Peter Sass

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.

AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control

The Clp protease complex degrades a multitude of substrates, which are engaged by a AAA+ chaperone such as ClpX and subsequently digested by the dynamic, barrel-shaped ClpP protease. Acyldepsipeptides (ADEPs) are natural product-derived antibiotics that activate ClpP for chaperone-independent protein digestion. Here we show that both protein and small-molecule activators of ClpP allosterically control the ClpP barrel conformation. We dissect the catalytic mechanism with chemical probes and show that ADEP in addition to opening the axial pore directly stimulates ClpP activity through cooperative binding. ClpP activation thus reaches beyond active site accessibility and also involves conformational control of the catalytic residues. Moreover, we demonstrate that substoichiometric amounts of ADEP potently prevent binding of ClpX to ClpP and, at the same time, partially inhibit ClpP through conformational perturbance. Collectively, our results establish the hydrophobic binding pocket as a major conformational regulatory site with implications for both ClpXP proteolysis and ADEP-based anti-bacterial activity.

Atropisomeric Dihydroanthracenones as Inhibitors of Multiresistant Staphylococcus aureus

Two bisdihydroanthracenone atropodiastereomeric pairs, including homodimeric flavomannin A (1) and the previously unreported flavomannin B (2), two new unsymmetrical dimers (3 and 4), and two new mixed dihydroanthracenone/anthraquinone dimers (5 and 6) were isolated from Talaromyces wortmannii , an endophyte of Aloe vera . The structures of 2-6 were elucidated by extensive NMR and mass spectrometric analyses. The axial chirality of the biaryls was determined using TDDFT ECD and VCD calculations, the combination of which however did not allow the assignment of the central chirality elements of 1. The compounds exhibited antibacterial activity against Staphylococcus aureus , including (multi)drug-resistant clinical isolates. Reporter gene analyses indicated induction of the SOS response for some of the derivatives, suggesting interference with DNA structure or metabolism. Fluorescence microscopy demonstrated defective segregation of the bacterial chromosome and DNA degradation. Notably, the compounds showed no cytotoxic activity, encouraging their further evaluation as potential starting points for antibacterial drug development.

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.

Bacterial cell division as a target for new antibiotics.

Bacterial resistance to currently applied antibiotics complicates the treatment of infections and demands the evaluation of new strategies to counteract multidrug-resistant bacteria. In recent years, the inhibition of the bacterial divisome, mainly by targeting the central cell division mediator FtsZ, has been recognized as a promising strategy for antibiotic attack. New antibiotics were shown to either interfere with the natural dynamics and functions of FtsZ during the cell cycle or to activate a bacterial protease to degrade FtsZ and thus bring about bacterial death in a suicidal manner. Their efficacy in animal models of infection together with resistance-breaking properties prove the potential of such drugs and validate the inhibition of bacterial cell division as an attractive approach for antibiotic intervention.

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.

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.

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.

A highly asynchronous developmental program triggered during germination of dormant akinetes of filamentous diazotrophic cyanobacteria

Germination of akinetes of filamentous heterocyst-forming cyanobacteria of the order Nostocales is an essential process that ensures survival and recolonization after long periods of unfavorable conditions, as desiccation, cold and low light. We studied the morphological, physiological and metabolic changes that occur during germination of akinetes in two model species of cell differentiation, Anabaena variabilis ATCC 29413 and Nostoc punctiforme ATCC 29133, which live in different habitats. We characterized the akinete envelopes and showed their similarity to envelopes of N2-fixing heterocysts. Akinete germination started inside the envelopes and was dependent on light intensity but independent of nitrogen supply. During the germination of A. variabilis akinetes, cell division and heterocyst differentiation were highly accelerated. The energy for cell division was initially supplied by respiration of glycogen and subsequently by photosynthesis. By contrast, during germination of N. punctiforme akinetes, cell division and heterocyst differentiation were slow. During the initial 15-20 h, N. punctiforme akinetes increased in volume and some burst. Only then did intact akinetes start to divide and fully germinate, possibly fueled by nutrients released from dead akinetes. The different strategies used by these different cyanobacteria allow successful germination of dormant cells and recolonization under favorable conditions.

Taking a Fresh Look at the Skull Base in Otorhinolaryngology With Web-Based Simulation Student's Interactive Skull-Base Trainer (SISTer)

IMPORTANCE The increasing amount of medical knowledge and necessity for time-effective teaching and learning have given rise to emerging online, or e-learning, applications. The base of the skull is a challenging anatomic area in the otorhinolaryngology (ORL) department-for both students and lecturers. Technology-enhanced learning might be an expedient approach to benefit both learners and lecturers. OBJECTIVE To investigate and create for advanced medical students a self-assessed adaptive e-learning application for the skull base within our curriculum of otolaryngology at the University Medical Center of Heinrich Heine University, Düsseldorf, Germany. DESIGN, SETTING, AND PARTICIPANTS Pilot approach with prospective evaluation of a newly implemented web-based e-learning simulation. The e-learning application (Students Interactive Skull-Base Trainer) was made accessible as an elective course to a total of 269 enrolled medical students during the first 2 semesters after web launch. INTERVENTIONS Spatiotemporal independent e-learning application for the skull base. MAIN OUTCOMES AND MEASURES Self-assessed evaluation with focus on general acceptance and personal value as well as usage data analysis. RESULTS The application was well accepted by the learners. More than 80% of the participating students found the application to be a beneficial tool for enhancing their analytical and clinical problem-solving skills. Although the general matter of the skull base seemed to be of lesser interest, the concept of anchored instructions with the use of high-end, interactive, multimedia-based content was considered to be particularly suitable for this challenging topic. Most of the students would have appreciated an extension of optional e-learning modules. CONCLUSIONS AND RELEVANCE With this pilot approach we were able to implement a useful and now well-accepted tool for blended learning. We showed that it is possible to raise interest even in this very specialized subspecialty of ORL with overall individual learning benefit for the students. There is a demand for more e-learning and web-based simulation to support the existing curricula in a hybrid, blended way.