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Journal of Bacteriology, November 2005, p. 7853-7856, Vol. 187, No. 22
0021-9193/05/$08.00+0     doi:10.1128/JB.187.22.7853-7856.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Chlamydia pneumoniae Type III Secretion-Related lcrH Gene Clusters Are Developmentally Expressed Operons

Scot P. Ouellette, Yasser M. AbdelRahman, Robert J. Belland, and Gerald I. Byrne^*

Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, Tennessee 38163

Received 11 July 2005/ Accepted 24 August 2005

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Two chlamydial homologues of the Yersinia lcrH chaperone for type III secretion system structural components are present within separate gene clusters. Quantitative transcriptional analyses demonstrated that each cluster is differentially regulated and expressed as an operon using major sigma factor elements, suggesting the presence of more elaborate developmental regulation mechanisms in chlamydiae.

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Chlamydia trachomatis and C. pneumoniae are obligate intracellular bacterial pathogens causing trachoma (10), sexually transmitted diseases (14), and community-acquired pneumonia (9). These unusual pathogens alternate between two functionally and morphologically distinct forms, namely, the infectious, metabolically inert elementary body (EB) and the noninfectious, metabolically active reticulate body (RB) (see reference 1 for a review). As a consequence of this biphasic life cycle, transcriptional patterns can be broadly categorized into the following three classes: early cycle (important for EB-to-RB differentiation), mid-cycle (RB growth and division), and late cycle (critical for RB-to-EB transition) (15).

Type III secretion systems (T3SS) are well-recognized virulence factors in gram-negative intracellular or host cell-associated pathogens. T3SS allow direct effector protein injection into the host cell cytosol (3), creating an environment favorable for bacterial growth and survival. Components of the T3SS have been characterized and are conserved among organisms possessing them. Unlike many bacterial pathogens whose T3SS genes are grouped into large genomic islands, the chlamydial apparatus components are scattered in several clusters throughout the genome, making it difficult to identify effector genes. The presence of two lcrH homologues (chaperones for apparatus components [13]) in Chlamydia is intriguing and suggests that this organism, like Salmonella, may have two T3SS and/or two sets of type III effectors, with one set associated with entry and the other associated with intracellular survival.

We have undertaken reverse transcription-PCR (RT-PCR) analyses of the genes present in the lcrH clusters and examined their promoters to show that they are differentially expressed operons. Total RNA was collected from C. pneumoniae-infected HEp-2 cells at select times over the developmental cycle by using Trizol (2). Host poly(A) RNA was removed (Oligotex; QIAGEN), and the remaining RNA was further purified (RNeasy; QIAGEN) and thoroughly digested with DNase (Invitrogen). Total DNA from duplicate samples was also isolated (DNeasy; QIAGEN). Figure 1A shows a representation of the lcrH gene clusters. Standard RT-PCR assays (SuperScript; Invitrogen) with 60-h RNA samples designed to overlap intergenic regions within each lcrH cluster and presumably to extend beyond potential transcriptional start sites or downstream untranslated regions demonstrated products of the correct size (Fig. 1B and C) compared to PCR products amplified from genomic DNA (data not shown). No product was detected when intergenic regions between the cluster and genes exterior to it were amplified. In addition, increasingly larger transcript sizes could be detected within the lcrH_1 (also designated scc2) cluster (Fig. 1D), but not within the lcrH_2 (scc3) cluster, which is expressed at about 10-fold lower levels than lcrH_1 at 60 h postinfection. These data provide strong support for the occurrence of polycistronic messages for each T3SS gene cluster. Attempts to detect a message at earlier time points were unsuccessful because the amount of chlamydial mRNA was below the detection limit for the PCR analysis.

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FIG. 1. Transcriptional analysis of the lcrH_1 and lcrH_2 gene clusters in C. pneumoniae. (A) Illustration of PCR products generated from cDNA. Bars indicate the locations and approximate sizes (not to scale) of PCR products if the mRNA extended over these regions; closed bars represent detected products, while open bars represent undetected ones. Numbers above bars reflect the lane numbers in panels B to D. (B) RT-PCR across intergenic regions of the clusters. Random hexamers were used to generate cDNAs that were subsequently amplified using the primer pairs indicated. Asterisks indicate no-RT control wells. Products of the expected sizes are present for amplifications within the clusters but not for genes exterior to the cluster. All lanes S are DNA ladders. Lanes for lcrH_1 cluster: lanes 1, primers 7 and 8, as noted in Table S1 of the supplemental material (predicted size, 380 bp); lanes 2, primers 5 and 6 (400 bp); lanes 3, primers 3 and 4 (405 bp); lanes 4, primers 1 and 2 (469 bp). (C) lcrH_2 cluster. Lanes 5, primers 9 and 10 (581 bp); lanes 6, primers 11 and 12 (435 bp); lanes 7, primers 13 and 14 (443 bp); lanes 8, primers 15 and 16 (442 bp). (D) RT-PCR for detection of polycistronic message. Products of increasing size were generated as predicted for lcrH_1 but not for lcrH_2. Lane 9, primers 1 and 4 (1,816 bp); lane 10, primers 1 and 6 (2,373 bp); lane 11, primers 1 and 8 (3,038 bp); lane 12, primers 9 and 12 (1,867 bp); lane 13, primers 9 and 14 (2,619 bp); lane 14, primers 9 and 16 (4,159 bp).

To further strengthen the designation of these gene clusters as operons, we performed quantitative RT-PCR on all genes within the clusters. TaqMan primer and probe sets were designed and tested (Applied Biosystems) against genomic DNA. Quantitative assessments of transcripts for genes within the lcrH clusters indicated that the lcrH_1 and lcrH_2 gene clusters are differentially expressed during the C. pneumoniae developmental cycle. 16S rRNA transcripts are often used as a measure of the growth rate and/or the number of organisms and are considered a useful means for the normalization of transcriptional data. However, using genomes to normalize transcript data eliminates any variability between these two parameters, which is especially important since 16S rRNA levels varied as much as 10-fold during the developmental cycle when normalized to genomes (Fig. 2A), indicating that genomic normalization of transcriptional data is preferable to other methods. These normalization considerations are likely to have broader applicability for quantitative transcriptional studies of other organisms, especially those undergoing developmental changes.

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FIG. 2. Quantitative real-time RT-PCR data for control genes and lcrH gene clusters. (A) Expression of control early (euo)-, mid (ompA and 16S rRNA)-, and late (omcB)-stage genes during the C. pneumoniae developmental cycle normalized to genomes. (B) Expression of each gene of the lcrH_1 gene cluster. (C) Expression of each gene of the lcrH_2 gene cluster. These data are representative of three separate experiments. Standard deviations are <5% of the sample values.

The RNA level was determined for each gene at each time point, using the TaqMan One-Step RT-PCR system (Applied Biosystems). Genomes were quantified from duplicate DNA samples using quantitative PCR directed against an unrelated gene (Cpn0423). Chlamydial genomes were normalized to host cell genomes (ß-actin) to control for differences in infection between samples. The expression of control early (euo)-, mid (ompA)-, and late (omcB)-stage genes shows a temporal separation for each, as determined by the time point at which transcription peaks (Fig. 2A). The lcrH_1 gene cluster (Fig. 2B) is expressed late in the developmental cycle, at the time when RBs are redifferentiating to EBs. Maximal expression occurs at 48 h postinfection (p.i.) and is similar to that of the known late gene omcB (Fig. 2A), which encodes an EB cysteine-rich outer envelope structural protein. In contrast, the lcrH_2 gene cluster (Fig. 2C) is expressed primarily during the RB growth phase, with maximal expression occurring at 24 h p.i., similar to the major outer membrane protein encoded by ompA and to ribosomal 16S rRNA. Cotranscription for each gene within each cluster adds further support to their designation as operons.

Chlamydia has three sigma factors, including a housekeeping factor, ⁶⁶, and two alternative sigma factors, ²⁸ and ⁵⁴. These sigma factors and their putative regulators are differentially regulated during the chlamydial developmental cycle (5, 11). A known late gene, hctB, has a ²⁸ promoter sequence (17), which suggests that late gene expression may be regulated by alternative sigma factors. Because of the differences in temporal expression of the T3SS operons, transcriptional start sites for each gene cluster were mapped using a 5' rapid amplification of cDNA ends method (Clontech/BD Biosciences) to determine sequence information for sigma factor use that may be important in regulating the developmental expression of these operons. A cDNA containing an upstream, untranslated message was generated using gene-specific primers and purified RNA. The cDNA was cloned, amplified, and sequenced. Putative ⁶⁶ promoter sequences could be identified upstream from the transcriptional start sites of –37 from the ATG for the lcrH_1 operon and –22 for the lcrH_2 operon (Fig. 3). Our data, in agreement with those of Fahr et al. (7), who also mapped ⁶⁶ late gene promoters, suggest that the developmental regulation of gene expression relies on more complex mechanisms than alternative sigma factors. For example, unknown regulators currently designated hypothetical regulatory RNAs or T3SS-specific regulators may be involved in developmental regulation. However, at this time, actual mechanisms for developmental regulation in chlamydiae remain unknown, and no obvious homologues have been identified.

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FIG. 3. Detection of transcriptional start sites and identification of promoter sequences. 5' rapid amplification of cDNA ends was used to generate a cDNA containing the transcriptional start site that was then cloned, amplified, and sequenced. An analysis of upstream DNA sequences identified promoter elements, which are marked. The E. coli 70 consensus sequence is shown. (A) Transcriptional start site and promoter sequence for the lcrH_1 operon. (B) Transcriptional start site and promoter sequence for the lcrH_2 operon. TSS, transcriptional start site.

For the lcrH_1 operon, a minor population of clones (<20%) gave another transcriptional start site, at –75, with ⁶⁶ promoter sequences upstream as well (data not shown). It is not known if each of these upstream sequences is a functional promoter, if the major promoter we identified is a processed form of the upstream promoter similar to what has been reported for the ompA transcript (6), or if the minor promoter upstream of the –75 transcriptional start site is an artifact. We cannot distinguish between these possibilities at this time, although our data argue against the last one because the identified promoter element upstream of –75 is well conserved. In contrast, the start site of lcrH_2 was easily identified, but the promoter structure was unusual in that the distance between the transcriptional start site and the –10 sequence was slightly longer than is typically found (11 bases versus 7 or 8 bases). Additionally, there is no well-conserved ribosome-binding site upstream of the ATG of Cpn1022. These differences in promoter elements and ribosome-binding sites may also be important for the developmental regulation of these operons, but this possibility requires further investigation.

We have demonstrated that the C. pneumoniae lcrH gene clusters are developmentally expressed operons putatively regulated by the major chlamydial sigma factor (⁶⁶), as evidenced by the presence of a ⁶⁶ promoter upstream of each operon. Because of the difficulty in isolating large amounts of intact, low-abundance polycistronic messages from chlamydial RNA from a pool of contaminating host cell RNA, we used RT-PCR, which avoids problems associated with Northern blotting, to determine the operon structure. This method, coupled with quantitative RT-PCR, gives a clear picture that these gene clusters are developmentally regulated operons. Our data can likely be extended to include lcrH genes from other chlamydiae since the organization of the lcrH gene clusters in C. trachomatis is the same as and their sequences are orthologously related to those of C. pneumoniae. This conservation between organisms is indicative of conserved function, which is not surprising given the relatedness of their developmental cycles.

Fields et al. (8) have demonstrated that the T3SS apparatus is present on EBs and is functional early in the developmental cycle: a model was proposed that the EB T3SS mediates the secretion of effectors early in an infection cycle, while RB expression of T3SS genes allows for replenishment of the T3SS on newly formed EBs. Similarly, the differential expression patterns of the lcrH genes support an EB-associated T3SS that secretes effectors (e.g., Tarp [4]) after attachment to the host cell and an RB-associated T3SS that secretes effectors from the inclusion into the cytosol. This is analogous to the case for Salmonella, where the SPI-1 T3SS secretes effectors into the host cell after attachment that induce membrane ruffling and uptake into the Salmonella-containing vacuole and the SPI-2 T3SS secretes effectors from the Salmonella-containing vacuole into the host cell cytosol. While Chlamydia probably lacks multiple structural T3SS since there are no obvious homologues elsewhere in the genome, this organism clearly developmentally regulates type III secretion based on the differential expression of the LcrH chaperones. Consequently, the protein products of the lcrH_1 operon likely function in the context of attachment since these genes are expressed when EBs are forming and protein can be detected in the EBs (12, 16), while the protein products of the lcrH_2 operon likely function in intracellular survival.

 
We thank Tim Higgins for his hard work and patience in preparing figures and illustrations.

This work was funded in part by NIH grants AI42790 and NIAID-DMID-03-05 to G.I.B. and HL71735 to G.I.B. and R.J.B.

 
* Corresponding author. Mailing address: Department of Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Ave., Memphis, TN 38163. Phone: (901) 448-6150. Fax: (901) 448-3330. E-mail: gbyrne{at}utmem.edu.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, November 2005, p. 7853-7856, Vol. 187, No. 22
0021-9193/05/$08.00+0     doi:10.1128/JB.187.22.7853-7856.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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• Hefty, P. S., Stephens, R. S. (2007). Chlamydial Type III Secretion System Is Encoded on Ten Operons Preceded by Sigma 70-Like Promoter Elements. J. Bacteriol. 189: 198-206 [Abstract] [Full Text]  

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