An Unambiguous Assignment and Structural Analysis Using Solution NMR Experiments of O-Antigen from Escherichia coli ATCC23505 (Serotype O9)

Abstract

Bacterial lipopolysaccharides (LPSs) are major surfaceexposed structural components of the outer membrane of Gram-negative bacteria. LPSs consist of lipid A, a core region, and an O-antigen region in many bacteria.1) LPSs play an important role in the pathogenicity of Gram-negative infections. In experimental animals, purified LPSs induce numerous pathophysiological activities that can lead to shock and death, a condition called "Sepsis".2) Most of these biological activities, including the induction of inflammatory cytokines, are expressed by the lipid A moiety, a ubiquitous component of LPSs that is structurally similar and serologically cross-reacting among Enterobacteriaceae, via toll-like receptor 4 (TLR4). [3][4][5] However, numerous recent studies have revealed that LPSs possessing the mannose homopolymer (MHP) as the O-antigen region, such as the LPS from Escherichia coli (E. coli) O9 (O9 LPS), exhibit several characteristic immunological activities differ from those of the well-known endotoxic shock. In addition, we previously described that the mannan moieties of Candida albicans water-soluble fraction, CAWS, from fungi shows similar immunological properties to Gramnegative bacterial O9 LPS.6) These characteristic activities should be exhibited by recognizing mannose residues via lectins, such as mannose-binding lectin (MBL). 7,8) Therefore, analyzing the structure of O-antigens, especially mannoserich regions, is important for understanding some of the biological activities of LPSs.The O-antigen structure of O9 LPS has been studied using methylation analysis, smith degradation, optical rotation and enzymatic methods, [9][10][11] however, a precise assignment by NMR spectroscopy has not been conducted.In general, focusing on the anomeric proton as the starting point for assignment, TOCSY and HSQC-TOCSY experiments are useful for the analysis of individual sugar components in polysaccharides.12) However, in some cases, the assignment can be empirically based due to signals that heavily overlap and small J coupling constants. For the reasons mentioned above, we attempted to apply recently developed pulse sequences, the heteronuclear 2-bond correlation (H2BC) experiments [13][14][15] in order to achieve an unambiguous assignment of the high mannose type polysaccharide, the O-antigen from O9 LPS.In the present study, we report on the structure of the Oantigen of O9 LPS, and completely assign all protons and carbons using 1D-, 2D-NMR spectroscopy including the DQF-COSY, TOCSY, NOESY, HSQC, H2BC, HSQC-TOCSY, and HMBC methods. ExperimentalBacterial Strain and Conditions for Growth E. coli ATCC23505 was obtained from American Type Culture Collection (ATCC). The strain was grown in Luria Bertani (LB) medium.Preparation of LPS O9 LPS was extracted from E. coli ATCC23505 (O9:K9B:H12) by the phenol water method.16) The acetone-dried cells (10 g) cultured in LB medium were suspended in 175 ml of distilled water (preheated at 65°C), and then 175 ml of 90% liquid phenol (preheated at 65°C) was added under vigorous st...

Structure analysis and antimutagenic activity of a novel salt‐soluble polysaccharide from Auricularia polytricha

“…c), in order to separate the neighboring protons in the 13 C dimension. In other words, the overlaid HSQC and HMQC spectra showed the entire intra‐ring assignment, as reported previously . In the spectrum of SSP, the five cross‐peaks (i.e.…”

“…In other words, the overlaid HSQC and HMQC spectra showed the entire intra-ring assignment, as reported previously. 21,22 In the spectrum of SSP, the five crosspeaks (i.e.…”

Structural characterisation and biological activities of a unique type β-d-glucan obtained from Aureobasidium pullulans

“…We further studied these ambiguously assigned protons by using the overlaid spectra of 13 C-edited HSQC and H2BC, in order to separate the neighbouring protons in the 13 C dimension. That is, the overlaid HSQC and H2BC spectra traced out the entire intra-ring assignment, as reported previously [23,27]. Thus, all the protons in AP-FBG were assigned unambiguously.…”

An unambiguous structural elucidation of a 1,3-β-d-glucan obtained from liquid-cultured Grifola frondosa by solution NMR experiments

“…In other words, the overlaid HSQC and H2BC spectra showed the entire intra-ring assignment, as reported previously. 23,24 Thus, all the protons in GRN-LE were assigned unambiguously.…”