Tools for the analysis of SplB under infection-mimicking conditions

Staphylococcus aureus, a prevalent nosocomial pathogen, exhibits genomic heterogeneity across strains. Extracellular proteases, including serine-like proteases (Spls), play a crucial role in the pathogen's survival within the host (Singh V. et al. Med Microbiol Immunol. 2019). Interestingly, distribution of these proteases among individual S. aureus strains differs significantly.

Our research objective is to investigate the genomic and proteomic differences of Spls among S. aureus strains. To this end, we initially examined levels of SplB and other proteases by mass spectrometry and Western blots in different media in the stationary growth phase across different strains.

In order to explore the temporal expression of spls in cell culture infection experiments and the different regulators involved we designed a specific reporter system. We adapted the vector pTricolor to integrate different measurements during an infection experiment. In brief, the cfp reporter, expressed from a SigA promoter, facilitates normalization and copy number control. The spl promoter region, driving gfp reporter expression, enables time resolved recording of spl expression at single cell level. Finally, dtomato expressed from promoters controlled by specific regulators of Spls, reveals the drivers of spl operon expression in an infection experiment.

Our findings reveal variations in the amount of the secreted protease among different strains, and even within a specific S. aureus lineage (NCTC8325). The molecular background of regulation of spl expression and its impact during infection are currently being analyzed.

The results obtained so far suggest that Spl regulation is more intricate than previously assumed.