Discovery and characterization ofde novosialic acid biosynthesis in the phylumFusobacterium

Abstract

Sialic acids are nine-carbon backbone carbohydrates found in prominent outermost positions of glycosylated molecules in mammals. Mimicry of sialic acid (N-acetylneuraminic acid, Neu5Ac) enables some pathogenic bacteria to evade host defenses. Fusobacterium nucleatum is a ubiquitous oral bacterium also linked with invasive infections throughout the body. We employed multidisciplinary approaches to test predictions that F. nucleatum engages in de novo synthesis of sialic acids. Here we show that F. nucleatum sbsp. polymorphum ATCC10953 NeuB (putative Neu5Ac synthase) restores Neu5Ac synthesis to an Escherichia coli neuB mutant. Moreover, purified F. nucleatum NeuB participated in synthesis of Neu5Ac from N-acetylmannosamine and phosphoenolpyruvate in vitro. Further studies support the interpretation that F. nucleatum ATCC10953 NeuA encodes a functional CMP-sialic acid synthetase and suggest that it may also contain a C-terminal sialic acid O-acetylesterase. We also performed BLAST queries of F. nucleatum genomes, revealing that only 4/31 strains encode a complete pathway for de novo Neu5Ac synthesis. Biochemical studies including mass spectrometry were consistent with the bioinformatic predictions, showing that F. nucleatum ATCC10953 synthesizes high levels of Neu5Ac, whereas ATCC23726 and ATCC25586 do not express detectable levels above background. While there are a number of examples of sialic acid mimicry in other phyla, these experiments provide the first biochemical and genetic evidence that a member of the phylum Fusobacterium can engage in de novo Neu5Ac synthesis.

Structure of the LPS O-chain from Fusobacterium nucleatum strain 10953, containing sialic acid

“…We have been undertaking a study to establish O-antigen structures of the F. nucleatum LPS in order to develop a better understanding of the surface glycochemistry of this emerging pathogen and recently established the O-antigen repeating unit structure for F. nucleatum subspecies nucleatum strain 25586 [16]. In this study, we performed structural characterization of the O-antigen of F. nucleatum subspecies polymorphum strain 10953 and identified sialic acid as a component of the O-antigen repeat, consistent with the recent identification of de novo sialic acid biosynthesis in this strain [17]. …”

Survival Mechanisms of Campylobacter hepaticus Identified by Genomic Analysis and Comparative Transcriptomic Analysis of in vivo and in vitro Derived Bacteria

“…Sialic acid has been demonstrated to shield pathogens from host immune responses by interacting with the sialic acid-binding proteins of the host. For example, Group B Streptococcus (GBS) can evade host responses and proliferate in blood due to capsular polysaccharide displaying sialic acid residues (Chang et al, 2014; Lewis et al, 2016). C. hepaticus harbors a sialic acid biosynthetic gene locus (UDP-GlcNAc converts to ManNAc, then converts to Neu5 Ac, followed by CMP-Neu5Ac, with the action of NeuC (EC 5.1.3.14), NeuB (EC 2.5.1.56) and NeuA (N-Acetylneuraminate cytidylyltransferase, EC 2.7.7.43, respectively).…”

Biological functions of sialic acid as a component of bacterial endotoxin

“…Free sialic acid (released by hydrolysis to the environment), can be used by bacteria in catabolic fermentation or oxidation (Vimr, 2013). Some bacteria such as E. coli K1, certain serotypes of N. meningitidis (Vimr, 2013), Fusobacterium (Lewis et al, 2016), or Campylobacter jejuni (Thomas, 2016) are able to synthesize sialic acid de novo, independently from the host.…”