Disruption of the ESX-5 system of Mycobacterium tuberculosis causes loss of PPE protein secretion, reduction of cell wall integrity and strong attenuation

The chromosome of Mycobacterium tuberculosis encodes five type VII secretion systems (ESX-1-ESX-5). While the role of the ESX-1 and ESX-3 systems in M. tuberculosis has been elucidated, predictions for the function of the ESX-5 system came from data obtained in Mycobacterium marinum, where it transports PPE and PE_PGRS proteins and modulates innate immune responses. To define the role of the ESX-5 system in M. tuberculosis, in this study, we have constructed five M. tuberculosis H37Rv ESX-5 knockout/deletion mutants, inactivating eccA(5), eccD(5), rv1794 and esxM genes or the ppe25-pe19 region. Whereas the Mtbrv1794ko displayed no obvious phenotype, the other four mutants showed defects in secretion of the ESX-5-encoded EsxN and PPE41, a representative member of the large PPE protein family. Strikingly, the MtbeccD(5) ko mutant also showed enhanced sensitivity to detergents and hydrophilic antibiotics. When the virulence of the five mutants was evaluated, the MtbeccD(5) ko and MtbΔppe25-pe19 mutants were found attenuated both in macrophages and in the severe combined immune-deficient mouse infection model. Altogether these findings indicate an essential role of ESX-5 for transport of PPE proteins, cell wall integrity and full virulence of M. tuberculosis, thereby opening interesting new perspectives for the study of this human pathogen.     

Complexes of Streptavidin-Fused Antigens with Biotinylated Antibodies Targeting Receptors on Dendritic Cell Surface: A Novel Tool for Induction of Specific T-Cell Immune Responses

The choice of tools that enable efficient targeting of exogenous antigens (Ag) for processing and presentation by professional Ag-presenting cells (APC) remains limited. This represents, indeed, a bottleneck in development of vaccines inducing specific T-cell responses. Here, we describe a novel strategy of Ag delivery into APCs. The Ag of choice is fused to the N- or C-terminus of streptavidin (SA) and tetrameric Ag-SA or SA-Ag fusion proteins are produced in E. coli and purified by 2-Iminobiotin-Agarose affinity chromatography. Alternatively, Ag-SA proteins are purified from urea extracts of E. coli inclusion bodies and refolded in vitro into functional tetramers. Complexes with biotinylated antibodies targeting cell surface receptors are formed and used to deliver the Ags of choice for processing and presentation by APCs and induction of Ag-specific CD4+ and CD8+ T-cell responses in vitro and in vivo.     

Full-length structural model of RET3 and SEC21 in COPI: identification of binding sites on the appendage for accessory protein recruitment motifs

COPI, a 600 kD heptameric complex (consisting of subunits α, β, γ, δ, ε, ζ, and β') "coatomer," assembles non-clathrin-coated vesicles and is responsible for intra-Golgi and Golgi-to-ER protein trafficking. Here, we report the three-dimensional structures of the entire sequences of yeast Sec21 (γ-COPI mammalian ortholog), yeast Ret3 (ζ-COPI mammalian ortholog), and the results of successive molecular dynamics investigations of the subunits and assembly based on a protein-protein docking experiment. The three-dimensional structures of the subunits in their complexes indicate the residues of the two subunits that impact on assembly, the conformations of Ret3 and Sec21, and their binding orientations in the complexed state. The structure of the appendage domain of Sec21, with its two subdomains--the platform and the β-sandwich, was investigated to explore its capacity to bind to accessory protein recruitment motifs. Our study shows that a binding site on the platform is capable of binding the Eps15 DPF and epsin DPW2 peptides, whereas the second site on the platform and the site on the β-sandwich subdomain were found to selectively bind to the amphiphysin FXDXF and epsin DPW1 peptides, respectively. Identifying the regions of both the platform and sandwich subdomains involved in binding each peptide motif clarifies the mechanism through which the appendage domain of Sec21 engages with the accessory proteins during the trafficking process of non-clathrin-coated vesicles.     

Toll-like receptors pathway in common variable immune deficiency (CVID) and X-linked agammaglobulinemia (XLA)

Common variable immunodeficiency (CVID) and X-linked agammaglobulinemia (XLA) are two major humoral immunodeficiencies, causing a high rate of early age mortality in children. In order to identifiy the possible factors involved in the pathogenesis of CVID and XLA, recent studies have focused on Toll-like receptors (TLRs) and demonstrate the defects in different TLR pathways in immune cells of CVID and XLA patients. Herein, we measured TLR-4 and TLR-9 RNA levels and consequently TNF-α and IFN-α production in peripheral blood mononuclear cells (PBMCs) of patients with CVID and XLA. Contrary to healthy individuals, TLR-9 expression was not significantly increased after ligand stimulation, whereas ligand-induced TLR-4 expression was not significantly different from that in healthy control PBMCs. Lipopolysaccharide (LPS)-stimulated TNF-α production was significantly reduced in patients compared to controls, whereas IFN-α production was increased in all groups after CpG stimulation without any significant inter-group difference. Our data suggest that defects in TLR-9 activated pathways may be a result of the decreased TLR-9 expression, although TLR-9 is not the only modulator of IFN-α production in these patients. On the other hand, impaired signaling in TLR-4 activated pathways which results in significant reduction in TNF-α production are not related to a defect in TLR-4 expression.     

Biodegradation of gasoline and BTEX in a microaerophilic biobarrier

Continuous bioremediation of gasoline-contaminatedwater in a packed-bed biobarrier system underoxygen-limited conditions is discussed. This studywas part of an extensive effort to develop analternative technology for the in situbioremediation of hydrocarbons where there is alimited supply of oxygen. Protruded stainless steelpieces and granulated peat moss were used as packingmaterial to support microbial growth in twobiobarriers. The inoculum was an enrichment culture ofan indigenous microbial population from a soil sample.The biobarriers' inlet gasoline concentrations and thelinear liquid velocities were similar to thosecommonly found at in situ conditions. Gasolineremoval efficiencies ranged from 94% to 99.9% in thestainless steel-packed biobarrier, and from 86.6% to99.6% in the peat moss-packed biobarrier. Effluentgasoline concentrations below 0.03 mg/l were obtainedat gasoline loading rates less than 27.5 mg/l.d in thestainless steel-packed biobarrier. The remainingfraction of gasoline in the effluent consisted mainlyof three aliphatic compounds and not the aromaticcompounds. Both biobarrier packings supported nearcomplete removal of the most soluble aromatichydrocarbons of gasoline (BTEX) under all theconditions examined. The consumption of sulfate andthe presence of sulfate-reducing microorganismssuggested the presence of anaerobic metabolism duringthe degradation of gasoline. Up to 92% gasoline wasremoved during the first 3 cm of the biobarriers'length.     

Recognition of functional genetic polymorphism using ESE motif definition: a conservative evolutionary approach to CYP2D6/CYP2C19 gene variants

Genetica - Although predicting the effects of variants near intron-exon boundaries is relatively straightforward, predicting the functional Exon Splicing Enhancers (ESEs) and the possible effects...     

Construction of miRNA‐lncRNA‐mRNA co‐expression network affecting EMT‐mediated cisplatin resistance in ovarian cancer

Platinum resistance is one of the major concerns in ovarian cancer treatment. Recent evidence shows the critical role of epithelial–mesenchymal transition (EMT) in this resistance. Epithelial‐like ovarian cancer cells show decreased sensitivity to cisplatin after cisplatin treatment. Our study prospected the association between epithelial phenotype and response to cisplatin in ovarian cancer. Microarray dataset {"type":"entrez-geo","attrs":{"text":"GSE47856","term_id":"47856"}}GSE47856 was acquired from the GEO database. After identifying differentially expressed genes (DEGs) between epithelial‐like and mesenchymal‐like cells, the module identification analysis was performed using weighted gene co‐expression network analysis (WGCNA). The gene ontology (GO) and pathway analyses of the most considerable modules were performed. The protein–protein interaction network was also constructed. The hub genes were specified using Cytoscape plugins MCODE and cytoHubba, followed by the survival analysis and data validation. Finally, the co‐expression of miRNA‐lncRNA‐TF with the hub genes was reconstructed. The co‐expression network analysis suggests 20 modules relating to the Epithelial phenotype. The antiquewhite4, brown and darkmagenta modules are the most significant non‐preserved modules in the Epithelial phenotype and contain the most differentially expressed genes. GO, and KEGG pathway enrichment analyses on these modules divulge that these genes were primarily enriched in the focal adhesion, DNA replication pathways and stress response processes. ROC curve and overall survival rate analysis show that the co‐expression pattern of the brown module''s hub genes could be a potential prognostic biomarker for ovarian cancer cisplatin resistance.