Recent progress: In the past two years, we have published seven papers demonstrating the power of our approaches to make significant advances. These include those documenting a role for intrinsic genetic interactions in shaping glycan evolution in protein glycosylation systems (B?rud et al, PNAS 2011), a new perspective on the functional correlates of bacterial protein glycosylation and glycoform diversification (Vik et al, Mol Microbiol 2012) and a novel approach to defining glycoproteomes (Anonsen et al, J Proteome Res, in press).
1) Glycosylation in pathogenic and commensal Neisseria strains
Supervisor: Bente B?rud
There is a high degree of polymorphism at the level of pgl gene content as some pgl genes are restricted to certain species. The ancient pgl locus is composed of pglF, pglG, pglH, pglB/B2, pglC and pglD. Pgl loci including pglG and pglH exists within N. meningitidis, N. gonorrhoeae and commensal strains, while pglB2 is found among all neisserial species except N. gonorrhoeae. On the other hand pglA and pglE are absent in most commensal species except N. lactamica. Several of the pgl genes such as pglI, pglA, pglE, pglG and pglH, have polynucleotide stretches that may undergo phase variation so that the bacteria can easily change their glycans.
More details are needed to understand a possible connection between pgl genotype/phenotype relationships in host-pathogenic versus host-commensal interactions. This master project will include bioinformatics to compare pgl gene content between pathogenic and commensal strains and it also aims at identifying glycosylation in available commensal strains.
1B) Recoding and phase variation in the pglA glycosyltransferase gene
Supervisor: Bente B?rud
We are currently investigating the molecular mechanisms behind glycan diversity in Neisseria. One of the pgl genes, the gene encoding the glycosyltransferase PglA, was recently shown to give rise to varying levels of glycosylation even though the different alleles were in phase off configuration. Further analyses revealed that many pglA phase-off variants were associated with unexpected high levels of the disaccharide glycoform generated by PglA. The relatively high level of glycosylation observed and preliminary colony lift assays suggest that this is not due to a small population that turns on the pglA gene, instead we hypothesize that this phenotype is due to nonstandard decoding involving programmed ribosomal frameshifting and/or programmed transcriptional realignment.
This master project aims at elucidating the molecular mechanisms behind recoding in the polyG tract within the pglA gene. This will be done by introducing different numbers of nucleotides in the polyG tract as well as doing mutagenesis on flanking sequences and then quantify the level of glycosylation. Also other pgl genes have poly nucleotide tracts and the study can be extended to include one or several of these.
Methods
The master student will make use of standard methods in microbiology and molecular biology such as cloning, subcloning, PCR, site-directed mutagenesis, transformasjon of E. coli and N. gonorrhoeae, Western blotting as well as preparing samples for mass spectrometric analysis.
The master project involves making recombinant plasmids that will be used to generate mutant strains of Neisseria where glycan status will be thoroughly investigated. In order to assess the level of glycan diversity in these systems as well as to elucidate its genetic basis, we have developed a simple system in which distinct pgl genes can be introduced into defined mutants of the N. gonorrhoeae strain N400. We use glycan-specific antibodies and Western blotting as well as mass spectrometric (MS) analyses of glycoproteins from N. gonorrhoeae mutants to define glycan status.
2) Identification of a methyltransferase responsible for phosphocholine modification of glycoproteins in N. gonorrhoeae.
Supervisors: Marina Aspholm and ?shild Vik
The Koomey lab has shown that the phospho-form molecules phosphoethanolamine (PE) and phosphocholine (PC) can appear as post-translational modifications (PTMs) directly linked to proteins (Hegge et al, PNAS 2004). So far all neisserial phospho-form target proteins have turned out to be glycosylated, and we have observed an intriguing interplay between these modifications (Anonsen et al, Infect. Immun. 2012). Yet, the functional relevance of these modifications remains to be determined. It is clear, however, that the enzymatic machinery necessary for the phospho-form modifications is restricted to the pathogenic species of Neisseria and are missing from all known non-pathogenic species. Recently, similar discoveries have been made in other bacterial systems where a clear link to virulence has been demonstrated (Mukherjee et al, Nature 2011; Cullen and Trent, PNAS 2010). Given the emerging interest in this field coupled with our experience and access to cutting edge methodologies for detection of PTMs we believe we are in a unique position to contribute to a broader understanding of the impact these modifications may have on neisserial virulence. In this context we are looking for a master student to identify a putative methyltransferase involved in generation of the phosphocholine modification. We have solid biochemical evidence that this activity exists and will use a candidate gene approach to try to identify the gene. Further work will involve characterization of the enzyme and its functional relevance (particularly in relation to its connection with glycosylation).
The practical part of the projects will be supervised by experienced researchers and postdocs who have driven the projects. During this project you will gain work experience in a wide variety of practical techniques, including recombinant DNA technology and mutagenesis, PCR, protein expression and purification, protein biochemistry, enzymatic assays, Western blotting, mammalian cell culture, and cell biology experiments.