The neuraminidase of Vibrio cholerae

Ollar, Robert A (1983) The neuraminidase of Vibrio cholerae. MSc(R) thesis, University of Glasgow.

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Abstract

Over the past 25 years research on the pathogenesis of cholera has focused on the role of enterotoxin, largely to the neglect of the other extracellular components such as neuraminidase, mucinase and proteinase. The main object of this thesis has been to study the effect of Vibrio cholerae neuraminidase on the histochemistry of the sialomucin layer, and the binding of the bacteria and of the enterotoxin to gut mucosa. Both laboratory-prepared and commercial sources of neuraminidase were utilised. For the former, V. cholerae NCTC 10732 was grown in proteose peptone broth containing bovine colostrum. The culture supernate, which contained the enzyme, was precipitated with methanol and the methanolic precipitate filtered on Sephadex G 100. The peak of neuraminidase activity in the eluate from the gel column did not coincide with the main protein peak as measured by either 280 nm absorption or the Lowry method. The partially purified neuraminidase preparations contained endoglycosidase, phospholipase-G and proteinase activity (as expected from previous studies) but lacked aldolase activity. Neuraminidase activity was determined both by the thiobarbituric acid assay (T.B.A.) and also by the methoxyphenyl neuraminic acid method (M.P.N.). Both procedures had advantages and limitations. The advantages of the M.P.N. method were greater sensitivity and better stability of the colour complex, especially at low enzyme concentrations. The main drawback of the method was its dependence on the synthetic substrate M.P.N. which is not part of the mucin of the small-gut. The main virtue of the T.B.A, assay was its applicability to detecting N-acetylneuraminic acid (NANA)-containing moieties liberated from a variety of natural substrates. However, the procedure could not determine whether the NANA was liberated as monosaccharides (indicative of neuraminidase activity) or as more complex NANA-containing entities (indicative either of endoglycosidase activity or simple solubilisation of sialomucin). A further drawback of the T.B.A. method is that its colour complex is generated not only by NANA but by 2-oxo-3-deoxy sugar acids. In order to investigate the possible effect of neuraminidase on rat intestinal mucosa in vitro, it was first necessary to find a tissue fixation procedure which preserved the sialomucin layer in tissue segments and frozen sections against loss during exposure to aqueous environments. Neither formal-saline nor Bouin's fluid were satisfactory but the paraformaldehyde vapour method cited by Culling gave adequate retention of the mucin layer. The susceptibility of the NANA moiety of mucin in paraformaldehyde vapour-fixed frozen sections to neuraminidase activity was monitored by Alcian Blue-Periodic Acid Schiff (PAS) staining. In agreement with previous published observations, it was noted that treatment of the sections with V. cholerae neuraminidase caused a general reduction in Alcian blue staining and a corresponding increase in PAS staining. This was interpreted as being due to loss of NANA (detected by Alcian blue) coupled with retention of the general muco- substance (detected by PAS). Quantitation of this phenomenon was obtained by determining the proportion of goblet cells staining with Alcian blue or PAS. The proportion of PAS-staining goblet cells was related to enzyme activity and thus provided a histochemical assay for neuraminidase, Although V. cholerae neuraminidase pretreatment of fixed rat ileal tissue caused significant histochemical change in the sialomucin layer, as revealed by the Alcian blue/PAS staining, it did not affect the adherence capacity of the tissue for live V. cholerae organisms. This result emerged from 8 experiments in which the numbers of these bacteria adhering to standardised tissue segments showed no significant difference when exposed to highly purified commercial V. cholerae neuraminidase compared with enzyme-free buffer.

Item Type: Thesis (MSc(R))
Qualification Level: Masters
Additional Information: Adviser: D ES Stewart-Tull
Keywords: Microbiology
Date of Award: 1983
Depositing User: Enlighten Team
Unique ID: glathesis:1983-73249
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jun 2019 08:56
Last Modified: 14 Jun 2019 08:56
URI: http://theses.gla.ac.uk/id/eprint/73249

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