Vibrio cholerae ParE2 toxin modulates its operon transcription by stabilization of an antitoxin DNA ruler

The parDE2 operon of Vibrio cholerae encodes a type II TA system, which is one of three loci in the superintegron of small chromosome II that show modest similarity to the parDE operon of plasmid RK2. ParE2, like plasmid RK2-encoded ParE, inhibits DNA gyrase, an essential topoisomerase that is also the target of quinolone antibacterial agents. Mechanistic understanding on ParE2 toxin inhibition by direct interaction with its cognate antitoxin and transcriptional autoregulation of the TA system are currently lacking. ParD2, the ribbon-helix-helix (RHH) antitoxin, auto-represses the parDE2 promoter. This repression is enhanced by ParE2, which therefore functions as a transcriptional co-repressor. Here we present protein-DNA interaction studies and high-resolution X-ray structures of the ParD2:ParE2 complex and isolated ParD2 antitoxin, revealing the basis of toxin inhibition and autoregulation of the TA operon by conditional cooperativity. Native mass spectrometry, SAXS and MALS studies confirm the presence of different oligomerization states of ParD2 in solution and the role of the DNA-binding hexameric ParD26:ParE22 assembly in transcriptional repression.

Toxin–Antitoxin Systems in Pathogenic Bacteria

Toxin–antitoxin (TA) systems, which are ubiquitously present in plasmids, bacterial and archaeal genomes, are classified as types I to VI, according to the nature of the antitoxin and to the mode of toxin inhibition [...]

Phage proteins block and trigger retron toxin/antitoxin systems

ABSTRACTBacteria carry dozens of Toxin/Antitoxin systems (TAs) in their chromosomes. Upon growth, the antitoxin is co-expressed and neutralizes the toxin. TAs can be activated and inhibit growth, but when and how this occurs has largely remained enigmatic, hindering our understanding of their physiological roles. We developed TIC/TAC (Toxin Inhibition/Activation Conjugation), a high-throughput reverse genetics approach, to systematically identify molecular blockers and triggers of TAs. By applying TIC/TAC to a tripartite TA, the retron-Sen2 of Salmonella Typhimurium, we have identified multiple blockers and triggers of phage origin. We demonstrate that diverse phage functionalities are sensed by the DNA-part of the antitoxin and ultimately activate the retron toxin. Phage-origin proteins can circumvent activation by directly blocking the toxin. Some identified triggers and blockers also act on an E. coli retron-TA, Eco9. We propose that retron-TAs act as abortive-infection anti-phage defense systems, and delineate mechanistic principles by which phages trigger or block them.

whISOBAXTM Inhibits Bacterial Pathogenesis and Enhances the Effect of Antibiotics

As bacteria are becoming more resistant to commonly used antibiotics, alternative therapies are being sought. whISOBAX (WH) is a witch hazel extract that is highly stable (tested up to 2 months in 37 °C) and contains a high phenolic content, where 75% of it is hamamelitannin and traces of gallic acid. Phenolic compounds like gallic acid are known to inhibit bacterial growth, while hamamelitannin is known to inhibit staphylococcal pathogenesis (biofilm formation and toxin production). WH was tested in vitro for its antibacterial activity against clinically relevant Gram-positive and Gram-negative bacteria, and its synergy with antibiotics determined using checkerboard assays followed by isobologram analysis. WH was also tested for its ability to suppress staphylococcal pathogenesis, which is the cause of a myriad of resistant infections. Here we show that WH inhibits the growth of all bacteria tested, with variable efficacy levels. The most WH-sensitive bacteria tested were Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecium and Enterococcus faecalis, followed by Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Streptococcus agalactiae and Streptococcus pneumoniae. Furthermore, WH was shown on S. aureus to be synergistic to linezolid and chloramphenicol and cumulative to vancomycin and amikacin. The effect of WH was tested on staphylococcal pathogenesis and shown here to inhibit biofilm formation (tested on S. epidermidis) and toxin production (tested on S. aureus Enterotoxin A (SEA)). Toxin inhibition was also evident in the presence of subinhibitory concentrations of ciprofloxacin that induces pathogenesis. Put together, our study indicates that WH is very effective in inhibiting the growth of multiple types of bacteria, is synergistic to antibiotics, and is also effective against staphylococcal pathogenesis, often the cause of persistent infections. Our study thus suggests the benefits of using WH to combat various types of bacterial infections, especially those that involve resistant persistent bacterial pathogens.

Molecular structures of the human Slo1 K+ channel in complex with β4

Slo1 is a Ca2+- and voltage-activated K+ channel that underlies skeletal and smooth muscle contraction, audition, hormone secretion and neurotransmitter release. In mammals, Slo1 is regulated by auxiliary proteins that confer tissue-specific gating and pharmacological properties. This study presents cryo-EM structures of Slo1 in complex with the auxiliary protein, β4. Four β4, each containing two transmembrane helices, encircle Slo1, contacting it through helical interactions inside the membrane. On the extracellular side, β4 forms a tetrameric crown over the pore. Structures with high and low Ca2+ concentrations show that identical gating conformations occur in the absence and presence of β4, implying that β4 serves to modulate the relative stabilities of ‘pre-existing’ conformations rather than creating new ones. The effects of β4 on scorpion toxin inhibition kinetics are explained by the crown, which constrains access but does not prevent binding.

Intrinsic Toxin-Derived Peptides Destabilize and Inactivate Clostridium difficile TcdB

ABSTRACT Clostridium difficile infection (CDI) is a major cause of hospital-associated, antibiotic-induced diarrhea, which is largely mediated by the production of two large multidomain clostridial toxins, TcdA and TcdB. Both toxins coordinate the action of specific domains to bind receptors, enter cells, and deliver a catalytic fragment into the cytosol. This results in GTPase inactivation, actin disassembly, and cytotoxicity. TcdB in particular has been shown to encode a region covering amino acids 1753 to 1851 that affects epitope exposure and cytotoxicity. Surprisingly, studies here show that several peptides derived from this region, which share the consensus sequence 1769NVFKGNTISDK1779, protect cells from the action of TcdB. One peptide, PepB2, forms multiple interactions with the carboxy-terminal region of TcdB, destabilizes TcdB structure, and disrupts cell binding. We further show that these effects require PepB2 to form a higher-order polymeric complex, a process that requires the central GN amino acid pair. These data suggest that TcdB1769–1779 interacts with repeat sequences in the proximal carboxy-terminal domain of TcdB (i.e., the CROP domain) to alter the conformation of TcdB. Furthermore, these studies provide insights into TcdB structure and functions that can be exploited to inactivate this critical virulence factor and ameliorate the course of CDI. IMPORTANCE Clostridium difficile is a leading cause of hospital-associated illness that is often associated with antibiotic treatment. To cause disease, C. difficile secretes toxins, including TcdB, which is a multidomain intracellular bacterial toxin that undergoes conformational changes during cellular intoxication. This study describes the development of peptide-based inhibitors that target a region of TcdB thought to be critical for structural integrity of the toxin. The results show that peptides derived from a structurally important region of TcdB can be used to destabilize the toxin and prevent cellular intoxication. Importantly, this work provides a novel means of toxin inhibition that could in the future develop into a C. difficile treatment. IMPORTANCE Clostridium difficile is a leading cause of hospital-associated illness that is often associated with antibiotic treatment. To cause disease, C. difficile secretes toxins, including TcdB, which is a multidomain intracellular bacterial toxin that undergoes conformational changes during cellular intoxication. This study describes the development of peptide-based inhibitors that target a region of TcdB thought to be critical for structural integrity of the toxin. The results show that peptides derived from a structurally important region of TcdB can be used to destabilize the toxin and prevent cellular intoxication. Importantly, this work provides a novel means of toxin inhibition that could in the future develop into a C. difficile treatment.