为探究鸭疫里默氏杆菌(RA)IX型分泌系统(T9SS)分泌的假定蛋白AS87_05150的功能,本研究采用自杀质粒同源重组的方法构建RA Yb2株AS87_05150基因缺失株Yb2Δ5150及其回补株cYb2Δ5150,经PCR及测序鉴定,结果显示AS87_05150基因缺失株Yb2Δ5...为探究鸭疫里默氏杆菌(RA)IX型分泌系统(T9SS)分泌的假定蛋白AS87_05150的功能,本研究采用自杀质粒同源重组的方法构建RA Yb2株AS87_05150基因缺失株Yb2Δ5150及其回补株cYb2Δ5150,经PCR及测序鉴定,结果显示AS87_05150基因缺失株Yb2Δ5150和回补株cYb2Δ5150均正确构建,缺失株的表型为16S r RNA^(+)AS87_05150 ORF^(-),回补株的表型为16S r RNA^(+)AS87_05150 ORF^(+)。采用荧光定量PCR(qPCR)检测Yb2、Yb2Δ5150和cYb2Δ5150株AS87_05150基因的转录水平;根据上述3株菌不同时间的OD_(600nm)值绘制各菌株的生长曲线;采用微量结晶紫法检测各菌株生物被膜(BF)的形成能力;将10^(7)cfu/孔的Yb2、Yb2Δ5150和cYb2Δ5150株分别感染Vero细胞,检测各菌株对Vero细胞的黏附和侵袭力。q PCR检测结果显示,cYb2Δ5150株中AS87_05150 m RNA相对转录水平比Yb2株高约19倍(P<0.0001),Yb2Δ5150株未扩增到AS87_05150基因;生长曲线结果显示,Yb2Δ5150与Yb2株的生长曲线基本一致,但cYb2Δ5150生长速度与Yb2Δ5150株相比极显著下降(P<0.01);BF测定结果显示,Yb2Δ5150和cYb2Δ5150株BF的形成能力与Yb2株相比均极显著降低(P<0.0001);黏附和侵袭力结果显示,这3株菌对Vero细胞的黏附和侵袭力均无显著差异。将3株菌分别以10^(8)cfu/只~10^(4)cfu/只、10^(10)cfu/只~10^(6)cfu/只和10^(9)cfu/只~10^(5)cfu/只的剂量经肌肉注射接种雏鸭,感染后观察雏鸭的发病情况,并采用Reed-Muench法计算3株菌对雏鸭的半数致死量(LD_(50));将3株菌均以10^(7)cfu/只经肌肉注射感染雏鸭,24 h后剖杀,无菌采集各组鸭血液、肝脏和脑组织,作相应处理后涂板采用平板计数法测定载菌量。根据各组鸭的死亡情况,计算Yb2、Yb2Δ5150和cYb2Δ5150株的LD_(50)分别为6.85×10^(5)cfu、8.75×10^(7)cfu和4.681×10^(8)cfu,即缺失株LD_(50)比野生株高约127倍,但回补株的LD_(50)比缺失株高约5倍。载菌量测定结果显示,Yb2Δ5150株和cYb2Δ5150株感染鸭血液、肝脏、脑组织中的细菌载量比Yb2株感染鸭的载菌量均极显著降低(P<0.001),但前两者之间上述指标均无显著差异。本实验结果表明AS87_05150与RA Yb2株的BF形成和致病性相关,但回补株对雏鸭的毒力和致病性均未回复到野生株的水平。本研究为进一步解析RA AS87_05150的功能及分子致病机制等奠定了基础。展开更多
The recently discovered type IX secretion system(T9SS)is limited to the Bacteroidetes phylum.Cytophaga hutchin-sonii,a member of the Bacteroidetes phylum widely spread in soil,has complete orthologs of T9SS components...The recently discovered type IX secretion system(T9SS)is limited to the Bacteroidetes phylum.Cytophaga hutchin-sonii,a member of the Bacteroidetes phylum widely spread in soil,has complete orthologs of T9SS components and many T9SS substrates.C.hutchinsonii can efficiently degrade crystalline cellulose using a novel strategy,in which bacterial cells must be in direct contact with cellulose.It can rapidly glide over surfaces via unclear mech-anisms.Studies have shown that T9SS plays an important role in cellulose degradation,gliding motility,and ion assimilation in C.hutchinsonii.As reported recently,T9SS substrates are N-or O-glycosylated at their C-terminal domains(CTDs),with N-glycosylation being related to the translocation and outer membrane anchoring of these proteins.These findings have deepened our understanding of T9SS in C.hutchinsonii.In this review,we focused on the research progress on diverse substrates and functions of T9SS in C.hutchinsonii and the glycosylation of its substrates.A model of T9SS functions and the glycosylation of its substrates was proposed.展开更多
文摘为探究鸭疫里默氏杆菌(RA)IX型分泌系统(T9SS)分泌的假定蛋白AS87_05150的功能,本研究采用自杀质粒同源重组的方法构建RA Yb2株AS87_05150基因缺失株Yb2Δ5150及其回补株cYb2Δ5150,经PCR及测序鉴定,结果显示AS87_05150基因缺失株Yb2Δ5150和回补株cYb2Δ5150均正确构建,缺失株的表型为16S r RNA^(+)AS87_05150 ORF^(-),回补株的表型为16S r RNA^(+)AS87_05150 ORF^(+)。采用荧光定量PCR(qPCR)检测Yb2、Yb2Δ5150和cYb2Δ5150株AS87_05150基因的转录水平;根据上述3株菌不同时间的OD_(600nm)值绘制各菌株的生长曲线;采用微量结晶紫法检测各菌株生物被膜(BF)的形成能力;将10^(7)cfu/孔的Yb2、Yb2Δ5150和cYb2Δ5150株分别感染Vero细胞,检测各菌株对Vero细胞的黏附和侵袭力。q PCR检测结果显示,cYb2Δ5150株中AS87_05150 m RNA相对转录水平比Yb2株高约19倍(P<0.0001),Yb2Δ5150株未扩增到AS87_05150基因;生长曲线结果显示,Yb2Δ5150与Yb2株的生长曲线基本一致,但cYb2Δ5150生长速度与Yb2Δ5150株相比极显著下降(P<0.01);BF测定结果显示,Yb2Δ5150和cYb2Δ5150株BF的形成能力与Yb2株相比均极显著降低(P<0.0001);黏附和侵袭力结果显示,这3株菌对Vero细胞的黏附和侵袭力均无显著差异。将3株菌分别以10^(8)cfu/只~10^(4)cfu/只、10^(10)cfu/只~10^(6)cfu/只和10^(9)cfu/只~10^(5)cfu/只的剂量经肌肉注射接种雏鸭,感染后观察雏鸭的发病情况,并采用Reed-Muench法计算3株菌对雏鸭的半数致死量(LD_(50));将3株菌均以10^(7)cfu/只经肌肉注射感染雏鸭,24 h后剖杀,无菌采集各组鸭血液、肝脏和脑组织,作相应处理后涂板采用平板计数法测定载菌量。根据各组鸭的死亡情况,计算Yb2、Yb2Δ5150和cYb2Δ5150株的LD_(50)分别为6.85×10^(5)cfu、8.75×10^(7)cfu和4.681×10^(8)cfu,即缺失株LD_(50)比野生株高约127倍,但回补株的LD_(50)比缺失株高约5倍。载菌量测定结果显示,Yb2Δ5150株和cYb2Δ5150株感染鸭血液、肝脏、脑组织中的细菌载量比Yb2株感染鸭的载菌量均极显著降低(P<0.001),但前两者之间上述指标均无显著差异。本实验结果表明AS87_05150与RA Yb2株的BF形成和致病性相关,但回补株对雏鸭的毒力和致病性均未回复到野生株的水平。本研究为进一步解析RA AS87_05150的功能及分子致病机制等奠定了基础。
基金The work is supported by the National Natural Science Foundation of China(No.31770080)National Key Research and Development Program of China(No.2021YFC2100500).
文摘The recently discovered type IX secretion system(T9SS)is limited to the Bacteroidetes phylum.Cytophaga hutchin-sonii,a member of the Bacteroidetes phylum widely spread in soil,has complete orthologs of T9SS components and many T9SS substrates.C.hutchinsonii can efficiently degrade crystalline cellulose using a novel strategy,in which bacterial cells must be in direct contact with cellulose.It can rapidly glide over surfaces via unclear mech-anisms.Studies have shown that T9SS plays an important role in cellulose degradation,gliding motility,and ion assimilation in C.hutchinsonii.As reported recently,T9SS substrates are N-or O-glycosylated at their C-terminal domains(CTDs),with N-glycosylation being related to the translocation and outer membrane anchoring of these proteins.These findings have deepened our understanding of T9SS in C.hutchinsonii.In this review,we focused on the research progress on diverse substrates and functions of T9SS in C.hutchinsonii and the glycosylation of its substrates.A model of T9SS functions and the glycosylation of its substrates was proposed.
