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Low‐oxygen tensions found in Salmonella‐infected gut tissue boost Salmonella replication in macrophages by impairing antimicrobial activity and augmenting Salmonella virulence
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Boas Felmy Kathrin Gendera Valentin Schatz Monika Nowottny Gregor Liebsch Michael Hensel Wolf‐Dietrich Hardt Roman G. Gerlach Jonathan Jantsch 《Cellular microbiology》2015,17(12):1833-1847
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Beatriz Mesa‐Pereira Carlos Medina Eva María Camacho Amando Flores Eduardo Santero 《Microbial biotechnology》2015,8(1):169-176
In order to increase the cytotoxic activity of a Salmonella strain carrying a salicylate‐inducible expression system that controls cytosine deaminase production, we have modified both, the vector and the producer bacterium. First, the translation rates of the expression module containing the Escherichia coli codA gene cloned under the control of the Pm promoter have been improved by using the T7 phage gene 10 ribosome binding site sequence and replacing the original GUG start codon by AUG. Second, to increase the time span in which cytosine deaminase may be produced by the bacteria in the presence of 5‐fluorocytosine, a 5‐fluorouracyl resistant Salmonella strain has been constructed by deleting its upp gene sequence. This new Salmonella strain shows increased cytosine deaminase activity and, after infecting tumour cell cultures, increased cytotoxic and bystander effects under standard induction conditions. In addition, we have generated a purD mutation in the producer strain to control its intracellular proliferation by the presence of adenine and avoid the intrinsic Salmonella cell death induction. This strategy allows the analysis and comparison of the cytotoxic effects of cytosine deaminase produced by different Salmonella strains in tumour cell cultures. 相似文献
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PERP,a host tetraspanning membrane protein,is required for Salmonella‐induced inflammation
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Kelly N. Hallstrom C. V. Srikanth Terence A. Agbor Christopher M. Dumont Kristen N. Peters Luminita Paraoan James E. Casanova Erik J. Boll Beth A. McCormick 《Cellular microbiology》2015,17(6):843-859
Salmonella enterica Typhimurium induces intestinal inflammation through the activity of type III secreted effector (T3SE) proteins. Our prior results indicate that the secretion of the T3SE SipA and the ability of SipA to induce epithelial cell responses that lead to induction of polymorphonuclear transepithelial migration are not coupled to its direct delivery into epithelial cells from Salmonella. We therefore tested the hypothesis that SipA interacts with a membrane protein located at the apical surface of intestinal epithelial cells. Employing a split ubiquitin yeast‐two‐hybrid screen, we identified the tetraspanning membrane protein, p53 effector related to PMP‐22 (PERP), as a SipA binding partner. SipA and PERP appear to have intersecting activities as we found PERP to be involved in proinflammatory pathways shown to be regulated by SipA. In sum, our studies reveal a critical role for PERP in the pathogenesis of S. Typhimurium, and for the first time demonstrate that SipA, a T3SE protein, can engage a host protein at the epithelial surface. 相似文献
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Salmonella enterica is a facultative intracellular pathogen residing in a unique host cell‐derived membrane compartment, termed Salmonella‐containing vacuole or SCV. By the activity of effector proteins translocated by the SPI2‐endoced type III secretion system (T3SS), the biogenesis of the SCV is manipulated to generate a habitat permissive for intracellular proliferation. By taking control of the host cell vesicle fusion machinery, intracellular Salmonella creates an extensive interconnected system of tubular membranes arising from vesicles of various origins, collectively termed Salmonella‐induced tubules (SIT). Recent work investigated the dynamic properties of these manipulations. New host cell targets of SPI2‐T3SS effector proteins were identified. By applying combinations of live cell imaging and ultrastructural analyses, the detailed organization of membrane compartments inhabited and modified by intracellular Salmonella is now available. These studies provided unexpected new details on the intracellular environments of Salmonella. For example, one kind of SIT, the LAMP1‐positive Salmonella‐induced filaments (SIF), are composed of double‐membrane tubules, with an inner lumen containing host cell cytosol and cytoskeletal filaments, and an outer lumen containing endocytosed cargo. The novel findings call for new models for the biogenesis of SCV and SIT and give raise to many open questions we discuss in this review. 相似文献
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Bistable flagellar and virulence gene expression generates specialized Salmonella subpopulations with distinct functions. Repressing flagellar genes allows Salmonella to evade caspase‐1 mediated host defenses and enhances systemic colonization. By definition, bistability arises when intermediate states of gene expression are rendered unstable by the underlying genetic circuitry. We demonstrate sustained bistable fliC expression in virulent Salmonella 14028 and document dynamic control of the distribution, or single‐cell census, of flagellar gene expression by the mutually repressing repressors YdiV and FliZ. YdiV partitions cells into the fliC‐OFF subpopulation, while FliZ partitions cells into the fliC‐HIGH subpopulation at late time points during growth. Bistability of ΔfliZ populations and ydiV‐independent FliZ control of flagellar gene expression provide evidence that the YdiV‐FliZ mutually repressing repressor circuit is not required for bistability. Repression and activation by YdiV and FliZ (respectively) can shape the census of fliC expression independently, and bistability collapses into a predominantly intermediate population in the absence of both regulators. Metered expression of YdiV and FliZ reveals variable sensitivity to these regulators and defines conditions where expression of FliZ enhances fliC expression and where FliZ does not alter the fliC census. Thus, this evolved genetic circuitry coordinates multiple layers of regulatory heterogeneity into a binary response. 相似文献
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Terence A. Agbor Zachary Demma Randall J. Mrsny Antonio Castillo Erik J. Boll Beth A. McCormick 《Cellular microbiology》2014,16(9):1339-1353
Neutrophil (polymorphonuclear leucocytes; PMN) transmigration across mucosal surfaces contributes to dysfunction of epithelial barrier properties, a characteristic underlying many mucosal inflammatory diseases. Using Salmonella enterica serovar Typhimurium (S. Typhimurium) as a prototypic proinflammatory insult, we have previously reported that the eicosanoid hepoxilin A3 (HXA3), an endogenous product of 12‐lipoxygenase (12‐LOX) activity, is secreted from the apical surface of the intestinal epithelium to establish a chemotactic gradient that guides PMN across the epithelial surface. Since little is known regarding the molecular mechanisms that regulate 12‐LOX during S. Typhimurium infection, we investigated this pathway. We found that expression of phospholipid glutathione peroxidase (GPX4), which is known to have an inhibitory effect on 12‐LOX activity, is significantly decreased at both the mRNA and protein level during infection with S. Typhimurium. Moreover, employing intestinal epithelial cell monolayers expressing siRNA against GPX4 mRNA, S. Typhimurium‐induced PMN migration was significantly increased compared with the non‐specific siRNA control cells. Conversely, in cells engineered to overexpress GPX4, S. Typhimurium‐induced PMN migration was significantly decreased, which is consistent with the finding that partial depletion of GPX4 by RNAi resulted in a significant increase in HXA3 secretion during S. Typhimurium infection. Mechanistically, although we found Salmonella entry not to be required for the induced decrease in GPX4, the secreted effector, SipA, which is known to induce epithelial responses leading to stimulation of HXA3, governed the decrease in GPX4 in a process that does not lead to an overall increase in the levels of ROS. Taken together, these results suggest that S. Typhimurium induces apical secretion of HXA3 by decreasing the expression of phospholipid GPX, which in turn leads to an increase in 12‐LOX activity, and hence HXA3 synthesis. 相似文献
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Multicopy integration of mini‐Tn7 transposons into selected chromosomal sites of a Salmonella vaccine strain
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Chromosomal integration of expression modules for transgenes is an important aspect for the development of novel Salmonella vectors. Mini‐Tn7 transposons have been used for the insertion of one such module into the chromosomal site attTn7, present only once in most Gram‐negative bacteria. However, integration of multiple mini‐Tn7 copies might be suitable for expression of appropriate amounts of antigen or combination of different modules. Here we demonstrate that integration of a 9.6 kb mini‐Tn7 harbouring the luciferase luxCDABE (lux) occurs at the natural attTn7 site and simultaneously other locations of the Salmonella chromosome, which were engineered using λ‐Red recombinase to contain one or two additional artificial attTn7 sites (a‐attTn7). Multicopy integration even at closely spaced attTn7 sites was unexpected in light of the previously reported distance‐dependent Tn7 target immunity. Integration of multiple copies of a mini‐Tn7 containing a gfp cassette resulted in increasing green fluorescence of bacteria. Stable consecutive integration of two mini‐Tn7 encoding lacZ and lux was achieved by initial transposition of lacZ‐mini‐Tn7, subsequent chromosomal insertion of a‐attTn7 and a second round of transposition with lux‐mini‐Tn7. Mini‐Tn7 thus constitutes a versatile method for multicopy integration of expression cassettes into the chromosome of Salmonella and possibly other bacteria. 相似文献
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The invasion of polarized epithelial cells by Salmonella enterica requires the cooperative activity of the Salmonella pathogenicity island (SPI) 1‐encoded type III secretion system (T3SS) and the SPI4‐encoded giant non‐fimbrial adhesin SiiE. SiiE is a highly repetitive protein composed of 53 bacterial Ig (BIg) domains and mediates binding to the apical side of polarized epithelial cells. We analysed the binding properties of SiiE and observed lectin‐like activity. SiiE‐dependent cell invasion can be ablated by chemical or enzymatic deglycosylation. Lectin blockade experiments revealed that SiiE binding is specific for glycostructures with terminal N‐acetyl‐glucosamine (GlcNAc) and/or α 2,3‐linked sialic acid. In line with these data, we found that SiiE‐expressing Salmonella bind to the GlcNAc polymer chitin. Various recombinant SiiE fragments were analysed for host cell binding. We observed that C‐terminal portions of SiiE bind to the apical side of polarized cells and the intensity of binding increases with the number of BIg domains present in the recombinant proteins. Based on these results, we propose that SiiE mediates multiple interactions per molecule with glycoproteins and/or glycosylated phospholipids present in the apical membrane of polarized epithelial cells. Thisintimate binding enables the subsequent function of the SPI1‐T3SS, resulting in host cell invasion. 相似文献
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Electrostatic potentials of the S‐locus F‐box proteins contribute to the pollen S specificity in self‐incompatibility in Petunia hybrida
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Junhui Li Yue Zhang Yanzhai Song Hui Zhang Jiangbo Fan Qun Li Dongfen Zhang Yongbiao Xue 《The Plant journal : for cell and molecular biology》2017,89(1):45-57
Self‐incompatibility (SI) is a self/non‐self discrimination system found widely in angiosperms and, in many species, is controlled by a single polymorphic S‐locus. In the Solanaceae, Rosaceae and Plantaginaceae, the S‐locus encodes a single S‐RNase and a cluster of S‐locus F‐box (SLF) proteins to control the pistil and pollen expression of SI, respectively. Previous studies have shown that their cytosolic interactions determine their recognition specificity, but the physical force between their interactions remains unclear. In this study, we show that the electrostatic potentials of SLF contribute to the pollen S specificity through a physical mechanism of ‘like charges repel and unlike charges attract’ between SLFs and S‐RNases in Petunia hybrida. Strikingly, the alteration of a single C‐terminal amino acid of SLF reversed its surface electrostatic potentials and subsequently the pollen S specificity. Collectively, our results reveal that the electrostatic potentials act as a major physical force between cytosolic SLFs and S‐RNases, providing a mechanistic insight into the self/non‐self discrimination between cytosolic proteins in angiosperms. 相似文献
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The COPII complex and lysosomal VAMP7 determine intracellular Salmonella localization and growth
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José Carlos Santos Magalie Duchateau Jennifer Fredlund Allon Weiner Adeline Mallet Christine Schmitt Mariette Matondo Véronique Hourdel Julia Chamot‐Rooke Jost Enninga 《Cellular microbiology》2015,17(12):1699-1720
Salmonella invades epithelial cells and survives within a membrane‐bound compartment, the Salmonella‐containing vacuole (SCV). We isolated and determined the host protein composition of the SCV at 30 min and 3 h of infection to identify and characterize novel regulators of intracellular bacterial localization and growth. Quantitation of the SCV protein content revealed 392 host proteins specifically enriched at SCVs, out of which 173 associated exclusively with early SCVs, 124 with maturing SCV and 95 proteins during both time‐points. Vacuole interactions with endoplasmic reticulum‐derived coat protein complex II vesicles modulate early steps of SCV maturation, promoting SCV rupture and bacterial hyper‐replication within the host cytosol. On the other hand, SCV interactions with VAMP7‐positive lysosome‐like vesicles promote Salmonella‐induced filament formation and bacterial growth within the late SCV. Our results reveal that the dynamic communication between the SCV and distinct host organelles affects both intracellular Salmonella localization and growth at successive steps of host cell invasion. 相似文献
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MYCN is a novel oncogenic target in adult B‐ALL that activates the Wnt/β‐catenin pathway by suppressing DKK3
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Desheng Kong Linlin Zhao Lili Sun Shengjin Fan Huibo Li Yanqiu Zhao Zhibo Guo Leilei Lin Lin Cui Ke Wang Wenjia Chen Yihui Zhang Jin Zhou Yinghua Li 《Journal of cellular and molecular medicine》2018,22(7):3627-3637