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.

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. Bacterial Clp proteases constitute compartmentalized macromolecular machines. On the molecular level, Clp proteases form large complexes that can be separated into two major components: a proteolytic core formed by a barrel-shaped tetradecamer of ClpP subunits 1 that has to associate with regulatory AAA+ Clp-ATPases (e.g. ClpX and ClpA in Escherichia coli, or ClpX and ClpC in Staphylococcus aureus) to allow for substrate recognition and proteolytic activity 2. In most bacteria including E. coli, S. aureus and Bacillus subtilis, 14 ClpP monomers arrange as two homo-heptameric rings, which stack vis-à-vis to form a cylindrical structure of about 90 Å in both diameter and height. Inside of the compartmentalized ClpP barrel, a spacious degradation chamber of approx. 50 Å width secludes the active sites of the protease located close to the equatorial plane of the ClpP barrel, which comprise 14 catalytic triads with the canonical residues typical for serine proteases (Ser, His, Asp). The compartmentalized structure of the ClpP tetradecamer effectively shields the active sites from potential protein substrates in the cytoplasmic environment, which can only be accessed by small peptides through narrow entry pores at the apical and distal surfaces of the ClpP barrel. ClpP itself is almost free of substrate specificity and is unable to degrade proteins on its own under natural conditions due to restricted substrate access to the inner proteolytic chamber of the ClpP barrel. Only small peptides that readily diffuse through the entrance pores are degraded 3. As such, the ClpP tetradecamer by itself should be considered as a peptidase but constitutes the dormant core of the larger proteolytic Clp complex. For proteolytic activity, the ClpP barrel has to associate with designated Clp-ATPases, hexameric unfoldases, which bind via distinct loops to the buried hydrophobic pockets at the apical sides of the ClpP barrel. The Clp-ATPases select natural Clp substrates, ...

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.

No 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.