Exploring the role of P. aeruginosa virulence factor AaaA in chronic skin wound isolates using novel biofilm models
Publication Date
December 11, 2024
Creator
Abstract
Pseudomonas aeruginosa is intrinsically multi-drug resistant with alarming rates of acquired antimicrobial resistance (AMR) developing and forms chronic infections that are difficult to eradicate in immunocompromised individuals. This causes high patient mortality, and a global burden of disease. P. aeruginosa is the leading cause of death following infection of the lungs of people with cystic fibrosis, with 90% of deaths associated with chronic Pseudomonas infection. It is also one of the main bacteria associated with chronic wound infections which have life-changing consequences for 1-2% of the population with patient management costing the NHS over £5.3 billion a year. The persistent and chronic nature of P. aeruginosa infections can be attributed to its arsenal of virulence factors along with the ability to form complex biofilms which provide a reservoir for the transmission of AMR genes. This project aims to determine how one of the virulence factors, an arginine specific aminopeptidase of P. aeruginosa (AaaA) functions, because AaaA is crucial for chronic infections. AaaA is an attractive vaccine or antimicrobial target because it (i) has a distinct active site pocket with arginine specific aminopeptidase activity, (ii) is tethered to the surface of P. aeruginosa, and thus accessible, and (iii) is encoded by a gene that is highly conserved within clinical P. aeruginosa strains. This study aimed to probe the gene expression and function of AaaA in conditions which mimic the host infection site as closely as possible. This was done by using clinical strains isolated from chronic wound infections in two different optimised chronic wound biofilms models.
In this study, it was shown that AaaA activity varies among clinical isolates biopsied from different chronic wounds and that AaaA is more highly produced and active in the synthetic chronic wound (SCW) model than in planktonic LB culture. It was also shown that AaaA has a role in biofilm formation and cell dispersion within the SCW. Taken together, it can be concluded that the SCW model is a viable option for the study of AaaA, and AaaA plays an important role in both chronic infection and biofilm formation. Competition biofilms between WT and ΔaaaA mutant revealed that the ΔaaaA mutant could be acting as a cheater, using the cleaved L-arginine provided by the WT in its own metabolic pathways.
The SCW model was then used to probe the regulatory network of aaaA. Surprisingly, in anaerobic conditions, there was a reduction in aaaA expression and in both lab-adapted and clinically isolated strains AaaA activity was lower. AaaA also did not provide a survival advantage after 24 hrs in the SCW model when incubated in the anaerobic cabinet, despite ΔaaaA mutants being unable to survive extended periods in anaerobic conditions in LB media. Studying the wider regulatory network of AaaA revealed a potential indirect link with c-di-GMP, which could help explain the role AaaA has on biofilm formation and cell localisation within the SCW model, as the c-di-GMP pathway regulates biofilm formation. Using RT-qPCR after 24 hrs in the SCW model, arginine-metabolism related genes were also shown to be upregulated in a laboratory strain compared with a derived ΔaaaA mutant strain.
To study the effect of AaaA in a dual species infection, the modified chronic wound biofilm (mCWB) model incorporating P. aeruginosa and S. aureus was optimised to allow growth of paired clinical isolates. AaaA activity varied depending on the strain tested, with the highest, consistent levels of AaaA activity being seen in a clinical strain isolated from a long-term infection. Imaging of the biofilms also implied AaaA plays a role in biofilm formation and cell dispersion in a dual species chronic wound infection. Using matched and mismatched clinical isolate pairs it was determined that co-colonisation fosters a synergistic relationship between S. aureus and P. aeruginosa, with mismatched strains unable to maintain a stable dual species association and instead exhibiting P. aeruginosa domination. However, this trend was independent of the presence of aaaA.
In summary, this study has developed and optimised two different biofilm models for studying AaaA in a realistic infection background. The function of AaaA was explored in clinical isolates from chronic wound infections, through the construction of mutants, to allow the regulation and function of AaaA to be explored in more realistic conditions. Combined, this study has developed, optimised, and validated a background representative of native conditions for the continued study of AaaA. The results of this study, along with results from Luckett et al (2012), imply AaaA could play an important role in in vivo infection. Due to its role in biofilm formation and potential role as an immune modulator, AaaA aids bacterial pathogenicity and the ability for P. aeruginosa to form chronic infections.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Life Sciences
eprints ID
79760
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