RNAi screen of endoplasmic reticulum-associated host factors reveals a role for IRE1alpha in supporting Brucella replication.
RNAi screen of endoplasmic reticulum-associated host factors reveals a role for IRE1alpha in supporting Brucella replication.
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DOI：
10.1371/journal.ppat.1000110
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发表时间：
2008-07-25
期刊：
影响因子：
6.7
通讯作者：
de Figueiredo P
中科院分区：
文献类型：
--
作者：
Qin QM;Pei J;Ancona V;Shaw BD;Ficht TA;de Figueiredo P
布鲁氏菌是引起布鲁氏菌病的兼性细胞内细菌病原体，布鲁氏菌病是一种具有深远意义的全球性人畜共患病。虽然最近的研究表明，布鲁氏菌属。在含有内质网(ER)驻留蛋白的细胞内复制，病原体确保这种复制生态位的分子机制仍然不清楚。在这里，我们通过利用果蝇S2细胞和RNA干扰(RNAi)技术来解决这个问题，以开发一种遗传上易于处理的系统，概括哺乳动物细胞感染的关键方面。在通过证明在两个宿主细胞系统中都需要肌醇磷脂3-激酶(PI3K)活性来支持布鲁氏菌感染来验证这一系统后，我们进行了240个基因的RNAi筛选，其中包括110个ER相关基因，以寻找介导细菌与ER相互作用的分子。我们发现了52个进化上保守的宿主因子，当耗尽时，它们抑制或增加布鲁氏菌感染。值得注意的是，这些因素中有29个以前没有被认为支持宿主细胞的细菌感染。其中最耐人寻味的是肌醇需求酶1(IRE1)，这是一种调节真核生物未折叠蛋白反应(UPR)的跨膜激酶。我们利用IRE1α−/−小鼠胚胎成纤维细胞(MEF)来证明该蛋白在支持布鲁氏菌感染哺乳动物细胞中的作用，从而验证了果蝇S2细胞系统在发现新的布鲁氏菌宿主因子方面的有效性。最后，我们提出了一个模型，在该模型中，IRE1α和在我们的筛选中发现的其他ER相关基因通过促进自噬小体的生物发生来介导布鲁氏菌的复制。布鲁氏菌属是兼性的细胞内病原体，在包括人类在内的广泛宿主中引起布鲁氏菌病。羊布鲁氏菌、流产布鲁氏菌和猪布鲁氏菌具有很高的传染性，很容易以雾化形式传播，目前还没有针对布鲁氏菌病的人类疫苗。因此，这些病原体被认为是潜在的生物恐怖因子。由于目前还没有研究宿主-布鲁氏菌相互作用的遗传系统，因此人们对介导感染的宿主因素知之甚少。在这里，我们证明了一个果蝇S2细胞系统和RNA干扰可以用来研究进化上保守的布鲁氏菌宿主蛋白在这些过程中所起的作用。我们还表明，这个系统提供了宿主因子的鉴定和表征，这些宿主因子介导了布鲁氏菌与宿主细胞内质网的相互作用。事实上，我们确定了52个宿主因素，当耗尽时，抑制或增加布鲁氏菌感染。在已确定的布鲁氏菌宿主因素中，有29个以前没有被证明支持细菌感染。最后，我们证明了新的宿主因子肌醇需要酶1(IRE1)及其哺乳动物同源基因(IRE1α)分别是布鲁氏菌感染果蝇S2和哺乳动物细胞所必需的。因此，这项工作有助于我们了解宿主因素对布鲁氏菌感染的影响。
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. 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.
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影响因子：
8.6
作者：
de Bagues, MPJ;Dudal, S;Gross, A
通讯作者：
Gross, A
影响因子：
3.4
作者：
Cheng, LW;Portnoy, DA
通讯作者：
Portnoy, DA
DOI：
10.1073/pnas.0506461102
发表时间：
2005-09-20
影响因子：
11.1
作者：
Cheng, LW;Viala, JPM;Portnoy, DA
通讯作者：
Portnoy, DA
影响因子：
3.1
作者：
Kahl-McDonagh, M. M.;Ficht, T. A.
通讯作者：
Ficht, T. A.
DOI：
10.1084/jem.20030088
发表时间：
2003-08-18
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Celli J;de Chastellier C;Franchini DM;Pizarro-Cerda J;Moreno E;Gorvel JP
通讯作者：
Gorvel JP