Construction of pSPI12-cured Salmonella enterica serovar Pullorum and identification of IpaJ as an immune response modulator
Construction of pSPI12-cured Salmonella enterica serovar Pullorum and identification of IpaJ as an immune response modulator
复制标题
pSPI12 治愈的肠沙门氏菌白痢血清型的构建及 IpaJ 作为免疫反应调节剂的鉴定
DOI：
10.1080/03079457.2018.1471195
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发表时间：
2018-01-01
期刊：
影响因子：
2.8
通讯作者：
Jiao, Xinan
中科院分区：
文献类型：
--
作者：
Yin, Chao;Xu, Lijuan;Jiao, Xinan
在沙门氏菌中，质粒参与了许多涉及毒力、代谢和抗生素耐药性的途径。为了研究肠沙门氏菌血清型Pullorum （S. Pullorum）中常见的多拷贝质粒pSPI12中ipaJ基因的功能，我们建立了一种消除质粒的方法，并利用自杀载体pDM4构建了质粒治愈菌C79-13-ΔpSPI12。简单地说，pSPI12的500 bp片段ipaJU被克隆到pDM4中，并通过共轭转移转化到S. Pullorum C79-13中。同源重组后，将自杀载体插入pSPI12中，得到pSPI12- pdm4 - ipaju。在自杀载体中诱导sacB基因的表达杀死了携带质粒的细菌，而失去质粒的后代在蔗糖的培养皿中存活。与野生型菌株和携带ipaJ的互补菌株相比，质粒固化菌株对鸡巨噬细胞HD11细胞和LMH肝上皮细胞的感染能力显著降低。此外，经质粒固化的菌株感染的HD11细胞和鸡脾脏中IFN-γ mRNA水平均上调，但IL-4水平在3株菌株间无显著差异。与野生型菌株相比，将ipaJ转化为肠炎链球菌也降低了感染巨噬细胞或鸡脾脏中促炎细胞因子的表达。这些结果表明pSPI12中的ipaJ基因参与了白糜杆菌感染，ipaJ蛋白调节了免疫应答。
ABSTRACT In Salmonella, plasmids participate in many pathways involved in virulence, metabolism, and antibiotic resistance. To investigate the function of the ipaJ gene in a multi-copy plasmid pSPI12 prevalent in Salmonella enterica serovar Pullorum (S. Pullorum), we established a method to eliminate the plasmid and constructed the plasmid-cured bacteria C79-13-ΔpSPI12 by using the suicide vector pDM4. Briefly, a 500 bp fragment ipaJU from pSPI12 was cloned into pDM4 and transformed into S. Pullorum C79-13 by conjugative transfer. After homologous recombination, the suicide vector was inserted into pSPI12 to produce pSPI12-pDM4-ipaJU. Induction of the expression of the sacB gene in the suicide vector killed the bacteria harbouring plasmid, while the progeny losing the plasmid survived in the plate with sucrose. The plasmid-cured strain showed extremely decreased ability to infect chicken macrophage HD11 cells and LMH hepatic epithelial cells compared to wild type strain and complementary strain carrying ipaJ. Additionally, IFN-γ mRNA levels were up-regulated in HD11 cells or chicken spleens infected by plasmid-cured strain, but no difference was detected in IL-4 among the three strains. Transforming ipaJ into S. Enteritidis also decreased expression of proinflammatory cytokines in infected macrophages or chicken spleens compared to wild type strain. These results suggest that the ipaJ gene in pSPI12 is involved in S. Pullorum infection and that IpaJ protein modulates immune response.