Beare Paul A, Howe Dale, Cockrell Diane C, Heinzen Robert A
Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, 903 S. 4th St., Hamilton, MT 59840, USA.
Appl Environ Microbiol. 2007 Jun;73(12):4048-54. doi: 10.1128/AEM.00411-07. Epub 2007 Apr 27.
Coxiella burnetii is an obligate intracellular bacterium that replicates in a large lysosome-like parasitophorous vacuole (PV). Current methods of cloning C. burnetii are laborious and technically demanding. We have developed an alternative cloning method that involves excision of individual C. burnetii-laden PVs from infected cell monolayers by micromanipulation. To demonstrate the cloning utility and efficiency of this procedure, we coinfected Vero cells with isogenic variants of the Nine Mile strain of C. burnetii. Coinhabited PVs harboring Nine Mile phase II (NMII) and Nine Mile phase I (NMI) or Nine Mile crazy (NMC) were demonstrated by immunofluorescence. PVs were then randomly excised from cells coinfected with NMI and NMC by micromanipulation, and PVs harboring both strains were identified by PCR. Fresh Vero cells were subsequently infected with organisms from coinhabited PVs, and the PV excision and PCR screening process was repeated. Without exception, PVs obtained from second-round excisions contained clonal populations of either NMII or NMC, demonstrating that micromanipulation is an efficient and reproducible procedure for obtaining C. burnetii clones.
伯纳特柯克斯体是一种专性细胞内细菌，在一个大的类似溶酶体的寄生泡（PV）中进行复制。目前克隆伯纳特柯克斯体的方法费力且技术要求高。我们开发了一种替代克隆方法，该方法涉及通过显微操作从感染的细胞单层中切除单个载有伯纳特柯克斯体的PV。为了证明该程序的克隆效用和效率，我们用伯纳特柯克斯体九英里菌株的同基因变体共感染了Vero细胞。通过免疫荧光证实了含有九英里II期（NMII）和九英里I期（NMI）或九英里疯狂型（NMC）的共栖PV。然后通过显微操作从感染了NMI和NMC的细胞中随机切除PV，并通过PCR鉴定含有两种菌株的PV。随后用来自共栖PV的生物体感染新鲜的Vero细胞，并重复PV切除和PCR筛选过程。无一例外，第二轮切除获得的PV包含NMII或NMC的克隆群体，这表明显微操作是获得伯纳特柯克斯体克隆的一种有效且可重复的程序。
