Identification and functional characterization of the NanH extracellular sialidase from Corynebacterium diphtheriae

Integrative Omics Research Center, Korea Research Institute of Bioscience and Biotechnology, 52 Eoeun-dong, Yuseong-gu, Daejeon, 305-333, Korea.
Journal of Biochemistry (Impact Factor: 2.58). 12/2009; 147(4):523-33. DOI: 10.1093/jb/mvp198
Source: PubMed


Corynebacterium diphtheriae, a pathogenic Gram-positive bacterium, contains sialic acids on its cell surface, but no genes related to sialic acid decoration or metabolism have been reported in C. diphtheriae. In the present study, we have identified a putative sialidase gene, nanH, from C. diphtheriae KCTC3075 and characterized its product for enzyme activity. Interestingly, the recombinant NanH protein was secreted as a catalytically active sialidase into the periplasmic space in Escherichia coli, while the short region at its C-terminus was truncated by proteolysis. We reconstructed a truncated NanH protein (His(6)-NanH(DeltaN)) devoid of its signal sequence as a mature enzyme fused with the 6xHis tag at the N-terminal region. The purified His(6)-NanH(DeltaN) can cleave alpha-2,3- and alpha-2,6-linked sialic acid from sialic acid-containing substrates. In addition, even though the efficiency was low, the recombinant His(6)-NanH(DeltaN) was able to catalyse the transfer of sialic acid using several sialoconjugates as donor, suggesting that the reversible nature of C. diphtheriae NanH can be used for the synthesis of sialyl oligosaccharides via transglycosylation reaction.

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    • "In fact, C. diphtheriae exposes sialic acids on its outer surface [82]. Sialidase activity was first identified in a crude preparation of diphtheria toxin [106], and sialidase production and composition of cell surface carbohydrates of C. diphtheriae seem to be directly depending on iron concentration in the medium [107–109]. A putative exosialidase, designated NanH, was identified in C. diphtheriae. "
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    ABSTRACT: To date the genus Corynebacterium comprises 88 species. More than half of these are connected to human and animal infections, with the most prominent member of the pathogenic species being Corynebacterium diphtheriae, which is also the type species of the genus. Corynebacterium species are characterized by a complex cell wall architecture: the plasma membrane of these bacteria is followed by a peptidoglycan layer, which itself is covalently linked to a polymer of arabinogalactan. Bound to this, an outer layer of mycolic acids is found which is functionally equivalent to the outer membrane of Gram-negative bacteria. As final layer, free polysaccharides, glycolipids, and proteins are found. The composition of the different substructures of the corynebacterial cell envelope and their influence on pathogenicity are discussed in this paper.
    01/2013; 2013(5):935736. DOI:10.1155/2013/935736
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    • "Some enzymatic properties of this enzyme were characterized in a previous study indicating that this thermo-labile protein has a temperature optimum of 37°C and hydrolyses substrates such as horse serum glycoproteins [51]. The homologous enzyme from C. diphtheriae was characterized previously and shown to contain neuraminidase and trans-sialidase activities [52,53]. In principle, neuraminidases are a distinct class of glycosyl hydrolases that catalyze the removal of terminal sialic acids from various glycoconjugates and contribute to the recognition of sialic acids exposed on host cell surfaces, whereas trans-sialidases can be used for the decoration of various acceptor molecules on the cell surface to enable the invasion of host cells under certain conditions [54]. "
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    ABSTRACT: Corynebacterium ulcerans has been detected as a commensal in domestic and wild animals that may serve as reservoirs for zoonotic infections. During the last decade, the frequency and severity of human infections associated with C. ulcerans appear to be increasing in various countries. As the knowledge of genes contributing to the virulence of this bacterium was very limited, the complete genome sequences of two C. ulcerans strains detected in the metropolitan area of Rio de Janeiro were determined and characterized by comparative genomics: C. ulcerans 809 was initially isolated from an elderly woman with fatal pulmonary infection and C. ulcerans BR-AD22 was recovered from a nasal sample of an asymptomatic dog. The circular chromosome of C. ulcerans 809 has a total size of 2,502,095 bp and encodes 2,182 predicted proteins, whereas the genome of C. ulcerans BR-AD22 is 104,279 bp larger and comprises 2,338 protein-coding regions. The minor difference in size of the two genomes is mainly caused by additional prophage-like elements in the C. ulcerans BR-AD22 chromosome. Both genomes show a highly similar order of orthologous coding regions; and both strains share a common set of 2,076 genes, demonstrating their very close relationship. A screening for prominent virulence factors revealed the presence of phospholipase D (Pld), neuraminidase H (NanH), endoglycosidase E (EndoE), and subunits of adhesive pili of the SpaDEF type that are encoded in both C. ulcerans genomes. The rbp gene coding for a putative ribosome-binding protein with striking structural similarity to Shiga-like toxins was additionally detected in the genome of the human isolate C. ulcerans 809. The molecular data deduced from the complete genome sequences provides considerable knowledge of virulence factors in C. ulcerans that is increasingly recognized as an emerging pathogen. This bacterium is apparently equipped with a broad and varying set of virulence factors, including a novel type of a ribosome-binding protein. Whether the respective protein contributes to the severity of human infections (and a fatal outcome) remains to be elucidated by genetic experiments with defined bacterial mutants and host model systems.
    BMC Genomics 07/2011; 12(1):383. DOI:10.1186/1471-2164-12-383 · 3.99 Impact Factor
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    • "Neuraminidases, or sialidases, belong to a class of glycosyl hydrolases that catalyze the removal of terminal sialic acid residues from a variety of glycoconjugates and can contribute to the recognition of sialic acids exposed on host cell surfaces [82,83]. The homologous counterpart of NanH was recently characterized in C. diphtheriae KCTC3075 and shown to be a protein containing neuraminidase and trans-sialidase activities [84]. Trans-sialidases located on the bacterial cell surface can be used for the decoration of sugar moiety acceptors with sialic acid to enable the invasion of hosts under certain conditions. "
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    ABSTRACT: Corynebacterium pseudotuberculosis is generally regarded as an important animal pathogen that rarely infects humans. Clinical strains are occasionally recovered from human cases of lymphadenitis, such as C. pseudotuberculosis FRC41 that was isolated from the inguinal lymph node of a 12-year-old girl with necrotizing lymphadenitis. To detect potential virulence factors and corresponding gene-regulatory networks in this human isolate, the genome sequence of C. pseudotuberculosis FCR41 was determined by pyrosequencing and functionally annotated. Sequencing and assembly of the C. pseudotuberculosis FRC41 genome yielded a circular chromosome with a size of 2,337,913 bp and a mean G+C content of 52.2%. Specific gene sets associated with iron and zinc homeostasis were detected among the 2,110 predicted protein-coding regions and integrated into a gene-regulatory network that is linked with both the central metabolism and the oxidative stress response of FRC41. Two gene clusters encode proteins involved in the sortase-mediated polymerization of adhesive pili that can probably mediate the adherence to host tissue to facilitate additional ligand-receptor interactions and the delivery of virulence factors. The prominent virulence factors phospholipase D (Pld) and corynebacterial protease CP40 are encoded in the genome of this human isolate. The genome annotation revealed additional serine proteases, neuraminidase H, nitric oxide reductase, an invasion-associated protein, and acyl-CoA carboxylase subunits involved in mycolic acid biosynthesis as potential virulence factors. The cAMP-sensing transcription regulator GlxR plays a key role in controlling the expression of several genes contributing to virulence. The functional data deduced from the genome sequencing and the extended knowledge of virulence factors indicate that the human isolate C. pseudotuberculosis FRC41 is equipped with a distinct gene set promoting its survival under unfavorable environmental conditions encountered in the mammalian host.
    BMC Genomics 12/2010; 11(1):728. DOI:10.1186/1471-2164-11-728 · 3.99 Impact Factor
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