Post-translational modification of proteins secreted by Pseudomonas aeruginosa and its role in bacterial physiology

Pseudomonas aeruginosa is a multi-drug resistant, opportunistic pathogen capable of causing serious and potentially life-threatening infections worldwide. An important component of its infection strategy is the secretion of an extensive arsenal of virulence factors. These virulence factors cause considerable tissue damage and are also involved in attenuation of the host immune response, as well as inter-kingdom defences. Maintaining control over the production, secretion and activity of these proteins is therefore essential and is likely to lie not only at the transcriptional level, but also through post-translational modification (PTM). By understanding the nature of PTM in extracellular proteins and how these may affect protein function and stability, a future platform for alternative anti-virulence interventions can be developed. In this dissertation I first looked at how environmental factors affect protein secretion in Pseudomonas aeruginosa. Although factors including nutrient availability, temperature, growth stage and strain type were found to modulate the overall proteomic output, the presence of charge variant protein isoforms proved to be a prominent and consistent biological phenomenon. Several key virulence factors presenting as charge variants were then selected for further analysis, and the nature of their PTMs was characterised by LC-MS/MS. All protein isoforms investigated were found to be multi-modified by acetylation and methylation, with a considerable level of overlap between these modifications in many cases. In the final part, site-directed mutagenesis of residues that are modified in the P. aeruginosa elastase enabled initial insights into how PTM may affect this protein’s function. Phenotypic analyses revealed that all mutants displayed reduced elastinolytic activity, as well as a reduced ability to swarm for two of these mutants. In particular, mutation of arginine 226 appeared to have the most prominent effect, with potential consequences in the processing of other secreted virulence factors. In conclusion, several principal virulence factors produced by P. aeruginosa were found to be consistently secreted as charge variable isoforms. These isoforms displayed extensive modification by acetylation and methylation and the potential roles these PTMs may have is discussed.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2021

Author dc:creator

• Forrest, Suzanne Laura

Advisor dc:contributor.advisor

• Welch, Martin

• Pseudomonas aeruginosa
• methylation
• post-translational modification
• acetylation
• secreted protein
• virulence factor

Licence

• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.80224

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/332789