About the Project
Neisseria meningitidis is the major agent responsible for bacterial meningitis but is also a frequent commensal of the human oropharyngeal tissues. Many surface structures of this bacterial pathogen are subject to stochastic, reversible ON and OFF switches in gene expression (termed phase variation, PV) due to mutations in simple sequence repeat tracts. Individual and combinatorial switches in expression of these structures impact on key virulence traits of this organism including adhesion to host tissues, cytotoxicity, iron uptake and survival of immune effectors. Understanding the genetic basis for these virulence traits is critical for improving effective implementation of vaccine strategies and use of therapeutic treatments.
In 2008-2009, Bayliss and colleagues performed a study of persistent carriage of meningococci in University of Nottingham students. A large collection of isolates from each carrier and from multiple carriers was obtained. The PV status of eight outer membrane proteins in multiple isolates from persistent carriers were analysed and shown to exhibit trends towards reduced expression as a function of persistent carriage. Concomitant serum samples from these carriers indicated the presence of a robust antibody response against selected surface antigens indicating that observed trends were due to immune escape. The PV of twelve further genes and whole genome sequences have also been analysed for this collection of isolates making it one of the most highly characterised sets of meningococcal carriage isolates.
In recent work, we have observed that three isogenic isolates from the same carrier differ in their cytotoxic effects on eukaryotic cells and in induction of cytokine responses. Differences in cytotoxicity correlate with PV of PilC proteins that control retraction of the type IV pilus. This study provides strong evidence of a profound impact of phase variation on the virulence traits of meningococci.
The aim of this project is to utilise this well-characterised set of isolates to understand how PV may impact on the disease-causing ability of meningococci.
The specific objectives are:-
1) Examine the cytotoxic and protective effects of multiple meningococcal carriage isolates on eukaryotic cells.
Infection of Calu3 cells (a bronchial epithelial cell line) in trans-well plates will be utilised as a model system. Carriage isolates will be assessed for adherence to, invasion of and cytotoxicity to these eukaryotic cells. The integrity of the cell monolayer will be assessed using chromatic assays and microscopy. Production of cytokines and activation of pathways of cell toxicity will be evaluated using ELISA, qRT-PCR, protein chips and RNASeq. The assays will compare multiple isolates from individual carriers and from different serogroups/clonal complexes (e.g. MenY-cc23 and MenW-cc11).
2) Determine the sensitivity to complement of meningococcal carriage isolates.
Comparison of isolates for differences in sensitivity to human serum in serum bactericidal assays and for deposition of complement effector protein on the cell surface by FACS. This assay is a key correlate of protection and is used for assessing whether vaccine-elicited sera will protect against a particular strain.
3) Investigation of immune evasion.
Hypervirulent isolates of the MenW clone will be tested for escape of bactericidal antibodies to specific antigens in a modified serum bactericidal assay developed by Bayliss et al. Strains with different tract lengths and modified to remove repeat tract will be compared in these assay to determine whether this clone has evolved a higher capacity to evade host antibody responses.
4) Comparative analysis of disease and carriage isolates.
Oldfield et al. indicates that our cc23 MenY carriage isolates are highly similar to invasive isolates. A similar scenario exists with the hypervirulent MenW clone. In both cases, disease isolates, obtained from the Meningococcal Reference Unit will be tested for variation in disease traits relative to the carriage isolates using assays outlined in objectives 1-3.
Funding Notes
This 3 year-funded studentship is available in the Department of Genetics & Genome Biology for start in September 2018. The studentship is open to Home/EU students at the standard Research Councils UK rate.
Applicants should hold or expected to hold a First or Upper Second class degree in a relevant field by September 2018. Those holding a Lower Second class degree plus a Master’s degree or with 3+ years relevant post-graduate experience may also be eligible. Applications from candidates with degrees from abroad may be eligible if their qualifications are deemed equivalent.
References
1. E.R. Moxon, C.D. Bayliss and D.W. Hood (2007). Bacterial contingency loci: the role of simple sequence DNA repeats in bacterial adaptation. Annual Review of Genetics. 40:307-333.
2. C.D. Bayliss (2009). Determinants of phase variation rate and the fitness implications of differing rates for bacterial pathogens and commensals. FEMS Microbiological Reviews 33: 504-520.
3. Bayliss (2009). Determinants of phase variation rate and the fitness implications of differing rates for bacterial pathogens and commensals. FEMS Microbiological Reviews 33: 504-520.
4. Alamro M, Bidmos FA, Chan H, Oldfield NJ, Newton E, Bai X, Aidley J, Care R, Mattick C, Turner DP, Neal KR, Ala'aldeen DA, Feavers I, Borrow R, Bayliss CD (2014). Phase variation mediates reductions in expression of surface proteins during persistent meningococcal carriage. Infect. Imm. 82: 2472-84 (PMID:24686058).
5. Bidmos FA, Neal KR, Oldfield NJ, Turner DP, Ala'Aldeen DA, Bayliss CD (2011). Persistence, replacement and rapid clonal expansion of meningococcal carriage isolates in a 2008 university student cohort. Journal Clin. Micro. 49: 506-512.
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