Genetic determinants of genus-level glycan diversity in a bacterial protein glycosylation system

Abstract

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 interspecies 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.Bacteria express a remarkable diversity of sugars and oligosaccharides in conjunction with protein glycosylation systems. Currently however, little is known about the evolutionary processes and selective forces shaping glycan biosynthetic pathways. The closely related bacterial pathogens Neisseria gonorrhoeae and Neisseria meningitidis remain serious sources of human disease and these species express antigenically variable oligosaccharides as components of their broad-spectrum, O-linked protein glycosylation (pgl) systems. With the exception of isolates of Neisseria elongata subspecies glycolytica, the status of such post-translational modifications in related commensal species colonizing humans remains largely undefined. Here, we exploit new data from further studies of protein glycosylation in Neisseria elongata subspecies glycolytica to address these concerns. Employing comparative genomics and glycan phenotyping, we show that related pgl systems are indeed expressed by all human-restricted Neisseria species but identify unique gene gain and loss events as well as loss-of-function polymorphisms that accommodate a dramatic shift in glycoform structure occurring across the genus. These findings constitute ...

Open Database Searching Enables the Identification and Comparison of Bacterial Glycoproteomes without Defining Glycan Compositions Prior to Searching

“…Recently, we reported that a single-loci was responsible for the generation of the O-linked glycans in B. Cenocepacia and that this loci is conserved across the Burkholderia (23). Although these results support that Burkholderia species use similar glycans, it has been noted that within other bacterial genera extensive glycan heterogeneity exists (25,26,29,32). As glycan heterogeneity can be challenging to predict, we reasoned that open searching would provide a means to assess the similarities in glycans used across Burkholderia species.…”

What Are We Missing by Using Hydrophilic Enrichment? Improving Bacterial Glycoproteome Coverage Using Total Proteome and FAIMS Analyses

“…Over the last decade, glycopeptide-based enrichment approaches have increasingly been used to track and characterise microbial glycosylation events [42,44,64,76]. Although enrichment strategies enable high-throughput analysis of glycosylation, we demonstrate here that hydrophilic enrichment provides only partial access to some microbial glycoproteomes.…”

Open database searching enables the identification and comparison of bacterial glycoproteomes without defining glycan compositions prior to searching

“…Recently, we reported that a single-loci was responsible for the generation of the O -linked glycans in B. Cenocepacia and that this loci is conserved across the Burkholderia [23]. Although these results support that Burkholderia species utilise similar glycans, it has been noted that within other bacterial genera extensive glycan heterogeneity exists [25, 26, 29, 32]. As glycan heterogeneity can be challenging to predict, we reasoned that open searching would provide a means to assess the similarities in glycans used across Burkholderia species.…”