Abstract
Subversion of the plasminogen activation system is implicated in the virulence of group A streptococci (GAS). GAS displays receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M-like protein (PAM). The plasminogen binding domain of PAM is highly variable, and this variation has been linked to host selective immune pressure. Site-directed mutagenesis of full-length PAM 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 lysine residues in the a1 and a2 repeats (Lys98 and Lys111) did not abrogate plasminogen binding by PAM nor did additional mutagenesis of Arg101 and His102 and Glu104, which have previously been implicated in plasminogen binding. 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.
Highlights
The ability of the organism to migrate from cutaneous and mucosal surfaces to deep tissue sites, resulting in severe invasive disease
A newly developed model of group A streptococci (GAS) infection using mice expressing a human plasminogen transgene indicates that plasminogen plays a critical role in GAS infection, with a significant increase in mortality observed in transgenic mice when compared with wild-type littermate control mice [5]
The PAM-positive GAS isolate AP53 exhibited a 60% increase in mortality in transgenic mice when compared with wild-type littermates, and a PAM-negative isogenic mutant of this strain showed only minimal virulence in both wild-type and human plasminogen transgenic murine backgrounds [5]
Summary
The ability of the organism to migrate from cutaneous and mucosal surfaces to deep tissue sites, resulting in severe invasive disease. The PAM-positive GAS isolate AP53 exhibited a 60% increase in mortality in transgenic mice when compared with wild-type littermates, and a PAM-negative isogenic mutant of this strain showed only minimal virulence in both wild-type and human plasminogen transgenic murine backgrounds [5]. It appears that for a subset of GAS isolates, the ability of PAM to focus plasminogen at the GAS cell surface is crucial for virulence. For site-directed mutants containing two or more mutations not encoded in a single primer, mutations were sequentially introduced using previously mutated DNA as a template
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