Improved Quantification, Propagation, Purification and Storage of the Obligate Intracellular Human Pathogen Orientia tsutsugamushi
Improved Quantification, Propagation, Purification and Storage of the Obligate Intracellular Human Pathogen Orientia tsutsugamushi
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DOI：
10.1371/journal.pntd.0004009
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发表时间：
2015-08-01
影响因子：
3.8
通讯作者：
Salje, Jeanne
中科院分区：
文献类型：
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作者：
Giengkam, Suparat;Blakes, Alex;Salje, Jeanne
恙虫病是东南亚农村地区严重发热性疾病的主要病因。病原体恙虫病东方体是一种专性细胞内细菌，通过纤恙螨叮咬传播给人类。对O.恙虫病已经落后于其他重要的人类病原体。原因之一是O.恙虫病是一种只能在哺乳动物细胞中培养的专性细胞内细菌，需要特定的繁殖和分析方法。在这里，我们进行了一系列工作，旨在改进这种重要但被忽视的人类病原体的定量、繁殖、纯化和长期储存方法。这些结果对其他研究O.恙虫病和其他专性细胞内病原体，如立克次体和衣原体科。恙虫病在培养的小鼠胚胎成纤维细胞（L929）中生长。使用O.恙虫病特异性qPCR测定。在培养的哺乳动物细胞中生长后，根据对细菌细胞数量的影响来监测导致活力和生长改善的条件。关键结果开发标准化生长测定以定量体外细菌复制和活力。定量比较不同的DNA提取方法。定量FBS浓度、柔红霉素补充、培养基组成对生长的影响，细菌纯化的优化包括宿主细胞裂解方法，纯化温度，细菌产量计算和不同离心速度下的细菌沉淀。在一系列条件下（包括不同冷冻条件）长期储存和冷冻后细菌活力损失的定量缓冲液和不同的冷冻速率。结论在这里，我们提出了一个标准化的方法比较O。经纯化、处理和在各种条件下繁殖后的恙虫病。两者合计，我们提出了一个身体的数据，以支持这种生物体的繁殖，纯化和储存的改进技术。这些数据将有助于提高临床分离率以及进行体外细胞生物学实验。
BackgroundScrub typhus is a leading cause of serious febrile illness in rural Southeast Asia. The causative agent, Orientia tsutsugamushi, is an obligate intracellular bacterium that is transmitted to humans by the bite of a Leptotrombidium mite. Research into the basic mechanisms of cell biology and pathogenicity of O. tsutsugamushi has lagged behind that of other important human pathogens. One reason for this is that O. tsutsugamushi is an obligate intracellular bacterium that can only be cultured in mammalian cells and that requires specific methodologies for propagation and analysis. Here, we have performed a body of work designed to improve methods for quantification, propagation, purification and long-term storage of this important but neglected human pathogen. These results will be useful to other researchers working on O. tsutsugamushi and also other obligate intracellular pathogens such as those in the Rickettsiales and Chlamydiales families.MethodologyA clinical isolate of O. tsutsugamushi was grown in cultured mouse embryonic fibroblast (L929) cells. Bacterial growth was measured using an O. tsutsugamushi-specific qPCR assay. Conditions leading to improvements in viability and growth were monitored in terms of the effect on bacterial cell number after growth in cultured mammalian cells.Key resultsDevelopment of a standardised growth assay to quantify bacterial replication and viability in vitro.Quantitative comparison of different DNA extraction methods.Quantification of the effect on growth of FBS concentration, daunorubicin supplementation, media composition, host cell confluence at infection and frequency of media replacement.Optimisation of bacterial purification including a comparison of host cell lysis methods, purification temperature, bacterial yield calculations and bacterial pelleting at different centrifugation speeds.Quantification of bacterial viability loss after long term storage and freezing under a range of conditions including different freezing buffers and different rates of freezing.ConclusionsHere we present a standardised method for comparing the viability of O. tsutsugamushi after purification, treatment and propagation under various conditions. Taken together, we present a body of data to support improved techniques for propagation, purification and storage of this organism. This data will be useful both for improving clinical isolation rates as well as performing in vitro cell biology experiments.