Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Mon, 31 Dec 2018 09:38:25 The VC1777–VC1779 proteins are members of a sialic acid-specific subfamily of TRAP transporters (SiaPQM) and constitute the sole route of sialic acid uptake in the human pathogen Vibrio cholerae Nityananda Chowdhury, 1 Jessica Norris, 2 Erin McAlister, 2 S. Y. Kathy Lau, 2 Gavin H. Thomas 2 and E. Fidelma Boyd 1 Correspondence E. Fidelma Boyd fboyd@udel.edu Gavin H. Thomas gavin.thomas@york.ac.uk Received 28 March 2012 Revised 27 April 2012 Accepted 30 April 2012 1 Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA 2 Department of Biology (Area 10), University of York, York YO10 5YW, UK Sialic acids are nine-carbon amino sugars that are present on all mucous membranes and are often used by bacteria as nutrients. In pathogenic Vibrio the genes for sialic acid catabolism (SAC) are known to be important for host colonization, yet the route for sialic acid uptake is not proven. Vibrio cholerae contains a tripartite ATP-independent periplasmic (TRAP) transporter, SiaPQM (VC1777–VC1779), encoded by genes within the Vibrio pathogenicity island-2 (VPI-2), which are adjacent to the SAC genes nanA, nanE and nanK. We demonstrate a correlation of the occurrence of VPI-2 and the ability of Vibrio to grow on the common sialic acid N-acetyl- neuraminic acid (Neu5Ac), and that a V. cholerae N16961 mutant defective in vc1777, encoding the large membrane protein component of the TRAP transporter, SiaM, is unable to grow on Neu5Ac as the sole carbon source. Using the genome context and known structures of the SiaP protein component of the TRAP transporter, we define a subfamily of Neu5Ac-specific TRAP transporters, of which the vc1777–vc1779 genes are the only representatives in V. cholerae.A recent report has suggested that an entirely different TRAP transporter (VC1927–VC1929) is the Neu5Ac transporter in V. cholerae. Bioinformatics and genomic analysis suggest strongly that this is a C 4 -dicarboxylate-specific TRAP transporter, and indeed disruption of vc1929 results in a defect in growth on C 4 -dicarboxylates but not Neu5Ac. Together these data demonstrate unequivocally that the siaPQM-encoded TRAP transporter within VPI-2 is the sole sialic acid transporter in V. cholerae. INTRODUCTION Sialic acids are monocarboxylates of a family of nine- carbon a-keto amino monosaccharides, which are also known as neuraminic or nonulosonic acids. The most common member of this group, which is ubiquitous and widely studied, is N-acetylneuraminic acid (Neu5Ac or NANA). In eukaryotes, sialic acids are chemically bonded at the termini of numerous glycans and are found extensively in mucus-rich environments such as the oral cavity and respiratory, intestinal and vaginal tracts. Sialic acids in eukaryotes play pivotal roles in a diverse array of cell–cell and cell–molecule interactions, such as stabilizing glycoconjugates and cell membranes, and acting as che- mical messengers (Varki, 1992, 2008; Vimr et al., 2004). Sialic acids are also utilized by commensal and pathogenic bacteria in a number of ways; for example, several patho- genic species of bacteria have been shown to sialylate their cell surfaces to mask them from the host immune system (Varki, 1992, 2008; Vimr & Lichtensteiger, 2002; Vimr et al., 2004). Many bacteria, including Vibrio cholerae, are also able to utilize sialic acid as a sole carbon, nitrogen and energy source (Nees et al., 1976; Vimr & Troy, 1985; Chang et al., 2004; Severi et al., 2005; Steenbergen et al., 2005; Almagro-Moreno & Boyd, 2009a, b; Brigham et al., 2009). Vimr and colleagues showed that three key catabolic enzymes, (i) Neu5Ac lyase/aldolase (NanA), (ii) N-acetylmannosamine kinase (NanK) and (iii) ManNAc-6-phosphate epimerase (NanE), act sequentially to convert sialic acid (Neu5Ac) into N-acetylglucosamine 6-phosphate (GlcNAc-6-P) (Vimr & Troy, 1985; Vimr et al., 2004). GlcNAc-6-P is then converted by two enzymes encoded by nagA and nagB into fructose Abbreviations: Cm, chloramphenicol; DAP, diaminopimelic acid; Neu5Ac, N-acetylneuraminic acid; SAC, sialic acid catabolism; SAT, sialic acid transporter; SBP, substrate binding-dependent; Sm, streptomycin; SOE, splicing by overlap extension; TRAP transporter, tripartite ATP-independent periplasmic transporter; VPI-2, Vibrio pathogenicity island-2. Supplementary methods and five supplementary figures are available with the online version of this paper. Microbiology (2012), 158, 2158–2167 DOI 10.1099/mic.0.059659-0 2158 059659 G 2012 SGM Printed in Great Britain