Identification and functional analysis of sialic acid metabolism in Vibrio vulnificus

Lubin, Jean-Bernard
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University of Delaware
Vibrio vulnificus is a Gram-negative, rod shaped, Gammaproteobacteria, found in estuarine and coastal waters throughout the world. V. vulnificus is a disease causing agent in fish and humans. It causes a severe and rapid septicemia, contracted primarily through raw oyster consumption, with a mortality rate of over 50% in susceptible individuals. The mechanisms by which V. vulnificus colonizes the host gastrointestinal tract and survives and proliferates in the host bloodstream are poorly understood. Sialic acids (neuraminic acids) also known as nonulosonic acids, are a diverse family of nine carbon amino sugars, the most common and widely studied being N -acetylneuraminic acid, or Neu5Ac. Crucial to cell to cell communication and self-recognition in metazoans, sialic acids are typically positioned at the terminal end of glycoconjugates allowing them to interact with the external environment. The self-recognition function of sialic acid has been shown to be crucial as a modulator of immune function. Sialic acid is also a major component of mucin and thus is found throughout the mucosal layer of the gastrointestinal tract of mammalian species. Bacteria have the ability to catabolize sialic acids as a sole carbon, nitrogen and energy source. This ability is overwhelmingly confined to commensal and pathogen species, where it has been shown to confer a competitive advantage in the host environment. Within bacteria, the ability to synthesize sialic acid has been demonstrated in a number of human pathogens and is phylogenetically widespread through diverse lineages. In addition to the canonical Neu5Ac, bacterial specific sialic acid-like molecules, legionaminic acid, and pseudaminic acid, are known. Several organisms relevant in human disease have been shown to decorate their surfaces with sialic acid to aid in host colonization and avoidance of immune responses. Preliminary investigations revealed that V. vulnificus could catabolize sialic acid and that disruption in this pathway lead to defects in intestinal colonization. It was also determined that V. vulnificus contained a putative sialic acid biosynthetic gene cluster. We hypothesized that sialic acid catabolism in V. vulnificus plays an important role in this organism's pathogenicity. We also believed that V. vulnificus utilizes sialic acid biosynthesis to decorate its cell surface, promoting survival in the host bloodstream. Initial genomic analysis of V. vulnificus clinical strains found the sialic acid catabolism (SAC) and transport (SAT) cluster present on chromosome II, and unlike Vibrio cholerae , is not associated with a pathogenicity island. However, the region was present predominantly among lineage I of V. vulnificus , which is comprised mainly of clinical isolates. We demonstrated that the isolates that contain this region can catabolize sialic acid as a sole carbon source and that the tripartite ATP-independent periplasmic (TRAP) transporter SiaPQM is essential for sialic acid uptake. Expression analysis of the SAT and SAC genes indicates that sialic acid is an inducer of expression. Overall, our study demonstrates that the ability to catabolize and transport sialic acid is predominately lineage specific in V. vulnificus , found predominantly in genotypically clinical isolates. Next we investigated the distribution, diversity and function of sialic acid biosynthesis in V. vulnificus . We found that 44% of the Vibrionaceae species examined contained sialic acid biosynthetic ( nab ) clusters. Evidence of duplication, divergence, horizontal transfer, and recombination was widespread in this region and biochemical analyses confirmed production of di-N -acetylated sialic acid-like sugars. V. vulnificus clinical isolates CMCP6 and YJ016, were found to contain two highly divergent clusters. PCR genotyping found the CMCP6-like cluster to be found overwhelmingly in the clinical, lineage I, isolates. Isolates containing the CMCP6-like alleles produced 40-fold higher levels of sialic acids than YJ016-like alleles associated with environmental isolates. Studies into the functional significance of biosynthesis in V. vulnificus found the LPS to be decorated with sialic acid residues. Expression of sialic acid was found to be is required for optimal motility, flagellar formation and biofilm formation. Competition experiments in a mouse model of septicemia reveal a significant 300-fold decrease in survival of the sialic acid synthase ( nab2 ) mutant strain versus the CMCP6 wild type. Competition experiments conducted in the YJ016 background, which produces less sialic acid, revealed significantly less attenuation in the mutant strain when compared to CMCP6. In summary, these data demonstrate a key biological function of sialic acid molecules in V. vulnificus and provides insight on how this species survives in the host bloodstream.