A proteomic model to investigate the effect of ClpX deficiency in Staphylococcus aureus in infection processes

Host-mediated stressors affect bacterial pathogens during infection processes. In response to those stressors, bacteria adapt their gene expression as well as ultimately proteome profile and activity. In Staphylococcus aureus protein homeostasis is largely mediated by the Clp system consisting of the ClpP peptidase, the unfoldases ClpX and ClpC, and the chaperones ClpB and ClpL. The proteases ClpXP and ClpCP are crucial for general and targeted proteolysis, which rely on the unfoldases interacting with specific targets. ClpX is particularly involved in overall regulation of bacterial virulence and fitness and it is the most conserved Clp unfoldase. However, the majority of S. aureus ClpX targets remains elusive. 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 using the newly developed plasmid systems pSauSE (allele exchange plasmid) and pTripleTREP (controllable expression plasmid). When tracking infection processes, the S. aureus mutant strain HG001 ∆clpX revealed a less virulent phenotype in a Galleria model compared to the HG001 wild type and drastically low intracellular replication in in-vitro HBE cell culture infection experiments. The effect of ClpX deficiency under infection-relevant conditions was recorded after cultivating HG001 wild type, the HG001 ∆clpX mutant and the complemented strain HG001 ∆clpX pTripleTREP_clpX under iron limitation and microaerobic conditions by mass spectrometry in data independent mode. To address differences in proteome composition caused by the strain background and growth conditions, a normalization strategy based on an external standard was employed. The standard consists of 15N-labeled Bacillus subtilis proteins enabling reliable distinction between standard and sample proteins and providing robust relative quantification of sample proteins. The proteomic profiles revealed specific ClpX- and condition-dependent changes as well as entanglement of those. An iModulon-inspired analysis resulted in 36 independent components, unravelling the different influences. Known ClpX-targets such as Spx and Sle1 were confirmed as robust targets and new targets such as NikA and SAOUHSC_00671 were suggested. Out of the two investigated infection-relevant conditions, iron limitation generally induced the Fur regulon, and under microaerobic condition the Rex regulon was induced. Moreover, ClpX-dependent condition-specific effects, such as a subregulon of the known Fur regulon, which is less responsive to the provoked iron limitation in the ∆clpX mutant, were also observed. With our proteomics data, we provide a global insight into ClpX-dependent adaptation of S. aureus physiology under infection-relevant conditions.