High-Throughput Analysis of Gene Function in the Bacterial Predator Bdellovibrio bacteriovorus
High-Throughput Analysis of Gene Function in the Bacterial Predator Bdellovibrio bacteriovorus
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DOI：
10.1128/mbio.01040-19
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发表时间：
2019-05-01
期刊：
影响因子：
6.4
通讯作者：
Camilli, Andrew
中科院分区：
文献类型：
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作者：
Duncan, Miles C.;Gillette, Rebecca K.;Camilli, Andrew
噬菌蛭弧菌是一种能够杀死大多数革兰氏阴性细菌（包括耐药病原体）并在其中复制的细菌捕食者。尽管这种生物体作为一种潜在的治疗方法越来越受到关注，但它的许多基因仍然没有被描述。在这里，我们用B进行高通量遗传筛选。利用转座子测序（Tn-seq）对食菌性昆虫进行了研究，以探索捕食的遗传要求。201个基因被认为是在没有猎物的情况下生长所必需的，而超过100个基因被发现是人类病原体霍乱弧菌和大肠杆菌在寄生虫和生物膜状态下捕食性生长所必需的。为了进一步开展这项工作，我们在B中创建了一个有序敲除库。bacteriovorus和开发新的高通量技术来表征突变体的阶段的不足，在捕食者的生命周期。使用显微镜和流式细胞仪，我们确认了10个突变体缺陷的猎物附着和8个突变体缺陷的猎物四舍五入。这些基因中的大多数是假设的，以前没有特征。最后，我们对B组提出了新的命名法。根据它们的捕食缺陷阶段，噬菌菌体突变体分为几类。这些结果有助于我们对细菌捕食的基本理解，并可能有助于治理B。重要性噬菌蛭弧菌（Bdellovibrio bacteriovorus）是一种捕食性细菌，可以杀死广泛的革兰氏阴性细菌，包括许多人类病原体。鉴于全球抗生素耐药性的上升和过去30年发现的新抗生素的缺乏，这种捕食者有可能成为传统抗生素的替代品。多年来，B。由于缺乏遗传工具，对食菌动物的研究受到了阻碍，而且直到最近才开始建立捕食的遗传机制。在这里，我们全面识别和描述了杀死细菌猎物所需的捕食者基因，以及干扰这一过程的基因，这可能使我们能够设计出更好的治疗性捕食者。根据我们的研究，我们和其他研究人员最终可能能够通过基因工程改造菌株，提高杀伤率，针对特定的猎物物种，或优先针对浮游或生物膜状态的猎物。
Bdellovibrio bacteriovorus is a bacterial predator capable of killing and replicating inside most Gram-negative bacteria, including antibiotic-resistant pathogens. Despite growing interest in this organism as a potential therapeutic, many of its genes remain uncharacterized. Here, we perform a high-throughput genetic screen with B. bacteriovorus using transposon sequencing (Tn-seq) to explore the genetic requirements of predation. Two hundred one genes were deemed essential for growth in the absence of prey, whereas over 100 genes were found to be specifically required for predative growth on the human pathogens Vibrio cholerae and Escherichia coli in both planktonic and biofilm states. To further this work, we created an ordered-knockout library in B. bacteriovorus and developed new high-throughput techniques to characterize the mutants by their stage of deficiency in the predator life cycle. Using microscopy and flow cytometry, we confirmed 10 mutants defective in prey attachment and eight mutants defective in prey rounding. The majority of these genes are hypothetical and previously uncharacterized. Finally, we propose new nomenclature to group B. bacteriovorus mutants into classes based on their stage of predation defect. These results contribute to our basic understanding of bacterial predation and may be useful for harnessing B. bacteriovorus to kill harmful pathogens in the clinical setting.IMPORTANCE Bdellovibrio bacteriovorus is a predatory bacterium that can kill a wide range of Gram-negative bacteria, including many human pathogens. Given the global rise of antibiotic resistance and dearth of new antibiotics discovered in the past 30 years, this predator has potential as an alternative to traditional antibiotics. For many years, B. bacteriovorus research was hampered by a lack of genetic tools, and the genetic mechanisms of predation have only recently begun to be established. Here, we comprehensively identify and characterize predator genes required for killing bacterial prey, as well as genes that interfere in this process, which may allow us to design better therapeutic predators. Based on our study, we and other researchers may ultimately be able to genetically engineer strains that have improved killing rates, target specific species of prey, or preferentially target prey in the planktonic or biofilm state.