The striking differences between the clinical symptoms of tetanus and botulism have been ascribed to the different fate of the parental neurotoxins once internalised in motor neurons. Tetanus toxin (TeNT) is known to undergo transcytosis into inhibitory interneurons and block the release of inhibitory neurotransmitters in the spinal cord, causing a spastic paralysis. In contrast, botulinum neurotoxins (BoNTs) block acetylcholine release at the neuromuscular junction, therefore inducing a flaccid paralysis. Whilst overt experimental evidence supports the sorting of TeNT to the axonal retrograde transport pathway, recent findings challenge the established view that BoNT trafficking is restricted to the neuromuscular junction by highlighting central effects caused by these neurotoxins. These results suggest a more complex scenario whereby BoNTs also engage long-range trafficking mechanisms. However, the intracellular pathways underlying this process remain unclear. We sought to fill this gap by using primary motor neurons either in mass culture or differentiated in microfluidic devices to directly monitor the endocytosis and axonal transport of full length BoNT/A and BoNT/E and their recombinant binding fragments. We show that BoNT/A and BoNT/E are internalised by spinal cord motor neurons and undergo fast axonal retrograde transport. BoNT/A and BoNT/E are internalised in non-acidic axonal carriers that partially overlap with those containing TeNT, following a process that is largely independent of stimulated synaptic vesicle endo-exocytosis. Following intramuscular injection in vivo, BoNT/A and TeNT displayed central effects with a similar time course. Central actions paralleled the peripheral spastic paralysis for TeNT, but lagged behind the onset of flaccid paralysis for BoNT/A. These results suggest that the fast axonal retrograde transport compartment is composed of multifunctional trafficking organelles orchestrating the simultaneous transfer of diverse cargoes from nerve terminals to the soma, and represents a general gateway for the delivery of virulence factors and pathogens to the central nervous system.

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. Delta toxin was characterized to be cytotoxic for cells expressing the ganglioside GM2 in their membrane. Here we report the genetic characterization of Delta toxin and its pore forming activity in lipid bilayers. Delta toxin consists of 318 amino acids, its 28 N-terminal amino acids corresponding to a signal peptide. The secreted Delta toxin (290 amino acids; 32619 Da) is a basic protein (pI 9.1) which shows a significant homology with C. perfringens Beta toxin (43% identity), with C. perfringens NetB (40% identity) and, to a lesser extent, with Staphylococcus aureus alpha toxin and leukotoxins. Recombinant Delta toxin showed a preference for binding to GM2, in contrast to Beta toxin, which did not bind to gangliosides. It is hemolytic for sheep red blood cells and cytotoxic for HeLa cells. In artificial diphytanoyl phosphatidylcholine membranes, Delta and Beta toxin formed channels. Conductance of the channels formed by Delta toxin, with a value of about 100 pS to more than 1 nS in 1 M KCl and a membrane potential of 20 mV, was higher than those formed by Beta toxin and their distribution was broader. The results of zero-current membrane potential measurements and single channel experiments suggest that Delta toxin forms slightly anion-selective channels, whereas the Beta toxin channels showed a preference for cations under the same conditions. C. perfringens Delta toxin shows a significant sequence homolgy with C. perfringens Beta and NetB toxins, as well as with S. aureus alpha hemolysin and leukotoxins, but exhibits different channel properties in lipid bilayers. In contrast to Beta toxin, Delta toxin recognizes GM2 as receptor and forms anion-selective channels.

The botulinum neurotoxin A C-terminal fragment (Hc

Botulinum neurotoxins are known to be among the most toxic known substances. They produce severe paralysis by preventing the release of acetylcholine at the neuromuscular junction. Thus, new strategies for efficient production of safe and effective anti-botulinum neurotoxin antisera have been a high priority. Here we describe the use of DNA electrotransfer into the skeletal muscle to enhance antiserum titers against botulinum toxin serotypes A, B, and E in mice. We treated animals with codon-optimized plasmid DNA encoding the nontoxic but highly immunogenic C-terminal heavy chain fragment of the toxin. By employing both codon optimization and the electrotransfer procedure, the immune response and corresponding neutralizing antiserum titers were markedly increased. The cellular localization of the antigen and the immunization regimens were also shown to increase neutralizing titers to >100 IU/ml. This study demonstrates that DNA electrotransfer is an effective procedure for raising neutralizing antiserum titers to remarkably high levels.

Entamoeba histolytica is the anaerobic protozoan parasite responsible for human amoebiasis, the third most deadly parasitic disease worldwide. This highly motile eukaryotic cell invades human tissues and constitutes an excellent experimental model of cell motility and cell shape deformation. The absence of extranuclear microtubules in Entamoeba histolytica means that the actin-rich cytoskeleton takes on a crucial role in not only amoebic motility but also other processes sustaining pathogenesis, such as the phagocytosis of human cells and the parasite's resistance of host immune responses. Actin is highly conserved among eukaryotes, although diverse isoforms exist in almost all organisms studied to date. However, E. histolytica has a single actin protein, the structure of which differs significantly from those of its human homologs. Here, we studied the expression, structure and dynamics of actin in E. histolytica. We used molecular and cellular approaches to evaluate actin gene expression during intestinal invasion by E. histolytica trophozoites. Based on a three-dimensional structural bioinformatics analysis, we characterized protein domains differences between amoebic actin and human actin. Fine-tuned molecular dynamics simulations enabled us to examine protein motion and refine the three-dimensional structures of both actins, including elements potentially accounting for differences changes in the affinity properties of amoebic actin and deoxyribonuclease I. The dynamic, multifunctional nature of the amoebic cytoskeleton prompted us to examine the pleiotropic forms of actin structures within live E. histolytica cells; we observed the cortical cytoskeleton, stress fibers, “dot-like” structures, adhesion plates, and macropinosomes. In line with these data, a proteomics study of actin-binding proteins highlighted the Arp2/3 protein complex as a crucial element for the development of macropinosomes and adhesion plaques.

SummaryInactivation of different small GTPases upon their glucosylation by lethal toxin from Clostridium sordellii strain IP82 (LT-82) is already known to lead to cell rounding, adherens junction (AJ) disorganization and actin depolymerization. In the present work, we observed that LT-82 induces a rapid dephosphorylation of paxillin, a protein regulating focal adhesion (FA), independently of inactivation of paxillin kinases such as Src, Fak and Pyk2. Among the small GTPases inactivated by this toxin, including Rac, Ras, Rap and Ral, we identified Rac1, as responsible for paxillin dephosphorylation using cells overexpressing Rac1 V12. Rac1 inactivation by LT-82 modifies interactions between proteins from AJ and FA complexes as shown by pull-down assays. We showed that in Triton X-100-insoluble membrane proteins from these complexes, namely E-cadherin, b-catenin, p120-catenin and talin, are decreased upon LT-82 intoxication, a treatment that also induces a rapid decrease in cell phosphoinositide content. Therefore, we proposed that Rac inactivation by LT-82 alters phosphoinositide metabolism leading to FA and AJ complex disorganization and actin depolymerization. Introductionc mi_1392 217..232

Cell motility is governed by a complex molecular machinery that converts physico-chemical cues into whole-cell movement. Understanding the underlying biophysical mechanisms requires the ability to measure physical quantities inside the cell in a simple, reproducible and preferably non-invasive manner. To this end, we developed BioFlow, a computational mechano-imaging method and associated software able to extract intracellular measurements including pressure, forces and velocity everywhere inside freely moving cells in two and three dimensions with high spatial resolution in a non-invasive manner. This is achieved by extracting the motion of intracellular material observed using fluorescence microscopy, while simultaneously inferring the parameters of a given theoretical model of the cell interior. We illustrate the power of BioFlow in the context of amoeboid cell migration, by modelling the intracellular actin bulk flow of the parasite Entamoeba histolytica using fluid dynamics, and report unique experimental measures that complement and extend both theoretical estimations and invasive experimental measures. Thanks to its flexibility, BioFlow is easily adaptable to other theoretical models of the cell, and alleviates the need for complex or invasive experimental conditions, thus constituting a powerful tool-kit for mechano-biology studies. BioFlow is open-source and freely available via the Icy software.

The development of amoebiasis is influenced by the expression of the lysine and glutamic acid rich protein 1 (KERP1), a virulence factor involved in Entamoeba histolytica adherence to human cells. Up to date, it is unknown how the protein transits the parasite cytoplasm towards the plasma membrane, specially because this organism lacks a well-defined endoplasmic reticulum (ER) and Golgi apparatus. In this work we demonstrate that KERP1 is present at the cell surface and in intracellular vesicles which traffic in a pathway that is independent of the ER-Golgi anterograde transport. The intracellular displacement of vesicles enriched in KERP1 relies on the actin-rich cytoskeleton activities. KERP1 is also present in externalized vesicles deposited on the surface of human cells. We further report the interactome of KERP1 with its association to endomembrane components and lipids. The model for KERP1 traffic here proposed hints for the first time elements of the endocytic and exocytic paths of E. histolytica.