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RNAi screen of endoplasmic reticulum-associated host factors reveals a role for IRE1alpha in supporting Brucella replication

التفاصيل البيبلوغرافية
العنوان:	RNAi screen of endoplasmic reticulum-associated host factors reveals a role for IRE1alpha in supporting Brucella replication
المؤلفون:	Paul de Figueiredo, Qing-Ming Qin, Veronica Ancona, Thomas A. Ficht, Brian D. Shaw, Jianwu Pei
المصدر:	PLoS Pathogens PLoS Pathogens, Vol 4, Iss 7, p e1000110 (2008)
سنة النشر:	2008
مصطلحات موضوعية:	lcsh:Immunologic diseases. Allergy, Autophagosome, Transcription, Genetic, Immunology, Brucella abortus, Brucella, Protein Serine-Threonine Kinases, Endoplasmic Reticulum, Microbiology, Infectious Diseases/Bacterial Infections, 03 medical and health sciences, Mice, Phosphatidylinositol 3-Kinases, Intracellular Calcium-Sensing Proteins, RNA interference, Virology, Endoribonucleases, Genetics, Animals, Humans, Genetic Testing, RNA, Small Interfering, lcsh:QH301-705.5, Molecular Biology, Gene, 030304 developmental biology, 0303 health sciences, Microbiology/Microbial Growth and Development, biology, 030306 microbiology, Schneider 2 cells, Endoplasmic reticulum, Intracellular parasite, Macrophages, Microbiology/Medical Microbiology, Cell Biology, biology.organism_classification, Cell biology, lcsh:Biology (General), Host-Pathogen Interactions, Unfolded protein response, Parasitology, RNA Interference, lcsh:RC581-607, Microbiology/Cellular Microbiology and Pathogenesis, HeLa Cells, Research Article
الوصف:	Brucella species are facultative intracellular bacterial pathogens that cause brucellosis, a global zoonosis of profound importance. Although recent studies have demonstrated that Brucella spp. replicate within an intracellular compartment that contains endoplasmic reticulum (ER) resident proteins, the molecular mechanisms by which the pathogen secures this replicative niche remain obscure. Here, we address this issue by exploiting Drosophila S2 cells and RNA interference (RNAi) technology to develop a genetically tractable system that recapitulates critical aspects of mammalian cell infection. After validating this system by demonstrating a shared requirement for phosphoinositide 3-kinase (PI3K) activities in supporting Brucella infection in both host cell systems, we performed an RNAi screen of 240 genes, including 110 ER-associated genes, for molecules that mediate bacterial interactions with the ER. We uncovered 52 evolutionarily conserved host factors that, when depleted, inhibited or increased Brucella infection. Strikingly, 29 of these factors had not been previously suggested to support bacterial infection of host cells. The most intriguing of these was inositol-requiring enzyme 1 (IRE1), a transmembrane kinase that regulates the eukaryotic unfolded protein response (UPR). We employed IRE1α−/− murine embryonic fibroblasts (MEFs) to demonstrate a role for this protein in supporting Brucella infection of mammalian cells, and thereby, validated the utility of the Drosophila S2 cell system for uncovering novel Brucella host factors. Finally, we propose a model in which IRE1α, and other ER-associated genes uncovered in our screen, mediate Brucella replication by promoting autophagosome biogenesis. Author Summary Brucella spp. are facultative intracellular pathogens that cause brucellosis in a broad range of hosts, including humans. Brucella melitensis, B. abortus, and B. suis are highly infectious and can be readily transmitted in aerosolized form, and a human vaccine against brucellosis is unavailable. Therefore, these pathogens are recognized as potential bioterror agents. Because genetic systems for studying host–Brucella interactions have been unavailable, little is known about the host factors that mediate infection. Here, we demonstrate that a Drosophila S2 cell system and RNA interference can be exploited to study the role that evolutionarily conserved Brucella host proteins play in these processes. We also show that this system provides for the identification and characterization of host factors that mediate Brucella interactions with the host cell endoplasmic reticulum. In fact, we identified 52 host factors that, when depleted, inhibited or increased Brucella infection. Among the identified Brucella host factors, 29 have not been previously shown to support bacterial infection. Finally, we demonstrate that the novel host factor inositol-requiring enzyme 1 (IRE1) and its mammalian ortholog (IRE1α) are required for Brucella infection of Drosophila S2 and mammalian cells, respectively. Therefore, this work contributes to our understanding of host factors mediating Brucella infection.
تدمد:	1553-7374
الوصول الحر:	https://explore.openaire.eu/search/publication?articleId=doi_dedup___::fed76de362edbe6b9245cdcac3d57d69Test https://pubmed.ncbi.nlm.nih.gov/18654626Test
حقوق:	OPEN
رقم الانضمام:	edsair.doi.dedup.....fed76de362edbe6b9245cdcac3d57d69
قاعدة البيانات:	OpenAIRE

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Although recent studies have demonstrated that Brucella spp. replicate within an intracellular compartment that contains endoplasmic reticulum (ER) resident proteins, the molecular mechanisms by which the pathogen secures this replicative niche remain obscure. Here, we address this issue by exploiting Drosophila S2 cells and RNA interference (RNAi) technology to develop a genetically tractable system that recapitulates critical aspects of mammalian cell infection. After validating this system by demonstrating a shared requirement for phosphoinositide 3-kinase (PI3K) activities in supporting Brucella infection in both host cell systems, we performed an RNAi screen of 240 genes, including 110 ER-associated genes, for molecules that mediate bacterial interactions with the ER. We uncovered 52 evolutionarily conserved host factors that, when depleted, inhibited or increased Brucella infection. Strikingly, 29 of these factors had not been previously suggested to support bacterial infection of host cells. The most intriguing of these was inositol-requiring enzyme 1 (IRE1), a transmembrane kinase that regulates the eukaryotic unfolded protein response (UPR). We employed IRE1α−/− murine embryonic fibroblasts (MEFs) to demonstrate a role for this protein in supporting Brucella infection of mammalian cells, and thereby, validated the utility of the Drosophila S2 cell system for uncovering novel Brucella host factors. Finally, we propose a model in which IRE1α, and other ER-associated genes uncovered in our screen, mediate Brucella replication by promoting autophagosome biogenesis.<br />Author Summary Brucella spp. are facultative intracellular pathogens that cause brucellosis in a broad range of hosts, including humans. Brucella melitensis, B. abortus, and B. suis are highly infectious and can be readily transmitted in aerosolized form, and a human vaccine against brucellosis is unavailable. Therefore, these pathogens are recognized as potential bioterror agents. Because genetic systems for studying host–Brucella interactions have been unavailable, little is known about the host factors that mediate infection. Here, we demonstrate that a Drosophila S2 cell system and RNA interference can be exploited to study the role that evolutionarily conserved Brucella host proteins play in these processes. We also show that this system provides for the identification and characterization of host factors that mediate Brucella interactions with the host cell endoplasmic reticulum. In fact, we identified 52 host factors that, when depleted, inhibited or increased Brucella infection. Among the identified Brucella host factors, 29 have not been previously shown to support bacterial infection. Finally, we demonstrate that the novel host factor inositol-requiring enzyme 1 (IRE1) and its mammalian ortholog (IRE1α) are required for Brucella infection of Drosophila S2 and mammalian cells, respectively. 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