Inhibition of Anthrax Lethal Toxin-Induced Cytolysis of RAW264.7 Cells by Celastrol

BACKGROUND: Bacillus anthracis is the bacterium responsible for causing anthrax. The ability of B. anthracis to cause disease is dependent on a secreted virulence factor, lethal toxin, that promotes survival of the bacteria in the host by impairing the immune response. A well-studied effect of lethal toxin is the killing of macrophages, although the molecular mechanisms involved have not been fully characterized. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate that celastrol, a quinone methide triterpene derived from a plant extract used in herbal medicine, inhibits lethal toxin-induced death of RAW264.7 murine macrophages. Celastrol did not prevent cleavage of mitogen activated protein kinase kinase 1, a cytosolic target of the toxin, indicating that it did not inhibit the uptake or catalytic activity of lethal toxin. Surprisingly, celastrol conferred almost complete protection when it was added up to 1.5 h after intoxication, indicating that it could rescue cells in the late stages of intoxication. Since the activity of the proteasome has been implicated in intoxication using other pharmacological agents, we tested whether celastrol blocked proteasome activity. We found that celastrol inhibited the proteasome-dependent degradation of proteins in RAW264.7 cells, but only slightly inhibited proteasome-mediated cleavage of fluorogenic substrates in vitro. Furthermore, celastrol blocked stimulation of IL-18 processing, indicating that celastrol acted upstream of inflammasome activation. CONCLUSIONS/SIGNIFICANCE: This work identifies celastrol as an inhibitor of lethal toxin-mediated macrophage lysis and suggests an inhibitory mechanism involving inhibition of the proteasome pathway

Proteolytic Processing of Nlrp1b Is Required for Inflammasome Activity

Nlrp1b is a NOD-like receptor that detects the catalytic activity of anthrax lethal toxin and subsequently co-oligomerizes into a pro-caspase-1 activation platform known as an inflammasome. Nlrp1b has two domains that promote oligomerization: a NACHT domain, which is a member of the AAA+ ATPase family, and a poorly characterized Function to Find Domain (FIIND). Here we demonstrate that proteolytic processing within the FIIND generates N-terminal and C-terminal cleavage products of Nlrp1b that remain associated in both the auto-inhibited state and in the activated state after cells have been treated with lethal toxin. Functional significance of cleavage was suggested by the finding that mutations that block processing of Nlrp1b also prevent the ability of Nlrp1b to activate pro-caspase-1. By using an uncleaved mutant of Nlrp1b, we established the importance of cleavage by inserting a heterologous TEV protease site into the FIIND and demonstrating that TEV protease processed this site and induced inflammasome activity. Proteolysis of Nlrp1b was shown to be required for the assembly of a functional inflammasome: a mutation within the FIIND that abolished cleavage had no effect on self-association of a FIIND-CARD fragment, but did reduce the recruitment of pro-caspase-1. Our work indicates that a post-translational modification enables Nlrp1b to function

Analysis of ribonucleoprotein complexes involved in transcriptional antitermination

grantor: University of TorontoThe stable association of the 'N' gene transcriptional antiterminator protein of bacteriophage lambda with transcribing RNA polymerase requires a 'nut' site ('boxA' + 'boxB ') in the nascent transcript, and the 'Escherichia coli' factors NusA, NusB, NusG and ribosomal protein S10. In this thesis, it is shown that N binds 'boxB' RNA and that the subsequent association of NusA with the N-'nut' site complex is facilitated by both boxA and 'boxB'. In the presence of N, NusA, and RNA polymerase, ribonucleoprotein complexes containing NusB, NusG, and S10 are assembled on nut RNA. The assembly of NusB, NusG, and S10 is impaired by mutations in nucleotides 2-7 of 'boxA'. The effects on assembly of mutations in 'boxA', 'boxB', NusA and RNA polymerase define weak protein-protein and protein-RNA interactions that contribute to the overall stability of the complex. Interaction of each component of the complex with two or more other components can explain the many observed cooperative binding associations in the DNA-independent assembly of a stable antitermination complex. A series of N fragments has been used to define regions of N that are important for its activity. The amino-terminal arginine-rich region of N (amino acids 1-22) was shown previously to recognize 'boxB' RNA; it is shown here that amino acids 34-47 of N bind NusA and are important for antitermination. The 'boxB'- and NusA-binding regions of N are sufficient to reverse the termination-enhancing effect of NusA, but insufficient to support a maximal level of antitermination. Instead, a region carboxy-terminal to the NusA-binding region can bind RNA polymerase directly and is also critical for antitermination. Antitermination at the ribosomal RNA operons of 'E. coli' is similar to N-mediated antitermination except that only 'boxA' RNA is required. A protein from a crude 'E. coli' extract was purified on the basis of its 'boxA'-binding activity. This protein was identified as ribosomal protein S1 and was shown to bind specifically to 'boxA' RNA. Although S1 competes with NusB and S10 for binding to 'boxA', it does not autonomously regulate antitermination ' in vitro'.Ph.D

The Cytoplasmic Domain of Anthrax Toxin Receptor 1 Affects Binding of the Protective Antigen▿

The protective antigen (PA) component of anthrax toxin binds the I domain of the receptor ANTXR1. Integrin I domains convert between open and closed conformations that bind ligand with high and low affinities, respectively; this process is regulated by signaling from the cytoplasmic domains. To assess whether intracellular signals might influence the interaction between ANTXR1 and PA, we compared two splice variants of ANTXR1 that differ only in their cytoplasmic domains. We found that cells expressing ANTXR1 splice variant 1 (ANTXR1-sv1) bound markedly less PA than did cells expressing a similar level of the shorter splice variant ANTXR1-sv2. ANTXR1-sv1 but not ANTXR1-sv2 associated with the actin cytoskeleton, although disruption of the cytoskeleton did not affect binding of ANTXR-sv1 to PA. Introduction of a cytoplasmic domain missense mutation found in the related receptor ANTXR2 in a patient with juvenile hyaline fibromatosis impaired actin association and increased binding of PA to ANTXR1-sv1. These results suggest that ANTXR1 has two affinity states that may be modulated by cytoplasmic signals

Synthesis of Potent Inhibitors of Anthrax Toxin Based on Poly-l-Glutamic Acid

Telah dilakukan penelitian mengenai pengujian aktivitas antibakteri dari ektrak air daun kecombrang (Etlingera elatior) yang bertujuan memberikan bukti ilmiah keunggulan tanaman kecombrang sebagai bahan pangan fungsional. Pengujian antibakteri dilakukan dengan metode difusi cakram dan identifikasi komponen kimia dengan alat GCMS. Ekstrak air daun kecombrang bersifat antibakteri E. Coli (zona hambat 10 mm/100%), S. Aureus (zona hambat 8,663 mm/20%.  Ekstrak air daun kecombrang memiliki komponen utama 2,3-butanadiol (tR=5,28, area=29,38, kemiripan 90 %) dan fenol (tR= 6,83,area=2,26, kemiripan 90 %). Kata Kunci: kecombrang (Etlingera elatior), pangan fungsional dan antibakter

Binding of Filamentous Actin to Anthrax Toxin Receptor 1 Decreases Its Association with Protective Antigen

ANTXR1 is a type I membrane protein that binds the protective antigen (PA) component of anthrax toxin. The cytosolic domain of ANTXR1 has a novel actin-binding region that influences the interaction of the ectodomain with PA. Here, we have investigated features of the cytosolic domain of ANTXR1 that reduce the association of the receptor with PA. We mutated a stretch of conserved acidic amino acids adjacent to the actin-binding region and found that the mutation increased the affinity for monomeric actin in vitro. ANTXR1 bearing this mutation exhibited increased association with the cytoskeleton and bound less PA compared to the wild-type receptor, confirming the inverse correlation between the two interactions. To determine whether binding of actin is sufficient to regulate the ectodomain, we replaced the actin-binding region of ANTXR1 with that from the yeast protein abp140 and with the WH2 domain of WAVE2. Although both of these domains bound monomeric actin in vitro, only the sequence from abp140 reduced binding of PA to a hybrid receptor. The actin binding regions of ANTXR1 and abp140, but not the WH2 domain, colocalized with actin stress fibers, which suggested that filamentous actin regulates ANTXR1. Consistent with this notion, disruption of actin filaments using latrunculin A increased the amount of PA bound to cells. This work provides evidence that cytoskeletal dynamics regulate ANTXR1 function