Luo Shengdong, Lu Shanshan, Fan Huahao, Sun Zhihui, Hu Yan, Li Ruisheng, An Xiaoping, Uversky Vladimir N, Chen Zeliang, Tong Yigang, Song Lihua
Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China.
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.
J Bacteriol. 2021 May 1;203(9). doi: 10.1128/JB.00588-20. Epub 2021 Feb 8.
strains carry one of four large, conserved, autonomously replicating plasmids (QpH1, QpRS, QpDV, and QpDG) or a QpRS-like chromosomally integrated sequence of unknown function. Here we report the characterization of the QpH1 plasmid of Nine Mile phase II by making QpH1-deficient strains. A shuttle vector pQGK containing the CBUA0036-0039a region (predicted as being required for the QpH1 maintenance) was constructed. The pQGK vector can be stably transformed into the Nine Mile II and maintained at a similar low copy like QpH1. Importantly, transformation with pQGK cured the endogenous QpH1 due to plasmid incompatibility. Compared to a Nine Mile II transformant of a RSF1010-ori based vector, the pQGK transformant shows a similar growth curve in both axenic media and Buffalo green monkey kidney cells, a variable growth defect in macrophage-like THP-1 cells depending on the origin of inoculum, and dramatically reduced ability of colonizing wild-type bone marrow-derived murine macrophages. Furthermore, we found CBUA0037-0039 ORFs are essential for plasmid maintenance, and CBUA0037-0038 ORFs account for plasmid compatibility. And plasmid-deficient can be isolated by using CBUA0037 or -0038 deletion vectors. Furthermore, QpH1-deficient strains caused a lesser extent of splenomegaly in SCID mice but, intriguingly, they had significant growth in SCID mouse-sourced macrophages. Taken together, our data suggest that QpH1 encodes factor(s) essential for colonizing murine, not human, macrophages. This study suggests a critical role of QpH1 for persistence in rodents and expands the toolkit for the genetic studies in All isolates carry one of four large, conserved, autonomously replicating plasmids or a plasmid-like chromosomally integrated sequence. The plasmid is a candidate virulence factor of unknown function. Here we describe the construction of novel shuttle vectors that allow making plasmid-deficient mutants. With this plasmid-curing approach, we characterized the role of the QpH1 plasmid in and infection models. We found that the plasmid plays a critical role for growth in murine macrophages. Our work suggests an essential role of the QpH1 plasmid for the acquisition of colonizing capability in rodents by This study represents a major step toward unravelling the mystery of the cryptic plasmids.
菌株携带四种大型、保守、自主复制的质粒（QpH1、QpRS、QpDV和QpDG）之一，或一个功能未知的类似QpRS的染色体整合序列。在此，我们通过构建QpH1缺陷菌株，报告了对九英里二期QpH1质粒的特性分析。构建了一个包含CBUA0036 - 0039a区域（预测为QpH1维持所必需）的穿梭载体pQGK。pQGK载体可稳定转化到九英里二期菌株中，并以与QpH1相似的低拷贝数维持。重要的是，由于质粒不相容性，用pQGK转化可消除内源性QpH1。与基于RSF1010 - ori载体的九英里二期转化体相比，pQGK转化体在无菌培养基和水牛绿猴肾细胞中显示出相似的生长曲线，在巨噬细胞样THP - 1细胞中的生长缺陷因接种物来源而异，并且在定殖野生型骨髓来源的小鼠巨噬细胞方面能力显著降低。此外，我们发现CBUA0037 - 0039开放阅读框对于质粒维持至关重要，CBUA0037 - 0038开放阅读框决定质粒相容性。并且可以使用CBUA0037或 - 0038缺失载体分离质粒缺陷菌株。此外，QpH1缺陷菌株在SCID小鼠中引起的脾肿大程度较小，但有趣的是，它们在SCID小鼠来源的巨噬细胞中生长显著。综上所述，我们的数据表明QpH1编码定殖小鼠而非人类巨噬细胞所必需的因子。这项研究表明QpH1在立克次氏体在啮齿动物中的持续存在中起关键作用，并扩展了立克次氏体基因研究的工具包。所有分离株携带四种大型、保守、自主复制的质粒之一或一个类似质粒的染色体整合序列。该质粒是一种功能未知的候选毒力因子。在此，我们描述了新型穿梭载体的构建，这些载体可用于构建质粒缺陷的立克次氏体突变体。通过这种质粒消除方法，我们在立克次氏体感染模型中表征了QpH1质粒的作用。我们发现该质粒在立克次氏体在小鼠巨噬细胞中的生长中起关键作用。我们的工作表明QpH1质粒对于立克次氏体在啮齿动物中获得定殖能力至关重要。这项研究代表了揭开立克次氏体隐蔽质粒之谜的重要一步。
