Báo cáo Y học: The structures of the lipooligosaccharide and capsule polysaccharide of Campylobacter jejuni genome sequenced strain NCTC 11168

The structures of the lipooligosaccharide and capsule polysaccharide of Campylobacter jejuni genome sequenced strain NCTC 11168

Tác giả: Frank St. Michael, Christine M. Szymanski, Jianjun Li, Kenneth H. Chan, Nam Huan Khieu, Suzon Larocque, Warren W. Wakarchuk, Jean-Robert Brisson and Mario A. Monteiro

Lĩnh vực: Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada

Nội dung tài liệu: Nghiên cứu này tập trung vào việc điều tra cấu trúc phân tử của lipooligosaccharide (LOS) và polysaccharide vỏ (capsule polysaccharide) của chủng Campylobacter jejuni NCTC 11168. LOS được chứng minh là có cấu trúc carbohydrate tương tự các epitope carbohydrate GM1a và GM2 của ganglioside người. Polysaccharide vỏ có khối lượng phân tử cao, bao gồm các đơn vị D-Rib, D-GalfNAc, một acid uronic được amid hóa, và 6-O-methyl-D-glycero-a-L-gluco-heptopyranose như một nhánh bên. Thông tin cấu trúc này sẽ hỗ trợ việc nhận diện và đặc trưng hóa các enzyme chuyên biệt tham gia vào quá trình sinh tổng hợp các cấu trúc này, đồng thời có thể dẫn đến khám phá các mục tiêu điều trị tiềm năng. Hơn nữa, mối tương quan giữa cấu trúc carbohydrate và bộ gen sẽ giúp làm sáng tỏ vai trò của các carbohydrate bề mặt này trong bệnh sinh của C. jejuni.

Mục lục chi tiết:

• The structures of the lipooligosaccharide and capsule polysaccharide of Campylobacter jejuni genome sequenced strain NCTC 11168
• Introduction
• EXPERIMENTAL PROCEDURES
• Bacterial strains and plasmids
• Media and growth conditions
• Generation of LOS and capsule
• Sugar composition and methylation linkage analysis
• Smith degradation
• CE-ESI-MS and CE-ESI-MS/MS
• Construction and characterization of insertional mutants
• Reverse transcriptase-polymerase chain reaction
• Deoxycholate-PAGE analysis and silver staining of polysaccharides
• Nuclear magnetic resonance
• Molecular modeling
• RESULTS
• Structural determination of the LOS
• Table 1. Methylation linkage analysis of C. jejuni NCTC 11168 intact LOS, core OS and Smith degradation products.
• Table 2. Negative ion ESI-MS data and proposed compositions for C. jejuni NCTC 11168 core OS and de-O-acylated LOS (masses include the addition of water)ª.
• Table 3. Interpretation of m/z ions in the FAB-MS spectrum of the methylated core OS from C. jejuni NCTC 11168.
• Fig. 1. Electron spray ionization-mass spectrometry. C. jejuni NCTC 11168 core OS showing a heterogeneous mixture (a). CE-MS/MS (+ ion mode, produces ions of m/z 1026) analysis of C. jejuni NCTC 11168 core OS (b). CE-MS/MS (+ ion mode, produces ions of m/z 1005) analysis of LOS from C. jejuni NCTC 11168 core OS (c).
• Fig. 2. FAB-MS spectra of the methylated C. jejuni NCTC 11168 core OS.
• Fig. 3. The complete structure of C. jejuni NCTC 11168 LOS.
• Table 3. (Continued).
• Fig. 4. CE-ESI-MS and CE-MS/MS analysis. CE-ESI-MS of PS-1 after acid hydrolysis (1 м HCl, 100 °C for 5 min) and Bio-Gel P-2 purification (a), CE-MS/MS of m/z 791 (b), CE-MS/MS of m/z 762 (c).
• Fig. 5. Proton spectra of C. jejuni whole cells and isolated polysaccharides. HR-MAS spectrum of C. jejuni whole cells (a) and its isolated polysaccharide (b). HR-MAS spectrum of C. jejuni Cj1428c mutant whole cells (c) and its isolated polysaccharide (d). The anomeric resonances are labeled according to the structures shown in Fig. 6.
• Fig. 6. Structure of polysaccharides PS-1 and PS-2 from C. jejuni, and labeling of the residues and atoms. Residue A is β-D-Ribf, residue B is the amide of α-D-GlcpA with ethanolamine at C-6 for PS-2 and with 2-amino-2-deoxyglycerol at C-6 for PS-1, residue C is β-D-GalfNAc, and residue D is D-glycero-α-L-gluco-heptopyranose.
• Fig. 7. NMR spectrum of PS-2. Spin simulated spectra for residue A (a), residue B (b), residue C (c) and the substituent at C-6 of residue B (d), along with the resolution enhanced proton spectrum (e). In (f) is the HMQC spectrum of the ring protons. In (g) is the HMBC spectrum showing the C=O region for assignment of C-6B and C-7C. The HMBC spectrum in (h) shows the interglycosidic ³J(C,H) correlations. In (i) the proton spectrum, NOESY and HMBC correlations for the NH resonances obtained in 90% H2O are shown.
• Table 4. NMR data for PS-1 and PS-2. The CH3 signal of acetone was set at 2.225 p.p.m. for ¹H and 31.07 p.p.m. for ¹³C. Average error of ± 0.2 p.p.m. for δc, ± 0.01 p.p.m. for δн, and ± 0.5 Hz for J. J-values were obtained from the spin-simulated spectra. Vicinal ³J values are positive and geminal ²J values are negative.
• Fig. 8. MALDI-MS of the methylated PS-2 from the Cj1428c mutant showing the repeat of the polysaccharide.
• Fig. 9. NMR spectra for PS-1. (a) 1D-TOCSY of H-1D resonance at 5.60 p.p.m. with 150 ms mixing time. (b) 1D-NOESY with 200 ms mixing time of the OMe (H-8D) resonance at 3.56 p.p.m. (c) Subsequent 1D-NOESY-TOCSY (H-8D, 200 ms; H-6D, 60 ms) for detection of the H-7D and H-7’D resonances. (d) 1D-TOCSY-NOESY (H-4D, H-8D, 30 ms; H-5D, 200 ms) for the detection of NOEs for H-5D. (e) 1D-TOCSY-NOESY (H-5D, 40 ms; H-4D, 400 ms) for detection of the NOEs for H-4D. (f) 1D-TOCSY-NOESY (H-2D, 40 ms; H-3D, 400 ms) for detection of NOEs for H-3D. (g) HMQC spectrum and assignment of ¹³C resonances. (h) HMBC spectra for location of -NH-CH-(CH2OH)2 substituent on residue B and OMe group on residue D. (i) NOESY spectrum and deter-mination of the sequence from inter-residue NOE correlations.
• Fig. 10. Molecular model of residues BCD for PS 2 with residue D being a D-glycero-L-gluco-heptopyranose in (a) and an L-glycero-D-gluco- heptopyranose in (b). Residue B was modeled as a glucuronic acid. The hydroxyl protons are not shown and the exocyclic chain of residue C is not shown in (a). The absolute configuration of residue D was estab-lished to be D-glycero-L-gluco-heptopyranose, consistent with the 5D-77’D NOEs and the inter-residue 3D-2C, 3D-4C, 5D-2C, 5D-4C NOES.
• Genetic analysis of the capsule polysaccharide
• Fig. 11. Mutagenesis and analysis of capsule mutants of C. jejuni NCTC 11168. (a) Schematic of the capsule gene cluster with gray arrows representing genes mentioned in the text. The kps genes involved in capsule transport and assembly are shown along with possible hypervariable regions within the cluster (due to homopolymeric C tracts or a 21-bp repeat, R) that may be responsible for the structural variability described. The constructs used in this study along with the direction of the KmR cassette insertion are shown. (b) Silver-stained deoxycholate-PAGE of proteinase K whole cell digests of wild-type NCTC 11168 and isogenic mutants. Lane 1: wild-type NCTC 11168; lane 2: kpsM mutant; lane 3: 1428c mutant; lane 4: 1439c mutant. The capsular repeat is indicated by the arrow.
• DISCUSSION
• ACKNOWLEDGMENTS
• REFERENCES