Post-streptococcal glomerulonephritis (PSGN) can result from an antecedent group A streptococcal infection. Previous investigations have demonstrated that PSGN-associated streptococci secrete a plasminogen activator (streptokinase), which has affinity for glomerular structures. Although the majority of group A streptococci secrete streptokinase (Skase), of which there are at least 6 classes, only those from PSGN-associated strains can induce experimental PSGN. It is proposed that these nephrotropic Skases initiate PSGN by facilitating the glomerular deposition of a Skase-plasmin complex; deposited plasmin in this complex then activates the complement resulting in the complement deposition (C3b) and induction of an inflammatory response. Although all group A Skases can activate plasminogen, those which induce experimental PSGN essentially differ from non-inducers by the presence of an internal 82 amino acid domain. This application will focus on determining if this domain is the glomerular binding domain and to what extent plasminogen activation is central to pathogenesis of this molecule. This will be accomplished by constructing chimeric Skase genes by the Polymerase Chain Reaction (PCR) and inserting these into an appropriate plasmid expression vector such as pMAL. This construct will permit the synthesis of foreign peptides fused to a maltose binding protein (MBP) which can then be affinity purified on amylose-agarose. Incubation with Factor Xa will cleave the fused peptide from the MBP. These polypeptides will be administered to rabbits by osmotic pumps to assess their ability to induce experimental PSGN.These chimeric genes will be constructed to reflect proteins which will have altered domains representing those which bind or do not bind to glomeruli, and those which are necessary for plasminogen binding and activation. This will be done by a combination of PCR-directed site specific mutagenesis and recombination of nucleotide domains between PSGN+ and PSGN- associated streptokinases. This study may also result in a greater understanding of the nature of streptokinase plasminogen interaction.
| Caballero, A R; Lottenberg, R; Johnston, K H (1999) Cloning, expression, sequence analysis, and characterization of streptokinases secreted by porcine and equine isolates of Streptococcus equisimilis. Infect Immun 67:6478-86 |
