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 characterize the NADase-inactive SPN, we
firstly attempted to clone the NADase-inactive spn allele in Escherichia coli. Although 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 mutagenized plasmid DNAs were used to
transform both E. coli and S. pyogenes strains. We observed
successful nucleotide substitutions back to the original spn nucleotide sequence
in S. pyogenes transformants, but not in E. coli transformants. 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
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