Glycoproteomic and Proteomic analysis of Burkholderia cenocepacia reveals glycosylation events within FliF and MotB are dispensable for motility. - HILIC enriched samples

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呈现高度保守性。尽管已有研究表明，糖基化抑制会对洋葱伯克霍尔德菌（Burkholderia cenocepacia，简称B. cenocepacia）的毒力产生不利影响，但学界对糖基化在伯克霍尔德菌糖蛋白中所发挥的作用仍知之甚少。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，以鉴定出那些功能依赖于糖基化的糖蛋白。本研究针对三种常见培养培养基（LB培养基、胰蛋白胨大豆肉汤（TSB）以及模拟囊性纤维化痰液环境的人工痰液培养基）开展糖蛋白组分析，证实洋葱伯克霍尔德菌K56-2菌株中至少存在141种糖基化修饰的糖蛋白。基于上述发现，我们采用数据非依赖采集（Data-Independent Acquisition，DIA）技术，针对不同培养基与生长阶段的洋葱伯克霍尔德菌开展糖蛋白组定量分析，结果显示绝大多数洋葱伯克霍尔德菌糖蛋白在所有实验条件下均有表达。通过探究糖基化缺失对糖蛋白组的影响，本研究发现仅有少量糖蛋白（BCAL1086、BCAL2974、BCAL0525、BCAM0505与BCAL0127）的修饰水平会因糖基化缺失而发生改变。通过与糖基化缺失菌株对比，评估上述糖蛋白缺失所带来的蛋白质组与表型影响后，我们发现BCAL0525的缺失，以及程度较轻的BCAL0127缺失，所引发的蛋白质组变化与糖基化整体缺失时观察到的效应高度一致。最后，我们证实，BCAL0525在丝氨酸358位点（Serine-358）的糖基化缺失，会同时导致菌株运动能力丧失，并对鞭毛装置（flagellar apparatus）引发蛋白质组层面的影响，这与鞭毛装置稳定性丧失的表型相符。综上，本研究证实，BCAL0525的O-连接糖基化修饰在洋葱伯克霍尔德菌中具有重要的生物学功能。