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

Abstract

Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within the Neisseria genus O‐linked protein glycosylation is conserved yet closely related Neisseria species express O‐oligosaccharyltransferases (PglOs) with distinct targeting activities. Within this work, we explore the targeting capacity of different PglOs using Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) fractionation and Data‐Independent Acquisition (DIA) to allow the characterization of the impact of changes in glycosylation on the proteome of Neisseria gonorrhoeae. We demonstrate FAIMS expands the known glycoproteome of wild type N. gonorrhoeae MS11 and enables differences in glycosylation to be assessed across strains expressing different pglO allelic chimeras with unique substrate targeting activities. Combining glycoproteomic insights with DIA proteomics, we demonstrate that alterations within pglO alleles have widespread impacts on the proteome of N. gonorrhoeae. Examination of peptides known to be targeted by glycosylation using DIA analysis supports alterations in glycosylation occupancy occurs independently of changes in protein levels and that the occupancy of glycosylation is generally low on most glycoproteins. This work thus expands our understanding of the N. gonorrhoeae glycoproteome and the roles that pglO allelic variation may play in governing genus‐level protein glycosylation.

Sweet complexity: O-linked protein glycosylation in pathogenic Neisseria

“…PilE glycosylation has also been shown to impact on type IV pilin functions such as autoagglutination, the efficiency of pilin subunit polymerization, and the dynamics of pilus extension - retraction ( Vik et al., 2012 ; Gault et al., 2015 ). In addition to PilE, several additional type IV biogenesis or related proteins (PilQ, PilN, PilH, PilI, PilJ, PilV, ComP) are glycosylated ( Hadjineophytou et al., 2022 , 2024 ) ( Table 1 ). In contrast, the more distant commensal neisserial species have O -OTases that supported glycosylation of other proteins, but not pilin ( Hadjineophytou et al., 2022 ).…”

“…Altogether, over 50 glycoproteins have been identified in N. gonorrhoeae ( Vik et al., 2009 ; Anonsen et al., 2012b ; Hadjineophytou et al., 2024 ). The glycoproteins identified are mainly lipoproteins or transmembrane – domain containing proteins localized extracytoplasmically (as summarized in Table 1 ).…”

“…It is therefore important to understand the molecular and phenotypic mechanisms involved. The recent identification of additional glycoproteins revealed that several of those are involved in antimicrobial resistance mechanisms ( Table 1 ) ( Hadjineophytou et al., 2024 ). The detected glycoproteins are involved in efflux (MtrC, MtrD, MacA, MacB, FarA) and influx (PorB) of antimicrobials.…”

“…By examining known glycopeptides in N. gonorrhoeae MS11 replicates using Data-Independent Acquisition (DIA) analysis, different glycosylation occupancy frequencies were found without affecting the protein abundance ( Hadjineophytou et al., 2024 ). This study also discovered that the glycan occupancy on glycoproteins was often low.…”

“…Studies of protein glycosylation have revealed that Neisseria species encompass numerous glycoproteins and display high intra- and interstrain glycoform variability ( Ku et al., 2009 ; Vik et al., 2009 ; Børud et al., 2010 , 2011 ; Anonsen et al., 2012b ; Børud et al., 2014 ; Anonsen et al., 2017 ; Hadjineophytou et al., 2024 ). To date, it has been shown that one strain has the capacity to express between 7 and 15 different glycoforms by differing combinations of glycosyltransferases and the glycan O -acetylation ( Anonsen et al., 2017 ; Wang et al., 2021 ).…”