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The Chlamydia trachomatis plasmid-encoded Pgp4 is a transcriptional regulator of virulence associated chromosomal genes

ABSTRACT: Chlamydia trachomatis causes chronic inflammatory diseases of the eye and genital tract of global medical importance. The chlamydial plasmid plays an important role in the pathophysiology of these diseases as plasmid-deficient organisms are highly attenuated. The plasmid encodes both noncoding RNAs and eight conserved ORFs of undefined function. To understand plasmid gene function we generated plasmid shuttle vectors with deletions in each of the eight ORFs. The individual deletion mutants were used to transform chlamydiae and the transformants were characterized in terms of plasmid biology and transcriptional profiling. We show that pgp1-2, -6 and -8 are essential for plasmid maintenance while the other ORFs can be deleted and the plasmid stably maintained. We further show that a pgp4 knockout mutant exhibits an in vitro phenotype similar to its isogenic plasmid-less strain in terms of abnormal inclusion morphology and lack of glycogen accumulation. Microarray and qRT-PCR analysis revealed that pgp4 is involved in transcriptional regulation of multiple chromosomal genes; including the glycogen synthase gene glgA. Based on our results, we propose that Pgp1 is a plasmid replicative helicase, Pgp2 is a plasmid replication protein, Pgp4 is a transcriptional regulator of virulence associated chromosomal genes, and Pgp6-8 are plasmid partitioning proteins. These findings have important implications for understanding the plasmid’s role in chlamydial pathogenesis and the development of novel antigenically multivalent live-attenuated chlamydial vaccines.

ORGANISM(S): Staphylococcus epidermidis RP62A Rickettsia rickettsii Chlamydia pneumoniae AR39 Coxiella burnetii RSA 493 Granulibacter bethesdensis Staphylococcus epidermidis ATCC 12228 Coxiella burnetii Chlamydia muridarum Chlamydia trachomatis D/UW-3/CX Staphylococcus haemolyticus JCSC1435 Chlamydia trachomatis Borreliella burgdorferi B31 Staphylococcus aureus subsp. aureus MW2 Chlamydia caviae GPIC

PROVIDER: GSE40979 | GEO | 2013/02/28

SECONDARY ACCESSION(S): PRJNA175480

REPOSITORIES: GEO

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Project description:Both prokaryotic and eukaryotic organisms take deterministic decisions to reprogram cell fates. A macroscopic manifestation of such an event is the remodelling of the cellsâ morphology and it typically is governed at the molecular scale by massive reorganization of the cellular transcriptome. With the regulation at the level of transcription initiation representing the most common form for such developmental reprogramming, cells typically rely on one or several master regulators to coordinate the the activity of hundreds of genes simultaneously by directly binding to their promoters. Despite the apparent simplicity of prokaryotes and their reduced genome size compared to that of their eukaryotic counterparts, free-living bacteria typically encode hundreds of transcription factors (TFs) in their genomes that could act as master TFs. By contrast, obligate intracellular bacteria such as Chlamydiae have a drastically reduced genome due to their intimate association with the host and thus a smaller number of TF genes. The genomes of members of the Chlamydiaceae family, which include the well-known bacterial pathogens Chlamydia trachomatis and Chlamydia pneumoniae, are only 1-1.2 Mbp. By contrast, members of the environmental Chlamydiae Waddlia chondrophila and Parachlamydia acanthamoebae have a 2-fold and 3-fold larger genome, respectively, likely allowing for an expansion of the host range while still retaining their host dependence and parasitic life style. Moreover, they all exhibit a characteristic chlamydial developmental cycle via two functionally specialized morphotypes, the infectious non-dividing elementary bodies (EBs) and the non-infectious dividing reticulate bodies (RBs). This developmental cycle is usually divided in three stages: the early stage during which EBs enter host cells and differentiate into RBs; the mid-stage where RBs proliferate inside a vacuole called inclusion and the late stage where RBs differentiate back into EBs and are released after exocytosis or cell lysis. Chlamydial genes are thus classified into three different temporal classes (early, mid and late expressed genes), likely reflecting the need of these transcripts in each of the three developmental stages. W. chondrophila, an emerging pathogen implicated in abortion in bovine and miscarriage in humans, encodes less than 20 TFs, 10 of which are conserved among the Chlamydiae. In light of this low TF multiplicity in the chlamydial pan-genome along with the common developmental cycle and parasitic life style, we aimed to define the regulatory pan-genome of each these conserved TFs to identify the elusive chlamydial master regulator and to characterize underling specificity for its target promoters using chromatin-immunoprecipitation followed by deep-sequencing (ChIP-Seq) of chlamydial cells growing inside the host. The immunochemistry of ChIP-Seq has the advantage of minimizing the contaminating nucleic acids compared to chlamydial transcriptome studies, it has the added benefit of providing the first unambiguous glimpse into the regulatory landscape of a bacterium inside host, offering a solid framework in understanding the stochastic and/or deterministic switches that bacteria rely on during infections. Examination of the regulatory network of an intracellular pathogen

Project description:Both prokaryotic and eukaryotic organisms take deterministic decisions to reprogram cell fates. A macroscopic manifestation of such an event is the remodelling of the cells’ morphology and it typically is governed at the molecular scale by massive reorganization of the cellular transcriptome. With the regulation at the level of transcription initiation representing the most common form for such developmental reprogramming, cells typically rely on one or several master regulators to coordinate the the activity of hundreds of genes simultaneously by directly binding to their promoters. Despite the apparent simplicity of prokaryotes and their reduced genome size compared to that of their eukaryotic counterparts, free-living bacteria typically encode hundreds of transcription factors (TFs) in their genomes that could act as master TFs. By contrast, obligate intracellular bacteria such as Chlamydiae have a drastically reduced genome due to their intimate association with the host and thus a smaller number of TF genes. The genomes of members of the Chlamydiaceae family, which include the well-known bacterial pathogens Chlamydia trachomatis and Chlamydia pneumoniae, are only 1-1.2 Mbp. By contrast, members of the environmental Chlamydiae Waddlia chondrophila and Parachlamydia acanthamoebae have a 2-fold and 3-fold larger genome, respectively, likely allowing for an expansion of the host range while still retaining their host dependence and parasitic life style. Moreover, they all exhibit a characteristic chlamydial developmental cycle via two functionally specialized morphotypes, the infectious non-dividing elementary bodies (EBs) and the non-infectious dividing reticulate bodies (RBs). This developmental cycle is usually divided in three stages: the early stage during which EBs enter host cells and differentiate into RBs; the mid-stage where RBs proliferate inside a vacuole called inclusion and the late stage where RBs differentiate back into EBs and are released after exocytosis or cell lysis. Chlamydial genes are thus classified into three different temporal classes (early, mid and late expressed genes), likely reflecting the need of these transcripts in each of the three developmental stages. W. chondrophila, an emerging pathogen implicated in abortion in bovine and miscarriage in humans, encodes less than 20 TFs, 10 of which are conserved among the Chlamydiae. In light of this low TF multiplicity in the chlamydial pan-genome along with the common developmental cycle and parasitic life style, we aimed to define the regulatory pan-genome of each these conserved TFs to identify the elusive chlamydial master regulator and to characterize underling specificity for its target promoters using chromatin-immunoprecipitation followed by deep-sequencing (ChIP-Seq) of chlamydial cells growing inside the host. The immunochemistry of ChIP-Seq has the advantage of minimizing the contaminating nucleic acids compared to chlamydial transcriptome studies, it has the added benefit of providing the first unambiguous glimpse into the regulatory landscape of a bacterium inside host, offering a solid framework in understanding the stochastic and/or deterministic switches that bacteria rely on during infections.

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The Chlamydia trachomatis plasmid-encoded Pgp4 is a transcriptional regulator of virulence associated chromosomal genes

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