Project description:In the social amoebae (Dictyostelia) quorum sensing system mediates aggregation of single cells into multicellular aggregates by chemotactic movement towards gradients of diffusible molecules known as acrasins. The acrasin of P. violaceum is the unusual dipeptide N-propionyl-gamma-L-glutamyl-L-ornithine-delta-lactam-ethylester, known as glorin. Phylogenetic analysis has indicated that P. violaceum is more related to the most derived group 4 dictyostelids than to the ancient group 2 polysphondylids such as P. pallidum. Nevertheless it has been reported that P. pallidum cells respond to glorin in chemotaxis assays. This has led to the assumption that glorin-based communication may be the most ancient form of intercellular communication that Dictyostelia invented to organize early steps of multicellular development. In this study we show that glorin mediates rapid changes in gene expression at the transition from vegetative growth to aggregation, apparently without pronounced cross-talk with the cyclic AMP-based communication system that coordinates post-aggregation events in this species. We describe glorin-mediated changes in gene expression in the social amoeba Polysphondylium pallidum at the transition from unicellular growth to multicellular development. Comparison of gene expression in growing cells versus cells starving for 2 or 3 hours in the presence or absence of glorin.
Project description:In the social amoebae (Dictyostelia) quorum sensing system mediates aggregation of single cells into multicellular aggregates by chemotactic movement towards gradients of diffusible molecules known as acrasins. The acrasin of P. violaceum is the unusual dipeptide N-propionyl-gamma-L-glutamyl-L-ornithine-delta-lactam-ethylester, known as glorin. Phylogenetic analysis has indicated that P. violaceum is more related to the most derived group 4 dictyostelids than to the ancient group 2 polysphondylids such as P. pallidum. Nevertheless it has been reported that P. pallidum cells respond to glorin in chemotaxis assays. This has led to the assumption that glorin-based communication may be the most ancient form of intercellular communication that Dictyostelia invented to organize early steps of multicellular development. In this study we show that glorin mediates rapid changes in gene expression at the transition from vegetative growth to aggregation, apparently without pronounced cross-talk with the cyclic AMP-based communication system that coordinates post-aggregation events in this species. We describe glorin-mediated changes in gene expression in the social amoeba Polysphondylium pallidum at the transition from unicellular growth to multicellular development.
Project description:Treponema pallidum subspecies pallidum (T. pallidum) infection induces significant immune responses, resulting in tissue damage. Gene expression plays an essential role in regulating the progression of syphilis infection. However, little is known about the regulatory role of miRNAs in the immune response to T. pallidum infection. Here, we analyze the differential expression of miRNAs in peripheral blood mononuclear cells (PBMCs) between secondary syphilis (SS) patients and healthy controls and study the correlation between miRNAs expression and clinical features with bioinformatics.
Project description:We use ChIP-seq targeting histone 3 lysine 27-acetylation (H3K27ac) to identify putative enhancer sites genome-wide in the ventral pallidum cortex of adult prairie voles
Project description:Treponema pallidum ssp. pallidum, the causative agent of syphilis, can now be cultured continuously in vitro utilizing a tissue culture system, and the multiplication rates are similar to those obtained in experimental infection of rabbits. In this study, the RNA transcript profiles of the T. pallidum Nichols during in vitro culture and rabbit infection were compared to examine whether gene expression patterns differed in these two environments. To this end, RNA preparations were converted to cDNA and subjected to RNA-seq using high throughput Illumina sequencing; reverse transcriptase quantitative PCR was also performed on selected genes for validation of results. The transcript profiles in the in vivo and in vitro environments were remarkably similar, exhibiting a high degree of concordance overall. However, transcript levels of 94 genes (9%) out of the 1,063 predicted genes in the T. pallidum genome were significantly different during rabbit infection versus in vitro culture, varying by up to 8-fold in the two environments. Genes that exhibited significantly higher transcript levels during rabbit infection included those encoding multiple ribosomal proteins, several prominent membrane proteins, glycolysis-associated enzymes, replication initiator DnaA, rubredoxin, thioredoxin, two putative regulatory proteins, and proteins associated with solute transport. In vitro cultured T. pallidum had higher transcript levels of DNA repair proteins, cofactor synthesis enzymes, and several hypothetical proteins. The overall concordance of the transcript profiles may indicate that these environments are highly similar in terms of their effects on T. pallidum physiology and growth, and may also reflect a relatively low level of transcriptional regulation in this reduced genome organism.
Project description:The experiment was performed with the intention of collecting transcriptomic data from isolated cell types (spore, stalk and vegetative cells) in Dictyostelium lacteum as well as from cysts in Polyshpondlyium pallidum. By combining these data with similar cell-type specific RNA-Seq data from other organisms, and by examining the expression patterns of transcription factor genes, we tried to characterize how gene regulation for cell differentiation evolved in Dictyostelia. Specifically ,we dissociated and collected spore and stalk from the fruiting bodies of D. lacteum at 24 hours of development. We also collected exponentially growing vegetative cells of D. lacteum. For collecting P. pallidum cyst samples, cells were induced to encyst with sorbitol, and samples were collected at 0, 8, 16, and 24 h of incubation. RNA was extracted using the RNeasy kit (QIAGEN), and cDNA libraries were made using the Illumina TruSeq kit. The Illumina sequencing platforms (NextSeq500 for D. lacteum samples, and Hi-seq 2000 for P. pallidum samples) were used for sequencing.
Project description:Background A molecular diagnostic test that could directly evaluate Treponema pallidum subspecies pallidum antigens in urine would greatly facilitate the prompt diagnosis of syphilis. Methods A multi-platform high resolution shotgun proteomics approach was used to investigate the presence of T. pallidum proteins in male urine samples collected from individuals with and without syphilis. Study participants were included in an observational cohort study conducted at the Institute of Tropical Medicine, Antwerp, Belgium from 2014 to 2017. In total, N = 60 individuals were included in this sub-study, including n = 36 with early symptomatic stage (primary/secondary), n = 13 early latent and n = 5 late latent stage and n = 6 controls. Almost all participants were HIV-positive (56/60; 93 %) and identified as men who have sex with men (MSM) (59/60; 98 %). Sixteen (27 %) individuals were serum PCR positive for T. pallidum at the time of diagnosis. Ten pools containing urine from six participants (±1.5 mL per individual) were created based on clinical staging. Urine protein was extracted from the pools by a two-step molecular weight cut-off centrifugal filtration to remove large proteins and non-protein material. Samples were digested, subjected to multi-dimensional liquid chromatographic separation based on hydrophobicity and were analysed with matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and/or liquid chromatography- electrospray ionization quadruple ion mobility time-of-flight high-definition mass spectrometry (LC/ESI-Q-IM-TOF) in data dependent (MALDI-TOF/TOF) and data independent HDMSE (ESI-IM-Q-TOF) acquisition modes. Results In total, 26 peptides corresponding to four unique T. pallidum proteins were uncovered in four different pooled urine samples, from n = 3 symptomatic (primary/secondary stage; PSS) and n = 1 latent stage pool. Peptides from GTPase Obg (Tp0742) were detected in two separate PSS pooled samples. Protein designations are listed as in the currently annotated T. pallidum proteome available at the UniProt database (Proteome ID: UP000014259) which is related to the genome assembly and annotation ID GCA_000410535.2 listed on the European Nucleotide Archive. Eleven peptides from an uncharacterized protein (Tp0369) and 8 peptides from a protein annotated as the Borrelia-like p83/100 protein (Tp0486) were detected in two different PSS pooled samples. A protein annotated as an ABC superfamily transport protein (Tp0804) was uncovered in a latent stage pooled sample. BLAST analyses revealed low peptide sequence similarity with other pathogenic and commensal prokaryotes and human proteins. Conclusions High resolution mass spectrometric analyses of protein extracts from pooled urine samples resulted in the detection of four T. pallidum proteins. This is the first successful account of applying proteomics approaches to detect treponemal antigens in clinical samples. These proteins could represent potential biomarkers for downstream diagnostic applications.
Project description:We performed a comprehensive miRNA profiling analysis of exosomes by Treponema pallidum-stimulated microarrays. A total of 2×106 macrophages were obtained by THP-1 differentiation and grown in RPMI-1640 containing 10% exosome-free FBS. Exosomes were acquired from macrophage culture supernatants with (n = 7) or without (n = 3) T. pallidum. Briefly, macrophages were washed in PBS twice and further grown in fresh medium for 12 h (n = 2), 24 h (n = 2) and 48 h (n = 3) to collect exosomes. Exosomal miRNA microarray assays were carried out with Agilent Human miRNA (8*60K) array.