Year

2015

Degree Name

Doctor of Philosophy

Department

Illawarra Health and Medical Research Institute

Abstract

In this study 1086 GAS isolates collected from 31 countries representing 175 M-types were phylogenetically assessed on their full length mature M protein sequence, with the aim of developing a new cluster-based classification system which would support future vaccine development and functional studies. Phylogenetic analysis identified that 175 M-types could be grouped in to 2 clades, 2 sub-clades and 48 clusters, 16 of which encompassed 82% of all analysed M-types. To evaluate the utility of this novel classification system as a tool for identification of M protein function, 26 M proteins representing 24 distinct M-types encompassing all major cluster groups were recombinantly expressed and screened for binding against a select range of host proteins. Surface plasmon resonance (SPR) binding experiments identified that both fibrinogen and plasminogen binding function was restricted to clade-Y. Plasminogen binding was further restricted to cluster D4 whereby all M protein contained the highly predictive plasminogen binding motif. High affinity IgA binding was observed only by M proteins associated with emm-clusters E1 and E6 whereas IgG binding was observed by emm-clusters E1, E3, E4, E6, A-C3 and non-clustered M proteins M57 and M14. Complement inhibitor C4-binding protein was shown to only bind clade-X M protein representatives, specifically emm-clusters E1, E3, E4 and E6. Although all M proteins in study were shown to express the previously characterised human serum albumin binding domain, emm-cluster E4 and D4 M protein representatives M53 and M98 were shown not to bind human serum albumin. The ability of GAS vaccine candidate ‘Streptococcal vaccine 1’ (SV1) to effectively opsonise the C-repeat targeted domains of the M proteins in this study was assessed in the presence of IgG, IgA, plasminogen, fibrinogen and albumin. Competitive SPR binding analysis identified that α-SV1 IgG binding did not compete for binding with the respective host proteins. Only IgA binding by E1 M proteins was shown to both inhibit and outcompete α-SV1 IgG binding interactions. These results clearly demonstrate the utility of this GAS classification system based on full length M protein sequence with applications of facilitating future M protein functional studies, epidemiological surveillance and vaccine development.

This study clearly demonstrates the development and utility of the newly implemented GAS classification system. Furthermore, we have identified novel M protein functions which may contribute to the different stages during GAS infection. These results illustrate the homologous genetic and functional properties of distinct M-types in regions where the epidemiology of infection is diverse. A better understanding of M protein interaction with the host will broaden our knowledge of GAS disease progression, facilitating the development of future therapeutics.

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.