Modulation of Host Cell Death and Lysis Are Required for the Release of .

is a -like bacterium and emerging pathogen of the respiratory tract. It is an obligate intracellular bacterium with a biphasic developmental cycle, which replicates in a wide range of host cells. The life cycle of has been shown to proceed for more than 12 days, but little is known about the mechanisms that mediate the cellular release of these bacteria.

Central residues of the amphipathic β-hairpin loop control the properties of Clostridium perfringens epsilon-toxin channel.

Clostridium perfringens epsilon toxin (ETX) is a heptameric pore-forming toxin of the aerolysin toxin family. ETX is the most potent toxin of this toxin family and the third most potent bacterial toxin with high cytotoxic and lethal activities in animals. In addition, ETX shows a demyelinating activity in nervous tissue leading to devastating multifocal central nervous system white matter disease in ruminant animals.

Magnesium therapy improves outcome in Streptococcus pneumoniae meningitis by altering pneumolysin pore formation.

Background And Purpose: Streptococcus pneumoniae is the most common cause of bacterial meningitis in adults and is characterized by high lethality and substantial cognitive disabilities in survivors. Here, we have studied the capacity of an established therapeutic agent, magnesium, to improve survival in pneumococcal meningitis by modulating the neurological effects of the major pneumococcal pathogenic factor, pneumolysin.

Experimental Approach: We used mixed primary glial and acute brain slice cultures, pneumolysin injection in infant rats, a mouse meningitis model and complementary approaches such as Western blot, a black lipid bilayer conductance assay and live imaging of primary glial cells.

OmpW of Caulobacter crescentus Functions as an Outer Membrane Channel for Cations.

Caulobacter crescentus is an oligotrophic bacterium that lives in dilute organic environments such as soil and freshwater. This bacterium represents an interesting model for cellular differentiation and regulation because daughter cells after division have different forms: one is motile while the other is non-motile and can adhere to surfaces. Interestingly, the known genome of C.

The deletion of several amino acid stretches of Escherichia coli alpha-hemolysin (HlyA) suggests that the channel-forming domain contains beta-strands.

Escherichia coli α-hemolysin (HlyA) is a pore-forming protein of 110 kDa belonging to the family of RTX toxins. A hydrophobic region between the amino acid residues 238 and 410 in the N-terminal half of HlyA has previously been suggested to form hydrophobic and/or amphipathic α-helices and has been shown to be important for hemolytic activity and pore formation in biological and artificial membranes. The structure of the HlyA transmembrane channel is, however, largely unknown.

Solid-state NMR, electrophysiology and molecular dynamics characterization of human VDAC2.

The voltage-dependent anion channel (VDAC) is the most abundant protein of the outer mitochondrial membrane and constitutes the major pathway for the transport of ADP, ATP, and other metabolites. In this multidisciplinary study we combined solid-state NMR, electrophysiology, and molecular dynamics simulations, to study the structure of the human VDAC isoform 2 in a lipid bilayer environment. We find that the structure of hVDAC2 is similar to the structure of hVDAC1, in line with recent investigations on zfVDAC2.

Residues involved in the pore-forming activity of the Clostridium perfringens iota toxin.

Clostridium perfringens iota toxin is a binary toxin that is organized into enzyme (Ia) and binding (Ib) components. Ib forms channels in lipid bilayers and mediates the transport of Ia into the target cells. Here we show that Ib residues 334-359 contain a conserved pattern of alternating hydrophobic and hydrophilic residues forming two amphipathic β-strands involved in membrane insertion and channel formation.

Inhibitions of the translocation pore of Clostridium botulinum C2 toxin by tailored azolopyridinium salts protects human cells from intoxication.

C2 toxin from Clostridium botulinum represents the prototype of clostridial binary actin ADP-ribosylating toxins which destroy the actin-cytoskeleton of mammalian cells and cause severe enteric diseases in humans and animals. After receptor-mediated endocytosis of the C2 toxin complex, the binding/translocation component C2IIa forms a heptameric transmembrane pore in membranes of acidified endosomal vesicles. The separate ADP-ribosyltransferase component C2I translocates through this C2IIa-pore from the endosomal lumen into the cytosol.

Use of nonelectrolytes reveals the channel size and oligomeric constitution of the Borrelia burgdorferi P66 porin.

In the Lyme disease spirochete Borrelia burgdorferi, the outer membrane protein P66 is capable of pore formation with an atypical high single-channel conductance of 11 nS in 1 M KCl, which suggested that it could have a larger diameter than 'normal' Gram-negative bacterial porins. We studied the diameter of the P66 channel by analyzing its single-channel conductance in black lipid bilayers in the presence of different nonelectrolytes with known hydrodynamic radii. We calculated the filling of the channel with these nonelectrolytes and the results suggested that nonelectrolytes (NEs) with hydrodynamic radii of 0.

Deletion of β-strands 9 and 10 converts VDAC1 voltage-dependence in an asymmetrical process.

Voltage-dependent anion selective channel isoform1 maintains the permeability of the outer mitochondrial membrane. Its voltage-gating properties are relevant in bioenergetic metabolism and apoptosis. The N-terminal domain is suspected to be involved in voltage-gating, due to its peculiar localization.

Identification and characterization of a novel porin family highlights a major difference in the outer membrane of chlamydial symbionts and pathogens.

The Chlamydiae constitute an evolutionary well separated group of intracellular bacteria comprising important pathogens of humans as well as symbionts of protozoa. The amoeba symbiont Protochlamydia amoebophila lacks a homologue of the most abundant outer membrane protein of the Chlamydiaceae, the major outer membrane protein MOMP, highlighting a major difference between environmental chlamydiae and their pathogenic counterparts. We recently identified a novel family of putative porins encoded in the genome of P.

β-Barrel mobility underlies closure of the voltage-dependent anion channel.

The voltage-dependent anion channel (VDAC) is the major protein in the outer mitochondrial membrane, where it mediates transport of ATP and ADP. Changes in its permeability, induced by voltage or apoptosis-related proteins, have been implicated in apoptotic pathways. The three-dimensional structure of VDAC has recently been determined as a 19-stranded β-barrel with an in-lying N-terminal helix.

Interspecies differences in membrane-associated protease activities of thyrocytes and their relevance for thyroid cancer studies.

Background: To understand the role of proteases involved in human thyroid cancer progression and tissue invasion, thyrocytes from other species could potentially be used provided their characteristics are similar. It is not known whether dipeptidyl peptidase IV and aminopeptidase N activities, which are overexpressed in human thyroid cancer, are, as in human, also absent in normal thyrocytes of other species, making them suitable models for studies on the regulation of these proteases.

Methods: To assess the role of these proteases, activity was measured in thyroid tissue of human, mouse, rat, porcine, bovine and ovine origin.

Changes in astrocyte shape induced by sublytic concentrations of the cholesterol-dependent cytolysin pneumolysin still require pore-forming capacity.

Streptococcus pneumoniae is a common pathogen that causes various infections, such as sepsis and meningitis. A major pathogenic factor of S. pneumoniae is the cholesterol-dependent cytolysin, pneumolysin.

Extracellular calcium reduction strongly increases the lytic capacity of pneumolysin from streptococcus pneumoniae in brain tissue.

Background: Streptococcus pneumoniae causes serious diseases such as pneumonia and meningitis. Its major pathogenic factor is the cholesterol-dependent cytolysin pneumolysin, which produces lytic pores at high concentrations. At low concentrations, it has other effects, including induction of apoptosis.

Mutations affecting export and activity of cytolysin A from Escherichia coli.

Cytolysin A (known as ClyA, HlyE, and SheA) is a cytolytic pore-forming protein toxin found in several Escherichia coli and Salmonella enterica strains. The structure of its water-soluble monomeric form and that of dodecameric ClyA pores is known, but the mechanisms of ClyA export from bacterial cells and of pore assembly are only partially understood. Here we used site-directed mutagenesis to study the importance of different regions of the E.

Structural properties of pore-forming oligomers of alpha-synuclein.

Soluble oligomers are potent toxins in many neurodegenerative diseases, but little is known about the structure of soluble oligomers and their structure-toxicity relationship. Here we prepared on-pathway oligomers of the 140-residue protein alpha-synuclein, a key player in Parkinson's disease, at concentrations an order of magnitude higher than previously possible. The oligomers form ion channels with well-defined conductance states in a variety of membranes, and their beta-structure differs from that of amyloid fibrils of alpha-synuclein.

Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX).

Epsilon-toxin (ETX) is a potent toxin produced by Clostridium perfringens strains B and D. The bacteria are important pathogens in domestic animals and cause edema mediated by ETX. This toxin acts most likely by heptamer formation and rapid permeabilization of target cell membranes for monovalent anions and cations followed by a later entry of calcium.

Detergent-like activity and alpha-helical structure of warnericin RK, an anti-Legionella peptide.

Warnericin RK is the first antimicrobial peptide known to be active against Legionella pneumophila, a pathogen bacterium that is responsible for severe pneumonia. Strikingly, this peptide displays a very narrow range of antimicrobial activity, almost limited to the Legionella genus, and a hemolytic activity. A similar activity has been described for delta-lysin, a well-known hemolytic peptide of Staphylococci that has not been described as antimicrobial.

Clostridium septicum alpha-toxin forms pores and induces rapid cell necrosis.

Alpha-toxin is the unique lethal virulent factor produced by Clostridium septicum, which causes traumatic or non-traumatic gas gangrene and necrotizing enterocolitis in humans. Here, we analyzed channel formation of the recombinant septicum alpha-toxin and characterized its activity on living cells. Recombinant septicum alpha-toxin induces the formation of ion-permeable channels with a single-channel conductance of about 175pS in 0.

The major outer membrane protein OprG of Pseudomonas aeruginosa contributes to cytotoxicity and forms an anaerobically regulated, cation-selective channel.

OprG of Pseudomonas aeruginosa is a member of the very large and widely distributed but poorly characterized OmpW (PF0392) family of outer membrane proteins. It was established here that OprG was highly transcribed in anaerobic environments rich in iron via the ANR regulator. In the absence of OprG, P.

Oligomerization is involved in pore formation by Bordetella adenylate cyclase toxin.

The Bordetella adenylate cyclase-hemolysin (CyaA, ACT, or AC-Hly) is a multifunctional toxin. Simultaneously with promoting calcium ion entry, CyaA delivers into host cells an adenylate cyclase enzyme (AC) and permeabilizes cell membrane by forming small cation-selective pores. Indirect evidence suggested that these two activities were accomplished by different membrane-inserted CyaA conformers, one acting as an AC-delivering monomer and the other as an uncharacterized pore-forming oligomer.

Clostridium perfringens delta toxin is sequence related to beta toxin, NetB, and Staphylococcus pore-forming toxins, but shows functional differences.

Clostridium perfringens produces numerous toxins, which are responsible for severe diseases in man and animals. Delta toxin is one of the three hemolysins released by a number of C. perfringens type C and possibly type B strains.

Moraxella catarrhalis M35 is a general porin that is important for growth under nutrient-limiting conditions and in the nasopharynges of mice.

Moraxella catarrhalis is a gram-negative respiratory pathogen that is an important causative agent for otitis media and exacerbations of chronic obstructive pulmonary disease. We have previously predicted the outer membrane protein M35 to be a general porin, and in the current study, we have investigated the function of M35 and its importance for survival of M. catarrhalis in vivo.