Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.
Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.
复制标题
DOI：
10.1371/journal.pone.0121271
复制
发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Galyov EE
中科院分区：
文献类型：
--
作者：
Lazar Adler NR;Stevens MP;Dean RE;Saint RJ;Pankhania D;Prior JL;Atkins TP;Kessler B;Nithichanon A;Lertmemongkolchai G;Galyov EE
类鼻疽伯克霍尔德菌是严重热带疾病类鼻疽的病原体，通常表现为败血症。B。类鼻疽K96243基因组编码11个预测的自身转运蛋白，这是一个经常与毒力相关的分泌和外膜蛋白的多样性家族。在一个系统的研究，这些autotorporters，我们构建了插入突变体在每个基因预测编码的autotorporters和评估他们的三个发病机制相关的表型：在BALB/c小鼠腹腔内类鼻疽模型的毒力，净细胞内复制J774.2小鼠巨噬细胞样细胞和生存在45%（v/v）正常人血清。从11个自身转运蛋白突变体的完整库中，我们确定了8个突变体，其与同基因亲本菌株（bcaA，boaA，boaB，bpaA，bpaC，bpaE，bpaF和比马）相比，半数致死剂量增加1至2-log 10。四个突变体，所有证明毒力衰减，表现出减少净细胞内复制J774.2巨噬细胞样细胞（比马，boaB，bpaC和bpaE）。一个单一的突变体（bpaC）被确定为表现出显着降低血清存活相比，野生型。的bpaC突变体，表现出毒力和净细胞内复制衰减，是敏感的补体介导的杀伤通过经典和/或凝集素途径。反式互补挽救了血清抗性。随后，我们表达了四个预测的自身转运蛋白的乘客结构域的重组蛋白，它们代表了鉴定的每个表型组：毒力减弱的那些（BcaA），毒力和净细胞内复制减弱的那些（BpaE），具有毒力缺陷的BpaC突变体，净细胞内复制和血清抗性以及显示野生型表型的那些（BatA）。只有BcaA和BpaE在使用来自血清阳性供体的全血的再刺激测定中引起强烈的IFN-γ应答，并且被来自流行区的血清阳性人血清识别。总之，几种预测的自身转运蛋白对B有贡献。类鼻疽毒力和BpaC可以通过赋予对补体介导的杀伤的抗性来实现。
Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v) normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA). Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE). A single mutant (bpaC) was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA), those attenuated for virulence and net intracellular replication (BpaE), the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA). Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors and were recognised by seropositive human sera from the endemic area. To conclude, several predicted autotransporters contribute to B. pseudomallei virulence and BpaC may do so by conferring resistance against complement-mediated killing.
登录
查看更多内容
影响因子：
4.2
作者：
Lafontaine ER;Balder R;Michel F;Hogan RJ
通讯作者：
Hogan RJ
影响因子：
5.2
作者：
Lazar Adler NR;Stevens JM;Stevens MP;Galyov EE
通讯作者：
Galyov EE
影响因子：
3.6
作者：
DeShazer, D;Brett, PJ;Woods, DE
通讯作者：
Woods, DE
影响因子：
2.6
作者：
Anuntagool, N;Sirisinha, S
通讯作者：
Sirisinha, S
影响因子：
3.1
作者：
Campos, Cristine G.;Byrd, Matthew S.;Cotter, Peggy A.
通讯作者：
Cotter, Peggy A.