Is plasminogen deployed as a virulence factor by Northern Territory group A streptococcal isolates during invasive disease

Group A streptococcal ( S. pyogenes ; GAS) infection is endemic in the Northern Territory of Australia, and the rates of invasive GAS disease and post-infection sequelae are among the highest reported in the world. Plasminogen is a potent human protease sequestered to the GAS cell-surface by plasminogen and fibrinogen receptors and activated by GAS streptokinase and host plasminogen activators. The critical role of plasminogen in GAS invasion was recently demonstrated in a human plasminogen transgenic mouse model of infection. The aim of this study was to determine whether plasminogen is deployed as a virulence factor in invasive GAS disease, with particular reference to the Northern Territory of Australia. This question was first approached from an epidemiological perspective, comparing Northern Territory GAS isolates from invasive infections with those from uncomplicated infections for their interaction with the plasminogen system. streptococcal pyrogenic exotoxin B (SpeB) expression and activity; and acquisition of cell-surface plasmin in human plasma were characterised for 29 GAS isolates of known clinical origin from the Northern Territory. The second approach to determining the role of plasminogen in invasive disease in the Northern Territory was to investigate GAS infection using a human plasminogen transgenic mouse model. A subset of Northern Territory GAS isolates selected for different in vitro plasminogen activation characteristics was tested for virulence in this model. This study revealed that Northern Territory GAS isolates from invasive disease cases acquire more plasminogen than isolates from uncomplicated infections, however they do not produce more streptokinase nor acquire more cell-surface plasmin after incubation in human plasma in vitro . Presence of the gene for the plasminogen-binding group A streptococcal M-like protein (PAM) conferred upon GAS isolates a strikingly different profile of interaction with plasminogen, characterised by higher plasminogen binding and plasmin acquisition in plasma. Differences in the catalytic specificity of streptokinase secreted by pam- positive and pam- negative isolates were identified and their allelic determinants investigated. A new model of GAS cell-surface plasminogen activation is proposed for pam- positive isolates. To characterise the role of streptokinase and the cysteine protease SpeB in acquisition of cell-surface plasmin activity in human plasma, such acquisition was compared in GAS deletion mutants for genes encoding these proteins and the corresponding wildtype strains. The dramatic reduction of GAS cell-surface plasmin activity by SpeB may significantly disable GAS invasive potential. Infection studies in the human plasminogen transgenic mouse model revealed the critical role of plasminogen in virulence of some Northern Territory isolates, however in vitro plasminogen activation characteristics do not predict clinical phenotype nor virulence in the transgenic mouse model. The results suggest an important and complex interaction between plasminogen and other host and/or bacterial factors in establishing invasive GAS infection in the Northern Territory of Australia.