Byrne G I, Ouellette S P, Wang Z, Rao J P, Lu L, Beatty W L, Hudson A P
Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706, USA.
Infect Immun. 2001 Sep;69(9):5423-9. doi: 10.1128/IAI.69.9.5423-5429.2001.
Chlamydia pneumoniae causes community-acquired pneumonia and is associated with several chronic diseases, including asthma and atherosclerosis. The intracellular growth rate of C. pneumoniae slows dramatically during chronic infection, and such persistence leads to attenuated production of new elementary bodies, appearance of morphologically aberrant reticulate bodies, and altered expression of several chlamydial genes. We used an in vitro system to further characterize persistent C. pneumoniae infection, employing both ultrastructural and transcriptional activity measurements. HEp-2 cells were infected with C. pneumoniae (TW-183) at a multiplicity of infection of 3:1, and at 2 h postinfection gamma interferon (IFN-gamma) was added to the medium at 0.15 or 0.50 ng/ml. Treated and untreated cultures were harvested at several times postinfection. RNA was isolated and reverse transcribed, and reverse transcription (RT)-PCR analyses targeting primary transcripts from chlamydial rRNA operons as well as dnaA, polA, mutS, minD, ftsK, and ftsW mRNA were done. Some cultures were fixed and stained for electron microscopic analysis, and a real-time PCR assay was used to assess relative chlamydial chromosome accumulation under each culture condition. The latter assays showed that bacterial chromosome copies accumulated severalfold during IFN-gamma treatment of infected HEp-2 cells, although less accumulation was observed in cells treated with the higher dose. Electron microscopy demonstrated that high-dose IFN-gamma treatment elicited aberrant forms of the bacterium. RT-PCR showed that chlamydial primary rRNA transcripts were present in all IFN-gamma-treated and untreated cell cultures, indicating bacterial metabolic activity. Transcripts from dnaA, polA, mutS, and minD, all of which encode products for bacterial chromosome replication and partition, were expressed in IFN-gamma-treated and untreated cells. In contrast, ftsK and ftsW, encoding products for bacterial cell division, were expressed in untreated cells, but expression was attenuated in cells treated with low-dose IFN-gamma and absent in cells given the high dose of cytokine. Thus, the development of persistence included production of transcripts for DNA replication-related, but not cell division-related, genes. These results provide new insight regarding molecular activities that accompany persistence of C. pneumoniae, as well as suggesting requirements for reactivation from persistent to productive growth.
肺炎衣原体可引起社区获得性肺炎，并与包括哮喘和动脉粥样硬化在内的多种慢性疾病相关。在慢性感染期间，肺炎衣原体的细胞内生长速度显著减慢，这种持续性导致新的原体产生减少、形态异常的网状体出现以及几种衣原体基因表达改变。我们使用体外系统，通过超微结构和转录活性测量进一步表征肺炎衣原体的持续性感染。用感染复数为3:1的肺炎衣原体（TW-183）感染HEp-2细胞，在感染后2小时，向培养基中加入0.15或0.50 ng/ml的γ干扰素（IFN-γ）。在感染后的几个时间点收获经处理和未处理的培养物。分离RNA并进行逆转录，然后针对衣原体rRNA操纵子的初级转录本以及dnaA、polA、mutS、minD、ftsK和ftsW mRNA进行逆转录（RT）-PCR分析。一些培养物进行固定和染色以进行电子显微镜分析，并使用实时PCR测定法评估每种培养条件下衣原体染色体的相对积累。后一种测定表明，在感染的HEp-2细胞的IFN-γ处理期间，细菌染色体拷贝数积累了几倍，尽管在高剂量处理的细胞中观察到的积累较少。电子显微镜显示高剂量IFN-γ处理引发了细菌的异常形态。RT-PCR表明，在所有IFN-γ处理和未处理的细胞培养物中都存在衣原体初级rRNA转录本，表明细菌具有代谢活性。dnaA、polA、mutS和minD的转录本，所有这些都编码细菌染色体复制和分配的产物，在IFN-γ处理和未处理的细胞中均有表达。相比之下，编码细菌细胞分裂产物的ftsK和ftsW在未处理的细胞中有表达，但在低剂量IFN-γ处理的细胞中表达减弱，在高剂量细胞因子处理的细胞中则无表达。因此，持续性的发展包括产生与DNA复制相关而非细胞分裂相关基因的转录本。这些结果为伴随肺炎衣原体持续性的分子活动提供了新的见解，也暗示了从持续性生长重新激活为增殖性生长的条件。
