About the Project
Lipoprotein precursors are translocated from the cytoplasm across the inner membrane to the periplasm, as directed by their N-terminal signal peptide. Here the precursor protein is sequentially processed i.e., the signal peptide is cleaved and the new N terminus formed is lipidated. The mature lipoprotein is typically translocated to the outer membrane and some are further surface displayed.
We recently published an extraordinary finding that the majority of invasive meningococcal isolates circulating in the UK contain one or two non-synonymous mutations in their signal peptide of FHbp which results in their failure to be processed. Surprisingly some of the precursor was still translocated to the outer membrane but is unlikely to be presented in the same manner as mature FHbp which is anchored to the outer membrane via its lipid moiety. The presentation of lipoproteins by clinical isolates is important for recognition by antibodies in individuals vaccinated by lipoprotein-based vaccines.
The signal peptide of individual lipoproteins varies in amino acid sequence between strains and it is important to determine which amino acids are essential for signal peptides to function and allow lipoprotein precursor proteins to become mature lipoproteins. With this knowledge, more accurate lipoprotein prediction programmes can be deployed, vaccine formulations based on lipoproteins improved to incorporate both precursor and lipidated forms where needed and new lipoprotein vaccines designed more intuitively.
Deployment of multiple diverse lipoprotein prediction tools suggest the meningococcus encodes 70 lipoproteins. This project sets out to experimentally confirm each one by cloning the corresponding genes, over-expressing them in N. meningitidis and performing Western immunoblots to evaluate their size; precursor size or processed size. Confocal microscopy will then be conducted to determine which lipoproteins are surface exposed. By comparing the amino acid sequence of each signal peptide of experimentally confirmed lipoproteins with those shown to remain as precursors, important correlations can be established allowing us to understand which residues and properties of signal peptides are important. This PhD will open up other avenues of interest for the PhD student to explore and in so doing, develop independence.
The PhD student will work in a CL2 laboratory and will be supervised by a strong team of 3 academics, all with extensive experience working with N. meningitidis. The student will be trained in multiple techniques.
da Silva RAG, Churchward CP, Karlyshev AV, Eleftheriadou O, Snabaitis AK, Longman MR, Ryan A, Griffin R. The role of apolipoprotein N-acyl transferase, Lnt, in the lipidation of factor H binding protein of Neisseria meningitidis strain MC58 and its potential as a drug target. Br J Pharmacol. 2017;174(14):2247-60.
da Silva RAG, Karlyshev AV, Oldfield NJ, Wooldridge KG, Bayliss CD, Ryan A, Griffin R. Variant Signal Peptides of Vaccine Antigen, FHbp, Impair Processing Affecting Surface Localization and Antibody-Mediated Killing in Most Meningococcal Isolates. Front Microbiol. 2019 Dec 19;10:2847. doi: 10.3389/fmicb.2019.02847.
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