Consequences of ClpX deficiency in Staphylococcus aureus under infection-mimicking conditions – a proteomic investigation

Introduction: Pathogenic bacteria have to cope with host-mediated stressors such as iron limitation and micro aerobic conditions. To do so, bacteria have to adapt gene expression as well as proteome profile and activity. In the pathogen S. aureus, protein homeostasis, and especially protein degradation and folding, is largely mediated by the Clp system consisting of the ClpP peptidase and the unfoldases ClpX and ClpC. The Clp protease system is crucial for general and targeted proteolysis, which relies on unfoldases interacting with specific targets. In particular, ClpX is the most conserved Clp unfoldase and it is involved in regulation of virulence.
Goals: The majority of ClpX targets remains elusive in S. aureus despite the clear role of the unfoldases in virulence and fitness. We therefore aim to investigate the proteomic effect of ClpX deficiency under infection-mimicking conditions.
Methods: For investigation of the effect of ClpX deficiency the S. aureus mutant strain HG001 ∆clpX::km and the complemented strain HG001 ∆clpX::km pTripleTREP_clpX were constructed employing the newly developed plasmid systems pSauSE (allele exchange plasmid) and pTripleTREP (controllable expression plasmid). Mass spectrometry-based proteome profiles were then recorded for S. aureus HG001 and the constructed isogenic mutants under aerobic iron-rich control conditions, micro aerobic conditions and iron depleted conditions in exponential and stationary growth phase.
Results: The proteomic profiles reveal ClpX- and condition-dependent changes. Known ClpX-targets such as Spx, Sle1 and Spa were confirmed. Under infection-relevant conditions, iron limitation generally induced the Fur regulon and repressed the IsrR targetome, and under micro aerobic condition the NreBC regulon and the Rex regulon are induced, as expected. Moreover, ClpX-dependent condition-specific effects, such as a sub regulon of the known Fur regulon which is less responsive to the provoked iron limitation in the ∆clpX mutant, were also observed.
Summary: By presenting our proteomic study, we provide a global insight into ClpX-dependent adaptation of S. aureus physiology under infection-relevant conditions.