Brucella mechanisms of autophagy mediated egress

描述（由申请方提供）：布鲁氏菌病是一种由布鲁氏菌属引起的全球高度流行的细菌性人畜共患病，在流行地区造成重大的公共卫生负担，并由于布鲁氏菌的高传染性而引起生物防御问题。人类布鲁氏菌病的控制和治疗受到缺乏预防措施和长期抗生素治疗疗效不一的限制，突出了对替代疗法的需求。布鲁氏菌经历细胞内循环，支持其在哺乳动物宿主中的生存、增殖和持久存在，并且是发病所需的。该细菌存在于细胞内的膜结合区室中，即含布鲁氏菌的空泡（BCV），其从初始内体空泡（eBCV）到源自内质网（ER）的复制性细胞器（rBCV）的进化由布鲁氏菌VirB IV型分泌系统控制。VirB装置将调节宿主功能的效应蛋白递送到宿主细胞中，并且是rBCV生物发生和细菌复制所需的。复制后，rBCVs通过与细胞间传播相关的自噬相关过程转化为功能不同的空泡（aBCVs），表明aBCVs介导细菌的外出和传播。尽管细菌外流在传播感染中具有明显的重要性，并且aBCVs在该过程中的作用已经确立，但对布鲁氏菌细胞内循环的这一特定阶段知之甚少。在这里，我们建议填补这一知识空白，并定义aBCV形成的机制和功能要求。具体地，我们将测试以下假设：i）VirB装置控制rBCV向aBCV的转化; ii）aBCV通过分泌性自噬介导细菌排出。首先，我们将利用一个可控的VirB表达系统，绕过VirB在周期中的早期要求，以测试其在aBCV形成中的作用，并定义这个特定阶段的细菌决定因素。其次，我们将使用活细胞成像方法来可视化aBCV形成和细菌排出的动力学，并确定aBCV是否介导细菌释放。最后，我们将通过siRNA介导的分泌性自噬蛋白的消耗来测试是否涉及这种特定的途径来定义aBCV形成和细菌排出的宿主决定因素。因此，拟议的研究将推进我们对布鲁氏菌VirB介导的宿主功能和细菌出口机制的颠覆的分子理解，以及这种病原体在感染过程中如何传播，最终为布鲁氏菌病的治疗干预提供新的靶点。

DESCRIPTION (provided by applicant): Brucellosis is a highly prevalent bacterial zoonosis worldwide caused by Brucella spp., inflicting significant public health burden in endemic areas and causing biodefense concerns due to Brucella high infectivity. Control and treatment of human brucellosis are limited by a lack of prophylactic measures and by the varying efficacy of long-term antibiotics treatments, highlighting the need for alternative therapies. Brucella undergoes an intracellular cycle that supports its survival, proliferation and persistence within mammalian hosts and is required for pathogenesis. The bacterium resides intracellularly within a membrane-bound compartment, the Brucella-containing vacuole (BCV), whose evolution from an initial endosomal vacuole (eBCV) to a replicative organelle (rBCV) derived from the endoplasmic reticulum (ER) is controlled by the Brucella VirB Type IV secretion system. The VirB apparatus delivers effector proteins into host cells that modulate host functions, and is required for rBCV biogenesis and bacterial replication. Following replication, rBCVs are converted into functionally distinct vacuoles (aBCVs) through an autophagy-related process associated with cell-to-cell spread, suggesting that aBCVs mediate bacterial egress and dissemination. Despite the obvious importance of bacterial egress in disseminating infections, and the established role of aBCVs in this process, little is known about this particular stage of the Brucella intracellular cycle. Here we propose to fill this knowledge gap and define the mechanisms and functional requirements of aBCV formation. Specifically, we will test the hypotheses that i) the VirB apparatus controls conversion of rBCV to aBCVs; ii) aBCVs mediate bacterial egress via secretory autophagy. First, we will take advantage of a controllable VirB expression system that circumvents VirB early requirements in the cycle to test its role in aBCV formation and define bacterial determinants of this specific stage. Second, we will use live cell imaging approaches to visualize the dynamics of aBCV formation and bacterial egress and establish whether aBCVs mediate bacterial release. Last, we will test via siRNA-mediated depletion of secretory autophagy proteins whether this particular pathway is involved to define the host determinants of aBCV formation and bacterial egress. The proposed studies will therefore advance our molecular understanding of Brucella VirB-mediated subversion of host functions and bacterial egress mechanisms, and how this pathogen may disseminate during infection, ultimately providing novel targets for therapeutic intervention against brucellosis.