Unraveling proteomic chaos by independent component analysis - ClpX proficiency promotes the iron and oxygen limitation responses of Staphylococcus aureus and affects the intracellular bacterial behavior

In the opportunistic pathogen Staphylococcus aureus , protein homeostasis is largely mediated by the Caseinolytic protease (Clp) system. The proteases ClpXP and ClpCP are crucial for general and targeted proteolysis, which rely on the unfoldases ClpX and ClpC interacting with specific targets. However, the global effect on the proteome especially under infection-relevant stresses is not well-understood. To assess the effect of ClpX deficiency during infection-related processes, mass spectrometry-based global proteome profiles of S. aureus HG001 wild-type, an isogenic Δ clpX mutant, and a clpX complemented strain were recorded under control conditions as well as iron and oxygen limitation. The proteomic profiles revealed specific ClpX- and stress-dependent changes. A set of 24 robust stress-independent ClpX modulated proteins was identified and the stress-dependent influences were unraveled by independent component analysis (using the iModulon approach). These analyses revealed a role of ClpX in e.g., cell division, cell envelope homeostasis, the quinone stress response and prophage activation. Moreover, ClpX-dependent stress-specific effects were observed in the Δ clpX mutant, e.g. reduced induction of the heme uptake system under iron limitation and a dampened Rex-controlled oxygen limitation response. This revealed in particular that ClpX is central for heme homeostasis in S. aureus . Furthermore, in a Galleria infection model, the S. aureus Δ clpX mutant was attenuated compared to the wild-type HG001. This is consistent with a drastically reduced intracellular replication of the Δ clpX -mutant in cell culture-based infection experiments, however, high intracellular persistence of the Δ clpX mutant was also observed. This highlights the relevance of ClpX for bacterial fitness and virulence.