Abhishek Kumar Singh (Greifswald / DE), Ole Plöhn (Greifswald / DE), Silva Holtfreter (Greifswald / DE), Karsten Becker (Greifswald / DE), Barbara M. Bröker (Greifswald / DE), Clemens Cammann (Greifswald / DE), Ulrike Seifert (Greifswald / DE)
Introduction
S. aureus is an important bacterial pathogen that causes a wide range of clinical manifestations. Post-translational modifications such as ubiquitination regulate host cell recognition of S. aureus by pattern recognition receptors and their respective downstream signalling pathways. Ubiquitination affects protein function, stability and localization, and is facilitated by ubiquitinating enzymes known as E3 ligases. Our aim was to analyse a selection of E3 ligases thought to be involved in regulating host cell responses to S. aureus infection.
Method
Macrophages are the frontline defenders of the innate immune response and are crucial in combating infections caused by S. aureus. To study the host cellular response, we infected THP-1 macrophage-like cells with different S. aureus strains and analysed E3 ligase expression at protein level via immunoblotting and RNA using qPCR. The role of selected E3 ubiquitin ligases was further investigated in the context of bacterial infection using the CRISPR-Cas9 knockout technique.
Results
This study revealed changes in the expression levels of selected E3 ligases in response to S. aureus infection in THP-1 cells. Furthermore, these changes could be confirmed by exposure of the cells to specific virulence factors such as staphylococcal protein A. In addition, an altered cytokine production was observed, suggesting a dynamic interplay between the virulence factors of the pathogen and the host immune response. Currently, the function of specific E3 ligases is analysed with respect to their impact on cellular signalling pathways, cell survival and cytokine production by comparing CRISR-CAS9-mediated knockout cells with their wild-type counterparts.
Discussion
In conclusion, our findings highlight the intricate molecular mechanisms involved in the ubiquitin-mediated host-pathogen interaction and provide potential approaches for targeted therapeutic intervention. Based on our results it will be of interest to deepen the knowledge on the function of selected E3 ligases. Furthermore, the observed changes in E3 ligase activity and cytokine profiles provide valuable insights into S. aureus pathogenicity.