Functional Characterisation of ClpP Mutations Conferring Resistance to Acyldepsipeptide Antibiotics in Firmicutes.

Imran T Malik,Peter Sass,Christian Mayer,Rebeca Pereira,Michael Groll,Marie‐Theres Vielberg,Kirsten Famulla,Heike Brötz‐Oesterhelt,Jan Straetener,Dhana Thomy,Helena Castro

doi:10.1002/cbic.201900787

Abstract

Acyldepsipeptide (ADEP) is an exploratory antibiotic with a novel mechanism of action. ClpP, the proteolytic core of the caseinolytic protease, is deregulated towards unrestrained proteolysis. Here, we report on the mechanism of ADEP resistance in Firmicutes. This bacterial phylum contains important pathogens that are relevant for potential ADEP therapy. For Staphylococcus aureus, Bacillus subtilis, enterococci and streptococci, spontaneous ADEP‐resistant mutants were selected in vitro at a rate of 10−6. All isolates carried mutations in clpP. All mutated S. aureus ClpP proteins characterised in this study were functionally impaired; this increased our understanding of the mode of operation of ClpP. For molecular insights, crystal structures of S. aureus ClpP bound to ADEP4 were determined. Well‐resolved N‐terminal domains in the apo structure allow the pore‐gating mechanism to be followed. The compilation of mutations presented here indicates residues relevant for ClpP function and suggests that ADEP resistance will occur at a lower rate during the infection process.

Highlights

For Staphylococcus aureus, Bacillus subtilis, enterococci and streptococci, spontaneous ADEP-resistant mutants were selected in vitro at a rate of 10 6
Crystal structures of S. aureus ClpP bound to ADEP4 were determined
We focussed on S. aureus as a major target pathogen for potential future clinical application of the ADEP class but the findings presented here shed light on ClpP mutations that we obtained in other genera of Firmicutes

Summary

Introduction

For Staphylococcus aureus, Bacillus subtilis, enterococci and streptococci, spontaneous ADEP-resistant mutants were selected in vitro at a rate of 10 6. Introduction aureus, enterococci and streptococci, including multidrugresistant strains and non-growing cells.[10,11,12,13] ADEP acts by an unusual mechanism that does not require bacterial growth to take effect, its anti-persister activity.[11,13] The antibiotic deregulates ClpP, the proteolytic core of the bacterial caseinolytic protease and a new target for antibiotic and antivirulence therapy.[10,14,15,16] ClpP is ubiquitous in bacteria, mitochondria, and chloroplasts.[17,18] In its active tetradecameric conformation, ClpP forms the central proteolytic compartment of a larger AAA + protease machine.[19] Two rings of seven ClpP monomers stack and interact to form a secluded barrel-shaped tetradecamer harbouring the 14 catalytic sites.[20,21] Small entrance pores at the top and bottom of the barrel restrict access of proteins and allow isolated ClpP to degrade small peptides only.[22,23] For protein degradation, Clp-ATPases are required (in Firmicutes ClpX, ClpC, and ClpE) to unfold designated substrates and thread them through the entrance pores.[16,24]. Å in apo-ClpP and substantially widened in the ADEP-bound state up to 30 Å.[13,25,26,32,33] As a consequence, ADEP-activated

Results

Discussion

Conclusion