Abstract
In living cells intracellular proteolysis is crucial for protein homeostasis, and ClpP proteases are conserved between eubacteria and the organelles of eukaryotic cells. In Staphylococcus aureus, ClpP associates to the substrate specificity factors, ClpX and ClpC forming two ClpP proteases, ClpXP and ClpCP. To address how individual ClpP proteases impact cell physiology, we constructed a S. aureus mutant expressing ClpX with an I265E substitution in the ClpP recognition tripeptide of ClpX. This mutant cannot degrade established ClpXP substrates confirming that the introduced amino acid substitution abolishes ClpXP activity. Phenotypic characterization of this mutant showed that ClpXP activity controls cell size and is required for growth at low temperature. Cells expressing the ClpXI265E variant, in contrast to cells lacking ClpP, are not sensitive to heat-stress and do not accumulate protein aggregates showing that ClpXP is dispensable for degradation of unfolded proteins in S. aureus. Consistent with this finding, transcriptomic profiling revealed strong induction of genes responding to protein folding stress in cells devoid of ClpP, but not in cells lacking only ClpXP. In the latter cells, highly upregulated loci include the urease operon, the pyrimidine biosynthesis operon, the betA-betB operon, and the pathogenicity island, SaPI5, while virulence genes were dramatically down-regulated.
Highlights
In all organisms ATP-dependent proteases are essential for maintaining protein homeostasis by disposing of damaged or unneeded proteins, as well as for the conditional degradation of functional proteins in response to external or internal signals[1]
To examine if S. aureus cells expressing the ClpXI265E variant are devoid of ClpXP proteolytic activity, we assessed if two known ClpXP substrates, the transcriptional regulator Spx and the Sle[1] autolysin, accumulate in the ClpXI265E mutant[22,23]
ClpP generally has the ability to associate to several Clp ATPases thereby forming different ClpP proteolytic complexes
Summary
In all organisms ATP-dependent proteases are essential for maintaining protein homeostasis by disposing of damaged or unneeded proteins, as well as for the conditional degradation of functional proteins in response to external or internal signals[1]. In order to degrade protein substrates, ClpP must associate to hexameric rings of one of several possible Clp ATPases[5]. In order to inactivate only the ClpXP protease, we constructed a S. aureus mutant variant of ClpX that cannot interact with ClpP, the constructed mutant retains ClpCP activity. This mutant cannot degrade established ClpXP substrates confirming that the introduced amino acid substitution abolishes ClpXP activity. Phenotypic characterization of this mutant supports that ClpXP and ClpCP perform different tasks in S. aureus, and that ClpC is superior to ClpX in targeting stress-damaged proteins for degradation by ClpP.
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