Structure of the capsule and lipopolysaccharide O-antigen from the channel catfish pathogen, Aeromonas hydrophila

Carbohydrate Research 486 (2019) 107858

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Structure of the capsule and lipopolysaccharide O-antigen from the channel catfish pathogen, Aeromonas hydrophila

T

Christian Heissa,∗, Zhirui Wanga, Charles M. Thurlowb, Mohammad J. Hossainb,1, Dawei Sunb, Mark R. Lilesb, Mark A. Saperc, Parastoo Azadia a

Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA, 30602, USA Department of Biological Sciences, Auburn University, 120 West Samford Avenue, Auburn, AL, 36849, USA c Department of Biological Chemistry, University of Michigan, 1150 W. Medical Center Drive, Ann Arbor, MI, 48109, USA b

ARTICLE INFO

ABSTRACT

Keywords: Aeromonas hydrophila Fish pathogen O-antigen capsule Lipopolysaccharide NMR Polysaccharide structure

A hypervirulent A. hydrophila (vAh) pathotype has been identified as the etiologic agent responsible for disease outbreaks in farmed carp species and channel catfish (Ictalurus punctatus) in China and the Southeastern United States, respectively. The possible route of infection has previously been unknown; however, virulence is believed to be multifactorial, involving the production/secretion of several virulence factors, including a high molecular weight group 4 capsular polysaccharide. Here we present chemical structural evidence of a novel capsule- and LPS-associated O-antigen found present in vAh isolated during these disease outbreaks. In this study, the chemical structure of the vAh O-antigen was determined by chemical analysis, Smith degradation, mass spectrometry, and 2D proton and carbon nuclear magnetic resonance (NMR) spectroscopy and found to be unique among described bacterial O-antigens. The O-antigen consists of hexasaccharide repeating units featuring a 4)-αL-Fucp-(1–3)-β-D-GlcpNAc-(1–4)-α-L-Fucp-(1–4)-β-D-Glcp-(1- backbone, substituted with single residue side chains of α-D-Glcp and α-D-Quip3NAc linked to O-3 of the two fucose residues. The polysaccharide is partially Oacetylated on O-6 of the 4-substituted β-Glcp residue.

1. Introduction The bacterial pathogen A. hydrophila is found widely in fresh, brackish, and seawater [1] and is the causative agent of motile Aeromonas septicemia (MAS) in channel catfish [2,3]. The hyper-virulent A. hydrophila (vAh) isolates affiliated with sequence type 251 (ST251) are responsible for epidemic MAS outbreaks in channel catfish and are highly virulent to channel catfish as compared to typical opportunistic A. hydrophila isolates from diseased fish [4]. Historically, A. hydrophila has been considered as an opportunistic pathogen in stressed or immunocompromised channel catfish. However, vAh strains apparently act as a primary pathogen in healthy channel catfish, with high mortality observed in mature, market-sized fish [5]. Comparative genomics [2] and phylogenetic [6] analyses of vAh strains isolated from Asian carp and channel catfish from China and the US, respectively, showed that they shared a recent common ancestor, with MAS outbreaks occurring in China in the late 1980s whereas the first documented occurrence in the US was a case in 2004 in Mississippi [6,7]. Since that time, MAS due to vAh strains has spread across the Southeastern United

States and has led to the mortality of over 20 million pounds of channel catfish in the State of Alabama alone [8]. vAh isolates are widespread in the US and China and pose a continuous threat for commercial fish farming in these countries. There have been a large number of predicted virulence factors seemingly acquired by horizontal transfer in vAh strains [2,5,7,8]; however, their roles in virulence have not been studied experimentally [2]. One putative virulence factor is the O-antigen capsule [9], as the capsular polysaccharides of other bacterial pathogens have been shown to play significant roles in bacterial pathogenesis by aiding host colonization [10], invasion [11] and/or preventing phagocytosis by host macrophages [12]. In Escherichia coli, capsules are classified in four different groups based on their assembly and secretion to the outer membrane [13]. The group 4 capsule (G4C) consists of oligosaccharide repeats comparable to that of the LPS-associated O-antigen, hence the name “O-antigen capsule” [13,14]. The G4C polysaccharides have been previously characterized in E. coli [14], Shigella sonnei [15], and Salmonella [16]. In S. sonnei the G4C was found to be responsible for virulence modulation in a rabbit infection model [15]. Given the

Corresponding author. E-mail address: [email protected] (C. Heiss). 1 Present address: The Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD. ∗

https://doi.org/10.1016/j.carres.2019.107858 Received 1 August 2019; Received in revised form 23 October 2019; Accepted 24 October 2019 Available online 26 October 2019 0008-6215/ © 2019 Published by Elsevier Ltd.

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Table 1 Glycosyl Composition Analysis of A. hydrophila ML09-119 O-antigen, expressed in mole per cent of total carbohydrate. Glycosyl residue

O-antigen (OPS)

Non-acetylated Product of Smith Degradation (OPS-SD)

Fucose (Fuc) 3-Acetamido-3,6-dideoxyglucose (Qui3NAc) Glucose (Glc) N-Acetylglucosamine (GlcNAc) Heptoses

29.0 15.7 31.5 19.7 4.0

48.7 25.8 0.7 24.8 n.d.

n.d. = not detected.

potential roles of the G4C in multiple aspects of bacterial pathogenesis, and the importance of the G4C structure in mediating host immune responses, we determined the glycosyl composition and structure of the A. hydrophila ML09-119 O-antigen, using chemical analysis, Smith degradation, mass spectrometry, and 2-dimensional NMR spectroscopy.

The 1-D proton NMR spectrum of the native OPS sample (Fig. 1A) showed at least seven anomeric signals of varying intensity between 4.5 and 5.4 ppm, at least three signals corresponding to 6-deoxyhexose methyl protons near 1.2 ppm, four signals in the acetyl region around 2 ppm, and a complex carbohydrate bulk region between 3.1 and 4.5 ppm. The correlation spectroscopy (COSY) and total correlation spectroscopy (TOCSY) spectra (Fig. S4) further resolved the peaks in the anomeric region, revealing that four of the monosaccharide spin systems were each present in two, similar variants. COSY and TOCSY, in combination with a heteronuclear single quantum coherence (HSQC) spectrum (Fig. 2) allowed the identification of some of the monosaccharide residues detected in the linkage analysis, but due to signal overlap, we were unable to determine the complete monosaccharide sequence from the native OPS sample. A small set of two methylene proton signals in HSQC, at a carbon chemical shift of 65.1 ppm and proton chemical shifts at 4.86 and 4.29 ppm suggested the presence of O-acylation on a secondary hydroxyl group. This was confirmed by the chemical shifts of one of the acetyl signals, which were consistent with O-acetyl, but not with N-acetyl methyl groups. Assuming that the presence of pairs of similar residues might be due to partial O-acetylation, we subjected the polysaccharide to de-O-acetylation conditions in order to obtain simpler spectra from the de-O-acetylated OPS (OPS-D) (Fig. 1B). We also noted the presence of only two residues in the linkage analysis theoretically susceptible to periodate oxidation (terminal α-Glc and 4-substituted β-Glc), which suggested Smith degradation as a potential method to partially depolymerize the polysaccharide and thus simplify the NMR spectra. The Smith degradation procedure yielded two main oligosaccharides that were analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). The resulting spectra of the two products from Smith degradation showed masses of 869 and 827 Da, respectively, along with minor signals arising from loss of Fuc (Δm = −146 Da) and loss of FucGlcNAc (Δm = −349 Da), as well as very small signals corresponding to the product of incomplete Smith degradation, still containing residual Glc (Δm = +162 Da) (Fig. S5). The 42 Da mass difference between the two main peaks corresponded to the presence or absence of one acetyl substituent. Thus, these two oligosaccharides were labeled OPS-S (for the acetylated oligosaccharide from Smith degradation) and OPS-SD (for the deacetylated oligosaccharide from Smith degradation). The molecular weight of OPS-SD was calculated from the MALDI mass to be 804 Da, in accordance with a composition of Fuc2-GlcNAcdHex3NAc-tetritol. Alditol acetate composition analysis of OPS-SD gave Fuc, dHex3NAc, and GlcNAc in 2:1:1 ratio (Table 1, Fig. S6A), and linkage analysis of this sample indicated the presence of terminal Fucp, 3,4-disubstituted Fucp, terminal dHexp3NAc, and 3-substituted GlcpNAc (Table 2, Fig. S6B). The two main oligosaccharides obtained by Smith degradation were separated by hydrophilic interaction chromatography (HILIC) and analyzed by NMR. The 1D proton NMR spectrum of OPS-SD (Fig. 1C) showed 3 signals in the anomeric region in a 1:2:1 ratio, indicating the presence of four anomeric protons and that OPS-SD was essentially a tetrasaccharide. The aliphatic region featured two signals between 1.1 and 1.3ppm in a 2:1 ratio. This chemical shift is indicative of the methyl groups of 6-deoxyhexoses, suggesting that three of the four

2. Results The A. hydrophila ML09-119 bacterial culture was grown from an isolate of a diseased catfish derived from an MAS outbreak. The LPS was isolated using phenol-water extraction, and the O-antigen was released by mild-acid hydrolysis of the LPS. The O-antigen was further purified by size-exclusion chromatography using a Superose 12 column. The glycosyl composition of the purified O-antigen (abbreviated OPS, for “O-polysaccharide”) was determined by gas chromatography-mass spectrometry (GC-MS) with electron-impact ionization (EI) of the alditol acetates, obtained by acid hydrolysis, reduction, and O-acetylation of the polysaccharide. The analysis (Fig. S1A) detected the presence of Fuc, Glc, GlcNAc, and a second amino sugar as the major constituents in a 2:2:1:1 ratio (Table 1), as well as two small peaks with a heptose (Hep) mass spectrum (Table 1). The mass spectrum of the second amino sugar was consistent with a 3-amino-3,6-dideoxyhexose (Fig. S1B). To confirm this assignment, we derivatized the constituent monosaccharides as TMS methyl glycosides and analyzed them by GC-MS with chemical ionization. The measured molecular weights of the unknown peaks were 363 Da (not shown), consistent with the TMS methyl glycoside of monoacetamido-dideoxyhexose. The monoacetamido-dideoxyhexose was further characterized by linkage analysis, using partially methylated alditol acetates (PMAAs) (Fig. S2), which demonstrated that this residue was terminal 3-acetamido-3,6-dideoxyhexose (dHex3NAc), i. e. a dHex3NAc that was not further glycosylated in the original polymer. Thus, it was either the non-reducing end residue of the backbone or the terminal residue of a side chain. Its abundance in the linkage analysis was comparable to that of the other residues, suggesting that this residue was part of the repeating unit and thus a side chain component. The linkage analysis further detected PMAAs corresponding to terminal glucopyranose, 3,4disubstituted fucopyranose, 4-substituted glucopyranose, and 3-substituted 2-acetamido-2-deoxyglucopyranose (3-GlcNAc) as main sugar residues (Fig. S2A, Table 2), in addition to several Hep residues, presumably D-glycero- and L-glycero-α-D-manno-heptose from the core oligosaccharide of the LPS [17,18]. The TMS glycosyl composition analysis (not shown) also gave evidence of the presence of some residual β-hydroxy fatty acids, indicating incomplete removal of lipid A, as well as a trace of Kdo. However, 1D-proton NMR spectra acquired before and after mild acid hydrolysis (Fig. S3) showed a drastic impact of the hydrolysis on the line shapes of the polysaccharides, in that they were very broad before (Fig. S3A) and sharp after mild-acid hydrolysis (Fig. S3B). The hydrolyzed material thus was suitable for 2D-NMR analysis. The molecular weight of the highest point of the O-antigen response included on a Superose 6 gel filtration column was measured around 600 kDa, but a significant amount was outside the exclusion limit (data not shown), indicating that some of the molecular weight distribution was larger than ~700 kDa. 2

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Table 2 Glycosyl linkage analysis of LPS. Values represent peak area %. Linkages

O-Antigen (OPS)

Non-Acetylated Product of Smith Degradation (OPS-SD)

Terminal fucopyranosyl residue (t-Fucp) Terminal glucopyranosyl residue (t-Glcp) 4-substituted fucopyranosyl residue (4-Fucp) 3-substituted fucopyranosyl residue (3-Fucp) 3,4-disubstituted fucopyranosyl residue (3,4-Fucp) 4-substituted glucopyranosyl residue (4-Glcp) 6-substituted galactopyranosyl residue (6-Galp) Terminal heptopyranosyl residue (t-Hepp) Terminal heptopyranosyl residue (t-Hepp) Terminal 3-acetamido-3,6-dideoxyglucopyranosyl residue (t-Quip3NAc) 2-substituted heptopyranosyl residue (2-Hepp) Terminal 2-acetamido-2-deoxyglucopyranosyl residue (t-GlcpNAc) 3.4-disubstituted glucopyranosyl residue (3,4-Glcp) 3-substituted 2-acetamido-2-deoxyglucopyranosyl residue (3-GlcpNAc) 6-substituted 2-acetamido-2-deoxyglucopyranosyl residue (6-GlcpNAc)

n.d. 14.2 0.4 1.3 28.5 16.7 1.1 1.4 0.7 19.1 0.8 0.2 1.3 13.9 0.5

13.6 n.d. n.d. n.d. 28.6 0.8 n.d. n.d. n.d. 35.7 n.d. 1.5 n.d. 19.8 n.d.

Fig. 2. Two-dimensional 1H–13C-HSQC NMR spectrum of OPS, showing the signals of residues from O-acetylated repeating units (blue labels), in addition to the signals of naturally present OPS-D (red labels). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

residue. The HMBC spectrum showed a correlation from H-1 to a signal at 69.6 ppm, which was associated with a proton chemical shift of 4.32 ppm in HSQC. In the COSY spectrum, the signal at 4.32 had a cross peak at 1.14 ppm, suggesting a 6-deoxyhexose, namely 6-deoxygalactose, i. e. fucose. Residue b had large 3J coupling constants around the ring (Table 4), suggesting β-gluco-configuration, and a C-2 chemical shift of 58.4 ppm, indicating 2-amino substitution. The downfield chemical shift of H-2 and the presence of a methyl signal near 2 ppm showed that this residue was β-GlcNAc, and the C-3 chemical shift of 83.0 ppm indicated glycosylation in this position. Residue c showed similar coupling constants to Residue a (Table 4) and also had a methyl group in the 6-position. The association of this methyl group with Residue c was found in the same way as the one of Residue a, with the help of an HMBC correlation between H-1 and C-5, which showed a prominent peak in the HSQC spectrum at 4.26/70.1 ppm (Fig. 3B). In contrast to Residue a, which was a terminal Fuc, C-3 and C-4 of Residue c resonated downfield, consistent with glycosylation in these positions, identifying Residue c as 3,4-disubstituted α-Fuc. Residue e was characterized by a small 3J1,2 coupling constant (Table 4), suggesting αanomeric configuration. Its C-3 resonated at 56.7 ppm, indicating an amino group in this position. The other 3J coupling constants around the ring were large (Table 4), consistent with gluco-configuration, and the upfield resonances of H-6 and C-6 indicated that Residue e was a 6-

Fig. 1. One-dimensional 1H NMR spectra of A. OPS, B. OPS-D, and C. OPS-SD.

monosaccharides present were 6-deoxyhexoses, consistent with the presence of one dHex3NAc and two Fuc residues. Two N-acetyl methyl signals of equal intensity near 2 ppm confirmed the two acetamido sugars, i. e. GlcNAc and dHex3NAc. The 2D 1H–13C HSQC NMR spectrum of OPS-SD (Fig. 3B) showed four anomeric signals, two of which were partially overlapped, two methine groups attached to nitrogen, so identified by their carbon chemical shifts below 60 ppm, two acetamido-methyl signals, and three peaks associated with the methyl groups of 6-deoxyhexoses, in addition to the signals in the carbohydrate bulk region. Tracing the connectivities shown in the COSY and TOCSY spectra and reading the carbon chemical shifts from the HSQC spectrum allowed the assignment of the residues to which the anomerics belonged (Table 3). Residue a (Table 3) had small 3J1,2 and 3J4,5 coupling constants (Table 4), consistent with α-galacto-configuration, i. e. having axial OH-1 and OH-4. Due to the lack of a COSY correlation beyond H4, it was necessary to consult the HMBC spectrum to identify this 3

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The 1D proton NMR spectrum of OPS-D (Fig. 1B) showed 6 anomeric signals, two signals of 6-deoxyhexose methyl groups (ratio 2:1), and two N-acetyl methyl signals of equal intensity. The HSQC spectrum of OPS-D (Fig. 5B) displayed six anomeric signals, two methine groups attached to nitrogen, two acetamido-methyl signals, and three peaks associated with the methyl groups of 6-deoxyhexoses, in addition to the signals in the carbohydrate bulk region. The spectrum was simplified in comparison to that of the native OPS and, together with the structure knowledge gained from the NMR analysis of OPS-SD, allowed the elucidation of the structural sequence of OPS. Whereas the anomeric protons of the two fucose residues in OPS-SD were overlapped and thus these two residues were hard to distinguish, all six anomeric proton signals were well separated in OPS-D, allowing complete assignment of the spectra (Table 3). Three of the residues assigned in OPS-D were very similar in both proton and carbon chemical shifts to those in OPS-SD, namely 3-β-GlcNAc, 3,4-α-Fuc, and α-Qui3NAc. However, the second Fuc residue in OPS-D was different in that its C-3 and C-4 resonated at significantly lower field than those of its counterpart in OPS-SD, indicating glycosylations on O-3 and O-4. Two anomeric signals that were present in OPS-D, but not in OPS-SD were associated with a 4-substituted β-Glc residue and a terminal α-Glc residue. These two residues were oxidized during the Smith degradation, leading to conversion of 4-substituted β-Glc to 2-substituted erythritol and to loss of the α-Glc residue. The nuclear Overhauser effect (NOESY) spectrum (Fig. 5A) showed inter-residue correlations between H-1 of F and H-3 of A (5.35/ 4.04 ppm), between H-1 of E and H-3 of C (5.23/4.01 ppm), between H1 of A and H-3 of B (5.05/3.77 ppm), between H-1 of C and H-4 of D (4.91/3.51 ppm), between H-1 of B and H-4 of C (4.74/4.14 ppm), and between H-1 of D and H-4 of A (4.55/4.15 ppm), establishing the sequence of OPS-D as {4-[F-3-]A-3-B-4-[E-3-]C-4-D-}n. The HMBC spectrum of OPS-D (Fig. 5C) showed inter-residue correlations between H-1 of F and C-3 of A (5.35/76.0 ppm), between H-1 of E and C-3 of C (5.23/76.3), between H-1 of A and C3 of B (5.05/82.7 ppm), between H-1 of B and C-4 of C (4.74/81.1 ppm), and between H-1 of D and C4 of A (4.55/81.6 ppm), as well as between H-3 of A and C-1 of F (4.04/ 102.0 ppm), between H-3 of C and C-1 of E (4.01/101.6 ppm), between H-3 of B and C-1 of A (3.77/102.2 ppm), between H-4 of D and C-1 of C (3.51/102.9 ppm), between H-4 of C and C-1 of B (4.14/103.6 ppm), and between H-4 of A and C-1 of D (4.15/105.5 ppm), completely defining the sequence of OPS-D and thus of OPS (Fig. 4B). The anomeric 1-bond C–H coupling constants (Table 3), obtained via HSQC without carbon decoupling during acquisition, confirmed the anomeric configurations of each residue, being near 170 Hz for the α-anomeric residues and near 160 for the β-anomeric residues [20]. The absolute configuration of Glc, Fuc, and GlcNAc determined by GC-MS analysis of TMS-derivatized S-(+)-2-butyl glycosides was found to be D-Glc, L-Fuc, and D-GlcNAc. The absolute configuration of the Qui3NAc residue was determined by NMR using carbon chemical shift displacements following a previously described protocol [21] (Table 3). The carbon chemical shifts of the Fuc residue glycosylated by Qui3NAc were not helpful for this determination because of the scarcity of observed 3,4-disubstituted fucose chemical shifts in the literature. However, the anomeric carbon chemical shift of Qui3NAc itself was highly diagnostic. The C-1 chemical shift of unsubstituted Qui3NAc has been measured at 94.5 ppm as described by Sidorczyk et al. [22] (corrected for different reference value) resulting in a glycosylation effect of the present Qui3NAc of +6.6 ppm in OPS-SD and of +7.1 ppm in OPS and OPS-D. This large effect is consistent with Qui3NAc having the opposite absolute configuration of L-Fuc [21], hence it was determined to have Dconfiguration. The stereochemistry of the Hep residue was not determined in this study as it was most likely part of the core oligosaccharide of the LPS. After establishing the main sequence of the polysaccharide (Fig. 4) based on the NMR data of OPS-D, we focused our attention again on the NMR spectra of the native OPS. Comparison of the COSY, TOCSY, and

Fig. 3. Two-dimensional NMR spectra of OPS-SD, demonstrating the sequence proposed in Fig. 4A. A. Partial 2D 1H–1H-ROESY spectrum; B. Partial 1H–13CHSQC spectrum with labels according to Table 3; C. Partial 1H–13C-HMBC spectrum. The solid lines represent inter-residue HMBC correlations; the dashed lines represent inter-residue NOE correlations.

deoxy sugar. Together with characteristic, upfield proton resonances of H-2 and H-4 [19], these results identified Residue e as α-3-acetamido3,6-dideoxyglucose (α-Qui3NAc). In addition to these 4 hexose residues, the HSQC spectrum contained signals consistent with 2-substituted erythritol, which was evidently the product from periodate oxidation of the 4-substituted glucose, followed by reduction and mild hydrolysis. The rotating-frame nuclear Overhauser effect (ROESY) spectrum (Fig. 3A) revealed inter-residue correlations between H-1 of Qui3NAc and H-3 of 3,4-Fuc and between H-1 of GlcNAc and H-4 of 3,4-Fuc. The NOE signals from H-1 of the two Fuc residues were not entirely conclusive due to overlap. There was a cross peak that seemed to be aligned with H-1 of Residue a and H-3 of Residue b, but no cross peak was apparently aligned with H-1 of Residue c. However, the heteronuclear multiple-bond coherence (HMBC) spectrum (Fig. 3C) revealed correlations between H-1 of the terminal Fuc residue and C-3 of GlcNAc and between H-1 of 3,4-Fuc and C-2 of erythritol. The fact that eythritol was connected to O-1 of Residue c explained the lack of an NOE correlation in the ROESY spectrum by invoking the flexibility of the open-chained erythritol, which precluded a strong NOE contact. Taken together, these data led to the conclusion that OPS-SD had the structure shown in Fig. 4A. OPS-S had nearly identical chemical shifts, except for the erythritol residue, whose H-4 signals were significantly shifted downfield (Table 3), in accordance with 4-O-acetylation. This indicated that its precursor (before Smith degradation), i. e. the 4-substituted glucose residue in OPS, was partially O-acetylated in the 6-position. 4

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Table 3 Chemical shift assignments (δ, ppm) and 1-bond C–H coupling constants of OPS-SD, OPS-S, OPS-D, and OPS. No.

a

Sugar Residue

b

3-β-GlcNAc

c

3,4-α-Fuc

d

2-Ery-ol

d’

2-(4-OAc)-Ery-ol

e

α-Qui3NAc

OPS-D A

3,4-α-Fuc

B

3-β-GlcNAc

C

3,4-α-Fuc

D

4-β-Glc

E

α-Qui3NAc

F

α-Glc 3,4-α-Fuc 1

JCH 3-β-GlcNAc

B′

1

JCH 3,4-α-Fuc

C′

1

JCH 4-(6-OAc)-β-Glc

D′

1

JCH α-Qui3NAc

E′

1

JCH α-Glc

F′

1

a

C NMR chemical shifts in italics.

Chemical shift (ppm)

OPS-SD/OPS-S a α-Fuc

OPS A′

13

JCH

Ac

1

2

3

4

5

6

5.00 102.4 4.67 103.8 4.99 101.7 3.78/3.74 63.5 3.77/3.72 63.7 5.28 101.0

3.68 70.8 3.91 58.4 4.05 71.0 3.66 82.0 3.80 81.1 3.58 72.9

3.83 72.2 3.64 83.0 4.09 75.2 3.88 73.1 4.35 77.8 4.13 56.7

3.79 74.5 3.49 71.5 4.15 80.6 3.75/3.68 64.9 4.07/4.07 68.9 3.15 76.2

4.32 69.6 3.44 78.7 4.26 70.1

1.14 18.1 3.91/3.91 63.5 1.24 19.0

2.03 25.0

3.99 70.8

1.25 19.0

2.06 25.0

5.05 102.2 4.74 103.6 4.91 102.9 4.55 105.5 5.23 101.6 5.35 102.0

4.02 71.2 3.89 58.3 4.11 71.2 3.36 76.7 3.60 73.2 3.58 75.0

4.04 76.0 3.77 82.7 4.01 76.3 3.49 78.6 4.09 57.0 3.85 74.4

4.15 81.6 3.52 71.7 4.14 81.1 3.51 80.8 3.17 76.3 3.49 71.6

4.37 70.3 3.48 78.5 4.42 70.0 3.39 78.9 3.98 71.1 3.97 74.4

1.27 17.8 3.91/3.77 63.5 1.25 18.5 4.00/3.85 63.6 1.26 19.5 3.90/3.83 63.2

5.05 102.2 170 Hz 4.74 103.6 159 Hz 4.86 103.5 171 Hz 4.55 106.3 159 Hz 5.25 101.6 174 Hz 5.33 102.0 172 Hz

4.07 71.4

4.05 76.1

4.07 81.6

4.37 70.3

1.27 17.8

3.89 58.3

3.77 82.7

3.52 71.7

3.48 78.5

3.91/3.77 63.5

4.11 71.3

4.07 76.0

4.14 81.1

4.42 70.0

1.22 18.5

3.36 76.7

3.51 78.2

3.46 81.5

3.60 76.1

4.86/4.29 65.1

2.17 23.1

3.58 73.2

4.10 57.0

3.16 76.7

3.99 71.6

1.26 19.5

2.06 25.1

3.55 75.2

3.72 74.6

3.45 72.2

3.87 74.5

3.87/3.78 63.5

NOE HMBC b3 c4 c4 d2

2.03 25.2

2.06 25.1

c3 c3 B3 B3 C4 C4 D4 A4 A4 C3 C3 A3 A3 B′3

2.03 25.2

C′4 D′4 A′4

A′3

The primed letters refer to residues belonging to O-acetylated repeating units.

Table 4 3 1 J H–1H Coupling constants in OPS-SD, determined from the 1D 1H spectrum. No.

Sugar Residue

3

a b c e

α-Fuc 3-β-GlcNAc 3,4-α-Fuc α-Qui3NAc

2.4 7.8 1.0 3.8

J1,2

3

3

3

10.4 ND 10.1 10.8

3.4 10.1 3.6 9.8

ND 8.6 <1 9.6

J2,3

J3,4

J4,5

3

J5,6

6.9 5.7, 2.6 6.7 6.3

ND = not determined.

HSQC spectra of OPS with those of OPS-D enabled us to identify the signals belonging to the portion of the polysaccharide that was nonacetylated in the native material. The remaining signals belonged to the O-acetylated polysaccharide, and four of its anomerics gave separate signals in HSQC (Fig. 2). We were able to completely assign these four residues, among which was a 6-O-acetylated 4-substituted β-Glc clearly distinguished by the downfield displacement of its H-6 protons. The remaining three residues showed only slightly displaced chemical

Fig. 4. A. Structure of the oligosaccharide obtained by Smith degradation. One of the two isolated fractions were acetylated on O4 of erythritol. B. Structure of the ML09-119 O-antigen polysaccharide.

5

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Fig. 5. Two-dimensional NMR spectra of OPS-D, demonstrating the sequence proposed in Fig. 4B. A. Partial 2D 1H–1H-NOESY spectrum; B. Partial 1H–13CHSQC spectrum with labels according to Table 3; C. Partial 1H–13C-HMBC spectrum. The solid lines represent inter-residue HMBC correlations; the dashed lines represent inter-residue NOE correlations.

Fig. 6. Comparison of A. hydrophila ML09-119 O-antigen with similar O-antigen structures from other Aeromonas spp. strains (Ah = A. hydrophila; As = A. salmonicida) [49,50]. To facilitate comparison, we added a second subunit printed in gray to the previously published structures.

shows structural similarity with several other Aeromonas spp. strains (Fig. 6), it has a larger and more complex subunit structure. Previously, a 26.5 kb novel O-antigen biosynthesis gene cluster had been identified in vAh isolated from initial channel catfish outbreaks [2]. This cluster was shown to be clonal in all vAh isolates tested. Further, this cluster shared homology with only one of the traditional A. hydrophila isolates and had been previously uncharacterized amongst the reported O-antigen structures of published A. hydrophila strains [2]. The analysis of this gene cluster indicated that this difference in homology could be derived from lateral gene transfer, which may explain why there is a larger complexity observed in the subunit of vAh when compared to other Aeromonas spp [2].

shifts. The NOESY spectrum of OPS (not shown) confirmed the connectivities found in OPS-D and assisted in the assignment of the Oacetylated repeat. The complete assignment is listed in Table 3. We estimated the degree of acetylation to be about 25% by integrating the anomeric signals in the HSQC spectrum. Taken together, the results demonstrate that the structure of the OPS consists of a hexasaccharide repeating unit, O-acetylated on 25% of Residue D (Fig. 4B). We also partially purified the non-LPS associated capsular polysaccharide (CPS) for comparison by 1D proton NMR (Fig. S3C). Although the residual contaminants obscure some of the polysaccharide peaks, it is apparent that the same peaks are present in CPS as in OPS, suggesting that CPS repeats the same polysaccharide structure as LPS. The degree of Oacetylation seems to be higher in the CPS, but this may be because the CPS fractions were not subjected to acid hydrolysis as was OPS, which may have led to partial removal of O-acetyl groups in OPS. Structural analysis of the vAh O-antigen, which included NMR and glycosyl composition and linkage analyses by GC-MS, revealed a structure that has not been previously observed in any other bacterial species (Fig. 4), although some similarities can be observed by comparison with polysaccharides from related organisms. For example, 3substituted β-D-GlcNAc has been established as constituent backbone residue in the O-antigens of A. hydrophila strains AH-1 [23], K691 [24], and SJ-44 [25], and α-D-Qui3NAc has been found in the O-antigen of A. veronii strain Bs19 [26]. While the O-antigen of A. hydrophila ML09-119

3. Discussion Recent results from our labs [9] showed that genes important for group 4 capsule assembly appear essential for the highly virulent pathotype of vAh. Furthermore, vAh mutants lacking gfcD, a predicted outer membrane protein for group 4 polysaccharide export, had decreased virulence, lacked biofilm-forming capacity, and decreased buoyancy compared to wild-type vAh, yet still expressed LPS. Structural characterization of the LPS O-antigen repeat in vAh presented here demonstrate that the same O-antigen repeating unit is also found in the secreted high molecular weight capsular polysaccharide confirming its 6

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identity as group 4 capsule. Thus, the group 4 capsular polysaccharide is the primary virulence factor and not the LPS. Several recent examples from other Gram-negative pathogens show that O-antigen capsular polysaccharides appear to shield binding of immunoglobulin to LPS and inhibit complement-mediated bacterial killing [15,27,28]. vAh capsule mutants have reduced capability of forming biofilms, another virulence factor [29]. Moreover, the presence of LPS and G4C containing the same O-antigen repeat is not uncommon amongst fish pathogens and was present in both Listonella anguillarum and V. ordalii [30]. Although the route of vAh infection is not known, it is likely to involve mucous-covered tissues such as gills, skin or intestine. Interestingly the vAh O-antigen structure described here is polar but uncharged. This is similar to other G4Cs characterized from other enteric pathogens, for example, Salmonella [16,31], enteropathogenic and enterohemorrhagic E. coli [32,33], but not Shigella sonnei, which is zwitterionic [15]. One hypothesis is that the uncharged G4C mimics the mucins secreted by the intestines [34]. This may also enhance adhesion to the mucous-containing surfaces. This mimicry may also enable the bacteria to evade the host immune response. We estimated the size of the vAh G4C to be greater than 700 kDa. This would correspond to at least 700 repeats of the O-antigen subunit reported here. This size is much larger than usual LPS O-antigens. However, our evidence from SEC clearly demonstrates the large size of the polysaccharide, and NMR before and after mild acid hydrolysis (Fig. S3) strongly suggests linkage through Kdo to a lipid anchor. This is consistent with what has been described as “KLPS” or, if there is no LPS of different structure, as “O-antigen capsule” [35]. The molecular weight we observed is comparable to high molecular weight capsules reported elsewhere [30,35–38]. For example, the O-antigen capsule produced by Salmonella enteritidis is estimated to be at least 3000 repeat units in size based on chromatography with a 2 × 106 Da dextran standard [31]. The high molecular weight O-antigen capsule may contribute to vAh's continued virulence in the presence of immune surveillance [34].

0.5 ml DI water, and subjected to ultracentrifugation at 86,000×g for 1 h, and the pellet was recovered to yield clean LPS. 4.2.2. Mild acid hydrolysis of LPS Cleavage of lipid A from the OPS was performed by modification of a previously described method [29]. A. hydrophila ML09-119 LPS was incubated in 1% acetic acid for 4 h at 100 °C, followed by centrifugation at 10,000×g for 30 min. The supernatant containing the O-antigen was taken into another tube and freeze-dried. 4.2.3. Gel-filtration chromatography The OPS was purified by passage through a Superose-12 10/300 GL column (GE Healthcare), using 50 mM ammonium acetate at pH 5 as eluent at a flow rate of 0.5 ml/min on an Agilent Infinity 1260 liquid chromatograph, equipped with refractive index detector. 4.2.4. Extraction of the O-antigen capsule (G4C) The capsule was extracted from A. hydrophila ML09-119 following previously described methods with modifications [14]. For preparation of the total cellular polysaccharides, ML09-119 was grown overnight in TSB at 30 °C with shaking. The resulting culture of A. hydrophila ML09119 was diluted 1:100 into 300 ml of fresh TSB and incubated statically at 30 °C for 48 h, and then the culture was subjected to centrifugation at 8000×g for 10 min and resuspended in 3 ml of sterile 1 × phosphatebuffered saline. An equal volume of saturated phenol (pH = 8.0) was added, and the mixture was incubated for 30 min at 70 °C with occasional mixing. After 30 min, the mixture was subjected to centrifugation at 10,000×g for 1 h. The aqueous layer was aspirated and mixed with 2 vol of 100% ethanol and incubated at 70 °C for 1 h to precipitate polysaccharides. The mixture was then subjected to centrifugation at 12,000×g for 30 min. The supernatant was aspirated, and the pellet was washed with 500 μl of 70% ethanol, centrifuged at 12,000×g, dried, and stored at −80 °C until further separation of the capsule polysaccharide. To separate the capsule polysaccharide, the dried total polysaccharide preparations were resuspended in 500 μl of sterile nuclease free water and subjected to ultracentrifugation at 86,000×g for 1 h. Following the ultracentrifugation, the supernatant containing the capsule polysaccharide was aspirated. To remove any residual LPS contamination from the supernatant, Triton X-114 was added to the supernatant to a final concentration of 1%. This mixture was incubated at 4 °C for 1 h with constant inversion to ensure a homogenous solution. To separate the Triton X-114 and LPS micelles, the mixture was then incubated at 37 °C for 10 min and subjected to centrifugation and 18,000×g at 25 °C for 1 h. The resulting aqueous phase containing the capsule was centrifuged one more time and lyophilized for NMR analysis.

4. Experimental 4.1. Growth of bacteria A. hydrophila strain ML09-119, which was isolated from a diseased catfish derived from an epidemic outbreak of MAS in channel catfish in Alabama in 2009 [2], was used in this study as a well characterized vAh strain. A. hydrophila ML09-119 was routinely grown on trypticase soy broth (TSB) and trypticase soy agar (TSA) at 30 °C with shaking at 200 rpm for 16 h. 4.2. Isolation of the O-antigen

4.3. Structural analysis of A. hydrophila ML09-119 O-antigen and the Oantigen capsule

4.2.1. Isolation of LPS LPS was extracted by the hot phenol-water method as described previously [40] with some modifications. Briefly, bacterial suspensions were centrifuged at 10,000×g for 5 min. The pellets were washed twice in PBS (pH = 7.2) (0.15 M) containing 0.15 mM CaCl2 and 0.5 mM MgCl2. Pellets were then resuspended in 10 ml PBS and sonicated for 10 min on ice. Protein was digested with proteinase K (100 μg/ml) (GBiosciences, St. Louis, MO) for 1 h at 65 °C. Nucleic acids were removed by treatment with RNase (40 μg/ml) (Amresco, Solon, OH) and DNase (20 μg/ml) (Millipore, Burlington, MA) in the presence of 1 μL/ml 20% MgSO4 and 4 μL/ml chloroform at 37 °C overnight. Subsequently, an equal volume of hot (70 °C) 90% phenol was added to the mixtures, followed by incubation at 70 °C for 15 min with shaking. After cooling, the sample was centrifuged (8000×g), the layers were separated, and the aqueous layer was mixed with 10 vol of 95% ethanol. After storing overnight at −20 °C, the precipitated polysaccharides were collected by centrifugation (2000×g, 4 °C), resuspended in water and dialyzed to remove residual phenol. The sample was lyophilized, resuspended in

4.3.1. Glycosyl composition analysis A) Alditol acetate method: Glycosyl composition analysis by the alditol acetate method was carried out using a modification of a previously described method [41]. A 200-μg portion of the sample was mixed with 20 μg myo-inositol as internal standard and hydrolyzed with 2 M trifluoroacetic acid for 2 h at 121 °C. The sample was then dried with a stream of dry nitrogen, dissolved in 1 M ammonium hydroxide and reduced with 10 mg/ml sodium borodeuteride at 25 °C for 2 h. After drying, the sample was acetylated with 200 μl each of acetic anhydride and pyridine for 1 h at 100 °C. The sample was dried and dissolved in dichloromethane for GC-MS analysis, which was performed on an Agilent 7890 N GC interfaced to a 5975C MSD (mass selective detector, electron impact ionization mode), using a Supelco Equity-1 fused silica capillary column (30 m × 0.25 mm ID). B) TMS Methyl Glycoside method: Glycosyl composition analysis by the TMS method was carried out as previously described [41,42]. The polysaccharide 7

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was depolymerized and converted to methyl glycosides by methanolysis of the dry samples for 16 h in 1 M HCl in methanol at 80 °C. For the detection of amino sugars, re-N-acetylation was conducted with pyridine and acetic anhydride in methanol. The samples were then per-Otrimethylsilylated by treatment with Tri-Sil (Pierce, ThermoFisher Scientific, USA) for 30 min at 80 °C. GC/MS analysis of the TMS methyl glycosides was performed on an Agilent 7890 N GC interfaced to a 5975C MSD (mass selective detector, electron impact ionization mode), using a Grace EC-1 fused silica capillary column (30 m × 0.25 mm ID). Additionally, GC-MS of TMS methyl glycosides was also performed on a Shimadzu QP2010 with chemical ionization, using a Grace EC-1 fused silica capillary column (30 m × 0.25 mm ID).

(1 kDa) against water. Then, 7 mg NaBH4 was added, and the mixture was incubated at room temperature for 16 h. After neutralization with AcOH and dialysis (1 kDa) the sample was lyophilized, dissolved in 1 M HCl, and allowed to react for 24 h at room temperature. The sample was then diluted with water and lyophilized [44]. 4.3.7. Hydrophilic interaction chromatography (HILIC) Separation of the Smith fragments was performed on a Shodex Asahipak NH2P-50 column (4.6 × 250 mm) by isocratic elution using 65% acetonitrile at a flow rate of 0.8 ml/min [45]. Seven injections of 10 μl were made in total. 4.3.8. MALDI-TOF mass spectrometry MALDI-TOF MS was acquired on a Bruker Microflex mass spectrometer in linear positive ion mode. The sample solution (1 μl) was mixed with 1 μl matrix (10 mg/ml 2,5-dihydroxybenzoic acid in 50% acetonitrile), and 0.5 μl of the mixture was spotted on the MALDI plate. A laser energy of 65% was used, and 100 scans were signal-averaged [46].

4.3.2. Glycosyl linkage analysis For glycosyl linkage analysis, the sample was permethylated, depolymerized, reduced, and acetylated; and the resultant partially methylated alditol acetates (PMAAs) were analyzed by gas chromatography-mass spectrometry (GC-MS) as described [42]. Initially, an aliquot of the sample was suspended in about 200 μl of dimethyl sulfoxide and placed on a magnetic stirrer for 3 d. Each sample was then permethylated as previously described [41], which involved treatment of the samples with a sodium hydroxide slurry and methyl iodide in dry DMSO. Briefly, permethylation was effected by two rounds of treatment with sodium hydroxide (15 min) and methyl iodide (40 min). The permethylated material was hydrolyzed using 2 M TFA (2 h in sealed tube at 121 °C), reduced with NaBD4, and acetylated using acetic anhydride/ TFA. The resulting PMAAs were analyzed on an Agilent 7890 GC interfaced to a 5975C MSD; separation was performed on a Supelco 2330 silica capillary column (30 m × 0.25 mm ID). The PMAAs were also analyzed on a Shimadzu QP2010 with chemical ionization, using a Supelco Equity-1 fused silica capillary column (30 m × 0.25 mm ID).

4.3.9. NMR spectroscopy Samples (10 mg of OPS, 15 mg of OPS-D, 1.7 mg of OPS-SD) were deuterium exchanged by dissolving in D2O (99.9% D, Sigma) and lyophilizing. The samples were then dissolved in 0.28 ml D2O (99.996% D, Cambridge Isotope Laboratories) and transferred to 5-mm precision NMR tubes with D2O-matched magnetic susceptibility plugs (Shigemi). 1D Proton and 2D TOCSY and NOESY NMR spectra, run with water presaturation, and gradient enhanced COSY, HSQC, and HMBC spectra were acquired on a Varian Inova-600 MHz spectrometer, equipped with a cryoprobe, at 50 °C. The TOCSY spinlock time was 80 ms, and the ROESY/NOESY mixing time was 200 ms. The 1-bond C–H coupling constant in HSQC was set at 146 Hz, and the multiple-bond coupling constant in HMBC at 8 Hz. Other experimental details are listed in the supplemental material (Table S1). An HSQC spectrum without decoupling during acquisition was collected to measure the anomeric 1-bond C–H coupling constants, using otherwise identical parameters to the first HSQC. Chemical shifts were referenced relative to sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) at 0.00 ppm, using the methyl signal of internal acetone (δH = 2.218 ppm) for the proton dimension [47] and calculating the carbon chemical shifts using the frequency ratio Ξ for 13C/1H of 0.25144953 for DSS in D2O in the unified chemical shift scale, according to IUPAC recommendations [48].

4.3.3. Absolute configuration of O-antigen components The absolute configuration (D or L) of monosaccharides other than α-3-acetamido-3,6-dideoxyglucose (Qui3NAc) was determined using combined gas chromatography/mass spectrometry (GC/MS) of the S(+)-2-butyl glycosides of the monosaccharides produced from the sample by acidic hydrolysis [42]. An aliquot of the sample was placed into a test tube with 400 μl of 2 M TFA and heated at 121 °C for 1.5 h. Once cooled, the sample was dried under nitrogen. Butylation was conducted using 1 M HCl in S-(+)-2-butanol (Fluka), which was prepared by adding the appropriate amount of acetyl chloride to the alcohol and carried out at 80 °C for 16 h. The sample was then dried and re-N-acetylated. Finally, the sample was per-O-trimethylsilylated by treatment with Tri-Sil at 80 °C (0.5 h). GC/MS analysis of the trimethylsilylated 2-butyl glycosides was performed on an Agilent 7890 A GC interfaced to a 5975C MSD, using an Agilent DB-1 fused silica capillary column (30 m × 0.25 mm ID).

Funding This research was supported by grants from the Alabama Agriculture Experiment Station (Project # ALA021-1-09005) and by the United States Department of Agriculture's Agriculture and Food Research Initiative (Project #2013-67015-21313). In addition, this work was supported by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, U.S. Department of Energy grant (DE-SC0015662) to Parastoo Azadi at the Complex Carbohydrate Research Center.

4.3.4. Determination of molecular weight of the LPS O-antigen The molecular weight of the O-antigen was measured by size-exclusion chromatography, using an Agilent Infinity 1260 high performance liquid chromatograph, equipped with a refractive index detector and a Superose 6 10/300 GL (GE Healthcare) column. The eluent was 50 mM ammonium acetate, pH 5 at a flow rate of 0.5 ml/min.

Author contributions Conception of the study: MRL, PA, CH; performing of experiments: CMT, MJH, DS, ZW, CH; interpretation of data: ZW, CH, DS, MJH; writing manuscript: CH, MRL, MAS, CMT.

4.3.5. De-O-acetylation An aqueous solution of the polysaccharide (~10 mg/ml) was brought to pH 11 by addition of concentrated ammonium hydroxide and left to stand at room temperature for 18 h, followed by lyophilization [43].

Declaration of competing interest The authors declare that they have no conflict of interest.

4.3.6. Smith degradation Polysaccharide (4.5 mg) was treated with 0.45 ml of 20 mg/ml sodium metaperiodate and kept in the dark at room temperature for 20 h. Ethylene glycol (6.8 μl) was added, and the mixture was dialyzed

Acknowledgments We thank the members of the Liles laboratory, in particular Ms. 8

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Nancy Capps for her support in this research, and we thank Dr. Russ Carlson for helpful discussions.

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