Autophagy is an evolutionarily conserved cellular recycling mechanism that occurs at a basal level in all cells. It can be further induced by various stimuli including starvation, hypoxia, and ...treatment with cytokines such as IFNG/IFNγ and TGFB/TGFβ. Type I IFNs are proteins that induce an antiviral state in cells. They also have antiproliferative, proapoptotic and immunomodulatory activities. We investigated whether type I IFN can also induce autophagy in multiple human cell lines. We found that treatment with IFNA2c/IFNα2c and IFNB/IFNβ induces autophagy by 24 h in Daudi B cells, as indicated by an increase of autophagy markers MAP1LC3-II, ATG12-ATG5 complexes, and a decrease of SQSTM1 expression. An increase of MAP1LC3-II was also detected 48 h post-IFNA2c treatment in HeLa S3, MDA-MB-231, T98G and A549 cell lines. The presence of autophagosomes in selected cell lines exposed to type I IFN was confirmed by electron microscopy analysis. Increased expression of autophagy markers correlated with inhibition of MTORC1 in Daudi cells, as well as inhibition of cancer cell proliferation and changes in cell cycle progression. Concomitant blockade of either MTOR or PI3K-AKT signaling in Daudi and T98G cells treated with IFNA2c increased the level of MAP1LC3-II, indicating that the PI3K-AKT-MTORC1 signaling pathway may modulate IFN-induced autophagy in these cells. Taken together, our findings demonstrated a novel function of type I IFN as an inducer of autophagy in multiple cell lines.
To determine whether Lassa virus was circulating in southern Mali, we tested samples from small mammals from 3 villages, including Soromba, where in 2009 a British citizen probably contracted a ...lethal Lassa virus infection. We report the isolation and genetic characterization of Lassa virus from an area previously unknown for Lassa fever.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Poxviruses are enveloped DNA viruses that replicate within the cytoplasm. The first viral structures are crescents and spherical particles, with a lipoprotein membrane bilayer, that are thought to be ...derived from the ER. We determined that A17, a conserved viral transmembrane protein essential for crescent formation, forms homo-oligomers and shares topological features with cellular reticulon-like proteins. The latter cell proteins promote membrane curvature and contribute to the tubular structure of the ER. When the purified A17 protein was incorporated into liposomes, 25 nm diameter vesicles and tubules formed at low and high A17 concentrations, respectively. In addition, intracellular expression of A17 in the absence of other viral structural proteins transformed the ER into aggregated three-dimensional (3D) tubular networks. We suggest that A17 is a viral reticulon-like protein that contributes to curvature during biogenesis of the poxvirus membrane.
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•Predicted structure of poxvirus A17 protein resembles ER tubule-forming reticulons•Purified A17 protein interacts with lipids to form 25 nm vesicles and tubules•Overexpression of A17 transforms the ER into a highly curved, tightly packed network•A17 contributes to curvature during viral membrane biogenesis from the ER
A17, an essential poxvirus membrane protein, shares predictable features with cellular proteins responsible for the ER tubular structure. A17 interacts with lipids to form 25 nm vesicles and tubules and increases the curvature and tight packing of the ER. These findings suggest that A17 has a reticulon-like role in viral membrane biogenesis.
Chlamydiae replicate intracellularly within a unique vacuole termed the inclusion. The inclusion circumvents classical endosomal/lysosomal pathways but actively intercepts a subset of Golgi-derived ...exocytic vesicles containing sphingomyelin (SM) and cholesterol. To further examine this interaction, we developed a polarized epithelial cell model to study vectoral trafficking of lipids and proteins to the inclusion. We examined seven epithelial cell lines for their ability to form single monolayers of polarized cells and support chlamydial development. Of these cell lines, polarized colonic mucosal C2BBe1 cells were readily infected with Chlamydia trachomatis and remained polarized throughout infection. Trafficking of (6-((N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)hexanoyl)sphingosine) (NBD-C₆-ceramide) and its metabolic derivatives, NBD-glucosylceramide (GlcCer) and NBD-SM, was analyzed. SM was retained within L2-infected cells relative to mock-infected cells, correlating with a disruption of basolateral SM trafficking. There was no net retention of GlcCer within L2-infected cells and purification of C. trachomatis elementary bodies from polarized C2BBe1 cells confirmed that bacteria retained only SM. The chlamydial inclusion thus appears to preferentially intercept basolaterally-directed SM-containing exocytic vesicles, suggesting a divergence in SM and GlcCer trafficking. The observed changes in lipid trafficking were a chlamydia-specific effect because Coxiella burnetii-infected cells revealed no changes in GlcCer or SM polarized trafficking.
Transmission of plague by fleas depends on infection of the proventricular valve in the insect's foregut by a dense aggregate of Yersinia pestis. Proventricular infection requires the Y. pestis hemin ...storage (hms) genes; here, we show that the hms genes are also required to produce an extracellular matrix and a biofilm in vitro, supporting the hypothesis that a transmissible infection in the flea depends on the development of a biofilm on the hydrophobic, acellular surface of spines that line the interior of the proventriculus. The development of biofilm and proventricular infection did not depend on the 3 Y. pestis quorum-sensing systems. The extracellular matrix enveloping the Y. pestis biofilm in the flea appeared to incorporate components from the flea's blood meal, and bacteria released from the biofilm were more resistant to human polymorphonuclear leukocytes than were in vitro-grown Y. pestis. Enabling arthropod-borne transmission represents a novel function of a bacterial biofilm.
Salmonella invade non-phagocytic cells by inducing massive actin rearrangements, resulting in membrane ruffle formation and phagocytosis of the bacteria. This process is mediated by a cohort of ...effector proteins translocated into the host cell by type III secretion system 1, which is encoded by genes in the Salmonella pathogenicity island (SPI) 1 regulon. This network is precisely regulated and must be induced outside of host cells. In vitro invasive Salmonella are prepared by growth in synthetic media although the details vary. Here, we show that culture conditions affect the frequency, and therefore invasion efficiency, of SPI1-induced bacteria and also can affect the ability of Salmonella to adapt to its intracellular niche following invasion. Aerobically grown late-exponential-phase bacteria were more invasive and this was associated with a greater frequency of SPI1-induced, motile bacteria, as revealed by single-cell analysis of gene expression. Culture conditions also affected the ability of Salmonella to adapt to the intracellular environment, since they caused marked differences in intracellular replication. These findings show that induction of SPI1 under different pre-invasion growth conditions can affect the ability of Salmonella to interact with eukaryotic host cells.
Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for virus infectivity and binds to a cellular receptor, insulin-degrading enzyme (IDE), through its unique amino terminal extracellular ...domain. Previous work has shown IDE plays an important role in VZV infection and virus cell-to-cell spread, which is the sole route for VZV spread in vitro. Here we report that a recombinant soluble IDE (rIDE) enhances VZV infectivity at an early step of infection associated with an increase in virus internalization, and increases cell-to-cell spread. VZV mutants lacking the IDE binding domain of gE were impaired for syncytia formation and membrane fusion. Pre-treatment of cell-free VZV with rIDE markedly enhanced the stability of the virus over a range of conditions. rIDE interacted with gE to elicit a conformational change in gE and rendered it more susceptible to proteolysis. Co-incubation of rIDE with gE modified the size of gE. We propose that the conformational change in gE elicited by IDE enhances infectivity and stability of the virus and leads to increased fusogenicity during VZV infection. The ability of rIDE to enhance infectivity of cell-free VZV over a wide range of incubation times and temperatures suggests that rIDE may be useful for increasing the stability of varicella or zoster vaccines.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background Mastocytosis associated with germline KIT activating mutations is exceedingly rare. We report the unique clinicopathologic features of a patient with systemic mastocytosis caused by a de ...novo germline KIT K509I mutation. Objectives We sought to investigate the effect of the germline KIT K509I mutation on human mast cell development and function. Methods Primary human mast cells derived from CD34+ peripheral blood progenitors were examined for growth, development, survival, and IgE-mediated activation. In addition, a mast cell transduction system that stably expressed the KIT K509I mutation was established. Results KIT K509I biopsied mast cells were round, CD25− , and well differentiated. KIT K509I progenitors cultured in stem cell factor (SCF) demonstrated a 10-fold expansion compared with progenitors from healthy subjects and developed into mature hypergranular mast cells with enhanced antigen-mediated degranulation. KIT K509I progenitors cultured in the absence of SCF survived but lacked expansion and developed into hypogranular mast cells. A KIT K509I mast cell transduction system revealed SCF-independent survival to be reliant on the preferential splicing of KIT at the adjacent exonic junction. Conclusion Germline KIT mutations associated with mastocytosis drive a well-differentiated mast cell phenotype distinct to that of somatic KIT D816V disease, the oncogenic potential of which might be influenced by SCF and selective KIT splicing.
Spirochetes are bacteria characterized in part by rotating periplasmic flagella that impart their helical or flat-wave morphology and motility. While most other bacteria rely on a transcriptional ...cascade to regulate the expression of motility genes, spirochetes employ post-transcriptional mechanism(s) that are only partially known. In the present study, we characterize a spontaneous non-motile mutant of the relapsing fever spirochete Borrelia hermsii that was straight, non-motile and deficient in periplasmic flagella. We used next generation DNA sequencing of the mutant's genome, which when compared to the wild-type genome identified a 142 bp deletion in the chromosomal gene encoding the flagellar export apparatus protein FliH. Immunoblot and transcription analyses showed that the mutant phenotype was linked to the posttranscriptional deficiency in the synthesis of the major periplasmic flagellar filament core protein FlaB. Despite the lack of FlaB, the amount of FlaA produced by the fliH mutant was similar to the wild-type level. The turnover of the residual pool of FlaB produced by the fliH mutant was comparable to the wild-type spirochete. The non-motile mutant was not infectious in mice and its inoculation did not induce an antibody response. Trans-complementation of the mutant with an intact fliH gene restored the synthesis of FlaB, a normal morphology, motility and infectivity in mice. Therefore, we propose that the flagellar export apparatus protein regulates motility of B. hermsii at the post-transcriptional level by influencing the synthesis of FlaB.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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