Cloning and characterization of neuraminidase from Salmonella typhimurium LT-2 and its relationship to neuraminidase from other bacteria
Hoyer, Lois Lawrisuk
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https://hdl.handle.net/2142/21308
Description
Title
Cloning and characterization of neuraminidase from Salmonella typhimurium LT-2 and its relationship to neuraminidase from other bacteria
Author(s)
Hoyer, Lois Lawrisuk
Issue Date
1989
Doctoral Committee Chair(s)
Vimr, Eric R.
Department of Study
Veterinary Medicine
Discipline
Veterinary Biosciences
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Genetics
Biology, Microbiology
Language
eng
Abstract
Bacterial neuraminidases (NANases) have been proposed to be bacterial virulence factors because of their ability to cleave sialic acid residues from host glycoconjugates. The gene encoding NANase (nanH) from Salmonella typhimurium has been cloned and its protein product purified. The purified NANase is 41.3 kD with an isoelectric point of approximately 9.0. The enzyme does not require Ca$\sp{++}$ for maximal activity and is not affected by EDTA or EGTA. S. typhimurium NANase is insensitive to N-acetylneuraminic acid (NeuNAc) and dithiothreitol. Compared to many previously studied NANases, the S. typhimurium NANase is relatively unaffected by the competitive inhibitor, 2-deoxy-2, 3-dehydro-NeuNAc. NANase activity is maximal at a sodium chloride concentration of approximately 100 mM and is not altered in the presence of increasing ionic strength. K$\sb{\rm m}$ and V$\sb{\rm max}$ values for the synthetic substrate 4-methylumbelliferyl-NeuNAc are 0.24 mM and 5200 nmol/min, respectively. In general, $\alpha$-(2-3)-linked sialic acid glycosides are preferentially cleaved by the S. typhimurium NANase. The enzyme hydrolyzes ganglioside substrates more rapidly than the glycoprotein or polysaccharide substrates assayed.
S. typhimurium nanH consists of 1128 nucleotides and encodes a 376 amino acid protein. Cellular localization studies indicate that 78% of this enzyme is cytoplasmic and 21% periplasmic. The amino acid sequence of S. typhimurium NANase shares 33% identity with the amino acid sequence of the small NANase of C. perfringens. Secondary structure predictions indicate that both NANases are rich in $\beta$-sheet structure. The only previously described NANase of molecular weight similar to the S. typhimurium and C. perfringens enzymes is that of C. sordellii.
Preliminary experiments to study the regulation of NANase production in S. typhimurium demonstrate that the enzyme is positively regulated by the catabolite repression system. Tn1000 insertions located 3.5 to 5.5 kb 5$\sp\prime$ of nanH decrease NANase activity by approximately 95%. Polarity has been ruled out as the cause of this decrease, suggesting complex regulation of the gene. The data from these studies will be useful for future investigations into the regulation of nanH and the role neuraminidase plays in S. typhimurium.
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