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-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.

FIG. 1

Results of quantitative real-time PCR assays to determine the relative level of accumulation of C. pneumoniae chromosomal DNA over time during infection of untreated (−Cx) and cycloheximide-treated (+Cx) HEp-2 cells. Cells were infected with C. pneumoniae TW-183 as described in Materials and Methods, and cultures were harvested at the times indicated. Total DNA was prepared, and a real-time PCR assay system was employed to determine the relative level of chlamydia DNA at each time point. Input into each quantitative assay was normalized to the host 18S rRNA genes, as described in Materials and Methods. Data are triplicate means plus standard errors and are expressed as fold increase in chlamydial DNA over time relative to the value obtained at 12 h postinfection.

FIG. 2

Representative RT-PCR analyses targeting C. pneumoniae genes whose products are required for chromosomal DNA replication and cell division, as a function of time in infection of untreated (−CX) or cycloheximide-treated (+CX) HEp-2 cells. Cells were infected with chlamydiae and harvested at the indicated times, and RNA was prepared from each harvested culture (see Material and Methods). RT-PCR analyses were performed using primers given in Table 1. (A) Primary rDNA transcripts; (B) adt1 transcript; (C) dnaA transcript; (D) polA transcript; (E) ftsK transcript. Lanes: C+, positive PCR controls for each primer set, using C. pneumoniae DNA as the amplification template; C−, negative RT-PCR controls using cDNA from uninfected HEp-2 cells as the amplification template; RT−, negative control showing the results of PCR with each primer set in the absence of RT of RNA preparations used; ACT, amplification product from an RT-PCR assay targeting host β-actin mRNA. Input into each assay was normalized to the host β-actin transcript. Sizes of the amplification products: primary rRNA, 609 bp; adt1, 433 bp; host actin, 550 bp; dnaA, 410 bp; polA, 405 bp; ftsK, 238 bp.

FIG. 3

Effect of IFN-γ on C. pneumoniae growth in HEp-2 cells. Cells were treated with the indicated amounts of IFN-γ 2 h postinfection and assayed for infectivity after 3 days of incubation. Results are means for individual samples from each treatment group as a function of the averaged untreated control. All samples were assayed in triplicate, and standard deviations are shown. Samples from cells exposed to 0.15 and 0.5 ng of IFN-γ per ml were used for ultrastructural analyses.

FIG. 4

Ultrastructural analysis by transmission EM of HEp-2 cells persistently infected for 48 h with C. pneumoniae. (A) Treatment with low levels of IFN-γ (0.15 ng/ml) resulted in the development of typical inclusions containing EB and RB. Normal RB are indicated by arrows. (B) Treatment with 0.50 ng of IFN-γ per ml to induce persistence engenders development of grossly enlarged RB (arrowheads). N, nucleus. Bar = 1 μm.

FIG. 5

Representative RT-PCR analyses targeting primary transcripts from the C. pneumoniae rRNA operons (A) and adt1 (B) in untreated infected HEp-2 cells and infected HEp-2 cells treated with low or high doses of rIFN-γ. Cells were infected with chlamydiae and harvested at 48 h posttreatment, and RNA was prepared from each harvested culture (see Material and Methods). RT-PCR analyses were performed using primers given in Table 1. Lanes: C+, positive PCR control for each primer set, using C. pneumoniae DNA as the amplification template. C−, negative RT-PCR control using cDNA from uninfected HEp-2 cells as the amplification template; RT−, negative control showing the results of PCR with each primer set in the absence of RT of RNA preparations used; Act, amplification product from an RT-PCR assay targeting host β-actin mRNA; EB, amplification product from pure EB RNA. Sizes of the amplification products are given in the legend to Fig. 1.

FIG. 6

Results from quantitative real-time PCR assays to determine the relative level of accumulation of C. pneumoniae chromosomal DNA during infection of untreated infected HEp-2 cells and infected HEp-2 cells treated with either 0.15 or 0.50 ng of rIFN-γ per ml. Cells were infected with C. pneumoniae TW-183 as described in Materials and Methods. Both treated and untreated cultures were grown without cycloheximide and were harvested at 48 h postinfection or posttreatment. DNA was prepared, and a real-time PCR assay system was used to determine the relative level of Chlamydia DNA from each preparation. Input into each assay was normalized to the host 18S rRNA genes, as described in Materials and Methods. Data are triplicate mean values relative to the mean value obtained for untreated cells at 12 h postinfection (not shown). Standard errors are indicated.

FIG. 7

Representative RT-PCR analyses targeting transcripts from C. pneumoniae DNA replication- and cell division-related genes in untreated infected HEp-2 cells and infected HEp-2 cells treated with low or high doses of rIFN-γ. Cells were infected with chlamydiae and harvested at 48 h posttreatment, and RNA was prepared from each harvested culture (see Material and Methods). RT-PCR analyses were performed using primers given in Table 1. (A) primary rRNA transcripts; (B) dnaA; (C) polA; (D) ftsK. Lanes: C+, positive PCR control for each primer set, using C. pneumoniae DNA as the amplification template; C−, negative RT-PCR control, using cDNA from uninfected HEp-2 cells as the amplification template; RT−, negative control showing the results of PCR with each primer set in the absence of RT of RNA preparations used; ACT, amplification product from an RT-PCR assay targeting host β-actin mRNA. Input was normalized to the host β-actin mRNA. Sizes of the amplification products are given in the legend to Fig. 1.