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

Abstract

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.

Combining FAIMS based glycoproteomics and DIA proteomics reveals widespread proteome alterations in response to glycosylation occupancy changes in Neisseria gonorrhoeae

“…Interestingly while previous studies have explored the impact of the abolishment of glycosylation, few studies have assessed how changes in glycosylation occupancy or glycosylation patterns impact bacterial proteomes. Recently it was demonstrated that within Burkholderia species the silencing of glycosylation using CRISPRi reduced occupancy but resulted in only modest proteomic impacts [84] yet in contrast our findings here support the N. gonorrhoeae proteome is extensively impacted by changes in glycosylation patterns. While O‐ linked glycosylation within B. cenocepacia has been shown to be important for the stability of multiple proteins [83, 85], we observed no change in the protein abundance of glycoproteins within this work suggesting that within N. gonorrhoeae O‐ linked glycosylation may exert its effects by modulating protein interactions or protein activities.…”

Glycan-Tailored Glycoproteomic Analysis Reveals Serine is the Sole Residue Subjected to O-Linked Glycosylation in Acinetobacter baumannii

“…By reducing the triggering of ETD/EThcD events to only potential glycopeptide precursors optimal ETD reaction times and/or extended ion filling times can be undertaken. However, while this approach has been widely utilized for the study of bacterial glycopeptides ,, we recently noted two examples within Moraxella osloensis and N. gonorrheae , wherein alterations within EThcD triggering was essential to allow site localization due to the lack of HexNAc residues within glycopeptides derived from these species. The diversity within the glycan structures utilized for O -linked glycosylation by A. baumannii strains supports the potential benefit of alternative ETD-triggering approaches to further improve glycoproteomic analysis of A. baumannii strains.…”