Abstract:
Across the Burkholderia genus O-linked protein glycosylation is highly conserved. While the inhibition of glycosylation has been shown to be detrimental for virulence in Burkholderia cepacia complex species, such as Burkholderia cenocepacia, little is known about how specific glycosylation sites impact protein functionality. Within this study, we sought to improve our understanding of the breadth, dynamics, and requirement for glycosylation across the B. cenocepacia O-glycoproteome. Assessing the B. cenocepacia glycoproteome across different culture media using complementary glycoproteomic approaches, we increase the known glycoproteome to 141 glycoproteins. Leveraging this repertoire of glycoproteins, we quantitively assessed the glycoproteome of B. cenocepacia using Data-Independent Acquisition (DIA) revealing the B. cenocepacia glycoproteome is largely stable across conditions with most glycoproteins constitutively expressed. Examination of how the absence of glycosylation impacts the glycoproteome reveals that the protein abundance of only five glycoproteins (BCAL1086, BCAL2974, BCAL0525, BCAM0505, and BCAL0127) are altered by the loss of glycosylation. Assessing ΔfliF (ΔBCAL0525), ΔmotB (ΔBCAL0127), and ΔBCAM0505 strains, we demonstrate the loss of FliF, and to a lesser extent MotB, mirror the proteomic effects observed in the absence of glycosylation in ΔpglL. While both MotB and FliF are essential for motility, we find loss of glycosylation sites in MotB or FliF does not impact motility supporting these sites are dispensable for function. Combined this work broadens our understanding of the B. cenocepacia glycoproteome supporting that the loss of glycoproteins in the absence of glycosylation is not an indicator of the requirement for glycosylation for protein function.
OBJECTIVE: Burkholderia cenocepacia is an opportunistic pathogen of concern within the Cystic Fibrosis community. Despite a greater appreciation of the unique physiology of B. cenocepacia gained over the last 20 years a complete understanding of the proteome and especially the O-glycoproteome, is lacking. In this study, we utilize systems biology approaches to expand the known B. cenocepacia glycoproteome as well as track the dynamics of glycoproteins across growth phases, culturing media and in response to the loss of glycosylation. We show that the glycoproteome of B. cenocepacia is largely stable across conditions and that the loss of glycosylation only impacts five glycoproteins including the motility associated proteins FliF and MotB. Examination of MotB and FliF shows, while these proteins are essential for motility, glycosylation is dispensable. Combined this work supports that B. cenocepacia glycosylation can be dispensable for protein function and may influence protein properties beyond stability.
摘要:
整个伯克霍尔德菌属的O-连接蛋白糖基化是高度保守的。虽然糖基化的抑制已被证明对洋葱伯克霍尔德氏菌复合物种的毒力有害,比如伯克霍尔德氏菌,关于特定的糖基化位点如何影响蛋白质功能知之甚少。在这项研究中,我们试图提高我们对广度的理解,动力学,以及对穿过隐血芽孢杆菌O-糖蛋白组的糖基化的要求。使用互补的糖蛋白质组学方法评估跨不同培养基的白头蛇糖蛋白质组,我们将已知的糖蛋白质组增加到141个糖蛋白。利用这些糖蛋白,我们使用数据独立采集(DIA)定量评估了白头蛇的糖蛋白质组,揭示了在大多数糖蛋白组成型表达的条件下,白头蛇的糖蛋白质组在很大程度上是稳定的.对不存在糖基化如何影响糖蛋白质组的研究表明,只有五种糖蛋白(BCAL1086,BCAL2974,BCAL0525,BCAM0505和BCAL0127)的蛋白质丰度因糖基化丧失而改变。评估ΔfliF(ΔBCAL0525),ΔmotB(ΔBCAL0127),和ΔBCAM0505菌株,我们证明了FliF的损失,在较小程度上,反映了在ΔpglL中不存在糖基化时观察到的蛋白质组效应。虽然MotB和FliF对于运动都是必不可少的,我们发现MotB或FliF中糖基化位点的丢失不会影响运动性,支持这些位点对于功能是不必要的。结合这项工作拓宽了我们对隐血芽孢杆菌糖蛋白组的理解,支持在不存在糖基化的情况下糖蛋白的损失不是蛋白质功能需要糖基化的指标。
目的:伯克霍尔德氏菌是囊性纤维化社区中值得关注的机会性病原体。尽管在过去的20年中,人们对B.cenocepacia的独特生理学有了更多的了解,但对蛋白质组尤其是O-糖蛋白组的全面了解,缺乏。在这项研究中,我们利用系统生物学方法来扩展已知的白头蛇芽孢杆菌糖蛋白组,并跟踪跨生长阶段的糖蛋白的动力学,培养基和响应糖基化的丧失。我们表明,白头蛇的糖蛋白质组在各种条件下基本上是稳定的,并且糖基化的丧失仅影响五种糖蛋白,包括运动性相关蛋白FliF和MotB。检查MotB和FliF显示,虽然这些蛋白质对运动至关重要,糖基化是可有可无的。结合这项工作,可以支持B.cenepacia糖基化对于蛋白质功能是可有可无的,并且可能会影响蛋白质的稳定性。