Activated ClpP kills persisters and eradicates a chronic biofilm infection

Abstract

Chronic infections are difficult to treat with antibiotics but are caused primarily by drug-sensitive pathogens. Dormant persister cells that are tolerant to killing by antibiotics are responsible for this apparent paradox. Persisters are phenotypic variants of normal cells and pathways leading to dormancy are redundant, making it challenging to develop anti-persister compounds. Biofilms shield persisters from the immune system, suggesting that an antibiotic for treating a chronic infection should be able to eradicate the infection on its own. We reasoned that a compound capable of corrupting a target in dormant cells will kill persisters. The acyldepsipeptide antibiotic (ADEP4) has been shown to activate the ClpP protease, resulting in death of growing cells. Here we show that ADEP4-activated ClpP becomes a fairly nonspecific protease and kills persisters by degrading over 400 proteins, forcing cells to self-digest. Null mutants of clpP arise with high probability, but combining ADEP4 with rifampicin produced complete eradication of Staphylococcus aureus biofilms in vitro and in a mouse model of a chronic infection. Our findings indicate a general principle for killing dormant cells-activation and corruption of a target, rather than conventional inhibition. Eradication of a biofilm in an animal model by activating a protease suggests a realistic path towards developing therapies to treat chronic infections.

Figure 1. Quantitative proteomic analysis of S. aureus cells treated with ADEP4 reveals extensive protein degradation

S. aureus cells were treated with ADEP4 in biological duplicates and submitted toa global quantitative proteomic analysis. The dispersion graphs show the relative abundances (treated/untreated) of a, total proteins and b, partiallytryptic peptides in different biological replicates. The significant changes in abundances (p≤0.05 and >2 fold) are represent in red circles. c, Function-enrichment analysis of proteins degraded by ADEP4. Functions overrepresented among proteins degraded by ADEP4 were annotated using Database for Annotation, Visualization and Integrated Discovery (DAVID) and the overrepresented pathways compared to the genome background are shown as columns, whereas their p-values are represented by the black dots.

Figure 2. ADEP4 kills persisters and in combination with rifampicin or linezolid eradicates stationary phase S. aureus

a, ADEP4 kills persisters isolated following ciprofloxacin treatment. b, Conventional antibiotics are inactive against stationary phase S. aureus. c, ADEP4 activity against stationary S. aureus. d, ADEP4 in combination with rifampicin or linezolid eradicates stationary phase S. aureus to the detection limit in 72h in MHB and e, 24h in chemically defined media. f, ADEP4 resistant mutants are less tolerant to rifampicin and linezolid than the parent wild type strain. The X axis is the limit of detection.* represents eradication to the limit of detection.

Figure 3. ADEP4 kills S. aureus biofilm and in combination with rifampicin eradicates the population

The X axis is the limit of detection.* represents eradication to the limit of detection.

Figure 4. ADEP4 in combination with rifampicin eradicates a deep-seated mouse thigh infection

a, Single day treatments with rifampicin and vancomycin reveal the deep-seated nature of the mouse thigh lesion. A second day of vancomycin treatment (vancomycin 48h) reveals an antibiotic tolerant subpopulation. b, Single day ADEP4 rifampicin combination eradicates S. aureus in the deep-seated infection. * represents eradication to the limit of detection c, Histopathology of S. aureus infected thighs reveals the presence of a biofilm. Left panel, Gram staining 80 × magnification; right panel, electron micrograph 8000 × magnification.

Extended data Figure 1

ADEP4 resistant strains are heat sensitive. Wild type ATCC 33591 and 9 ADEP4 resistant isolates with mutations in clpP were grown for 20 hours in MHB at 44°C in 96-well polystyrene plates.

Extended data Figure 2

ADEP4/rifampicin eradicates a variety of S. aureus strains. S. aureus was grown in MHB for 16 hours and challenged with 10 × MIC of ADEP4 and rifampicin. Colony counts were performed every 24 hours. Limit of detection = 2.

Extended data Figure 3

ADEP4/rifampicin eradicates S. aureus in a hollow fiber infection model. The concentration of ADEP was varied over time to match the pharmacokinetics in the mouse model. Limit of detection = 2.

Extended data Figure 4