Host cell responses to Chlamydia pneumoniae in gamma interferon-induced persistence overlap those of productive infection and are linked to genes involved in apoptosis, cell cycle, and metabolism

Abstract

The respiratory pathogen Chlamydia (Chlamydophila) pneumoniae is associated with chronic diseases, including atherosclerosis and giant-cell arteritis, which are accompanied by the occurrence of these obligate intracellular bacteria in blood vessels. There, C. pneumoniae seems to be present in a persistent state. Persistence is characterized by modified bacterial metabolism and morphology, as well as a reversible arrest of chlamydial development. In cell culture, this persistent state can be induced by gamma interferon (IFN-gamma). To elucidate this long-term interaction between chlamydiae and their host cells, microarray screening on epithelial HeLa cells was performed. Transcription of persistently (and productively) infected cells was compared with that of mock-infected cells. Sixty-six host cell genes were regulated at 24 h and/or 96 h of IFN-gamma-induced persistence. Subsequently, a set of 17 human host cell genes related to apoptosis, cell cycle, or metabolism was identified as permanently up- or down-regulated by real-time PCR. Some of these chlamydia-dependent host cell responses were diminished or even absent in the presence of rifampin. However, other expression patterns were not altered by the inhibition of bacterial RNA polymerase, suggesting two different modes of host cell activation. Thus, in the IFN-gamma model, the persisting bacteria cause long-lasting changes in the expression of genes coding for functionally important proteins. They might be potential drug targets for the treatment of persistent C. pneumoniae infections.

FIG. 1.

Typical changes in morphology of the RB-like aberrant forms of C. pneumoniae in IFN-γ-induced persistence (right) compared to productive infection (left) determined by transmission electron microscopy in HeLa cells at 48 h postinfection. A statistical evaluation can be found in Table 1.

FIG. 2.

Determination of DNA for ompA of C. pneumoniae demonstrating similar bacterial loads in the three biologically independent infection experiments (A, B, and C) that were analyzed by real-time PCR. The amount of ompA DNA was determined in triplicate for each of the three infection experiments. Depicted are means plus standard deviations.

FIG. 3.

Host cell gene regulation induced by C. pneumoniae in productive infection (24 h) and IFN-γ-induced persistence (24 and 96 h postinfection). Depicted are means plus standard deviations (SD) of the normalized log₁₀ of the relative amount of mRNA of genes determined by two-step real-time PCR in mock-infected and C. pneumoniae-infected HeLa cells. The figures summarize the results of three independent infection experiments (A, B, and C). In each experiment, the real-time PCR was performed in quadruplicate for the 17 candidate genes and in triplicate for the 3 genes that were used for normalization (GUS, TBP, and 18S rRNA). For better comparison, the amount of mRNA determined after 24 h of productive infection was set to 1. To provide some orientation, dotted lines are drawn at relative mRNA amounts of 2.0 and 0.5. Additionally, the calculated factor of regulation (mean ± SD), in comparing chlamydia-infected cells and mock-infected cells, can be found above the corresponding pair of columns. Calculated factors of differential regulation above 2.0 and below 0.5 are indicated in boldface. An asterisk indicates a significant change (P ≤ 0.05). There was no regulation of HASPA1-A and HSPA1-B as confirmed by Northern blotting (data not shown). Therefore, the regulation observed in the microarray was most likely an artifact.

FIG. 4.

Host cell gene regulation induced by C. pneumoniae in IFN-γ-induced persistence at the most relevant time point, 96 h postinfection with an MOI of 3, confirms the data obtained with an MOI of 30. Depicted are means plus standard deviations (SD) of the normalized log₁₀ of the relative amounts of mRNA of a selection of genes determined by two-step real-time PCR in mock-infected and infected (MOI = 3) HeLa cells. The figures summarize the results of two independent infection experiments. In each experiment, the real-time PCR was performed in quadruplicate for the candidate genes and in triplicate for the three genes that were used for normalization (GUS, TBP, and 18S rRNA). Calculated factors of regulation (mean ± SD), comparing chlamydia-infected and mock-infected cells above 2.0 and below 0.5, are indicated in boldface. An asterisk indicates significant changes (P ≤ 0.05). As examples, three genes with a high factor of regulation at an MOI of 30 (OASL, DKK1, and KRT17), as well as three genes with relatively low factors of regulation (BNIP3, CA9, and LOX), were selected for the experiment. Four of these genes were down-regulated in the high-MOI real-time PCR 96 h postinfection in the persistence model, and two were up-regulated. The genes are in the same order as in Fig. 5.

FIG. 5.

The effect of rifampin on host cell gene regulation induced by C. pneumoniae in productive infection (24 h) and IFN-γ-induced persistence (24 and 96 h postinfection [p.i.]). Depicted are means plus standard deviations (SD) of the normalized log₁₀ of the relative amounts of mRNA of genes determined by two-step real-time PCR in mock-infected and infected HeLa cells. The figures summarize the results of three independent infection experiments (A, B, and C). In each experiment, the real-time PCR was performed in triplicate for seven selected candidate genes and in duplicate for the two genes that were used for normalization (TBP and 18S rRNA). For better comparison of the rifampin effect, the amount of mRNA determined for each experimental condition was set to 1. The results for mock-infected cells and the corresponding C. pneumoniae-infected cells are depicted as pairs for each condition. The changes (n-fold) of the mock control in the absence of rifampin caused by 24 h or 96 h of IFN-γ treatment were as follows: 0.9/1.1 for DKK1, 2.6/2.0 for OASL, 1.2/2.5 for BNIP3, 0.8/6.2 for CA9, 0.8/5.3 for DACT1, 3.5/8.4 for KRT17, and 1.4/3.7 for LOX. To provide some orientation, dotted lines are drawn at relative mRNA amounts of 2.0 and 0.5. An asterisk indicates a significant change (P ≤ 0.05). The two genes with the highest factor of regulation are in the top row; the others are in alphabetical order.