MOLECULAR CHARACTERIZATION OF A MAJOR SEROTYPE M49 GROUP-A STREPTOCOCCAL DNASE GENE (SDAD)

Group A streptococci (GAS) express up to four types of secreted DNases . Although GAS infections are correlated with the production of anti-D Nase B antibodies, the roles of DNases in the pathogenesis of GAS infe ctions remain unclear. From a lambda library of serotype M49 strain CS 101 GAS genome, a 2,147-bp fragment expressing DNase activity on an in dicator agar,vas identified and sequenced. One 1,155-bp open reading f rame (ORF) was identified in this fragment. This ORF was found to be 4 8% identical on the amino acid level to group C streptococcal DNase (S dc). The regions of highest homology corresponded to amino acid residu es that were also identified as part of the active site in staphylococ cal nuclease. Transcription analysis revealed a specific 1.3-kb mRNA, which corresponded to the size predicted by the promoter and transcrip tion termination signature sequences and indicated a monocistronic mod e of transcription. Allelic replacement of the ORF rendered a M49 muta nt devoid of extracellular DNase activity when cultured on indicator a gar. Virulence parameters such as resistance to phagocytosis were not affected by the mutation. The sda gene was cloned and expressed in Esc herichia coli as a thioredoxin fusion. By performing Ouchterlony immun odiffusion on the recombinant protein and by using protein preparation s from culture supernatants of wild-type bacteria and the DNase mutant , the results of immunoreactivity with DNase type-specific polyclonal rabbit antisera classified the DNase as a type D enzyme. Fifty percent of patients with sera exhibiting high titers of antistreptolysin or a nti-DNase B antibodies also had SdaD-reactive antibodies in comparison with <10% of serologically normal controls. While the value of recomb inant SdaD for diagnostic purposes needs to be clarified, the isogenic DNase mutant pair of M49 should allow the significance of GAS DNase D as a bacterial virulence factor to be determined.