The Dual Mode of Antibacterial Action of the Synthetic Small Molecule DCAP Involves Lipid II Binding.

The synthetic small molecule DCAP is a chemically well-characterized compound with antibiotic activity against Gram-positive and Gram-negative bacteria, including drug-resistant pathogens. Until now, its mechanism of action was proposed to rely exclusively on targeting the bacterial membrane, thereby causing membrane depolarization, and increasing membrane permeability (Eun et al. 2012, J. Am. Chem. Soc. 134 (28), 11322-11325; Hurley et al. 2015, ACS Med. Chem. Lett. 6, 466-471). Here, we show that the antibiotic activity of DCAP results from a dual mode of action that is more targeted and multifaceted than previously anticipated. Using microbiological and biochemical assays in combination with fluorescence microscopy, we provide evidence that DCAP interacts with undecaprenyl pyrophosphate-coupled cell envelope precursors, thereby blocking peptidoglycan biosynthesis and impairing cell division site organization. Our work discloses a concise model for the mode of action of DCAP which involves the binding to a specific target molecule to exert pleiotropic effects on cell wall biosynthetic and divisome machineries.

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.

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.

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.

A new host cell internalisation pathway for SadA-expressing staphylococci triggered by excreted neurochemicals.

Staphylococcus aureus is a facultative intracellular pathogen that invades a wide range of professional and nonprofessional phagocytes by triggering internalisation by interaction of surface-bound adhesins with corresponding host cell receptors. Here, we identified a new concept of host cell internalisation in animal-pathogenic staphylococcal species. This new mechanism exemplified by Staphylococcus pseudintermedius ED99 is not based on surface-bound adhesins but is due to excreted small neurochemical compounds, such as trace amines (TAs), dopamine (DOP), and serotonin (SER), that render host cells competent for bacterial internalisation. The neurochemicals are produced by only one enzyme, the staphylococcal aromatic amino acid decarboxylase (SadA). Here, we unravelled the mechanism of how neurochemicals trigger internalisation into the human colon cell line HT-29. We found that TAs and DOP are agonists of the α2-adrenergic receptor, which, when activated, induces a cascade of reactions involving a decrease in the cytoplasmic cAMP level and an increase in F-actin formation. The signalling cascade of SER follows a different pathway. SER interacts with 5HT receptors that trigger F-actin formation without decreasing the cytoplasmic cAMP level. The neurochemical-induced internalisation in host cells is independent of the fibronectin-binding protein pathway and has an additive effect. In a sadA deletion mutant, ED99ΔsadA, internalisation was decreased approximately threefold compared with that of the parent strain, and treating S. aureus USA300 with TAs increased internalisation by approximately threefold.

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.

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.

Acyldepsipeptide antibiotics kill mycobacteria by preventing the physiological functions of the ClpP1P2 protease.

The Clp protease complex in Mycobacterium tuberculosis is unusual in its composition, functional importance and activation mechanism. Whilst most bacterial species contain a single ClpP protein that is dispensable for normal growth, mycobacteria have two ClpPs, ClpP1 and ClpP2, which are essential for viability and together form the ClpP1P2 tetradecamer. Acyldepsipeptide antibiotics of the ADEP class inhibit the growth of Gram-positive firmicutes by activating ClpP and causing unregulated protein degradation. Here we show that, in contrast, mycobacteria are killed by ADEP through inhibition of ClpP function. Although ADEPs can stimulate purified M. tuberculosis ClpP1P2 to degrade larger peptides and unstructured proteins, this effect is weaker than for ClpP from other bacteria and depends on the presence of an additional activating factor (e.g. the dipeptide benzyloxycarbonyl-leucyl-leucine in vitro) to form the active ClpP1P2 tetradecamer. The cell division protein FtsZ, which is a particularly sensitive target for ADEP-activated ClpP in firmicutes, is not degraded in mycobacteria. Depletion of the ClpP1P2 level in a conditional Mycobacterium bovis BCG mutant enhanced killing by ADEP unlike in other bacteria. In summary, ADEPs kill mycobacteria by preventing interaction of ClpP1P2 with the regulatory ATPases, ClpX or ClpC1, thus inhibiting essential ATP-dependent protein degradation.

The cell wall precursor lipid II acts as a molecular signal for the Ser/Thr kinase PknB of Staphylococcus aureus.

The assembly of the bacterial cell wall requires synchronization of a multitude of biosynthetic machineries and regulatory networks. The eukaryotic-like serine/threonine kinase PknB has been implicated in coordinating cross-wall formation, autolysis and cell division in Staphylococcus aureus. However, the signal molecule sensed by this kinase remained elusive so far. Here, we provide compelling biochemical evidence that PknB interacts with the ultimate cell wall precursor lipid II, triggering kinase activity. Moreover, we observed crosstalk of PknB with the two component system WalKR and identified the early cell division protein FtsZ as another PknB phosphorylation substrate in S. aureus. In agreement with the implied role in regulation of cell envelope metabolism, we found PknB to preferentially localize to the septum of S. aureus and the PASTA domains to be crucial for recruitment to this site. The data provide a model for the contribution of PknB to control cell wall metabolism and cell division.

[E-learning in ENT: Usage in University Medical Centers in Germany]. / Einsatz von E-Learning an deutschen Universitäts-HNO-Kliniken.

E-learning is an essential part of innovative medical teaching concepts. The challenging anatomy and physiology in ENT is considered particularly suitable for self-assessed and adaptive e-learning. Usage and data on daily experience with e-learning in German ENT-university hospitals are currently unavailable and the degree of implementation of blended learning including feed-back from medical students are currently not known. We investigated the current need and usage of e-learning in academic ENT medical centers in Germany. We surveyed students and chairs for Otorhinolaryngology electronically and paperbased during the summer semester 2015. Our investigation revealed an overall heterogenous picture on quality and quantity of offered e-learning applications. While the overall amount of e-learning in academic ENT in Germany is rather low, at least half of the ENT-hospitals in medical faculties reported that e-learning had improved their own teaching activities. More collaboration among medical faculties and academic ENT-centers may help to explore new potentials, overcome technical difficulties and help to realize more ambitious projects.

Generation of a vancomycin-intermediate Staphylococcus aureus (VISA) strain by two amino acid exchanges in VraS.

OBJECTIVES: Staphylococcus aureus is a notorious bacterial pathogen and antibiotic-resistant isolates complicate current treatment strategies. We characterized S. aureus VC40, a laboratory mutant that shows full resistance to glycopeptides (vancomycin and teicoplanin MICs ≥32 mg/L) and daptomycin (MIC = 4 mg/L), to gain deeper insights into the underlying resistance mechanisms. METHODS: Genomics and transcriptomics were performed to characterize changes that might contribute to development of resistance. The mutations in vraS were reconstituted into a closely related parental background. In addition, antimicrobial susceptibility testing, growth analyses, transmission electron microscopy, lysostaphin-induced lysis and autolysis assays were performed to characterize the phenotype of resistant strains. RESULTS: Genome sequencing of strain VC40 revealed 79 mutations in 75 gene loci including genes encoding the histidine kinases VraS and WalK that control cell envelope-related processes. Transcriptomics indicated the increased expression of their respective regulons. Although not reaching the measured MIC for VC40, reconstitution of the L114S and D242G exchanges in VraS(VC40) into the susceptible parental background (S. aureus NCTC 8325) resulted in increased resistance to glycopeptides and daptomycin. The expression of VraS(VC40) led to increased transcription of the cell wall stress stimulon, a thickened cell wall, a decreased growth rate, reduced autolytic activity and increased resistance to lysostaphin-induced lysis in the generated mutant. CONCLUSIONS: We show that a double mutation of a single gene locus, namely vraS, is sufficient to convert the vancomycin-susceptible strain S. aureus NCTC 8325 into a vancomycin-intermediate S. aureus.

Bacterial caseinolytic proteases as novel targets for antibacterial treatment.

Bacterial Clp proteases are important for protein turnover and homeostasis in order to maintain vital cellular functions particularly under stress conditions. Apart from their crucial role in general protein quality control by degrading abnormally folded or otherwise aberrant or malfunctioning proteins, their temporally and spatially precise proteolysis of key regulatory proteins additionally guides several developmental processes like cell motility, genetic competence, cell differentiation, sporulation as well as important aspects of virulence. Due to their apparent relevance for many physiological processes and their conservation among diverse bacterial species including human pathogens, Clp proteases have attracted considerable attention as targets for antibacterial action in recent years. Particularly a novel class of potent acyldepsipeptide antibiotics unleashes ClpP, the uniform proteolytic core unit of the degradative Clp complexes, to bring about bacterial death via uncontrolled proteolysis of proteins that are essential for bacterial viability. In addition, covalent inhibition of the catalytic center of ClpP by another class of small molecule inhibitors is investigated in the context of virulence inhibition. Both antibacterial mechanisms constitute innovative approaches with the potential to control infections caused by multi-resistant bacterial pathogens due to the lack of cross-resistance to established antibiotic classes.

Antibiotic acyldepsipeptides activate ClpP peptidase to degrade the cell division protein FtsZ.

The worldwide spread of antibiotic-resistant bacteria has lent urgency to the search for antibiotics with new modes of action that are devoid of preexisting cross-resistances. We previously described a unique class of acyldepsipeptides (ADEPs) that exerts prominent antibacterial activity against Gram-positive pathogens including streptococci, enterococci, as well as multidrug-resistant Staphylococcus aureus. Here, we report that ADEP prevents cell division in Gram-positive bacteria and induces strong filamentation of rod-shaped Bacillus subtilis and swelling of coccoid S. aureus and Streptococcus pneumoniae. It emerged that ADEP treatment inhibits septum formation at the stage of Z-ring assembly, and that central cell division proteins delocalize from midcell positions. Using in vivo and in vitro studies, we show that the inhibition of Z-ring formation is a consequence of the proteolytic degradation of the essential cell division protein FtsZ. ADEP switches the bacterial ClpP peptidase from a regulated to an uncontrolled protease, and it turned out that FtsZ is particularly prone to degradation by the ADEP-ClpP complex. By preventing cell division, ADEP inhibits a vital cellular process of bacteria that is not targeted by any therapeutically applied antibiotic so far. Their unique multifaceted mechanism of action and antibacterial potency makes them promising lead structures for future antibiotic development.

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.

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.

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.