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, ...

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.

Fast adaptation to environmental changes ensures bacterial survival, and proteolysis represents a key cellular process in adaptation. The Clp protease system is a multi-component machinery responsible for protein homoeostasis, protein quality control, and targeted proteolysis of transcriptional regulators in prokaryotic cells and prokaryote-derived organelles of eukaryotic cells. A functional Clp protease complex consists of the tetradecameric proteolytic core ClpP and a hexameric ATP-consuming Clp-ATPase, several of which can associate with the same proteolytic core. Clp-ATPases confer substrate specificity by recognising specific degradation tags, and further selectivity is conferred by adaptor proteins, together allowing for a fine-tuned degradation process embedded in elaborate regulatory networks. This review focuses on the contribution of the Clp protease system to prokaryotic survival and summarises the current state of knowledge for exemplary bacteria in an increasing degree of interaction with eukaryotic cells. Starting from free-living bacteria as exemplified by a non-pathogenic and a pathogenic member of the Firmicutes, i.e., <i>Bacillus subtilis</i> and <i>Staphylococcus aureus</i>, respectively, we turn our attention to facultative and obligate intracellular bacterial pathogens, i.e., <i>Mycobacterium tuberculosis, Listeria monocytogenes,</i> and <i>Chlamydia trachomatis</i>, and conclude with mitochondria. Under stress conditions, the Clp protease system exerts its pivotal role in the degradation of damaged proteins and controls the timing and extent of the heat-shock response by regulatory proteolysis. Key regulators of developmental programmes like natural competence, motility, and sporulation are also under Clp proteolytic control. In many pathogenic species, the Clp system is required for the expression of virulence factors and essential for colonising the host. In accordance with its evolutionary origin, the human mitochondrial Clp protease strongly resembles its bacterial counterparts, taking a central role in protein quality control and homoeostasis, energy metabolism, and apoptosis in eukaryotic cells, and several cancer cell types depend on it for proliferation.

An endophytic fungus isolated from the inflorescence axis of Aster tataricus is proposed as a new species. Phylogenetic analyses based on sequences from the ribosomal DNA cluster (the ITS1+5.8S+ITS2, 18S, and 28S regions) and the RPB2 gene revealed a relationship between the unknown fungus and the Stictidaceae lineage of the Ostropales. The new species, Cyanodermella asteris, grows in standard fungal growth media as a fluffy, pink filamentous fungus. Asexual and sexual sporulation has not yet been observed on media or in the plant.

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.