Doctor of Philosophy
School of Biological Sciences - Faculty of Science
Sanderson-Smith, Martina Louise, Investigation of the role of the plasminogen-binding group A streptococcal M-like protein (PAM) in the pathogenesis of Streptococcus pyogenes, PhD thesis, School of Biological Sciences, University of Wollongong, 2006. http://ro.uow.edu.au/theses/602
The Gram positive bacterium Streptococcus pyogenes (group A streptococcus; GAS) is the major etiological agent of a variety of skin and mucosal infections in humans. Whilst the majority of GAS infection results in only mild, uncomplicated disease, the migration of GAS from superficial to deep tissue sites can result in invasive infection. In recent years, there has been a resurgence in severe GAS disease, however, the details of GAS pathogenesis have yet to be fully elucidated. Increasingly, subversion of the host plasminogen activation system is being implicated in the virulence of S. pyogenes. GAS display receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M-like protein (PAM). PAM has been implicated in the pathogenesis of certain GAS isolates, but the mechanism of plasminogen binding by PAM, and the role of this interaction in the pathogenesis of GAS, requires further investigation. Thus, the focus of this thesis has been to characterise plasminogen binding by PAM and a number of naturally occurring PAM variants. Characterisation of PAM genes from 13 GAS isolates revealed that whilst these molecules are highly conserved in the c and d repeat domains, they display significant variation within the plasminogen binding repeat motifs (a1/a2). Percent identity to the prototype PAM a1/a2 repeat sequence ranged from 52% to 100% amongst the variants studied here. No correlation was seen between the presence of a PAM gene, or variation within the sequence of PAM, and site of GAS isolation. In order to determine the impact of sequence variation on protein function, recombinant proteins representing six naturally occurring variants of PAM, together with a recombinant M1 protein were expressed and purified. Equilibrium dissociation constants for the interaction of PAM variants with biotinylated glu-plasminogen ranged from 1.58 nM to 7.58 nM. Effective concentrations of prototype PAM required for 50% inhibition of plasminogen binding to immobilised PAM variants ranged from 0.34 nM to 22.06 nM. These results suggest that while variation in the a1/a2 region of the PAM protein does affect the comparative affinity of PAM variants, the functional capacity to bind plasminogen at physiologically relevant concentrations is conserved. Additionally, a potential role for the a region of PAM in eliciting a protective immune response was investigated using a mouse model for GAS infection. The a1 region of PAM was found to protect immunised mice challenged with a homologous PAM-positive GAS strain. These data suggest a link between selective immune pressure against the plasminogen-binding repeats and the functional conservation of the binding domain in PAM variants. Site-directed mutagenesis of full length PAMNS13 protein from an invasive GAS isolate was undertaken to assess the contribution of residues in the a1 and a2 repeat domains to plasminogen binding function. Mutagenesis to alanine of key plasminogen binding site lysine residues in the a1 and a2 repeats (Lys98 and Lys111) did not abrogate plasminogen binding by PAM, nor did additional mutagenesis of Arg101, His102 and Glu104, which have previously been implicated in plasminogen binding by PAM. Plasminogen binding was only abolished with the additional mutagenesis of Arg114 and His115 to alanine. Furthermore, mutagenesis of both arginine (Arg101 and Arg114) and histidine (His102 and His115) residues abolished interaction with plasminogen despite the presence of Lys98 and Lys111 in the binding repeats. This study shows for the first time that residues Arg101, Arg114, His102 and His115 in both the a1 and a2 repeat domains of PAM can mediate high affinity plasminogen binding. These data suggest that highly conserved arginine and histidine residues may compensate for variation elsewhere in the a1 and a2 plasminogen binding repeats, and may explain the maintenance of high affinity plasminogen binding by naturally occurring variants of PAM. Initial sequence characterisation of PAM variants in this study revealed a phylogenetically distinct PAM variant, PAMNS88.2. This variant binds plasminogen with high affinity (Kd = 7.58 nM), despite displaying only 52% identity to the classical a1/a2 repeat domain of PAM. It was therefore of interest to characterise the putative plasminogen binding domain of PAMNS88.2. Additionally, the association of GAS strain NS88.2, from which PAMNS88.2 was isolated, with the invasive disease bacteraemia, makes it a candidate for virulence studies employing the recently developed human plasminogen transgenic mouse. Site-directed mutagenesis of the putative plasminogen binding site indicated that as with PAMNS13, PAMNS88.2 does not interact with plasminogen exclusively via lysine residues. Mutagenesis to alanine of lysine residues Lys96 and Lys101 reduced but did not abrogate plasminogen binding by PAMNS88.2. Plasminogen binding was only abolished with the additional mutagenesis of Arg107 and His108 to alanine. Furthermore, mutagenesis of Arg107 and His108 abolished plasminogen binding by PAMNS88.2 despite the presence of Lys96 and Lys101 in the binding site. Given that GAS strain NS88.2 is associated with the invasive disease bacteraemia, and is virulent in the humanised plasminogen transgenic mouse, the successful abrogation of plasminogen binding by PAMNS88.2 may facilitate the development of a PAMNS88.2 allelic replacement isogenic mutant for use in future studies involving this model. This study examines in detail the interaction of PAM and PAM variants with the human zymogen plasminogen. The maintenance of plasminogen-binding function in spite of binding site sequence variation suggests that the ability to interact with plasminogen is evolutionarily advantageous to a subset of GAS isolates. Additionally, this study provides previously unreported details of the ability of PAM to interact with plasminogen independently of binding site lysine residues. These findings have implications for both the future identification of novel plasminogen binding proteins, and may facilitate both the understanding of the role of PAM in GAS disease, and the development of therapeutics to assist in the treatment and prevention of streptococcal infection.
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