Abstract
C. albicans is a commensal fungi which populates most healthy individuals’ microflora, but can turn opportunistic in immunocompromised individuals. Opportunistic C. albicans can cause severe disease and is linked with high rates of mortality. With limited therapeutic options and increasing resistance to antifungals, novel treatment strategies for C. albicans infections is paramount.Macrophages are a key immune cell which mounts an immediate response to C. albicans infection. Macrophages both sequester C. albicans infection through phagocytosis and produce immune signalling and antifungal responses. All of which aid in killing C. albicans pathogen and alerting other immune cells of localised infection.
Herein, murine bone marrow derived macrophages (BMDM) were used to study the in vitro response to C. albicans infection. The BMDM response to C. albicans infection was compared with other conventional agonists like LPS, fungal cell wall component Zymosan and LPS containing gram negative bacteria P. aeruginosa. Overall, live C. albicans infection was unexpectedly able to avoid inducing a strong inflammatory response in isolated macrophages, compared to these conventional agonists.
TMT phosphoproteomics was used as a discovery screen for BMDM signalling response to C. albicans and P. aeruginosa infection. Overall, C. albicans infection induced a muted signalling response compared to P. aeruginosa infection.
Misidentification of peptides was evaluated through in silico and experimental infection models. Misidentifications may arise from infected samples containing two peptide sources: mouse macrophages and C. albicans yeast. In silico trypsin digest of Mus musculus and C. albicans showed limited homology between the two species. Experimentally, a library free workflow using direct DIA with the default search settings incorrectly identified over 1,000 C. albicans peptides in the non-spiked (mouse BMDM only) containing samples. Thus, search parameters were optimised in Spectronaut to minimise the number of misidentifications, i.e. C. albicans peptides detected in the Mouse only samples, at a reduced penalty to overall proteome coverage. These settings provide an option for high-stringency, robust identification results for library-free DIA for which were used for total proteome analysis.
The total proteome landscape of BMDM response to C. albicans was studied through two avenues. The BMDM response during WT C. albicans infection (which can morphologically change into virulent hyphal filaments) was compared with a yeast locked (less virulent) Hgc1 KO C. albicans strain. Proteins induced more so in WT than Hgc1 KO C. albicans infection had roles in inflammation. 2) C. albicans was co-treated with LPS or P. aeruginosa. C. albicans was found to broadly down regulate LPS and P. aeruginosa regulated protein responses in co-treated groups compared to LPS or P. aeruginosa infections alone. In summary, the work presented in this thesis demonstrates the BMDM response to C. albicans infection has an immune suppressive effect when compared to a conventional agonist like LPS or P. aeruginosa bacterial infection.
This thesis presents multiple proteomic datasets which can continue to be used for future study of BMDM response to C. albicans infection.
| Date of Award | 2024 |
|---|---|
| Original language | English |
| Sponsors | Wellcome Trust |
| Supervisor | Simon Arthur (Supervisor) & Albena Dinkova-Kostova (Supervisor) |