chrishad

• Hadjineophytou, Chris; Loh, Edmund; Koomey, Michael & Scott, Nichollas E. (2024). Combining FAIMS based glycoproteomics and DIA proteomics reveals widespread proteome alterations in response to glycosylation occupancy changes in Neisseria gonorrhoeae. Proteomics. ISSN 1615-9853. 24(14). doi: 10.1002/pmic.202300496. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Hadjineophytou, Chris; Anonsen, Jan Haug; Svingerud, Tina; Mortimer, Tatum D.; Grad, Yonatan H. & Nichollas, Sscott E. [Vis alle 7 forfattere av denne artikkelen] (2022). Sculpting the Bacterial O-Glycoproteome: Functional Analyses of Orthologous Oligosaccharyltransferases with Diverse Targeting Specificities. mBio. ISSN 2161-2129. 13(3). doi: 10.1128/mbio.03797-21. Fulltekst i vitenarkiv Vis sammendrag

  Protein glycosylation systems are widely recognized in bacteria, including members of the genus Neisseria. In most bacterial species, the molecular mechanisms and evolutionary contexts underpinning target protein selection and the glycan repertoire remain poorly understood. Broad-spectrum O-linked protein glycosylation occurs in all human-associated species groups within the genus Neisseria, but knowledge of their individual glycoprotein repertoires is limited. Interestingly, PilE, the pilin subunit of the type IV pilus (Tfp) colonization factor, is glycosylated in Neisseria gonorrhoeae and Neisseria meningitidis but not in the deeply branching species N. elongata subsp. glycolytica. To examine this in more detail, we assessed PilE glycosylation status across the genus and found that PilEs of commensal clade species are not modified by the gonococcal PglO oligosaccharyltransferase. Experiments using PglO oligosaccharyltransferases from across the genus expressed in N. gonorrhoeae showed that although all were capable of broad-spectrum protein glycosylation, those from a deep-branching group of commensals were unable to support resident PilE glycosylation. Further glycoproteomic analyses of these strains using immunoblotting and mass spectrometry revealed other proteins differentially targeted by otherwise remarkably similar oligosaccharyltransferases. Finally, we generated pglO allelic chimeras that begin to localize PglO protein domains associated with unique substrate targeting activities. These findings reveal previously unappreciated differences within the protein glycosylation systems of highly related bacterial species. We propose that the natural diversity manifest in the neisserial protein substrates and oligosaccharyltransferases has significant potential to inform the structure-function relationships operating in these and related bacterial protein glycosylation systems.

• Wang, Nelson; Anonsen, Jan Haug; Hadjineophytou, Chris; Reinar, William Brynildsen; Børud, Bente & Vik, Åshild [Vis alle 7 forfattere av denne artikkelen] (2020). Allelic polymorphisms in a glycosyltransferase gene shape glycan repertoire in the O-linked protein glycosylation system of Neisseria. Glycobiology. ISSN 0959-6658. doi: 10.1093/glycob/cwaa073. Fulltekst i vitenarkiv Vis sammendrag

  Glycosylation of multiple proteins via O-linkage is well documented in bacterial species of Neisseria of import to human disease. Recent studies of protein glycosylation (pgl) gene distribution established that related protein glycosylation systems occur throughout the genus including nonpathogenic species. However, there are inconsistencies between pgl gene status and observed glycan structures. One of these relates to the widespread distribution of pglG, encoding a glycosyltransferase that in Neisseria elongata subsp. glycolytica is responsible for the addition of di-N-acetyl glucuronic acid at the third position of a tetrasaccharide. Despite pglG residing in strains of N. gonorrhoeae, N. meningitidis and N. lactamica, no glycan structures have been correlated with its presence in these backgrounds. Moreover, PglG function in N. elongata subsp. glycolytica minimally requires UDP-glucuronic acid (GlcNAcA), and yet N. gonorrhoeae, N. meningitidis and N. lactamica lack pglJ, the gene whose product is essential for UDP-GlcNAcA synthesis. We examined the functionality of pglG alleles from species spanning the Neisseria genus by genetic complementation in N. elongata subsp. glycolytica. The results indicate that select pglG alleles from N. meningitidis and N. lactamica are associated with incorporation of an N-acetyl-hexosamine at the third position and reveal the potential for an expanded glycan repertoire in those species. Similar experiments using pglG from N. gonorrhoeae failed to find any evidence of function suggesting that those alleles are missense pseudogenes. Taken together, the results are emblematic of how allelic polymorphisms can shape bacterial glycosyltransferase function and demonstrate that such alterations may be constrained to distinct phylogenetic lineages.

• Hadjineophytou, Chris; Anonsen, Jan Haug; Wang, Nelson; Ma, Kevin; Viburiene, Raimonda & Vik, Åshild [Vis alle 10 forfattere av denne artikkelen] (2019). Genetic determinants of genus-level glycan diversity in a bacterial protein glycosylation system. PLoS Genetics. ISSN 1553-7390. 15(12), s. 1–24. doi: 10.1371/journal.pgen.1008532. Fulltekst i vitenarkiv Vis sammendrag

  The human pathogens N. gonorrhoeae and N. meningitidis display robust intra- and interstrain glycan diversity associated with their O-linked protein glycosylation (pgl) systems. In an effort to better understand the evolution and function of protein glycosylation operating there, we aimed to determine if other human-restricted, Neisseria species similarly glycosylate proteins and if so, to assess the levels of glycoform diversity. Comparative genomics revealed the conservation of a subset of genes minimally required for O-linked protein glycosylation glycan and established those pgl genes as core genome constituents of the genus. In conjunction with mass spectrometric–based glycan phenotyping, we found that extant glycoform repertoires in N. gonorrhoeae, N. meningitidis and the closely related species N. polysaccharea and N. lactamica reflect the functional replacement of a progenitor glycan biosynthetic pathway. This replacement involved loss of pgl gene components of the primordial pathway coincident with the acquisition of two exogenous glycosyltransferase genes. Critical to this discovery was the identification of a ubiquitous but previously unrecognized glycosyltransferase gene (pglP) that has uniquely undergone parallel but independent pseudogenization in N. gonorrhoeae and N. meningitidis. We suggest that the pseudogenization events are driven by processes of compositional epistasis leading to gene decay. Additionally, we documented instances where inter-species recombination influences pgl gene status and creates discordant genetic interactions due ostensibly to the multi-locus nature of pgl gene networks. In summary, these findings provide a novel perspective on the evolution of protein glycosylation systems and identify phylogenetically informative, genetic differences associated with Neisseria species.

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• Hadjineophytou, Chris; Anonsen, Jan Haug & Koomey, Michael (2019). Genus-wide assessment of pilus glycosylation in the genus Neisseria. Fulltekst i vitenarkiv
• Hadjineophytou, Chris; Anonsen, Jan Haug & Koomey, Michael (2018). A genus-wide assessment of neisserial Tfp pilin subunit glycosylation: a systems level approach reveals distribution and governing co-factors. Fulltekst i vitenarkiv
• Hadjineophytou, Chris & Koomey, Michael (2018). A genus-wide assessment of neisserial Tfp pilin subunit glycosylation: a systems level approach reveals distribution and governing co-factors. Fulltekst i vitenarkiv

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