Ca2+ and the interaction of pore-formers with membranes

The interaction of pore-forming agents, such as Sendai virus, influenza virus (at pH 5 3), activated complement,Staphylococcus aureus α-toxin, melittin and polylysine, with the surface membrane of cells has been studied. In each case the following changes are initiated: collapse of membrane potential, leakage of ions, and leakage of phosphorylated metabolites. The changes can be inihibited by extracellular Ca²⁺ at physiological concentration; Mg²⁺ is less effective, and Zn²⁺ is more effective, than Ca²⁺ Ca²⁺ appears to act at a stage subsequent to the binding of pore-forming agent to cells. It is concluded that divalent cations are able to protect cells against the damaging effects of certain viruses, toxins or the components of activated complement in a manner that is worthy of further investigation.     

Urokinase-targeted recombinant bacterial protein toxins — a rationally designed and engineered anticancer agent for cancer therapy

Urokinase-targeted recombinant bacterial protein toxins are a sort of rationally designed and engineered anticancer recombinant fusion proteins representing a novel class of agents for cancer therapy. Bacterial protein toxins have long been known as the primary virulence factor(s) for a variety of pathogenic bacteria and are the most powerful human poisons. On the other hand, it has been well documented that urokinase-type plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR), making up the uPA system, are over-expressed in a variety of human tumors and tumor cell lines. The expression of uPA system is highly correlated with tumor invasion and metastasis. To exploit these characteristics in the design of tumor cell-selective cytotoxins, two prominent bacterial protein toxins, i.e., the diphtheria toxin and anthrax toxin are deliberately engineered through placing a sequence targeted specifically by the uPA system to form anticancer recombinant fusion proteins. These uPA system-targeted bacterial protein toxins are activated selectively on the surface of uPA system-expressing tumor cells, thereby killing these cells. This article provides a review on the latest progress in the exploitation of these recombinant fusion proteins as potent tumoricidal agents. It is perceptible that the strategies for cancer therapy are being innovated by this novel therapeutic approach.     

Urokinase-targeted recombinant bacterial protein toxins-a rationally designed and engineered anticancer agent for cancer therapy

Urokinase-targeted recombinant bacterial protein toxins are a sort of rationally designed and engineered anticancer recombinant fusion proteins representing a novel class of agents for cancer therapy.Bacterial protein toxins have long been known as the primary virulence factor(s) for a variety of pathogenic bacteria and are the most powerful human poisons.On the other hand,it has been well documented that urokinase-type plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR),making up the uPA system,are overexpressed in a variety of human tumors and tumor cell lines.The expression of uPA system is highly correlated with tumor invasion and metastasis.To exploit these characteristics in the design of tumor cell-selective cytotoxins,two prominent bacterial protein toxins,i.e.,the diphtheria toxin and anthrax toxin are deliberately engineered through placing a sequence targeted specifically by the uPA system to form anticancer recombinant fusion proteins.These uPA system-targeted bacterial protein toxins are activated selectively on the surface of uPA systemexpressing tumor cells,thereby killing these cells.This article provides a review on the latest progress in the exploitation of these recombinant fusion proteins as potent tumoricidal agents.It is perceptible that the strategies for cancer therapy are being innovated by this novel therapeutic approach.     

Purification of bacterial exotoxins. The case of botulinum, tetanus, anthrax, pertussis and cholera toxins.

Bacterial protein toxins and their fragments have been isolated and purified for various reasons, including the development of efficient vaccines and for methods of identification of bacterial agents causing disease. This activity continues today but a new area of bacterial protein toxin research has recently emerged. Since it was shown that toxin molecules comprise several types of biological activity within their structural domains, it was suggested to use these domains (and their combinations) as biochemical tools for developing novel agents for disease imaging and and/or relieving. In this way eukaryotic cell-receptor specific fusion toxins have been developed to prevent malignancy in human. While human clinical trials of these preparations have only recently begun, the preliminary clinical findings are promising. Also fusion proteins which combine independent immunodominant epitopes from different antigens have also been developed thus opening a way for the generation of new vaccines for both human and veterinary use. Receptor binding fragments of microbial toxins when combined with other molecules may be useful in delivering these molecules into the cell. In this way novel agents may be developed with a potential for inducing specific changes at the molecular level for the correction of metabolic disorders causing human and animal diseases. Bacterial protein toxins such as anthrax, botulinum, cholera, pertussis and tetanus for which considerable progress has been achieved in structure-function analysis are promising candidates for such research. Particularly exciting appears the idea of extending this research to the cells of the nervous system, exploiting the unique specificity of the botulinum or tetanus toxin fragments which may bring long desired methods for treatment of various disorders of the nervous system. Data on functional domains of these toxins as well as methods of purification of the whole toxins and their fragments are considered in this review as they form a base for their further structure-function analysis and engineering applications.     

Common action of certain viruses,toxins, and activated complement: pore formation and its prevention by extracellular Ca2+

Haemolysis by Sendal virus, -toxin, and activated complement is inhibited by high concentrations of divalent cations. In Daudi cells, sublytic amounts of these agents induce the following changes: collapse of surface membrane potential, uptake of Na⁺ and loss of K⁺ from cells, and leakage of phosphorylated metabo-tites from cells. The changes induced by Sendal virus and complement are sensitive to physiological concentrations of extracellular Ca²⁺. It is concluded that fluctuations in plasma Ca²⁺ concentration may affect the damaging action of certain pore-forming agents on susceptible cells.     

Bacterial toxins modifying the actin cytoskeleton.

Numerous bacterial toxins recognize the actin cytoskeleton as a target. The clostridial binary toxins (Iota and C2 families) ADP-ribosylate the actin monomers causing the dissociation of the actin filaments. The large clostridial toxins from Clostridium difficile, Clostridium sordellii and Clostridium novyi inactivate, by glucosylation, proteins from the Rho family that regulate actin polymerization. In contrast, the cytotoxic necrotic factor from Escherichia coli activates Rho by deamidation and increases the formation of actin filaments. The enterotoxin of Bacteroides fragilis is a protease specific for E-cadherin and it promotes the reorganization of the actin cytoskeleton. The bacterial toxins that modify the actin cytoskeleton induce various cell disfunctions including changes in cell barrier permeability and disruption of intercellular junctions.     

Molecular and Structural Damage to Escherichia coli Produced by Antibody, Complement, and Lysozyme Systems

The antibacterial action of antibody (normal and hyperimmune), complement, and lysozyme has been studied by correlating the ultrastructural and biochemical changes that they cause in smooth Escherichia coli. Both normal and hyperimmune antibody, in the absence of lysozyme, produced complement-dependent release, into the suspending medium, of 63 to 72% of the (32)P-labeled phospholipid and 74 to 85% of the small molecular bacterial constituents. Macromolecular nucleic acid labeled with (32)P was not released. By phase microscopy, these cells appeared as bacilli but their ultrastructure showed general swelling, with smoothing of the normally wrinkled outer cell wall layers. Cytoplasmic membranes were damaged and the internal cell structure was disorganized. Membranous spherules, apparently from the outermost putatively lipopolysaccharide cell layer, were released into the medium. When lysozyme was added to antibody and complement, (32)P-labeled macromolecular constituents were released from the cells. Damage to ultrastructure then included loss of cell wall rigidity, cell wall breakage, and some spheroplast formation. Characteristic fibrillar fragmentation was seen in cell wall mucopeptide layers. The relationships between antibody-complement dependent release of bacterial phospholipid, loss of selective cell permeability, and increase in sensitivity to lysozyme are discussed.     

Coenzyme depletion by members of the aerolysin family of pore-forming toxins leads to diminished ATP levels and cell death

Recent studies demonstrated that a variety of bacterial pore-forming toxins induce cell death through a process of programmed necrosis characterized by the rapid depletion of cellular ATP. However, events leading to the necrosis and depletion of ATP are not thoroughly understood. We demonstrate that ATP-depletion induced by two pore-forming toxins, the Clostridium perfringens epsilon-toxin and the Aeromonas hydrophila aerolysin toxin, is associated with decreased mitochondrial membrane potential and opening of the mitochondrial permeability transition pore. To gain further insight into the toxin-induced metabolic changes contributing to necrosis and depletion of ATP, we analyzed the biochemical profiles of 251 distinct compounds by GC/MS or LC/MS/MS following exposure of a human kidney cell line to the epsilon-toxin. As expected, numerous biochemicals were seen to increase or decrease in response to epsilon-toxin. However, the pattern of these changes was consistent with the toxin-induced disruption of major energy-producing pathways in the cell including disruptions to the beta-oxidation of lipids. In particular, treatment with epsilon-toxin led to decreased levels of key coenzymes required for energy production including carnitine, NAD (and NADH), and coenzyme A. Independent biochemical assays confirmed that epsilon-toxin and aerolysin induced the rapid decrease of these coenzymes or their synthetic precursors. Incubation of cells with NADH or carnitine-enriched medium helped protect cells from toxin-induced ATP depletion and cell death. Collectively, these results demonstrate that members of the aerolysin family of pore-forming toxins lead to decreased levels of essential coenzymes required for energy production. The resulting loss of energy substrates is expected to contribute to dissipation of the mitochondrial membrane potential, opening of the mitochondrial permeability transition pore, and ultimately cell death.     

Animal models used to test the interactions between infectious agents and products of cigarette smoked implicated in sudden infant death syndrome

Animal test systems are reviewed that have relevance to sudden infant death syndrome (SIDS) are reviewed. These test interactions between infectious agents (or their toxins) and products of cigarette smoke. Infectious agents implicated in SIDS include members of the enterobacteria and clostridia, Staphylococcus aureus and Streptococcus pyogenes. Smoking is thought to be the single most preventable cause of SIDS. Tobacco smoke contains many extremely toxic products including cyanide and nicotine. Many animal test systems are available to examine the potency of bacterial toxins and smoke-derived components. These include mice, hamsters, rats and chick embryos. Such systems reveal synergy between bacterial toxins, especially endotoxin and superantigens. They have also demonstrated potentiation of low levels of bacterial toxin by low levels of both nicotine and its primary metabolite, cotinine. These findings suggest a possible causal explanation for the fact that passive exposure to cigarette smoke is a risk factor in sudden infant death syndrome.     

Multifaceted role of Rho, Rac, Cdc42 and Ras in intercellular junctions, lessons from toxins

Tight junctions (TJs) and adherens junctions (AJs) are dynamic structures linked to the actin cytoskeleton, which control the paracellular permeability of epithelial and endothelial barriers. TJs and AJs are strictly regulated in a spatio-temporal manner by a complex signaling network, including Rho/Ras-GTPases, which have a pivotal role. Rho preferentially regulates TJs by controlling the contraction of apical acto-myosin filaments, whereas Rac/Cdc42 mainly coordinate the assembly-disassembly of AJ components. However, a subtle balance of Rho/Ras-GTPase activity and interplay between these molecules is required to maintain an optimal organization and function of TJs and AJs. Conversely, integrity of intercellular junctions generates signals through Rho-GTPases, which are involved in the regulation of multiple cellular processes. Rho/Ras-GTPases and the control of intercellular junctions are the target of various bacterial toxins responsible for severe diseases in man and animals, and are part of their mechanism of action. This review focuses on the regulation of TJs and AJs by Rho/Ras-GTPases through molecular approaches and bacterial toxins.     

Potassium and tetraphenylphosphonium ion-selective electrodes for monitoring changes in the permeability of bacterial outer and cytoplasmic membranes

A tetraphenylphosphonium ion (TPP(+))-selective electrode, originally developed as a membrane potential indicator, is useful for measuring increases in the permeability of bacterial outer membranes induced by antimicrobial agents. The combination of this electrode with a potassium ion-selective electrode enabled us to determine changes in the permeability of bacterial outer and cytoplasmic membranes simultaneously. Outer membrane permeabilization induced by antimicrobial agents, chlorhexidine and polyhexamethylene biguanide (PHMB), as monitored with the TPP(+) electrode, correlated closely with the ability of the agents to release lipopolysaccharide (LPS) from the outer membrane.     

The magic glue hyaluronan and its eraser hyaluronidase: a biological overview

Hyaluronan (HA) is a multifunctional high molecular weight polysaccharide found throughout the animal kingdom, especially in the extracellular matrix (ECM) of soft connective tissues. HA is thought to participate in many biological processes, and its level is markedly elevated during embryogenesis, cell migration, wound healing, malignant transformation, and tissue turnover. The enzymes that degrade HA, hyaluronidases (HAases) are expressed both in prokaryotes and eukaryotes. These enzymes are known to be involved in physiological and pathological processes ranging from fertilization to aging. Hyaluronidase-mediated degradation of HA increases the permeability of connective tissues and decreases the viscosity of body fluids and is also involved in bacterial pathogenesis, the spread of toxins and venoms, acrosomal reaction/ovum fertilization, and cancer progression. Furthermore, these enzymes may promote direct contact between pathogens and the host cell surfaces. Depolymerization of HA also adversely affects the role of ECM and impairs its activity as a reservoir of growth factors, cytokines and various enzymes involved in signal transduction. Inhibition of HA degradation therefore may be crucial in reducing disease progression and spread of venom/toxins and bacterial pathogens. Hyaluronidase inhibitors are potent, ubiquitous regulating agents that are involved in maintaining the balance between the anabolism and catabolism of HA. Hyaluronidase inhibitors could also serve as contraceptives and anti-tumor agents and possibly have antibacterial and anti-venom/toxin activities. Additionally, these molecules can be used as pharmacological tools to study the physiological and pathophysiological role of HA and hyaluronidases.     

In vitro cytochemical and cytophysiological study of in vivo activated mouse peritoneal macrophages

The morphological, cytochemical (acid phosphatase activity) and cytophysiological (phagocytosis) features of mouse peritoneal macrophages activated in vivo by two bacterial agents. Corynebacterium parvum parvum and Polidin, were investigated in vitro. Both immunostimulants induced an increase in cytochemical and phagocytic activities of the activated peritoneal macrophages but in different degrees, the changes being more extensive in the case of Corynebacterium parvum-activated macrophages.     

Palmitate lipotoxicity in enteric glial cells: Lipid remodeling and mitochondrial ROS are responsible for cyt c release outside mitochondria

Enteric glial cells (EGCs) are components of the enteric nervous system, an organized structure that controls gut functions. EGCs may be vulnerable to different agents, such as bacterial infections that could alter the intestinal epithelial barrier, allowing bacterial toxins and/or other agents possessing intrinsic toxic effect to access cells. Palmitate, known to exhibit lipotoxicity, is released in the gut during the digestion process. In this study, we investigated the lipotoxic effect of palmitate in cultured EGCs, with particular emphasis on palmitate-dependent intracellular lipid remodeling. Palmitate but not linoleate altered mitochondrial and endoplasmic reticulum lipid composition. In particular, the levels of phosphatidic acid, key precursor of phospholipid synthesis, increased, whereas those of mitochondrial cardiolipin (CL) decreased; in parallel, phospholipid remodeling was induced. CL remodeling (chains shortening and saturation) together with palmitate-triggered mitochondrial burst, caused cytochrome c (cyt c) detachment from its CL anchor and accumulation in the intermembrane space as soluble pool. Palmitate decreased mitochondrial membrane potential and ATP levels, without mPTP opening. Mitochondrial ROS permeation into the cytosol and palmitate-induced ER stress activated JNK and p38, culminating in Bim and Bax overexpression, factors known to increase the outer mitochondrial membrane permeability. Overall, in EGCs palmitate produced weakening of cyt c-CL interactions and favoured the egress of the soluble cyt c pool outside mitochondria to trigger caspase-3-dependent viability loss. Elucidating the mechanisms of palmitate lipotoxicity in EGCs may be relevant in gut pathological conditions occurring in vivo such as those following an insult that may damage the intestinal epithelial barrier.     

Convergent use of RhoGAP toxins by eukaryotic parasites and bacterial pathogens

Inactivation of host Rho GTPases is a widespread strategy employed by bacterial pathogens to manipulate mammalian cellular functions and avoid immune defenses. Some bacterial toxins mimic eukaryotic Rho GTPase-activating proteins (GAPs) to inactivate mammalian GTPases, probably as a result of evolutionary convergence. An intriguing question remains whether eukaryotic pathogens or parasites may use endogenous GAPs as immune-suppressive toxins to target the same key genes as bacterial pathogens. Interestingly, a RhoGAP domain-containing protein, LbGAP, was recently characterized from the parasitoid wasp Leptopilina boulardi, and shown to protect parasitoid eggs from the immune response of Drosophila host larvae. We demonstrate here that LbGAP has structural characteristics of eukaryotic RhoGAPs but that it acts similarly to bacterial RhoGAP toxins in mammals. First, we show by immunocytochemistry that LbGAP enters Drosophila immune cells, plasmatocytes and lamellocytes, and that morphological changes in lamellocytes are correlated with the quantity of LbGAP they contain. Demonstration that LbGAP displays a GAP activity and specifically interacts with the active, GTP-bound form of the two Drosophila Rho GTPases Rac1 and Rac2, both required for successful encapsulation of Leptopilina eggs, was then achieved using biochemical tests, yeast two-hybrid analysis, and GST pull-down assays. In addition, we show that the overall structure of LbGAP is similar to that of eukaryotic RhoGAP domains, and we identify distinct residues involved in its interaction with Rac GTPases. Altogether, these results show that eukaryotic parasites can use endogenous RhoGAPs as virulence factors and that despite their differences in sequence and structure, eukaryotic and bacterial RhoGAP toxins are similarly used to target the same immune pathways in insects and mammals.     

Human L-ficolin,a Recognition Molecule of the Lectin Activation Pathway of Complement,Activates Complement by Binding to Pneumolysin,the Major Toxin of Streptococcus pneumoniae

The complement system is an essential component of the immune response, providing a critical line of defense against different pathogens including S. pneumoniae. Complement is activated via three distinct pathways: the classical (CP), the alternative (AP) and the lectin pathway (LP). The role of Pneumolysin (PLY), a bacterial toxin released by S. pneumoniae, in triggering complement activation has been studied in vitro. Our results demonstrate that in both human and mouse sera complement was activated via the CP, initiated by direct binding of even non-specific IgM and IgG3 to PLY. Absence of CP activity in C1q^−/− mouse serum completely abolished any C3 deposition. However, C1q depleted human serum strongly opsonized PLY through abundant deposition of C3 activation products, indicating that the LP may have a vital role in activating the human complement system on PLY. We identified that human L-ficolin is the critical LP recognition molecule that drives LP activation on PLY, while all of the murine LP recognition components fail to bind and activate complement on PLY. This work elucidates the detailed interactions between PLY and complement and shows for the first time a specific role of the LP in PLY-mediated complement activation in human serum.     

Thiol-dependent cytolytic bacterial toxins: streptolysin O and prominent toxins

Streptolysin O is the prototype of fifteen bacterial cytolytic protein toxins elaborated by gram-positive bacteria of species Streptococcus, Clostridium, Bacillus and Listeria. These toxins share a number of common properties: they are antigenically related as shown by cross-neutralization and immunoprecipitation; their cytolytic and other reducing agents; these toxins are inactivated by cholesterol and certain related sterols. This group of oxygen-labile cytolytic toxins has been named sulfyhdryl-activated toxins or thiol-activated cytolysins. The mechanism of action of these toxins is very likely identical or at least closely similar.     

Environmental sensing by chromatin: an epigenetic contribution to evolutionary change

Chromatin structure and function are regulated by families of protein-modifying enzymes that are sensitive to a variety of metabolic and environmental agents. These enzymes, and proteins that read the modifications they maintain, constitute a system by which environmental agents, such as chemical toxins and dietary components, can directly regulate patterns of gene expression. This review describes this environmental sensing system from an evolutionary perspective. It is proposed that persistent environmentally-induced changes in gene expression patterns can cause changes in phenotype that are acted upon by natural selection, and that epigenetic processes can potentially play central roles in evolution.     

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

The pore-formation activity of monomeric and oligomeric forms of different Cry1 toxins (from Cry1A to Cry1G) was analyzed by monitoring ionic permeability across Manduca sexta brush border membrane vesicles. The membrane vesicles were isolated from microvilli structures, showing a high enrichment of apical membrane markers and low intrinsic K⁺ permeability. A fluorometric assay performed with 3,3′-dipropylthiodicarbocyanine fluorescent probe, sensitive to changes in membrane potential, was used. Previously, it was suggested that fluorescence determinations with this dye could be strongly influenced by the pH, osmolarity and ionic strength of the medium. Therefore, we evaluated these parameters in control experiments using the K⁺-selective ionophore valinomycin. We show here that under specific ionic conditions changes in fluorescence can be correlated with ionic permeability without effects on osmolarity or ionic strength of the medium. It is extremely important to attenuate the background response due to surface membrane potential and the participation of the endogenous permeability of the membrane vesicles. Under these conditions, we analyzed the pore-formation activity induced by monomeric and oligomeric structures of different Cry1 toxins. The Cry1 toxin samples containing oligomeric structures correlated with high pore activity, in contrast to monomeric samples that showed marginal pore-formation activity, supporting the hypothesis that oligomer formation is a necessary step in the mechanism of action of Cry toxins.     

Exploiting cell death pathways by an E. coli cytotoxin: autophagy as a double-edged sword for the host

Cytotoxic necrotizing factor 1 is a bacterial protein toxin from Escherichia coli that is able to activate the Rho GTPases and to hinder apoptosis and mitotic catastrophe. Upon exposure to toxin, cells undergo a complex framework of changes, including cytoskeleton remodeling and multinucleation. These cells also show a high survival rate for long periods of time and improve both their macropinocytotic scavenging activities and microautophagy. Only at the very end, probably when "feeding" materials are exhausted, do these cells die by autophagy. Taking into account the complex role of bacterial protein toxins in the infectious processes, we indicate the CNF1 activity as a Janus-faced paradigm of those bacteria that hijack cell fate to their own benefit. This could somehow be linked to the hypothesized connection between certain bacterial toxins and cancer onset.