| Streptococcus suis serotype 2(SS2)has been reported to be a highly invasive pathogen in swine and a zoonotic agent for human which causes severe infections like meningitis,arthritis,sepsis and acute death.It caused serious economic losses to the porcine industry,threatened the public health safety and also attracted worldwide attention.So far,the pathogenesis of SS2 is still unclear,so it is necessary to do further exploration about SS2 virulence factors.In our previous study,comparative genomics analysis was taken among 17 strains of different virulent SS2,and a gene cluster named nmt was identified which exists in SS2 virulent strain specifically,suggesting it is related to the virulence of SS2.The nmt gene cluster is composed of three consecutive genes nadR-pnuC-mutT that located at the same operon.In this study,the distribution and bioinformatics of nmt gene cluster in different SS2 strains were analyzed.Based on this,the functions of each gene in the cluster on the pathogenesis of SS2 were studied,so as to provide new insights for the prevention and treatment of streptococcus suis disease.1.Distribution and bioinformatics analysis of nmt gene cluster in SS2In our previous study,the nmt gene cluster has been identified by comparative genomics analysis,it only exists in virulent SS2 strains.In order to further verify the distribution of the cluster in SS2 different virulent strains,we chose 17 SS2 high virulent strains and 14 low virulent strains for PCR detection.The results showed that nmt gene cluster exists in SS2 virulent strain specifically,which was consistent with the results of comparative genomics analysis,indicating it was a potential virulence-associated gene in SS2.Bioinformatics analysis revealed that nadR,pnuC and mutT were all encoded by negative chain,and they located at the same operon and transcribed together.The analysis of the conserved structural domain of proteins showed that the three genes jointly encoded the nicotinic acid salvage pathway in the bacterial NAD metabolic pathway.Because NAD metabolism is closely related to bacterial virulence,it further suggests that nmt gene cluster might play an important role in the virulence of SS2,providing an experimental and theoretical basis for the subsequent studies.2.Effect of nmt gene cluster on the virulence of SS2In order to further confirm the gene cluster effect on SS2 virulence,we constructed a deletion mutant with nadR-pnuC-mutT named ?nmt in SS2 high virulent strain ZY05719.Growth curve results revealed that the growth ability of ?nmt was inhibited in logarithmic phase,and recovered to the same level as the wild-type strain ZY05719 in stationary phase.The in vivo infection experiments with zebrafish and mice model confirmed that ?nmt significantly decreased the virulence of SS2.All the results indicated that nmt gene cluster is closely related to SS2 virulence.For exploring the mechanism on virulence changes,we will verify the function of individual genes in nmt gene cluster.3.The role of nadR in SS2 pathogenesisThe protein encoded by nadR gene has been firstly reported as a bifunctional protein in Salmonella typhimurium.It utilized as enzyme catalytic and transcriptional regulation in NAD metabolic pathway,and indirectly participates in bacterial virulence.But the role of nadR in SS2 has not been reported.So we constructed a deletion mutant strain ?nadR in SS2 high virulent strain ZY05719.The results showed ?nadR significantly inhibited cell growth and formed shorter chain compared with the wild-type strain ZY05719.Besides,by using Artificial Culture Medium(CDM),we confirmed that NadR participated in transporting the substrates nicotinamide mononucleotide(NMN)and Nicotinamide riboside(RNam)in NAD metabolic pathway.Both zebrafish and BALB/c mice in vivo virulent experiments results showed ?nadR dramatically attenuated the virulence of SS2,and the ability to colonize tissues of ?nadR was notably reduced in comparison with ZY05719 in the BALB/c mice infection model.RNA-seq results showed AnadR has 790 differentially expressed genes compared with ZY05719,which accounts for about 38.64%of the total number of genes.All these genes covered the majority of life activities,such as nutrient transport and metabolism,protein synthesis and modification,DNA replication and repair,cell division and differentiation,signal transduction and defense mechanism,indicating it functions as a global regulation in SS2.In summary,nadR is involved in the utilization of NMN and RNam enzymes in SS2 NAD metabolic pathway and functions as a global regulator,affecting the growth and virulence of SS2 by regulating the majority of bacterial life activities.4.The role of mutT in SS2 pathogenesismutT gene encodes Nudix-like hydrolase,which has been reported to self-inhibited regulate through binding to promoter region of its own operon in SS2,but missing the activity of enzyme.In order to further confirm the role of mutT in SS2 virulence,we constructed a deletion mutant strain ?mutT in SS2 high virulent strain ZY05719.The results showed the growth ability of ?mutT has no obvious changes.Animal infection experiments showed?mutT significantly attenuated the virulence of SS2 both in zebrafish and BALB/c mice model,and the ability to colonize tissues was notably reduced in comparison with ZY05719 in the BALB/c mice infection model.All the results indicate,mutT did not influence the growth ability of SS2,but can affect the virulence of SS2 by participating in the survival ability in host tissues and blood.In summary,this study proved that nmt gene cluster was widely distributed in SS2 high virulent strains,encoded the nicotinic acid salvage pathway in SS2 NAD metabolic pathway,and related to the virulence of SS2.Among them,nadR gene is involved in the utilization of NMN and RNam enzymes in SS2 NAD metabolic pathway,and functions as a global regulator,affecting the growth and virulence of SS2 by regulating the majority of bacterial life activities.mutT gene changed the virulence of SS2 by affecting the survival of bacteria in host tissues and blood.Another gene pnuC in this gene cluster was also confirmed to be involved in SS2 oxidative stress tolerance and affect bacterial virulence in our previous study. |
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