Chlamydia pneumoniae expresses genes required for DNA replication but not cytokinesis during persistent infection of HEp-2 cells.
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.
Byrne
G I
GI
Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706, USA. gibyrne@facstaff.wisc.edu
Ouellette
S P
SP
Wang
Z
Z
Rao
J P
JP
Lu
L
L
Beatty
W L
WL
Hudson
A P
AP
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division / Q000235:genetics*; D016993:Chlamydophila pneumoniae / Q000235:genetics* / Q000254:growth & development* / Q000648:ultrastructure; D002876:Chromosomes, Bacterial / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics*; D004269:DNA, Bacterial / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D007371:Interferon-gamma