Fluorescence Activated Cell Sorting of Rickettsia prowazekii-Infected Host Cells Based on Bacterial Burden and Early Detection of Fluorescent Rickettsial Transformants.
Fluorescence Activated Cell Sorting of Rickettsia prowazekii-Infected Host Cells Based on Bacterial Burden and Early Detection of Fluorescent Rickettsial Transformants.
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DOI：
10.1371/journal.pone.0152365
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Wood DO
中科院分区：
文献类型：
--
作者：
Driskell LO;Tucker AM;Woodard A;Wood RR;Wood DO
普氏立克次体是一种专性胞内细菌，仅在真核宿主细胞的胞质溶胶中复制。尽管这种生活方式对遗传操作造成了障碍，但通过转座子插入和同源重组机制已经产生了立克次体突变体。然而，鉴定和分离R所需的时间长度阻碍了进展。prowazekii转化体。为了减少每个转化实验所需的时间和与立克次体繁殖和收获相关的可变性，使用表征的冷冻原液来产生电感受态立克次体。采用这些立克次体的转化实验建立了荧光立克次体群体可以在电穿孔后一周内使用荧光激活细胞分选仪进行鉴定。随着转化DNA量的增加，早期检测得到改善。此外，我们证明了立克次体感染的细胞的异质群体可以根据每个细胞的立克次体数量分为不同的亚群。总之，我们的数据表明，荧光报告和细胞分选的组合代表了一个重要的技术进步，将有助于分离不同的R。prowazekii突变体，并允许在各种感染负荷下更仔细地检查感染对宿主细胞的影响。
Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that replicates only within the cytosol of a eukaryotic host cell. Despite the barriers to genetic manipulation that such a life style creates, rickettsial mutants have been generated by transposon insertion as well as by homologous recombination mechanisms. However, progress is hampered by the length of time required to identify and isolate R. prowazekii transformants. To reduce the time required and variability associated with propagation and harvesting of rickettsiae for each transformation experiment, characterized frozen stocks were used to generate electrocompetent rickettsiae. Transformation experiments employing these rickettsiae established that fluorescent rickettsial populations could be identified using a fluorescence activated cell sorter within one week following electroporation. Early detection was improved with increasing amounts of transforming DNA. In addition, we demonstrate that heterogeneous populations of rickettsiae-infected cells can be sorted into distinct sub-populations based on the number of rickettsiae per cell. Together our data suggest the combination of fluorescent reporters and cell sorting represent an important technical advance that will facilitate isolation of distinct R. prowazekii mutants and allow for closer examination of the effects of infection on host cells at various infectious burdens.