Structural Studies of the Lipopolysaccharide Isolated from Plesiomonas shigelloides O22:H3 (CNCTC 90/89)

Plesiomonas shigelloides is a Gram-negative, rod-shaped bacterium which causes foodborne intestinal infections, including gastroenteritis. It is one of the most frequent causes of travellers’ diarrhoea. Lipopolysaccharide (LPS, endotoxin), an important virulence factor of the species, is in most cases characterised by a smooth character, demonstrated by the presence of all regions, such as lipid A, core oligosaccharide, and O-specific polysaccharide, where the latter part determines O-serotype. P. shigelloides LPS is still a poorly characterised virulence factor considering a “translation” of the particular O-serotype into chemical structure. To date, LPS structure has only been elucidated for 15 strains out of 102 O-serotypes. Structures of the new O-specific polysaccharide and core oligosaccharide of P. shigelloides from the Czechoslovak National Collection of Type Cultures CNCTC 90/89 LPS (O22), investigated by chemical analysis, mass spectrometry, and 1H,13C nuclear magnetic resonance (NMR) spectroscopy, have now been reported. The pentasaccharide repeating unit of the O-specific polysaccharide is built of one d-QuipNAc and is rich in four d-GalpNAcAN residues. Moreover, the new core oligosaccharide shares common features of other P. shigelloides endotoxins, i.e., the lack of phosphate groups and the presence of uronic acids.


Introduction
Plesiomonas shigelloides is the only species in the genus Plesiomonas and belongs to the Enterobacteriaceae family. It is usually isolated from fish, crabs, prawns, mussels, and oysters, as well as birds, amphibians, and mammals. The species is a Gram-negative, oxidise-positive, rod-shaped bacterium which causes intestinal infections associated with consumption of seafood, uncooked food, and contaminated water. Foreign travel, particularly to Latin America, the Caribbean, and South and Southeast Asia, is a second major risk factor associated with Plesiomonas infections in humans [1]. Recently, the species was pointed out as an emerging agent of gastroenteritis that varies from a secretory or watery diarrhoea to more severe dysentery-like symptoms [2]. P. shigelloides infection used to be omitted during diagnostics, since it occurred as a concurrent infection with well-established stool pathogens such as Salmonella spp., Campylobacter jejuni, Shigella spp., or Vibrio spp. [2]. Extra-intestinal infections caused by P. shigelloides, most notably, meningitidis in neonates, bacteraemia, sepsis, and septic shock, were uncommonly reported for this, but sepsis and meningitidis are related with the serious course and high fatality rate [3,4].
The pathogenicity of P. shigelloides is poorly understood. Lately, a repeats-in-toxin (RTX) cytolysin, lysophospholipase, a twin-arginine translocation (Tat) system, and the antibacterial type VI secretion effector phospholipase A1 were described [5] among known P. shigelloides virulence factors, such as

P. Shigelloides O22:H3 (Strain CNCTC 90/89) Showed the Prevailing Smooth-Rough Phenotype
The P. shigelloides O22:H3 (strain CNCTC 90/89) LPS was isolated by the phenol/water extraction [31] and purified as previously reported [32]. Both phenol and water phases were collected, however the LPS was obtained only from the water phase (yield 2.86% of dry bacterial mass). The mild acid hydrolysis of the LPS (200 mg) yielded four fractions (1)(2)(3)(4) separated by gel filtration on Bio-Gel P-10, with relatively low content of the O-specific polysaccharide (fraction 1). The fraction 1 yielded only 0.89 mg of the O-specific polysaccharide, substituting a core region, contrary to significantly higher amounts of two oligosaccharide fractions, 2 (18.9 mg) and 3 (42.2 mg). Thus, elution profile suggested a smooth-rough phenotype of the analysed LPS.

Structures of the O-Specific Polysaccharide and Core Oligosaccharide
Sugar and methylation analyses of the N-acetylated fractions combined with determination of the absolute configuration revealed the presence of 2,3,7-trisubstituted l,d-Hepp, 3,4-disubstituted l,d-Hepp, 7-substituted l,d-Hepp, terminal d-GalpN, 6-substituted d-Glcp, terminal d-Glcp, and terminal d-Galp for fraction 3 and an additional 4-substituted d-GalpNAc and 2-acetamido-2,6-dideoxy-d-glucopyranose (d-QuipNAc, observed only in the absolute configuration analysis) for fraction 2, whereas the last residue was also a constituent of the O-specific polysaccharide (fraction 1). All fractions analysed by NMR spectroscopy and mass spectrometry were finally identified as the O-specific polysaccharide linked to the core OS (fraction 1), the core OS fraction substituted by one repeating unit (RU) of the O-specific polysaccharide (fraction 2-OSRU), and the unsubstituted core OS (fraction 3-OS). Fraction 4 consisted of a mixture of low molecular weight oligosaccharides containing 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) released during mild acid hydrolysis of the LPS (data not shown). An initial NMR analysis of the fraction 1 showed the presence of signals characteristic both for the O-specific polysaccharide (QuipNAc, GalpNAcAN) and the core OS (high-intensity signals), thus it was too heterogenic for a complete NMR analysis (data not shown). The fractions 2 and 3 were analysed by one-dimensional (1D) 1 H NMR spectra, as shown in Figure 1.
L,D-Hepp, 7-substituted L,D-Hepp, terminal D-GalpN, 6-substituted D-Glcp, terminal D-Glcp, and terminal D-Galp for fraction 3 and an additional 4-substituted D-GalpNAc and 2-acetamido-2,6dideoxy-D-glucopyranose (D-QuipNAc, observed only in the absolute configuration analysis) for fraction 2, whereas the last residue was also a constituent of the O-specific polysaccharide (fraction 1). All fractions analysed by NMR spectroscopy and mass spectrometry were finally identified as the O-specific polysaccharide linked to the core OS (fraction 1), the core OS fraction substituted by one repeating unit (RU) of the O-specific polysaccharide (fraction 2-OSRU), and the unsubstituted core OS (fraction 3-OS). Fraction 4 consisted of a mixture of low molecular weight oligosaccharides containing 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) released during mild acid hydrolysis of the LPS (data not shown). An initial NMR analysis of the fraction 1 showed the presence of signals characteristic both for the O-specific polysaccharide (QuipNAc, GalpNAcAN) and the core OS (highintensity signals), thus it was too heterogenic for a complete NMR analysis (data not shown). The fractions 2 and 3 were analysed by one-dimensional (1D) 1 H NMR spectra, as shown in Figure 1.  The oligosaccharides OS and OSRU were further analysed using one-and two-dimensional (1D and 2D) 1 H, 13 C-NMR spectroscopy. All the spin systems were assigned using COSY and TOCSY with different mixing times, and HSQC-DEPT, HSQC-TOCSY, and HMBC spectra. Chemical shift values for the OS and OSRU are shown in Table 1 as average values for the OS residues together with the identified inter-residue connectivities. Chemical shift values (Table 1) were compared with previously published NMR data for respective monosaccharides [18,20,26].  Structural analysis indicated that the core OS ( Figure 1a,d and Table 1) is similar to the previously identified core oligosaccharide of P. shigelloides strain CNCTC 78/89 [22]. Both structures were undecasaccharides and differed by only one sugar residue in the outer core region. In the strain 90/89, 6-substituted Glcp (residue G) was identified instead of the 6-substituted GlcpN in the strain 78/89. Since these two core OS structures were very similar, we have focused on the detailed description of sugar residues present in the outer core region only (residues G and H) as a part of the core OS and OSRU fractions. The NMR analysis of the OSRU showed eleven monosaccharides of the core OS (residues A, B, C, D, E, F, G, H, I, J, and L) and an additional five sugars building the RU of the O-specific polysaccharide (residues K, M, N, O, and P) (Figure 1a-c and Table 1) The d configuration of all identified 2-acetamido-2-deoxy-galacturonamides (GalNAcAN) was deduced from the 13 C NMR data using the known regularities in the glycosylation effects [34,35].
Additionally, the chemical shifts of the H-5/C-5 signals of M, N, P, and O were not pH-sensitive, while the chemical shifts of H-5/C-5 of the galacturonic acids (residue F and B present in the core OS) were shifted with the change of pH from 4.0 to 8.0 (data not shown) as a characteristic feature of uronic acids with free carboxyl group. No pH-dependence suggested that residues M, N, P, and O were primary amides of GalpNAcA. A location of the amino groups at position C-6 of GalpNAcAN residues was further supported by HMBC spectra measured in a 9:1 H 2 O:D 2 O mixture that enabled detection of NH protons (data not shown). Resonances for NH protons were assigned by COSY and TOCSY experiments. The signals at 8. 38, 8.34, 8.17, 8.15, 8.10, and 8.07 ppm were assigned as (N2)H of residues K, M, P, N, H, and O, respectively. Furthermore, signals for NH 2 protons at C-6 of residue M (7.99 and 7.71 ppm), residue N (7.86 and 7.51 ppm), residue P (7.83 and 7.48 ppm), and residue O (7.60 and 7.32 ppm) were also identified.
For the OSRU, the residue H with the H-1/C-1 signals at 4.90/98.0 ppm (J C-1,H-1~1 76 Hz) was assigned as the 4-substituted α-d-GalpNAc based on the characteristic signal of C-2 (50.8 ppm) and the downfield shift of the C-4 signal (76.4 ppm). The presence of the 4-substituted GalpNAc in OSRU instead of the unsubstituted GalpNAc in the OS indicated the linkage between the core oligosaccharide and the O-specific polysaccharide. Moreover, the 4-substituted α-d-GalpNAc was also identified by the OS as a place of substitution by the single terminal β-d-QuipNAc residue (K).
Residue G with the H-1/C-1 signals at δ 4.98/100.9 (J C-1, H-1~1 73 Hz) was recognized as the 6-substituted α-d-Glcp based on the large vicinal couplings between all ring protons and the characteristic downfield shift of the C-6 signal (66.0 ppm).
The remaining residues of the outer and inner core region (A, B, C, D, E, F, I, J, and L) have been described in Table 1. The HMBC 1 H-31 P correlation NMR spectrum of the OSRU fraction indicated the lack of phosphate groups. The sequence of sugars in the OSRU was identified by HMBC ( Figure 2, Table 1) and NOESY (Table 1) experiments, indicating the structure presented in Figure 1a 13 C HSQC-DEPT and HMBC spectra of the OSRU. The capital letters refer to carbohydrate residues, as shown in Figure 1 and Table 1. The Arabic numerals refer to protons and carbons in respective residues. Letter C with a prime sign denotes residues →2,3,7)-L-α-D-Hepp-(1→ instead of the →3,7)-L-α-D-Hepp-(1→ (C residue). Residues B' and J' are variants of residues B and J respectively, present in the core OS containing t-Glc (L residue).

Mass Spectrometry Analysis of the Core OS and OSRU Structures and a Glycine Substituent
The elucidated structures of the OS and OSRU isolated from P. shigelloides CNCTC 90/89 LPS were confirmed by mass spectrometry. The MALDI-TOF mass spectrum of the core OS (Figure 3a Figure 1 and Table 1. The Arabic numerals refer to protons and carbons in respective residues. Letter C with a prime sign denotes residues →2,3,7)-l-α-d-Hepp-(1→ instead of the →3,7)-l-α-d-Hepp-(1→ (C residue). Residues B' and J' are variants of residues B and J respectively, present in the core OS containing t-Glc (L residue).

Mass Spectrometry Analysis of the Core OS and OSRU Structures and a Glycine Substituent
The elucidated structures of the OS and OSRU isolated from P. shigelloides CNCTC 90/89 LPS were confirmed by mass spectrometry. The MALDI-TOF mass spectrum of the core OS (Figure 3a Table 2.  The Hex stands for t-Glc, t-Gal, and 6-substituted Glc residues in the structure, as shown in Figure 1a. The Gly stands for glycine residue. The presence of the core OS glycoforms containing a single QuiNAc and the lack of NMR support for fraction 1 called into question this sugar residue as an integral part of the O-specific polysaccharide RU. Thus, a partial hydrolysis of the O-specific polysaccharide combined with MALDI-TOF MS analysis was used to confirm the pentasaccharide structure of the RU (Figure 4 and Table 3). Furthermore, fragments attributed to 2RU and 3RU characterised by similar heterogeneity patterns were also identified.   The MALDI-TOF spectrum showed that the ion at m/z 1074.39 [M-H 2 O+H, Na] + corresponded to four GalNAcAN molecules and one QuiNAc as components of the RU. It was accompanied by ions of higher m/z values (m/z 2125.79 and 3195.20). These ions indicated fragments built of two and three RUs respectively, proving that QuiNAc is an integral component of the O-specific polysaccharide. Furthermore, fragments attributed to 2RU and 3RU characterised by similar heterogeneity patterns were also identified.

Discussion
Lipopolysaccharides of P. shigelloides play an important role in the pathogenesis of infections as pathogen-associated molecular patterns triggering toll-like receptor 4 and components of a cytotoxin complex. These virulence factors of P. shigelloides are still poorly characterized macromolecules. To date, only 15 structures of Plesiomonas LPSs out of 102 O-serotypes have been reported. Although, for some Plesiomonas strains, LPSs were extracted [14,17,20,24,25] both from phenol and water phases during phenol-water extraction, and the analysed LPS of P. shigelloides 90/89 was recovered only from the water phase.
The analysed LPS showed the prevailing smooth-rough phenotype, since only a trace amount of the O-specific polysaccharide fraction was isolated among poly-and oligosaccharides obtained after mild acid hydrolysis of LPS. Due to this limitation and the heterogeneity of fraction 1, only the first RU structure was elucidated as the pentasaccharide composed of the β-d-QuipNAc and four α-d-GalpNAcAN residues (Figure 1b). A rare monosaccharide, GalpNAcAN, has been found previously in the structure of the O-antigen of Francisella novicida (U112) [37], F. tularensis [38], Escherichia coli O35 [39] and O121 [40], Pseudomonas aeruginosa O6 [41], Pseudomonas chlororaphis subsp. aureofaciens UCM B-306 [42], and Shigella dysenteriae type 7 [34]. Amidation of the carboxyl group of hexuronic acids, including d-GalNAcA, has been reported as a feature involved in an adjustment of the optimal charge of the cell surface [42]. The elucidated RU structure constitutes a single biological repeating unit linked to the core OS by the →3)-βd -QuipNAc substituting the outer core →4)-α-d-GalpNAc. Due to the heterogeneity of the fraction 1, anomeric configuration of the QuiNAc could not be established for subsequent O-specific polysaccharide repeating units. The study supported significant variability of P. shigelloides O-specific polysaccharides structures presented by 102 O-serotypes, pointing out the core oligosaccharides as possible targets for developed active and passive immunisation.
Moreover, a new core oligosaccharide composed of an undecasaccharide is reported herein, which represents the new core type among known P. shigelloides LPS. It is characterised by some heterogeneity corresponding to the absence of the terminal β-d-Glcp residue, that is characteristic for core oligosaccharides of P. shigelloides. Minor glycoforms represented the complete core OS with one additional β-d-QuipNAc residue and a nonstoichiometric Gly substitution. The presence of the core OS glycoform substituted by the terminal QuipNAc cannot be explained by the in-source OSRU fragmentation (Figure 3b), thus its origin cannot be explained based on the collected data. This new type of core OS shares features that are typical for the majority of P. shigelloides core regions: an inner region consisting of GalA, Glc, Gal, Kdo, and three Hep residues. Characteristic lack of phosphate groups in the core oligosaccharide structure distinguished it from the known LPS structure of bacteria from the Enterobacteriaceae family. Phosphate groups are very important carriers of negative charges, playing an important role in stabilising the conformation of the entire LPS molecule, and thus the bacterial membrane, by interacting with doubly charged Ca 2+ and Mg 2+ ions. The role of negatively charged residues in the investigated core oligosaccharide is played by GalA instead.
The core OS of P. shigelloides 90/89 is similar to the previously reported core oligosaccharide of P. shigelloides strain CNCTC 78/89 [22], differing only by one sugar residue in the outer core, that is the 6-substituted Glc instead of the 6-substituted GlcN. In the core OS of P. shigelloides O33 [20], both glycoforms, containing 6-substituted GlcN or 6-substituted Glc and O-acetylation of the terminal GalNAc, were reported. The core OS of P. shigelloides 90/89 is also similar to the core OS of serotype O33, that contained the 6-substituted Glc, but was devoid of O-acetyl.
The results presented herein broaden the knowledge about P. shigellodes lipopolysaccharides and may form the basis for further research, to clarify the relationship between the structure and biological activity of endotoxins. Structural analysis is the first step in understanding the unusual physicochemical properties of LPS, which can be used in the future research on the relationships between the structure of LPS and the type of aggregates formed in the aquatic environment, which may have an impact on the pathogenicity of P. shigelloides bacteria.

Bacteria
P. shigelloides serovar O22:H3 (strain CNCTC 90/89) was obtained from the collection of the Institute of Hygiene and Epidemiology, Prague, Czech Republic. Bacteria were grown and harvested as described previously [22].

Lipopolysaccharide and Oligosaccharides OS and OSRU
LPS was extracted from bacterial cells by the hot phenol/water method [31] and purified as previously reported [32]. Both phenol and water phases were collected and dialysed extensively against de-ionised water and purified by ultracentrifugation. The LPS obtained from the water phase (200 mg) was hydrolysed with 1.5% acetic acid at 100 • C for 30 min. The reaction mixture was freeze-thaw cycled, and then incubated at 100 • C for 15 min, and finally centrifuged (40,000× g, 20 min). The core OS (43.8 mg, average yield) was separated from core oligosaccharides substituted with one repeating unit of the O-antigen (19.48 mg, average yield) by size-exclusion chromatography, on a Bio-Gel P-10 column (1.6 × 100 cm) equilibrated with 0.05 M pyridine/acetic acid buffer of pH 5.6. A Knauer differential refractometer was used for monitoring of eluates. After, all fractions were freeze-dried and checked by 1 H NMR spectroscopy and MALDI-TOF mass spectrometry.

Partial Acid Hydrolysis
A sample of the O-specific polysaccharide (fraction 1) was hydrolysed with 48% hydrofluoric acidat −20 • C. Progress of hydrolysis was checked by MALDI-TOF MS. The products obtained by hydrolysis after 24 h were lyophilised for further MALDI-TOF MS analysis.

Analytical Methods
Monosaccharides were examined as their alditol acetates by GC-MS [32]. Partially methylated alditol acetates were analysed according to the method of Ciucanu and Kerek [43] by GC-MS using a Thermo Scientific ITQ system using a ZebronTM ZB-5HT (Thermo Fisher Scientific, Waltham, MA, USA) GC Capillary Column (30 m × 0.25 mm × 0.25 µm), and with temperature program gradient from 150 to 270 • C at 8 • C/min. The absolute configurations of the monosaccharides were determined using (−)-2-butanol for the formation of 2-butyl glycosides [44,45].

NMR Spectroscopy
NMR spectra of OS and OSRU were obtained on a Bruker 600 MHz spectrometer. The sample was first repeatedly exchanged with D 2 O (99%) with intermediate lyophilisation. NMR spectra were obtained for D 2 O solutions or 9:1 D 2 O:H 2 O mixture (for identification of the exchangeable protons of the amide groups) at 25 • C. Internal calibration was applied using acetone (2.225 ppm, 31.05 ppm). The NMR signals were assigned by one-and two-dimensional experiments (COSY, TOCSY, NOESY, HMBC, HSQC-TOCSY, and HSQC-DEPT). The J C-1,H-1 constant values were achieved from a non-decoupled HSQC-DEPT experiment. The TOCSY experiments were recorded with the mixing times 30, 60, and 100 ms. The delay time in the HMBC experiment was set to 60 ms and the mixing time in the NOESY experiment was 200 ms. For observation of phosphate groups, a two-dimensional (2D) 1 H, 31 P HMBC NMR spectrum was recorded. The spectra were acquired and processed with the help of standard Bruker software (Bruker BioSpin GmbH, Rheinstetten, Germany). The 2D spectra were assigned using the NMRFAM-SPARKY program [46].

Mass Spectrometry
The MALDI-TOF MS spectra were obtained on an Ultraflextreme (Bruker, Bremen, Germany) instrument in a positive ion mode. Samples were dissolved in water (1 mg/mL). The 2,5-Dihydroxybenzoic acid (10 mg/mL in 1:1 AcN/0.2 M citric acid [v/v]) was used as a matrix for analyses. For MALDI TOF/TOF MS analysis using the LIFT mode, the ion source voltage was set at 7.5 kV with a precursor ion mass window of 4 Da. The precursor ion was accelerated at 19.0 kV in the LIFT cell. The reflector voltage was set at 29.5 kV. External calibration was applied using the Peptide Calibration Standard II (Bruker Daltonics, Germany). Ions were interpreted according to the nomenclature of Domon and Costello [36].

Conclusions
Structural studies of P. shigelloides 90/89 lipopolysaccharide have been presented. This study reported the structure of the pentasaccharide repeating unit constituting the O-specific polysaccharide of P. shigelloides CNCTC 90/89 (O22) LPS. It contains rare monosaccharides such as β-d-QuipNAc and four GalpNAcAN residues. Moreover, a new type of the core oligosaccharide composed of an undecasaccharide was presented, that shares common P. shigelloides features: the lack of phosphate groups and the presence of uronic acids.