Fiorella Tonello

Glutamate exocytosis from astrocytes controls synaptic strength

The release of transmitters from glia influences synaptic functions. The modalities and physiological functions of glial release are poorly understood. Here we show that glutamate exocytosis from astrocytes of the rat hippocampal dentate molecular layer enhances synaptic strength at excitatory synapses between perforant path afferents and granule cells. The effect is mediated by ifenprodil-sensitive NMDA ionotropic glutamate receptors and involves an increase of transmitter release at the synapse. Correspondingly, we identify NMDA receptor 2B subunits on the extrasynaptic portion of excitatory nerve terminals. The receptor distribution is spatially related to glutamate-containing synaptic-like microvesicles in the apposed astrocytic processes. This glial regulatory pathway is endogenously activated by neuronal activity–dependent stimulation of purinergic P2Y1 receptors on the astrocytes. Thus, we provide the first combined functional and ultrastructural evidence for a physiological control of synaptic activity via exocytosis of glutamate from astrocytes.

Anthrax toxins suppress T lymphocyte activation by disrupting antigen receptor signaling

Anthrax is an infection caused by pathogenic strains of Bacillus anthracis, which secretes a three-component toxic complex consisting of protective antigen (PA), edema factor (EF), and lethal factor (LF). PA forms binary complexes with either LF or EF and mediates their entry into host cells. Although the initial phases of bacterial growth occur in the lymph node, the host fails to mount an effective immune response. Here, we show that LT and ET are potent suppressors of human T cell activation and proliferation triggered through the antigen receptor. Both LT and ET inhibit the mitogen-activated protein and stress kinase pathways, and both toxins inhibit activation of NFAT and AP-1, two transcription factors essential for cytokine gene expression. These data identify a novel strategy of immune evasion by B. anthracis, based on both effector subunits of the toxic complex, and targeted to a key cellular component of adaptive immunity.

The Helicobacter pylori neutrophil‐activating protein is an iron‐binding protein with dodecameric structure

The neutrophil‐activating protein (HP‐NAP) of Helicobacter pylori is a major 17 kDa antigen of the immune response of infected individuals. Amino acid sequence comparison indicated a high similarity between HP‐NAP and both bacterial DNA‐protecting proteins (Dps) and ferritins. The structure prediction and spectroscopic analysis presented here indicate a close similarity between HP‐NAP and Dps. Electron microscopy revealed that HP‐NAP forms hexagonal rings of 9–10 nm diameter with a hollow central core as seen in Dps proteins, clearly different from the 12 nm icositetrameric (24 subunits) ferritins. However, HP‐NAP is resistant to thermal and chemical denaturation similar to the ferritin family of proteins. In addition, HP‐NAP binds up to 40 atoms of iron per monomer and does not bind DNA. We therefore conclude that HP‐NAP is an unusual, small, ferritin that folds into a four‐helix bundle that oligomerizes into dodecamers with a central hole capable of binding up to 500 iron atoms per oligomer.

Pharmacology: Screening inhibitors of anthrax lethal factor

The disease anthrax is caused by lethal factor, an enzyme component of the toxin produced by the spore-forming bacterium Bacillus anthracis. Here we describe substrate molecules for this factor that offer a means for high-throughput screening of potential inhibitors for use in anthrax treatment. Our assay should help to answer the urgent call for new and specific therapies to combat this pathogen after its recent emergence as a terrorist bioweapon.

Screening inhibitors of anthrax lethal factor

The disease anthrax is caused by lethal factor, an enzyme component of the toxin produced by the spore-forming bacterium Bacillus anthracis. Here we describe substrate molecules for this factor that offer a means for high-throughput screening of potential inhibitors for use in anthrax treatment. Our assay should help to answer the urgent call for new and specific therapies to combat this pathogen after its recent emergence as a terrorist bioweapon.

The anthrax lethal factor and its MAPK kinase-specific metalloprotease activity

The anthrax lethal factor is a multi-domain protein toxin released by Bacillus anthracis which enters cells in a process mediated by the protective antigen and specific cell receptors. In the cytosol, the lethal factor cleaves the N-terminal tail of many MAPK kinases, thus deranging a major cell signaling pathway. The structural features at the basis of these activities of LF are reviewed here with particular attention to the proteolytic activity and to the identification of specific inhibitors. A significant similarity between the metalloprotease domain of the lethal factor and of that of the clostridial neurotoxins has been noted and is discussed.

Cell entry and cAMP imaging of anthrax edema toxin

The entry and enzymatic activity of the anthrax edema factor (EF) in different cell types was studied by monitoring EF‐induced changes in intracellular cAMP with biochemical and microscopic methods. cAMP was imaged in live cells, transfected with a fluorescence resonance energy transfer biosensor based on the protein kinase A regulatory and catalytic subunits fused to CFP and YFP, respectively. The cAMP biosensor was located either in the cytosol or was membrane‐bound owing to the addition of a tag determining its myristoylation/palmitoylation. Real‐time imaging of cells expressing the cAMP biosensors provided the time course of EF catalytic activity and an indication of its subcellular localization. Bafilomycin A1, an inhibitor of the vacuolar ATPase proton pump, completely prevented EF activity, even when added long after the toxin. The time course of appearance of the adenylate cyclase activity and of bafilomycin A1 action suggests that EF enters the cytosol from late endosomes. EF remains associated to these compartments and its activity shows a perinuclear localization generating intracellular cAMP concentration gradients from the cell centre to the periphery.

Intracellular Targets and Metalloprotease Activity of Tetanus and Botulism Neurotoxins

Tetanus and botulism neurotoxins (TeNT and BoNT, respectively) produced by Clostridia are the most toxic substances known: the mouse LD50 of highly purified preparations is between 0.1 and 1 ng/kg. They block the release of neuro- transmitters either at the peripheral (BoNT) or central (TeNT) nervous system. This tremendous potency derives from two essential features of these bacterial toxins: (a) their absolute neurospecificity and (b) their intracellular catalytic activity. By concentrating their action on a limited number of cells, whose complete functionality is essential to the survival of very complex animals such as the vertebrates, neurotoxins lead to animal death with a minimal amount of toxic molecules. The basis of this cell selectivity resides on receptors uniquely present on neuronal cells (see Halpern and Neale, this volume). Although questioned by a number of researchers, it was not unexpected that clostridial neurotoxins are enzymes, acting in the neuron cytosol (Schiavo et al. 1993a). In fact, an enzyme can modify one after another all the target molecules present in the system and hence one single molecule of an enzymic toxin is able to intoxicate a synapse. TeNT and the seven BoNTs, A-G, are zinc endopeptidases specific for protein components of the neuroexocytosis apparatus. This enzymatic activity is the subject of this chapter.

Potent inhibitors of anthrax lethal factor from green tea

The anthrax lethal factor (LF) has a major role in the development of anthrax. LF is delivered by the protective antigen (PA) inside the cell, where it exerts its metalloprotease activity on the N‐terminus of MAPK‐kinases. PA+LF are cytotoxic to macrophages in culture and kill the Fischer 344 rat when injected intravenously. We describe here the properties of some polyphenols contained in green tea as powerful inhibitors of LF metalloproteolytic activity, and how the main catechin of green tea, (−)epigallocatechin‐3‐gallate, prevents the LF‐induced death of macrophages and Fischer 344 rats.

Anthrax toxins inhibit immune cell chemotaxis by perturbing chemokine receptor signalling

Pathogenic strains of Bacillus anthracis produce two potent toxins, lethal toxin (LT), a metalloprotease that cleaves mitogen‐activated protein kinase kinases, and oedema toxin (ET), a calcium/calmodulin‐dependent adenylate cyclase. Emerging evidence indicates a role for both toxins in suppressing the initiation of both innate and adaptive immune responses, which are essential to keep the infection under control. Here we show that LT and ET inhibit chemotaxis of T‐cells and macrophages by subverting signalling by both CXC and CC chemokine receptors. The data highlight a novel strategy of immunosuppression by B. anthracis based on inhibition of immune cell homing.