In silico design and in vitro validation of a novel PCR-RFLP assay for determination of phylogenetic clusters of streptokinase gene alleles in streptococci groups.

Streptokinase (SK), a heterogeneous plasminogen (Pg) activator protein secreted by groups A, C and G streptococci (GAS/GCS/GGS) is a virulence factor composed of three structural domains; SKα/SKß/SKγ. Phylogenetic analysis of the major variable region of SKß (sk-V1; nucleotides 448-791; 343bp) which classifies the SK alleles into SK1/SK2 clusters and SK2a/SK2b sub-clusters, is an approved assay to categorize clinical/natural streptococcal-isolates into co-related functional/pathogenesis groups. Herein, we describe a novel PCR-RFLP assay that in combination with Numerical Taxonomy and multivariate analysis System (NTSYS) resulted to dendrograms with complete adaption to that of the phylogenetic analysis of sk-V1-based clustering. In silico analyses by 30 restriction enzymes on GenBank-acquired sk-V1 sequences of known streptococcal clusters, resulted to the selection of "BsrI, MseI and Tsp45I″ enzymes that produced proper patterns to construct the expected dendrograms. In vitro analysis of the selected enzymes on clinical isolates of GAS/GCS/GGS validated the production of the same in silico-observed digestion patterns. Comparison of the constructed dendrogram and phylogenetic trees of selected GenBank and clinical isolates of streptococci indicated complete adaptation. Assessment of Pg-activation activity in selected clinical isolates indicated the expected co-related functionalities of the classified SK-clusters by the invented PCR-RFLP/NTSYS method. The simplicity of the assay relieves the need of sequencing/phylogenetic analyses for SK-clustering.

Characterization of a novel streptokinase produced by Streptococcus equisimilis of non-human origin.

Streptokinases are proteins with plasminogen activator activity produced by certain hemolytic streptococci. We previously identified equine streptococcal isolates which produced streptokinases (ESKs) that bound both human and equine plasminogen but only readily activated equine plasminogen (14). This property was exploited to purify a representative ESK produced by Streptococcus equisimilis strain 87-542-W. Affinity chromatography with human plasminogen resulted in the isolation of a M(r) approximately 49,000 molecule with two isoforms. This ESK was subsequently compared to well characterized streptokinases (HSKs) that efficiently activate human plasminogen. Differences in streptokinases were identified in the highly conserved amino-terminal amino acid sequence, peptide maps, and antigenic properties, and these differences were supported by DNA hybridization studies. These results indicate that the family of proteins identified as streptokinases has much greater diversity than previously appreciated.

Streptokinase gene variable region classification in streptococci: lack of correlation with post-streptococcal glomerulonephritis.

To investigate a possible causal role of streptokinase (SKase) in acute post-streptococcal glomerulonephritis (APSGN), the major variable region of SKase genes of Streptococcus pyogenes strains isolated from patients with and without APSGN were analyzed using the polymerase chain reaction, restriction enzyme analysis and the direct sequencing of SKase genes. In the APSGN-associated strains, six of nine revealed mutant classes corresponding to the nephritogenic classes I and II proposed by Johnston et al. [1992], the remaining three belonged to non-nephritogenic classes. In twenty strains not associated with APSGN, seventeen belonged to classes I and II, while three were from other classes. The major variable region of the SKase gene shows no apparent relation with induction of APSGN in humans, suggesting that unique classes of streptococcal SKase do not play a role in the pathogenesis of APSGN.

Streptokinase gene polymorphism in group A streptococci isolated from Ethiopian children with various disease manifestations.

Certain variants of streptokinase from group A streptococci have been associated with acute post-streptococcal glomerulonephritis (APSGN). The streptokinase gene (ska) has previously been grouped into nine different polymorphic genotypes of which ska1, ska2, ska6, and ska9 were identified in group A streptococci associated with clinical and experimental APSGN. A total of 53 group A streptococci isolated from Ethiopian children: five from acute rheumatic fever, 18 from APSGN, ten each from tonsillitis, impetigo and healthy carriers, were analyzed for ska gene polymorphism using polymerase chain reaction (PCR) and restriction enzyme analysis. The frequency of the nephritis-associated streptokinase genotypes was 83% among the APSGN isolates and 74% in the non-ASPGN isolates. ska2 was the most commonly found genotype with a frequency of 64% among all isolates, 66% among the APSGN isolates, and 63% among the non-APSGN isolates. ska1 was identified in 13% among all isolates and 17% among the APSGN isolates. Seventeen non-APSGN isolates from Scandinavian countries were studied for comparison and all carried either ska1 or ska2. The other nephritis-associated ska6 and ska9 were not detected among the 53 Ethiopian isolates. ska1 was exclusively associated with serum opacity reaction (SOR) producers. ska2 was evenly distributed among SOR-positive and SOR-negative isolates. The other genotypes were detected only among SOR-negative strains. The findings of the present study showed an even distribution of the nephritis-associated streptokinase gene among group A streptococcal isolates with no correlation to disease pattern. Thus additional factors must also be operative in the pathogenesis of APSGN.