Developmental stage specific metabolic and transcriptional activity of chlamydial elementary bodies and reticulate bodies in an axenic medium
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ABSTRACT:
ORGANISM(S): Staphylococcus epidermidis RP62A Rickettsia rickettsii Coxiella burnetii RSA 493 Granulibacter bethesdensis Staphylococcus epidermidis ATCC 12228 Coxiella burnetii Chlamydia trachomatis D/UW-3/CX Chlamydia trachomatis Staphylococcus haemolyticus JCSC1435 Borreliella burgdorferi B31 Chlamydia pneumoniae AR39 Staphylococcus aureus subsp. aureus MW2 Chlamydia caviae GPIC Chlamydia muridarum
PROVIDER: GSE39530 | GEO | 2012/11/20
SECONDARY ACCESSION(S): PRJNA171054
REPOSITORIES: GEO
Similar Datasets
Project description:Chlamydia trachomatis is a significant human pathogen yet their obligate intracellular nature severe restrictions upon research. Chlamydiae undergo a complex developmental cycle characterized by an infectious cell type known as the elementary body (EB) and an intracellular active replicative form called the reticulate body (RB). EBs have historically been described as metabolically dormant. A cell-free (axenic) culture system was developed which showed high levels of metabolic and biosynthetic activity from both EBs and RBs. EBs preferentially utilized glucose-6-phosphate as an energy source whereas RBs required ATP. Both developmental forms showed improved activity when incubated under microaerobic conditions. Incorporation of isotopically-labeled amino acids into proteins from both developmental forms indicated unique expression profiles which were confirmed by genome-wide transcriptional analysis. The described axenic culture system will greatly enhance biochemical and physiological analyses of chlamydiae. Chlamydia axenic metabolic activity
2012-11-20 | E-GEOD-39530 | biostudies-arrayexpress
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
2016-02-19 | E-GEOD-68059 | biostudies-arrayexpress
Project description:The obligate intracellular developmental cycle of Chlamydia trachomatis presents significant challenges in defining its proteome. In this study we have applied quantitative proteomics to both the intracellular reticulate body (RB) and the extracellular elementary body (EB) from C. trachomatis. We used C. trachomatis L2 which is a model chlamydial isolate for such a study since it has a high infectivity: particle ratio and there is an excellent quality genome sequence. EBs and RBs (>99% pure) were quantified by chromosomal and plasmid copy number using PCR to determine the concentrations of chlamydial proteins per bacterial cell. RBs harvested at 15h post infection (PI) were purified by three successive rounds of gradient centrifugation. This is the earliest possible time to obtain purified RBs, free from host cell components in quantity, within the constraints of the technology, EBs were purified at 48h PI. We then used two-dimensional reverse phase UPLC to fractionate RB or EB peptides before mass spectroscopic analysis, providing absolute amount estimates of chlamydial proteins.
2016-06-28 | PXD003025 | Pride
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.
2016-02-19 | GSE68059 | GEO
Project description:The obligate intracellular bacterium Chlamydia has a unique developmental cycle that alternates between two contrasting cell types. With a hardy envelope and highly condensed genome, the small elementary body (EB) maintains limited metabolic activities yet can survive in an extracellular environment and is infectious. After entering host cells, EBs differentiate into larger and proliferating reticulate bodies (RBs). Progeny EBs are derived from RBs in late developmental stages and eventually exit host cells. How the expression of the chlamydial genome consisting of nearly 1000 genes governs the chlamydial developmental cycle is unclear. A previous microarray study identified only 29 immediately early genes, defined as genes activated by the first hour postinoculation, in C. trachomatis. By performing RNA sequencing analysis for C. trachomatis cultures with high multiplicities of infection (i.e., MOI of 50 and 200), we observed that 730 C. trachomatis genes underwent 2- to 900-fold activation within one hour postinoculation. By conducting quantitative reverse transcription real-time PCR (qRT-PCR) analysis for 48 of the 730 genes using an MOI of 1, we confirmed the expression increases in 46 genes. Our results demonstrate that the immediate early transcriptome is tens of times more extensive than previously realized. Gene ontology analysis indicates that the activation spans across all functional categories. We conclude that a supermajority of the C. trachomatis genes are activated almost immediately after EBs are inside host cells to initiate the differentiation toward RBs and to establish an intracellular niche conducive for chlamydial development and growth. RNA-Seq analysis was performed for Chlamydia trachomatis L2
2024-01-30 | GSE248988 | GEO
Project description:Chlamydiae are obligate intracellular bacteria comprising well-known human pathogens and ubiquitous symbionts of protists, which are characterized by a unique developmental cycle. Here we comprehensively analyzed gene expression dynamics of Protochlamydia amoebophila during infection of its Acanthamoeba host by RNA sequencing. This revealed a highly dynamic transcriptional landscape, where major transcriptional shifts are conserved among chlamydial symbionts and pathogens. Our data served to propose a time-resolved model for type III protein secretion during the developmental cycle, and we provide evidence for a biphasic metabolism of P. amoebophila during infection, which involves energy parasitism and amino acids as carbon source during initial stages and a post-replicative switch to endogenous glucose-based ATP production. This fits well with major transcriptional changes in the amoeba host, where upregulation of complex sugar breakdown precedes the P. amoebophila metabolic switch. The biphasic chlamydial metabolism represents a unique adaptation to exploit eukaryotic host cells, which likely contributed to the evolutionary success of this group of microbes.
2017-04-26 | GSE93891 | GEO
Project description:The bacteria in the chlamydiales order are obligate intracellular parasites of eukaryotic cells. They are reliant on an infectious cycle consisting of at least three phenotypically distinct cell forms termed the reticulate body (RB), the intermediate body (IB) and the elementary body (EB). The EB is infectious but does not replicate. The RB replicates in the host cell but is non-infectious, while the IB is an intermediate form that transitions to the EB form. Within this order, the genus Chlamydia contains the causative agents of a number of important pathogens of humans. C. psittaci causes zoonotic infections resulting in pneumonia, while C. pneumoniae is a human pathogen that causes respiratory disease and is linked to atherosclerosis. Biovars of C. trachomatis are the causative agents of trachoma, the leading cause of preventable blindness worldwide, as well as sexually transmitted infections with the potential to cause pelvic inflammatory disease and infertility. Irrespective of the resulting disease, all chlamydial species share the same obligate intracellular life cycle and developmental cell forms. In this study we investigated the role of four transcriptional regulators on chlamydial gene expression and completion of the developmental cycle. We ectopically expressed the transcriptional repressor Euo, each of the two nucleoid associated proteins HctA and HctB and the two component sensor kinase CtcB in the RB. All four blocked development of infectious EBs. Transcriptional analysis using RNA-seq and differential expression clustering revealed three classes of gene expression. FISH analysis demonstrated that these classes corresponded to distinct cell forms revealing the gene expression profile of the RB, IB and EB developmental forms. Additionally, these data revealed that the genes for the T3SS were cell type restricted. The structural genes for the T3SS were expressed primarily in the IB while the two translocons were expressed in distinct cell types one in the RB and the other in the EB. Overall these data show that the chlamydial developmental cycle produces three distinct regulons corresponding to the RB, IB and EB cell forms and that the T3SS is expressed in a cell form specific manner suggesting defined functional roles for the T3SS in specific cell forms.
2024-09-11 | GSE276506 | GEO
Project description:The bacteria in the chlamydiales order are obligate intracellular parasites of eukaryotic cells. They are reliant on an infectious cycle consisting of at least three phenotypically distinct cell forms termed the reticulate body (RB), the intermediate body (IB) and the elementary body (EB). The EB is infectious but does not replicate. The RB replicates in the host cell but is non-infectious, while the IB is an intermediate form that transitions to the EB form. Within this order, the genus Chlamydia contains the causative agents of a number of important pathogens of humans. C. psittaci causes zoonotic infections resulting in pneumonia, while C. pneumoniae is a human pathogen that causes respiratory disease and is linked to atherosclerosis. Biovars of C. trachomatis are the causative agents of trachoma, the leading cause of preventable blindness worldwide, as well as sexually transmitted infections with the potential to cause pelvic inflammatory disease and infertility. Irrespective of the resulting disease, all chlamydial species share the same obligate intracellular life cycle and developmental cell forms. In this study we investigated the role of four transcriptional regulators on chlamydial gene expression and completion of the developmental cycle. We ectopically expressed the transcriptional repressor Euo, each of the two nucleoid associated proteins HctA and HctB and the two component sensor kinase CtcB in the RB. All four blocked development of infectious EBs. Transcriptional analysis using RNA-seq and differential expression clustering revealed three classes of gene expression. FISH analysis demonstrated that these classes corresponded to distinct cell forms revealing the gene expression profile of the RB, IB and EB developmental forms. Additionally, these data revealed that the genes for the T3SS were cell type restricted. The structural genes for the T3SS were expressed primarily in the IB while the two translocons were expressed in distinct cell types one in the RB and the other in the EB. Overall these data show that the chlamydial developmental cycle produces three distinct regulons corresponding to the RB, IB and EB cell forms and that the T3SS is expressed in a cell form specific manner suggesting defined functional roles for the T3SS in specific cell forms.
2024-04-01 | GSE262652 | GEO
Project description:The inability to propagate obligate intracellular pathogens under axenic (host cell-free) culture conditions imposes severe experimental constraints that have negatively impacted progress in understanding pathogen virulence and disease mechanisms. Coxiella burnetii, the causative agent of human Q (Query) fever, is an obligate intracellular bacterial pathogen that replicates exclusively in an acidified, lysosome-like vacuole. To define conditions that support C. burnetii growth, we systematically evaluated the organismâ??s metabolic requirements using expression microarrays, genomic reconstruction, and metabolite typing. This led to development of a complex nutrient medium that supported substantial growth (~ 3 log10) of C. burnetii in a 2.5% oxygen environment. Importantly, axenically grown C. burnetii were highly infectious for Vero cells and exhibited developmental forms characteristic of in vivo grown organisms. Axenic cultivation of C. burnetii will facilitate studies of the organismâ??s pathogenesis and genetics, and aid development of Q fever preventatives such as an effective subunit vaccine. Furthermore, the systematic approach used here may be broadly applicable to development of axenic media that support growth of other medically important obligate intracellular pathogens. Host cell-free growth, Vero cell growth and carryover baseline of Coxiella burnetii
2010-06-20 | E-GEOD-13338 | biostudies-arrayexpress
Project description:The inability to propagate obligate intracellular pathogens under axenic (host cell-free) culture conditions imposes severe experimental constraints that have negatively impacted progress in understanding pathogen virulence and disease mechanisms. Coxiella burnetii, the causative agent of human Q (Query) fever, is an obligate intracellular bacterial pathogen that replicates exclusively in an acidified, lysosome-like vacuole. To define conditions that support C. burnetii growth, we systematically evaluated the organism’s metabolic requirements using expression microarrays, genomic reconstruction, and metabolite typing. This led to development of a complex nutrient medium that supported substantial growth (~ 3 log10) of C. burnetii in a 2.5% oxygen environment. Importantly, axenically grown C. burnetii were highly infectious for Vero cells and exhibited developmental forms characteristic of in vivo grown organisms. Axenic cultivation of C. burnetii will facilitate studies of the organism’s pathogenesis and genetics, and aid development of Q fever preventatives such as an effective subunit vaccine. Furthermore, the systematic approach used here may be broadly applicable to development of axenic media that support growth of other medically important obligate intracellular pathogens.
2009-10-21 | GSE13338 | GEO