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).

Keywords: Edwardsiellaea; NMR spectroscopy; core oligosaccharide; genomic.

Figure 1

Comparative genomic analysis using progressive Mauve to identify the waa cluster on the chromosomes of E. hoshinae ATCC35051 (track 1), E. tarda KC-Pc-HB1 (track 2), E. tarda EIB202 (track 3), E. piscicida 18EpOKYJ (track 4), E. anguillarum ET080813 (track 5) and E. ictaluri S07-698 (track 6). Matching colors indicate homologous segments that are connected across genomes. Chromosomal regions inside blue squares contain waa clusters, and red squares show differences in comparison to E. tarda EIB202.

Figure 2

Phylogenetic tree generated by the neighbor-joining method on the basis of the waa cluster sequence.

Figure 3

(A) Comparison of the proteins present in the waa gene cluster of four different Edwardsiella species. (B) Schematic comparison of waa gene cluster models of Edwardsiella. Black arrows and dark gray color between sequences indicate identities higher than 80.0%. Gray arrows and a light gray color between sequences indicate identities higher than 70.0%. White arrows and no color between sequences indicate no identities. Striped arrows indicate deleted genes.

Figure 4

Comparative genomic analysis using progressive Mauve to identify the chromosomal region containing orthologous to wapG of E. tarda EIB202 in the chromosomes of E. hoshinae ATCC35051 (track 1), E. anguillarum ET080813 (track 2), E. tarda KC-Pc-HB1 (track 3), E. tarda EIB202 (track 4), E. piscicida 18EpOKYJ (track 5), E. anguillarum C-5-1 (track 6) and E. ictaluri S07-698 (track 7). Matching colors indicate homologous segments connected across genomes. Chromosomal regions inside black squares contain orthologous to wapG.

Figure 5

Edwardsiella lipopolysaccharides analyzed by tricine SDS-PAGE. E. tarda EIB202 (lane 1), E. piscicida HL9.1 (lane 2), E. ictaluri AL-15-01-CATFISH, (lane 3), E. hoshinae DSMZ 13771^T (lane 4) and E. anguillarum 205/03 (lane 5).

Figure 6

Anomeric regions of ¹H−¹³C HSQC-DEPT spectra of E. anguillarum 205/03, E. hoshinae DSMZ 13771^T and E. ictaluri AL-15-01-CATFISH core oligosaccharides.

Figure 7

(A,B) Selected regions of the ¹H−¹³C HSQC-DEPT and (C) ¹H−¹H NOESY spectra of the fraction OSVI of E. hoshinae DSMZ 13771^T core oligosaccharide.

Figure 8

(A,B) Selected regions of the ¹H−¹³C HSQC-DEPT and (C) ¹H−¹H NOESY spectra of the fraction OSVII of E. ictaluri AL-15-01-CATFISH core oligosaccharide.

Figure 9

(1) Chemical structure of the core oligosaccharide of E. piscicida HL9.1 and E. anguillarum 205/03, (2) E. ictaluri AL-15-01-CATFISH, and (3) E. hoshinae DSMZ 13771^T with presumptive assignment of genes involved in its biosynthesis.