Hypomorphic Glycosyltransferase Alleles and Recoding at Contingency Loci Influence Glycan Microheterogeneity in the Protein Glycosylation System of Neisseria Species

Abstract

As more bacterial protein glycosylation systems are identified and characterized, a central question that arises is, what governs the prevalence of particular glycans associated with them? In addition, accumulating evidence shows that bacterial protein glycans can be subject to the phenomenon of microheterogeneity, in which variant glycan structures are found at specific attachment sites of a given glycoprotein. Although factors underlying microheterogeneity in reconstituted expression systems have been identified and modeled, those impacting natural systems largely remain enigmatic. On the basis of a sensitive and specific glycan serotyping system, microheterogeneity has been reported for the broad-spectrum, O-linked protein glycosylation system in species within the genus Neisseria. To elucidate the mechanisms involved, a genetic approach was used to identify a hypomorphic allele of pglA (encoding the PglA galactosyltransferase) as a significant contributor to simultaneous expression of multiple glycoforms. Moreover, this phenotype was mapped to a single amino acid polymorphism in PglA. Further analyses revealed that many pglA phase-off variants (containing out-of-frame configurations in simple nucleotide repeats within the open reading frame) were associated with disproportionally high levels of the N,N=-diacetylbacillosamine-Gal disaccharide glycoform generated by PglA. This phenotype is emblematic of nonstandard decoding involving programmed ribosomal frameshifting and/or programmed transcriptional realignment. Together, these findings provide new information regarding the mechanisms of neisserial protein glycan microheterogeneity and the anticipatory nature of contingency loci. P rotein glycosylation systems based on both N-and O-linked modifications are well documented in eubacteria. In addition to the distinctions based on the nature of site attachment, bacterial protein glycosylation systems can be differentiated on the basis of whether they are dedicated to a single class of proteins or target a broad spectrum of protein substrates. These systems can also be classified as to the degree of glycan diversity observed within and between related species. For example, in the type IV pilus (Tfp) O-linked glycosylation in Pseudomonas aeruginosa, some strains utilize the endogenous O-antigen unit found in lipopolysaccharide (LPS; which in itself varies between strains), while others employ D-arabinofuranose moieties synthesized by an apparently dedicated biosynthetic machinery (11, 28). In the N-linked, broad-spectrum protein glycosylation system of Campylobacter jejuni, a conserved heptasaccharide has been presumed to be highly disseminated. Major disparities in protein glycan diversification have been observed for flagellum-associated, O-linked systems, with those in Campylobacter, Helicobacter, and Clostridium exhibiting high levels of intra-and interstrain variation, while the system in P. aeruginosa appears to be limited to two major glycoforms (4,10,13,26). Why protein-associated glycans display high intr...

The Sweet Tooth of Bacteria: Common Themes in Bacterial Glycoconjugates

“…As this would result in a metabolic conflict with PglA, a conserved deletion inactivates PglH in most strains. This results in reduced glycan diversity, as the action of PglH results in unique disaccharide products modified with glucose residues instead of galactose (480,481). Taken together, PglD, -C, and -B synthesize the UDP-sugars, and PglB, -A, -E, and -H form the core locus of glycan assembly, while the PglO OST transfers the finished glycan en bloc to the substrate (476,480) (Fig.…”

“…The N. gonorrhoeae enzyme can be constitutively expressed or shows phase variability in some cases; the meningococcal enzyme, in contrast, is always phase variable (473). As mentioned above, N. meningitidis strains can also express PglH, a GT for which the activity results in unique glycans but which is inactive in most strains (480,481).…”

“…For example, PglE and -F are present in all strains studied, in contrast to PglG, -H, and -B2, which are not present in C311 but are expressed in a large number of clinical isolates. Moreover, PglG, -H, and -B2 are potentially phase variable (481,492,495). Together with the extreme promiscuity of the PglL OST, this results in an enormous glycan repertoire, which confers an evolutionary advantage to N. meningitidis (66,496).…”

Characterization of a Unique Tetrasaccharide and Distinct Glycoproteome in the O -Linked Protein Glycosylation System of Neisseria elongata subsp. glycolytica

“…Thus, individual strains can express anywhere between 1 and 7 glycoforms ranging in size from mono-to trisaccharides, and within meningococcal strains, 18 distinct protein-targeted glycans have been defined (9). Furthermore, relatively high levels of microheterogeneity have been observed, which result from the expression of hypomorphic alleles of glycosyltransferase genes, thus altering the relative abundance of specific glycoforms (10,15).…”

The glycan alphabet is not universal: a hypothesis

“…The importance of glycans, especially in prokaryotes, is well documented. Establishing the specific role of glycans and studying structure-function relationship is largely hindered by factors such as non-availability of high-throughput sequencing methods, inadequate information as to which genes are involved in, non-template driven biosynthesis, phase variation (16) and microheterogeneity (17). In this study, completely sequenced prokaryotic genomes were searched for monosaccharide biosynthesis enzymes using sequence homology-based approach.…”