The NrtR Transcriptional Factor Regulates Homeostasis Of Bacterial NAD~+ Cofactor

Nicotinamide adenine dinucleotide(NAD+)is an essential biological cofactor for metabolic conversions of NAD-dependent enzymes.However,little is known about NAD biosynthesis in the opportunistic pathogen Streptococcus suis(S.suis).An in silico search for relevant orthologs in the S.suis genome identified the nadR-punC-nrtR operon as likely encoding an NAD salvage pathway.The key regulator of this pathway is the Nudix family transcription regulator NrtR,which is composed of an N-terminal Nudix-like effector domain and a C-terminal DNA-binding wHTH-like domain.NrtR binding sites were predicted in the intergenic region upstream of the operon indicating that NrtR may serve as an autoregulator.The S.suis 2 NrtR(SsNrtR)exhibits high sequence conservation with other orthologs in both streptococci and other more distantly related organisms.Using chemical cross-linking assays,we also observed homodimer formation by SsNrtR in addition to other higher MW multimers.Electrophoretic mobility shift assays(EMSA)showed that,NrtR efficiently bound the DNA probe in a dose-dependent manner and ADPR should serve as an anti-repressor of the NAD salvage operon.Presumably,the cell interprets the accumulation of ADPR as a signal to replenish the NAD cofactor pool.The putative residues critical for SsNrtR DNA binding were predicted from a multiple sequence alignment and 3 SsNrtR mutants(H223A,K246A and R248A)were constructed to test their functions.EMSA results showed that,both lysine 246 and arginine 248 were essential for DNA binding,whereas the H223A mutant was unaffected.Compared to the wild type,nadR-lacZ expression was clearly increased in the AnrtR background,whereas its expression mirrored wild type levels in the complemented mutant.Thus,we conclude that that NrtR indeed functions as an autoregulatory transcription repressor of the nadR-punC-nrtR operon.Members of NrtR family may have lost their Nudix catalytic activity due to an apparent lack of conservation of a strictly conserved Nudix hydrolase signature sequence,GX5EX7REUXEEXGU.To examine whether the low Nudix activity of SsNrtR was due to a substitution in the Nudix motif,we created a K92E mutation to restore the consensus Nudix motif.The K92E mutation resulted in a significant increase in NrtR catalysis of ADPR,NADH,and NADPH.Interestingly,the mutation triggered no obvious impact upon NrtR DNA binding,suggesting that the N-terminal Nudix domain and C-terminal wHTH binding domain are largely independent.We compared genome data from a variety of S.suis clinical isolates in NCBI and found that nrtR is encoded in a strain-specific manner(i.e.not a component of the core genome).This may explain the lack of Nudix catalysis from SsNrtR,as it is apparently not a conserved component of S.suis NAD+ metabolism.Likewise,the ?nrtR mutant grew normally.Thus,we were curious whether SsNrtR might play a role in virulence.Intriguingly,S.suis genome comparisons did reveal an obvious correlation between the existence of nrtR and the reported virulence of the strain,especially among S.suis serotype 2 strains.Consequently,an experimental mouse infection model was tested to assess the potential relationship between nrtR and S.suis 2 virulence.All SPF mice inoculated intravenously with the WT strain developed serious clinical symptoms,such as high fever,swollen joints,shivering,and central nervous system failure within 24 h.Lethality was 100%<40 hr.post-inoculation.In contrast,mice challenged with the?nrtR strain survived longer overall and three mice even survived the entire 20-day observation period.Taken together,our results support a role for SsNrtR as a virulence factor,especially for S.suis 2 pathogenesis.Nicotinamide adenine dinucleotides(NAD+ and NADP+)plays a crucial role in living organisms because it acts as the primary biological cofactor for numerous redox reactions in cellular metabolism.NAD+ biosynthesis in the food-borne pathogen,Vibrio cholerae(V cholerae)has yet not been experimentally explored.The in silico search for orthologs in the V.cholerae genome identified the pncB,nadD,nadE to encode the NAD+ synthase of salvage pathway.The regulon of this pathway is NrtR homolog from the Nudix family,composed of an N-terminal Nudix-like effector domain and a C-terminal DNA-binding HTH-like domain.Complex formation between the catalytically inactive Nudix protein NrtR and its DNA binding site was suppressed by the antirepressor ADP-ribose.V.cholerae NrtR perhaps make no contribution to bacterial virulence,whereas nrtR mutant beared a higher MIC,predicting one new mechanism of pathogen evolution exists.Besides PncB salvage pathway,the in silico search for orthologs in the V.cholerae genome identified the nadB,nadA&naaC gene to encode the NAD+ synthase of de novo pathway.Three NrtR binding sites were predicted in the intergenic region between nrtR and pncB.NrtR regulons and their binding sites were widely distributed in Vibrio and co-localized with the NAD+ metabolism related gene cluster.Multiple sequence alignment analyses suggested that NrtR Orthologues in different Vibrio species shared a high similarity.Gel shift assays showed that NrtR efficiently bound the pncB probe and nrtR probe in a dose-dependent manner.The shifed bands completely disappeared when 300?M ADPR was added to the incubation system.NrtR-DNA interaction was reversed by ADP-ribose(ADPR)in a dose-dependent manner.ADPP modulates NrtR repressor function through a Nudix switch.Binding of ADPR to NrtR Nudix domain would promote dissociation of NrtR-DNA complexes,leading to de-repression of transcription of NAD biosynthetic genes.Thus,as an anti-repressor of NAD synthesis,ADPR is based on the assumption that the cell may interpret the accumulation of ADPR as a signal to replenish the NAD cofactor pool.The reporter plasmid(pTL61T-PnrtR,pTL61T-PpncB)was introduced into wild type strain V.cholerae,?nrtR,C?nrtR.Compared to the wild-type,the expression of lacZ was sharply promoted under the promoter of pncB in ?nrtR strain,and?-galactosidase activity has reduced in functional complementation strain C?nrtR.This phenomenon perhaps due to the transcription sites were strongly bound by NrtR in wild type strain.In contrast,the transcription could be easily initiated when nrtR was knocked out.Whereas the expression of lacZ has sharply reduced under the promoter of nrtR in ?nrtR strain,and ?-galactosidase activity has increased in functional complementation strain C?nrtR.This phenomenon indicated that nrtR gene is probablely autoregulated and the transcription could not be initiated when nrtR was knocked out.These results strongly suggest that NrtR indeed functions as a transcriptional repressor of pncB and auto-activation to affect the expression of NAD+synthesis operons.We tested the sensitivity of wild type and ?nrtR to a dozen of various antibiotics.Both wild type and ?nrtR were resistant to ampicillin,kanamycin,erythromycin,penicillin and ?nrtR exhibited higer minimum inhibitory concentration(MIC)compared to WT.The fact that no significant differences were found in the general biological characteristics between wild-type and mutant,perhaps suggests that NrtR was passably not required for the intracellular growth/survival and virulence.