Structural Diversity among Edwardsiellaceae Core Oligosaccharides

Abstract

The Edwardsiella genus presents five different pathogenic species: Edwardsiella tarda, E. anguillarum, E. piscicida, E. hoshinae and E. ictaluri. These species cause infections mainly in fish, but they can also infect reptiles, birds or humans. Lipopolysaccharide (endotoxin) plays an important role in the pathogenesis of these bacteria. For the first time, the chemical structure and genomics of the lipopolysaccharide (LPS) core oligosaccharides of E. piscicida, E. anguillarum, E. hoshinae and E. ictaluri were studied. The complete gene assignments for all core biosynthesis gene functions were acquired. The structure of core oligosaccharides was investigated by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. The structures of E. piscicida and E. anguillarum core oligosaccharides show the presence of →3,4)-L-glycero-α-D-manno-Hepp, two terminal β-D-Glcp, →2,3,7)-L-glycero-α-D-manno-Hepp, →7)-L-glycero-α-D-manno-Hepp, terminal α-D-GlcpN, two →4)-α-D-GalpA, → 3)-α-D-GlcpNAc, terminal β-D-Galp and →5-substituted Kdo. E. hoshinare core oligosaccharide shows only one terminal β-D-Glcp, and instead of terminal β-D-Galp a terminal α-D-GlcpNAc. E. ictaluri core oligosaccharide shows only one terminal β-D-Glcp, one →4)-α-D-GalpA and do not have terminal α-D-GlcpN (see complementary figure).

1. Introduction

One of the less frequently encountered pathogenic genera in the order Enterobacteriales is the genus Edwardsiella [1], which was established in 1965 by Ewing et al. [2]. It belongs to the order and family Hafniaceae based upon biochemical and physiologic characteristics, the presence of the enterobacterial common antigen, and different molecular signatures identified through comprehensive comparative genomic analyses [3,4]. E. tarda, E. ictaluri and E. hoshinae have been the traditional causative agents of Edwardsiellosis until 2012; however, intensive studies have just recently revealed two other species, E. piscicida and E. anguillarum [5].

Edwardsiellosis is a serious disease affecting a wide range of cultured fish species both in marine and freshwater environments [6]. It usually occurs under imbalanced environmental conditions, such as high water temperatures, poor water quality and high organic content. Fish infected show abnormal swimming behavior, loss of pigmentation, swelling of the abdominal surface, rectal hernia and other symptoms [7]. This disease may become a significant health issue for fish and humans, accounting for severe economic losses in the aquaculture industries [8].

E. tarda is the best studied species in this genus, having a wide ecological niche and host range including fish, birds, amphibians, reptiles, marine mammals and humans [5]. In humans, is the only recognized pathogenic species primarily associated with sporadic cases of gastroenteritis. In rare instances, has also been reported to cause extraintestinal diseases, involving cases of septicaemia and bacteremia [1]. E. ictaluri causes enteric septicaemia in cultured channel and white catfish, which appear to be the primary host species of this pathogen, as well as tilapia and other warm-water species [9]. E. hoshinae has been most often isolated from reptiles and birds [10]. Two new species have been recently added to the Edwardsiella genus, first E. piscicida and later E. anguillarum. E. piscicida comprises exclusively pathogenic strains isolated from fish but shares many phenotypic characteristics identical to E. tarda. E. anguillarum is a microorganism potentially pathogenic to eels and distinguishable from the other species of the genus for the capacity to produce acetoin from glucose and to ferment arabinose [5].

The LPS is an amphiphilic molecule located in the outer leaflet of the outer membrane of gram-negative bacteria that confers stability, integrity and organization to the outer membrane. Furthermore, it maintains the barrier function against bacteriophages and the action of certain antibiotics, as well as against the host defense mechanism during infections [11]. The LPS molecule consists of a polysaccharide and a lipid A part. The lipid A is the hydrophobic part and has endotoxic and pyrogenic properties, among others. The polysaccharide is the hydrophilic part and can be subdivided into the O-specific chain (O-antigen), which is more external and variable, and the core oligosaccharide, which is internal and conserved [11]. The core oligosaccharide can be further subdivided into the inner core and outer core. The inner core is bound to the lipid A and commonly contains two or three heptoses and 2-keto-3-deoxyoctulosonic acid (Kdo), and in many bacteria, it contains non-carbohydrate components, such as phosphate, amino acids and ethanolamine substituents. The outer core usually contains hexoses or hexosamines and is the part bound to the O-antigen. Unlike the inner core, which tends to be evolutionary conserved within a taxonomic family or genus, the outer core shows more variability [12]. The O-antigen, which may be present (Smooth-LPS) or not (Rough-LPS), is the external component of the LPS and consists of repeating oligosaccharide units that may be linear or branched. It shows the largest variation between species and evokes a specific response [13,14].

The genes for the core oligosaccharide of LPS are mainly organized into clusters of contiguous genes. In several Enterobacteriaceae, such as Escherichia coli, Salmonella enterica and Klebsiella pneumoniae, genes involved in LPS core biosynthesis are found clustered in a region of the chromosome, the waa gene cluster [15]. However, in other species such as Proteus mirabilis, Yersinia enterocolitica and Plesiomonas shigelloides, belonging to different families of Enterobacteriales, some genes involved in LPS core biosynthesis are not clustered and located outside the waa gene cluster [16,17,18].

As a first, the genome of E. tarda EIB202 was fully sequenced, and a clear region with the waa gene cluster was identified. As in the majority of Enterobacteriales, the first gene of the cluster is hldD (also referred to as rfaD), which codes for the ADP-L-glycero-D-manno-heptose-6-epimerase (encoded protein ETAE_0083), and at the 3′ end of the cluster, the coaD (encoded protein ETAE_0071) codifies for phosphopantetheine adenylyltransferase. The function of genes found in this cluster seems to be in agreement with the chemical structure of the core oligosaccharide of E. tarda EIB202: →5-substituted Kdop, →3,4)-L-glycero-α-D-manno-Hepp, →2,3,7)-L-glycero-α-D-manno-Hepp, two terminal β-D-Glcp, →7)-L-glycero-α-D-manno-Hepp, two →4)-α-D-GalpA, terminal α-D-GlcpN, →3)-α-D-GlcpNAc and terminal β-D-Galp, which had been previously described [19]. However, the LPS motif β-D-Glcp-(1→2)-α-L-HeppII is not encoded by any of the glycosyltransferases found in the waa cluster. This motif is highly similar to the WapG of P. shigelloides and is encoded by a gene outside this cluster, ETAE_RS09105 [19].

The complete structural analysis of the core oligosaccharides is of high importance for a better understanding of LPS biological activity and is a prerequisite for strategies aimed at the treatment of endotoxicosis. As the Edwardsiella genus has four other species reported, we characterized the chemical structure of the core oligosaccharide of E. piscicida, E. anguillarum, E. hoshinae and E. ictaluri, supported by genomic analysis.

2. Results

2.1. Comparative Genomics of Edwardsiella Strains

In E. tarda, as in many Enterobacteriales, the waa region starts from hldD (also referred to as rfaD) to coaD gene. Analysis of seven E. tarda, thirteen E. piscicida, two E. anguillarum, six E. ictaluri and two E. hoshinae strains with complete genome sequences available on the NCBI website, using the E. tarda EIB202 coaD and hldD, allowed us to locate the waa gene cluster in each genome. Comparative genomics of this region using Mauve software version 20150226 show that they are highly conserved in all species, with some differences in E. ictaluri and E. hoshinae, as well as in some E. tarda strains (Figure 1).

A phylogenetic tree generated by the neighbor-joining method on the basis of the waa cluster (hldD to coaD) sequence shows two different types of waa clusters in Edwardsiella (Figure 2). One is found in all E. piscicida, E. anguillarum and E. ictaluri strains tested, and in three E. tarda strains, including the EIB202 strain. The other type is found in all E. hoshinae tested and in four E. tarda strains.

To identify orthologs common in both species, reciprocal BLASTp (Figure 3) compared the complete set of predicted proteins of E. tarda EIB202 with that of the other. Although the proteins encoded by some genes in the waa cluster of E. piscicida and E. anguillarum have been annotated with different names, all of them show identical or similar protein size (nº of amino acids) and identities higher than 93.0% of those of orthologous genes in the E. tarda EIB202 waa cluster (Figure 3). These data suggest that E. piscicida and E. anguillarum probably have the same core-oligosaccharide structure as E. tarda EIB202.

The E. ictaluri waa cluster does not contain either the wapC or part of the wapB. The remaining wapB encodes a peptide of 129 amino acids that shows 96.6% identity with the carboxy-terminal end of WapB. Furthermore, the WaaL of E. ictaluri is smaller (218 amino acids) than the WaaL of E. tarda, E. piscicida and E. anguillarum (377 amino acids), but shows an identity of 91.7% compared to E. tarda EIB202.

For E. hoshinae, which also presents differences in this genomic region, it can be seen that it lacks the wabK gene, and the downstream of waaC presents a new gene, which encodes a glycosyltransferase belonging to the glycosyltransferase family 2. This new glycosyltransferase has no homology to WabK, so they are different proteins. They show 84.0% identity to the hyaluronan synthase (HyaD) of E. tarda NCTC13561, which is present in the waa cluster of this strain, and also 40.5% identity with WapD of P. shigelloides. These homologies suggest that the function of this new glycosyltransferase (named WahX) could be N-acetylhexosamine. Curiously, glycosyltransferases orthologous to WahX were found in some E. tarda strains such as KC-Pc-HB1, AT98-87 and FL95-01 (85.0, 70.9 and 85.1% identities, respectively) that do not have orthologous to WabK. Furthermore, as in E. ictaluri, waaL encodes a smaller protein (265 amino acids) than the WaaL of E. tarda EIB202, E. piscicida and E. anguillarum. This protein does not show homology with the WaaL of any E. piscicida or E. anguillarum strain, nor to E. tarda EIB202. However, it shows 75.5 to 78.5% homology to the WaaL of E. tarda strains containing the wahX in the waa cluster. These data suggest that E. ictaluri has a different and smaller core oligosaccharide structure than the other four Edwardsiella species. The data also suggest that the E. hoshinae core-oligosaccharide structure is different than that of E. tarda EIB202, E. piscicida and E. anguillarum but similar to E. tarda strains that lack the wabK gene (Figure 2).

Finally, it is known that the E. tarda EIB202 LPS motif β-D-Glc-(1→2)-α-L-HepII, corresponding to the WapG (ETAE_RS09105), is not encoded by any of the glycosyltransferases present in the waa cluster. To find orthologous proteins to this one, we analyzed the complete genomes of E. piscicida, E. anguillarum, E. ictaluri and E. hoshinae strains using BLASTp. Comparative genomic analyses using Mauve show that the genomic region containing this gene is highly variable in the Edwardsiella species (Figure 4). Orthologous to this protein were found in all E. piscicida strains, in the E. anguillarum strain C-5-1 and in E. tarda strains, whose waa cluster contains the wabK gene. However, neither strain of E. ictaluri, E. hoshinae nor E. tarda strains containing the wahX gene possess alleles orthologous to WapG. In order to confirm it, five contiguous genes above and under the wapG of E. tarda EIB202 were checked in these Edwardsiella species, and the results were either low query coverage and a high E-value or no significant similarity found.

2.2. Structural Analysis of Lipopolysaccharide of Edwardsiella Species

The LPSs of four Edwardsiella species were visually characterized by electrophoresis-gel tricine SDS-PAGE. The analyses showed that while E. tarda EIB202, E. piscicida HL9.1 and E. anguillarum 205/03 LPSs are smooth (S-LPS), E. ictaluri AL-15-01-CATFISH and E. hoshinae DSMZ 13771^T LPSs are smooth/rough (SR-LPS). However, E. ictaluri appears to have a significantly lower amount and number of repeating units than others (Figure 5).

The yields of LPSs from E. piscicida HL9.1, E. anguillarum 205/03, E. hoshinae DSMZ 13771^T and E. ictaluri AL-15-01-CATFISH bacterial masses were 0.5%, 1.0%, 0.9% and 1.1%, respectively. The mild acid hydrolysis of the E. piscicida and E. ictaluri LPSs yielded seven fractions, and the other E. anguillarum and E. hoshinae LPSs yielded eight fractions. The fractions consisting of unsubstituted core oligosaccharide (OS) were identified: in E. piscicida and E. hoshinae as fraction VI (OSVI), in E. anguillarum and E. ictaluri as fraction VII (OSVII). All investigations were carried out on OSVI and OSVII fractions isolated from Edwardsiella LPSs.

The comparison of anomeric regions of Edwardsiella cores (Figure 6) has shown different numbers of sugar residues.

2.2.1. Structural Analysis of E. piscicida HL9.1 and E. anguillarum 205/03 Core Oligosaccharides

The E. piscicida OSVI had the same structure as the E. anguillarum OSVII; thus, the data for OSVI is not presented herein to avoid unnecessary duplication. The ¹H−¹³C HSQC-DEPT spectrum of E. anguilarum OSVII contained main signals for eleven residues belonging to the core-oligosaccharide structure. The structural analysis showed the presence of →3,4)-L-glycero-α-D-manno-Hepp (residue B), two terminal β-D-Glcp (residues C and H), →2,3,7)-L-glycero-α-D-manno-Hepp (residue D), →7)-L-glycero-α-D-manno-Hepp (residue E), terminal α-D-GlcpN (residue G), two →4)-α-D-GalpA (residues F and I), → 3)-α-D-GlcpNAc (residue K), terminal β-D-Galp (residue M) and →5-substituted Kdop (residue A) (Figure 6,

Table 1. Chemical shifts of the E. anguillarum 205/03 core oligosaccharide (OSVII).

Residues	Chemical Shifts (ppm)
	H1/C1	H2/C2	H3(H3ax,eq)/C3	H4/C4	H5/C5	H6,6’/C6	H7,7’/C7	H8,8’/C8 (NAc)
A →5)-Kdo	nd	97.6	1.95, 2,32 34.6	4.19 66.3	4.25 75.3	3.77 69.8	3.92 66.7	3.70, 3.89 64.4
B →3,4)-L-glycero-α-D-manno-Hepp-(1→	5.17 101.3	4.14 69.8	4.32 75.0	4.31 74.2	4.25 71.9	4.15 70.2	3.82 63.7
C β-D-Glcp-(1→	4.55 103.3	3.38 74.4	3.51 76.3	3.49 69.9	3.45 76.5	3.86, 3.89 62.0
D →2,3,7)-L-glycero-α-D-manno-Hepp-(1→	5.44 99.6	4.34 78.5	4.11 80.0	4.11 66.6	3.69 73.3	4.28 69.3	3.63, 4.01 73.3
E →7)−L-glycero-α-D-manno-Hepp-(1→	4.97 103.4	4.05 71.1	3.95 72.3	3.93 71.4	3.72 73.4	4.23 69.5	3.69, 3.88 72.0
F →4)-α-D-GalpA-(1→	5.42 99.4	4.09 70.0	4.20 68.5	4.62 77.6	4.44 70.4	176.5
G α-D-GlcpN-(1→	5.27 95.5	3.36 55.4	4.00 70.4	3.65 68.3	4.14 73.3	3.68, 3.95 60.4
H β-D-Glcp-(1→	4.63 103.1	3.40 74.8	3.56 71.3	3.33 71.3	3.62 76.4	3.67, 3.92 62.0
I →4)-α-D-GalpA-(1→	5.42 102.5	3.92 69.9	4.29 72.2	4.48 80.9	4.63 72.4	175.5
J α-D-GlcpN-(1→	5.29 97.2	3.39 55.6	3.98 70.6	3.63 70.5	4.28 72.5	3.70, 3.98 62.5
K →3)-α-D-GlcpNAc-(1→ M β-D-Galp-(1→	5.20 99.6 4.53 103.7	4.27 51.5 3.61 71.3	3.78 75.4 3.79 72.2	3.64 71.1 3.90 71.2	4.03 71.3 3.59 75.3	3.82, 3.85 60.1 3.73, 3.77 63.1		2.13 22.7, 175.7
Gly	168.2	3.92, 4.07 41.8

ax, axial position; eq, equatorial position. nd, not detected.

).

The ¹H−¹H NOESY spectrum showed strong inter-residue cross-peaks between the transglycosidic protons: H−1 of B/H−5 of A, H−1 of D/H−3 of B, H−1 of C/H−4 of B, H−1 of E/H−7 of D, H−1 of H/H−2 of D, H−1 of F/H−7 of E, H−1 of G/H−4 of F, H−1 of I/H−3 of D, H−1 of J/H−4 of I, H−1 of K/H−4 of I and H−1 of M/H−3 of K. The presence of heterogeneity in OSVII was due to the partial presence of a structure with terminal α-D-GlcpN (residue J) instead of β-D-Galp-(1→3)-α-D-GlcpNAc-(1→ fragment (residues M and K). These analyses allowed for the establishment of the same structure of E. piscicida and E. anguillarum core oligosaccharides as were identified in E. tarda EIB202 [19].

2.2.2. Structural Analysis of E. hoshinae DSMZ 13771^T Core Oligosaccharide

The ¹H−¹³C HSQC-DEPT spectrum of the E. hoshinae OSVI contained main signals for ten anomeric protons and carbons and Kdo spin systems, respectively. The analysis of E. hoshinae OSVI spectra showed the presence of →3,4)-L-glycero-α-D-manno-Hepp (residue B), β-D-Glcp (residue C), →3,7)-L-glycero-α-D-manno-Hepp (residue D), →7)-L-glycero-α-D-manno-Hepp (residue E), two →4)-α-D-GalpA (residues F and I), two terminal α-D-GlcpN (residues G and J), →4)-α-D-GlcpNAc (residue K), terminal α-D-GlcpNAc (residue L) and →5-substituted Kdop (residue A) (Figure 6 and Figure 7,

Table 2. Chemical shifts of the E. hoshinae DSMZ 13771^T core oligosaccharide (OSVI).

Residues	Chemical Shifts (ppm)
	H1/C1	H2/C2	H3(H3ax,eq)/C3	H4/C4	H5/C5	H6,6’/C6	H7,7’/C7	H8,8’/C8 (NAc)
A →5)-Kdo	nd	nd	1.93, 2,26 34.2	4.12 66.0	4.21 74.7	3.73 69.8	3.90 66.4	3.74, 3.85 64.3
B →3,4)-L-glycero-α-D-manno-Hepp-(1→	5.20 101.0	4.15 69.8	4.29 75.3	4.32 74.5	4.20 71.7	4.17 70.3	3.81 63.4
C β-D-Glcp-(1→	4.48 103.5	3.38 74.7	3.53 76.0	3.39 70.1	3.29 75.9	3.76, 3.90 62.2
D →3,7)-L-glycero-α-D-manno-Hepp-(1→	5.42 99.7	4.32 74.8	4.13 80.2	4.12 66.9	3.71 73.3	4.24 69.3	3.64, 4.01 73.1
E →7)−L-glycero-α-D-manno-Hepp-(1→	4.96 102.8	4.06 71.3	3.91 72.2	3.97 71.2	3.79 73.1	4.25 69.1	3.71, 3.80 72.2
F →4)-α-D-GalpA-(1→	5.23 99.7	4.13 70.1	4.23 68.2	4.59 77.4	4.47 70.3	176.2
G α-D-GlcpN-(1→	5.33 96.1	3.34 55.0	4.04 70.0	3.69 68.1	4.11 73.2	3.71, 3.98 60.3
I →4)-α-D-GalpA-(1→	5.50 102.0	3.93 70.0	4.25 72.1	4.49 80.2	4.63 72.6	175.8
J α-D-GlcpN-(1→	5.29 96.5	3.42 55.7	3.86 70.3	3.68 70.2	4.20 72.3	3.72, 3.99 62.4
K →4)-α-D-GlcpNAc-(1→	5.11 99.6	4.29 52.5	3.64 73.3	3.58 74.6	4.261 71.9	3.91,3.94 60.8		2.09 22.7, 175.8
L α-D-GlcpNAc-(1→	5.21 99.7	4.36 51.6	3.62 71.5	3.64 73.4	4.11 71.1	3.85, 3.87 60.7		2.13 22.7, 175.8
Gly	168.2	3.92, 4.07 41.8

ax, axial position; eq, equatorial position. nd, not detected.

).

Residue A was identified as the 5-substituted Kdo on the basis of characteristic deoxy proton signals at δ_H 1.93 ppm (H−3ax) and δ_H 2.26 ppm (H−3eq), as well as a high chemical shift of the C−5 signal (δ_C 74.7 ppm). Residue B (δ_H/δ_C 5.20/101.0 ppm, ¹J_C-1,H-1 ~176 Hz) was recognized as the 3,4-disubstituted L-glycero-α-D-manno-Hepp residue on the basis of the small vicinal couplings between H−1, H−2 and H−3 and the relatively high chemical shifts of the C−3 (δ_C 75.3 ppm) and the C−4 (δ_C 74.5 ppm) signals. Residue C (δ_H/δ_C 4.48/103.5 ppm, ¹J_C-1,H-1 ~162 Hz) was recognized as the β-D-Glcp on the basis of the large vicinal couplings between all protons in the sugar ring. Residue D (δ_H/δ_C 5.42/99.7 ppm, ¹J_C-1,H-1 ~176 Hz) was recognized as the 3,7-disubstituted L-glycero-α-D-manno-Hepp residue from the ¹H and ¹³C chemical shift values, small vicinal couplings between H−1, H−2 and H−3, and relatively high chemical shift values of the C−3 (δ_C 80.2 ppm), and C−7 (δ_C 73.1 ppm) signals. Residue E (δ_H/δ_C 4.96/102.8 ppm, ¹J_C-1,H-1 ~172 Hz) was recognized as the 7-substituted L-glycero-α-D-manno-Hepp from the ¹H and ¹³C chemical shifts, the small vicinal couplings between H−1, H−2 and H−3, and the relatively high chemical shift value of the C−7 (δ_C 72.2 ppm) signal. Residue F (δ_H/δ_C 5.23/99.7 ppm, ¹J_C-1,H-1 ~176 Hz) was recognized as the 4-substituted α-D-GalpA based on the characteristic five proton spin system, the high chemical shifts of the H-3 (δ_H 4.23 ppm), H-4 (δ_H 4.59), H-5 (δ_H 4.47) and C-4 (δ_C 77.4 ppm) signals, the large vicinal couplings between H-2 and H-3 and small vicinal coupling between H−3, H−4 and H−5. Residue I (δ_H/δ_C 5.50/102.0 ppm, ¹J_C-1,H-1 ~176 Hz) was also recognized as the 4-substituted α-D-GalpA residue based on the similar characteristic five proton spin system. Residue G (δ_H/δ_C 5.33/96.1 ppm, ¹J_C-1,H-1 ~176 Hz) was recognized as the terminal α-D-GlcpN due to the large coupling between H−1, H−2 and H−3 and the small vicinal coupling between H−3, H−4 and H−5, as well as the chemical shift value of the C−2 (δ_C 55.0). Residue J (δ_H/δ_C 5.29/96.5 ppm, ¹J_C-1,H-1 ~176 Hz) was recognized as the terminal α-D-GlcpN due to the large coupling between H−1, H−2 and H−3 and the small vicinal coupling between H−3, H−4 and H−5, as well as the characteristic chemical shift value of the C−2 (δ_C 55.7 ppm). Residue K (δ_H/δ_C 5.11/99.6 ppm, ¹J_C-1,H-1 ~174 Hz) was recognized as the 4-substituted α-D-GlcpNAc from a low ¹³C chemical shift of the C-2 signal (δ_C 52.5 ppm), and of the C−4 signal (δ_C 75.7 ppm), and the large vicinal couplings between all ring protons. The N-acetyl group at δ_H/δ_C 2.09/22.7 ppm (δ_C 175.8 ppm) was identified. The terminal residue L (δ_H/δ_C 5.06/99.8 ppm, ¹J_C-1,H-1 ~172 Hz) was recognized as the α-D-GlcpNAc from a low ¹³C chemical shift of the C−2 signal (δ_C 51.6 ppm), and the large vicinal couplings between all ring protons. The N-acetyl group at δ_H/δ_C 2.13/22.7 ppm (δ_C 175.8 ppm) was identified. The presence of heterogeneity in OSVI was due to partial replacement of α-D-GlcpN (residue J) by α-D-GlcpNAc (residue K). The ³¹P NMR spectra showed no indication of phosphate groups in the OSVI.

In the HSQC-DEPT spectra of OSVI (at δ_H/δ_C 3.92, 4.07/41.8 ppm), additional negative CH₂ signals were detected. These resonances showed a correlation with carbonyl carbon signals at δ_C 168.1 ppm in the HMBC spectra, suggesting the presence of glycine.

The ¹H−¹H NOESY spectrum showed strong inter-residue cross-peaks between the following transglycosidic protons: H−1 of B/H−5 of A, H−1 of D/H−3 of B, H−1 of E/H−7 of D, H−1 of F/H−7 of E, H−1 of G/H−4 of F, H−1 of I/H−3 of D, H−1 of J/H−4 of I, H−1 of K/H−4 I, H−1 of L/H−4 of K. The linkage between H−1 of C/H−4 of B was not observed. Despite this the HMBC spectrum of OSVI confirmed the substitution positions of all monosaccharide residues. The cross-peaks between H−1 of K/H−4 of I, and H−1 of J/H−4 of I showed heterogeneity due to the presence of a core-oligosaccharide structure with terminal α-D-GlcpN (residue J) instead α-D-GlcpNAc-(1→4)-α-D-GlcpNAc-(1→) fragment (residues L and K).

2.2.3. Structural Analysis of E. ictaluri AL-15-01-CATFISH Core Oligosaccharide

The analysis of E. ictaluri OSVII showed the presence of →3,4)-L-glycero-α-D-manno–Hepp (residue B), terminal β-D-Glcp (residue C), →3,7)-L-glycero-α-D-manno-Hepp (residue D), L-glycero-α-D-manno-Hepp (residue E), →4-α-D-GalpA (residues I), →3-α-D-GlcpNAc (residue K), terminal β-D-Galp (residue M) and →5-substituted Kdop (residue A) (Figure 8, Table 3).

Residue A was identified as the 5-substituted Kdo on the basis of characteristic deoxy proton signals at δ_H 1.92/2.27 ppm (H-3ax/H-3eq) as well as a high chemical shift of the C−5 signal (δ_C 74.7 ppm). Residue B (δ_H/δ_C 5.06/102.2 ppm, ¹J_C-1,H-1 ~176 Hz) was recognized as the 3,4-disubstituted L-glycero-α-D-manno-Hepp residue on the basis of the small vicinal couplings between H−1, H−2 and H−3, and relatively high chemical shifts of the C−3 (δ_C 75.2 ppm) and the C−4 (δ_C 76.1 ppm) signals. Residue C (δ_H/δ_C 4.48/103.3 ppm, ¹J_C-1,H-1 ~162 Hz) was recognized as the β-D-Glcp on the basis of the large vicinal couplings between all protons in the sugar ring. Residue D (δ_H/δ_C 5.36/99.1 ppm, ¹J_C-1,H-1 ~176 Hz) was recognized as the 3,7-disubstituted L-glycero-α-D-manno-Hepp residue from the ¹H and ¹³C chemical shift values, small vicinal couplings between H−1, H−2 and H−3, and relatively high chemical shift values of the C−3 (δ_C 76.3 ppm), and C−7 (δ_C 70.8 ppm) signals. Residue E (δ_H/δ_C 4.96/101.5 ppm, ¹J_C-1,H-1 ~172 Hz) was recognized as the L-glycero-α-D-manno-Hepp from the ¹H and ¹³C chemical shifts, the small vicinal couplings between H−1, H−2 and H−3, and the relatively low chemical shift value of the C−7 (δ_C 63.9 ppm) signal. Residue K (δ_H/δ_C 5.00/98.4 ppm, ¹J_C-1,H-1 ~172 Hz) was recognized as the 3-substituted α-D-GlcpNAc from a low ¹³C chemical shift of the C−2 signal (δ_C 54.3 ppm), C−3 (δ_C 79.2 ppm) and the large vicinal couplings between all ring protons. The N-acetyl group at δ_H/δ_C 2.19/22.6 ppm (δ_C 175.2 ppm) was identified. The residue M (δ_H/δ_C 4.54/103.7 ppm, ¹J_C-1,H-1 ~162 Hz) was recognized as the terminal β-D-Galp due to the large vicinal couplings between H−1, H−2 and H−3 and the small vicinal couplings between H−3, H−4 and H−5. The ³¹P NMR spectra showed no indication of phosphate groups in the OSVI.

In the ¹H-¹³C HSQC-DEPT spectrum of E. ictaluri OSVII (at δ_H/δ_C 3.90, 4.09/41.6 ppm) additional negative CH₂ signals were detected. These resonances showed a correlation with carbonyl carbon signals at δ_C 168.2 ppm in the HMBC spectra, suggesting the presence of glycine.

¹H−¹H NOESY spectra showed strong inter-residue cross-peaks between the following transglycosidic protons: H−1 of D/H−3 of B, H−1 of E/H−7 of D, H−1 of I/H−3 of D, H−1 of K/H−4 of I and H−1 of M/H−3 of K (Figure 8C).

Table 3. Chemical shifts of the E. ictaluri AL-15-01-CATFISH core oligosaccharide (OSVII).

Residues	Chemical Shifts (ppm)
	H1/C1	H2/C2	H3(H3ax,eq)/C3	H4/C4	H5/C5	H6,6’/C6	H7,7’/C7	H8,8’/C8 (NAc)
A →5)-Kdo	nd	nd	1.92, 2,27 34.1	4.14 64.6	4.14 74.7	4.01 71.1	3.92 66.9	3.69, 3.89 63.8
B →3,4)-L-glycero-α-D-manno-Hepp-(1→	5.18 101.2	4.15 70.4	4.25 75.2	4.28 76.1	4.11 71.0	4.11 69.5	3.79 63.8
C β-D-Glcp-(1→	4.48 103.3	3.33 73.4	3.52 75.8	3.52 69.7	3.40 76.9	3.75, 3.88 61.2
D →3,7)-L-glycero-α-D-manno-Hepp-(1→	5.36 99.1	4.26 68.4	4.18 76.3	4.11 67.4	3.79 71.0	4.05 69.5	3.67, 3.89 70.8
E L-glycero-α-D-manno-Hepp-(1→	4.96 101.5	4.00 70.7	3.96 72.7	3.97 69.3	3.87 71.1	4.13 69.3	3.72, 3.92 63.9
I →4)-α-D-GalpA-(1→	5.57 100.8	3.93 69.7	4.18 68.7	4.11 77.9	4.76 71.3	175.1
K →3)-α-D-GlcpNAc-(1→ M β-D-Galp-(1→	5.00 98.4 4.54 103.7	4.06 54.3 3.62 71.5	3.84 79.2 3.72 73.3	4.01 71.1 3.97 69.2	4.30 71.3 3.67 75.9	3.82, 3.96 60.4 3.70, 3.73 61.1		(2.19) (22.6, 175.2)
Gly	168.4	3.90, 4.09 41.6

ax, axial position; eq, equatorial position; nd, not detected.

Table 3. Chemical shifts of the E. ictaluri AL-15-01-CATFISH core oligosaccharide (OSVII).

Residues	Chemical Shifts (ppm)
	H1/C1	H2/C2	H3(H3ax,eq)/C3	H4/C4	H5/C5	H6,6’/C6	H7,7’/C7	H8,8’/C8 (NAc)
A →5)-Kdo	nd	nd	1.92, 2,27 34.1	4.14 64.6	4.14 74.7	4.01 71.1	3.92 66.9	3.69, 3.89 63.8
B →3,4)-L-glycero-α-D-manno-Hepp-(1→	5.18 101.2	4.15 70.4	4.25 75.2	4.28 76.1	4.11 71.0	4.11 69.5	3.79 63.8
C β-D-Glcp-(1→	4.48 103.3	3.33 73.4	3.52 75.8	3.52 69.7	3.40 76.9	3.75, 3.88 61.2
D →3,7)-L-glycero-α-D-manno-Hepp-(1→	5.36 99.1	4.26 68.4	4.18 76.3	4.11 67.4	3.79 71.0	4.05 69.5	3.67, 3.89 70.8
E L-glycero-α-D-manno-Hepp-(1→	4.96 101.5	4.00 70.7	3.96 72.7	3.97 69.3	3.87 71.1	4.13 69.3	3.72, 3.92 63.9
I →4)-α-D-GalpA-(1→	5.57 100.8	3.93 69.7	4.18 68.7	4.11 77.9	4.76 71.3	175.1
K →3)-α-D-GlcpNAc-(1→ M β-D-Galp-(1→	5.00 98.4 4.54 103.7	4.06 54.3 3.62 71.5	3.84 79.2 3.72 73.3	4.01 71.1 3.97 69.2	4.30 71.3 3.67 75.9	3.82, 3.96 60.4 3.70, 3.73 61.1		(2.19) (22.6, 175.2)
Gly	168.4	3.90, 4.09 41.6

ax, axial position; eq, equatorial position; nd, not detected.

3. Disscussion

The Edwardsiella genus belongs to the order Enterobacteriales and family Hafniaceae. At present, this genus includes five species: E. tarda, E. piscicida, E. anguillarum, E. hoshinae and E. ictaluri. However, the description of E. piscicida [20] and E. anguillarum [21] resulted from a reclassification of diverse isolates previously identified as E. tarda. Until now, only the chemical analysis and genomics of the core oligosaccharide from E. tarda EIB202 have been described [19]. In this report, the chemical structure of the complete LPS-core of E. piscicida, E. anguillarum, E. hoshinae and E. ictaluri and the genomic regions (waa cluster) involved in its biosynthesis were presented. We identified orthologous genes to E. tarda EIB202 coaD and hldD in seven E. tarda, thirteen E. piscicida, two E. anguillarum, six E. ictaluri and two E. hoshinae strains with complete genome sequences available on the NCBI website to locate the waa gene cluster in each genome. As expected, the genes and genomic organization of this cluster were highly conserved in all species, encoding all the activities required for outer core assembly and the transferases needed for inner core oligosaccharide synthesis. However, comparative genomics of this region by Mauve software and a phylogenetic tree showed three different types of waa clusters in Edwardsiella. Two types contained a gene that encodes a glycosyltransferase orthologous to WabK and were found in all E. piscicida, E. anguillarum and E. ictaluri strains tested, as well as in three E. tarda strains. In E. tarda EIB202, WabK was described as a galactosyltransferase that incorporates a Galp residue in a β(1→4) to GlcpNAc. However, the E. ictaluri waa cluster had a deletion between wabK and wabH, so the cluster did not contain either wapC or part of wapB. The loss of this genomic fragment was detected in all the E. ictaluri strains whose genomes had been completely sequenced. The third type contained a gene that encodes a glycosyltransferase orthologous to WahX and was found in all E. hoshinae strains tested and in four E. tarda strains. Furthermore, the antigen O ligase encoded for the waaL included in these three waa clusters was not orthologous, and this could be related to the differential glycosyltransferase (WabK or WahX) encoded in each of these clusters.

In the last year, a revision of the taxonomic position of the isolates previously identified as E. tarda has been carried out, and some strains were reassigned as E. piscicida or E. anguillarum. This has been the case for E. tarda EIB202, FL6-60 and ET-001, which have been reclassified as E. piscicida [5]. The reclassification of these strains was in agreement with the phylogenetic tree generated by the neighbor-joining method on the basis of the waa cluster sequence that clusters them with E. piscicida strains. However, the remaining E. tarda strains genomically analyzed were clustered with E. hoshinae, and their waa clusters contain the same wahX gene as E. hoshinae strains.

In E. tarda EIB202, the ETAE_RS09105 gene was located outside the waa cluster and encodes the WapG transferase, which was involved in the LPS motif β-D-Glcp-(1→2)-α-L-HeppII. Orthologous to this protein only were found in E. piscicida or E. tarda, reclassified as E. piscicida, and in some E. anguillarum strains (C-5-1 strain). Comparative genomics showed that this chromosomal region was conserved in E. piscicida and E. anguillarum strains containing the orthologous ETAE_RS09105 gene but was highly variable in other Edwardsiella species.

The chemical structure of the core oligosaccharides carried out in E. piscicida HL9.1 and E. anguillarum 205/03 seems to be in agreement with the genomic studies, which determined that both strains have the same LPS-core as E. tarda EIB202 [19].

Genomic studies conducted by comparison with the BLASTp algorithm showed that E. ictaluri strains, such as E. piscicida and E. anguillarum, had an orthologous to the WabK but did not contain the genes encoding the WapC, WapB or WapG. WapB is an enzyme that transfers α-D-GlcpNAc to α-D-GalpA; WapC transfers α-D-GalpA to a Hepp; and WapG transfers β-D-Glcp to a Hepp, all of them in different acceptor substrates of LPS-core in a α(1→4), α(1→7) and α(1→2) linkage, respectively. The chemical structure of the core oligosaccharide carried out in E. ictaluri AL-15-01-CATFISH seems to be in agreement with the absence of these three genes.

Genomic studies conducted by comparison with the BLASTp algorithm showed that E. hoshinae strains did not have a gene that encodes a protein orthologous to WabK, in contrast to E. piscicida, E. anguillarum and E. ictaluri. However, they had a gene that encodes a N-acetylhexosamine glycosyltransferase (named WahX) that could be involved in the transfer of α-D-GlcpNAc to a α-D-GlcpNAc or α-D-GlcN in a different acceptor substrate of LPS-core in a α(1→4) linkage (Figure 6). Furthermore, E. hoshinae strains, such as E. ictaluri, did not have a gene encoding WapG. The presence of an orthologous to WahX and the absence of WapG were also determined in the E. tarda strains KC-Pc-HB1, FL95-01, ATCC15947 and AL98-87. The chemical structure of the core oligosaccharide carried out in E. hoshinae DSMZ 13771^T seemed to be in agreement with the absence of WabK and the presence of WahX.

Figure 9 shows the presumptive chemical structure and assignment of genes involved in the three Edwardsiella core oligosaccharide types. The first type was found in E. piscicida and some E. anguillarum strains, as well as in E. tarda EIB202. In E. anguillarum ET080813, no orthologous to WapG was detected, and maybe it does not have the H residue. The second type was found in E. ictaluri, and the third type was found in E. hoshinae strains.

4. Materials and Methods

4.1. Bacterial Strains and Culture Conditions

Bacterial strains from the Edwardsiella genus used in these experiments are described as follows: E. tarda EIB202 was obtained from the Y. Zhang laboratory [22]; E. piscicida HL9.1, E. hoshinae DSMZ 13771^T and E. anguillarum 205/03 strains were provided by the B. Magariños group in the University of Santiago de Compostela; and the E. ictaluri AL-15-01-CATFISH strain was provided by the C. R. Arias group in Auburn University. Edwardsiella strains were grown in nutrient broth (NB). While E. tarda and E. piscicida were grown at 37 °C, E. anguillarum, E. hoshinae and E. ictalurid were grown at 26 °C.

4.2. LPS Isolation and Electrophoresis

For screening purposes, LPS was obtained after proteinase K digestion of whole cells, and the LPS samples were separated by SDS-Tricine-PAGE as previously described and visualized by silver staining [23,24]. Two different methods based on the hot phenol-water method described by Westphal were performed in order to extract the LPS from bacterial cells [25]. The first variation was performed with E. piscicida and E. anguillarum, which produce an O-antigen with many repeating units. The LPS was extracted from the crude-cell envelope fraction according to the Westphal method, modified by Osborn [25,26].

4.3. Core Oligosaccharide Isolation

The LPS (20 mg) was heated with 1.5% acetic acid at 100 °C for 45 min. The precipitate (lipid A) was removed by centrifugation (20 min/12,000 rpm/4 °C). The supernatants, containing the mixture of poly- and oligosaccharides, were fractionated using a Bruker chromatographic system (amaZon SL, Berlin, Germany) on a Superdex G2500 column (7.5 mm × 60 cm) equilibrated with 0.05 M acetic acid and with a flow rate of 1 mL/min each fraction. Eluates were monitored with a Knauer differential refractometer, and all fractions were checked by NMR spectroscopy.

4.4. Instrumental Method

NMR spectroscopy. All NMR spectra were recorded on a Bruker Avance III 600 MHz spectrometer equipped with a 5 mm QCI cryoprobe with z-gradient. The measurements were performed at 298 K without simple spinning and using the acetone signal (𝛿_H/𝛿_C 2.225/31.05 ppm) as an internal reference. In the TOCSY experiments, the mixing times were 30, 60 and 100 ms. The NOESY experiment was performed with a mixing time of 200 ms, and the HMBC experiment with a delay of 80 ms. The data were acquired and processed using standard Bruker software (TopSpin 3.0). The processed spectra were assigned with the help of the SPARKY program [27].

4.5. Comparative Genomics

The complete assembled genome sequences of seven E. tarda, thirteen E. piscicida, two E. anguillarum, six E. ictaluri and two E. hoshinae strains available on the National Center for Biotechnology Information (NCBI) genome website were retrieved, and we performed a local tblastn of E. tarda EIB202 coaD and hldD (also referred to as rfaD) to locate the waa gene cluster in each genome. We also performed a local tblastn of E. tarda EIB202 ETAE_RS09105. The genomic regions containing these genes were compared through progressive Mauve software (https://darlinglab.org/mauve/mauve.html accessed on 3 November 2022) using the default parameters [28] and genes in the selected region were predicted with Glimmer v3.0.2 [29]. Alignment of genomic regions containing the waa cluster was performed by MUSCLE [30] of the EMBL-EBI website [31]. For each strain, the Genome Assembly and Annotation report was of interest in order to see the proteins corresponding to the core oligosaccharide part of the LPS. Protein domains were determined with the NCBI Conserved Domain Database (CDD) [32], and identities were inspected using the BLASTp network service at NCBI. Phylogenetic tree diagrams were generated by the neighbor-joining method using the MEGA-X software version 10.0.4 [33].