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首页> 外文期刊>Infection and immunity >High-Throughput, Signature-Tagged Mutagenic Approach To Identify Novel Virulence Factors of Yersinia pestis CO92 in a Mouse Model of Infection
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High-Throughput, Signature-Tagged Mutagenic Approach To Identify Novel Virulence Factors of Yersinia pestis CO92 in a Mouse Model of Infection

机译：高通量，签名标记的诱变方法，以识别鼠疫鼠疫CO92感染模型中的新型毒力因子。

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The identification of new virulence factors in Yersinia pestis and understanding their molecular mechanisms during an infection process are necessary in designing a better vaccine or to formulate an appropriate therapeutic intervention. By using a high-throughput, signature-tagged mutagenic approach, we created 5,088 mutants of Y. pestis strain CO92 and screened them in a mouse model of pneumonic plague at a dose equivalent to 5 50% lethal doses (LD50) of wild-type (WT) CO92. From this screen, we obtained 118 clones showing impairment in disseminating to the spleen, based on hybridization of input versus output DNA from mutant pools with 53 unique signature tags. In the subsequent screen, 20/118 mutants exhibited attenuation at 8 LD50 when tested in a mouse model of bubonic plague, with infection by 10/20 of the aforementioned mutants resulting in 40% or higher survival rates at an infectious dose of 40 LD50. Upon sequencing, six of the attenuated mutants were found to carry interruptions in genes encoding hypothetical proteins or proteins with putative functions. Mutants with in-frame deletion mutations of two of the genes identified from the screen, namely, rbsA, which codes for a putative sugar transport system ATP-binding protein, and vasK, a component of the type VI secretion system, were also found to exhibit some attenuation at 11 or 12 LD50 in a mouse model of pneumonic plague. Likewise, among the remaining 18 signature-tagged mutants, 9 were also attenuated (40 to 100%) at 12 LD50 in a pneumonic plague mouse model. Previously, we found that deleting genes encoding Braun lipoprotein (Lpp) and acyltransferase (MsbB), the latter of which modifies lipopolysaccharide function, reduced the virulence of Y. pestis CO92 in mouse models of bubonic and pneumonic plague. Deletion of rbsA and vasK genes from either the Δlpp single or the Δlpp ΔmsbB double mutant augmented the attenuation to provide 90 to 100% survivability to mice in a pneumonic plague model at 20 to 50 LD50. The mice infected with the Δlpp ΔmsbB ΔrbsA triple mutant at 50 LD50 were 90% protected upon subsequent challenge with 12 LD50 of WT CO92, suggesting that this mutant or others carrying combinational deletions of genes identified through our screen could potentially be further tested and developed into a live attenuated plague vaccine(s).
机译：在设计更好的疫苗或制定适当的治疗干预措施时，必须鉴定鼠疫耶尔森菌中的新毒力因子并了解其在感染过程中的分子机制。通过使用高通量，带有特征标签的诱变方法，我们创建了鼠疫耶尔森氏菌CO92的5,088个突变体，并在小鼠肺炎鼠疫模型中以相当于5-50％致死剂量（LD50）的野生鼠疫的剂量对其进行了筛选（WT）型CO92。从该筛选中，基于来自具有53个独特标记标签的突变体池中输入DNA与输出DNA的杂交，我们获得了118个克隆，这些克隆在向脾脏扩散方面表现出障碍。在随后的筛选中，当在鼠疫鼠模型中进行测试时，有20/118个突变体在8 LD50处表现出衰减，感染10/20的上述突变体在40 LD50的感染剂量下可导致40％或更高的存活率。测序后，发现六个减毒突变体在编码假设蛋白质或具有推定功能的蛋白质的基因中带有中断。还发现了从筛选中鉴定出的两个基因的框内缺失突变的突变体，即编码假定的糖转运系统ATP结合蛋白的rbsA和VI型分泌系统的组成部分vasK。在肺鼠疫的小鼠模型中，在11点或12点LD50处显示出一些衰减。同样，在肺鼠疫鼠模型中，在剩下的18个带标签标记的突变体中，有9个在12 LD50时也被减弱（40％至100％）。以前，我们发现删除编码布劳恩脂蛋白（Lpp）和酰基转移酶（MsbB）的基因（后者可修饰脂多糖功能）可降低鼠疫和肺鼠疫小鼠模型中的鼠疫耶尔森氏菌CO92的毒力。从Δlpp单突变或ΔlppΔmsbB双突变体中删除rbsA和vasK基因会增加衰减，从而在20至50 LD50的肺鼠疫模型中为小鼠提供90％至100％的存活率。在随后用WT CO92的12 LD50攻击后，在50 LD50处感染ΔlppΔmsbBΔrbsA三重突变体的小鼠受到90％的保护，这表明该突变体或其他带有通过我们的筛选鉴定出的基因组合缺失的突变体可能会得到进一步测试和开发制成减毒活鼠疫疫苗。

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