CRISPRi-Mediated Silencing of Burkholderia O‑Linked Glycosylation Systems Enables the Depletion of Glycosylation Yet Results in Modest Proteome Impacts

The process of O-linked
protein glycosylation is highly conserved
across the Burkholderia genus and mediated
by the oligosaccharyltransferase PglL. While our understanding of Burkholderia glycoproteomes has increased in recent
years, little is known about how Burkholderia species respond to modulations in glycosylation. Utilizing CRISPR
interference (CRISPRi), we explored the impact of silencing of O-linked
glycosylation across four species of Burkholderia; Burkholderia cenocepacia K56-2, Burkholderia diffusa MSMB375, Burkholderia
multivorans ATCC17616, and Burkholderia
thailandensis E264. Proteomic and glycoproteomic analyses
revealed that while CRISPRi enabled inducible silencing of PglL, this
did not abolish glycosylation, nor recapitulate phenotypes such as
proteome changes or alterations in motility that are associated with
glycosylation null strains, despite inhibition of glycosylation by
nearly 90%. Importantly, this work also demonstrated that CRISPRi
induction with high levels of rhamnose leads to extensive impacts
on the Burkholderia proteomes, which
without appropriate controls mask the impacts specifically driven
by CRISPRi guides. Combined, this work revealed that while CRISPRi
allows the modulation of O-linked glycosylation with reductions up
to 90% at a phenotypic and proteome levels, Burkholderia appears to demonstrate a robust tolerance to fluctuations in glycosylation
capacity.

O-连接蛋白质糖基化（O-linked protein glycosylation）过程在伯克霍尔德菌属（Burkholderia）中高度保守，并由寡糖转移酶PglL（oligosaccharyltransferase PglL）介导。尽管近年来我们对伯克霍尔德菌糖蛋白质组的认知有所提升，但对于伯克霍尔德菌物种如何响应糖基化调控的机制仍知之甚少。本研究利用CRISPR干扰（CRISPR interference，CRISPRi）技术，探究了沉默O-连接糖基化对四种伯克霍尔德菌物种的影响：即唐菖蒲伯克霍尔德菌K56-2（Burkholderia cenocepacia K56-2）、弥漫伯克霍尔德菌MSMB375（Burkholderia diffusa MSMB375）、多噬伯克霍尔德菌ATCC17616（Burkholderia multivorans ATCC17616）以及泰国伯克霍尔德菌E264（Burkholderia thailandensis E264）。蛋白质组学与糖蛋白质组学分析结果显示，尽管CRISPRi可诱导沉默PglL基因，但即便糖基化抑制率接近90%，该技术既未能完全消除糖基化，也未能复现糖基化缺失菌株所具有的蛋白质组变化、运动能力改变等相关表型。值得注意的是，本研究同时证实，高浓度鼠李糖（rhamnose）诱导CRISPRi会对伯克霍尔德菌的蛋白质组产生广泛影响，若未设置恰当的对照，这类影响会掩盖CRISPRi向导RNA所特异性介导的调控效应。综上，本研究表明，尽管CRISPRi可实现O-连接糖基化的调控，使糖基化水平最高降低90%，并体现在表型与蛋白质组层面，但伯克霍尔德菌似乎对糖基化能力的波动表现出极强的耐受性。