Molecular characterization of a major serotype M49 group A streptococcal DNase gene (sdaD)

Abstract

Group A streptococci (GAS) express up to four types of secreted DNases. Although GAS infections are correlated with the production of anti-DNase B antibodies, the roles of DNases in the pathogenesis of GAS infections remain unclear. From a lambda library of serotype M49 strain CS101 GAS genome, a 2,147-bp fragment expressing DNase activity on an indicator agar was identified and sequenced. One 1,155-bp open reading frame (ORF) was identified in this fragment. This ORF was found to be 48% identical on the amino acid level to group C streptococcal DNase (Sdc). The regions of highest homology corresponded to amino acid residues that were also identified as part of the active site in staphylococcal nuclease. Transcription analysis revealed a specific 1.3-kb mRNA, which corresponded to the size predicted by the promoter and transcription termination signature sequences and indicated a monocistronic mode of transcription. Allelic replacement of the ORF rendered a M49 mutant devoid of extracellular DNase activity when cultured on indicator agar. Virulence parameters such as resistance to phagocytosis were not affected by the mutation. The sda gene was cloned and expressed in Escherichia coli as a thioredoxin fusion. By performing Ouchterlony immunodiffusion on the recombinant protein and by using protein preparations 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 anti-DNase B antibodies also had SdaD-reactive antibodies in comparison with <10% of serologically normal controls. While the value of recombinant 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.

Potential virulence and antimicrobial susceptibility of Aeromonas popoffii recovered from freshwater and seawater

“…All strains had the genes for DNases, GCAT and lipases and showed DNase and lipase activity. Although the role of DNases is unknown in Aeromonas pathogenicity, these genes are involved in Streptococcus infections [25], and are considered important for bacterial nutrition [7]. GCAT had only been previously investigated by PCR in isolates of A. salmonicida [9].…”

Nuclease A (Gbs0661), an extracellular nuclease of Streptococcus agalactiae, attacks the neutrophil extracellular traps and is needed for full virulence

“…The above results led us to test respiration-permissive and non-permissive GBS culture supernatants for DNase activity by using Methyl green-DNA indicator plates (Mgreen plates) (Ferrieri et al, 1980;Podbielski et al, 1996). As shown in Fig.…”

Invasive M1T1 group A Streptococcus undergoes a phase‐shift in vivo to prevent proteolytic degradation of multiple virulence factors by SpeB

“…In addition, we found a protein secreted by the M1T1 clone that bears considerable homology to Sda from M49 and M18, and Sdn from M3. This protein, which is not found in the SF370 strain, may represent an allelic variant of streptodornase D characterized by Podbielski et al (1996). Using reverse genomics we were able to confirm the presence of Sda in the M1T1 clone (data not shown).…”