Transcriptome analysis of chlamydial growth during IFN-gamma-mediated persistence and reactivation

Abstract

Chlamydia trachomatis is an obligatory intracellular prokaryotic parasite that causes a spectrum of clinically important chronic inflammatory diseases of humans. Persistent infection may play a role in the pathophysiology of chlamydial disease. Here we describe the chlamydial transcriptome in an in vitro model of IFN-gamma-mediated persistence and reactivation from persistence. Tryptophan utilization, DNA repair and recombination, phospholipid utilization, protein translation, and general stress genes were up-regulated during persistence. Down-regulated genes included chlamydial late genes and genes involved in proteolysis, peptide transport, and cell division. Persistence was characterized by altered but active biosynthetic processes and continued replication of the chromosome. On removal of IFN-gamma, chlamydiae rapidly reentered the normal developmental cycle and reversed transcriptional changes associated with cytokine treatment. The coordinated transcriptional response to IFN-gamma implies that a chlamydial response stimulon has evolved to control the transition between acute and persistent growth of the pathogen. In contrast to the paradigm of persistence as a general stress response, our findings suggest that persistence is an alternative life cycle used by chlamydiae to avoid the host immune response.

Fig. 1.

Model of IFN-γ chlamydial persistence and reactivation from persistence. Shown are one-step growth curves with corresponding ultrastructural images from normal, IFN-γ-treated, and reactivation from IFN-γ treatment cells. (A) Untreated chlamydia-infected HeLa 229 cells. (A1) One-step growth curve showing a 16-h latency period followed by a rapid burst in recoverable IFU. Typical RB developmental forms are present at 12 (A2) and 24 (A3)hPI.By48hPI(A4) the inclusion contains a mixture of characteristic RBs and EBs. (B) IFN-γ-treated chlamydia-infected HeLa cells. A similar latency period is observed, but, unlike untreated cells, there is no infectious burst (B1). RBs appear similar to untreated cultures at 12 h PI (B2); however, at 24 (B3) and 48 (B4) h PI, the RBs are distinctly different, appearing as large aberrant noninfectious forms. (C) Reactivation from persistence. There was a rapid recovery in infectious EB after removal of IFN-γ (C1); recoverable IFU was 96% of control (untreated) infections. At 6 h, reactivation chlamydiae remained aberrant (C2), but by 18 (C3) and 24 (C4) h after removal of IFN-γ, inclusions were typical with morphologically normal EBs and RBs. (Scale bar, 1 μm.)

Fig. 2.

Microarray analysis of C. trachomatis gene expression in infected HeLa 229 cells cultured in the presence and absence of IFN-γ.(A) Gene expression differences between infections in the absence and presence of IFN-γ for 12 h. (B) Gene expression differences between infections in the absence and presence of IFN-γ for 24 h. (C) Gene expression differences between an infection in the absence of IFN-γ for 48 h PI and an infection in the presence of IFN-γ for 24 h, followed by the removal of IFN-γ and tryptophan supplementation for 24 h. Data are presented in an array layout format representing a linear map of the bacterial genome, i.e., starting from the origin of replication in the upper left corner and proceeding from CT001 to CT035 in the top row, CT036 to CT070 in the second row, and proceeding until the final row, which contains the last chromosomal ORF (CT876), the eight plasmid ORFs, and nine negative controls. Fold change in expression is given for each gene according to the colored legend as IFN-γ-treated/control. Correlation between the fold changes (+IFN-γ vs. -IFN-γ at 24 h PI) measured by qPCR and microarray is shown in D. Genes exhibiting a ±1.5-fold difference in expression were plotted based on fold change at 12 and 24 h PI, and after reactivation from IFN-γ treatment (E and F). Up-regulated genes numbered 202, 204, and 217 for the 12-h, 24-h, and reactivation samples, respectively. Fold changes were greatest at 24 h (1.5–6.5) and minimal in reactivation (1.5–3.0). A similar pattern was also observed for the down-regulated genes: 163, 210, and 138 genes for the 12-h, 24-h, and reactivation samples, respectively, with fold changes ranging from 1.5 to 8.1 (24 h), 1.5 to 5.3 (12 h), and 1.5 to 2.7 (reactivation).

Fig. 3.

Quantitative RT-PCR analysis of gene expression. (Left) Up-regulated genes. (Right) Down-regulated genes. Copy numbers of gene transcripts per bacterial cell are given for control cultures (open bars), IFN-γ-treated cultures (filled bars), and reactivated cultures at 3, 6, 12, 18, and 24 h post-IFN-γ removal (hatched bars). Copy number differences at 24 h PI for control and IFN-γ-treated cultures are indicated by an asterisk if their differences are considered significant at a level of P < 0.005.