Functional genomic approaches to manipulating Bordetella pertussis outer membrane synthesis for vaccine development.

  • David Mcculloch

Student thesis: Doctoral ThesisPhD


Bacterial growth and replication are dependent upon the expression of cell wall biosynthesis genes, many of whom are essential for bacterial viability. The mre/mrd operon is a set of such genes which carries out synthesis of the peptidoglycan (PG) sacculus during bacterial elongation. The manipulation of the mre/mrd operon in gram-negative bacteria is lethal under unmodified growth conditions, resulting in them being classified as essential for bacterial viability. However, this does not appear to be the case in B. pertussis.

In B. pertussis the mre/mrd operon is conditionally essential during in vitro plate growth, dependent on the activation state of the BVG two component system. This poses the questions of:
- whether mre/mrd mutation induces phenotypic changes in B. pertussis as observed in other gram-negative bacteria?
- how a fastidious bacteria like B. pertussis tolerates the loss of these classically essential cell wall biosynthesis genes?
-whether disruption of the peptidoglycan sacculus in mre/mrd mutants results in increased outer membrane vesicle (OMV) production, which have shown promise as vaccine antigens against B. pertussis?

Analysis of mre/mrd mutant strains growth phenotypes displayed that essentiality was only observed during in vitro plate growth under BVG- modulating conditions. Conversely the mre/mrd operon was non-essential during bacterial growth in in vitro liquid culture independent of BVG activation state. Interestingly, this was despite the change in bacterial morphology, which was observed upon mutation of mre/mrd in other gram-negative bacteria, being present in B. pertussis.
Utilising transcriptomic profiling of BP536 wildtype B. pertussis during growth in plate and liquid culture mediums, I investigated how B. pertussis could tolerate the loss of classically essential cell wall biosynthesis genes. This analysis identified genes involved in glycine betaine transport, a well characterised osmoprotectant, to be significantly upregulated during BVG- broth growth, where mutants are viable, when compared to BVG- plate growth, where mutants are non-viable. This analysis suggests that the bacteria’s ability to regulate osmolarity, through betaine uptake, facilitates tolerance of mre/mrd mutations. This finding was validated by betaine supplementation rescuing BP536ΔmreB BVG- plate viability. However, BP536ΔmreB broth viability was not altered upon loss of the ATPase of the betaine glycine uptake system, suggesting conditional viability dependent upon BVG- growth medium is multifactorial.

Characterisation of the vesiculation phenotype of mre/mrd mutants displayed hypervesiculation was induced upon loss of elongasome peptidoglycan biosynthesis, with comparable levels of vesiculation to other hypervesiculating B. pertussis strains. Characterisation of OMV phenotypes displayed mre/mrd mutants OMVs had antigen profiles like that of OMVs produced by wildtype strains. Promisingly, the average size of OMVs produced by mutant strains were in the optimum range for uptake by immune cells. Overall, this analysis suggests that mre/mrd mutant strains are promising for OMV production to be utilised as vaccine antigens.
Date of Award13 Sept 2023
Original languageEnglish
Awarding Institution
  • University of Bath
SupervisorAndrew Preston (Supervisor) & Brian Jones (Supervisor)

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