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 l...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 SPN.展开更多
Prion disease represents a group of fatal neurogenerative diseases in humans and animals that are associated with energy loss,axonal degeneration,and mitochondrial dysfunction.Axonal degeneration is an early hallmark ...Prion disease represents a group of fatal neurogenerative diseases in humans and animals that are associated with energy loss,axonal degeneration,and mitochondrial dysfunction.Axonal degeneration is an early hallmark of neurodegeneration and is triggered by SARM1.We found that depletion or dysfunctional mutation of SARM1 protected against NAD+loss,axonal degeneration,and mitochondrial functional disorder induced by the neurotoxic peptide PrP106-126.NAD+supplementation rescued prion-triggered axonal degeneration and mitochondrial dysfunction and SARM1 overexpression suppressed this protective effect.NAD+supplementation in PrP106-126-incubated N2a cells,SARM1 depletion,and SARM1 dysfunctional mutation each blocked neuronal apoptosis and increased cell survival.Our results indicate that the axonal degeneration and mitochondrial dysfunction triggered by PrP^(106-126) are partially dependent on SARM1 NADase activity.This pathway has potential as a therapeutic target in the early stages of prion disease.展开更多
文摘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 SPN.
基金supported by the National Natural Science Foundation of China,No.31972641the National Key Research and Development Program of China,No.2017YFC1200500(both to LFY).
文摘Prion disease represents a group of fatal neurogenerative diseases in humans and animals that are associated with energy loss,axonal degeneration,and mitochondrial dysfunction.Axonal degeneration is an early hallmark of neurodegeneration and is triggered by SARM1.We found that depletion or dysfunctional mutation of SARM1 protected against NAD+loss,axonal degeneration,and mitochondrial functional disorder induced by the neurotoxic peptide PrP106-126.NAD+supplementation rescued prion-triggered axonal degeneration and mitochondrial dysfunction and SARM1 overexpression suppressed this protective effect.NAD+supplementation in PrP106-126-incubated N2a cells,SARM1 depletion,and SARM1 dysfunctional mutation each blocked neuronal apoptosis and increased cell survival.Our results indicate that the axonal degeneration and mitochondrial dysfunction triggered by PrP^(106-126) are partially dependent on SARM1 NADase activity.This pathway has potential as a therapeutic target in the early stages of prion disease.
