Temporal analysis of Coxiella burnetii morphological differentiation
Temporal analysis of Coxiella burnetii morphological differentiation
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DOI：
10.1128/jb.186.21.7344-7352.2004
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发表时间：
2004-11-01
影响因子：
3.2
通讯作者：
Heinzen, RA
中科院分区：
文献类型：
--
作者：
Coleman, SA;Fischer, ER;Heinzen, RA
贝氏柯克斯体经历了一个不明确的发育周期，产生形态上不同的小细胞变体（SCV）和大细胞变体（LCV）。我们开发了一个模型来研究C。贝氏体形态发生，其使用用从老化的感染细胞培养物收获的同质SCV（II期Nine Mile株）同步感染的Vero细胞。在8天的细胞内生长的时间过程中的透射电子显微镜分析进行了评价，结合一步生长曲线相关联的形态分化与生长周期阶段。滞后期发生在感染后的前2天（p.i.）并且主要由SCV到LCV的形态发生组成。在接下来的4天中，LCV形式占主导地位，在此期间观察到指数增长。在指数期计算的世代时间为10.2小时（通过定量PCR测定）和11.7小时（通过重新铺板荧光焦点形成单位测定）。稳定期在感染后约6天开始。并且与SCV的再现一致，其在感染后8天数量增加。定量逆转录酶-PCR证实scvA的最大表达，其编码SCV特异性蛋白，在感染后8天，而免疫金透射电子显微镜显示ScvA在整个滞后期和指数期降解，在稳定期开始时观察到表达增加。总的来说，这些结果表明，贝氏隐孢子虫的整个生长周期是一个封闭的细菌系统的特点，生物体的复制形式是LCV。本报告中描述的实验模型将允许一个全球转录组和蛋白质组分析的C。Burnetii发育型。
Coxiella burnetii undergoes a poorly defined developmental cycle that generates morphologically distinct small-cell variants (SCV) and large-cell variants (LCV). We developed a model to study C. burnetii morphogenesis that uses Vero cells synchronously infected with homogeneous SCV (Nine Mile strain in phase II) harvested from aged infected cell cultures. A time course transmission electron microscopic analysis over 8 days of intracellular growth was evaluated in conjunction with one-step growth curves to correlate morphological differentiations with growth cycle phase. Lag phase occurred during the first 2 days postinfection (p.i.) and was primarily composed of SCV-to-LCV morphogenesis. LCV forms predominated over the next 4 days, during which exponential growth was observed. Calculated generation times during exponential phase were 10.2 h (by quantitative PCR assay) and 11.7 h (by replating fluorescent focus-forming unit assay). Stationary phase began at approximately 6 days p.i. and coincided with the reappearance of SCV, which increased in number at 8 days p.i. Quantitative reverse transcriptase-PCR demonstrated maximal expression of scvA, which encodes an SCV-specific protein, at 8 days p.i., while immunogold transmission electron microscopy revealed degradation of ScvA throughout lag and exponential phases, with increased expression observed at the onset of stationary phase. Collectively, these results indicate that the overall growth cycle of C burnetii is characteristic of a closed bacterial system and that the replicative form of the organism is the LCV. The experimental model described in this report will allow a global transcriptome and proteome analysis of C. burnetii developmental forms.