Dataset Information

Quantitative proteomics of the infectious and replicative forms of Chlamydia trachomatis L2

ABSTRACT: 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.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Chlamydia Trachomatis Serovar L2 (strain 434/bu / Atcc Vr-902b)

TISSUE(S): Prokaryotic Cell

DISEASE(S): Chlamydia Trachomatis Infectious Disease

SUBMITTER: Paul Skipp

LAB HEAD: Paul James Skipp

PROVIDER: PXD003025 | Pride | 2016-06-28

REPOSITORIES: Pride

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Publications

Quantitative Proteomics of the Infectious and Replicative Forms of Chlamydia trachomatis.

Skipp Paul J S PJ Hughes Chris C McKenna Thérèse T Edwards Richard R Langridge James J Thomson Nicholas R NR Clarke Ian N IN

PloS one 20160212 2

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 as a model chlamydial isolate for our study since it has a high infectivity:particle ratio and there is an excellent quality genome sequence. EBs and RBs (>99% pure) were quantifi ...[more]

PMID: 26871455

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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.

Project description:Directing differentiation of human embryonic stem cells (hESC) into specific cell types using an easy and reproducible protocol is a perquisite for the clinical use of hESC in regenerative medicine protocols. Here, we report the generation of mesodermal cells with differentiation potential to myocytes, osteoblasts, chondrocytes and adipocytes. We demonstrate that during hESC differentiation as embryoid bodies (EB), inhibition of TGF-b/Activin/Nodal signaling using SB-431542 (SB) markedly up-regulated paraxial mesodermal markers (TBX6, TBX5), early myogenic transcriptional factors (Myf5, Pax7) as well as myocyte committed markers (NCAM, CD34, Desmin, MHC (fast), alpha-smooth muscle actin, Nkx2.5, cTNT). Establishing EB outgrowth cultures (SB-OG) in the presence of SB (1 uM) led to further enrichment of cells expressing markers for myocyte progenitor cell: CD34+ (33%), NCAM+ (CD56) (73%), PAX7 (25%) and mature myocyte proteins (MYOD1, tropomyocin, fast MHC an; d SERCA1). Further analysis using DNA microarray revealed differential up-regulation of 117 genes (>2-fold compared to control cells) annotated to myogenic development and function. During ex vivo culture, contracting myocytes were observed (80% of the population) and the cells formed myofibres when implanted intramuscularly in vivo. Furthermore, in the presence of fetal bovine serum (10% FBS), SB-OG cells developed morphologically and phenotypically into a homogeneous stromal (mesenchymal) stem cell (MSC)-like population expressing characteristic MSC CD markers: CD44 (100%), CD73 (98%), CD146 (96%) and CD166 (88%). They were karyotypically normal and were able to differentiate ex vivo and in vivo into osteoblasts, adipocytes and chondrocytes. Experiment Overall Design: Human ESCs were differentiated as EBs for 10 days, where after EBs were plated onto fibronectin coated petri dishes. At confluency the outgrowth culture was passaged, at passage 5 3 samples were taken from both control-OG and SB-OG. the samples were mixed and frozen.

Dataset Information

Quantitative proteomics of the infectious and replicative forms of Chlamydia trachomatis L2

Publications

Quantitative Proteomics of the Infectious and Replicative Forms of Chlamydia trachomatis.

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