Characterization of structurally diverse nonulosonic acids biosynthesized by Vibrio vulnificus
Date
2018
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Nonulosonic acids (NulOs) are a diverse family of nine carbon α-keto acid sugars that are involved in a wide range of functions across all branches of life. The family of NulOs is divided into two subgroups: sialic acids and prokaryotic-specific NulOs. The most common sialic acid is N-acetylneuraminic acid (Neu5Ac), although there are over 40 known structural variants of sialic acids characterized. The sialic acids are an important group of carbohydrates, and are involved in many cell-cell communications and innate immunity regulation within humans. Many bacteria and archaea biosynthesize the sialic acid Neu5Ac and the ability to produce this sugar and incorporation into cell surface structures has been implicated in a variety of host-pathogen interactions. In addition to sialic acid, bacteria have the ability to biosynthesize prokaryotic specific NulOs, of which there are seven known isomers characterized. These prokaryotic NulOs are similar in structure to Neu5Ac but little is known regarding their role in host-pathogen interactions. ☐ Nonulosonic acids are present in a large number of non-pathogenic species and little is known regarding their function in an environmental setting. In particular, all strains of the marine species Vibrio vulnificus are capable of NulO biosynthesis with at least three distinct NulO gene clusters identified within the species. Previous work has shown that NulOs decorate the lipopolysaccharide of two V. vulnificus strains and deletion of NulO biosynthesis genes results in defects in optimal motility, biofilm formation and antimicrobial peptide resistance. Despite this, the structural and functional diversity of the V. vulnificus NulO glycans are unknown. We demonstrate that the NulO produced by a clinical V. vulnificus strain CMCP6 is 5-N-acetyl-7-N-acetyl-D-alanyl-legionaminic acid (Leg5Ac7AcAla). The biosynthesis of Leg5Ac7AcAla contributed to maintaining membrane integrity, as mutant strains unable to produce or incorporate Leg5Ac7AcAla into the LPS have increased membrane permeability, sensitivity to bile-salts and antimicrobial peptides, and defects in biofilm formation. Using the crustacean model, Artemia franciscana, we demonstrate that Leg5Ac7AcAla-deficient bacteria have decreased virulence potential compared with wild type. Additionally, bioinformatics analysis suggested that the Leg5Ac7AcAla biosynthesis appears to be present in several bacterial species. Together, our data indicate that different V. vulnificus strains produce multiple NulOs and that the modified legionaminic acid Leg5Ac7AcAla plays a critical role in the physiology, survivability and pathogenicity of V. vulnificus CMCP6. ☐ Next, we characterize genetically and functionally the enzymes involved in the biosynthetic pathway of Leg5Ac7AcAla in V. vulnificus CMCP6. A putative N-acetyltransferase encoded by nab5 was identified and predicted to be involved in the modification of the legionaminic acid in V. vulnificus. Deletion of the nab5 gene resulted in the loss of Leg5Ac7AcAla production suggesting this enzyme is required for the biosynthesis of this NulO. Physiological characterizations of the nab5 deletion mutant demonstrated defects in motility and biofilm formation. Additionally, this mutant has increased sensitivity to the AMP polymyxin B but interestingly, this defect was not as significant as what is observed for other mutants in the NulO biosynthesis pathway of CMCP6. Bioinformatics analyses of the putative Leg5Ac7AcAla pathway provided evidence for the function for the remaining enzymes in the pathway as well as three enzymes of unknown function. ☐ We investigated the significance of bacterial catabolism of the sialic acid Neu5Ac. It was demonstrated that the major human pathogen V. cholerae, the causative agent of the gastrointestinal disease cholera, can grow efficiently on intestinal mucus and its component sialic acids. A tripartite ATP-independent periplasmic SiaPQM strain, transporter deficient mutant, was attenuated for colonization using a streptomycin-pretreated adult mouse model. The transporter mutant was also significantly outcompeted in in vitro competition assays in mouse intestinal mucus, indicating that sialic acid uptake is essential for fitness. Phylogenetic analyses demonstrated that the ability to utilize sialic acid was distributed among 452 bacterial species from eight phyla. The majority of species belonged to four phyla, Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria, mostly commensals and/or pathogens. Overall, our data demonstrate that the ability to take up host-derived sugars and sialic acid specifically allows V. cholerae a competitive advantage in intestinal colonization and that this is a trait that is sporadic in its occurrence and phylogenetic distribution and ancestral in some genera but horizontally acquired in others.