Project description:As Trypanosoma cruzi, the etiological agent of Chagas disease, multiplies in the cytoplasm of nucleated host cells, infection with this parasite is highly likely to affect host cells. We performed an exhaustive transcriptome analysis of T. cruzi-infected HeLa cells using an oligonucleotide microarray containing probes for greater than 47,000 human gene transcripts. In comparison with uninfected cells, those infected with T. cruzi showed greater than threefold up-regulation of 41 genes and greater than threefold down-regulation of 23 genes. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) of selected, differentially expressed genes confirmed the microarray data. Many of these up- and down-regulated genes were related to cellular proliferation, including seven up-regulated genes encoding proliferation inhibitors and three down-regulated genes encoding proliferation promoters, strongly suggesting that T. cruzi infection inhibits host cell proliferation, which may allow more time for T. cruzi to replicate and produce its intracellular nests. These findings provide new insight into the molecular mechanisms by which intracellular T. cruzi infection influences the host cell, leading to pathogenicity. Experiment Overall Design: Three replicates of infected and uninfected HeLa cell were analyzed. To examine the extent of cross hybridization between T. cruzi cRNA and Human chip, trypomastigote cRNA was hybridized with the same chip.
Project description:Trypanosoma cruzi is an obligate intracellular protozoan parasite that causes human Chagas’ disease, a leading cause of heart failure in Latin America. Using Affymetrix oligonucleotide arrays we screened phenotypically diverse human cells (foreskin fibroblasts, microvascular endothelial cells and vascular smooth muscle cells) for a common transcriptional response signature to T. cruzi. A common feature was a prominent type I interferon response, indicative of a secondary response to secreted cytokines. Using transwell plates to distinguish cytokine-dependent and -independent gene expression profiles in T. cruzi-infected cells, a core cytokine-independent response was identified in fibroblasts and endothelial cells that featured metabolic and signaling pathways involved in cell proliferation, amino acid catabolism and response to wounding. Significant downregulation of genes involved in mitotic cell cycle and cell division predicted that T. cruzi infection impedes cell cycle progression in the host cell. Overall design: Primary human foreskin fibroblasts and human microvascular endothelial cells were grown in transwell dishes to reach 80% confluency. The cells in the bottom of the transwell were infected with T. cruzi for 24 hours, while the cells on the top of the transwel were not infected but exposed to the same culture medium bathing the infected cells. Transwells containing uninfected cells in both levels were also included as controls. Vascular smooth muscle cells were omitted from the transwell experiments due to difficulties obtaining adequate RNA from cells plated on the top chamber. Instead they were cultred in Petri dishes and mock/infected with T. cruzi for 24 or 48 hours.Total RNA was extracted and gene expression levels analyzed with Affymetrix microarrays. Two independent biological replicates were included for each type of treatment.
Project description:The intracellular pathogen Trypanosoma cruzi secretes an activity that blocks TGF-β-dependent induction of connective tissue growth factor (CTGF/CCN2). Here, we address the mechanistic basis for T. cruzi-mediated interference of CTGF/CCN2 expression by examining host cell signaling pathways and the global inhibitory effect on TGF-β-dependent gene expression. We show that the expression of a discrete subset of TGF-β-inducible genes involved in cell proliferation, wound repair, and immune regulation are blocked by the soluble T. cruzi activity, demonstrating that this parasite-derived activity has broad, but specific effects on fibroblast gene regulation. Overall design: Primary human fibroblasts were treated with TGF-β, T. cruzi conditioned medium (PCM) and TGF-β/ PCM simultaneously. Untreated cells were also included as controls. Total RNA was extracted and gene expression levels analyzed with Affymetrix microarrays. Three independent biological replicates were included for each type of treatment.
Project description:Host cell infection by the intracellular pathogen, Trypanosoma cruzi, involves activation of signaling pathways, cytoskeletal reorganization, and targeted recruitment of host cell lysosomes. To determine the consequences of T. cruzi invasion on host cell gene expression, high density microarrays consisting of approximately 27,000 human cDNAs were hybridized with fluorescent probes generated from T. cruzi-infected human fibroblasts (HFF) at early time points following infection (2-24 h). Surprisingly, no genes were induced > or =2-fold in HFF between 2 and 6 h post-infection (hpi) in repeated experiments while immediate repression of six host cell transcripts was observed. A significant increase in transcript abundance for 106 host cell genes was observed at 24 hpi. Among the most highly induced is a set of interferon-stimulated genes, indicative of a type I interferon (IFN) response to T. cruzi. In support of this, T. cruzi-infected fibroblasts begin to secrete IFNbeta at 18 hpi following the induction of IFNbeta transcripts. As compared with global transcriptional responses evoked by other intracellular pathogens, T. cruzi is a stealth parasite that elicits few changes in host cell transcription during the initiation of infection. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set Overall design: Computed