Chlamydia pneumoniae Expresses Genes Required for DNA Replication but Not Cytokinesis during Persistent Infection of HEp-2 Cells

Abstract

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-␥) 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-␥ 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-␥ treatment elicited aberrant forms of the bacterium. RT-PCR showed that chlamydial primary rRNA transcripts were present in all IFN-␥-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-␥-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-␥ 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.Chlamydia pneumoniae and Chlamydia trachomatis are intracellular bacterial pathogens that function as etiologic agents of important human diseases. The former, for example, causes community-acquired pneumonia and has recently been associated with various chronic diseases such as asthma and atherosclerosis (9, 16). C. trachomatis remains a significant cause of infectious, preventable blindness (trachoma) in the developing world (26) and is a sexually transmitted pathogen known to cause fertility deficits in women (26), as well as chronic arthritis in both sexes (for a review, see refer...

Proteomic Analysis of Differentially Expressed Chlamydia pneumoniae Genes during Persistent Infection of HEp-2 Cells

“…A molecular profile of C. pneumoniae persistence is beginning to emerge based on two current reports examining mRNA levels (3,11) and the protein patterns obtained in this study.…”

“…Stimulation of host cells by IFN-␥ inhibits growth of C. pneumoniae primarily by induction of indoleamine 2,3-dioxygenase (IDO) activity, which deprives the organism of tryptophan (12,14,22). In a fashion similar to that used with Chlamydia trachomatis, our group (15) and others (3,11) have shown that subinhibitory concentrations of IFN-␥ can lead to the formation of a persistent state of C. pneumoniae in vitro, a phenomenon that is characterized by the formation of pleomorphic reticulate bodies (RBs) that are maintained in a viable but culture-negative state.…”

“…Only limited amounts of data have characterized gene expression profiles of C. pneumoniae under persistent infection (3,11). Using reverse transcriptase PCR, Byrne et al (3) have shown that DNA replication genes (i.e., dnaA, polA, mutS, and minD) are expressed at normal levels in C. pneumoniae during IFN-␥-mediated persistent infection; however, expression of cell division-related genes (i.e., ftsK and ftsW) is downregulated.…”

Hydroxymethylglutaryl Coenzyme A Reductase Inhibition Reduces Chlamydia pneumoniae -Induced Cell Interaction and Activation

“…IFN-␥ stimulates C pneumoniae to enter the persistent state characteristic of chronic infection. 9 Thus, by stimulating RANTES secretion in VSMCs, C pneumoniae may not only contribute to the trafficking and homing of lymphoid cells but also may induce its own persistence. The RANTES production trigger has not yet been identified.…”

Comparison of Eleven Commercial Tests for Chlamydia pneumoniae -Specific Immunoglobulin G in Asymptomatic Healthy Individuals

“…The development of persistence in vitro is characterized by a reduction of the intracellular growth rate of C. pneumoniae and by the appearance of morphologically aberrant reticulate bodies (27). Persistent C. pneumoniae has been shown to be metabolically active (1,10,32), but the period of persistence as well as the influence on the antibody titer of the host is unknown. There is emerging evidence that persistent C. pneumoniae might be implicated in the pathogenesis of several chronic and sometimes destructive diseases of the lung and the nervous system and atherosclerosis (13, 29, 41-43, 48, 52).…”