Self-resistance mechanism to acyldepsipeptide antibiotics in the Streptomyces producer.

Clp proteases are ubiquitous in bacteria and play an important role in regulatory proteolysis and in maintaining protein homeostasis within the bacterial cell. They consist of a tetradecameric, proteolytic ClpP core and associated AAA+ Clp-ATPases. The Clp system of Streptomyces is unusually complex, comprising up to five ClpP homologs (ClpP1-ClpP5) and three Clp-ATPases (ClpX, ClpC1, and ClpC2). Streptomycetes produce a plethora of secondary metabolites, including potent acyldepsipeptide (ADEP) antibiotics, which target ClpP. We have previously reported on the operation mode of the Streptomyces ClpP1P2 protease and identified a novel clpP gene (named clpPADEP) as a resistance determinant encoded near the ADEP biosynthesis gene cluster. However, the molecular function of ClpPADEP remains enigmatic. Here, we report on the molecular self-resistance mechanism to ADEP via ClpPADEP and its interaction with the Clp system in the ADEP producer Streptomyces hawaiiensis NRRL 15010. By combining cell-based and in vitro studies, we show that ClpPADEP interferes with the formation of the ClpP1P2 complex and inhibits the proteolytic activity of ClpP1. Moreover, ClpPADEP forms a functional complex with ClpP2 and Clp-ATPases. By these means, ClpPADEP protects the producer cell against ADEP in a two-pronged approach. On the one hand, it prevents ADEP from corrupting ClpP1 to degrade delicate essential proteins and polypeptides. On the other hand, ClpPADEP ensures the survival of the producer cells by maintaining the housekeeping function of the Clp protease in regulatory proteolysis.IMPORTANCEAcyldepsipeptide (ADEP) antibiotics kill bacteria using an unusual mechanism of action, that is, the multilayered deregulation and activation of ClpP, the proteolytic core of the bacterial Clp protease. ADEP is highly effective in killing Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Considering the elaborate mechanism of action of ADEP as well as the complexity of the essential Clp system in Streptomyces with up to five ClpP homologs as potential ADEP targets, the question arises: how does the producer ensure self-resistance in such a complex system? Here, we describe the molecular mechanism of self-resistance to ADEP in the producer Streptomyces hawaiiensis NRRL 15010, which is based on the presence of a phylogenetically distinct ClpP protein in the genome of the ADEP producer strain.