Structure of the lipopolysaccharide isolated from the novel species Uruburuella suis

Carbohydrate Research 357 (2012) 75–82

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Structure of the lipopolysaccharide isolated from the novel species Uruburuella suis Alba Silipo a,⇑, Luisa Sturiale b, Cristina De Castro a, , Rosa Lanzetta a, , Michelangelo Parrilli a, , Domenico Garozzo b, Antonio Molinaro a,  a b

Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Napoli, Italy Istituto di Chimica e Tecnologia dei Polimeri—ICTP, CNR, Via P. Gaifami 18, 95126 Catania, Italy

a r t i c l e

i n f o

Article history: Received 28 March 2012 Received in revised form 10 May 2012 Accepted 13 May 2012 Available online 22 May 2012 Keywords: Uruburuella suis Neisseriaceae Lipooligosaccharide NMR spectroscopy MS spectrometry

a b s t r a c t Uruburuella suis is a novel species isolated from lungs and heart of pigs with pneumonia and pericarditis. Phenotypic and phylogenetic evidences showed that it represented a hitherto unknown subline within the family Neisseriaceae. In the present work we defined the whole structure of the LPS isolated from Uruburuella suis. The structural determination, which was achieved by chemical, spectroscopic and spectrometric approaches, indicates a novel rough type lipopolysaccharide rich in negatively charged groups in the lipid A-inner core region. The elucidation of the structural features of the LPS from Uruburuella suis is a first step toward the comprehension of the characteristics of the cell envelope in such new and interesting microorganisms. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Lipopolysaccharides1,2 are heat-stable complex amphiphilic macromolecules indispensable for the growth and the survival of Gram-negative bacteria, for the correct assembly of the external membrane and the right positioning of porins. The very low fluidity of the highly ordered structure of the LPS monolayer contributes to form a defensive barrier which helps the bacteria to resist antimicrobial compounds and environmental stresses. LPS are also called endotoxins because they are cell-bound and, once released, can play a key role in the pathogenesis of Gram-negative infections in both plant and animal hosts,3 in which they trigger the activation of both the innate and adaptative immune systems. Lipopolysaccharides are composed of three distinct domains: a hydrophilic polysaccharide, the O-specific chain, covalently linked to an oligosaccharide named core which in turn is connected to the glycolipid portion, the lipid A. In the core oligosaccharide, inner and outer regions are usually distinguished: the inner core, proximal to the lipid A, consists of peculiar monose residues like heptoses and an acidic sugar termed Kdo (3-deoxy-D-manno-oct-2ulosonic acid) which is a marker of LPS together with 3-OH fatty acids. The inner core region can be decorated with negatively charged substituents, often present in not stoichiometric amounts, ⇑ Corresponding author. Fax: +39 (0)81 674393.  

E-mail address: [email protected] (A. Silipo). Fax: +39 (0)81 674393.

0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carres.2012.05.010

like phosphate (P), pyrophosphate (PP), pyrophosphorylethanolamine (PPEtN), phospho-arabinosamine (PAra4N), and uronic acids (as GalpA). The outer core region is more variable and is usually composed by hexoses. Bacteria can also biosynthesize LPS without O-specific chain and in this case LPS is defined of R-type (rough type) or lipooligosaccharide (LOS) and confers a rough appearance to the colony morphology. The lipid A4,5 is a vital component of the microbial metabolism and has crucial functions of protection and defense, acting as a strong stimulator of the innate immune system. Lipid A is the less variable portion of LPS and possesses a rather conservative structure usually consisting of a b-(1?6) disaccharide backbone usually consisting of glucosamine, phosphorylated at positions 1 and 40 and acylated with 3-hydroxy fatty acids at positions 2 and 3 of both GlcNs via amide and ester linkage. These acyl chains, defined primarily because they are directly linked to the sugar backbone, are further acylated to their hydroxy groups by secondary acyl moieties. Kdo, or derivative of this sugar, is linked to the vicinal glucosamine of lipid A backbone at the primary function at position 60 . Uruburuella suis has been isolated from lungs and heart of pigs with pneumonia and pericarditis.6 Phenotypic and phylogenetic evidences showed that the new unidentified strain represented a hitherto unknown subline within the family Neisseriaceae, a family of Gram-negative, parasitic bacteria including several important human pathogens. The understanding of the molecular mechanisms involved in the inflammatory process requires the knowledge of the structure of the bacteria derived inflammatory

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molecules, and among these LPS plays a key role. No structural data are so far available for LPS structure for such a new species. Moreover, the LPS structural characterization is the first step to the design of antimicrobial compounds and therapeutic strategies. These considerations, together with the intrinsic interest derived from its nature of clinical isolate belonging to a new species and genus within the Neisseriaceae family, prompted us to start the isolation of the pure LPS molecules to determine the whole structure. 2. Results 2.1. Isolation and compositional analysis of LPS isolated from Uruburuella suis LPS fraction was extracted from and analyzed by SDS electrophoresis. The isolated and purified LPS was found to have the typical run to the bottom of the gel as R-type LPS (LOS). The compositional analysis of the isolated LOS revealed the presence of L-Rha, D-GlcN, D-Glc, L-glycero-D-manno-heptose (L,DHep), 3-deoxy-D-manno-oct-2-ulopyranosonic acid (D-Kdo). Methylation analysis revealed the presence of terminal Rha, 2,3-substituted Rha, 3,4-substituted Hep, 2,3,7-substituted Hep, terminal Hep, 4-substituted Glc, terminal Glc, terminal GlcN, 6-substituted GlcN, 4,5-substituted Kdo, terminal Kdo. Furthermore, at high retention time a heptose disaccharide Hep-(1?7)-Hep was found (Fig. 1). Fatty acids analysis revealed the presence of (R)-3-hydroxytetradecanoic (C14:0(3-OH)) in amide linkage and (R)-3-hydroxydodecanoic (C12:0(3-OH)) acid and dodecanoic acid (C12:0) in ester linkage. In order to achieve the primary structure of the core portion of Uruburuella suis, LOS was fully de-acylated to obtain an oligosaccharide fraction OS that, by gel-permeation chromatography, was further purified. The compositional analysis of the isolated OS confirmed the presence of the sugar residues found in the intact LOS fraction.

2.2. Structural characterization of OS product The 1H NMR spectrum of OS is shown in Figure 2. A combination of homo- and heteronuclear 2D NMR experiments (DQF-COSY, TOCSY, ROESY, 1H–31P, 1H–13C HSQC and 1H–13C HMBC) was executed in order to assign all the spin systems of OS and the monosaccharide sequence. In the anomeric region of the 1H NMR spectrum (Fig. 2) 11 anomeric signals were identified (A–M, Table 1, Fig. 2a); furthermore, signals resonating at 1.80/2.04 and 1.69/ 2.09 ppm were identified as the H-3 methylene protons of two Kdo residues, K1 and K2. The anomeric configuration of each monosaccharide unit was assigned on the basis of the 3JH1,H2 coupling constants obtained by the DQF-COSY and the intra-residual NOE contacts observable in the ROESY spectrum, whereas the values of the vicinal 3JH,H coupling constants allowed the identification of the relative configuration of each residue. The proton resonances of all spin systems were obtained by DQF-COSY and TOCSY spectra and were used to assign the carbon resonances in the HSQC spectrum. Residues A and B were both identified as composing the disaccharide backbone of the lipid A. The gluco configuration was indicated by high 3JH,H ring proton values (all around 8–10 Hz). In details, residue A was plainly identified as the a-GlcN of lipid A skeleton probed by the correlation of H-2 with a nitrogen bearing carbon signal at 55.2 ppm, testifying its nature of amino-sugar whose C-6 carbon signal, downfield shifted at 69.5 ppm, was proof of glycosyl substitution at this position (Table 1). The high-field shift of proton resonances of H-2 was indicative of the absence of acylation at these positions. The chemical shifts values of H-1/C1, the low 3JH1,H2 coupling constant and the intra-residual NOE contact of H-1 with H-2 were all in agreement with a-anomeric configuration of A. Analogously, spin system B was identified as GlcN; the HSQC spectrum showed the correlation of H-2 with nitrogen bearing carbon signals; the high-field shift of proton resonances of H-2 was indicative of the absence of acylation. The chemical shifts of H-1/C-1, the 3JH1,H2 coupling constant (8.0 Hz)

CHDOAc OAc

MeO 101

OMe

OMe O

MeO

4600

OAc

CH2OMe

OAc

MeO OMe

4200 43

O

3800

392

CH2

88

Abundance

3400 3000 2600 2200 1800

45

89 75

1400 1000

199

59 117

600

157 170 138

200

231 60

100

140

180

220

262 263 260

290 300

340

380

m/z

Figure 1. Electron impact mass spectrum of the terminal Hep-(1?7)-Hep disaccharide isolated via methylation analysis; the structure and the main fragments are shown in the inset.

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A. Silipo et al. / Carbohydrate Research 357 (2012) 75–82

a

H I L G

C A F

B

M E D

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

ppm

b

1.0

1.5

2.0

2.5

3.0

I1-G4 M1-F7

3.5

C1-I4 C1-I3

4.0 H1-F2 I2-L1

B1-A6

4.5

D1-E6

I1-G6 I1-G4

L1-I2 D1-E4

F1-E3

5.0

4.5

5.0

M1-F7

E1-K15

H1-F1

G1-F3

4.0

3.5

3.0

2.5

ppm

1

Figure 2. (a) H NMR spectrum of product OS. Anomeric signals of spin system are designated as in Table 1. (b) Zoom of the TOCSY (gray) and T-ROESY (black) spectra in which the key inter-residual NOE contacts are indicated.

and the intra-residual NOE contact of H-1 with H-3 and H-5 were all in agreement with a b-anomeric configuration of B. Residue C was recognized as b-GlcN as indicated by large ring coupling constants 3JH,H and by intra-residual NOE contact of H-1 with H-3 and H-5. The amino function was inferred by the correlation of H-2 C with a nitrogen bearing carbon signal at 57.0 ppm. Because of the absence of the anomeric proton signal, the spin system of Kdo residues K1 and K2 were both assigned starting from the diastereotopic H-3methylene protons (H-3ax and H-3eq, respectively, Fig. 2). The a-configuration was attributed by the chemical

shift values of H-3 and by the values of 3JH7, H8a and 3JH7, H8b coupling constants.7,8 Spin systems E, F, and M (Table 1, Fig. 2), were all identified as a-heptose residues, as indicated by their 3JH1,H2 and 3JH2,H3 coupling constants (below 3 Hz) and by the intra-residual NOE of H1 with H-2. The 13C chemical shift value of C-6 of these heptose residues (all below 70 ppm) allowed us to identify them as L-glycero-D-manno heptose, in accordance with the chemical analysis. Spin systems D, G, and L (Table 1, Fig. 2) were identified as glucose residues, as indicated by their large ring 3JH,H coupling

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A. Silipo et al. / Carbohydrate Research 357 (2012) 75–82

Table 1 1 H, 13C and

31

P (in bold) NMR chemical shifts of the oligosaccharide derived from alkaline treatment of the LOS from Uruburuella suis gen. nov., sp. nov. Chemical shift d (1H/13C)

Unit

1

2

3

4

5

6

A 6-a-GlcN

5.28 94.4 1.51 4.30 102.5 4.38 105.6 4.97 101.6 5.10 99.3

2.59 55.2

3.52 72.2

3.28 69.8

3.96 71.7

4.20/3.60 69.5

2.57 56.1 2.45 57.0 3.01 74.2 3.97 70.4

3.45 76.0 3.26 75.5 3.49 75.8 4.13 74.0

3.34 69.7 3.33 69.6 3.16 70.6 4.18 73.9

3.54 74.5 3.25 75.7 3.49 76.8 ND ND

5.23 98.1 5.30 100.2 4.72 97.3 4.85 98.9 4.88 97.9 4.85 101.2

4.07 73.8 3.46 71.9 3.90 70.2 4.09 76.6 3.41 71.4 3.91 70.0

ND

ND

ND

ND

ND

ND

3.97 76.5 3.64 71.5 3.79 69.7 3.70 79.6 3.64 72.5 3.7 70.4 1.80/2.04 34.3 1.69/2.09 34.8

3.53 77.8 3.30 72.3 3.54 71.3 3.44 68.9 3.56 71.5 4.10 70.3 4.00 71.7

3.72 70.9 3.92 68.8 3.98 69.2 4.00 71.5 ND ND 4.08 76.7 3.93 65.8

3.33/3.59 63.0 3.74/3.71 60.6 3.70/3.78 61.6 4.43 72.5 1.17 3.92 68.7 3.86/3.68 59.8 1.23 18.0 1.17 16.2 3.74 59.8 4.00 69.2 3.96 71.5 3.51 72.2

B 6-b-GlcN C t-b-GlcN D t-b-Glc E 3,4-a-Hep F 2,3,7-a-Hep G t-a-Glc H t-a-Rha I 2,3-a-Rha L t-a-Glc M t-a-Hep K1 4,5-a-Kdo K2 t-a-Kdo

7

8

3.93/3.86 64.8 3.66/3.62 70.7

3.89 63.1 3.90 69.9 3.91 69.9

3.88/3.63 63.1 3.77 63.5

residues G, O-2, O-3 and O-7 of F, O-2 and O-3 of I, O-4 and O-5 of K1, whereas residues C, D, H, L, M, and K2 were non-reducing terminal sugars, in full agreement with the methylation analysis data. The inter-residual NOE contacts (Fig. 2) and the long range correlations present in the HMBC spectrum yielded the oligosaccharide sequence. The linkage between glucosamine residues A and B of the lipid A backbone was validated by the inter-residual NOE contact of H-1 B with H-6a,b A. The weak down-field shift of C-6 B (63.0 ppm) was in agreement with the a-(2?6) ketosidic linkage of Kdo K1 with residue B of b-GlcN. The linkage of heptose E to O-5 of Kdo K1 was proven by the downfield shifts of the signal for its C-5 (a-Kdo, 76.7 ppm) and the NOE connectivity between H-1 of heptose and H-5 of Kdo. Furthermore, the K2-(2?4)-K1 linkage between the two Kdo residues

constants. The strong intra-residue NOE contacts of H-1 with H-3 and H-5 together with the 3JH1,H2 coupling constant (7 Hz) was diagnostic of b-configuration of residue D whereas the intra-residue NOE contact of H-1 with H-2 and the 3JH1,H2 coupling constant (3 Hz) were indicative of a-anomeric configuration of residue G and L. Residues H and I were recognized as a-rhamnose residues. Actually, in TOCSY spectrum scalar correlations of the ring protons with methyl signals in the shielded region at, respectively, 1.23 and 1.17 ppm were visible. The manno configuration was established from the 3JH1,H2 and 3JH2,H3 small values, the a-configuration was assigned by the intra-residual NOE contact of H-1 with H-2 and upfield chemical shift of its H-5 and C-5. The down-field shift of carbon resonances identified the glycosylated positions: O-6 of residues A and B, O-3 and O-4 of E, O-4 of

ppm -2.4

-2.0

-1.6

A1

A2

-1.2

E6 -0.8

-0.4

0.0 5.5

5.0

4.5

Figure 3.

4.0 31

1

3.5

P– H HSQC spectrum of OS.

3.0

2.5

ppm

A. Silipo et al. / Carbohydrate Research 357 (2012) 75–82

79

was proven by the presence of the characteristic inter-residue NOE contact between H3eq of Kdo moiety K1 and H-6 of Kdo K29 (Fig. 2b). Residue E was substituted at O-3 and O-4. The NOE contact (Fig. 2b) of H-4 and H-6 E with H-1 D evidenced that O-4 of a-heptose E was glycosylated by the b-glucose D. Residue E was further substituted at O-3 by residue F, according to the NOE (Fig. 2b) of H-3 and H-4 E with H-1 F. Residue F, identified as the 2,3,7-trisubstituted a-heptose, was substituted at position 2 by the terminal aRhamnose H as indicated by the NOE contacts of H1 H with H1 and H2 F and ( Fig. 2b). The NOE correlation of H-3 F with H-1 G (Fig. 2) gave evidence of substitution of residue F at O-3 by a-glucose G. Furthermore, residue F was glycosylated at O-7 by the heptose residue M, as demonstrated by the NOE contact of H-7 F with H-1 M. Residue G was substituted at O-4 by a-rhamnose I as indicated by the related NOE contact (Fig. 2). Furthermore, a-rhamnose was glycosylated at position 2 by the terminal a-glucose L as proven by the NOE contact of H1 L with H2 I, and at position 3 by the terminal b-glucosamine C, as testified by the NOE contact of H-3 I with H-1 C (4.38 ppm). The HMBC spectrum confirmed the assigned structure, since it contained all the required long-range correlations to demonstrate the proximity of the residues. The 31P–1H HSQC spectrum (Fig. 3) showed two cross peaks, whose 31P chemical shifts were in accordance with the presence of two phosphate groups. The 31P signal resonating at 1.51 ppm correlated with the proton signal at 5.28 identified as H-1 of aGlcN A composing the lipid A backbone. The second phosphate group was linked at O-6 of a-Hep E, as evident by the cross peak between the 31P signal at 1.17 ppm and H-6 E at 4.43 ppm. Thus, all data were in agreement to indicate the following oligosaccharide structure:

with those previously emerged from structural characterization of the OS species, produced by the strong alkaline treatment required for the complete LOS deacylation. Such treatment evidently caused the cleavage of the acetamido and phosphoethanolamine groups. We have also tried to characterize the product after hydrazinolysis, which leaves amide and pyrophosphate unaffected, but unfortunately, the high heterogeneity of the hydrazine treated LOS prevented any attempt to assign by NMR a location to both groups. By homology with several LPS core oligosaccharides, we can tentatively assign the acetyl group at the GlcN residue whereas the PEtN group could only be located on the O-6 of heptose residue, which is the only phosphorylated position of the core oligosaccharide. Additional ions, also found in the low mass-range of the same mass spectrum, were assigned to the lipid A moieties. The main lipid A species, at m/z 1792.8, was consistent with a hexa-acylated disaccharide backbone carrying two amide linked 14:0(3-OH), two 12:0(3-OH), two C12:0, both ester linked, and three phosphate groups, located by combining the info derived from either MS and above NMR data (see below). The main LOS species at m/z 3913.0, showed in Figure 4a, derived by a combination of the core OS ion peak at m/z 2120.2 and the lipid A species at m/z 1792.8. The position of the secondary fatty acids on the GlcN backbone was thereafter established by MALDI analysis of the lipid A isolated by acetate buffer hydrolysis from the intact LOS and subsequent treatment with ammonium hydroxide.11 This approach allowed the location of the amide-bound acyloxyacyl moieties left unaltered by this mild hydrolysis. The MALDI spectrum registered in negative ion mode of the ammonium hydroxide product (Fig. 4b) contained a main ion peak at m/z 1236.2 related to a tetra-acylated monophosphorylated lipid A species with two amide-linked 14:0(3-OH) residues, both substituted by a C12:0.

A MALDI mass spectrum of the oligosaccharide mixture OS fully confirmed the above structural hypotheses (not shown).

In conclusion, lipid A was mainly constituted by a symmetrically substituted hexa-acylated species carrying two 12:0 as secondary fatty acids, both linked to the primary amide linked C14:0(3-OH), and a pyrophosphate group on the non reducing GlcN. In summary, the complete structure of the lipo-oligosaccharide from the virulent strain of Uruburuella suis carried out by chemical analyses, MALDI MS and 2D NMR spectroscopy, is reported in Figure 5.

2.3. Structural characterization by MALDI mass spectrometry of the intact LOS and lipid A In order to confirm the structure of the lipid A-core saccharide region and gain more information on the non carbohydrate substituents, the intact LOS and the lipid A were analyzed by MALDI MS. The negative ion MALDI mass spectrum of intact LOS ( Fig. 4a) showed at higher molecular masses (between 3000 and 4000 Da) a series of [MH] ions related to the native LOS mixture. It was made up of a main LOS ion at m/z 3913.0 as well as of species differing from this principal component as they lack terminal monosaccharide unit(s), namely Hep, Hex, and HexNAc, and/or a phosphate group. Besides these peaks, ion fragments arising from the very labile glycoside bond cleavage between Kdo and the lipid A moiety,10 were also present at lower mass-ranges. Such LOS fragmentation (b-elimination) yields both oligosaccharide(s) as B-type ions and lipid A as Y-type ions. In the present spectrum (Fig. 4a) the ion fragment at m/z 2120.2 corresponded to the intact core oligosaccharide composed of three Hep, two Kdo, two dHex, three Hex, one HexN, an acetyl group and an additional phosphoethanolamine group (PEtN = 123 Da). This finding was perfectly in line

3. Conclusions Uruburuella suis is a novel microbial species isolated from lungs and heart of pigs with pneumonia and pericarditis6 and is also characterized as a subline within the family Neisseriaceae, a family of Gram-negative, parasitic bacteria including several important pathogens of humans. This is the first report on the primary structure of the R-LPS from U. suis, which turned out to possess a novel oligosaccharide structure. The oligosaccharide core region is, in wild type R-LPS, the most external part of the molecule, thus plays a key role in the bacterial cell interaction with external environment. From the chemical point of view, the R-LPS can be built of several monosaccharides which can be arranged giving either a linear or a branched architecture. In U. suis, as in almost the totality of Gram-negative

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A. Silipo et al. / Carbohydrate Research 357 (2012) 75–82

a

LOS OS:3Hep 2Kdo 2dHex 3Hex 1HexNAc 1PEtN Δ m/z 2120.2

3913.0

20000

Hexa-acylated Lipid A Δ m/z 1792.8 15000

Hexa-acylated Lipid A

Hex+HexNAc

Counts

1792.8

10000

3720.7

Hep 3833.0 1712.9

P 5000

Hep

OS

1765.07 1900.4 2120.2 1614.4 2076.3

P

3548.0 3355.8 3641.1

Hep

0 1500

2000

2500

3000 Mass (m/z)

3500

b

4000

4500

OH O R'O HO

O

O HO HO NH

tetra-acylated monophosphorylated lipid A

H 2O

O

O

NH OR

O

1236.2

O

O

O

10000 R=H, R'=P or R=P, R'=H

1434.4

Counts

8000

C12:0 (3-OH)

6000

14 12

14

12

1252.2

4000

C12:0 (3-OH)

1633.7

2000

0 1100

1200

1300

1400 Mass (m/z)

1500

1600

1700

Figure 4. (a) Negative ion MALDI TOF mass spectrum of the intact LOS from LOS from Uruburuella suis obtained in linear mode. (b) Negative MALDI TOF mass spectrum, acquired in reflector mode; of lipid A after treatments with ammonium hydroxide.

bacteria, the linkage of core oligosaccharide to lipid A occurs between an a-Kdo residue and the 60 -hydroxymethylene of the GlcN residue; the first Kdo unit bears at its O-5 position a heptose trisaccharide fragment: a-L,D-Hepp-(1-7)-a-L,D-Hepp-(1-3)-a-L,D-Hepp. Kdo in turn carries another negative charged substituent at its O4 position, a further a-Kdo unit. The first heptose residue of the inner core region at position O-6 is substituted by a phosphate, likely derivatised as PEtN. Neisseria meningitidis LPS possesses variable oligosaccharide chains12 lacking O-chain portion, the conserved inner core consisting of a heptose disaccharide, in which the distal heptose unit (Hep II) is generally substituted by GlcNAc and can carry PEtn at the 3 or 6 position or not at all. The proximal heptose residue (Hep I) can provide a point of attachment for the outer-core and is substituted at the 4 position by glucose residue, the starting sugar of long, hexose rich outer core region. Sialylation of the LPS can occurs at terminal galactopyranosyl residue, this last being an example of molecular mimicry. No deoxysugar was found. Despite the

phylogenetic relationship with Neisseriaceae, no strong similarities with the core structures could be found. As for lipid A, it showed higher similarities with Neisseriaceae, since both are constituted by symmetrically hexa-acylated species, carrying C14:0 (3-OH) as primary amide linked acyl chains and C12:0 (3-OH) and C12:O as ester linked primary and secondary fatty acids. Lipid A of Neisseria meningitidis also carries two PPEtN groups at both positions of the disaccharide backbone.

4. Experimental 4.1. LPS extraction The freeze dried cells were extracted three times with a mixture of aq 90% phenol/chloroform/petroleum ether (2:5:8 v/v/v) as described.13 After removal of organic solvents under vacuum, the LOS fraction was precipitated from concentrated phenol solution

A. Silipo et al. / Carbohydrate Research 357 (2012) 75–82

4.3. Isolation of lipid A

OH HO

O

L

HO OH

O O

C

Free lipid A was obtained by hydrolysis of the LOS (with 10 mM sodium acetate buffer pH 4.4, (100 °C, 3 h). The solution was extracted three times with CHCl3/MeOH/H2O (100:100:30 v/v/v) and centrifuged (4 °C, 5000g, 15 min). The organic phase contained the lipid A and the water phase contained the core oligosaccharide.

OH

HO HO

81

O

HO NHAc O

I

4.4. General and analytical methods OH

HO HO

O OH

O

G

HO

HO OH

O

H

O

OH

O

F

HO

OH O

O

E

OH

OH OH O

HO

OH O

P

O

D

OH

NH2

OH COOH

HOOC

K1 O

O O

O

HOH2C

O O O

P

OH

HO O O

K2

HO

O

O

HO

O

OH O M

OH

OH OH OH

CH2OH

B

A

O

P O

O

O

HO

O

O

NH

NH O

O

O

O

HO

P

OH

O

4.5. NMR spectroscopy

O

O

OH OH

Determination of sugars residues and of their absolute configuration, GLC and GLC–MS were all carried out as described.15–18 Monosaccharides were identified as acetylated O-methyl glycosides derivatives. After methanolysis (2 M HCl/MeOH, 85°, 24 h) and acetylation with acetic anhydride in pyridine (85°, 30 min) the sample was analyzed by GLC–MS. Linkage analysis was carried out by methylation of the complete core region as described. The sample was hydrolyzed with 4 M trifluoroacetic acid (100 °C, 4 h), carbonyl-reduced with NaBD4, carboxy-methylated, carboxyl-reduced, acetylated and analyzed by GLC–MS. Total fatty acid content was obtained by acid hydrolysis. LOS was first treated with HCl 4 M (4 h, 100 °C) and then with NaOH 5 M (30 min, 100 °C). Fatty acids were then extracted in CHCl3, methylated with diazomethane, and analyzed by GLC–MS. The ester bound fatty acids were selectively released by base-catalyzed hydrolysis with NaOH 0.5 M/MeOH (1:1 v/v, 85°, 2 h), then the product was acidified, extracted in CHCl3, methylated with diazomethane, and analyzed by GLC–MS.

OH

O

O

O

For structural assignments of OS, 1D and 2D 1H NMR spectra were recorded in 0.5 mL of D2O at 300 K, pD 7 (uncorrected value) on Bruker 600 DRX equipped with a cryo probe. Spectra were calibrated with internal acetone [dH 2.225, dC 31.45]. 31P NMR experiments were carried out using a Bruker DRX-400 spectrometer, aqueous 85% phosphoric acid was used as external reference (0.00 ppm). Rotating frame Overhauser enhancement spectroscopy (ROESY), total correlation spectroscopy (TOCSY), double quantumfiltered phase-sensitive correlation spectroscopy (DQF-COSY), heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments were performed and processed as described.19,20

12

12

4.6. MALDI TOF mass spectrometry

14 12

14

12

Figure 5. Structure of the LOS from Uruburuella suis. Positions of pyrophosphate and phosphoethanolamine groups are tentative.

with water, the precipitate was washed with aqueous 80% phenol and then three times with cold acetone and then lyophilized. The LOS fractions were analyzed by SDS–polyacrylamide gel electrophoresis on 16% gels, which were stained with silver nitrate.14 4.2. Isolation of oligosaccharide OS For isolation of OS, LOS (40 mg) was with anhydrous hydrazine (2 ml), stirred at 37 °C for 90 min, cooled, poured into ice-cold acetone (20 ml), and allowed to precipitate. The precipitate was then centrifuged (3000g, 30 min), washed twice with ice-cold acetone, dried, dissolved in water and lyophilized. OS product was de-Nacylated with 4 M KOH as described. Salts were removed by gel permeation chromatography with Sephadex G-10 (Pharmacia) column (50  1.5 cm) to yield the resulting oligosaccharide OS.15

MALDI-TOF mass spectra were recorded in the negative polarity on a Perseptive (Framingham, MA, USA) Voyager STR equipped with delayed extraction technology. Ions formed by a pulsed UV laser beam (nitrogen laser, k = 337 nm) were accelerated by 24 kV. The mass spectra reported are the result of 256 laser shots. OS, LOS and lipid A sample preparation: The oligosaccharide mixture was analyzed in linear mode using dihydroxybenzoic acid (DHB) 50 mg/mL in TFA 0.1%- ACN 80/20 as matrix solution. R-type LPS MALDI preparation was performed as recently reported in detail.10 MALDI preparation of lipid A was performed as described:21 samples were dissolved in CHCl3/CH3OH (1:1 v/v), whereas matrix solution was prepared by dissolving 2,4,6-trihydroxyacetophenone (THAP) in CH3OH/0.1% TFA/CAN (7:2:1). A sample/matrix solution mixture (1:1 v/v) was deposited (1 lL) onto the MALDI plate and left to dry at room temperature. Lipid A MALDI mass spectra were acquired both in linear and in reflector mode. References 1. Alexander, C.; Rietschel, E. T. J. Endotoxin Res. 2001, 7, 167–202.

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