Identification of Coxiella burnetii surface-exposed and cell envelope associated proteins using a combined bioinformatics plus proteomics strategy
Identification of Coxiella burnetii surface-exposed and cell envelope associated proteins using a combined bioinformatics plus proteomics strategy
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DOI：
10.1002/pmic.201300338
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发表时间：
2014-08-01
期刊：
影响因子：
3.4
通讯作者：
Skultety, Ludovit
中科院分区：
文献类型：
--
作者：
Flores-Ramirez, Gabriela;Jankovicova, Barbora;Skultety, Ludovit
革兰氏阴性病原体贝氏柯克斯体是一种在真核细胞的吞噬溶酶体空泡内复制的细胞内细菌。这种病原体可以感染广泛的宿主，是人类Q热的病原体。表面暴露和细胞包膜相关蛋白被认为是重要的发病机制和保护性免疫。在此，我们提出了一种补充策略，包括（i）计算机模拟预测和（ii）使用三种富集方法结合蛋白质鉴定来清点蛋白质组组成。经典的Triton X-114相分区的效率进行了比较，两个新的程序，分离碱性蛋白质的液相IEF，和细胞表面酶剃须使用生物功能磁珠。在使用七种不同的生物信息学算法分析的2026个蛋白质序列中，预测有157个是外膜蛋白（OMP）和/或脂蛋白（LP）。使用这三种富集方案，我们确定了196个非冗余蛋白，包括39个预测的OMP和/或LP，32个未知或特征不佳的蛋白质，和17个IV型分泌系统的效应物。我们还确定了八个蛋白与兼职活动，和几个蛋白显然与周边的整体或锚定OMP和/或LP。
The Gram-negative pathogen Coxiella burnetii is an intracellular bacterium that replicates within the phagolysosomal vacuoles of eukaryotic cells. This pathogen can infect a wide range of hosts, and is the causative agent of Q fever in humans. Surface-exposed and cell envelope associated proteins are thought to be important for both pathogenesis and protective immunity. Herein, we propose a complementary strategy consisting of (i) in silico prediction and (ii) inventory of the proteomic composition using three enrichment approaches coupled with protein identification. The efficiency of classical Triton X-114 phase partitioning was compared with two novel procedures; isolation of alkaline proteins by liquid-phase IEF, and cell surface enzymatic shaving using biofunctional magnetic beads. Of the 2026 protein sequences analyzed using seven distinct bioinformatic algorithms, 157 were predicted to be outer membrane proteins (OMP) and/or lipoproteins (LP). Using the three enrichment protocols, we identified 196 nonredundant proteins, including 39 predicted OMP and/or LP, 32 unknown or poorly characterized proteins, and 17 effectors of the Type IV secretion system. We additionally identified eight proteins with moonlighting activities, and several proteins apparently peripherally associated with integral or anchored OMP and/or LP.