Table_1_Membrane Proteocomplexome of Campylobacter jejuni Using 2-D Blue Native/SDS-PAGE Combined to Bioinformatics Analysis.docx

Campylobacter is the leading cause of the human bacterial foodborne infections in the developed countries. The perception cues from biotic or abiotic environments by the bacteria are often related to bacterial surface and membrane proteins that mediate the cellular response for the adaptation of Campylobacter jejuni to the environment. These proteins function rarely as a unique entity, they are often organized in functional complexes. In C. jejuni, these complexes are not fully identified and some of them remain unknown. To identify putative functional multi-subunit entities at the membrane subproteome level of C. jejuni, a holistic non a priori method was addressed using two-dimensional blue native/Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in strain C. jejuni 81–176. Couples of acrylamide gradient/migration-time, membrane detergent concentration and hand-made strips were optimized to obtain reproducible extraction and separation of intact membrane protein complexes (MPCs). The MPCs were subsequently denatured using SDS-PAGE and each spot from each MPCs was identified by mass spectrometry. Altogether, 21 MPCs could be detected including multi homo-oligomeric and multi hetero-oligomeric complexes distributed in both inner and outer membranes. The function, the conservation and the regulation of the MPCs across C. jejuni strains were inspected by functional and genomic comparison analyses. In this study, relatedness between subunits of two efflux pumps, CmeABC and MacABputC was observed. In addition, a consensus sequence CosR-binding box in promoter regions of MacABputC was present in C. jejuni but not in Campylobacter coli. The MPCs identified in C. jejuni 81–176 membrane are involved in protein folding, molecule trafficking, oxidative phosphorylation, membrane structuration, peptidoglycan biosynthesis, motility and chemotaxis, stress signaling, efflux pumps and virulence.

弯曲杆菌（Campylobacter）是发达国家引发人类细菌性食源性感染的首要致病菌。该类细菌感知生物或非生物环境的信号线索，往往与介导空肠弯曲杆菌（Campylobacter jejuni）环境适应细胞应答的细菌表面及膜蛋白密切相关。这类蛋白极少以单一独立实体行使功能，通常会组装为功能性复合物。在空肠弯曲杆菌中，这类复合物尚未被完全鉴定，其中部分仍未被发现。为了在空肠弯曲杆菌的膜亚蛋白质组（membrane subproteome）层面鉴定推定的功能性多亚基实体，本研究以空肠弯曲杆菌81–176菌株为研究对象，采用基于二维蓝色天然/十二烷基硫酸钠（Sodium dodecyl sulfate, SDS）聚丙烯酰胺凝胶电泳（polyacrylamide gel electrophoresis, PAGE）的非预设性整体研究策略。研究优化了丙烯酰胺梯度/迁移时间、膜蛋白去垢剂浓度以及手工制备胶条的参数，以实现完整膜蛋白复合物（membrane protein complexes, MPCs）的可重复提取与分离。随后通过SDS-PAGE对膜蛋白复合物进行变性处理，并利用质谱技术对每个膜蛋白复合物对应的各个斑点进行鉴定。总计可检测到21种膜蛋白复合物，包括分布于内膜与外膜的多同源寡聚体与多异源寡聚体复合物。通过功能与基因组比较分析，对空肠弯曲杆菌不同菌株间膜蛋白复合物的功能、保守性及调控机制进行了考察。本研究中，观察到两种外排泵（efflux pumps）CmeABC与MacABputC的亚基之间存在相关性。此外，在空肠弯曲杆菌的MacABputC基因启动子区域存在CosR结合框（CosR-binding box）的共有序列，而在结肠弯曲杆菌（Campylobacter coli）中则未检测到该序列。本研究在空肠弯曲杆菌81–176的膜组分中鉴定得到的膜蛋白复合物，参与了蛋白质折叠、分子转运、氧化磷酸化（oxidative phosphorylation）、膜结构构建、肽聚糖（peptidoglycan）生物合成、运动与趋化、应激信号传导、外排泵系统以及毒力相关生物学过程。