Generation of Yersinia pestis attenuated strains by signature-tagged mutagenesis in search of novel vaccine candidates
Generation of Yersinia pestis attenuated strains by signature-tagged mutagenesis in search of novel vaccine candidates
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DOI：
10.1128/iai.72.2.908-915.2004
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发表时间：
2004-02-01
影响因子：
3.1
通讯作者：
Cohen, S
中科院分区：
文献类型：
--
作者：
Flashner, Y;Mamroud, E;Cohen, S
为了寻找用于鼠疫病原体--鼠疫耶尔森氏菌的新型减毒疫苗候选者，对皮下感染的小鼠模型使用了一种签名标记突变策略，并对其进行了优化。建立了鼠疫杆菌金伯利53强毒株的标记突变体文库。通过连续两个循环对300个突变株进行筛选，筛选出16个在感染后48小时在脾中未检测到的突变株。通过与野生型菌株的竞争和接种100CFU(相当于野生型菌株的100-50%致死剂量[LD50])后的毒力试验，在体内评估了这些突变体。获得了广泛的衰减范围，从竞争指数为10(-5)到10(-7)的无毒突变体到平均死亡时间延迟的强毒突变体，或者在两种检测中与野生型难以区分的突变体。对所选突变体的表型和基因类型的鉴定导致了毒力相关基因的鉴定，这些基因编码参与全球细菌生理的因子(例如PurH、Purk、dna E和greA)或假想的多肽，以及毒力调节基因lcrF。其中一个无毒突变菌株(LD50，>；10(7)CFU)被发现在pcm基因上被破坏，这可能与细菌对环境胁迫的反应有关。这个金伯利53pcm突变株优于EV76活疫苗株，因为它诱导了对保护性V和F1抗原的抗体效价提高了10到100倍，而且因为它提供了有效的保护性免疫。
In a search for novel attenuated vaccine candidates for use against Yersinia pestis, the causative agent of plague, a signature-tagged mutagenesis strategy was used and optimized for a subcutaneously infected mouse model. A library of tagged mutants of the virulent Y. pestis Kimberley53 strain was generated. Screening of 300 mutants through two consecutive cycles resulted in selection of 16 mutant strains that were undetectable in spleens 48 h postinfection. Each of these mutants was evaluated in vivo by assays for competition against the wild-type strain and for virulence following inoculation of 100 CFU (equivalent to 100 50% lethal doses [LD50] of the wild type). A wide spectrum of attenuation was obtained, ranging from avirulent mutants exhibiting competition indices of 10(-5) to 10(-7) to virulent mutants exhibiting a delay in the mean time to death or mutants indistinguishable from the wild type in the two assays. Characterization of the phenotypes and genotypes of the selected mutants led to identitication of virulence-associated genes coding for factors involved in global bacterial physiology (e.g., purH, purK, dnaE, and greA) or for hypothetical polypeptides, as well as for the virulence regulator gene lcrF. One of the avirulent mutant strains (LD50, > 10(7) CFU) was found to be disrupted in the pcm locus, which is presumably involved in the bacterial response to environmental stress. This Kimberley53pcm mutant was superior to the EV76 live vaccine strain because it induced 10- to 100-fold-higher antibody titers to the protective V and F1 antigens and because it conferred efficacious protective immunity.