Development of a Proximity Labeling System to Map the Chlamydia trachomatis Inclusion Membrane.
Development of a Proximity Labeling System to Map the Chlamydia trachomatis Inclusion Membrane.
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DOI：
10.3389/fcimb.2017.00040
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发表时间：
2017
影响因子：
5.7
通讯作者：
Ouellette SP
中科院分区：
文献类型：
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作者：
Rucks EA;Olson MG;Jorgenson LM;Srinivasan RR;Ouellette SP
衣原体生长在一个被称为包涵体的膜结合的空泡内。支持这种病原体特异性寄生细胞器的生物发生和完整性的细胞过程尚不清楚。衣原体分泌称为Inc的整合膜蛋白，插入衣原体包涵体膜（IM）。Inc含有至少两个两侧为末端的疏水跨膜结构域，其大小不同并暴露于宿主胞质溶胶。此外，Inc在衣原体发育周期中暂时表达。检查Inc功能的数据有限，因为（i）难以使用疏水蛋白和（ii）IM固有的脆弱性。我们假设Incs的功能协作，以保持完整的衣原体列入小Incs组织IM和较大的Incs与宿主细胞机械接口。为了研究这一假设，我们已经适应了接近标记策略，使用APEX 2，突变的大豆抗坏血酸过氧化物酶，生物素化的相互作用和近端蛋白质在几分钟内在过氧化氢及其外源性底物，生物素苯酚的存在下。我们成功地表达，从诱导型背景，APEX 2单独，或融合蛋白的IncATM（TM =跨膜结构域只），印加，和IncF与APEX 2沙眼衣原体血清型L2。IncF-APEX 2、IncATM-APEX 2和IncA-APEX 2定位于IM，而缺乏分泌信号的APEX 2仍然与细菌相关。我们确定了过表达对包涵体直径、质粒稳定性和高尔基体源性鞘磷脂获得的影响。虽然存在诱导构建体表达的总体影响，但IncF-APEX 2过表达对这些测量产生最大的负面影响。重要的是，在生物素-苯酚存在下的Inc-APEX 2表达导致IM的生物素化。这些数据表明，Inc表达受到调节以控制最佳IM生物发生。我们随后定义了溶解已知Inc并且与下拉条件相容的裂解条件。重要的是，我们已经创建了强大的工具，可以直接检查IM的动态组成，这将为促进衣原体生长和发育的关键相互作用提供新的见解。
Chlamydia grows within a membrane-bound vacuole termed an inclusion. The cellular processes that support the biogenesis and integrity of this pathogen-specified parasitic organelle are not understood. Chlamydia secretes integral membrane proteins called Incs that insert into the chlamydial inclusion membrane (IM). Incs contain at least two hydrophobic transmembrane domains flanked by termini, which vary in size and are exposed to the host cytosol. In addition, Incs are temporally expressed during the chlamydial developmental cycle. Data examining Inc function are limited because of (i) the difficulty in working with hydrophobic proteins and (ii) the inherent fragility of the IM. We hypothesize that Incs function collaboratively to maintain the integrity of the chlamydial inclusion with small Incs organizing the IM and larger Incs interfacing with host cell machinery. To study this hypothesis, we have adapted a proximity-labeling strategy using APEX2, a mutant soybean ascorbate peroxidase that biotinylates interacting and proximal proteins within minutes in the presence of H2O2 and its exogenous substrate, biotin-phenol. We successfully expressed, from an inducible background, APEX2 alone, or fusion proteins of IncATM (TM = transmembrane domain only), IncA, and IncF with APEX2 in Chlamydia trachomatis serovar L2. IncF-APEX2, IncATM-APEX2, and IncA-APEX2 localized to the IM whereas APEX2, lacking a secretion signal, remained associated with the bacteria. We determined the impact of overexpression on inclusion diameter, plasmid stability, and Golgi-derived sphingomyelin acquisition. While there was an overall impact of inducing construct expression, IncF-APEX2 overexpression most negatively impacted these measurements. Importantly, Inc-APEX2 expression in the presence of biotin-phenol resulted in biotinylation of the IM. These data suggest that Inc expression is regulated to control optimal IM biogenesis. We subsequently defined lysis conditions that solubilized known Incs and were compatible with pulldown conditions. Importantly, we have created powerful tools to allow direct examination of the dynamic composition of the IM, which will provide novel insights into key interactions that promote chlamydial growth and development within the inclusion.
影响因子：
--
作者：
Moore, Elizabeth R
通讯作者：
Moore, Elizabeth R