An investigation into the factors that affect membrane vesicle composition in bacteria

Outer membrane vesicles (OMVs) are nano-sized structures that are formed when portions of the bacterial membrane bud and pinch off from the cell in a process called vesiculation. This process entraps a diverse range of bacterial products within the vesicles (including virulence factors) which are later released into the environment. The main project aims were to gain a fundamental understanding of vesiculation in a range of bacterial species and to enable targeted expression of recombinant proteins and other molecules for delivery and inclusion in OMVs. OMVs were isolated and characterised from various strains of Escherichia coli and Pseudomonas aeruginosa. It was found that OMVs from different bacterial strains are similar in appearance but have very different compositions and cargo. The proteins FimA and Flagellin were found to be heavily enriched within E. coli K-12 OMVs in a mutually exclusive way. They are known virulence factors that have been shown to be reciprocally regulated in E. coli cells but not in OMVs. FimA has previously been found to have an anti-inflammatory effect on human immune cells whereas Flagellin has a pro-inflammatory effect, which may be the reason that the two proteins are not packaged within OMVs together. This was further explored by purification of OMVs from a series of E. coli gene knockouts and clinical isolates to compare the protein profiles of the OMVs. Lastly, an E. coli strain containing GFP fused to FimA was trialled as a method of targeted delivery within OMVs. This method was successful as the GFP-FimA protein fusion was detected in the OMVs purified from this strain. Recombinant protein fusions such as this could allow use of E. coli OMVs for therapeutic applications such as drug delivery and vaccines. Furthermore, the packaging of FimA and Flagellin into E. coli OMVs may play a significant role in its pathogenicity and ability to modulate the host response to infection. These findings could highlight potential new drug targets against OMV-producing pathogens such as E. coli as well as providing further insight into using OMVs for drug delivery and vaccines.