Browse
Submit Data
Databases
API
Help

Dataset Information

12 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Crystal structure of Bacillus cereus HlyIIR, a transcriptional regulator of the gene for pore-forming toxin hemolysin II.

ABSTRACT: Production of Bacillus cereus and Bacillus anthracis toxins is controlled by a number of transcriptional regulators. Here we report the crystal structure of B. cereus HlyIIR, a regulator of the gene encoding the pore-forming toxin hemolysin II. We show that HlyIIR forms a tight dimer with a fold and overall architecture similar to the TetR family of repressors. A remarkable feature of the structure is a large internal cavity with a volume of 550 A(3) suggesting that the activity of HlyIIR is modulated by binding of a ligand, which triggers the toxin production. Virtual ligand library screening shows that this pocket can accommodate compounds with molecular masses of up to 400-500 Da. Based on structural data and previous biochemical evidence, we propose a model for HlyIIR interaction with the DNA.

SUBMITTER: Kovalevskiy OV

PROVIDER: S-EPMC1828608 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Properties of Bacillus cereus hemolysin II: a heptameric transmembrane pore.

Project description:The gene encoding hemolysin II (HlyII) was amplified from Bacillus cereus genomic DNA and a truncated mutant, HlyII(DeltaCT), was constructed lacking the 94 amino acid extension at the C terminus. The proteins were produced in an E. coli cell-free in vitro transcription and translation system, and were shown to assemble into SDS-stable oligomers on rabbit erythrocyte membranes and liposomes. The hemolytic activity of HlyII was measured with rabbit erythrocytes yielding an HC(50) value of 1.64 ng mL(-1), which is over 15 times more potent than staphylococcal alpha-hemolysin. HlyII(DeltaCT) was about eight times less potent than HlyII in this assay. Limited proteolysis of the oligomers formed by HlyII and HlyII(DeltaCT) on red cell membranes showed that the C-terminal extension is sensitive to digestion, while HlyII(DeltaCT) is protease resistant and migrates with an electrophoretic mobility similar to that of digested HlyII. HlyII forms moderately anion selective, rectifying pores (I(+80)/I(-80) = 0.57, 1 M KCl, pH 7.4) in planar lipid bilayers of diphytanoylphosphatidylcholine with a unitary conductance of 637 pS (1 M KCl, 5 mM HEPES, pH 7.4) and exhibits no gating over a wide range of applied potentials (-160 to +160 mV). In addition, it was demonstrated that HlyII forms a homoheptameric pore by using gel shift electrophoresis aided by a genetically encoded oligoaspartate tag. Although they share limited primary sequence identity (30%), these data confirm that HlyII is a structural and functional homolog of staphylococcal alpha-hemolysin.

| S-EPMC2373656 | biostudies-literature

The Pore-Forming Hemolysin BL Enterotoxin from Bacillus cereus: Subunit Interactions in Cell-Free Systems.

Project description:The tripartite enterotoxin Hemolysin BL (Hbl) has been widely characterized as a hemolytic and cytotoxic virulence factor involved in foodborne diarrheal illness caused by Bacillus cereus. Previous studies have described the formation of the Hbl complex and aimed to identify the toxin's mode of action. In this study, we analyzed the assembly of Hbl out of its three individual subunits L1, L2 and B in a soluble as well as a putative membrane bound composition using a Chinese hamster ovary (CHO) cell-free system. Subunits were either coexpressed or synthesized individually in separate cell-free reactions and mixed together afterwards. Hemolytic activity of cell-free synthesized subunits was demonstrated on 5% sheep blood agar and identified both synthesis procedures, coexpression as well as individual synthesis of each subunit, as functional for the synthesis of an active Hbl complex. Hbl's ability to perforate cell membranes was evaluated using a propidium iodide uptake assay. These data suggested that coexpressed Hbl subunits augmented cytotoxic activity with increasing concentrations. Further, a pre-pore-complex of L1-L2 showed cytotoxic effects suggesting the possibility of an interaction between the cell membrane and the pre-pore-complex. Overall, this study shows that cell-free protein synthesis is a fast and efficient way to study the assembly of multiple protein subunits in soluble as well as vesicular fractions.

| S-EPMC8623112 | biostudies-literature

X-ray crystal structure of the B component of Hemolysin BL from Bacillus cereus.

Project description:Bacillus cereus Hemolysin BL enterotoxin, a ternary complex of three proteins, is the causative agent of food poisoning and requires all three components for virulence. The X-ray structure of the binding domain of HBL suggests that it may form a pore similar to other soluble channel forming proteins. A putative pathway of pore formation is discussed.

| S-EPMC3220058 | biostudies-literature

Sequential CRISPR-Based Screens Identify LITAF and CDIP1 as the Bacillus cereus Hemolysin BL Toxin Host Receptors.

Project description:Bacteria and their toxins are associated with significant human morbidity and mortality. While a few bacterial toxins are well characterized, the mechanism of action for most toxins has not been elucidated, thereby limiting therapeutic advances. One such example is the highly potent pore-forming toxin, hemolysin BL (HBL), produced by the gram-positive pathogen Bacillus cereus. However, how HBL exerts its effects and whether it requires any host factors is unknown. Here, we describe an unbiased genome-wide CRISPR-Cas9 knockout screen that identified LPS-induced TNF-α factor (LITAF) as the HBL receptor. Using LITAF-deficient cells, a second, subsequent whole-genome CRISPR-Cas9 screen identified the LITAF-like protein CDIP1 as a second, alternative receptor. We generated LITAF-deficient mice, which exhibit marked resistance to lethal HBL challenges. This work outlines and validates an approach to use iterative genome-wide CRISPR-Cas9 screens to identify the complement of host factors exploited by bacterial toxins to exert their myriad biological effects.

| S-EPMC7486266 | biostudies-literature

Current Perspective on the Membrane-Damaging Action of Thermostable Direct Hemolysin, an Atypical Bacterial Pore-forming Toxin.

Project description:Thermostable direct hemolysin (TDH) is the major virulence determinant of the gastroenteric bacterial pathogen Vibrio parahaemolyticus. TDH is a membrane-damaging pore-forming toxin (PFT). TDH shares remarkable structural similarity with the actinoporin family of eukaryotic PFTs produced by the sea anemones. Unlike most of the PFTs, it exists as tetramer in solution, and such assembly state is crucial for its functionality. Although the structure of the tetrameric assembly of TDH in solution is known, membrane pore structure is not available yet. Also, the specific membrane-interaction mechanisms of TDH, and the exact role of any receptor(s) in such process, still remain unclear. In this mini review, we discuss some of the unique structural and physicochemical properties of TDH, and their implications for the membrane-damaging action of the toxin. We also present our current understanding regarding the membrane pore-formation mechanism of this atypical bacterial PFT.

| S-EPMC8343067 | biostudies-literature

NMR structure of the Bacillus cereus hemolysin II C-terminal domain reveals a novel fold.

Project description:In addition to multiple virulence factors, Bacillus cereus a pathogen that causes food poisoning and life-threatening wound infections, secretes the pore-forming toxin hemolysin II (HlyII). The HlyII toxin has a unique 94 amino acid C-terminal domain (HlyIIC). HlyIIC exhibits splitting of NMR resonances due to cis/trans isomerization of a single proline near the C-terminus. To overcome heterogeneity, we solved the structure of P405M-HlyIIC, a mutant that exclusively stabilizes the trans state. The NMR structure of HlyIIC reveals a novel fold, consisting of two subdomains ?A-?1-?2 and ?3-?4-?B-?5, that come together in a barrel-like structure. The barrel core is fastened by three layers of hydrophobic residues. The barrel end opposite the HlyIIC-core has a positively charged surface, that by binding negatively charged moieties on cellular membranes, may play a role in target-cell surface recognition or stabilization of the heptameric pore complex. In the WT domain, dynamic flexibility occurs at the N-terminus and the first ?-helix that connects the HlyIIC domain to the HlyII-core structure. In the destabilizing P405M mutant, increased flexibility is evident throughout the first subdomain, suggesting that the HlyIIC structure may have arisen through gene fusion.

| S-EPMC5468326 | biostudies-literature

Binding to The Target Cell Surface Is The Crucial Step in Pore Formation of Hemolysin BL from Bacillus cereus.

Project description:A major virulence factor involved in Bacillus cereus food poisoning is the three-component enterotoxin hemolysin BL. It consists of the binding component B and the two lytic components L1 and L2. Studying its mode of action has been challenging, as natural culture supernatants additionally contain Nhe, the second three-component enterotoxin, and purification of recombinant (r) Hbl components has been difficult. In this study, we report on pore-forming, cytotoxic, cell binding and hemolytic activity of recently generated rHbl components expressed in E. coli. It is known that all three Hbl components are necessary for cytotoxicity and pore formation. Here we show that an excess of rHbl B enhances, while an excess of rHbl L1 hinders, the velocity of pore formation. Most rapid pore formation was observed with ratios L2:L1:B = 1:1:10 and 10:1:10. It was further verified that Hbl activity is due to sequential binding of the components B - L1 - L2. Accordingly, all bioassays proved that binding of Hbl B to the cell surface is the crucial step for pore formation and cytotoxic activity. Binding of Hbl B took place within minutes, while apposition of the following L1 and L2 occurred immediately. Further on, applying toxin components simultaneously, it seemed that Hbl L1 enhanced binding of B to the target cell surface. Overall, these data contribute significantly to the elucidation of the mode of action of Hbl, and suggest that its mechanism of pore formation differs substantially from that of Nhe, although both enterotoxin complexes are sequentially highly related.

| S-EPMC6563250 | biostudies-literature

Swarming behavior of and hemolysin BL secretion by Bacillus cereus.

Project description:An association between swarming and hemolysin BL secretion was observed in a collection of 42 Bacillus cereus isolates (P=0.029). The highest levels of toxin were detected in swarmers along with swarm cell differentiation (P=0.021), suggesting that swarming B. cereus strains may have a higher virulence potential than nonswarming strains.

| S-EPMC1932716 | biostudies-literature

The pore-forming toxin ? hemolysin/cytolysin triggers p38 MAPK-dependent IL-10 production in macrophages and inhibits innate immunity.

Project description:Group B Streptococcus (GBS) is a leading cause of invasive bacterial infections in human newborns and immune-compromised adults. The pore-forming toxin (PFT) ? hemolysin/cytolysin (?h/c) is a major virulence factor for GBS, which is generally attributed to its cytolytic functions. Here we show ?h/c has immunomodulatory properties on macrophages at sub-lytic concentrations. ?h/c-mediated activation of p38 MAPK drives expression of the anti-inflammatory and immunosuppressive cytokine IL-10, and inhibits both IL-12 and NOS2 expression in GBS-infected macrophages, which are critical factors in host defense. Isogenic mutant bacteria lacking ?h/c fail to activate p38-mediated IL-10 production in macrophages and promote increased IL-12 and NOS2 expression. Furthermore, targeted deletion of p38 in macrophages increases resistance to invasive GBS infection in mice, associated with impaired IL-10 induction and increased IL-12 production in vivo. These data suggest p38 MAPK activation by ?h/c contributes to evasion of host defense through induction of IL-10 expression and inhibition of macrophage activation, a new mechanism of action for a PFT and a novel anti-inflammatory role for p38 in the pathogenesis of invasive bacterial infection. Our studies suggest p38 MAPK may represent a new therapeutic target to blunt virulence and improve clinical outcome of invasive GBS infection.

| S-EPMC3400567 | biostudies-literature

The UPEC pore-forming toxin α-hemolysin triggers proteolysis of host proteins to disrupt cell adhesion, inflammatory, and survival pathways.

Project description:Uropathogenic Escherichia coli (UPEC), which are the leading cause of both acute and chronic urinary tract infections, often secrete a labile pore-forming toxin known as α-hemolysin (HlyA). We show that stable insertion of HlyA into epithelial cell and macrophage membranes triggers degradation of the cytoskeletal scaffolding protein paxillin and other host regulatory proteins, as well as components of the proinflammatory NFκB signaling cascade. Proteolysis of these factors requires host serine proteases, and paxillin degradation specifically involves the serine protease mesotrypsin. The induced activation of mesotrypsin by HlyA is preceded by redistribution of mesotrypsin precursors from the cytosol into foci along microtubules and within nuclei. HlyA intoxication also stimulated caspase activation, which occurred independently of effects on host serine proteases. HlyA-induced proteolysis of host proteins likely allows UPEC to not only modulate epithelial cell functions, but also disable macrophages and suppress inflammatory responses.

| S-EPMC3266558 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data