III型分泌系统(type III secretion system,T3SS)是副溶血弧菌主要的毒力因子之一,能传递与致病有关的毒性因子来发挥病原菌的毒性,而温度是影响细菌基因表达等生命活动的关键生态因子。本文以ATCC17802和JA21作为实验菌株,分别提取其R...III型分泌系统(type III secretion system,T3SS)是副溶血弧菌主要的毒力因子之一,能传递与致病有关的毒性因子来发挥病原菌的毒性,而温度是影响细菌基因表达等生命活动的关键生态因子。本文以ATCC17802和JA21作为实验菌株,分别提取其RNA,在不同温度和温度应激条件下,以pvuA为内参基因,以副溶血弧菌T3SS1的vcrD1、vopS、vopD1和T3SS2的vscC2β、vcrD2β、vopP2β作为目的基因,运用荧光定量PCR(RT-PCR)技术检测T3SS相关基因的表达下,用ΔΔCt法计算基因表达差异。结果表明:副溶血弧菌(Vibrio parahaemolyticus)ATCC17802菌株的T3SS1基因在16℃表达量最高,T3SS2基因在25℃表达量最高。副溶血弧菌JA21菌株的T3SS1基因和T3SS2基因均在25℃表达量最高。2株副溶血弧菌从不同的培养温度转入到应激温度37℃后,T3SS1和T3SS2基因的表达量均被显著诱导,并表现出时间依赖性,呈现先升高后降低的趋势。这为进一步探究环境因素与副溶血弧菌致病力的关系提供了科学依据。展开更多
Xanthomonas oryzea pv.oryzae(Xoo)is the causal agent of bacterial blight of rice,which is a significant threat to many of rice-growing regions.The type Ⅲ secretion system(T3SS)is an essential virulence factor in Xoo....Xanthomonas oryzea pv.oryzae(Xoo)is the causal agent of bacterial blight of rice,which is a significant threat to many of rice-growing regions.The type Ⅲ secretion system(T3SS)is an essential virulence factor in Xoo.Expression of the T3SS is often induced in the host environment or in hrp-inducing medium but is repressed in nutrient-rich medium.The elucidation of molecular mechanism underlying induction of T3SS genes expression is a very important step to lift the veil on global virulence regulation network in Xoo.Thus,an efficient and reliable genetic tool system is required for detection of the T3SS proteins.In this study,we constructed a protein expression vector pH3-flag based on the backbone of pHM1,a most widely used vector in Xoo strains,especially a model strain PXO99A.This vector contains a synthesized MCS-FLAG cassette that consists of a multiple cloning site(MCS),containing a modified pUC18 polylinker,and Flag as a C-terminal tag.The cassette is flanked by transcriptional terminators to eliminate interference of external transcription enabling detection of accurate protein expression.We evaluated the potential of this expression vector as T3SS proteins detection system and demonstrated it is applicable in the study of T3SS genes expression regulation in Xoo.This improved expression system could be very effectively used as a molecular tool in understanding some virulence genes expression and regulation in Xoo and other Xanthomonas spp.展开更多
In order to enrich the research about type Ⅲ secretion system(T3SS) injectisome of Vibrio alginolyticus,vscX gene was cloned from V.alginolyticus strain HY9901 for bioinformatics analysis and expression analysis.Spec...In order to enrich the research about type Ⅲ secretion system(T3SS) injectisome of Vibrio alginolyticus,vscX gene was cloned from V.alginolyticus strain HY9901 for bioinformatics analysis and expression analysis.Specific primers were designed according to the full-length genome sequence of V.alginolyticus in GenBank.vscX gene(GenBank accession number:FR780679) contained a 378 bp open reading frame(ORF),encoding a putative protein of 125 amino acids.The theoretical molecular weight was 14.209 2 kD and theoretical pI was 5.75.By using Signal 4.1 Server and TMHMM Server 2.0,it was predicted that VscX protein had no transmembrane domain or signal peptide.The results of prediction using SoftBerry-Psite software showed that VscX protein contained three casein kinase Ⅱ phosphorylation sites,one N-myristoylation site and three C-terminal targeting signal sites.Subcellular localization revealed that VscX might be located in cytoplasm with the possibility of 56.5%.According to SMART prediction,VscX had one Pfam(1-125 aa) domain.Phylogenetic analysis revealed that VscX from V.alginolyticus and VscX from V.parahemolyticus were clustered into the same group.Network interaction analysis showed that vscX was adjacent to vscY,vopB and sycN.By real-time fluorescent quantitative PGR technique and 2^(-△△Ct) method,the differences in expression levels of VscX mRNA in V.alginolyticus strain HY9901,T3 SS deletion strain AvscO and complementary strain C-vscO at different growth stages were analyzed.The results showed that the expression levels of VscX mRNA in V.alginolyticus strain HY9901 and C-vscO were significantly up-regulated at stable growth stage(P<0.01);the expression levels of VscX mRNA in deletion strain AvscO were significantly up-regulated at late growth stage(P <0.01).This study provided the basis for revealing the transport mechanism of T3 SS injectisome of V.alginolyticus.展开更多
Pseudomonas aeruginosa is a significant pathogen mainly causing healthcare-associated infections(HAIs).Newly emerging high-risk clones of P.aeruginosa with elevated virulence profiles furtherly cause severe community-...Pseudomonas aeruginosa is a significant pathogen mainly causing healthcare-associated infections(HAIs).Newly emerging high-risk clones of P.aeruginosa with elevated virulence profiles furtherly cause severe community-acquired infections(CAIs).Usually,it is not common for P.aeruginosa to co-carry exoU and exoS genes,encoding two type III secretion system(T3SS)effectors.The pathogenicity mechanism of exoS+/exoU+strains of P.aeruginosa remains unclear.Here,we provide detailed evidence for a subset of hypervirulent P.aeruginosa strains,which abundantly co-express and secrete the T3SS effectors ExoS and ExoU.The exoS+/exoU+P.aeruginosa strains were available to cause both HAIs and CAIs.The CAI-associated strains could elicit severe inflammation and hemorrhage,leading to higher death rates in a murine acute pneumonia model,and had great virulence potential in establishing chronic infections,demonstrating hypervirulence when compared to PAO1(exoS+/exoU-)and PA14(exoS-/exoU+).Both ExoS and ExoU were co-expressed and co-secreted in abundance in exoS+/exoU+strains.Their abundant protein secretion could boost exoS+/exoU+strains’potentials for cytotoxicity in vitro and pathogenicity in vivo.Genomic evidence indicates that exoU acquisition is likely mediated by horizontal gene transfer(HGT)of the pathogenicity island PAPI-2,while deletion of exoU was sufficient to mitigate virulence in the exoS+/exoU+strains.Furthermore,bioinformatics analysis showed that such exoS+/exoU+P.aeruginosa strains turned out to be widely distributed across the globe.Overall,the research provide detailed evidence for the high virulence and epidemicity of exoS+/exoU+strains of P.aeruginosa,highlighting an urgent need for surveillance against these high-risk hypervirulent strains.展开更多
[Objectives]The paper was to clone and analyze bioinformatics of vscN gene from Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the vscN gene sequence of V.alginolyticus HY9901.The full ...[Objectives]The paper was to clone and analyze bioinformatics of vscN gene from Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the vscN gene sequence of V.alginolyticus HY9901.The full length of vscN gene was amplified by PCR and bioinformatics analysis was performed.[Results]The vscN gene was 1323 bp in total,encoding 440 amino acids,with the theoretical molecular weight of 47.86 kD and the theoretical pI value of 5.89.The online prediction showed that there was no signal peptide and no transmembrane region in VscN.The amino acid sequence had 10 N-myristoylation sites,8 phosphorylation sites(2 protein kinase C phosphorylation sites,6 casein kinase II phosphorylation sites),1 amidation site,11 microbody C-terminal target signal sites,1 ATP/GTP binding site motif A(P ring),and 1 ATPaseαandβsubunit specific site.Homology analysis showed that the VscN protein of V.alginolyticus had high homology with that of V.antiquarius,with a similarity of 95.14%.Phylogenetic tree analysis showed that the VscN of V.alginolyticus was clustered into the same subgroup as that of V.diabolicus and V.antiquarius.Functional domain analysis of VscN protein showed that it had Pfam and AAA domains,and involved in the regulation of bacterial virulence.The three-dimensional structure model of VscN simulated by SWISS-MODEL software was similar to the structure of flagellate-specific ATPase FliH-FliI complex.[Conclusions]The results lay a foundation for further study on the regulatory mechanism of VscN protein on bacterial virulence.展开更多
文摘III型分泌系统(type III secretion system,T3SS)是副溶血弧菌主要的毒力因子之一,能传递与致病有关的毒性因子来发挥病原菌的毒性,而温度是影响细菌基因表达等生命活动的关键生态因子。本文以ATCC17802和JA21作为实验菌株,分别提取其RNA,在不同温度和温度应激条件下,以pvuA为内参基因,以副溶血弧菌T3SS1的vcrD1、vopS、vopD1和T3SS2的vscC2β、vcrD2β、vopP2β作为目的基因,运用荧光定量PCR(RT-PCR)技术检测T3SS相关基因的表达下,用ΔΔCt法计算基因表达差异。结果表明:副溶血弧菌(Vibrio parahaemolyticus)ATCC17802菌株的T3SS1基因在16℃表达量最高,T3SS2基因在25℃表达量最高。副溶血弧菌JA21菌株的T3SS1基因和T3SS2基因均在25℃表达量最高。2株副溶血弧菌从不同的培养温度转入到应激温度37℃后,T3SS1和T3SS2基因的表达量均被显著诱导,并表现出时间依赖性,呈现先升高后降低的趋势。这为进一步探究环境因素与副溶血弧菌致病力的关系提供了科学依据。
基金supported by the National Key R&D Program of China (2017YFD0200400)the National Natural Science Foundation of China (31772122 and 31470235)
文摘Xanthomonas oryzea pv.oryzae(Xoo)is the causal agent of bacterial blight of rice,which is a significant threat to many of rice-growing regions.The type Ⅲ secretion system(T3SS)is an essential virulence factor in Xoo.Expression of the T3SS is often induced in the host environment or in hrp-inducing medium but is repressed in nutrient-rich medium.The elucidation of molecular mechanism underlying induction of T3SS genes expression is a very important step to lift the veil on global virulence regulation network in Xoo.Thus,an efficient and reliable genetic tool system is required for detection of the T3SS proteins.In this study,we constructed a protein expression vector pH3-flag based on the backbone of pHM1,a most widely used vector in Xoo strains,especially a model strain PXO99A.This vector contains a synthesized MCS-FLAG cassette that consists of a multiple cloning site(MCS),containing a modified pUC18 polylinker,and Flag as a C-terminal tag.The cassette is flanked by transcriptional terminators to eliminate interference of external transcription enabling detection of accurate protein expression.We evaluated the potential of this expression vector as T3SS proteins detection system and demonstrated it is applicable in the study of T3SS genes expression regulation in Xoo.This improved expression system could be very effectively used as a molecular tool in understanding some virulence genes expression and regulation in Xoo and other Xanthomonas spp.
基金Supported by National Natural Science Foundation of China(31402344,31572656)Major Program of Natural Science Foundation of Guangdong Province(2015A030308020)
文摘In order to enrich the research about type Ⅲ secretion system(T3SS) injectisome of Vibrio alginolyticus,vscX gene was cloned from V.alginolyticus strain HY9901 for bioinformatics analysis and expression analysis.Specific primers were designed according to the full-length genome sequence of V.alginolyticus in GenBank.vscX gene(GenBank accession number:FR780679) contained a 378 bp open reading frame(ORF),encoding a putative protein of 125 amino acids.The theoretical molecular weight was 14.209 2 kD and theoretical pI was 5.75.By using Signal 4.1 Server and TMHMM Server 2.0,it was predicted that VscX protein had no transmembrane domain or signal peptide.The results of prediction using SoftBerry-Psite software showed that VscX protein contained three casein kinase Ⅱ phosphorylation sites,one N-myristoylation site and three C-terminal targeting signal sites.Subcellular localization revealed that VscX might be located in cytoplasm with the possibility of 56.5%.According to SMART prediction,VscX had one Pfam(1-125 aa) domain.Phylogenetic analysis revealed that VscX from V.alginolyticus and VscX from V.parahemolyticus were clustered into the same group.Network interaction analysis showed that vscX was adjacent to vscY,vopB and sycN.By real-time fluorescent quantitative PGR technique and 2^(-△△Ct) method,the differences in expression levels of VscX mRNA in V.alginolyticus strain HY9901,T3 SS deletion strain AvscO and complementary strain C-vscO at different growth stages were analyzed.The results showed that the expression levels of VscX mRNA in V.alginolyticus strain HY9901 and C-vscO were significantly up-regulated at stable growth stage(P<0.01);the expression levels of VscX mRNA in deletion strain AvscO were significantly up-regulated at late growth stage(P <0.01).This study provided the basis for revealing the transport mechanism of T3 SS injectisome of V.alginolyticus.
基金supported by grants from the National Key R&D Program of China(2021YFC2302005)the Joint Funds of the International Development Research Center of Canada(109282-001)the National Key R&D Program of China(2021YFC2301004 and 2017YFE0125600).
文摘Pseudomonas aeruginosa is a significant pathogen mainly causing healthcare-associated infections(HAIs).Newly emerging high-risk clones of P.aeruginosa with elevated virulence profiles furtherly cause severe community-acquired infections(CAIs).Usually,it is not common for P.aeruginosa to co-carry exoU and exoS genes,encoding two type III secretion system(T3SS)effectors.The pathogenicity mechanism of exoS+/exoU+strains of P.aeruginosa remains unclear.Here,we provide detailed evidence for a subset of hypervirulent P.aeruginosa strains,which abundantly co-express and secrete the T3SS effectors ExoS and ExoU.The exoS+/exoU+P.aeruginosa strains were available to cause both HAIs and CAIs.The CAI-associated strains could elicit severe inflammation and hemorrhage,leading to higher death rates in a murine acute pneumonia model,and had great virulence potential in establishing chronic infections,demonstrating hypervirulence when compared to PAO1(exoS+/exoU-)and PA14(exoS-/exoU+).Both ExoS and ExoU were co-expressed and co-secreted in abundance in exoS+/exoU+strains.Their abundant protein secretion could boost exoS+/exoU+strains’potentials for cytotoxicity in vitro and pathogenicity in vivo.Genomic evidence indicates that exoU acquisition is likely mediated by horizontal gene transfer(HGT)of the pathogenicity island PAPI-2,while deletion of exoU was sufficient to mitigate virulence in the exoS+/exoU+strains.Furthermore,bioinformatics analysis showed that such exoS+/exoU+P.aeruginosa strains turned out to be widely distributed across the globe.Overall,the research provide detailed evidence for the high virulence and epidemicity of exoS+/exoU+strains of P.aeruginosa,highlighting an urgent need for surveillance against these high-risk hypervirulent strains.
基金Supported by Outstanding Graduate Entering Laboratory Project of College of FisheriesGuangdong Ocean University+3 种基金National Natural Science Foundation of China (32073015)Natural Science Foundation of Guangdong Province(2021A1515011078)Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University (CXXL2022005)Undergraduate Innovation Team of Guangdong Ocean University (CCTD201802)
文摘[Objectives]The paper was to clone and analyze bioinformatics of vscN gene from Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the vscN gene sequence of V.alginolyticus HY9901.The full length of vscN gene was amplified by PCR and bioinformatics analysis was performed.[Results]The vscN gene was 1323 bp in total,encoding 440 amino acids,with the theoretical molecular weight of 47.86 kD and the theoretical pI value of 5.89.The online prediction showed that there was no signal peptide and no transmembrane region in VscN.The amino acid sequence had 10 N-myristoylation sites,8 phosphorylation sites(2 protein kinase C phosphorylation sites,6 casein kinase II phosphorylation sites),1 amidation site,11 microbody C-terminal target signal sites,1 ATP/GTP binding site motif A(P ring),and 1 ATPaseαandβsubunit specific site.Homology analysis showed that the VscN protein of V.alginolyticus had high homology with that of V.antiquarius,with a similarity of 95.14%.Phylogenetic tree analysis showed that the VscN of V.alginolyticus was clustered into the same subgroup as that of V.diabolicus and V.antiquarius.Functional domain analysis of VscN protein showed that it had Pfam and AAA domains,and involved in the regulation of bacterial virulence.The three-dimensional structure model of VscN simulated by SWISS-MODEL software was similar to the structure of flagellate-specific ATPase FliH-FliI complex.[Conclusions]The results lay a foundation for further study on the regulatory mechanism of VscN protein on bacterial virulence.
