Genomic transcriptional profiling of the developmental cycle of Chlamydia trachomatis - PubMed (original) (raw)
Genomic transcriptional profiling of the developmental cycle of Chlamydia trachomatis
Robert J Belland et al. Proc Natl Acad Sci U S A. 2003.
Abstract
Chlamydia trachomatis is one of the most common bacterial pathogens and is the etiological agent of debilitating sexually transmitted and ocular diseases in humans. The organism is an obligate intracellular prokaryote characterized by a highly specialized biphasic developmental cycle. We have performed genomic transcriptional analysis of the chlamydial developmental cycle. This approach has led to the identification of a small subset of genes that control the primary (immediate-early genes) and secondary (late genes) differentiation stages of the cycle. Immediate-early gene products initiate bacterial metabolism and potentially modify the bacterial phagosome to escape fusion with lysosomes. One immediate early gene (CT147) is a homolog of the human early endosomal antigen-1 that is localized to the chlamydial phagosome; suggesting a functional role for CT147 in establishing the parasitophorous vacuole in a nonfusogenic pathway. Late gene products terminate bacterial cell division and constitute structural components and remodeling activities involved in the formation of the highly disulfide cross-linked outer-membrane complex that functions in attachment and invasion of new host cells. Many of the genes expressed during the immediate-early and late differentiation stages are Chlamydia-specific and have evolutionary origins in eukaryotic lineages.
Figures
Fig. 1.
Transcriptional profiling of the developmental cycle of C. trachomatis serovar D in HeLa 229 cells with accompanying transmission electron microscopy of bacterial inclusions at 1, 3, 8, 16, 24, and 40 h PI. Results are shown in an array layout format that is a linear representation of the bacterial genome (i.e., row 1, column 1 is ORF CT001; row 1, column 2 is CT002, etc...). The data shown are the normalized expression values for three independent experiments done in duplicate. The 1- and 3-h analyses were from infections using a MOI of 100, whereas the 8-, 16-, 24-, and 40-h analyses were from infections using a MOI of 1. Black circles represent genes that are considered transcriptionally inactive at the time point shown. For example, the 29 immediate-early ORFs are shown in the 1-h PI analysis, and their expression level, CT numbers, and chromosomal location are indicated by their position in the array layout. The complete data set is listed in Table 2, which is published as supporting information on the PNAS web site,
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Fig. 2.
Expression levels of immediate-early and late genes detected by quantitative RT-PCR. Quantitative RT-PCR (qRT) assays were performed by using TaqMan primer/probe sets specific for each gene by using RNA samples purified from infections done at a MOI of 1. The efficiency of amplification was comparable for all primer/probe sets as estimated by standard curves. Transcript levels increased for each immediate-early gene (8/8) by 1 h PI, demonstrating that new transcription had occurred. In contrast, all late genes analyzed showed decreased transcript levels at 1 h PI, indicating that EBs contained significant levels of mRNA but that these levels decreased during the early part of the developmental cycle. Late gene expression for all eight genes tested was up-regulated between 16 and 40 h PI. RNA samples from purified EBs (0 time) were used to control for “carry over” transcript levels.
Fig. 3.
Confocal fluorescence microscopy and radioimmune precipitation of C. trachomatis CT147. (A) Schematic comparison of human EEA1 and_C. trachomatis_ CT147. The proteins have nearly identical predicted molecular masses (≈162 kDa) and share N- and C-terminal Zn finger domains. The the EEA1 C-terminal domain is of the C2H2 type and is termed a FYVE domain (39). The central regions of the proteins have extensive coiled-coil domains (shown as blue rectangles) and two (CT147) or more leucine zipper motifs (shown as black bars). CT147 lacks homology with the region of EEA1 that has been shown to bind Rab5 (shown as the blue filled oval). (B) CT147 expression can be seen early in the developmental cycle (i.e., 8 h PI as indicated by red arrows) and continues throughout the cycle. Localization of RBs within the inclusion in infected cells is shown for comparison (i.e., at all times in the cycle, as indicated by green arrow). (C) CT147 is localized to the inclusion membrane as shown by immunofluorescent staining in comparison to RBs. (Upper) Costaining results usingα-CT147 andα-EB antisera are shown. α-CT147 staining is predominantly at the periphery of the inclusion, whereas the α-EB staining is found throughout the inclusion. The inclusions are shown in the differential interference contrast (DIC) microscopic images and are indicated with arrows. (Lower) The results of costaining with α-CT147 and α-IncG antisera are shown. Both antisera react with antigens found predominantly at the periphery of the inclusion, indicating that CT147 is localized to the inclusion membrane. (D) CT147 is first detected by RIP at 16 h PI. CT147 is indicated by the arrow (predicted Mr = 162 kDa). At subsequent time points the quantity of the full-length product decreased and the quantity and number of breakdown products increased (indicated by the bracket), indicating increased levels of posttranslational modification.
Fig. 4.
Schematic representation of the C. trachomatis developmental cycle. The infectious EB, after attachment and internalization by the eukaryotic cell, undergoes a 1° differentiation to the metabolically active RB. This phase of the developmental cycle is characterized by expression of immediate-early and early genes and involves cellular processes associated with vacuole modification and the initiation of bacterial metabolism (as indicated in the boxed region). Bacterial transcription and translation follow decondensation of the chromosome, leading to bacterial replication by binary fission (between ≈16 and 40 h PI). The majority of genes are expressed during this highly active stage of the cycle. Late in the developmental cycle the organism undergoes a 2° differentiation from RB to EB. This phase of the cycle is associated with expression of late genes and their corresponding products that give rise to the highly disulfide cross-linked OM complex (remodeling disulfide isomerases and proteases) and chromosome condensation (the histone-like proteins) as indicated in the boxed region. The eukaryotic cell then lyses, releasing infectious EBs to infect neighboring cells and continue the infectious process.
Comment in
- Chlamydia uncloaked.
Byrne GI. Byrne GI. Proc Natl Acad Sci U S A. 2003 Jul 8;100(14):8040-2. doi: 10.1073/pnas.1533181100. Epub 2003 Jun 30. Proc Natl Acad Sci U S A. 2003. PMID: 12835422 Free PMC article. No abstract available.
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