Introduction

Ubiquitin E3 ligases catalyse the transfer of ubiquitin moieties to protein substrates, affecting protein function, stability and localisation. During infection, ubiquitination is critical for regulating host cellular signalling pathways to recognise and eliminate pathogens. Intracellular pathogens are ubiquitinated by specific E3 ligases that target e.g. bacteria for autophagosomal degradation. However, knowledge of the E3 ligases involved is very limited, especially for Gram-positive bacteria such as Staphylococcus aureus.

Method

Lung epithelial cells are the first line of defence against infections of the respiratory tract. Therefore, we analysed A549 lung epithelial cells during S. aureus infection, focusing on the role of the E3 ligase LRSAM1. We used the CRISPR-Cas9 system to generate LRSAM1-deficient A549 cells and compared their cellular response to that of LRSAM1-expressing cells during S. aureus infection by monitoring the activation of cellular signalling pathways and the subsequent cytokine production, host cell death and the intracellular survival of S. aureus.

Results

The analysis of the LRSAM1-deficient A549 cells revealed a strong impact on the intracellular replication of S. aureus during infection which was accompanied by an increased host cell death compared to control cells. Moreover, by monitoring IκBα degradation an altered activation of the NFκB-pathway could be observed which resulted in elevated secretion of the pro-inflammatory cytokines IL-6 and IL-8. Currently LRSAM1 deficient cells are analysed for their capability to ubiquitinate intracellular S. aureus as well as the impact of LRSAM1 on autophagy.

Discussion

In conclusion, our results indicate a prominent role for LRSAM1 during S. aureus infection. Further experiments are needed to clarify its exact substrates and thus elucidate the mechanism by which this E3 ligase affects intracellular bacteria recognition and thus directly or indirectly impacts intracellular bacteria survival and host cell death. Understanding the molecular mechanisms underlying the role of LRSAM1 will help to develop new therapeutic strategies against host cell persistence of e.g. S. aureus.