A genomic safe haven for mutant complementation in Cryptococcus neoformans.
A genomic safe haven for mutant complementation in Cryptococcus neoformans.
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DOI：
10.1371/journal.pone.0122916
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Fraser JA
中科院分区：
文献类型：
--
作者：
Arras SD;Chitty JL;Blake KL;Schulz BL;Fraser JA
正如科赫的假设构成了早期传染病研究的基础一样，斯坦利·法科夫的分子科赫假设定义了确定特定基因是否对病原体的毒力起作用的最佳实践。从根本上说，这些分子假设表明，如果一个基因参与毒力，它的移除将损害毒力。同样，它的重新引入应该会恢复突变株的毒力。这些方法被广泛应用于新生隐球菌，其中通过生物转化删除基因是一种成熟的技术。然而，这些突变体的互补就不那么简单了。目前，将采取三种方法之一：在原始位置重新引入基因，将基因重新引入基因组中的随机位置，或者根本不对突变进行补充。根据使用的方法，突变可能会得到补充，但其他基因在这个过程中可能会被破坏。为了克服目前互补方法的缺点，我们创造了一种新的工具来帮助研究基因在毒力中的作用的这一关键步骤。我们已经在新生隐孢子虫基因组中鉴定了一个被称为“安全港”的小的无基因区域，并构建了一个针对这个预先选择的位置的DNA构建物的质粒载体。该质粒载体的整合频率很高，在不破坏相邻基因的情况下有效地补充了突变菌株。整合靶向载体后，对安全避风港两侧基因的QRT-PCR结果显示，它们的表达没有变化，在包括鼻腔感染小鼠模型在内的一系列表型分析中也没有观察到次级表型。综合起来，这些数据证实了我们已经成功地为隐球菌群创建了急需的分子资源，使分子科赫假设得以可靠地实现。
Just as Koch’s postulates formed the foundation of early infectious disease study, Stanley Falkow’s molecular Koch’s postulates define best practice in determining whether a specific gene contributes to virulence of a pathogen. Fundamentally, these molecular postulates state that if a gene is involved in virulence, its removal will compromise virulence. Likewise, its reintroduction should restore virulence to the mutant. These approaches are widely employed in Cryptococcus neoformans, where gene deletion via biolistic transformation is a well-established technique. However, the complementation of these mutants is less straightforward. Currently, one of three approaches will be taken: the gene is reintroduced at the original locus, the gene is reintroduced into a random site in the genome, or the mutant is not complemented at all. Depending on which approach is utilized, the mutant may be complemented but other genes are potentially disrupted in the process. To counter the drawbacks of the current approaches to complementation we have created a new tool to assist in this key step in the study of a gene’s role in virulence. We have identified and characterized a small gene-free region in the C. neoformans genome dubbed the “safe haven”, and constructed a plasmid vector that targets DNA constructs to this preselected site. The plasmid vector integrates with high frequency, effectively complementing a mutant strain without disrupting adjacent genes. qRT-PCR of the flanking genes on either side of the safe haven site following integration of the targeting vector revealed no changes in their expression, and no secondary phenotypes were observed in a range of phenotypic assays including an intranasal murine infection model. Combined, these data confirm that we have successfully created a much-needed molecular resource for the Cryptococcus community, enabling the reliable fulfillment of the molecular Koch’s postulates.
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通讯作者：
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