Abd El-Fatah Rofida M, Mesbah Noha M, Abo-Elmatty Dina M, Aly Khaled A
Department of Biochemistry, Faculty of Pharmacy and Pharmaceutical Industries, Sinai University, El-Arish, North Sinai, Egypt.
Department of Biochemistry, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt.
Toxicon. 2018 May;146:129-137. doi: 10.1016/j.toxicon.2018.02.047. Epub 2018 Mar 2.
The human pathogen Staphylococcus aureus encodes the ESAT6-like Secretion System (ESS). The ESS pathway secretes pathogenic substrates such as EsxA, EsxB, EsxC, EsxD and EssD that mediate staphylococcal establishment in persistent abscess lesions. The biochemical behavior of these substrates is not fully understood. EsxC is species-specific lysine-rich homodimer that lacks recognizable topogenic sequence. Studies have shown that EsxC is required for the secretion of other substrates, thereby revealing its biomedical importance. Here, EsxC self-association was investigated in the presence of several metal ion chelators. Results show that EsxC homodimerization is abolished in the presence of EDTA and EGTA, suggesting a role for calcium in mediating EsxC self-association. Complementation experiments confirm that EsxC homodimerization is calcium-dependent. N- and C-terminal truncations of EsxC were constructed, followed by bacterial two-hybrid screening. Results show that EsxC self-association is mediated by its C-terminal domain. Affinity purification of recombinant EsxC to apparent homogeneity, followed by chemical crosslinking and SDS-PAGE led to the detection of the monomeric and dimeric forms of the protein. In contrast and when a purified EsxC variant lacking the C-terminus was subjected to similar conditions, only the monomeric form was observed. These in vivo and in vitro data highlight the contribution of the C-terminus of the virulence factor EsxC to self-association, and document a previously unreported role for calcium in mediating protein-protein interactions in this pathogenic secretion system.
人类病原体金黄色葡萄球菌编码ESAT6样分泌系统（ESS）。ESS途径分泌致病性底物，如EsxA、EsxB、EsxC、EsxD和EssD，这些底物介导葡萄球菌在持续性脓肿病变中的定植。这些底物的生化行为尚未完全了解。EsxC是一种物种特异性的富含赖氨酸的同型二聚体，缺乏可识别的拓扑序列。研究表明，EsxC是其他底物分泌所必需的，从而揭示了其生物医学重要性。在此，研究了在几种金属离子螯合剂存在下EsxC的自我缔合。结果表明，在EDTA和EGTA存在下，EsxC同型二聚化被消除，表明钙在介导EsxC自我缔合中起作用。互补实验证实EsxC同型二聚化是钙依赖性的。构建了EsxC的N端和C端截短体，随后进行细菌双杂交筛选。结果表明，EsxC的自我缔合由其C端结构域介导。将重组EsxC亲和纯化至表观均一性，随后进行化学交联和SDS-PAGE，检测到该蛋白的单体和二聚体形式。相反，当对缺乏C端的纯化EsxC变体进行类似处理时，仅观察到单体形式。这些体内和体外数据突出了毒力因子EsxC的C端对自我缔合的贡献，并记录了钙在介导该致病分泌系统中蛋白质-蛋白质相互作用方面以前未报道的作用。
