Glycoproteomic and Proteomic analysis of Burkholderia cenocepacia reveals glycosylation events within FliF and MotB are dispensable for motility.-DDA delta BCAM0505

Within the Burkholderia genus O-linked protein glycosylation is now known to be highly conserved at the pathway and glycosylation substrate levels. While inhibition of glycosylation has been shown to be detrimental to virulence in B. cenocepacia, little is known about the role of glycosylation in Burkholderia glycoproteins. Within this study we have sought to improve our understanding of the breadth and dynamics of the B. cenocepacia O-glycoproteome to identify glycoproteins which require glycosylation for functionality. Assessing the glycoproteome across multiple common culturing media (LB, TSB, and artificial sputum medium to simulate cystic fibrosis sputum-like conditions) we demonstrate at least 141 glycoproteins are subjected to glycosylation within B. cenocepacia K56-2. Leveraging this insight, we quantitively assessed the glycoproteome of B. cenocepacia using Data-Independent Acquisition (DIA) across culturing media and growth phases revealing most B. cenocepacia glycoproteins are express under all conditions. Examination of how the absence of glycosylation impacts the glycoproteome reveals only a subset of the glycoproteome (BCAL1086, BCAL2974, BCAL0525, BCAM0505 and BCAL0127) appear impacted by the loss of glycosylation. Assessing the proteomic and phenotypic impacts of the loss of these glycoproteins compared to glycosylation null strains revealing the loss of BCAL0525, and to a lesser extend BCAL0127, mirror the proteomic effects observed in the absence of glycosylation. Finally, we demonstrate the loss of glycosylation within BCAL0525 at Serine-358 results in both loss of motility and proteomic impacts on flagellar apparatus consistent with the loss of apparatus stability. Combined this work demonstrates that O-linked glycosylation of BCAL0525 is functionally important within B. cenocepacia.

目前已知，伯克霍尔德菌属（Burkholderia）内的O-连接蛋白质糖基化（O-linked protein glycosylation）在通路与糖基化底物层面均呈现高度保守性。尽管已有研究证实糖基化抑制会对洋葱伯克霍尔德菌（B. cenocepacia）的毒力产生不利影响，但目前对于糖基化在伯克霍尔德菌糖蛋白中的功能仍知之甚少。本研究旨在加深我们对洋葱伯克霍尔德菌O-糖蛋白组（O-glycoproteome）的覆盖范围与动态变化的认知，以鉴定出那些依赖糖基化才能发挥功能的糖蛋白。通过在三种常见培养培养基（LB、TSB以及模拟囊性纤维化痰液环境的人工痰液培养基）中对糖蛋白组进行分析，我们证实洋葱伯克霍尔德菌K56-2中至少有141种糖蛋白发生了糖基化修饰。基于上述发现，我们采用数据非依赖性采集（Data-Independent Acquisition, DIA）技术，对不同培养培养基与生长阶段下的洋葱伯克霍尔德菌糖蛋白组进行定量分析，结果显示绝大多数洋葱伯克霍尔德菌糖蛋白在所有测试条件下均有表达。通过分析糖基化缺失对糖蛋白组的影响，我们发现仅有部分糖蛋白（BCAL1086、BCAL2974、BCAL0525、BCAM0505及BCAL0127）的表达会因糖基化缺失而受到影响。通过对比糖基化缺失菌株与上述糖蛋白缺失菌株的蛋白质组与表型变化，我们发现BCAL0525的缺失（以及程度较轻的BCAL0127缺失）能够重现糖基化整体缺失所带来的蛋白质组学效应。最后，我们证实BCAL0525在丝氨酸358位点（Serine-358）的糖基化缺失，会导致菌株运动能力丧失，并对鞭毛结构产生蛋白质组层面的影响，这与鞭毛结构稳定性丧失的表型一致。综上，本研究证实BCAL0525的O-连接糖基化在洋葱伯克霍尔德菌中具有重要的功能意义。