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Host Cell Response (host + cell_response)
Selected AbstractsIndications for cell stress in response to adenoviral and baculoviral gene transfer observed by proteome profiling of human cancer cellsELECTROPHORESIS, Issue 11 2010Christopher Gerner Abstract Gene transfer to cultured cells is an important tool for functional studies in many areas of biomedical research and vector systems derived from adenoviruses and baculoviruses are frequently used for this purpose. In order to characterize how viral gene transfer vectors affect the functional state of transduced cells, we applied 2-D PAGE allowing quantitative determination of protein amounts and synthesis rates of metabolically labeled cells and shotgun proteomics. Using HepG2 human hepatoma cells we show that both vector types can achieve efficient expression of green fluorescent protein, which accounted for about 0.1% of total cellular protein synthesis 72,h after transduction. No evidence in contrast was found for expression of proteins from the viral backbones. With respect to the host cell response, both vectors induced a general increase in protein synthesis of about 50%, which was independent of green fluorescent protein expression. 2-D PAGE autoradiographs identified a 3.6-fold increase of ,-actin synthesis in adenovirus transduced cells. In addition shotgun proteomics of cytoplasmic and nuclear extract fractions identified a slight induction of several proteins related to inflammatory activation, cell survival and chromatin function by both virus types. These data demonstrate that commonly used gene transfer vectors induce a response reminiscent of stress activation in host cells, which needs to be taken into account when performing functional assays with transduced cells. [source] IpgD, a protein secreted by the type III secretion machinery of Shigella flexneri, is chaperoned by IpgE and implicated in entry focus formationMOLECULAR MICROBIOLOGY, Issue 1 2000Kirsten Niebuhr Invasion of epithelial cells by Shigella flexneri involves entry and intercellular dissemination. Entry of bacteria into non-phagocytic cells requires the IpaA,D proteins that are secreted by the Mxi,Spa type III secretion machinery. Type III secretion systems are found in several Gram-negative pathogens and serve to inject bacterial effector proteins directly into the cytoplasm of host cells. In this study, we have analysed the IpgD protein of S. flexneri, the gene of which is located on the virulence plasmid at the 5, end of the mxi,spa locus. We have shown that IpgD (i) is stored in the bacterial cytoplasm in association with a specific chaperone, IpgE; (ii) is secreted by the Mxi,Spa type III secretion system in amounts similar to those of the IpaA,D proteins; (iii) is associated with IpaA in the extracellular medium; and (iv) is involved in the modulation of the host cell response after contact of the bacterium with epithelial cells. This suggests that IpgD is an effector that might be injected into host cells to manipulate cellular processes during infection. [source] H. pylori selectively blocks EGFR endocytosis via the non-receptor kinase c-Abl and CagACELLULAR MICROBIOLOGY, Issue 1 2009Bianca Bauer Summary Helicobacter pylori infection is a primary cause of peptic ulcers and is associated with gastric carcinogenesis. The H. pylori -induced pathophysiology may be linked to the deregulation of EGFR signalling. Elevated mucosal levels of EGF and the EGFR have been found in antral gastric biopsies of H. pylori -infected patients. A critical mechanism for regulating EGFR signalling is ligand-induced endocytosis. The internalized receptor recycles back to the plasma membrane for continued signalling or is targeted for degradation terminating receptor signalling. Here, we show that H. pylori blocks EGFR endocytosis and receptor degradation upon prolonged infection of gastric epithelial cells. Moreover, this inhibition occurs via a CagA-dependent, but CagA phosphorylation-independent activation of the non-receptor kinase c-Abl, which in turn phosphorylates the EGFR target site pY1173. This suggests a novel CagA-induced host cell response that is independent of CagA tyrosine phosphorylation. Our data indicate an intriguing strategy of H. pylori in host cell manipulations by altering selective receptor populations via a CagA-dependent endocytic mechanism. Furthermore, we identified a new role for c-Abl in phosphorylation of the EGFR target site pY1173 during H. pylori infection. [source] The Salmonella SPI1 effector SopB stimulates nitric oxide production long after invasionCELLULAR MICROBIOLOGY, Issue 1 2005Dan Drecktrah Summary The ability of Salmonella enterica to invade and replicate within host cells depends on two type III secretion systems (TTSSs) encoded on pathogenicity islands 1 and 2 (SPI1 and SPI2). The current paradigm holds that these systems translocate two classes of effectors that operate sequentially and independently. In essence, the SPI1 TTSS mediates early events (i.e. invasion) whereas the SPI2 TTSS mediates post-invasion processes (i.e. replication, vacuole maturation). Contrary to this model, we have found in infected macrophages that a SPI1 effector, SopB/SigD, increased inducible nitric oxide synthase levels and nitric oxide production, host cell process previously known only to be a target of the SPI2 TTSS. Furthermore, SopB protein and message persist many hours after invasion. Our findings reveal an unanticipated potential for dialogue between the SPI1 and SPI2 TTSS and the host cell response. [source] Diversity of human colonic butyrate-producing bacteria revealed by analysis of the butyryl-CoA:acetate CoA-transferase geneENVIRONMENTAL MICROBIOLOGY, Issue 2 2010Petra Louis Summary Butyrate-producing bacteria play an important role in the human colon, supplying energy to the gut epithelium and regulating host cell responses. In order to explore the diversity and culturability of this functional group, we designed degenerate primers to amplify butyryl-CoA:acetate CoA-transferase sequences from faecal samples provided by 10 healthy volunteers. Eighty-eight per cent of amplified sequences showed > 98% DNA sequence identity to CoA-transferases from cultured butyrate-producing bacteria, and these fell into 12 operational taxonomic units (OTUs). The four most prevalent OTUs corresponded to Eubacterium rectale, Roseburia faecis, Eubacterium hallii and an unnamed cultured species SS2/1. The remaining 12% of sequences, however, belonged to 20 OTUs that are assumed to come from uncultured butyrate-producing strains. Samples taken after ingestion of inulin showed significant (P = 0.019) increases in Faecalibacterium prausnitzii. Because several of the dominant butyrate producers differ in their DNA % G+C content, analysis of thermal melt curves obtained for PCR amplicons of the butyryl-CoA:acetate CoA-transferase gene provides a convenient and rapid qualitative assessment of the major butyrate producing groups present in a given sample. This type of analysis therefore provides an excellent source of information on functionally important groups within the colonic microbial community. [source] Neutrophil influx during non-typhoidal salmonellosis: who is in the driver's seat?FEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2006Çagla Tükel Abstract A massive neutrophil influx in the intestine is the histopathological hallmark of Salmonella enterica serovar Typhimurium-induced enterocolitis in humans. Two major hypotheses on the mechanism leading to neutrophil infiltration in the intestinal mucosa have emerged. One hypothesis suggests that S. enterica serovar Typhimurium takes an active role in triggering this host response by injecting proteins, termed effectors, into the host cell cytosol which induce a proinflammatory gene expression profile in the intestinal epithelium. The second hypothesis suggests a more passive role for the pathogen by proposing that bacterial invasion stimulates the innate pathways of inflammation because the pathogen-associated molecular patterns of S. enterica serovar Typhimurium are recognized by pathogen recognition receptors on cells in the lamina propria. A review of the current literature reveals that, while pathogen recognition receptors are clearly involved in eliciting neutrophil influx during S. enterica serovar Typhimurium infection, a direct contribution of effectors in triggering proinflammatory host cell responses cannot currently be ruled out. [source] Helicobacter pylori CagA: Analysis of Sequence Diversity in Relation to Phosphorylation Motifs and Implications for the Role of CagA as a Virulence FactorHELICOBACTER, Issue 3 2001Doyle J. Evans Jr Abstract CagA is transported into host target cells and subsequently phosphorylated. Clearly this is a mechanism by which Helicobacter pylori could take control of one or more host cell signal transduction pathways. Presumably the end result of this interaction favors survival of H. pylori, irrespective of eventual damage to the host cell. CagA is noted for its amino acid (AA) sequence diversity, both within and outside the variable region of the molecule. The primary purpose of this review is to examine how variation in the type and number of CagA phosphorylation sites might determine the outcome of infection by different strains of H. pylori. The answer to this question could help to explain the widely disparate results obtained when H. pylori CagA status has been compared to type and severity of disease outcome in different populations, that is in different countries. Analysis of all available CagA sequences revealed that CagA contains both tyrosine phosphorylation motifs (TPMs) and cyclic-AMP-dependent phosphorylation motifs (CPMs). There are two potential CPMs near the N-terminus of CagA and at least two in the repeat region; these are not all equally well conserved. We also defined a 48-residue AA sequence, which includes the N-terminal TPM at tyrosine (Y)-122, which distinguishes between Eastern (Hong Kong-Taiwan-Japan-Thailand) H. pylori isolates and those from the West (Europe-Africa-the Americas-Australia). All 28 of the Eastern type CagA proteins have a functional N-terminal TPM whereas 11 of 47 (23.4%) of the Western type contain an inactive motif, with threonine (T) replacing the critical aspartic acid (D) residue. Only 13 of 24 (54%) known CagA sequences have an active TPM in the repeat region and only one has two TPMs in this region. The potential TPM near the C-terminus of CagA is not likely to be important since only 3 of 24 (12.5%) sequences were found to be intact. Protein database searches revealed that the AA sequence immediately following the TPM at Y-122 in CagA is homologous with a pair of PDZ domains which are common in signal transducing proteins, particularly tyrosine phosphatases. This provides a theoretical link between CagA and many of the observed responses of host cells to H. pylori. In summary, not all CagA proteins are equal in their potential for initiating host cell responses via signal transduction pathways. The degree of functional diversity of this protein depends upon which phosphorylation motifs are critical to the biological activity of CagA. [source] Helicobacter pylori and gastric cancer: possible role of microRNAs in this intimate relationshipCLINICAL MICROBIOLOGY AND INFECTION, Issue 9 2009C. Belair Abstract Chronic infection by Helicobacter pylori is a major risk factor for gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. H. pylori possesses a set of virulence factors, including the CagA effector, which interferes with intracellular signalling pathways and mediates phenotypic alterations, strongly evoking neoplasic transformation. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression involved in development, cell proliferation and immune responses. miRNAs are frequently altered in cancers, revealing their functions as oncogenes or tumour suppressors. However, the role, if any, that miRNAs play in the host cell responses to H. pylori remains unknown. This review considers the possible involvement of some miRNAs, including miR-146, miR-155, miR-21, miR-27a, miR-106-93-25 and miR-221-222 clusters and the miR-200 family in H. pylori -induced infection and gastric cancers. Further exploration of miRNA-mediated gene silencing, taking into account the relationship between host targets and bacterial effectors, will most certainly bring new insights into the control of gene expression in human gastric cells chronically infected by H. pylori. [source] Chlamydia trachomatis -infected host cells resist dsRNA-induced apoptosisCELLULAR MICROBIOLOGY, Issue 9 2010Linda Böhme Summary Human pathogenic Chlamydia trachomatis have evolved sophisticated mechanisms to manipulate host cell signalling pathways in order to prevent apoptosis. We show here that host cells infected with C. trachomatis resist apoptosis induced by polyI:C, a synthetic double-stranded RNA that mimics viral infections. Infected cells displayed significantly reduced levels of PARP cleavage, caspase-3 activation and a decrease in the TUNEL positive population in the presence of polyI:C. Interestingly, the chlamydial block of apoptosis was upstream of the initiator caspase-8. Processing of caspase-8 was reduced in infected cells and coincided with a decrease in Bid truncation and downstream caspase-9 cleavage. Moreover, the enzymatic activity of caspase-8, measured by a luminescent substrate, was significantly reduced in infected cells. Caspase-8 inhibition by Chlamydia was dependent on cFlip as knock-down of cFlip decreased the chlamydial block of caspase-8 activation and consequently reduced apoptosis inhibition. Our data implicate that chlamydial infection interferes with the host cell's response to viral infections and thereby influences the fate of the cell. [source] | |||||||||||||