of-the-art chemical analysis (Logan et al, 2002;McNally et al, 2006McNally et al, , 2007Thibault et al, 2001) indicates the presence of O-linked carbohydrate residues. To date, the flagellin of Campylobacter jejuni strain 81-176 is known to be decorated predominantly with 5,7 diacetamido-3,5,7,9 tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid, Pse5Ac7Ac), which is attached to up to 19 different Ser/Thr residues in the flagellin.…”

Colonize, evade, flourish

“…6A). [67][68][69] H. pylori uses homologs of the pse genes to generate PseAc. The glycosylated flagellin subunits are then exported from the cytoplasm by the flagellar secretory machinery for integration into the emerging filament (Fig.…”