Investigation of the molecular basis of virulence of Streptococcus equi

Barrett, Lee (2012) Investigation of the molecular basis of virulence of Streptococcus equi. Masters thesis, Northumbria University.

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Abstract

Equine strangles is a purulent lymphadenitis of the head and neck and is caused by Streptococcus equi ssp equi remaining a worldwide, endemic infection that represents around 30% of all annually recorded incidents of equine disease. Despite much effort, current vaccination strategies have proved largely ineffective towards S. equi, ssp equi, with the current focus based on ‘reverse vaccinology’, using genome sequence data of S. equi ssp equi to identify surface exposed and secreted proteins.

Streptococcus equi shares much of its genome with the genetically closely related, S. equi ssp zooepidemicus. Despite this close genetic association, S. equi ssp zooepidemicus exhibits very distinct pathogenicity variations. S. equi ssp zooepidemicus can infect a wide
variety of vertebrate hosts showing a high degree of antigenic variability and a large amount of strain variability whereas S. equi is largely equine specific and processes a limited number of strain types. Understanding the molecular basis of virulence of these contrasting organisms therefore remains a key requirement if a suitable vaccination.

Identification of potential virulence factors has been greatly aided by the availability
of the full genome sequence of S. equi ssp equi (4047) and S. equi ssp zooepidemicus (H70).

In this study, we identified and investigated a range of putative virulence factors including the covalently attached surface protein SEQ2190, the secreted phospholipase
enzymes SlaA (SEQ0849) and SlaB (SEQ2155) and finally the lipoprotein, acid phosphatase SeLppC (SEQ0346) and its orthologue SzLppC (SZO16870). In all cases, using molecular
biology techniques the coding region representative for each of these proteins was cloning into expression constructs, expressed and purified and further investigations carried out.

Although efforts to obtain a 3-dimensional structure of SEQ2190 were unsuccessful, bioinformatic investigations have identified SEQ2190 as a unique protein in S. equi ssp equi
with a putative structure suggesting a role in bacteria-host interaction.

SlaA was demonstrated to be an active sPLA2 enzyme, active against a 1, 2-dithio analog of diheptanoyl phosphatidylcholine but not 2-deoxy-2-thio- Arachidonoyl
phosphatidylcholine. It was also demonstrated to have a specific requirement for the divalent ion, Ca2+ for activity, an optimum temperature higher than expected (40°C), a Km of 14.40 ± 7.866 mM and a specific activity of 5.06x10-2 ± 3.01x10-3 μmol/min/mg. SlaA was also shown to react to equine post-infection convalescent serum.

Although some characterisation had been previously undertaken regarding whole cell SeLppC extracts, in this study we produced expression constructs of SeLppC and its
orthologous pseudogene (SzLppC) found in the genome strain of S. equi ssp zooepidemicus. We used purified SeLppC and SzLppC to demonstrate activity against pNPP and two
biologically significant substrates (5’AMP and 5’UMP) and recorded the Km values for each. We also identified SeLppC to not possess a specific activity for the divalent ion Cu2+, a lower than expected pH optimum and a higher than expected temperature range. Furthermore we also demonstrated that SzLppC is not a pseudogene, indicating an error within the S. equi spp zooepidemicus genome strain.

Item Type: Thesis (Masters)
Uncontrolled Keywords: molecular biology, virulence factors, lipoproteins
Subjects: B400 Nutrition
C900 Others in Biological Sciences
Department: Faculties > Health and Life Sciences > Applied Sciences
University Services > Graduate School > Master of Philosophy
Related URLs:
Depositing User: Ay Okpokam
Date Deposited: 23 Aug 2012 08:53
Last Modified: 25 Oct 2017 08:34
URI: http://nrl.northumbria.ac.uk/id/eprint/8509

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