Characterization of NADase-Inactive NAD⁺ Glycohydrolase in <i>Streptococcus pyogenes</i>

Abstract

Background: Streptococcus pyogenes secretes NAD + glycohydrolase (NADase, also known as SPN or Nga). All S. pyogenes strains examined to date possess the gene that encodes SPN ( spn ), but some strains produce SPN that lacks detectable NADase activity. Although there is much evidence to support that SPN’s NADase activity contributes to virulence, there is very little evidence that NADase-inactive SPN has detectable functions. Results: In order to charac terize the NADase-inactive SPN, we firstly attempted to clone the NADase-inactive spn allele in Escherichia coli . Al though we obtained recombinants which were shown to have the correct size insert, all had some mutations in the spn allele. Therefore, we attempted to change the mutated nucleotides back to the original nucleotides. While a nucleotide mutagenesis (inverse PCR method) easily changed a target nucleotide of control genes back to the original nucleotides, the mutations of NADase-inactive spn allele were never successfully converted back to the original nucleotides. Finally the mutant spn alleles were sub-cloned into another vector (pLZ12-Km2), which is maintained in both E. coli and S. pyogenes . The resultant plasmids were subjected to nucleotide mutagenesis using inverse PCR; the resultant mu tagenized plasmid DNAs were used to transform both E. coli and S. pyogenes strains. We observed successful nucleo tide substitutions back to the original spn nucleotide sequence in S. pyogenes transformants, but not in E. coli transfor mants. Thus, the NADase-inactive spn allele was successfully cloned in S. pyogenes , but not in E. coli . However, we could not find an association with NADase-inactive spn allele and virulence in a mouse infection model. Conclusions: These results suggest that NADase-inactive spn allele has some toxic effect to E. coli , but not S. pyogenes . This effect may due to an as of yet unknown function attributable to NADase-inactive SPN.