Cloning and sequencing of a <i>Clostridium perfringens</i> sialidase gene

Roggentin, Peter; Rothe, Bernd; Lottspeich, Friedrich; Schauer, Roland

doi:10.1016/0014-5793(88)80219-9

Cited by 84 publications

(46 citation statements)

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“…A distinctive feature found in the M. viridifaciens neuraminidase is that it possesses a large C-terminal region downstream of the last Asp box. The amino acid sequence in the N-terminal region shows similarity to that of the C. perfringens neuraminidase (24).…”

Section: Resultsmentioning

confidence: 85%

“…Recently, neuraminidase genes were cloned from several pathogenic microorganisms, including Clostridium sordellii G12 (25), Clostridium perfringens A99 (24), Salmonella typhimurium (23), Vibrio cholerae 395 (29), and Bacteroides fragilis TAL2480 (26). From examination of their deduced amino acid sequences, a conserved sequence of 12 amino acids (Asp box) which was repeated at four or five positions was found (23).…”

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confidence: 99%

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Cloning, expression, and characterization of the Micromonospora viridifaciens neuraminidase gene in Streptomyces lividans

1992

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We have cloned the Micromonospora viridifaciens neuraminidase (EC 3.2.1.18) gene (nedA) in Streptomyces fividans. This was accomplished by using the vector pU7O2 and BgM-BclI libraries of M. viridifaciens chromosomal inserts created in S. fividans. The libraries were screened for the expression of neuraminidase by monitoring the cleavage of the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid. Positive clones (BG6, BG7, BC4, and BC8) contained the identical 2-kb BclI-BgI fragment and expressed neuraminidase efficiently and constitutively using its own promoter in the heterologous host. From the nucleotide sequence analysis, an open reading frame of 1,941 bp which encodes a polypeptide with an M, of 68,840 was detected. The deduced amino acid sequence has five Asp boxes, -Ser-X-Asp-X-Gly-XThr-Trp, showing great similarity to other bacterial and viral neuraminidases. We have also identified the catalytic domain by using truncated proteins produced in S. fividans. Neuraminidase (EC 3.2.1.18) is a glycosidase which cleaves an a-ketosidic linkage between sialic acid and the adjacent sugar residue on glycoproteins, glycolipids, and oligosaccharides. It is widely distributed in microorganisms such as viruses, bacteria, and actinomycetes and in various tissues from fowls and mammals. For myxovirus it is an important surface antigen and also plays a role in facilitating the movement of the virus both to and from the site of infection (1). Bacteria and actinomycetes which produce neuraminidase can release sialic acids from glycoconjugates for use as carbon and energy sources. In pathogenic microorganisms, neuraminidase activity has a strong correlation with pathogenesis (27).Recently, neuraminidase genes were cloned from several pathogenic microorganisms, including Clostridium sordellii G12 (25), Clostridium perfringens A99 (24), Salmonella typhimurium LT-2 (23), Vibrio cholerae 395 (29), and Bacteroides fragilis TAL2480 (26). From examination of their deduced amino acid sequences, a conserved sequence of 12 amino acids (Asp box) which was repeated at four or five positions was found (23). This conserved sequence shows similarity to the neuraminidase of influenza virus A H7N1 and H13N9. The structures of neuraminidases from nonpathogenic bacteria have not been characterized. In a recent report, neuraminidase was widely found in culture fluids of actinomycetes (3). For Micromonospora spp., 10 of the 39 strains examined exhibited neuraminidase activity. Micromonospora viridifaciens showed the highest neuraminidase activity among this group. Neuraminidase from M.viridifaciens was purified, and its properties were analyzed (2). Neuraminidase activity in M. viridifaciens was detected in the culture broth only when a substrate such as colominic acid or the product N-acetylneuraminic acid was present in the culture medium (2).We would like to understand the regulation of neuraminidase gene (ned4) expression and the properties of its * Corresponding author. product in M. viridifaciens and ...

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Section: Resultsmentioning

confidence: 85%

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confidence: 99%

Cloning, expression, and characterization of the Micromonospora viridifaciens neuraminidase gene in Streptomyces lividans

1992

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“…On the other hand, the NeuAc residues linked α2 3 to the terminal Gal of G M" and G M$ (Neu5Acα3Galβ4GlcβCer) as well as that linked α2 8 to another NeuAc are quite susceptible to various microbial and mammalian sialidases [1]. Among several well-studied microbial sialidases, those from Clostridium perfringens [4] and Vibrio cholerae [5] have been most commonly used for in itro cleavage of the NeuAc from sialoglycoconjugates. The sialidase activity in mammalian tissues was first reported by Warren and Spearing in 1960 [6] and the distribution of this enzyme in mammalian organs was surveyed by Carubelli as early as 1962 [7].…”

Section: Introductionmentioning

confidence: 99%

Degradation of GM1 and GM2 by mammalian sialidases

Moriya

et al. 2001

Biochemical Journal

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In mammalian tissues, the pathway known for the catabolism ofGM1[Galβ3GalNAcβ4(Neu5Acα3)Galβ4GlcCer;where Cer is ceramide] is the conversion of this ganglioside into GM2 [GalNAcβ4(Neu5Acα3)Galβ4GlcβCer] by β-galactosidase followed by the conversion of GM2 into GM3 (Neu5Acα3Galβ4GlcβCer) by β-N-acetylhexosaminidase A (Hex A). However, the question of whether or not GM1 and GM2 can also be respectively converted into asialo-GM1 (Galβ3GalNAcβ4Galβ4GlcCer; GA1) and asialo-GM2 (GalNAcβ4Galβ4GlcβCer, GA2) by mammalian sialidases has not been resolved. This is due to the fact that sialidases purified from mammalian tissues always contained detergents that interfered with the in vitro hydrolysis of GM1 and GM2 in the presence of an activator protein. The mouse model of human type B Tay–Sachs disease created by the disruption of the Hexa gene showed no neurological abnormalities, with milder clinical symptoms than the human counterpart, and the accumulation of GM2 in the brains of affected mice was only limited to certain regions [Sango, Yamanaka, Hoffmann, Okuda, Grinberg, Westphal, McDonald, Crawley, Sandhoff, Suzuki and Proia (1995) Nat. Genet. 11, 170–176]. These results suggest the possible presence of an alternative catabolic pathway (the GA2 pathway) in mouse to convert GM2 into GA2 by sialidase. To show the existence of this pathway, we have used recombinant mammalian cytosolic sialidase and membrane-associated sialidase to study the desialylation of GM1 and GM2. We found that the mouse membrane-bound sialidase was able to convert GM1 and GM2 into their respective asialo-derivatives in the presence of human or mouse GM2 activator protein. The cytosolic sialidase did not exhibit this activity. Our results suggest that, in vivo, the stable NeuAc of GM1 and GM2 may be removed by the mammalian membrane-associated sialidase in the presence of GM2 activator protein. They also support the presence of the GA2 pathway for the catabolism of GM2 in mouse.

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“…They are involved in recognition processes, connection with ECM components, and intercellular interactions (1 (2,36,42,48). Deprotected polymers promote further enzymatic degradation of the ECM to release potential bacterial nutrients (26,38). As predicted from the presence of nanI, the M. alligatoris genome also encodes a sialic acid lyase (nanA; GenBank accession number AY515696), which has not been found in any other mycoplasma characterized to date, that could catalyze the release of pyruvate from sialic acid, liberating N-acetylglucosamine for entry into glycolysis as described above.…”

Section: Discussionmentioning

confidence: 99%

Spreading Factors of Mycoplasma alligatoris , a Flesh-Eating Mycoplasma

Brown¹,

Zacher²,

Farmerie

2004

J Bacteriol

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Mycoplasma alligatoris causes lethal invasive disease of alligators and caimans. A homolog of the nagH gene, encoding a hyaluronidase secreted by Clostridium perfringens, and a C. perfringens hyaluronidase nagI or nagK pseudogene were discovered in the M. alligatoris genome. The nagH gene was detected by PCR in the closest relative of M. alligatoris, Mycoplasma crocodyli, but not in 40 other species representing the Mycoplasma hominis, Mycoplasma pneumoniae, and Spiroplasma phylogenetic clusters. The hyaluronidase activity in the cellular fraction of M. alligatoris and M. crocodyli SP4 broth cultures was equivalent to 10 ؊16 U of Streptomyces hyalurolyticus hyaluronidase CFU ؊1 . Negligible activity was present in the cell-free supernatant fraction. No chondroitinase activity was detected. There is also a novel homolog of the nanI gene, which encodes a sialidase secreted by C. perfringens, in the M. alligatoris genome. The signature YRIP and SXDXGXTW motifs and catalytic residues of the clostridial sialidase are conserved in the mycoplasmal gene, but the leader sequence necessary for its secretion by C. perfringens is absent. The gene was not detected by PCR in any other mycoplasma. Potent cell-associated sialidase activity was present in M. alligatoris colonies on agar but not in the cell-free supernatants of broth cultures or in M. crocodyli. The presence of hyaluronidase and sialidase in M. alligatoris is consistent with the rapid invasiveness and necrotizing effects of this organism, and the lack of sialidase in M. crocodyli is consistent with its comparatively attenuated virulence. This genetic and biochemical evidence suggests that the spreading factors hyaluronidase and sialidase, a combination unprecedented in mycoplasmas, are the basis of the virulence of M. alligatoris.Mycoplasma alligatoris causes a lethal invasive disease in adult alligators (Alligator mississippiensis) and closely related caimans (Caiman latirostris). The pathology observed as early as 1 week after infection by instillation via the glottis includes necrotizing pneumonia, severe pericarditis, necrotizing myocarditis, lymphocytic interstitial nephritis, lymphocytic periportal hepatitis, splenic hyperplasia, pyogranulomatous meningitis, and necrotizing synovitis (9,10,11,13,18,33). Hatchling alligators also rapidly developed disseminated M. alligatoris mycoplasmosis after intratracheal instillation, and the lesions are similar to those of adults and include acute multifocal brainstem hemorrhage (35). Mycoplasma crocodyli (23), the closest known relative of M. alligatoris (98% 16S rRNA gene similarity [9]), causes a similar necrotizing synovitis and occasionally subacute pneumonia in Nile crocodiles (Crocodylus niloticus) (27,28). The severity of the lesions correlates with the numbers of M. alligatoris cells in affected tissues, suggesting that a spreading factor(s) contributes to the virulence. The extracellular matrix (ECM)-degrading enzymes that act as bacterial spreading factors include hyaluronidases, sialidases, and mucinases (26...

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Cloning and sequencing of a Clostridium perfringens sialidase gene

Cited by 84 publications

References 11 publications

Cloning, expression, and characterization of the Micromonospora viridifaciens neuraminidase gene in Streptomyces lividans

Cloning, expression, and characterization of the Micromonospora viridifaciens neuraminidase gene in Streptomyces lividans

Degradation of GM1 and GM2 by mammalian sialidases

Spreading Factors of Mycoplasma alligatoris , a Flesh-Eating Mycoplasma

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