- Email: [email protected]
Calcium-induced folding of intrinsically disordered RTX proteins: Implications for RTX toxin physiology
Abstracts / Toxicon 75 (2013) 205–223
Clostridium botulinum C3 protein: A novel transporter for selective delivery of proteins into the cytosol of monocytes/macrophages L. Dmochewitz, H. Barth Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany
The C3 protein from Clostridium botulinum (C3bot) monoADP-ribosylates RhoA, -B and -C of eukaryotic cells. In low concentrations, C3bot is selectively taken up into the cytosol of monocytes/macrophages in less than 3 h and changes the actin cytoskeleton of such cells due to the C3-catalyzed inactivation of Rho (Fahrer et al., 2010). Most likely, C3bot is endocytosed and translocates from acidified early endosomes into the cytosol of monocytes/macrophages while it is not taken up into epithelial cells or fibroblasts under such conditions. The finding that monocytes/macrophages are target cells for C3bot contributes to understand the role of clostridial C3 ADP-ribosyltransferases in pathogenesis. Moreover, it might allow for the use of enzymatic inactive C3bot1E174Q as a carrier for the selective delivery of cargo proteins into the cytosol of monocytes/macrophages. To prove this hypothesis, the actin ADP-ribosylating C2I protein from Clostridium botulinum (w50 kDa) was genetically fused to C3bot1E174Q and C3bot1E174Q-C2I was expressed in Escherichia coli. The purified recombinant fusion toxin was recognized by specific antibodies against C2I as well as C3 and ADP-ribosylated actin in vitro indicating its C2I-specific enzyme activity. C3bot1E174Q-C2I was taken up into the cytosol of J774A.1 and RAW264.7 macrophages as indicated by sequential ADP-ribosylation of actin from such cells. In contrast, C3bot1E174Q-C2I was not taken up into the cytosol of HeLa and Vero epithelial cells. The biochemical data were confirmed by confocal fluorescence microscopy experiments with intact cells. In conclusion, the data indicate that C3bot1E174Q can be used for selective and specific delivery of foreign proteins into the cytosol of macrophages. Therefore, C3 fusion proteins might be attractive novel tools in experimental monocyte/macrophage pharmacology as well as potential novel therapeutics for treatment of monocyte/ macrophage-associated diseases. Fahrer, J., Kuban, J., Heine, K., Rupps, G., Kaiser, E., Felder, E., Benz, R., Barth, H., 2010. Selective and specific internalization of clostridial C3 ADPribosyltransferases into macrophages and monocytes. Cell Microbiol 12, 233-247.
209
the SNARE proteins. There is evidence that they are endocytosed inside acidic vesicles and that the pH gradient across the membrane drives the insertion and translocation of the metalloprotease L domain in the cytosol. This domain is linked to the rest of the molecule by a single interchain disulfide bridge that has to be reduced on the cytosolic side of the membrane to free its enzymatic activity. Using an assay that drive the entry of these neurotoxins from the plasma membrane of neurons in culture, we found that the translocation of the L chain is very fast, i.e. it is accomplished in minutes at 37 C and that it is highly temperature dependent. In addition by using specific inhibitors we identified the specific cytosolic protein disulfide reducing system that cleaves the interchain disulfide bridge. http://dx.doi.org/10.1016/j.toxicon.2013.08.014
Blocking botulism: A journey into modules and modulators M. Montal Section of Neurobiology, University of California San Diego, La Jolla, CA 92093-0366, USA
Botulinum neurotoxin (BoNT), the causative agent of botulism, is acknowledged to be one of the most feared biological weapons of the 21st century (CDC Category A). The imminent threat of a terrorist attack empowered by BoNT remains an issue of major concern nationally and globally. For BoNT, a modular nanomachine, functional complexity emerges from its modular design and the tight interplay between its component modules–a partnership with consequences that surpass the simple sum of the individual component's action. This presentation focuses on the dynamic interplay between modules, which is inextricably linked not only to the activity of the toxin but also to its sensitivity to translocation inhibitors which possess antibotulinal activity. Targeting the protein-conducting channel of BoNT is anticipated to open a new path for developing counter-measures, an endeavor of paramount significance to health science and biodefense. http://dx.doi.org/10.1016/j.toxicon.2013.08.015
http://dx.doi.org/10.1016/j.toxicon.2013.08.013
Membrane translocation of tetanus and botulinum neurotoxins a
a
a
b
M. Pirazzini , O. Rossetto , F. Bordin , C.C. Shone , T. Binz c, C. Montecucco a
Calcium-induced folding of intrinsically disordered RTX proteins: Implications for RTX toxin physiology A.C. Sotomayor-Pérez, D. Ladant, A. Chenal Institut Pasteur, Laboratoire de Biochimie des Interactions Macromoléculaires, 75015 Paris, France
a
Dipartimento di Scienze Biomediche and Istituto CNR di Neuroscienze, Università di Padova, Viale G. Colombo 3, 35131 Padova, Italy Health Protection Agency, Porton Down, Salisbury, Wiltshire SP4 OJG, UK c Institut für Biochemie, Medizinische Hochschule Hannover, D-30623 Hannover, Germany b
The Clostridial neurotoxins that cause tetanus or botulism act in the cytosol of nerve terminals by cleaving
The past decade has seen a fundamental reappraisal of the protein structure-to-function paradigm because it became evident that a significant fraction of polypeptides, commonly designated as natively or intrinsically disordered proteins, are lacking ordered structures under physiological conditions. Ligand-induced disorder-to-order transition plays a key role in the biological functions of
210
Abstracts / Toxicon 75 (2013) 205–223
many proteins that contain intrinsically disordered regions. This trait is exhibited by RTX (Repeat in ToXin) motifs found in more than 250 virulence factors secreted by Gramnegative pathogenic bacteria. We investigated several CyaA RTX polypeptides of different lengths. We showed that the RTX proteins exhibit the hallmarks of intrinsically disordered proteins in the absence of calcium: they exhibit a weak secondary structure content and a strong timeaveraged apparent hydration, due in part to the internal electrostatic repulsions between negatively charged residues, as revealed by the measured mean net charge. Calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of the RTX proteins. We propose that the intrinsically disordered character of the RTX proteins may facilitate the uptake and secretion of virulence factors through the bacterial secretion machinery. These results support the hypothesis that the calcium-induced folding reaction is achieved upon protein secretion and, in the case of proteins containing RTX motifs, could be finely regulated by the calcium gradient across bacterial cell wall. http://dx.doi.org/10.1016/j.toxicon.2013.08.016
Toxicological effects of palytoxins: An integrated in vivo and in vitro approach A. Tubaro a, G. Del Favero a, D. Beltramo b, P. Lorenzon a, M. Sciancalepore a, C. Florio a, M. Poli c, S. Sosa a a Department of Life Sciences, University of Trieste, Via A. Valerio 6, Trieste 34127, Italy b Istituto di Ricerche Biomediche “A. Marxer” RBM SpA, Via Ribes 1, 10010 Colleretto Giacosa, TO, Italy c U.S. Army Medical Research Institute of Infectious Diseases, Ft Detrick, MD 21701-5011, USA
Palytoxins (PLTXs) are potentially lethal, non-proteinaceous marine compounds which are being detected with increasing frequency in European temperate coastal waters. Adverse effects on human health ascribed to PLTX have been reported after oral intake of contaminated seafood in tropical regions. In addition, dermatological problems have occurred after skin exposures to marine aerosol and/or seawater during Ostreopsis blooms in temperate areas or through handling aquaria containing zoanthids. In the Mediterranean region, monitoring programs have detected PLTX-like molecules in edible mollusks and echinoderms along the Italian and French coasts, although no human poisonings have been yet reported. Epidemiological data suggests that consumption of PLTX-contaminated seafood is potentially lethal. Symptoms usually start with gastro-intestinal malaise, often accompanied by myalgia, muscular cramps, dyspnoea and, sometimes, cardiac alterations. In vivo toxicity studies of oral PLTX administration in mice confirmed the hypothesis that skeletal and cardiac muscles are very sensitive targets for these toxins. After acute oral administration, PLTX induced ultra-structural alterations visible as fiber disorganization and mitochondrial aggregations. Repeated oral administration caused more severe
alterations, especially at the cardiac level, visible by light microscopy: fiber degeneration and vacuolation were visible in a dose dependent manner after daily treatment for 7 days. Thus, the mechanism of toxic action was studied using primary cultures of mouse skeletal muscle cells and of rat ventricular myocytes, as cell models. PLTX was very toxic for both cells, causing concentrationdependent cytotoxicity in the nanomolar range, including severe morphological and functional alterations. Spontaneous contractile activity of cultured cardiac myocytes was lost after exposure to the toxin (2 nM). Similarly, the ability of skeletal myocytes to respond to the physiologic neurotransmitter acetylcholine was reduced after 30 minutes of incubations with 6 nM PLTX or overnight exposure to 0.1 nM. http://dx.doi.org/10.1016/j.toxicon.2013.08.017
Transcriptome analysis of expressed sequence tags (ESTs) from the venom glands of the ant species Tetramorium bicarinatum (Hymenoptera: Formicidae) W. Bouzid a, C. Klopp b, A. Vétillard a a Equipe venins et activités biologiques VACBIO EA 4357, PRES-Université de Toulouse, Centre universitaire Jean-François Champollion, Albi, France b Plateforme bioinformatique Genotoul, UR875 Biométrie et Intelligence Artificielle, INRA, 31326 Castanet-Tolosan, France
Animal venoms are a potential source of natural compounds that have selected a diverse range of biological targets. Nevertheless, most of the chemical diversity encoded within these animal venoms, and particularly that of arthropod groups, remains uncharacterized. To the best of our best knowledge, ant gland venom transcriptome has never been characterized and studies on venom ants have mainly concerned proteomic analyses. In the present work, mRNA from pooled venom glands of 200 individuals of the species Tetramorium bicarinatum were extracted and used to construct a differential cDNA library by SSH (Suppression subtractive hybridization) technology. This ant species was used as it is frequently found in tropical and subtropical areas and is thought to have an interesting diversity in its venom composition. A total of 400 ESTs (expressed sequence tags) independent clones were sequenced from which a set of 374 high quality ESTs were generated. These sequences were clustered and assembled into 269 contigs (198 multiple sequences and 71 singletons). About 72% (269) of the contigs matched BLASTx hits with an interesting diversity and unusual abundance (48%) of cellular transcripts related to gene and protein expression reflecting the specialization of this tissue. In addition, transcripts encoding transposase were relatively high expressed (14%). Their presence could witness the presence of transposable elements believed to be responsible for venom toxin diversity. About twenty per cent of the ESTs were categorized as putative toxins, being the major part represented by allergens (48% of the total venom toxins) such as pilosulin 5, sol i 3 and Myp p I and II. Intriguingly, neither hyaluronidase nor phospholipase A2, known to be
Clostridium botulinum C3 protein: A novel transporter for selective delivery of proteins into the cytosol of monocytes/macrophages L. Dmochewitz, H. Barth Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany
The C3 protein from Clostridium botulinum (C3bot) monoADP-ribosylates RhoA, -B and -C of eukaryotic cells. In low concentrations, C3bot is selectively taken up into the cytosol of monocytes/macrophages in less than 3 h and changes the actin cytoskeleton of such cells due to the C3-catalyzed inactivation of Rho (Fahrer et al., 2010). Most likely, C3bot is endocytosed and translocates from acidified early endosomes into the cytosol of monocytes/macrophages while it is not taken up into epithelial cells or fibroblasts under such conditions. The finding that monocytes/macrophages are target cells for C3bot contributes to understand the role of clostridial C3 ADP-ribosyltransferases in pathogenesis. Moreover, it might allow for the use of enzymatic inactive C3bot1E174Q as a carrier for the selective delivery of cargo proteins into the cytosol of monocytes/macrophages. To prove this hypothesis, the actin ADP-ribosylating C2I protein from Clostridium botulinum (w50 kDa) was genetically fused to C3bot1E174Q and C3bot1E174Q-C2I was expressed in Escherichia coli. The purified recombinant fusion toxin was recognized by specific antibodies against C2I as well as C3 and ADP-ribosylated actin in vitro indicating its C2I-specific enzyme activity. C3bot1E174Q-C2I was taken up into the cytosol of J774A.1 and RAW264.7 macrophages as indicated by sequential ADP-ribosylation of actin from such cells. In contrast, C3bot1E174Q-C2I was not taken up into the cytosol of HeLa and Vero epithelial cells. The biochemical data were confirmed by confocal fluorescence microscopy experiments with intact cells. In conclusion, the data indicate that C3bot1E174Q can be used for selective and specific delivery of foreign proteins into the cytosol of macrophages. Therefore, C3 fusion proteins might be attractive novel tools in experimental monocyte/macrophage pharmacology as well as potential novel therapeutics for treatment of monocyte/ macrophage-associated diseases. Fahrer, J., Kuban, J., Heine, K., Rupps, G., Kaiser, E., Felder, E., Benz, R., Barth, H., 2010. Selective and specific internalization of clostridial C3 ADPribosyltransferases into macrophages and monocytes. Cell Microbiol 12, 233-247.
209
the SNARE proteins. There is evidence that they are endocytosed inside acidic vesicles and that the pH gradient across the membrane drives the insertion and translocation of the metalloprotease L domain in the cytosol. This domain is linked to the rest of the molecule by a single interchain disulfide bridge that has to be reduced on the cytosolic side of the membrane to free its enzymatic activity. Using an assay that drive the entry of these neurotoxins from the plasma membrane of neurons in culture, we found that the translocation of the L chain is very fast, i.e. it is accomplished in minutes at 37 C and that it is highly temperature dependent. In addition by using specific inhibitors we identified the specific cytosolic protein disulfide reducing system that cleaves the interchain disulfide bridge. http://dx.doi.org/10.1016/j.toxicon.2013.08.014
Blocking botulism: A journey into modules and modulators M. Montal Section of Neurobiology, University of California San Diego, La Jolla, CA 92093-0366, USA
Botulinum neurotoxin (BoNT), the causative agent of botulism, is acknowledged to be one of the most feared biological weapons of the 21st century (CDC Category A). The imminent threat of a terrorist attack empowered by BoNT remains an issue of major concern nationally and globally. For BoNT, a modular nanomachine, functional complexity emerges from its modular design and the tight interplay between its component modules–a partnership with consequences that surpass the simple sum of the individual component's action. This presentation focuses on the dynamic interplay between modules, which is inextricably linked not only to the activity of the toxin but also to its sensitivity to translocation inhibitors which possess antibotulinal activity. Targeting the protein-conducting channel of BoNT is anticipated to open a new path for developing counter-measures, an endeavor of paramount significance to health science and biodefense. http://dx.doi.org/10.1016/j.toxicon.2013.08.015
http://dx.doi.org/10.1016/j.toxicon.2013.08.013
Membrane translocation of tetanus and botulinum neurotoxins a
a
a
b
M. Pirazzini , O. Rossetto , F. Bordin , C.C. Shone , T. Binz c, C. Montecucco a
Calcium-induced folding of intrinsically disordered RTX proteins: Implications for RTX toxin physiology A.C. Sotomayor-Pérez, D. Ladant, A. Chenal Institut Pasteur, Laboratoire de Biochimie des Interactions Macromoléculaires, 75015 Paris, France
a
Dipartimento di Scienze Biomediche and Istituto CNR di Neuroscienze, Università di Padova, Viale G. Colombo 3, 35131 Padova, Italy Health Protection Agency, Porton Down, Salisbury, Wiltshire SP4 OJG, UK c Institut für Biochemie, Medizinische Hochschule Hannover, D-30623 Hannover, Germany b
The Clostridial neurotoxins that cause tetanus or botulism act in the cytosol of nerve terminals by cleaving
The past decade has seen a fundamental reappraisal of the protein structure-to-function paradigm because it became evident that a significant fraction of polypeptides, commonly designated as natively or intrinsically disordered proteins, are lacking ordered structures under physiological conditions. Ligand-induced disorder-to-order transition plays a key role in the biological functions of
210
Abstracts / Toxicon 75 (2013) 205–223
many proteins that contain intrinsically disordered regions. This trait is exhibited by RTX (Repeat in ToXin) motifs found in more than 250 virulence factors secreted by Gramnegative pathogenic bacteria. We investigated several CyaA RTX polypeptides of different lengths. We showed that the RTX proteins exhibit the hallmarks of intrinsically disordered proteins in the absence of calcium: they exhibit a weak secondary structure content and a strong timeaveraged apparent hydration, due in part to the internal electrostatic repulsions between negatively charged residues, as revealed by the measured mean net charge. Calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of the RTX proteins. We propose that the intrinsically disordered character of the RTX proteins may facilitate the uptake and secretion of virulence factors through the bacterial secretion machinery. These results support the hypothesis that the calcium-induced folding reaction is achieved upon protein secretion and, in the case of proteins containing RTX motifs, could be finely regulated by the calcium gradient across bacterial cell wall. http://dx.doi.org/10.1016/j.toxicon.2013.08.016
Toxicological effects of palytoxins: An integrated in vivo and in vitro approach A. Tubaro a, G. Del Favero a, D. Beltramo b, P. Lorenzon a, M. Sciancalepore a, C. Florio a, M. Poli c, S. Sosa a a Department of Life Sciences, University of Trieste, Via A. Valerio 6, Trieste 34127, Italy b Istituto di Ricerche Biomediche “A. Marxer” RBM SpA, Via Ribes 1, 10010 Colleretto Giacosa, TO, Italy c U.S. Army Medical Research Institute of Infectious Diseases, Ft Detrick, MD 21701-5011, USA
Palytoxins (PLTXs) are potentially lethal, non-proteinaceous marine compounds which are being detected with increasing frequency in European temperate coastal waters. Adverse effects on human health ascribed to PLTX have been reported after oral intake of contaminated seafood in tropical regions. In addition, dermatological problems have occurred after skin exposures to marine aerosol and/or seawater during Ostreopsis blooms in temperate areas or through handling aquaria containing zoanthids. In the Mediterranean region, monitoring programs have detected PLTX-like molecules in edible mollusks and echinoderms along the Italian and French coasts, although no human poisonings have been yet reported. Epidemiological data suggests that consumption of PLTX-contaminated seafood is potentially lethal. Symptoms usually start with gastro-intestinal malaise, often accompanied by myalgia, muscular cramps, dyspnoea and, sometimes, cardiac alterations. In vivo toxicity studies of oral PLTX administration in mice confirmed the hypothesis that skeletal and cardiac muscles are very sensitive targets for these toxins. After acute oral administration, PLTX induced ultra-structural alterations visible as fiber disorganization and mitochondrial aggregations. Repeated oral administration caused more severe
alterations, especially at the cardiac level, visible by light microscopy: fiber degeneration and vacuolation were visible in a dose dependent manner after daily treatment for 7 days. Thus, the mechanism of toxic action was studied using primary cultures of mouse skeletal muscle cells and of rat ventricular myocytes, as cell models. PLTX was very toxic for both cells, causing concentrationdependent cytotoxicity in the nanomolar range, including severe morphological and functional alterations. Spontaneous contractile activity of cultured cardiac myocytes was lost after exposure to the toxin (2 nM). Similarly, the ability of skeletal myocytes to respond to the physiologic neurotransmitter acetylcholine was reduced after 30 minutes of incubations with 6 nM PLTX or overnight exposure to 0.1 nM. http://dx.doi.org/10.1016/j.toxicon.2013.08.017
Transcriptome analysis of expressed sequence tags (ESTs) from the venom glands of the ant species Tetramorium bicarinatum (Hymenoptera: Formicidae) W. Bouzid a, C. Klopp b, A. Vétillard a a Equipe venins et activités biologiques VACBIO EA 4357, PRES-Université de Toulouse, Centre universitaire Jean-François Champollion, Albi, France b Plateforme bioinformatique Genotoul, UR875 Biométrie et Intelligence Artificielle, INRA, 31326 Castanet-Tolosan, France
Animal venoms are a potential source of natural compounds that have selected a diverse range of biological targets. Nevertheless, most of the chemical diversity encoded within these animal venoms, and particularly that of arthropod groups, remains uncharacterized. To the best of our best knowledge, ant gland venom transcriptome has never been characterized and studies on venom ants have mainly concerned proteomic analyses. In the present work, mRNA from pooled venom glands of 200 individuals of the species Tetramorium bicarinatum were extracted and used to construct a differential cDNA library by SSH (Suppression subtractive hybridization) technology. This ant species was used as it is frequently found in tropical and subtropical areas and is thought to have an interesting diversity in its venom composition. A total of 400 ESTs (expressed sequence tags) independent clones were sequenced from which a set of 374 high quality ESTs were generated. These sequences were clustered and assembled into 269 contigs (198 multiple sequences and 71 singletons). About 72% (269) of the contigs matched BLASTx hits with an interesting diversity and unusual abundance (48%) of cellular transcripts related to gene and protein expression reflecting the specialization of this tissue. In addition, transcripts encoding transposase were relatively high expressed (14%). Their presence could witness the presence of transposable elements believed to be responsible for venom toxin diversity. About twenty per cent of the ESTs were categorized as putative toxins, being the major part represented by allergens (48% of the total venom toxins) such as pilosulin 5, sol i 3 and Myp p I and II. Intriguingly, neither hyaluronidase nor phospholipase A2, known to be