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

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.

The ADEP Biosynthetic Gene Cluster in Streptomyces hawaiiensis NRRL 15010 Reveals an Accessory clpP Gene as a Novel Antibiotic Resistance Factor.

The increasing threat posed by multiresistant bacterial pathogens necessitates the discovery of novel antibacterials with unprecedented modes of action. ADEP1, a natural compound produced by Streptomyces hawaiiensis NRRL 15010, is the prototype for a new class of acyldepsipeptide (ADEP) antibiotics. ADEP antibiotics deregulate the proteolytic core ClpP of the bacterial caseinolytic protease, thereby exhibiting potent antibacterial activity against Gram-positive bacteria, including multiresistant pathogens. ADEP1 and derivatives, here collectively called ADEP, have been previously investigated for their antibiotic potency against different species, structure-activity relationship, and mechanism of action; however, knowledge on the biosynthesis of the natural compound and producer self-resistance have remained elusive. In this study, we identified and analyzed the ADEP biosynthetic gene cluster in S. hawaiiensis NRRL 15010, which comprises two NRPSs, genes necessary for the biosynthesis of (4S,2R)-4-methylproline, and a type II polyketide synthase (PKS) for the assembly of highly reduced polyenes. While no resistance factor could be identified within the gene cluster itself, we discovered an additional clpP homologous gene (named clpPADEP) located further downstream of the biosynthetic genes, separated from the biosynthetic gene cluster by several transposable elements. Heterologous expression of ClpPADEP in three ADEP-sensitive Streptomyces species proved its role in conferring ADEP resistance, thereby revealing a novel type of antibiotic resistance determinant.IMPORTANCE Antibiotic acyldepsipeptides (ADEPs) represent a promising new class of potent antibiotics and, at the same time, are valuable tools to study the molecular functioning of their target, ClpP, the proteolytic core of the bacterial caseinolytic protease. Here, we present a straightforward purification procedure for ADEP1 that yields substantial amounts of the pure compound in a time- and cost-efficient manner, which is a prerequisite to conveniently study the antimicrobial effects of ADEP and the operating mode of bacterial ClpP machineries in diverse bacteria. Identification and characterization of the ADEP biosynthetic gene cluster in Streptomyces hawaiiensis NRRL 15010 enables future bioinformatics screenings for similar gene clusters and/or subclusters to find novel natural compounds with specific substructures. Most strikingly, we identified a cluster-associated clpP homolog (named clpPADEP) as an ADEP resistance gene. ClpPADEP constitutes a novel bacterial resistance factor that alone is necessary and sufficient to confer high-level ADEP resistance to Streptomyces across species.

Inducing secondary metabolite production by the endophytic fungus Fusarium tricinctum through coculture with Bacillus subtilis.

Coculturing the fungal endophyte Fusarium tricinctum with the bacterium Bacillus subtilis 168 trpC2 on solid rice medium resulted in an up to 78-fold increase in the accumulation in constitutively present secondary metabolites that included lateropyrone (5), cyclic depsipeptides of the enniatin type (6-8), and the lipopeptide fusaristatin A (9). In addition, four compounds (1-4) including (-)-citreoisocoumarin (2) as well as three new natural products (1, 3, and 4) were not present in discrete fungal and bacterial controls and only detected in the cocultures. The new compounds were identified as macrocarpon C (1), 2-(carboxymethylamino)benzoic acid (3), and (-)-citreoisocoumarinol (4) by analysis of the 1D and 2D NMR and HRMS data. Enniatins B1 (7) and A1 (8), whose production was particularly enhanced, inhibited the growth of the cocultivated B. subtilis strain with minimal inhibitory concentrations (MICs) of 16 and 8 µg/mL, respectively, and were also active against Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis with MIC values in the range 2-8 µg/mL. In addition, lateropyrone (5), which was constitutively present in F. tricinctum, displayed good antibacterial activity against B. subtilis, S. aureus, S. pneumoniae, and E. faecalis, with MIC values ranging from 2 to 8 µg/mL. All active compounds were equally effective against a multiresistant clinical isolate of S. aureus and a susceptible reference strain of the same species.

Antibiotic Acyldepsipeptides Stimulate the Streptomyces Clp-ATPase/ClpP Complex for Accelerated Proteolysis.

Clp proteases consist of a proteolytic, tetradecameric ClpP core and AAA+ Clp-ATPases. Streptomycetes, producers of a plethora of secondary metabolites, encode up to five different ClpP homologs, and the composition of their unusually complex Clp protease machinery has remained unsolved. Here, we report on the composition of the housekeeping Clp protease in Streptomyces, consisting of a heterotetradecameric core built of ClpP1, ClpP2, and the cognate Clp-ATPases ClpX, ClpC1, or ClpC2, all interacting with ClpP2 only. Antibiotic acyldepsipeptides (ADEP) dysregulate the Clp protease for unregulated proteolysis. We observed that ADEP binds Streptomyces ClpP1, but not ClpP2, thereby not only triggering the degradation of nonnative protein substrates but also accelerating Clp-ATPase-dependent proteolysis. The explanation is the concomitant binding of ADEP and Clp-ATPases to opposite sides of the ClpP1P2 barrel, hence revealing a third, so far unknown mechanism of ADEP action, i.e., the accelerated proteolysis of native protein substrates by the Clp protease. IMPORTANCE Clp proteases are antibiotic and anticancer drug targets. Composed of the proteolytic core ClpP and a regulatory Clp-ATPase, the protease machinery is important for protein homeostasis and regulatory proteolysis. The acyldepsipeptide antibiotic ADEP targets ClpP and has shown promise for treating multiresistant and persistent bacterial infections. The molecular mechanism of ADEP is multilayered. Here, we present a new way how ADEP can deregulate the Clp protease system. Clp-ATPases and ADEP bind to opposite sides of Streptomyces ClpP, accelerating the degradation of natural Clp protease substrates. We also demonstrate the composition of the major Streptomyces Clp protease complex, a heteromeric ClpP1P2 core with the Clp-ATPases ClpX, ClpC1, or ClpC2 exclusively bound to ClpP2, and the killing mechanism of ADEP in Streptomyces.

The functional ClpXP protease of Chlamydia trachomatis requires distinct clpP genes from separate genetic loci.

Clp proteases play a central role in bacterial physiology and, for some bacterial species, are even essential for survival. Also due to their conservation among bacteria including important human pathogens, Clp proteases have recently attracted considerable attention as antibiotic targets. Here, we functionally reconstituted and characterized the ClpXP protease of Chlamydia trachomatis (ctClpXP), an obligate intracellular pathogen and the causative agent of widespread sexually transmitted diseases in humans. Our in vitro data show that ctClpXP is formed by a hetero-tetradecameric proteolytic core, composed of two distinct homologs of ClpP (ctClpP1 and ctClpP2), that associates with the unfoldase ctClpX via ctClpP2 for regulated protein degradation. Antibiotics of the ADEP class interfere with protease functions by both preventing the interaction of ctClpX with ctClpP1P2 and activating the otherwise dormant proteolytic core for unregulated proteolysis. Thus, our results reveal molecular insight into ctClpXP function, validating this protease as an antibacterial target.