Abstract
Glycomics, the study of microbial polysaccharides and genes responsible for their formation, requires the continuous development of rapid and sensitive methods for the identification of glycan structures. In this study, methods for the direct analysis of sugars from 108 to 1010 cells are outlined using the human gastrointestinal pathogen, Campylobacter jejuni. Using capillary-electrophoresis coupled with sensitive electrospray mass spectrometry, we demonstrate variability in the lipid A component of C. jejuni lipooligosaccharides (LOSs). In addition, these sensitive methods have permitted the detection of phase-variable LOS core structures that were not observed previously. High resolution magic angle spinning (HR-MAS) NMR was used to examine capsular polysaccharides directly from campylobacter cells and showed profiles similar to those observed for purified polysaccharides analyzed by solution NMR. This method also exhibited the feasibility of campylobacter serotyping, mutant verification, and preliminary sugar analysis. HR-MAS NMR examination of growth from individual colonies of C. jejuni NCTC11168 indicated that the capsular glycan modifications are also phase-variable. These variants show different staining patterns on deoxycholate-PAGE and reactivity with immune sera. One of the identified modifications was a novel -OP=O(NH2)OMe phosphoramide, not observed previously in nature. In addition, HR-MAS NMR detected the N-linked glycan, GalNAc-alpha1,4-GalNAc-alpha1,4-[Glc-beta1,3-]GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac, where Bac is 2,4-diacetamido-2,4,6-trideoxy-d-glucopyranose, in C. jejuni and Campylobacter coli. The presence of this common heptasaccharide in multiple campylobacter isolates demonstrates the conservation of the N-linked protein glycosylation pathway in this organism and describes the first report of HR-MAS NMR detection of N-linked glycans on glycoproteins from intact bacterial cells.
Highlights
For methodologies that can analyze carbohydrate structures with increasing levels of sensitivity and simplicity
Using capillary-electrophoresis coupled with sensitive electrospray mass spectrometry, we demonstrate variability in the lipid A component of C. jejuni lipooligosaccharides (LOSs)
High resolution magic angle spinning (HR-MAS) NMR was used to examine capsular polysaccharides directly from campylobacter cells and showed profiles similar to those observed for purified polysaccharides analyzed by solution NMR
Summary
Bacterial Strains and Growth Conditions—C. jejuni NCTC11168 (HS:2) was isolated from a case of human enteritis [28] and later sequenced by Parkhill et al [12]. Preparation of O-Deacylated LOSs from Cells for CE-MS Analysis— Overnight growth of C. jejuni from one agar plate (ϳ1010 cells) was resuspended in phosphate-buffered saline, pH 7.4. The sample was placed in an ice bath, and the excess hydrazine was destroyed with cold acetone in dry ice. The deacylated LOS was isolated by centrifugation (16,000 ϫ g for 15 min), and the product was washed again with acetone, centrifuged, resuspended in water, and centrifuged, and the LOS-containing supernatant was lyophilized. Preparation of Cells for HR-MAS NMR—C. jejuni overnight growth from one agar plate (ϳ1010 cells) was harvested and suspended in 1 ml of 10 mM potassium-buffered saline (pH 7) made in D2O containing 10% sodium azide (w/v). The immunoblot was incubated with goat-anti-rabbit secondary antibody conjugated to alkaline phosphatase (1:2500 dilution, Sigma) and developed with the nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate detection system (Roche Applied Science)
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