Age- and Sex-Specific Reference Values for Renal Volume and Association with Risk Factors for Chronic Kidney Disease in a General Population-An MRI-Based Study.

BACKGROUND: Renal volume (RV) is associated with renal function and with a variety of cardiovascular risk factors (CVRFs). We analysed RV using magnetic resonance imaging (MRI) in a large population-based study (Study of Health in Pomerania; SHIP-TREND) to find sex- and age-specific reference values for RV and to test the influence of several markers on RV. The main objective is to describe reference values for RV in people from the general population without kidney disease. METHODS: 1815 participants without kidney disease (930 women) aged 21-81 years were included in our study. Right and left RV with and without body surface area (BSA) indexation were compared among three age groups (22-39 years, 40-59 years, 60-81 years) by median and interquartile range and tested separately in women and men. RESULTS: The estimated glomerular filtration rate (eGFR), serum uric acid, and right and left RV were higher in men compared to women (all p < 0.001). Left kidneys were larger than right kidneys (both sexes). With age, RV showed a continuously decreasing trend in women and an upside-down U-shaped relation in men. In multivariable linear regression models, current smoking (ß = 14.96, 95% CI 12.12; 17.79), BSA (ß = 97.66, 95% CI 90.4; 104.93), diastolic blood pressure (ß = 0.17, 95% CI 0.01; 0.32), and eGFR (ß = 0.57, 95% CI 0.50; 0.65) were positively associated with both left and right RV, whereas uric acid (ß = -0.03, 95% CI -0.05; -0.01) showed an inverse association with RV. Interestingly, the same eGFR correlated with higher RV in men compared to women. CONCLUSION: Reference values for RV are different for age groups and sex. For any given age, female kidneys are smaller than male kidneys. RV associates positively with eGFR, but for any chosen eGFR, renal volume in females is lower compared to males. RV decreases with age, but in men showed a U-shaped correlation. This may reflect hyperfiltration and glomerular hypertrophy associated with the presence of CVRF in middle-aged males.

Exploring the Translational Gap of a Novel Class of Escherichia coli IspE Inhibitors.

Discovery of novel antibiotics needs multidisciplinary approaches to gain target enzyme and bacterial activities while aiming for selectivity over mammalian cells. Here, we report a multiparameter optimisation of a fragment-like hit that was identified through a structure-based virtual-screening campaign on Escherichia coli IspE crystal structure. Subsequent medicinal-chemistry design resulted in a novel class of E. coli IspE inhibitors, exhibiting activity also against the more pathogenic bacteria Pseudomonas aeruginosa and Acinetobacter baumannii. While cytotoxicity remains a challenge for the series, it provides new insights on the molecular properties for balancing enzymatic target and bacterial activities simultaneously as well as new starting points for the development of IspE inhibitors with a predicted new mode of action.

Molecular Characterization of the ClpC AAA+ ATPase in the Biology of Chlamydia trachomatis.

Bacterial AAA+ unfoldases are crucial for bacterial physiology by recognizing specific substrates and, typically, unfolding them for degradation by a proteolytic component. The caseinolytic protease (Clp) system is one example where a hexameric unfoldase (e.g., ClpC) interacts with the tetradecameric proteolytic core ClpP. Unfoldases can have both ClpP-dependent and ClpP-independent roles in protein homeostasis, development, virulence, and cell differentiation. ClpC is an unfoldase predominantly found in Gram-positive bacteria and mycobacteria. Intriguingly, the obligate intracellular Gram-negative pathogen Chlamydia, an organism with a highly reduced genome, also encodes a ClpC ortholog, implying an important function for ClpC in chlamydial physiology. Here, we used a combination of in vitro and cell culture approaches to gain insight into the function of chlamydial ClpC. ClpC exhibits intrinsic ATPase and chaperone activities, with a primary role for the Walker B motif in the first nucleotide binding domain (NBD1). Furthermore, ClpC binds ClpP1P2 complexes via ClpP2 to form the functional protease ClpCP2P1 in vitro, which degraded arginine-phosphorylated ß-casein. Cell culture experiments confirmed that higher order complexes of ClpC are present in chlamydial cells. Importantly, these data further revealed severe negative effects of both overexpression and depletion of ClpC in Chlamydia as revealed by a significant reduction in chlamydial growth. Here, again, NBD1 was critical for ClpC function. Hence, we provide the first mechanistic insight into the molecular and cellular function of chlamydial ClpC, which supports its essentiality in Chlamydia. ClpC is, therefore, a potential novel target for the development of antichlamydial agents. IMPORTANCE Chlamydia trachomatis is an obligate intracellular pathogen and the world's leading cause of preventable infectious blindness and bacterial sexually transmitted infections. Due to the high prevalence of chlamydial infections along with negative effects of current broad-spectrum treatment strategies, new antichlamydial agents with novel targets are desperately needed. In this context, bacterial Clp proteases have emerged as promising new antibiotic targets, since they often play central roles in bacterial physiology and, for some bacterial species, are even essential for survival. Here, we report on the chlamydial AAA+ unfoldase ClpC, its functional reconstitution and characterization, individually and as part of the ClpCP2P1 protease, and establish an essential role for ClpC in chlamydial growth and intracellular development, thereby identifying ClpC as a potential target for antichlamydial compounds.

E. coli Toxin YjjJ (HipH) Is a Ser/Thr Protein Kinase That Impacts Cell Division, Carbon Metabolism, and Ribosome Assembly.

Protein Ser/Thr kinases are posttranslational regulators of key molecular processes in bacteria, such as cell division and antibiotic tolerance. Here, we characterize the E. coli toxin YjjJ (HipH), a putative protein kinase annotated as a member of the family of HipA-like Ser/Thr kinases, which are involved in antibiotic tolerance. Using SILAC-based phosphoproteomics we provide experimental evidence that YjjJ is a Ser/Thr protein kinase and its primary protein substrates are the ribosomal protein RpmE (L31) and the carbon storage regulator CsrA. YjjJ activity impacts ribosome assembly, cell division, and central carbon metabolism but it does not increase antibiotic tolerance as does its homologue HipA. Intriguingly, overproduction of YjjJ and its kinase-deficient variant can activate HipA and other kinases, pointing to a cross talk between Ser/Thr kinases in E. coli. IMPORTANCE Adaptation to growth condition is the key for bacterial survival, and protein phosphorylation is one of the strategies adopted to transduce extracellular signal in physiological response. In a previous work, we identified YjjJ, a putative kinase, as target of the persistence-related HipA kinase. Here, we performed the characterization of this putative kinase, complementing phenotypical analysis with SILAC-based phosphoproteomics and proteomics. We provide the first experimental evidence that YjjJ is a Ser/Thr protein kinase, having as primary protein substrates the ribosomal protein RpmE (L31) and the carbon storage regulator CsrA. We show that overproduction of YjjJ has a major influence on bacterial physiology, impacting DNA segregation, cell division, glycogen production, and ribosome assembly.

Antibiotics: Precious Goods in Changing Times.

Antibiotics represent a first line of defense of diverse microorganisms, which produce and use antibiotics to counteract natural enemies or competitors for nutritional resources in their nearby environment. For antimicrobial activity, nature has invented a great variety of antibiotic modes of action that involve the perturbation of essential bacterial structures or biosynthesis pathways of macromolecules such as the bacterial cell wall, DNA, RNA, or proteins, thereby threatening the specific microbial lifestyle and eventually even survival. However, along with highly inventive modes of antibiotic action, nature also developed a comparable set of resistance mechanisms that help the bacteria to circumvent antibiotic action. Microorganisms have evolved specific adaptive responses that allow to appropriately react to the presence of antimicrobial agents, thereby ensuring survival during antimicrobial stress. In times of rapid development and spread of antibiotic (multi-)resistance, new resistance-breaking strategies to counteract bacterial infections are desperately needed. This chapter is an update to Chapter 1 of the first edition of this book and intends to give an overview of common antibiotics and their target pathways. It will also present examples for new antibiotics with novel modes of action, illustrating that nature's repertoire of innovative new antimicrobial agents has not been fully exploited yet, and we still might find new drugs that help to evade established antimicrobial resistance strategies.

Microscopy-Based Multiwell Assay to Characterize Disturbed Bacterial Morphogenesis Upon Antibiotic Action.

The urgent need of new antimicrobial agents to combat life-threatening bacterial infections demands the identification and characterization of novel compounds that interfere with new and unprecedented target pathways or structures in multiresistant bacteria. Here, bacterial cell division has emerged as a new and promising target pathway for antibiotic intervention. Compounds, which inhibit division, commonly induce a characteristic filamentation phenotype of rod-shaped bacteria, such as Bacillus subtilis. Hence, this filamentation phenotype can be used to identify and characterize novel compounds that primarily target bacterial cell division. Since novel compounds of both synthetic and natural product origin are often available in small amounts only, thereby limiting the number of assays during mode of action studies, we here describe a semiautomated, microscopy-based approach that requires only small volumes of compounds to allow for the real-time observation of their effects on living bacteria, such as filamentation or cell lysis, in high-throughput 96-well-based formats. We provide a detailed workflow for the initial characterization of multiple compounds at once and further tools for the initial, microscopy-based characterization of their antibacterial mode of action.

Quantitative Analysis of Microscopy Data to Evaluate Bacterial Responses to Antibiotic Treatment.

Microscopy is a powerful method to evaluate the direct effects of antibiotic action on the single cell level. As with other methodologies, microscopy data is obtained through sufficient biological and technical replicate experiments, where evaluation of the sample is generally followed over time. Even if a single antibiotic is tested for a defined time, the most certain outcome is large amounts of raw data that requires systematic analysis. Although microscopy is a helpful qualitative method, the recorded information is stored as defined quantifiable units, the pixels. When this information is transferred to diverse bioinformatic tools, it is possible to analyze the microscopy data while avoiding the inherent bias associated to manual quantification. Here, we briefly describe methods for the analysis of microscopy images using open-source programs, with a special focus on bacteria exposed to antibiotics.

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.

Cervimycin-Resistant Staphylococcus aureus Strains Display Vancomycin-Intermediate Resistant Phenotypes.

Resistance to antibiotics is an increasing problem and necessitates novel antibacterial therapies. The polyketide antibiotics cervimycin A to D are natural products of Streptomyces tendae HKI 0179 with promising activity against multidrug-resistant staphylococci and vancomycin-resistant enterococci. To initiate mode of action studies, we selected cervimycin C- and D-resistant (CmR) Staphylococcus aureus strains. Genome sequencing of CmR mutants revealed amino acid exchanges in the essential histidine kinase WalK, the Clp protease proteolytic subunit ClpP or the Clp ATPase ClpC, and the heat shock protein DnaK. Interestingly, all characterized CmR mutants harbored a combination of mutations in walK and clpP or clpC. In vitro and in vivo analyses showed that the mutations in the Clp proteins abolished ClpP or ClpC activity, and the deletion of clpP rendered S. aureus but not all Bacillus subtilis strains cervimycin-resistant. The essential gene walK was the second mutational hotspot in the CmR S. aureus strains, which decreased WalK activity in vitro and generated a vancomycin-intermediate resistant phenotype, with a thickened cell wall, a lower growth rate, and reduced cell lysis. Transcriptomic and proteomic analyses revealed massive alterations in the CmR strains compared to the parent strain S. aureus SG511, with major shifts in the heat shock regulon, the metal ion homeostasis, and the carbohydrate metabolism. Taken together, mutations in the heat shock genes clpP, clpC, and dnaK, and the walK kinase gene in CmR mutants induced a vancomycin-intermediate resistant phenotype in S. aureus, suggesting cell wall metabolism or the Clp protease system as primary target of cervimycin. IMPORTANCE Staphylococcus aureus is a frequent cause of infections in both the community and hospital setting. Resistance development of S. aureus to various antibiotics is a severe problem for the treatment of this pathogen worldwide. New powerful antimicrobial agents against Gram-positives are needed, since antibiotics like vancomycin fail to cure vancomycin-intermediate resistant S. aureus (VISA) and vancomycin-resistant enterococci (VRE) infections. One candidate substance with promising activity against these organisms is cervimycin, which is an antibiotic complex with a yet unknown mode of action. In our study, we provide first insights into the mode of action of cervimycins. By characterizing cervimycin-resistant S. aureus strains, we revealed the Clp system and the essential kinase WalK as mutational hotspots for cervimycin resistance in S. aureus. It further emerged that cervimycin-resistant S. aureus strains show a VISA phenotype, indicating a role of cervimycin in perturbing the bacterial cell envelope.

Native metabolomics identifies the rivulariapeptolide family of protease inhibitors.

The identity and biological activity of most metabolites still remain unknown. A bottleneck in the exploration of metabolite structures and pharmaceutical activities is the compound purification needed for bioactivity assignments and downstream structure elucidation. To enable bioactivity-focused compound identification from complex mixtures, we develop a scalable native metabolomics approach that integrates non-targeted liquid chromatography tandem mass spectrometry and detection of protein binding via native mass spectrometry. A native metabolomics screen for protease inhibitors from an environmental cyanobacteria community reveals 30 chymotrypsin-binding cyclodepsipeptides. Guided by the native metabolomics results, we select and purify five of these compounds for full structure elucidation via tandem mass spectrometry, chemical derivatization, and nuclear magnetic resonance spectroscopy as well as evaluation of their biological activities. These results identify rivulariapeptolides as a family of serine protease inhibitors with nanomolar potency, highlighting native metabolomics as a promising approach for drug discovery, chemical ecology, and chemical biology studies.

The hypersusceptible antibiotic screening strain Staphylococcus aureus SG511-Berlin harbors multiple mutations in regulatory genes.

The genetic plasticity of Staphylococcus aureus has facilitated the evolution of many virulent and drug-resistant strains. Here we present the sequence of the 2.74 Mbp genome of S. aureus SG511-Berlin, which is frequently used for antibiotic screening. Although S. aureus SG511 and the related methicillin-resistant S. aureus MRSA252 share a high similarity in their core genomes, indicated by an average nucleotide identity (ANI) of 99.83%, the accessory genomes of these strains differed, as nearly no mobile elements and resistance determinants were identified in the genome of S. aureus SG511. Susceptibility testing showed that S. aureus SG511 was susceptible to most of the tested antibiotics of different classes. Intriguingly, and in contrast to the standard laboratory strain S. aureus HG001, S. aureus SG511 was even hyper-susceptible towards cell wall and membrane targeting agents, with the exception of the MurA-inhibitor fosfomycin. In depth comparative genome analysis revealed that, in addition to the loss of function mutation in the antibiotic sensor histidine kinase gene graS, further mutations had occurred in the lysyltransferase gene mprF, the structural giant protein gene ebh, and the regulator genes codY and saeR, which might contribute to antibiotic susceptibility. In addition, an insertion element in agrC abolishes Agr-activity in S. aureus SG511, and the spa and sarS genes, which encode the surface protein SpA and its transcriptional regulator, were deleted. Thus, the lack of mobile resistance genes together with multiple mutations affecting cell envelope morphology may render S. aureus SG511 hyper-susceptible towards most cell wall targeting agents.

Progression of the late-stage divisome is unaffected by the depletion of the cytoplasmic FtsZ pool.

Cell division is a central and essential process in most bacteria, and also due to its complexity and highly coordinated nature, it has emerged as a promising new antibiotic target pathway in recent years. We have previously shown that ADEP antibiotics preferably induce the degradation of the major cell division protein FtsZ, thereby primarily leading to a depletion of the cytoplasmic FtsZ pool that is needed for treadmilling FtsZ rings. To further investigate the physiological consequences of ADEP treatment, we here studied the effect of ADEP on the different stages of the FtsZ ring in rod-shaped bacteria. Our data reveal the disintegration of early FtsZ rings during ADEP treatment in Bacillus subtilis, indicating an essential role of the cytoplasmic FtsZ pool and thus FtsZ ring dynamics during initiation and maturation of the divisome. However, progressed FtsZ rings finalized cytokinesis once the septal peptidoglycan synthase PBP2b, a late-stage cell division protein, colocalized at the division site, thus implying that the concentration of the cytoplasmic FtsZ pool and FtsZ ring dynamics are less critical during the late stages of divisome assembly and progression.

Cell Division Protein FtsZ Is Unfolded for N-Terminal Degradation by Antibiotic-Activated ClpP.

Antibiotic acyldepsipeptides (ADEPs) deregulate ClpP, the proteolytic core of the bacterial Clp protease, thereby inhibiting its native functions and concomitantly activating it for uncontrolled proteolysis of nonnative substrates. Importantly, although ADEP-activated ClpP is assumed to target multiple polypeptide and protein substrates in the bacterial cell, not all proteins seem equally susceptible. In Bacillus subtilis, the cell division protein FtsZ emerged to be particularly sensitive to degradation by ADEP-activated ClpP at low inhibitory ADEP concentrations. In fact, FtsZ is the only bacterial protein that has been confirmed to be degraded in vitro as well as within bacterial cells so far. However, the molecular reason for this preferred degradation remained elusive. Here, we report the unexpected finding that ADEP-activated ClpP alone, in the absence of any Clp-ATPase, leads to an unfolding and subsequent degradation of the N-terminal domain of FtsZ, which can be prevented by the stabilization of the FtsZ fold via nucleotide binding. At elevated antibiotic concentrations, importantly, the C terminus of FtsZ is notably targeted for degradation in addition to the N terminus. Our results show that different target structures are more or less accessible to ClpP, depending on the ADEP level present. Moreover, our data assign a Clp-ATPase-independent protein unfolding capability to the ClpP core of the bacterial Clp protease and suggest that the protein fold of FtsZ may be more flexible than previously anticipated.IMPORTANCE Acyldepsipeptide (ADEP) antibiotics effectively kill multidrug-resistant Gram-positive pathogens, including vancomycin-resistant enterococcus, penicillin-resistant Streptococcus pneumoniae (PRSP), and methicillin-resistant Staphylococcus aureus (MRSA). The antibacterial activity of ADEP depends on a new mechanism of action, i.e., the deregulation of bacterial protease ClpP that leads to bacterial self-digestion. Our data allow new insights into the mode of ADEP action by providing a molecular explanation for the distinct bacterial phenotypes observed at low versus high ADEP concentrations. In addition, we show that ClpP alone, in the absence of any unfoldase or energy-consuming system, and only activated by the small molecule antibiotic ADEP, leads to the unfolding of the cell division protein FtsZ.

Cell division protein FtsZ: from structure and mechanism to antibiotic target.

Antimicrobial resistance to virtually all clinically applied antibiotic classes severely limits the available options to treat bacterial infections. Hence, there is an urgent need to develop and evaluate new antibiotics and targets with resistance-breaking properties. Bacterial cell division has emerged as a new antibiotic target pathway to counteract multidrug-resistant pathogens. New approaches in antibiotic discovery and bacterial cell biology helped to identify compounds that either directly interact with the major cell division protein FtsZ, thereby perturbing the function and dynamics of the cell division machinery, or affect the structural integrity of FtsZ by inducing its degradation. The impressive antimicrobial activities and resistance-breaking properties of certain compounds validate the inhibition of bacterial cell division as a promising strategy for antibiotic intervention.

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.

Staphylococcus aureus Lpl protein triggers human host cell invasion via activation of Hsp90 receptor.

Staphylococcus aureus is a facultative intracellular pathogen. Recently, it has been shown that the protein part of the lipoprotein-like lipoproteins (Lpls), encoded by the lpl cluster comprising of 10 lpls paralogue genes, increases pathogenicity, delays the G2/M phase transition, and also triggers host cell invasion. Here, we show that a recombinant Lpl1 protein without the lipid moiety binds directly to the isoforms of the human heat shock proteins Hsp90α and Hsp90ß. Synthetic peptides covering the Lpl1 sequence caused a twofold to fivefold increase of S. aureus invasion in HaCaT cells. Antibodies against Hsp90 decrease S. aureus invasion in HaCaT cells and in primary human keratinocytes. Additionally, inhibition of ATPase function of Hsp90 or silencing Hsp90α expression by siRNA also decreased the S. aureus invasion in HaCaT cells. Although the Hsp90ß is constitutively expressed, the Hsp90α isoform is heat-inducible and appears to play a major role in Lpl1 interaction. Pre-incubation of HaCaT cells at 39°C increased both the Hsp90α expression and S. aureus invasion. Lpl1-Hsp90 interaction induces F-actin formation, thus, triggering an endocytosis-like internalisation. Here, we uncovered a new host cell invasion principle on the basis of Lpl-Hsp90 interaction.

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.

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.

Consequences of dosing and timing on the antibacterial effects of ADEP antibiotics.

Antibiotic acyldepsipeptides (ADEPs) exert potent antibacterial activity in rodent models of bacterial infection and exceptional efficacy against persister cells of methicillin-resistant Staphylococcus aureus (MRSA). The mechanism of ADEP action is unusual in that the antibiotic releases the destructive capacity of over-activated ClpP, the proteolytic core of the bacterial Clp protease. The essential bacterial cell division protein FtsZ had emerged in a previous study as a preferred protein substrate of ADEP-activated ClpP but it is definitely not the only cellular substrate. In the current study, we set out to follow the morphological changes that lead to ADEP-mediated bacterial death in S. aureus and Bacillus subtilis, differentiating between antibacterial effects at low and high ADEP concentrations. Here, fluorescence and time-lapse microscopy data show that cells adopt a characteristic phenotype of cell division inhibition at ADEP levels close to the MIC, but retain the capacity to form viable daughter cells for a substantial period of time when transferred to ADEP-free growth medium. After extended exposure to low ADEP concentrations, nucleoids of B. subtilis started to disorganize and upon compound removal many cells failed to re-organize nucleoids, re-initiate cytokinesis and consequently died. Survival versus cell death of filamentous cells attempting recovery depended on the timing of completion of new septa in relation to the loss of cell envelope integrity. We show that the potential to recover after ADEP removal depends on the antibiotic concentration as well as the treatment duration. When exposed to ADEP at concentrations well above the MIC, biomass production ceased rapidly as did the potential to recover. In time-kill studies both long-time exposure to low ADEP levels as well as short-time exposure to high concentrations proved highly effective, while intermittent concentrations and time frames were not. We here provide new insights into the antimicrobial activity of ADEP antibiotics and the consequences of dosing and timing for bacterial physiology which should be considered in view of a potential therapeutic application of ADEPs.