A common origin for the bacterial toxin-antitoxin systems parD and ccd, suggested by analyses of toxin/target and toxin/antitoxin interactions.

PubMed

Smith, Andrew B; López-Villarejo, Juan; Diago-Navarro, Elizabeth; Mitchenall, Lesley A; Barendregt, Arjan; Heck, Albert J; Lemonnier, Marc; Maxwell, Anthony; Díaz-Orejas, Ramón

2012-01-01

Bacterial toxin-antitoxin (TA) systems encode two proteins, a potent inhibitor of cell proliferation (toxin) and its specific antidote (antitoxin). Structural data has revealed striking similarities between the two model TA toxins CcdB, a DNA gyrase inhibitor encoded by the ccd system of plasmid F, and Kid, a site-specific endoribonuclease encoded by the parD system of plasmid R1. While a common structural fold seemed at odds with the two clearly different modes of action of these toxins, the possibility of functional crosstalk between the parD and ccd systems, which would further point to their common evolutionary origin, has not been documented. Here, we show that the cleavage of RNA and the inhibition of protein synthesis by the Kid toxin, two activities that are specifically counteracted by its cognate Kis antitoxin, are altered, but not inhibited, by the CcdA antitoxin. In addition, Kis was able to inhibit the stimulation of DNA gyrase-mediated cleavage of DNA by CcdB, albeit less efficiently than CcdA. We further show that physical interactions between the toxins and antitoxins of the different systems do occur and define the stoichiometry of the complexes formed. We found that CcdB did not degrade RNA nor did Kid have any reproducible effect on the tested DNA gyrase activities, suggesting that these toxins evolved to reach different, rather than common, cellular targets.

The Binary Toxin CDT of Clostridium difficile as a Tool for Intracellular Delivery of Bacterial Glucosyltransferase Domains.

PubMed

Beer, Lara-Antonia; Tatge, Helma; Schneider, Carmen; Ruschig, Maximilian; Hust, Michael; Barton, Jessica; Thiemann, Stefan; Fühner, Viola; Russo, Giulio; Gerhard, Ralf

2018-06-01

Binary toxins are produced by several pathogenic bacteria. Examples are the C2 toxin from Clostridium botulinum , the iota toxin from Clostridium perfringens, and the CDT from Clostridium difficile . All these binary toxins have ADP-ribosyltransferases (ADPRT) as their enzymatically active component that modify monomeric actin in their target cells. The binary C2 toxin was intensively described as a tool for intracellular delivery of allogenic ADPRTs. Here, we firstly describe the binary toxin CDT from C. difficile as an effective tool for heterologous intracellular delivery. Even 60 kDa glucosyltransferase domains of large clostridial glucosyltransferases can be delivered into cells. The glucosyltransferase domains of five tested large clostridial glucosyltransferases were successfully introduced into cells as chimeric fusions to the CDTa adapter domain (CDTaN). Cell uptake was demonstrated by the analysis of cell morphology, cytoskeleton staining, and intracellular substrate glucosylation. The fusion toxins were functional only when the adapter domain of CDTa was N -terminally located, according to its native orientation. Thus, like other binary toxins, the CDTaN/b system can be used for standardized delivery systems not only for bacterial ADPRTs but also for a variety of bacterial glucosyltransferase domains.

Steady-state levels of G-protein beta-subunit expression are regulated by treatment of cells with bacterial toxins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watkins, D.C.; Northup, J.K.; Malbon, C.C.

1987-05-01

Cultures of 3T3-L1 cells were incubated with either 10 ng/ml cholera toxin or 10 ng/ml pertussis toxin from 4 days prior to the initiation of differentiation and throughout the subsequent incubation. Toxin concentrations were sufficient to completely prevent the labelling of alpha-subunits with (/sup 32/P)NAD/sup +/ and pertussis toxin and to prevent by more than 90% the labelling with (/sup 32/P)NAD/sup +/ and cholera toxin in membranes prepared from these cells. Neither toxin prevented the differentiation to the adipocyte phenotype. Neither toxin prevented the increases in the relative amounts of G-proteins which occur upon differentiation. Both toxins dramatically decreased themore » amount of beta-subunits. As measured by quantitative immunoblotting with antisera specific for both the 35 kDa and 36 kDa beta-subunits, levels of beta-subunit were decreased by more than 50% of steady-state level of control cells. Thus, bacterial toxins which modifies G-protein alpha-subunits are capable of modulating the levels of beta-subunits in vivo. The basis for the regulation of G-protein subunit expression by bacterial toxins is under study.« less

A Common Origin for the Bacterial Toxin-Antitoxin Systems parD and ccd, Suggested by Analyses of Toxin/Target and Toxin/Antitoxin Interactions

PubMed Central

Mitchenall, Lesley A.; Barendregt, Arjan; Heck, Albert J.; Lemonnier, Marc; Maxwell, Anthony; Díaz-Orejas, Ramón

2012-01-01

Bacterial toxin-antitoxin (TA) systems encode two proteins, a potent inhibitor of cell proliferation (toxin) and its specific antidote (antitoxin). Structural data has revealed striking similarities between the two model TA toxins CcdB, a DNA gyrase inhibitor encoded by the ccd system of plasmid F, and Kid, a site-specific endoribonuclease encoded by the parD system of plasmid R1. While a common structural fold seemed at odds with the two clearly different modes of action of these toxins, the possibility of functional crosstalk between the parD and ccd systems, which would further point to their common evolutionary origin, has not been documented. Here, we show that the cleavage of RNA and the inhibition of protein synthesis by the Kid toxin, two activities that are specifically counteracted by its cognate Kis antitoxin, are altered, but not inhibited, by the CcdA antitoxin. In addition, Kis was able to inhibit the stimulation of DNA gyrase-mediated cleavage of DNA by CcdB, albeit less efficiently than CcdA. We further show that physical interactions between the toxins and antitoxins of the different systems do occur and define the stoichiometry of the complexes formed. We found that CcdB did not degrade RNA nor did Kid have any reproducible effect on the tested DNA gyrase activities, suggesting that these toxins evolved to reach different, rather than common, cellular targets. PMID:23029540

Mass Spectrometric Detection of Bacterial Protein Toxins and Their Enzymatic Activity.

PubMed

Kalb, Suzanne R; Boyer, Anne E; Barr, John R

2015-08-31

Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A-G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin.

Antibody-based bacterial toxin detection

NASA Astrophysics Data System (ADS)

Menking, Darrell E.; Heitz, Jonathon M.; Anis, Nabil A.; Thompson, Roy G.

1994-03-01

Fiber optic evanescent fluorosensors are under investigation in our laboratory for the study of drug-receptor interactions for detection of threat agents and antibody-antigen interactions for detection of biological toxins. In a one step assay, antibodies against Cholera toxin or Staphylococcus Enterotoxin B were noncovalently immobilized on quartz fibers and probed with fluorescein-isothiocyanate (FITC)-labeled toxins. In the two-step assay, Cholera toxin or Botulinum toxoid A was immobilized onto the fiber, followed by incubation in an antiserum or partially purified antitoxin IgG. These were then probed with FITC-anti-IgG antibodies. Unlabeled toxins competed with labeled toxins or antitoxin IgG in a dose-dependent manner and the detection of the toxins was in the nanomolar range.

Mass Spectrometric Detection of Bacterial Protein Toxins and Their Enzymatic Activity

PubMed Central

Kalb, Suzanne R.; Boyer, Anne E.; Barr, John R.

2015-01-01

Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A–G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin. PMID:26404376

Human-specific bacterial pore-forming toxins induce programmed necrosis in erythrocytes.

PubMed

LaRocca, Timothy J; Stivison, Elizabeth A; Hod, Eldad A; Spitalnik, Steven L; Cowan, Peter J; Randis, Tara M; Ratner, Adam J

2014-08-26

A subgroup of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins (PFTs) has an unusually narrow host range due to a requirement for binding to human CD59 (hCD59), a glycosylphosphatidylinositol (GPI)-linked complement regulatory molecule. hCD59-specific CDCs are produced by several organisms that inhabit human mucosal surfaces and can act as pathogens, including Gardnerella vaginalis and Streptococcus intermedius. The consequences and potential selective advantages of such PFT host limitation have remained unknown. Here, we demonstrate that, in addition to species restriction, PFT ligation of hCD59 triggers a previously unrecognized pathway for programmed necrosis in primary erythrocytes (red blood cells [RBCs]) from humans and transgenic mice expressing hCD59. Because they lack nuclei and mitochondria, RBCs have typically been thought to possess limited capacity to undergo programmed cell death. RBC programmed necrosis shares key molecular factors with nucleated cell necroptosis, including dependence on Fas/FasL signaling and RIP1 phosphorylation, necrosome assembly, and restriction by caspase-8. Death due to programmed necrosis in RBCs is executed by acid sphingomyelinase-dependent ceramide formation, NADPH oxidase- and iron-dependent reactive oxygen species formation, and glycolytic formation of advanced glycation end products. Bacterial PFTs that are hCD59 independent do not induce RBC programmed necrosis. RBC programmed necrosis is biochemically distinct from eryptosis, the only other known programmed cell death pathway in mature RBCs. Importantly, RBC programmed necrosis enhances the growth of PFT-producing pathogens during exposure to primary RBCs, consistent with a role for such signaling in microbial growth and pathogenesis. In this work, we provide the first description of a new form of programmed cell death in erythrocytes (RBCs) that occurs as a consequence of cellular attack by human-specific bacterial toxins. By defining a new RBC

A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel.

PubMed

Tang, Cheng; Zhou, Xi; Nguyen, Phuong Tran; Zhang, Yunxiao; Hu, Zhaotun; Zhang, Changxin; Yarov-Yarovoy, Vladimir; DeCaen, Paul G; Liang, Songping; Liu, Zhonghua

2017-07-01

Voltage-gated sodium channels (Na V s) are activated by transiting the voltage sensor from the deactivated to the activated state. The crystal structures of several bacterial Na V s have captured the voltage sensor module (VSM) in an activated state, but structure of the deactivated voltage sensor remains elusive. In this study, we sought to identify peptide toxins stabilizing the deactivated VSM of bacterial Na V s. We screened fractions from several venoms and characterized a cystine knot toxin called JZTx-27 from the venom of tarantula Chilobrachys jingzhao as a high-affinity antagonist of the prokaryotic Na V s Ns V Ba (nonselective voltage-gated Bacillus alcalophilus ) and NaChBac (bacterial sodium channel from Bacillus halodurans ) (IC 50 = 112 nM and 30 nM, respectively). JZTx-27 was more efficacious at weaker depolarizing voltages and significantly slowed the activation but accelerated the deactivation of Ns V Ba, whereas the local anesthetic drug lidocaine was shown to antagonize Ns V Ba without affecting channel gating. Mutation analysis confirmed that JZTx-27 bound to S3-4 linker of Ns V Ba, with F98 being the critical residue in determining toxin affinity. All electrophysiological data and in silico analysis suggested that JZTx-27 trapped VSM of Ns V Ba in one of the deactivated states. In mammalian Na V s, JZTx-27 preferably inhibited the inactivation of Na V 1.5 by targeting the fourth transmembrane domain. To our knowledge, this is the first report of peptide antagonist for prokaryotic Na V s. More important, we proposed that JZTx-27 stabilized the Ns V Ba VSM in the deactivated state and may be used as a probe to determine the structure of the deactivated VSM of Na V s.-Tang, C., Zhou, X., Nguyen, P. T., Zhang, Y., Hu, Z., Zhang, C., Yarov-Yarovoy, V., DeCaen, P. G., Liang, S., Liu, Z. A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel. © FASEB.

Bacterial Signaling to the Nervous System through Toxins and Metabolites.

PubMed

Yang, Nicole J; Chiu, Isaac M

2017-03-10

Mammalian hosts interface intimately with commensal and pathogenic bacteria. It is increasingly clear that molecular interactions between the nervous system and microbes contribute to health and disease. Both commensal and pathogenic bacteria are capable of producing molecules that act on neurons and affect essential aspects of host physiology. Here we highlight several classes of physiologically important molecular interactions that occur between bacteria and the nervous system. First, clostridial neurotoxins block neurotransmission to or from neurons by targeting the SNARE complex, causing the characteristic paralyses of botulism and tetanus during bacterial infection. Second, peripheral sensory neurons-olfactory chemosensory neurons and nociceptor sensory neurons-detect bacterial toxins, formyl peptides, and lipopolysaccharides through distinct molecular mechanisms to elicit smell and pain. Bacteria also damage the central nervous system through toxins that target the brain during infection. Finally, the gut microbiota produces molecules that act on enteric neurons to influence gastrointestinal motility, and metabolites that stimulate the "gut-brain axis" to alter neural circuits, autonomic function, and higher-order brain function and behavior. Furthering the mechanistic and molecular understanding of how bacteria affect the nervous system may uncover potential strategies for modulating neural function and treating neurological diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

The DinJ/RelE Toxin-Antitoxin System Suppresses Bacterial Proliferation and Virulence of Xylella fastidiosa in Grapevine.

PubMed

Burbank, Lindsey P; Stenger, Drake C

2017-04-01

Xylella fastidiosa, the causal agent of Pierce's disease of grapes, is a slow-growing, xylem-limited, bacterial pathogen. Disease progression is characterized by systemic spread of the bacterium through xylem vessel networks, causing leaf-scorching symptoms, senescence, and vine decline. It appears to be advantageous to this pathogen to avoid excessive blockage of xylem vessels, because living bacterial cells are generally found in plant tissue with low bacterial cell density and minimal scorching symptoms. The DinJ/RelE toxin-antitoxin system is characterized here for a role in controlling bacterial proliferation and population size during plant colonization. The DinJ/RelE locus is transcribed from two separate promoters, allowing for coexpression of antitoxin DinJ with endoribonuclease toxin RelE, in addition to independent expression of RelE. The ratio of antitoxin/toxin expressed is dependent on bacterial growth conditions, with lower amounts of antitoxin present under conditions designed to mimic grapevine xylem sap. A knockout mutant of DinJ/RelE exhibits a hypervirulent phenotype, with higher bacterial populations and increased symptom development and plant decline. It is likely that DinJ/RelE acts to prevent excessive population growth, contributing to the ability of the pathogen to spread systemically without completely blocking the xylem vessels and increasing probability of acquisition by the insect vector.

Bacterial and fungal keratitis in Upper Egypt: In vitro screening of enzymes, toxins and antifungal activity

PubMed Central

Gharamah, Abdullah A; Moharram, Ahmed M; Ismail, Mady A; AL-Hussaini, Ashraf K

2014-01-01

Purpose: This work was conducted to study the ability of bacterial and fungal isolates from keratitis cases in Upper Egypt to produce enzymes, toxins, and to test the isolated fungal species sensitivity to some therapeutic agents. Materials and Methods: One hundred and fifteen patients clinically diagnosed to have microbial keratitis were investigated. From these cases, 37 bacterial isolates and 25 fungal isolates were screened for their ability to produce extra-cellular enzymes in solid media. In addition, the ability of fungal isolates to produce mycotoxins and their sensitivity to 4 antifungal agents were tested. Results: Protease, lipase, hemolysins, urease, phosphatase, and catalase were detected respectively in 48.65%, 37.84%, 59.46%, 43.24%, 67.57%, and 100% out of 37 bacterial isolates tested. Out of 25 fungal isolates tested during the present study, 80% were positive for protease, 84% for lipase and urease, 28% for blood hemolysis, and 100% for phosphatase and catalase enzymes. Thirteen fungal isolates were able to produce detectable amounts of 7 mycotoxins in culture medium (aflatoxins (B1, B2, G1, and G2), sterigmatocystin, fumagillin, diacetoxyscirpenol, zearalenone, T-2 toxin, and trichodermin). Among the antifungal agents tested in this study, terbinafine showed the highest effect against most isolates in vitro. Conclusion: In conclusion, the ability of bacterial and fungal isolates to produce extracellular enzymes and toxins may be aid in the invasion and destruction of eye tissues, which, in turn, lead to vision loss. PMID:24008795

The CDI toxin of Yersinia kristensenii is a novel bacterial member of the RNase A superfamily

DOE Office of Scientific and Technical Information (OSTI.GOV)

Batot, Gaëlle; Michalska, Karolina; Ekberg, Greg

Contact-dependent growth inhibition (CDI) is an important mechanism of inter-bacterial competition found in many Gram-negative pathogens. CDI+ cells express cell-surface CdiA proteins that bind neighboring bacteria and deliver C-terminal toxin domains (CdiA-CT) to inhibit target-cell growth. CDI+ bacteria also produce CdiI immunity proteins, which specifically neutralize cognate CdiA-CT toxins to prevent self-inhibition. Here, we present the crystal structure of the CdiA-CT/CdiI(Ykris) complex from Yersinia kris-tensenii ATCC 33638. CdiA-CTYkris adopts the same fold as angiogenin and other RNase A paralogs, but the toxin does not share sequence similarity with these nucleases and lacks the characteristic disulfide bonds of the superfamily. Consistentmore » with the structural homology, CdiA-CTYkris has potent RNase activity in vitro and in vivo. Structure-guided mutagenesis reveals that His175, Arg186, Thr276 and Tyr278 contribute to CdiA-CTYkris activity, suggesting that these residues participate in substrate binding and/or catalysis. CdiI(Ykris) binds directly over the putative active site and likely neutralizes toxicity by blocking access to RNA substrates. Significantly, CdiA-CTYkris is the first non-vertebrate protein found to possess the RNase A superfamily fold, and homologs of this toxin are associated with secretion systems in many Gram-negative and Gram-positive bacteria. These observations suggest that RNase Alike toxins are commonly deployed in inter-bacterial competition.« less

The ARTT motif and a unified structural understanding of substraterecognition in ADP ribosylating bacterial toxins and eukaryotic ADPribosyltransferases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, S.; Tainer, J.A.

2001-08-01

ADP-ribosylation is a widely occurring and biologically critical covalent chemical modification process in pathogenic mechanisms, intracellular signaling systems, DNA repair, and cell division. The reaction is catalyzed by ADP-ribosyltransferases, which transfer the ADP-ribose moiety of NAD to a target protein with nicotinamide release. A family of bacterial toxins and eukaryotic enzymes has been termed the mono-ADP-ribosyltransferases, in distinction to the poly-ADP-ribosyltransferases, which catalyze the addition of multiple ADP-ribose groups to the carboxyl terminus of eukaryotic nucleoproteins. Despite the limited primary sequence homology among the different ADP-ribosyltransferases, a central cleft bearing NAD-binding pocket formed by the two perpendicular b-sheet core hasmore » been remarkably conserved between bacterial toxins and eukaryotic mono- and poly-ADP-ribosyltransferases. The majority of bacterial toxins and eukaryotic mono-ADP-ribosyltransferases are characterized by conserved His and catalytic Glu residues. In contrast, Diphtheria toxin, Pseudomonas exotoxin A, and eukaryotic poly-ADP-ribosyltransferases are characterized by conserved Arg and catalytic Glu residues. The NAD-binding core of a binary toxin and a C3-like toxin family identified an ARTT motif (ADP-ribosylating turn-turn motif) that is implicated in substrate specificity and recognition by structural and mutagenic studies. Here we apply structure-based sequence alignment and comparative structural analyses of all known structures of ADP-ribosyltransfeases to suggest that this ARTT motif is functionally important in many ADP-ribosylating enzymes that bear a NAD binding cleft as characterized by conserved Arg and catalytic Glu residues. Overall, structure-based sequence analysis reveals common core structures and conserved active sites of ADP-ribosyltransferases to support similar NAD binding mechanisms but differing mechanisms of target protein binding via sequence variations within

Cytotoxicity of bacterial-derived toxins to immortal lung epithelial and macrophage cells.

PubMed

Peterson, Dianne E; Collier, Jayne M; Katterman, Matthew E; Turner, Rachael A; Riley, Mark R

2010-03-01

Health risks associated with inhalation and deposition of biological materials have been a topic of great concern due to highly publicized cases of inhalation anthrax, of new regulations on the release of particulate matter, and to increased concerns on the hazards of indoor air pollution. Here, we present an evaluation of the sensitivity of two immortal cell lines (A549, human lung carcinoma epithelia) and NR8383 (rat alveolar macrophages) to a variety of bacterial-derived inhalation hazards and simulants including etoposide, gliotoxin, streptolysin O, and warfarin. The cell response is evaluated through quantification of changes in mitochondrial succinate dehydrogenase activity, release of lactate dehydrogenase, initiation of apoptosis, and through changes in morphology as determined by visible light microscopy and scanning electron microscopy. These cells display dose-response relations to each toxin, except for triton which has a step change response. The first observable responses of the epithelial cells to these compounds are changes in metabolism for one toxin (warfarin) and alterations in membrane permeability for another (gliotoxin). The other four toxins display a similar time course in response as gauged by changes in metabolism and loss of membrane integrity. Macrophages are more sensitive to most toxins; however, they display a lower level of stability. This information can be used in the design of cell-based sensors responding to these and similar hazards.

Clostridium perfringens epsilon toxin: the third most potent bacterial toxin known.

PubMed

Alves, Guilherme Guerra; Machado de Ávila, Ricardo Andrez; Chávez-Olórtegui, Carlos Delfin; Lobato, Francisco Carlos Faria

2014-12-01

Epsilon toxin (ETX) is produced by Clostridium perfringens type B and D strains and causes enterotoxemia, a highly lethal disease with major impacts on the farming of domestic ruminants, particularly sheep. ETX belongs to the aerolysin-like pore-forming toxin family. Although ETX has striking similarities to other toxins in this family, ETX is often more potent, with an LD50 of 100 ng/kg in mice. Due to this high potency, ETX is considered as a potential bioterrorism agent and has been classified as a category B biological agent by the Centers for Disease Control and Prevention (CDC) of the United States. The protoxin is converted to an active toxin through proteolytic cleavage performed by specific proteases. ETX is absorbed and acts locally in the intestines then subsequently binds to and causes lesions in other organs, including the kidneys, lungs and brain. The importance of this toxin for veterinary medicine and its possible use as a biological weapon have drawn the attention of researchers and have led to a large number of studies investigating ETX. The aim of the present work is to review the existing knowledge on ETX from C. perfringens type B and D. Copyright © 2014 Elsevier Ltd. All rights reserved.

Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells.

PubMed

Ernst, Katharina; Schmid, Johannes; Beck, Matthias; Hägele, Marlen; Hohwieler, Meike; Hauff, Patricia; Ückert, Anna Katharina; Anastasia, Anna; Fauler, Michael; Jank, Thomas; Aktories, Klaus; Popoff, Michel R; Schiene-Fischer, Cordelia; Kleger, Alexander; Müller, Martin; Frick, Manfred; Barth, Holger

2017-06-02

Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.

The ng_ζ1 toxin of the gonococcal epsilon/zeta toxin/antitoxin system drains precursors for cell wall synthesis.

PubMed

Rocker, Andrea; Peschke, Madeleine; Kittilä, Tiia; Sakson, Roman; Brieke, Clara; Meinhart, Anton

2018-04-27

Bacterial toxin-antitoxin complexes are emerging as key players modulating bacterial physiology as activation of toxins induces stasis or programmed cell death by interference with vital cellular processes. Zeta toxins, which are prevalent in many bacterial genomes, were shown to interfere with cell wall formation by perturbing peptidoglycan synthesis in Gram-positive bacteria. Here, we characterize the epsilon/zeta toxin-antitoxin (TA) homologue from the Gram-negative pathogen Neisseria gonorrhoeae termed ng_ɛ1 / ng_ζ1. Contrary to previously studied streptococcal epsilon/zeta TA systems, ng_ɛ1 has an epsilon-unrelated fold and ng_ζ1 displays broader substrate specificity and phosphorylates multiple UDP-activated sugars that are precursors of peptidoglycan and lipopolysaccharide synthesis. Moreover, the phosphorylation site is different from the streptococcal zeta toxins, resulting in a different interference with cell wall synthesis. This difference most likely reflects adaptation to the individual cell wall composition of Gram-negative and Gram-positive organisms but also the distinct involvement of cell wall components in virulence.

[Alteration of cholera toxin biosynthesis in Vibrio cholerae 01 as a result of temperate phage 139 integration into bacterial chromosome].

PubMed

Eroshenko, G A; Smirnova, N I

2002-01-01

Infection of V. cholerae 01 (classical and eltor biovars) cells with the temperate cholera phage 139 derived from V. cholerae serogroup 0139 followed by integration of the phage genome into the bacterial chromosome significantly increased the production of cholera toxin, the main virulence factor. The level of toxin biosynthesis in the lysogenic V. cholerae classical strain increased 3-fold and that in V. eltor thirty times in comparison with the parental strains. Increased production of cholera toxin was not associated with an increase in the number of copies of genes involved in its biosynthesis but seemed to be due to changes in toxinogenesis regulation.

Colibactin: More Than a New Bacterial Toxin.

PubMed

Faïs, Tiphanie; Delmas, Julien; Barnich, Nicolas; Bonnet, Richard; Dalmasso, Guillaume

2018-04-10

Cyclomodulins are bacterial toxins that interfere with the eukaryotic cell cycle. A new cyclomodulin called colibactin, which is synthetized by the pks genomic island, was discovered in 2006. Despite many efforts, colibactin has not yet been purified, and its structure remains elusive. Interestingly, the pks island is found in members of the family Enterobacteriaceae (mainly Escherichia coli and Klebsiella pneumoniae ) isolated from different origins, including from intestinal microbiota, septicaemia, newborn meningitis, and urinary tract infections. Colibactin-producing bacteria induce chromosomal instability and DNA damage in eukaryotic cells, which leads to senescence of epithelial cells and apoptosis of immune cells. The pks island is mainly observed in B2 phylogroup E. coli strains, which include extra-intestinal pathogenic E. coli strains, and pks E. coli are over-represented in biopsies isolated from colorectal cancer. In addition, pks E. coli bacteria increase the number of tumours in diverse colorectal cancer mouse models. Thus, colibactin could have a major impact on human health. In the present review, we will focus on the biological effects of colibactin, the distribution of the pks island, and summarize what is currently known about its synthesis and its structure.

Colibactin: More Than a New Bacterial Toxin

PubMed Central

Faïs, Tiphanie; Delmas, Julien; Barnich, Nicolas; Bonnet, Richard

2018-01-01

Cyclomodulins are bacterial toxins that interfere with the eukaryotic cell cycle. A new cyclomodulin called colibactin, which is synthetized by the pks genomic island, was discovered in 2006. Despite many efforts, colibactin has not yet been purified, and its structure remains elusive. Interestingly, the pks island is found in members of the family Enterobacteriaceae (mainly Escherichia coli and Klebsiella pneumoniae) isolated from different origins, including from intestinal microbiota, septicaemia, newborn meningitis, and urinary tract infections. Colibactin-producing bacteria induce chromosomal instability and DNA damage in eukaryotic cells, which leads to senescence of epithelial cells and apoptosis of immune cells. The pks island is mainly observed in B2 phylogroup E. coli strains, which include extra-intestinal pathogenic E. coli strains, and pks E. coli are over-represented in biopsies isolated from colorectal cancer. In addition, pks E. coli bacteria increase the number of tumours in diverse colorectal cancer mouse models. Thus, colibactin could have a major impact on human health. In the present review, we will focus on the biological effects of colibactin, the distribution of the pks island, and summarize what is currently known about its synthesis and its structure. PMID:29642622

Nanoparticle-detained toxins for safe and effective vaccination

NASA Astrophysics Data System (ADS)

Hu, Che-Ming J.; Fang, Ronnie H.; Luk, Brian T.; Zhang, Liangfang

2013-12-01

Toxoid vaccines--vaccines based on inactivated bacterial toxins--are routinely used to promote antitoxin immunity for the treatment and prevention of bacterial infections. Following chemical or heat denaturation, inactivated toxins can be administered to mount toxin-specific immune responses. However, retaining faithful antigenic presentation while removing toxin virulence remains a major challenge and presents a trade-off between efficacy and safety in toxoid development. Here, we show a nanoparticle-based toxin-detainment strategy that safely delivers non-disrupted pore-forming toxins for immune processing. Using erythrocyte membrane-coated nanoparticles and staphylococcal α-haemolysin, we demonstrate effective virulence neutralization via spontaneous particle entrapment. Compared with vaccination with heat-denatured toxin, mice vaccinated with the nanoparticle-detained toxin showed superior protective immunity against toxin-mediated adverse effects. We find that the non-disruptive detoxification approach benefited the immunogenicity and efficacy of toxoid vaccines. We anticipate that this study will open new possibilities in the preparation of antitoxin vaccines against the many virulence factors that threaten public health.

Improved purification process for cholera toxin and its application to the quantification of residual toxin in cholera vaccines.

PubMed

Jang, Hyun; Kim, Hyo Seung; Kim, Jeong Ah; Seo, Jin Ho; Carbis, Rodney

2009-01-01

A simplified method for the purification of cholera toxin was developed. The 569B strain of Vibrio cholerae, a recognized hyper-producer of cholera toxin, was propagated in a bioreactor under conditions that promote the production of the toxin. The toxin was separated from the bacterial cells using 0.2-microm crossflow microfiltration, the clarified toxin was passed through the membrane into the permeate, and the bacterial cells were retained in the retentate. The 0.2-microm permeate was then concentrated 3-fold and diafiltered against 10 mM phosphate buffer, pH 7.6, using 30-kDa crossflow ultrafiltration. The concentrated toxin was loaded onto a cation exchange column, the toxin was bound to the column, and most of the impurities were passed unimpeded through the column. The toxin was eluted with a salt gradient of phosphate buffer, pH7.0, containing 1.0M NaCl. The peak containing the toxin was assayed for cholera toxin and protein and the purity was determined to be 92%. The toxin peak had a low endotoxin level of 3.1 EU/microg of toxin. The purified toxin was used to prepare antiserum against whole toxin, which was used in a G(M1) ganglioside-binding ELISA to determine residual levels of toxin in an oral inactivated whole-cell cholera vaccine. The G(M1) ganglioside-binding ELISA was shown to be very sensitive and capable of detecting as little as 1 ng/ml of cholera toxin.

Evolution of the deaminase fold and multiple origins of eukaryotic editing and mutagenic nucleic acid deaminases from bacterial toxin systems

PubMed Central

Iyer, Lakshminarayan M.; Zhang, Dapeng; Rogozin, Igor B.; Aravind, L.

2011-01-01

The deaminase-like fold includes, in addition to nucleic acid/nucleotide deaminases, several catalytic domains such as the JAB domain, and others involved in nucleotide and ADP-ribose metabolism. Using sensitive sequence and structural comparison methods, we develop a comprehensive natural classification of the deaminase-like fold and show that its ancestral version was likely to operate on nucleotides or nucleic acids. Consequently, we present evidence that a specific group of JAB domains are likely to possess a DNA repair function, distinct from the previously known deubiquitinating peptidase activity. We also identified numerous previously unknown clades of nucleic acid deaminases. Using inference based on contextual information, we suggest that most of these clades are toxin domains of two distinct classes of bacterial toxin systems, namely polymorphic toxins implicated in bacterial interstrain competition and those that target distantly related cells. Genome context information suggests that these toxins might be delivered via diverse secretory systems, such as Type V, Type VI, PVC and a novel PrsW-like intramembrane peptidase-dependent mechanism. We propose that certain deaminase toxins might be deployed by diverse extracellular and intracellular pathogens as also endosymbionts as effectors targeting nucleic acids of host cells. Our analysis suggests that these toxin deaminases have been acquired by eukaryotes on several independent occasions and recruited as organellar or nucleo-cytoplasmic RNA modifiers, operating on tRNAs, mRNAs and short non-coding RNAs, and also as mutators of hyper-variable genes, viruses and selfish elements. This scenario potentially explains the origin of mutagenic AID/APOBEC-like deaminases, including novel versions from Caenorhabditis, Nematostella and diverse algae and a large class of fast-evolving fungal deaminases. These observations greatly expand the distribution of possible unidentified mutagenic processes catalyzed by

Toxins, Targets, and Triggers: An Overview of Toxin-Antitoxin Biology.

PubMed

Harms, Alexander; Brodersen, Ditlev Egeskov; Mitarai, Namiko; Gerdes, Kenn

2018-06-07

Bacterial toxin-antitoxin (TA) modules are abundant genetic elements that encode a toxin protein capable of inhibiting cell growth and an antitoxin that counteracts the toxin. The majority of toxins are enzymes that interfere with translation or DNA replication, but a wide variety of molecular activities and cellular targets have been described. Antitoxins are proteins or RNAs that often control their cognate toxins through direct interactions and, in conjunction with other signaling elements, through transcriptional and translational regulation of TA module expression. Three major biological functions of TA modules have been discovered, post-segregational killing ("plasmid addiction"), abortive infection (bacteriophage immunity through altruistic suicide), and persister formation (antibiotic tolerance through dormancy). In this review, we summarize the current state of the field and highlight how multiple levels of regulation shape the conditions of toxin activation to achieve the different biological functions of TA modules. Copyright © 2018 Elsevier Inc. All rights reserved.

Clostridium spiroforme toxin is a binary toxin which ADP-ribosylates cellular actin.

PubMed

Popoff, M R; Boquet, P

1988-05-16

We have purified from Clostridium spiroforme strain 246 an heterogeneous population of proteins (Sa) ranging from 43 to 47 kilodaltons exhibiting ADP-ribosyl transferase activity as do C. botulinum C2 toxin component I or the ia chain of C. perfringens E iota toxin. C. spiriforme Sa had alone no activity upon injection in mice or inoculated to Vero cells. When spiroforme ADP ribosyl transferase were mixed with a trypsin activated protein (Sb) separated from C. spiroforme bacterial supernatant, a lethal effect in mice and cytotoxicity on Vero cells were recorded. The Sa cross-reacted immunologically with either the light chain of C. perfringens E iota toxin or the ADP-ribosyl transferase from C. difficile 196 strain. No immunological relatedness was observed between Sa and C2 toxin component I. C. spiroforme toxin is thus another binary toxin close to iota.

Prolonged exposure to bacterial toxins downregulated expression of toll-like receptors in mesenchymal stromal cell-derived osteoprogenitors

PubMed Central

Mo, Irene Fung Ying; Yip, Kevin Hak Kong; Chan, Wing Keung; Law, Helen Ka Wai; Lau, Yu Lung; Chan, Godfrey Chi Fung

2008-01-01

Background Human mesenchymal stromal cells (MSCs, also known as mesenchymal stem cells) are multipotent cells with potential therapeutic value. Owing to their osteogenic capability, MSCs may be clinically applied for facilitating osseointegration in dental implants or orthopedic repair of bony defect. However, whether wound infection or oral microflora may interfere with the growth and osteogenic differentiation of human MSCs remains unknown. This study investigated whether proliferation and osteogenic differentiation of MSCs would be affected by potent gram-positive and gram-negative derived bacterial toxins commonly found in human settings. Results We selected lipopolysaccharide (LPS) from Escherichia coli and lipoteichoic acid (LTA) from Streptococcus pyogenes as our toxins of choice. Our findings showed both LPS and LTA did not affect MSC proliferation, but prolonged LPS challenge upregulated the osteogenic differentiation of MSCs, as assessed by alkaline phosphatase activity and calcium deposition. Because toll-like receptors (TLRs), in particularly TLR4 and TLR2, are important for the cellular responsiveness to LPS and LTA respectively, we evaluated their expression profiles serially from MSCs to osteoblasts by quantitative PCR. We found that during osteogenic differentiation, MSC-derived osteoprogenitors gradually expressed TLR2 and TLR4 by Day 12. But under prolonged incubation with LPS, MSC-derived osteoprogenitors had reduced TLR2 and TLR4 gene expression. This peculiar response to LPS suggests a possible adaptive mechanism when MSCs are subjected to continuous exposure with bacteria. Conclusion In conclusion, our findings support the potential of using human MSCs as a biological graft, even under a bacterial toxin-rich environment. PMID:18799018

Toxin yet not toxic: Botulinum toxin in dentistry.

PubMed

Archana, M S

2016-04-01

Paracelsus contrasted poisons from nonpoisons, stating that "All things are poisons, and there is nothing that is harmless; the dose alone decides that something is a poison". Living organisms, such as plants, animals, and microorganisms, constitute a huge source of pharmaceutically useful medicines and toxins. Depending on their source, toxins can be categorized as phytotoxins, mycotoxins, or zootoxins, which include venoms and bacterial toxins. Any toxin can be harmful or beneficial. Within the last 100 years, the perception of botulinum neurotoxin (BTX) has evolved from that of a poison to a versatile clinical agent with various uses. BTX plays a key role in the management of many orofacial and dental disorders. Its indications are rapidly expanding, with ongoing trials for further applications. However, despite its clinical use, what BTX specifically does in each condition is still not clear. The main aim of this review is to describe some of the unclear aspects of this potentially useful agent, with a focus on the current research in dentistry.

Endoribonuclease type II toxin-antitoxin systems: functional or selfish?

PubMed

Ramisetty, Bhaskar Chandra Mohan; Santhosh, Ramachandran Sarojini

2017-07-01

Most bacterial genomes have multiple type II toxin-antitoxin systems (TAs) that encode two proteins which are referred to as a toxin and an antitoxin. Toxins inhibit a cellular process, while the interaction of the antitoxin with the toxin attenuates the toxin's activity. Endoribonuclease-encoding TAs cleave RNA in a sequence-dependent fashion, resulting in translational inhibition. To account for their prevalence and retention by bacterial genomes, TAs are credited with clinically significant phenomena, such as bacterial programmed cell death, persistence, biofilms and anti-addiction to plasmids. However, the programmed cell death and persistence hypotheses have been challenged because of conceptual, methodological and/or strain issues. In an alternative view, chromosomal TAs seem to be retained by virtue of addiction at two levels: via a poison-antidote combination (TA proteins) and via transcriptional reprogramming of the downstream core gene (due to integration). Any perturbation in the chromosomal TA operons could cause fitness loss due to polar effects on the downstream genes and hence be detrimental under natural conditions. The endoribonucleases encoding chromosomal TAs are most likely selfish DNA as they are retained by bacterial genomes, even though TAs do not confer a direct advantage via the TA proteins. TAs are likely used by various replicons as 'genetic arms' that allow the maintenance of themselves and associated genetic elements. TAs seem to be the 'selfish arms' that make the best use of the 'arms race' between bacterial genomes and plasmids.

Neutralization of Bacterial YoeBSpn Toxicity and Enhanced Plant Growth in Arabidopsis thaliana via Co-Expression of the Toxin-Antitoxin Genes

PubMed Central

Abu Bakar, Fauziah; Yeo, Chew Chieng; Harikrishna, Jennifer Ann

2016-01-01

Bacterial toxin-antitoxin (TA) systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP) fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP. Interestingly, the inducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells. PMID:27104531

AB toxins: a paradigm switch from deadly to desirable.

PubMed

Odumosu, Oludare; Nicholas, Dequina; Yano, Hiroshi; Langridge, William

2010-07-01

To ensure their survival, a number of bacterial and plant species have evolved a common strategy to capture energy from other biological systems. Being imperfect pathogens, organisms synthesizing multi-subunit AB toxins are responsible for the mortality of millions of people and animals annually. Vaccination against these organisms and their toxins has proved rather ineffective in providing long-term protection from disease. In response to the debilitating effects of AB toxins on epithelial cells of the digestive mucosa, mechanisms underlying toxin immunomodulation of immune responses have become the focus of increasing experimentation. The results of these studies reveal that AB toxins may have a beneficial application as adjuvants for the enhancement of immune protection against infection and autoimmunity. Here, we examine similarities and differences in the structure and function of bacterial and plant AB toxins that underlie their toxicity and their exceptional properties as immunomodulators for stimulating immune responses against infectious disease and for immune suppression of organ-specific autoimmunity.

AB Toxins: A Paradigm Switch from Deadly to Desirable

PubMed Central

Odumosu, Oludare; Nicholas, Dequina; Yano, Hiroshi; Langridge, William

2010-01-01

To ensure their survival, a number of bacterial and plant species have evolved a common strategy to capture energy from other biological systems. Being imperfect pathogens, organisms synthesizing multi-subunit AB toxins are responsible for the mortality of millions of people and animals annually. Vaccination against these organisms and their toxins has proved rather ineffective in providing long-term protection from disease. In response to the debilitating effects of AB toxins on epithelial cells of the digestive mucosa, mechanisms underlying toxin immunomodulation of immune responses have become the focus of increasing experimentation. The results of these studies reveal that AB toxins may have a beneficial application as adjuvants for the enhancement of immune protection against infection and autoimmunity. Here, we examine similarities and differences in the structure and function of bacterial and plant AB toxins that underlie their toxicity and their exceptional properties as immunomodulators for stimulating immune responses against infectious disease and for immune suppression of organ-specific autoimmunity. PMID:22069653

Clostridium difficile binary toxin CDT

PubMed Central

Gerding, Dale N; Johnson, Stuart; Rupnik, Maja; Aktories, Klaus

2014-01-01

Binary toxin (CDT) is frequently observed in Clostridium difficile strains associated with increased severity of C. difficile infection (CDI). CDT belongs to the family of binary ADP-ribosylating toxins consisting of two separate toxin components: CDTa, the enzymatic ADP-ribosyltransferase which modifies actin, and CDTb which binds to host cells and translocates CDTa into the cytosol. CDTb is activated by serine proteases and binds to lipolysis stimulated lipoprotein receptor. ADP-ribosylation induces depolymerization of the actin cytoskeleton. Toxin-induced actin depolymerization also produces microtubule-based membrane protrusions which form a network on epithelial cells and increase bacterial adherence. Multiple clinical studies indicate an association between binary toxin genes in C. difficile and increased 30-d CDI mortality independent of PCR ribotype. Further studies including measures of binary toxin in stool, analyses of CDI mortality caused by CDT-producing strains, and examination of the relationship of CDT expression to TcdA and TcdB toxin variants and PCR ribotypes are needed. PMID:24253566

Anti-bacterial effect of essential oil from Xanthium strumarium against shiga toxin-producing Escherichia coli.

PubMed

Sharifi-Rad, J; Soufi, L; Ayatollahi, S A M; Iriti, M; Sharifi-Rad, M; Varoni, E M; Shahri, F; Esposito, S; Kuhestani, K; Sharifi-Rad, M

2016-09-19

Shiga toxin-producing Escherichia coli (STEC) serotype O157:H7 is one of the most important human pathogenic microorganisms, which can cause life-threatening infections. Xanthium strumarium L. is a plant with anti-bacterial activity against gram-negative and gram-positive bacteria. This study aims to demonstrate in vitro efficacy of the essential oil (EO) extracted from Xanthium strumarium L. against E. coli O157:H7. Using the agar test diffusion, the effect of Xanthium strumarium L. EO (5, 10, 15, 30, 60, and 120 mg/mL) was verified at each of the four different growth phases of E. coli O157:H7. Cell counts of viable cells and colony forming unit (CFU) were determined at regular time points using Breed's method and colony counting method, respectively. No viable cell was detectable after the 1 hour-exposure to X. strumarium EO at 30, 60, and 120 mg/mL concentrations. No bacterial colony was formed after 1 h until the end of the incubation period at 24 h. At lower concentrations, the number of bacteria cells decreased and colonies could be observed only after incubation. At the exponential phase, the EO at 15 mg/mL was only bacteriostatic, while from 30 mg/mL started to be bactericidal. X. strumarium EO antibacterial activity against Shiga toxin-producing E. coli O157:H7 is dependent on EO concentration and physiological state of the microorganisms tested. The best inhibitory activity was achieved during the late exponential and the stationary phases.

Bacterial Pore-Forming Toxins Promote the Activation of Caspases in Parallel to Necroptosis to Enhance Alarmin Release and Inflammation During Pneumonia.

PubMed

Gonzalez-Juarbe, Norberto; Bradley, Kelley M; Riegler, Ashleigh N; Reyes, Luis F; Brissac, Terry; Park, Sang-Sang; Restrepo, Marcos I; Orihuela, Carlos J

2018-04-11

Pore-forming toxins are the most common virulence factor in pathogenic bacteria. They lead to membrane permeabilization and cell death. Herein, we show that respiratory epithelial cells (REC) undergoing bacterial pore-forming toxin (PFT)-induced necroptosis simultaneously experienced caspase activation independently of RIPK3. MLKL deficient REC treated with a pan-caspase inhibitor were protected in an additive manner against PFT-induced death. Subsequently, cleaved versions of caspases-2, -4 and -10 were detected within REC undergoing necroptosis by immunoblots and monoclonal antibody staining. Caspase activation was observed in lung samples from mice and non-human primates experiencing Gram-negative and Gram-positive bacterial pneumonia, respectively. During apoptosis, caspase activation normally leads to cell shrinkage, nuclear condensation, and immunoquiescent death. In contrast, caspase activity during PFT-induced necroptosis increased the release of alarmins to the extracellular milieu. Caspase-mediated alarmin release was found sufficient to activate resting macrophages, leading to Interleukin-6 production. In a mouse model of Gram-negative pneumonia, deletion of caspases -2 and -11, the mouse orthologue of caspase-4, reduced pulmonary inflammation, immune cell infiltration and lung damage. Thus, our study describes a previously unrecognized role for caspase activation in parallel to necroptosis, and indicates that their activity plays a critical pro-inflammatory role during bacterial pneumonia.

Biodegradation of polyether algal toxins--isolation of potential marine bacteria.

PubMed

Shetty, Kateel G; Huntzicker, Jacqueline V; Rein, Kathleen S; Jayachandran, Krish

2010-12-01

Marine algal toxins such as brevetoxins, okadaic acid, yessotoxin, and ciguatoxin are polyether compounds. The fate of polyether toxins in the aqueous phase, particularly bacterial biotransformation of the toxins, is poorly understood. An inexpensive and easily available polyether structural analog salinomycin was used for enrichment and isolation of potential polyether toxin degrading aquatic marine bacteria from Florida bay area, and from red tide endemic sites in the South Florida Gulf coast. Bacterial growth on salinomycin was observed in most of the enrichment cultures from both regions with colony forming units ranging from 0 to 6×10(7) per mL. The salinomycin biodegradation efficiency of bacterial isolates determined using LC-MS ranged from 22% to 94%. Selected bacterial isolates were grown in media with brevetoxin as the sole carbon source to screen for brevetoxin biodegradation capability using ELISA. Out of the two efficient salinomycin biodegrading isolates MB-2 and MB-4, maximum brevetoxin biodegradation efficiency of 45% was observed with MB-4, while MB-2 was unable to biodegrade brevetoxin. Based on 16S rRNA sequence similarity MB-4 was found have a match with Chromohalobacter sp.

Nε-Fatty acylation of Rho GTPases by a MARTX toxin effector.

PubMed

Zhou, Yan; Huang, Chunfeng; Yin, Li; Wan, Muyang; Wang, Xiaofei; Li, Lin; Liu, Yanhua; Wang, Zhao; Fu, Panhan; Zhang, Ni; Chen, She; Liu, Xiaoyun; Shao, Feng; Zhu, Yongqun

2017-10-27

The multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are a family of large toxins that are extensively distributed in bacterial pathogens. MARTX toxins are autocatalytically cleaved to multiple effector domains, which are released into host cells to modulate the host signaling pathways. The Rho guanosine triphosphatase (GTPase) inactivation domain (RID), a conserved effector domain of MARTX toxins, is implicated in cell rounding by disrupting the host actin cytoskeleton. We found that the RID is an N ε -fatty acyltransferase that covalently modifies the lysine residues in the C-terminal polybasic region of Rho GTPases. The resulting fatty acylation inhibited Rho GTPases and disrupted Rho GTPase-mediated signaling in the host. Thus, RID can mediate the lysine N ε -fatty acylation of mammalian proteins and represents a family of toxins that harbor N-fatty acyltransferase activities in bacterial pathogens. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

sRNA antitoxins: more than one way to repress a toxin.

PubMed

Wen, Jia; Fozo, Elizabeth M

2014-08-04

Bacterial toxin-antitoxin loci consist of two genes: one encodes a potentially toxic protein, and the second, an antitoxin to repress its function or expression. The antitoxin can either be an RNA or a protein. For type I and type III loci, the antitoxins are RNAs; however, they have very different modes of action. Type I antitoxins repress toxin protein expression through interacting with the toxin mRNA, thereby targeting the mRNA for degradation or preventing its translation or both; type III antitoxins directly bind to the toxin protein, sequestering it. Along with these two very different modes of action for the antitoxin, there are differences in the functions of the toxin proteins and the mobility of these loci between species. Within this review, we discuss the major differences as to how the RNAs repress toxin activity, the potential consequences for utilizing different regulatory strategies, as well as the confirmed and potential biological roles for these loci across bacterial species.

Artificial activation of toxin-antitoxin systems as an antibacterial strategy.

PubMed

Williams, Julia J; Hergenrother, Paul J

2012-06-01

Toxin-antitoxin (TA) systems are unique modules that effect plasmid stabilization via post-segregational killing of the bacterial host. The genes encoding TA systems also exist on bacterial chromosomes, and it has been speculated that these are involved in a variety of cellular processes. Interest in TA systems has increased dramatically over the past 5 years as the ubiquitous nature of TA genes on bacterial genomes has been revealed. The exploitation of TA systems as an antibacterial strategy via artificial activation of the toxin has been proposed and has considerable potential; however, efforts in this area remain in the early stages and several major questions remain. This review investigates the tractability of targeting TA systems to kill bacteria, including fundamental requirements for success, recent advances, and challenges associated with artificial toxin activation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Xylella fastidiosa plasmid-encoded PemK toxin is an endoribonuclease.

USDA-ARS?s Scientific Manuscript database

Stable inheritance of pXF-RIV11 in Xylella fastidiosa is conferred by the pemI/pemK plasmid addiction system. PemK serves as a toxin inhibiting bacterial growth; PemI is the corresponding antitoxin that blocks activity of PemK toxin by direct binding. PemK toxin and PemI antitoxin were over-expre...

Dissection of Host Susceptibility to Bacterial Infections and Its Toxins.

PubMed

Nashef, Aysar; Agbaria, Mahmoud; Shusterman, Ariel; Lorè, Nicola Ivan; Bragonzi, Alessandra; Wiess, Ervin; Houri-Haddad, Yael; Iraqi, Fuad A

2017-01-01

Infection is one of the leading causes of human mortality and morbidity. Exposure to microbial agents is obviously required. However, also non-microbial environmental and host factors play a key role in the onset, development and outcome of infectious disease, resulting in large of clinical variability between individuals in a population infected with the same microbe. Controlled and standardized investigations of the genetics of susceptibility to infectious disease are almost impossible to perform in humans whereas mouse models allow application of powerful genomic techniques to identify and validate causative genes underlying human diseases with complex etiologies. Most of current animal models used in complex traits diseases genetic mapping have limited genetic diversity. This limitation impedes the ability to create incorporated network using genetic interactions, epigenetics, environmental factors, microbiota, and other phenotypes. A novel mouse genetic reference population for high-resolution mapping and subsequently identifying genes underlying the QTL, namely the Collaborative Cross (CC) mouse genetic reference population (GRP) was recently developed. In this chapter, we discuss a variety of approaches using CC mice for mapping genes underlying quantitative trait loci (QTL) to dissect the host response to polygenic traits, including infectious disease caused by bacterial agents and its toxins.

Converting a Staphylococcus aureus toxin into effective cyclic pseudopeptide antibiotics.

PubMed

Solecki, Olivia; Mosbah, Amor; Baudy Floc'h, Michèle; Felden, Brice

2015-03-19

Staphylococcus aureus produces peptide toxins that it uses to respond to environmental cues. We previously characterized PepA1, a peptide toxin from S. aureus, that induces lytic cell death of both bacterial and host cells. That led us to suggest that PepA1 has an antibacterial activity. Here, we demonstrate that exogenously provided PepA1 has activity against both Gram-positive and Gram-negative bacteria. We also see that PepA1 is significantly hemolytic, thus limiting its use as an antibacterial agent. To overcome these limitations, we converted PepA1 into nonhemolytic derivatives. Our most promising derivative is a cyclic heptapseudopeptide with inconsequential toxicity to human cells, enhanced stability in human sera, and sharp antibacterial activity. Mechanistically, linear and helical PepA1 derivatives form pores at the bacterial and erythrocyte surfaces, while the cyclic peptide induces bacterial envelope reorganization, with insignificant action on the erythrocytes. Our work demonstrates that bacterial toxins might be an attractive starting point for antibacterial drug development. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular mechanisms of action of bacterial exotoxins.

PubMed

Balfanz, J; Rautenberg, P; Ullmann, U

1996-07-01

Toxins are one of the inventive strategies that bacteria have developed in order to survive. As virulence factors, they play a major role in the pathogenesis of infectious diseases. Recent discoveries have once more highlighted the effectiveness of these precisely adjusted bacterial weapons. Furthermore, toxins have become an invaluable tool in the investigation of fundamental cell processes, including regulation of cellular functions by various G proteins, cytoskeletal dynamics and neural transmission. In this review, the bacterial toxins are presented in a rational classification based on the molecular mechanisms of action.

Structure, Evolution, and Functions of Bacterial Type III Toxin-Antitoxin Systems.

PubMed

Goeders, Nathalie; Chai, Ray; Chen, Bihe; Day, Andrew; Salmond, George P C

2016-09-28

Toxin-antitoxin (TA) systems are small genetic modules that encode a toxin (that targets an essential cellular process) and an antitoxin that neutralises or suppresses the deleterious effect of the toxin. Based on the molecular nature of the toxin and antitoxin components, TA systems are categorised into different types. Type III TA systems, the focus of this review, are composed of a toxic endoribonuclease neutralised by a non-coding RNA antitoxin in a pseudoknotted configuration. Bioinformatic analysis shows that the Type III systems can be classified into subtypes. These TA systems were originally discovered through a phage resistance phenotype arising due to a process akin to an altruistic suicide; the phenomenon of abortive infection. Some Type III TA systems are bifunctional and can stabilise plasmids during vegetative growth and sporulation. Features particular to Type III systems are explored here, emphasising some of the characteristics of the RNA antitoxin and how these may affect the co-evolutionary relationship between toxins and cognate antitoxins in their quaternary structures. Finally, an updated analysis of the distribution and diversity of these systems are presented and discussed.

Pore-Forming Toxins Induce Macrophage Necroptosis during Acute Bacterial Pneumonia.

PubMed

González-Juarbe, Norberto; Gilley, Ryan Paul; Hinojosa, Cecilia Anahí; Bradley, Kelley Margaret; Kamei, Akinobu; Gao, Geli; Dube, Peter Herman; Bergman, Molly Ann; Orihuela, Carlos Javier

2015-12-01

Necroptosis is a highly pro-inflammatory mode of cell death regulated by RIP (or RIPK)1 and RIP3 kinases and mediated by the effector MLKL. We report that diverse bacterial pathogens that produce a pore-forming toxin (PFT) induce necroptosis of macrophages and this can be blocked for protection against Serratia marcescens hemorrhagic pneumonia. Following challenge with S. marcescens, Staphylococcus aureus, Streptococcus pneumoniae, Listeria monocytogenes, uropathogenic Escherichia coli (UPEC), and purified recombinant pneumolysin, macrophages pretreated with inhibitors of RIP1, RIP3, and MLKL were protected against death. Alveolar macrophages in MLKL KO mice were also protected during S. marcescens pneumonia. Inhibition of caspases had no impact on macrophage death and caspase-1 and -3/7 were determined to be inactive following challenge despite the detection of IL-1β in supernatants. Bone marrow-derived macrophages from RIP3 KO, but not caspase-1/11 KO or caspase-3 KO mice, were resistant to PFT-induced death. We explored the mechanisms for PFT-induced necroptosis and determined that loss of ion homeostasis at the plasma membrane, mitochondrial damage, ATP depletion, and the generation of reactive oxygen species were together responsible. Treatment of mice with necrostatin-5, an inhibitor of RIP1; GW806742X, an inhibitor of MLKL; and necrostatin-5 along with co-enzyme Q10 (N5/C10), which enhances ATP production; reduced the severity of S. marcescens pneumonia in a mouse intratracheal challenge model. N5/C10 protected alveolar macrophages, reduced bacterial burden, and lessened hemorrhage in the lungs. We conclude that necroptosis is the major cell death pathway evoked by PFTs in macrophages and the necroptosis pathway can be targeted for disease intervention.

Pore-Forming Toxins Induce Macrophage Necroptosis during Acute Bacterial Pneumonia

PubMed Central

González-Juarbe, Norberto; Gilley, Ryan Paul; Hinojosa, Cecilia Anahí; Bradley, Kelley Margaret; Kamei, Akinobu; Gao, Geli; Dube, Peter Herman; Bergman, Molly Ann; Orihuela, Carlos Javier

2015-01-01

Necroptosis is a highly pro-inflammatory mode of cell death regulated by RIP (or RIPK)1 and RIP3 kinases and mediated by the effector MLKL. We report that diverse bacterial pathogens that produce a pore-forming toxin (PFT) induce necroptosis of macrophages and this can be blocked for protection against Serratia marcescens hemorrhagic pneumonia. Following challenge with S. marcescens, Staphylococcus aureus, Streptococcus pneumoniae, Listeria monocytogenes, uropathogenic Escherichia coli (UPEC), and purified recombinant pneumolysin, macrophages pretreated with inhibitors of RIP1, RIP3, and MLKL were protected against death. Alveolar macrophages in MLKL KO mice were also protected during S. marcescens pneumonia. Inhibition of caspases had no impact on macrophage death and caspase-1 and -3/7 were determined to be inactive following challenge despite the detection of IL-1β in supernatants. Bone marrow-derived macrophages from RIP3 KO, but not caspase-1/11 KO or caspase-3 KO mice, were resistant to PFT-induced death. We explored the mechanisms for PFT-induced necroptosis and determined that loss of ion homeostasis at the plasma membrane, mitochondrial damage, ATP depletion, and the generation of reactive oxygen species were together responsible. Treatment of mice with necrostatin-5, an inhibitor of RIP1; GW806742X, an inhibitor of MLKL; and necrostatin-5 along with co-enzyme Q10 (N5/C10), which enhances ATP production; reduced the severity of S. marcescens pneumonia in a mouse intratracheal challenge model. N5/C10 protected alveolar macrophages, reduced bacterial burden, and lessened hemorrhage in the lungs. We conclude that necroptosis is the major cell death pathway evoked by PFTs in macrophages and the necroptosis pathway can be targeted for disease intervention. PMID:26659062

Neutralizing Monoclonal Antibodies against Disparate Epitopes on Ricin Toxin's Enzymatic Subunit Interfere with Intracellular Toxin Transport.

PubMed

Yermakova, Anastasiya; Klokk, Tove Irene; O'Hara, Joanne M; Cole, Richard; Sandvig, Kirsten; Mantis, Nicholas J

2016-03-07

Ricin is a member of the A-B family of bacterial and plant toxins that exploit retrograde trafficking to the Golgi apparatus and endoplasmic reticulum (ER) as a means to deliver their cytotoxic enzymatic subunits into the cytoplasm of mammalian cells. In this study we demonstrate that R70 and SyH7, two well-characterized monoclonal antibodies (mAbs) directed against distinct epitopes on the surface of ricin's enzymatic subunit (RTA), interfere with toxin transport from the plasma membrane to the trans Golgi network. Toxin-mAb complexes formed on the cell surface delayed ricin's egress from EEA-1(+) and Rab7(+) vesicles and enhanced toxin accumulation in LAMP-1(+) vesicles, suggesting the complexes were destined for degradation in lysosomes. Three other RTA-specific neutralizing mAbs against different epitopes were similar to R70 and SyH7 in terms of their effects on ricin retrograde transport. We conclude that interference with toxin retrograde transport may be a hallmark of toxin-neutralizing antibodies directed against disparate epitopes on RTA.

The Pathogenetic Effect of Natural and Bacterial Toxins on Atopic Dermatitis

PubMed Central

Park, Kyung-Duck; Pak, Sok Cheon; Park, Kwan-Kyu

2016-01-01

Atopic dermatitis (AD) is a common allergic skin disease that is associated with chronic, recurrent eczematous and pruritic lesions at the flexural folds caused by interacting factors related to environmental and immune system changes. AD results in dry skin, and immunoglobulin E-mediated allergic reactions to foods and environmental allergens. While steroids and anti-histamines temporarily relieve the symptoms of AD, the possibility of side effects from pharmacological interventions remains. Despite intensive research, the underlying mechanisms for AD have not been clarified. A study of Staphylococcus aureus (S. aureus) established the role of its toxins in the pathogenesis of AD. Approximately 90% of patients with AD experience S. aureus colonization and up to 50%–60% of the colonizing S. aureus is toxin-producing. Any damage to the protective skin barrier allows for the entry of invading allergens and pathogens that further drive the pathogenesis of AD. Some natural toxins (or their components) that have therapeutic effects on AD have been studied. In addition, recent studies on inflammasomes as one component of the innate immune system have been carried out. Additionally, studies on the close relationship between the activation of inflammasomes and toxins in AD have been reported. This review highlights the literature that discusses the pathogenesis of AD, the role of toxins in AD, and the positive and negative effects of toxins on AD. Lastly, suggestions are made regarding the role of inflammasomes in AD. PMID:28025545

Structure, Evolution, and Functions of Bacterial Type III Toxin-Antitoxin Systems

PubMed Central

Goeders, Nathalie; Chai, Ray; Chen, Bihe; Day, Andrew; Salmond, George P. C.

2016-01-01

Toxin-antitoxin (TA) systems are small genetic modules that encode a toxin (that targets an essential cellular process) and an antitoxin that neutralises or suppresses the deleterious effect of the toxin. Based on the molecular nature of the toxin and antitoxin components, TA systems are categorised into different types. Type III TA systems, the focus of this review, are composed of a toxic endoribonuclease neutralised by a non-coding RNA antitoxin in a pseudoknotted configuration. Bioinformatic analysis shows that the Type III systems can be classified into subtypes. These TA systems were originally discovered through a phage resistance phenotype arising due to a process akin to an altruistic suicide; the phenomenon of abortive infection. Some Type III TA systems are bifunctional and can stabilise plasmids during vegetative growth and sporulation. Features particular to Type III systems are explored here, emphasising some of the characteristics of the RNA antitoxin and how these may affect the co-evolutionary relationship between toxins and cognate antitoxins in their quaternary structures. Finally, an updated analysis of the distribution and diversity of these systems are presented and discussed. PMID:27690100

Anthrax lethal and edema toxins in anthrax pathogenesis

PubMed Central

Liu, Shihui; Moayeri, Mahtab; Leppla, Stephen H.

2014-01-01

The pathophysiological effects resulting from many bacterial diseases are caused by exotoxins released by the bacteria. Bacillus anthracis, a spore-forming bacterium, is such a pathogen, causing anthrax through a combination of bacterial infection and toxemia. B. anthracis causes natural infection in humans and animals and has been a top bioterrorism concern since the 2001 anthrax attacks in the USA. The exotoxins secreted by B. anthracis use CMG2 as the major toxin receptor and play essential roles in pathogenesis during the entire course of the disease. This review focuses on the activities of anthrax toxins and their roles in initial and late stages of anthrax infection. PMID:24684968

Sensitive detection of active Shiga toxin using low cost CCD based optical detector

USDA-ARS?s Scientific Manuscript database

To reduce the sources and incidence of food-borne illness there is a need to develop inexpensive sensitive devices for detection of active toxin, such as Shiga toxin type 2 (Stx2). This approach increases the availability of foodborne bacterial toxin diagnostics in regions where there are limited r...

Incorporation of a non-human glycan mediates human susceptibility to a bacterial toxin

PubMed Central

Byres, Emma; Paton, Adrienne W.; Paton, James C.; Löfling, Jonas C.; Smith, David F.; Wilce, Matthew C.J.; Talbot, Ursula M.; Chong, Damien C.; Yu, Hai; Huang, Shengshu; Chen, Xi; Varki, Nissi M.; Varki, Ajit; Rossjohn, Jamie; Beddoe, Travis

2009-01-01

AB5 toxins comprise an A subunit that corrupts essential eukaryotic cell functions, and pentameric B subunits that direct target cell uptake after binding surface glycans. Subtilase cytotoxin (SubAB) is an AB5 toxin secreted by Shiga toxigenic Escherichia coli (STEC)1, which causes serious gastrointestinal disease in humans2. SubAB causes haemolytic uraemic syndrome-like pathology in mice3 via SubA-mediated cleavage of BiP/GRP78, an essential endoplasmic reticulum chaperone4. Here we show that SubB has a strong preference for glycans terminating in the sialic acid N-glycolylneuraminic acid (Neu5Gc), a monosaccharide not synthesised in humans. Structures of SubB-Neu5Gc complexes revealed the basis for this specificity, and mutagenesis of key SubB residues abrogated in vitro glycan recognition, cell binding and cytotoxicity. SubAB specificity for Neu5Gc was confirmed using mouse tissues with a human-like deficiency of Neu5Gc and human cell lines fed with Neu5Gc. Despite human lack of Neu5Gc biosynthesis, assimilation of dietary Neu5Gc creates high-affinity receptors on human gut epithelia and kidney vasculature. This, together with the human lack of Neu5Gc-containing body fluid competitors, confers susceptibility to the gastrointestinal and systemic toxicities of SubAB. Ironically, foods rich in Neu5Gc are the most common source of STEC contamination. Thus a bacterial toxin’s receptor is generated by metabolic incorporation of an exogenous factor derived from food. PMID:18971931

Host adaptation of a bacterial toxin from the human pathogen Salmonella Typhi

PubMed Central

Deng, Lingquan; Song, Jeongmin; Gao, Xiang; Wang, Jiawei; Yu, Hai; Chen, Xi; Varki, Nissi; Naito-Matsui, Yuko; Galán, Jorge E.; Varki, Ajit

2014-01-01

Salmonella Typhi is an exclusive human pathogen that causes typhoid fever. Typhoid toxin is a S. Typhi virulence factor that can reproduce most of the typhoid fever symptoms in experimental animals. Toxicity depends on toxin binding to terminally sialylated glycans on surface glycoproteins. Human glycans are unusual because of the lack of CMAH, which in other mammals converts N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc). Here we report that typhoid toxin binds to and is toxic towards cells expressing glycans terminated in Neu5Ac (expressed by humans) over glycans terminated in Neu5Gc (expressed by other mammals). Mice constitutively expressing CMAH thus displaying Neu5Gc in all tissues are resistant to typhoid toxin. The atomic structure of typhoid toxin bound to Neu5Ac reveals the structural bases for its binding specificity. These findings provide insight into the molecular bases for Salmonella Typhi’s host specificity and may help the development of therapies for typhoid fever. PMID:25480294

Antibody-mediated inhibition of ricin toxin retrograde transport.

PubMed

Yermakova, Anastasiya; Klokk, Tove Irene; Cole, Richard; Sandvig, Kirsten; Mantis, Nicholas J

2014-04-08

Ricin is a member of the ubiquitous family of plant and bacterial AB toxins that gain entry into the cytosol of host cells through receptor-mediated endocytosis and retrograde traffic through the trans-Golgi network (TGN) and endoplasmic reticulum (ER). While a few ricin toxin-specific neutralizing monoclonal antibodies (MAbs) have been identified, the mechanisms by which these antibodies prevent toxin-induced cell death are largely unknown. Using immunofluorescence confocal microscopy and a TGN-specific sulfation assay, we demonstrate that 24B11, a MAb against ricin's binding subunit (RTB), associates with ricin in solution or when prebound to cell surfaces and then markedly enhances toxin uptake into host cells. Following endocytosis, however, toxin-antibody complexes failed to reach the TGN; instead, they were shunted to Rab7-positive late endosomes and LAMP-1-positive lysosomes. Monovalent 24B11 Fab fragments also interfered with toxin retrograde transport, indicating that neither cross-linking of membrane glycoproteins/glycolipids nor the recently identified intracellular Fc receptor is required to derail ricin en route to the TGN. Identification of the mechanism(s) by which antibodies like 24B11 neutralize ricin will advance our fundamental understanding of protein trafficking in mammalian cells and may lead to the discovery of new classes of toxin inhibitors and therapeutics for biodefense and emerging infectious diseases. IMPORTANCE Ricin is the prototypic member of the AB family of medically important plant and bacterial toxins that includes cholera and Shiga toxins. Ricin is also a category B biothreat agent. Despite ongoing efforts to develop vaccines and antibody-based therapeutics against ricin, very little is known about the mechanisms by which antibodies neutralize this toxin. In general, it is thought that antibodies simply prevent toxins from attaching to cell surface receptors or promote their clearance through Fc receptor (FcR)-mediated uptake

Diversification of Type VI Secretion System Toxins Reveals Ancient Antagonism among Bee Gut Microbes.

PubMed

Steele, Margaret I; Kwong, Waldan K; Whiteley, Marvin; Moran, Nancy A

2017-12-12

Microbial communities are shaped by interactions among their constituent members. Some Gram-negative bacteria employ type VI secretion systems (T6SSs) to inject protein toxins into neighboring cells. These interactions have been theorized to affect the composition of host-associated microbiomes, but the role of T6SSs in the evolution of gut communities is not well understood. We report the discovery of two T6SSs and numerous T6SS-associated Rhs toxins within the gut bacteria of honey bees and bumble bees. We sequenced the genomes of 28 strains of Snodgrassella alvi , a characteristic bee gut microbe, and found tremendous variability in their Rhs toxin complements: altogether, these strains appear to encode hundreds of unique toxins. Some toxins are shared with Gilliamella apicola , a coresident gut symbiont, implicating horizontal gene transfer as a source of toxin diversity in the bee gut. We use data from a transposon mutagenesis screen to identify toxins with antibacterial function in the bee gut and validate the function and specificity of a subset of these toxin and immunity genes in Escherichia coli Using transcriptome sequencing, we demonstrate that S. alvi T6SSs and associated toxins are upregulated in the gut environment. We find that S. alvi Rhs loci have a conserved architecture, consistent with the C-terminal displacement model of toxin diversification, with Rhs toxins, toxin fragments, and cognate immunity genes that are expressed and confer strong fitness effects in vivo Our findings of T6SS activity and Rhs toxin diversity suggest that T6SS-mediated competition may be an important driver of coevolution within the bee gut microbiota. IMPORTANCE The structure and composition of host-associated bacterial communities are of broad interest, because these communities affect host health. Bees have a simple, conserved gut microbiota, which provides an opportunity to explore interactions between species that have coevolved within their host over millions of

Horizontal gene transfer of chromosomal Type II toxin-antitoxin systems of Escherichia coli.

PubMed

Ramisetty, Bhaskar Chandra Mohan; Santhosh, Ramachandran Sarojini

2016-02-01

Type II toxin-antitoxin systems (TAs) are small autoregulated bicistronic operons that encode a toxin protein with the potential to inhibit metabolic processes and an antitoxin protein to neutralize the toxin. Most of the bacterial genomes encode multiple TAs. However, the diversity and accumulation of TAs on bacterial genomes and its physiological implications are highly debated. Here we provide evidence that Escherichia coli chromosomal TAs (encoding RNase toxins) are 'acquired' DNA likely originated from heterologous DNA and are the smallest known autoregulated operons with the potential for horizontal propagation. Sequence analyses revealed that integration of TAs into the bacterial genome is unique and contributes to variations in the coding and/or regulatory regions of flanking host genome sequences. Plasmids and genomes encoding identical TAs of natural isolates are mutually exclusive. Chromosomal TAs might play significant roles in the evolution and ecology of bacteria by contributing to host genome variation and by moderation of plasmid maintenance. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Toxin-Antitoxin Systems as Multilevel Interaction Systems

PubMed Central

Goeders, Nathalie; Van Melderen, Laurence

2014-01-01

Toxin-antitoxin (TA) systems are small genetic modules usually composed of a toxin and an antitoxin counteracting the activity of the toxic protein. These systems are widely spread in bacterial and archaeal genomes. TA systems have been assigned many functions, ranging from persistence to DNA stabilization or protection against mobile genetic elements. They are classified in five types, depending on the nature and mode of action of the antitoxin. In type I and III, antitoxins are RNAs that either inhibit the synthesis of the toxin or sequester it. In type II, IV and V, antitoxins are proteins that either sequester, counterbalance toxin activity or inhibit toxin synthesis. In addition to these interactions between the antitoxin and toxin components (RNA-RNA, protein-protein, RNA-protein), TA systems interact with a variety of cellular factors, e.g., toxins target essential cellular components, antitoxins are degraded by RNAses or ATP-dependent proteases. Hence, TA systems have the capacity to interact with each other at different levels. In this review, we will discuss the different interactions in which TA systems are involved and their implications in TA system functions and evolution. PMID:24434905

Defining the mRNA recognition signature of a bacterial toxin protein

DOE PAGES

Schureck, Marc A.; Dunkle, Jack A.; Maehigashi, Tatsuya; ...

2015-10-27

Bacteria contain multiple type II toxins that selectively degrade mRNAs bound to the ribosome to regulate translation and growth and facilitate survival during the stringent response. Ribosome-dependent toxins recognize a variety of three-nucleotide codons within the aminoacyl (A) site, but how these endonucleases achieve substrate specificity remains poorly understood. In this paper, we identify the critical features for how the host inhibition of growth B (HigB) toxin recognizes each of the three A-site nucleotides for cleavage. X-ray crystal structures of HigB bound to two different codons on the ribosome illustrate how HigB uses a microbial RNase-like nucleotide recognition loop tomore » recognize either cytosine or adenosine at the second A-site position. Strikingly, a single HigB residue and 16S rRNA residue C1054 form an adenosine-specific pocket at the third A-site nucleotide, in contrast to how tRNAs decode mRNA. Finally, our results demonstrate that the most important determinant for mRNA cleavage by ribosome-dependent toxins is interaction with the third A-site nucleotide.« less

Defining the mRNA recognition signature of a bacterial toxin protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schureck, Marc A.; Dunkle, Jack A.; Maehigashi, Tatsuya

Bacteria contain multiple type II toxins that selectively degrade mRNAs bound to the ribosome to regulate translation and growth and facilitate survival during the stringent response. Ribosome-dependent toxins recognize a variety of three-nucleotide codons within the aminoacyl (A) site, but how these endonucleases achieve substrate specificity remains poorly understood. In this paper, we identify the critical features for how the host inhibition of growth B (HigB) toxin recognizes each of the three A-site nucleotides for cleavage. X-ray crystal structures of HigB bound to two different codons on the ribosome illustrate how HigB uses a microbial RNase-like nucleotide recognition loop tomore » recognize either cytosine or adenosine at the second A-site position. Strikingly, a single HigB residue and 16S rRNA residue C1054 form an adenosine-specific pocket at the third A-site nucleotide, in contrast to how tRNAs decode mRNA. Finally, our results demonstrate that the most important determinant for mRNA cleavage by ribosome-dependent toxins is interaction with the third A-site nucleotide.« less

mRNA bound to the 30S subunit is a HigB toxin substrate

PubMed Central

Schureck, Marc A.; Maehigashi, Tatsuya; Miles, Stacey J.; Marquez, Jhomar; Dunham, Christine M.

2016-01-01

Activation of bacterial toxins during stress results in cleavage of mRNAs in the context of the ribosome. These toxins are thought to function as global translational inhibitors yet recent studies suggest each may have distinct mRNA specificities that result in selective translation for bacterial survival. Here we demonstrate that mRNA in the context of a bacterial 30S subunit is sufficient for ribosome-dependent toxin HigB endonucleolytic activity, suggesting that HigB interferes with the initiation step of translation. We determined the X-ray crystal structure of HigB bound to the 30S, revealing that two solvent-exposed clusters of HigB basic residues directly interact with 30S 16S rRNA helices 18, 30, and 31. We further show that these HigB residues are essential for ribosome recognition and function. Comparison with other ribosome-dependent toxins RelE and YoeB reveals that each interacts with similar features of the 30S aminoacyl (A) site yet does so through presentation of diverse structural motifs. PMID:27307497

Clostridial binary toxins: iota and C2 family portraits.

PubMed

Stiles, Bradley G; Wigelsworth, Darran J; Popoff, Michel R; Barth, Holger

2011-01-01

There are many pathogenic Clostridium species with diverse virulence factors that include protein toxins. Some of these bacteria, such as C. botulinum, C. difficile, C. perfringens, and C. spiroforme, cause enteric problems in animals as well as humans. These often fatal diseases can partly be attributed to binary protein toxins that follow a classic AB paradigm. Within a targeted cell, all clostridial binary toxins destroy filamentous actin via mono-ADP-ribosylation of globular actin by the A component. However, much less is known about B component binding to cell-surface receptors. These toxins share sequence homology amongst themselves and with those produced by another Gram-positive, spore-forming bacterium also commonly associated with soil and disease: Bacillus anthracis. This review focuses upon the iota and C2 families of clostridial binary toxins and includes: (1) basics of the bacterial source; (2) toxin biochemistry; (3) sophisticated cellular uptake machinery; and (4) host-cell responses following toxin-mediated disruption of the cytoskeleton. In summary, these protein toxins aid diverse enteric species within the genus Clostridium.

Clostridial Binary Toxins: Iota and C2 Family Portraits

PubMed Central

Stiles, Bradley G.; Wigelsworth, Darran J.; Popoff, Michel R.; Barth, Holger

2011-01-01

There are many pathogenic Clostridium species with diverse virulence factors that include protein toxins. Some of these bacteria, such as C. botulinum, C. difficile, C. perfringens, and C. spiroforme, cause enteric problems in animals as well as humans. These often fatal diseases can partly be attributed to binary protein toxins that follow a classic AB paradigm. Within a targeted cell, all clostridial binary toxins destroy filamentous actin via mono-ADP-ribosylation of globular actin by the A component. However, much less is known about B component binding to cell-surface receptors. These toxins share sequence homology amongst themselves and with those produced by another Gram-positive, spore-forming bacterium also commonly associated with soil and disease: Bacillus anthracis. This review focuses upon the iota and C2 families of clostridial binary toxins and includes: (1) basics of the bacterial source; (2) toxin biochemistry; (3) sophisticated cellular uptake machinery; and (4) host–cell responses following toxin-mediated disruption of the cytoskeleton. In summary, these protein toxins aid diverse enteric species within the genus Clostridium. PMID:22919577

HLH-30/TFEB-mediated autophagy functions in a cell-autonomous manner for epithelium intrinsic cellular defense against bacterial pore-forming toxin in C. elegans.

PubMed

Chen, Huan-Da; Kao, Cheng-Yuan; Liu, Bang-Yu; Huang, Shin-Whei; Kuo, Cheng-Ju; Ruan, Jhen-Wei; Lin, Yen-Hung; Huang, Cheng-Rung; Chen, Yu-Hung; Wang, Horng-Dar; Aroian, Raffi V; Chen, Chang-Shi

2017-02-01

Autophagy is an evolutionarily conserved intracellular system that maintains cellular homeostasis by degrading and recycling damaged cellular components. The transcription factor HLH-30/TFEB-mediated autophagy has been reported to regulate tolerance to bacterial infection, but less is known about the bona fide bacterial effector that activates HLH-30 and autophagy. Here, we reveal that bacterial membrane pore-forming toxin (PFT) induces autophagy in an HLH-30-dependent manner in Caenorhabditis elegans. Moreover, autophagy controls the susceptibility of animals to PFT toxicity through xenophagic degradation of PFT and repair of membrane-pore cell-autonomously in the PFT-targeted intestinal cells in C. elegans. These results demonstrate that autophagic pathways and autophagy are induced partly at the transcriptional level through HLH-30 activation and are required to protect metazoan upon PFT intoxication. Together, our data show a new and powerful connection between HLH-30-mediated autophagy and epithelium intrinsic cellular defense against the single most common mode of bacterial attack in vivo.

The action of the bacterial toxin microcin B17. Insight into the cleavage-religation reaction of DNA gyrase.

PubMed

Pierrat, Olivier A; Maxwell, Anthony

2003-09-12

We have examined the effects of the bacterial toxin microcin B17 (MccB17) on the reactions of Escherichia coli DNA gyrase. MccB17 slows down but does not completely inhibit the DNA supercoiling and relaxation reactions of gyrase. A kinetic analysis of the cleavage-religation equilibrium of gyrase was performed to determine the effect of the toxin on the forward (cleavage) and reverse (religation) reactions. A simple mechanism of two consecutive reversible reactions with a nicked DNA intermediate was used to simulate the kinetics of cleavage and religation. The action of MccB17 on the kinetics of cleavage and religation was compared with that of the quinolones ciprofloxacin and oxolinic acid. With relaxed DNA as substrate, only a small amount of gyrase cleavage complex is observed with MccB17 in the absence of ATP, whereas the presence of the nucleotide significantly enhances the effect of the toxin on both the cleavage and religation reactions. In contrast, ciprofloxacin, oxolinic acid, and Ca2+ show lesser dependence on ATP to stabilize the cleavage complex. MccB17 enhances the overall rate of DNA cleavage by increasing the forward rate constant (k2) of the second equilibrium. In contrast, ciprofloxacin increases the amount of cleaved DNA by a combined effect on the forward and reverse rate constants of both equilibria. Based on these results and on the observations that MccB17 only slowly inhibits the supercoiling and relaxation reactions, we suggest a model of the interaction of MccB17 with gyrase.

The role of toxins in Clostridium difficile infection.

PubMed

Chandrasekaran, Ramyavardhanee; Lacy, D Borden

2017-11-01

Clostridium difficile is a bacterial pathogen that is the leading cause of nosocomial antibiotic-associated diarrhea and pseudomembranous colitis worldwide. The incidence, severity, mortality and healthcare costs associated with C. difficile infection (CDI) are rising, making C. difficile a major threat to public health. Traditional treatments for CDI involve use of antibiotics such as metronidazole and vancomycin, but disease recurrence occurs in about 30% of patients, highlighting the need for new therapies. The pathogenesis of C. difficile is primarily mediated by the actions of two large clostridial glucosylating toxins, toxin A (TcdA) and toxin B (TcdB). Some strains produce a third toxin, the binary toxin C. difficile transferase, which can also contribute to C. difficile virulence and disease. These toxins act on the colonic epithelium and immune cells and induce a complex cascade of cellular events that result in fluid secretion, inflammation and tissue damage, which are the hallmark features of the disease. In this review, we summarize our current understanding of the structure and mechanism of action of the C. difficile toxins and their role in disease. Published by Oxford University Press on behalf of FEMS 2017.

Bacterial Toxin–Antitoxin Systems: More Than Selfish Entities?

PubMed Central

Van Melderen, Laurence; Saavedra De Bast, Manuel

2009-01-01

Bacterial toxin–antitoxin (TA) systems are diverse and widespread in the prokaryotic kingdom. They are composed of closely linked genes encoding a stable toxin that can harm the host cell and its cognate labile antitoxin, which protects the host from the toxin's deleterious effect. TA systems are thought to invade bacterial genomes through horizontal gene transfer. Some TA systems might behave as selfish elements and favour their own maintenance at the expense of their host. As a consequence, they may contribute to the maintenance of plasmids or genomic islands, such as super-integrons, by post-segregational killing of the cell that loses these genes and so suffers the stable toxin's destructive effect. The function of the chromosomally encoded TA systems is less clear and still open to debate. This Review discusses current hypotheses regarding the biological roles of these evolutionarily successful small operons. We consider the various selective forces that could drive the maintenance of TA systems in bacterial genomes. PMID:19325885

Foodborne pathogens and their toxins.

PubMed

Martinović, Tamara; Andjelković, Uroš; Gajdošik, Martina Šrajer; Rešetar, Dina; Josić, Djuro

2016-09-16

Foodborne pathogens, mostly bacteria and fungi, but also some viruses, prions and protozoa, contaminate food during production and processing, but also during storage and transport before consuming. During their growth these microorganisms can secrete different components, including toxins, into the extracellular environment. Other harmful substances can be also liberated and can contaminate food after disintegration of food pathogens. Some bacterial and fungal toxins can be resistant to inactivation, and can survive harsh treatment during food processing. Many of these molecules are involved in cellular processes and can indicate different mechanisms of pathogenesis of foodborne organisms. More knowledge about food contaminants can also help understand their inactivation. In the present review the use of proteomics, peptidomics and metabolomics, in addition to other foodomic methods for the detection of foodborne pathogenic fungi and bacteria, is overviewed. Furthermore, it is discussed how these techniques can be used for discovering biomarkers for pathogenicity of foodborne pathogens, determining the mechanisms by which they act, and studying their resistance upon inactivation in food of animal and plant origin. Comprehensive and comparative view into the genome and proteome of foodborne pathogens of bacterial or fungal origin and foodomic, mostly proteomic, peptidomic and metabolomic investigation of their toxin production and their mechanism of action is necessary in order to get further information about their virulence, pathogenicity and survival under stress conditions. Furthermore, these data pave the way for identification of biomarkers to trace sources of contamination with food-borne microorganisms and their endo- and exotoxins in order to ensure food safety and prevent the outbreak of food-borne diseases. Therefore, detection of pathogens and their toxins during production, transport and before consume of food produce, as well as protection against

Comparative Genomics Evidence That Only Protein Toxins are Tagging Bad Bugs

PubMed Central

Georgiades, Kalliopi; Raoult, Didier

2011-01-01

The term toxin was introduced by Roux and Yersin and describes macromolecular substances that, when produced during infection or when introduced parenterally or orally, cause an impairment of physiological functions that lead to disease or to the death of the infected organism. Long after the discovery of toxins, early genetic studies on bacterial virulence demonstrated that removing a certain number of genes from pathogenic bacteria decreases their capacity to infect hosts. Each of the removed factors was therefore referred to as a “virulence factor,” and it was speculated that non-pathogenic bacteria lack such supplementary factors. However, many recent comparative studies demonstrate that the specialization of bacteria to eukaryotic hosts is associated with massive gene loss. We recently demonstrated that the only features that seem to characterize 12 epidemic bacteria are toxin–antitoxin (TA) modules, which are addiction molecules in host bacteria. In this study, we investigated if protein toxins are indeed the only molecules specific to pathogenic bacteria by comparing 14 epidemic bacterial killers (“bad bugs”) with their 14 closest non-epidemic relatives (“controls”). We found protein toxins in significantly more elevated numbers in all of the “bad bugs.” For the first time, statistical principal components analysis, including genome size, GC%, TA modules, restriction enzymes, and toxins, revealed that toxins are the only proteins other than TA modules that are correlated with the pathogenic character of bacteria. Moreover, intracellular toxins appear to be more correlated with the pathogenic character of bacteria than secreted toxins. In conclusion, we hypothesize that the only truly identifiable phenomena, witnessing the convergent evolution of the most pathogenic bacteria for humans are the loss of metabolic activities, i.e., the outcome of the loss of regulatory and transcription factors and the presence of protein toxins, alone, or

Disease-enhancing antibodies improve the efficacy of bacterial toxin-neutralizing antibodies

PubMed Central

Chow, Siu-Kei; Smith, Cameron; MacCarthy, Thomas; Pohl, Mary Ann; Bergman, Aviv; Casadevall, Arturo

2013-01-01

SUMMARY During infection, humoral immunity produces a polyclonal response with various immunoglobulins recognizing different epitopes within the microbe or toxin. Despite this diverse response, the biological activity of an antibody (Ab) is usually assessed by the action of a monoclonal population. We demonstrate that a combination of monoclonal antibodies (mAbs) that are individually disease-enhancing or neutralizing to Bacillus anthracis protective antigen (PA), a component of anthrax toxin, results in significantly augmented protection against the toxin. This boosted protection is Fc gamma receptor (FcγR)-dependent and involves the formation of stoichiometrically defined mAb-PA complexes that requires immunoglobulin bivalence and simultaneous interaction between PA and the two mAbs. The formation of these mAb-PA complexes inhibits PA oligomerization, resulting in protection. These data suggest that functional assessments of single Abs may inaccurately predict how the same Abs will operate in polyclonal preparations and imply that potentially therapeutic mAbs may be overlooked in single Ab screens. PMID:23601104

RAB-5- and RAB-11-dependent vesicle-trafficking pathways are required for plasma membrane repair after attack by bacterial pore-forming toxin.

PubMed

Los, Ferdinand C O; Kao, Cheng-Yuan; Smitham, Jane; McDonald, Kent L; Ha, Christine; Peixoto, Christina A; Aroian, Raffi V

2011-02-17

Pore-forming toxins (PFTs) secreted by pathogenic bacteria are the most common bacterial protein toxins and are important virulence factors for infection. PFTs punch holes in host cell plasma membranes, and although cells can counteract the resulting membrane damage, the underlying mechanisms at play remain unclear. Using Caenorhabditis elegans as a model, we demonstrate in vivo and in an intact epithelium that intestinal cells respond to PFTs by increasing levels of endocytosis, dependent upon RAB-5 and RAB-11, which are master regulators of endocytic and exocytic events. Furthermore, we find that RAB-5 and RAB-11 are required for protection against PFT and to restore integrity to the plasma membrane. One physical mechanism involved is the RAB-11-dependent expulsion of microvilli from the apical side of the intestinal epithelial cells. Specific vesicle-trafficking pathways thus protect cells against an attack by PFTs on plasma membrane integrity, via altered plasma membrane dynamics. Copyright © 2011 Elsevier Inc. All rights reserved.

Dinophysis Toxins: Causative Organisms, Distribution and Fate in Shellfish

PubMed Central

Reguera, Beatriz; Riobó, Pilar; Rodríguez, Francisco; Díaz, Patricio A.; Pizarro, Gemita; Paz, Beatriz; Franco, José M.; Blanco, Juan

2014-01-01

Several Dinophysis species produce diarrhoetic toxins (okadaic acid and dinophysistoxins) and pectenotoxins, and cause gastointestinal illness, Diarrhetic Shellfish Poisoning (DSP), even at low cell densities (<103 cells·L−1). They are the main threat, in terms of days of harvesting bans, to aquaculture in Northern Japan, Chile, and Europe. Toxicity and toxin profiles are very variable, more between strains than species. The distribution of DSP events mirrors that of shellfish production areas that have implemented toxin regulations, otherwise misinterpreted as bacterial or viral contamination. Field observations and laboratory experiments have shown that most of the toxins produced by Dinophysis are released into the medium, raising questions about the ecological role of extracelular toxins and their potential uptake by shellfish. Shellfish contamination results from a complex balance between food selection, adsorption, species-specific enzymatic transformations, and allometric processes. Highest risk areas are those combining Dinophysis strains with high cell content of okadaates, aquaculture with predominance of mytilids (good accumulators of toxins), and consumers who frequently include mussels in their diet. Regions including pectenotoxins in their regulated phycotoxins will suffer from much longer harvesting bans and from disloyal competition with production areas where these toxins have been deregulated. PMID:24447996

Pore-forming toxin-mediated ion dysregulation leads to death receptor-independent necroptosis of lung epithelial cells during bacterial pneumonia

PubMed Central

González-Juarbe, Norberto; Bradley, Kelley Margaret; Shenoy, Anukul Taranath; Gilley, Ryan Paul; Reyes, Luis Felipe; Hinojosa, Cecilia Anahí; Restrepo, Marcos Ignacio; Dube, Peter Herman; Bergman, Molly Ann; Orihuela, Carlos Javier

2017-01-01

We report that pore-forming toxins (PFTs) induce respiratory epithelial cell necroptosis independently of death receptor signaling during bacterial pneumonia. Instead, necroptosis was activated as a result of ion dysregulation arising from membrane permeabilization. PFT-induced necroptosis required RIP1, RIP3 and MLKL, and could be induced in the absence or inhibition of TNFR1, TNFR2 and TLR4 signaling. We detected activated MLKL in the lungs from mice and nonhuman primates experiencing Serratia marcescens and Streptococcus pneumoniae pneumonia, respectively. We subsequently identified calcium influx and potassium efflux as the key initiating signals responsible for necroptosis; also that mitochondrial damage was not required for necroptosis activation but was exacerbated by MLKL activation. PFT-induced necroptosis in respiratory epithelial cells did not involve CamKII or reactive oxygen species. KO mice deficient in MLKL or RIP3 had increased survival and reduced pulmonary injury during S. marcescens pneumonia. Our results establish necroptosis as a major cell death pathway active during bacterial pneumonia and that necroptosis can occur without death receptor signaling. PMID:28387756

Pore-forming toxin-mediated ion dysregulation leads to death receptor-independent necroptosis of lung epithelial cells during bacterial pneumonia.

PubMed

González-Juarbe, Norberto; Bradley, Kelley Margaret; Shenoy, Anukul Taranath; Gilley, Ryan Paul; Reyes, Luis Felipe; Hinojosa, Cecilia Anahí; Restrepo, Marcos Ignacio; Dube, Peter Herman; Bergman, Molly Ann; Orihuela, Carlos Javier

2017-05-01

We report that pore-forming toxins (PFTs) induce respiratory epithelial cell necroptosis independently of death receptor signaling during bacterial pneumonia. Instead, necroptosis was activated as a result of ion dysregulation arising from membrane permeabilization. PFT-induced necroptosis required RIP1, RIP3 and MLKL, and could be induced in the absence or inhibition of TNFR1, TNFR2 and TLR4 signaling. We detected activated MLKL in the lungs from mice and nonhuman primates experiencing Serratia marcescens and Streptococcus pneumoniae pneumonia, respectively. We subsequently identified calcium influx and potassium efflux as the key initiating signals responsible for necroptosis; also that mitochondrial damage was not required for necroptosis activation but was exacerbated by MLKL activation. PFT-induced necroptosis in respiratory epithelial cells did not involve CamKII or reactive oxygen species. KO mice deficient in MLKL or RIP3 had increased survival and reduced pulmonary injury during S. marcescens pneumonia. Our results establish necroptosis as a major cell death pathway active during bacterial pneumonia and that necroptosis can occur without death receptor signaling.

Mass Spectrometric Identification and Differentiation of Botulinum Neurotoxins through Toxin Proteomics.

PubMed

Kalb, Suzanne R; Barr, John R

2013-08-01

Botulinum neurotoxins (BoNTs) cause the disease botulism, which can be lethal if untreated. There are seven known serotypes of BoNT, A-G, defined by their response to antisera. Many serotypes are distinguished into differing subtypes based on amino acid sequence and immunogenic properties, and some subtypes are further differentiated into toxin variants. Toxin characterization is important as different types of BoNT can respond differently to medical countermeasures for botulism, and characterization of the toxin can aid in epidemiologic and forensic investigations. Proteomic techniques have been established to determine the serotype, subtype, or toxin variant of BoNT. These techniques involve digestion of the toxin into peptides, tandem mass spectrometric (MS/MS) analysis of the peptides, and database searching to identify the BoNT protein. These techniques demonstrate the capability to detect BoNT and its neurotoxin-associated proteins, and differentiate the toxin from other toxins which are up to 99.9% identical in some cases. This differentiation can be accomplished from toxins present in a complex matrix such as stool, food, or bacterial cultures and no DNA is required.

Influence of bacterial toxins on the GTPase activity of transducin from bovine retinal rod outer segments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rybin, V.O.; Gureeva, A.A.

1986-05-10

The action of cholera toxin, capable of ADP-ribosylation of the activator N/sub s/ protein, and pertussis toxin, capable of ADP-ribosylation of the inhibitor N/sub i/ protein of the adenylate cyclase complex, on transducin, the GTP-binding protein of the rod outer segments of the retina, was investigated. It was shown that under the action of pertussis and cholera toxins, the GTPase activity of transducin is inhibited. Pertussin toxin inhibits the GTPase of native retinal rod outer segments by 30-40%, while GTPase of homogeneous transducin produces a 70-80% inhibition. The action of toxins on transducin depends on the presence and nature ofmore » the guanylic nucleotide with which incubation is performed. On the basis of the data obtained it is suggested that pertussis toxin interacts with pretransducin and with the transducin-GDP complex, while cholera toxin ADP-ribosylates the transducin-GTP complex and does not act on transducin lacking GTP.« less

Prokaryotic adenylate cyclase toxin stimulates anterior pituitary cells in culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cronin, M.J.; Evans, W.S.; Rogol, A.D.

1986-08-01

Bordetella pertussis synthesis a variety of virulence factors including a calmodulin-dependent adenylate cyclase (AC) toxin. Treatment of anterior pituitary cells with this AC toxin resulted in an increase in cellular cAMP levels that was associated with accelerated exocytosis of growth hormone (GH), prolactin, adrenocorticotropic hormone (ACTH), and luteinizing hormone (LH). The kinetics of release of these hormones, however, were markedly different; GH and prolactin were rapidly released, while LH and ACTH secretion was more gradually elevated. Neither dopamine agonists nor somatostatin changes the ability of AC toxin to generate cAMP (up to 2 h). Low concentrations of AC toxin amplifiedmore » the secretory response to hypophysiotrophic hormones. The authors conclude that bacterial AC toxin can rapidly elevate cAMP levels in anterior pituitary cells and that it is the response that explains the subsequent acceleration of hormone release.« less

Botulinum toxin in pain treatment.

PubMed

Colhado, Orlando Carlos Gomes; Boeing, Marcelo; Ortega, Luciano Bornia

2009-01-01

Botulinum toxin (BTX) is one of the most potent bacterial toxins known and its effectiveness in the treatment of some pain syndromes is well known. However, the efficacy of some of its indications is still in the process of being confirmed. The objective of this study was to review the history, pharmacological properties, and clinical applications of BTX in the treatment of pain of different origins. Botulinum toxin is produced by fermentation of Clostridium botulinum, a Gram-positive, anaerobic bacterium. Commercially, BTX comes in two presentations, types A and B. Botulinum toxin, a neurotoxin with high affinity for cholinergic synapses, blocks the release of acetylcholine by nerve endings without interfering with neuronal conduction of electrical signals or synthesis and storage of acetylcholine. It has been proven that BTX can selectively weaken painful muscles, interrupting the spasm-pain cycle. Several studies have demonstrated the efficacy and safety of BTX-A in the treatment of tension headaches, migraines, chronic lumbar pain, and myofascial pain. Botulinum toxin type A is well tolerated in the treatment of chronic pain disorders in which pharmacotherapy regimens can cause side effects. The reduction in the consumption of analgesics and length of action of 3 to 4 months per dose represent other advantages of its use. However, further studies are necessary to establish the efficacy of BTX-A in chronic pain disorders and its exact mechanism of action, as well as its potential in multifactorial treatments.

Excitable toxin-antitoxin modules coordinated through intracellular bottlenecks

NASA Astrophysics Data System (ADS)

Mather, William

Chronic infections and pathogenic biofilms present a serious threat to the health of humans by decreasing life expectancy and quality. The resilience of these microbial communities has been attributed to the spontaneous formation of persister cells, which constitute a small fraction of the population capable of surviving a wide range of environmental stressors. Gating of bacterial persistence has recently been linked to toxin-antitoxin (TA) modules, which are operons with an evolutionarily conserved motif that includes a toxin that halts cell growth and a corresponding antitoxin that neutralizes the toxin. While many such modules have been identified and studied in a wide range of organisms, little consideration of the interactions between multiple modules within a single host has been made. Moreover, the multitude of different antitoxin species are degraded by a relatively small number of proteolytic pathways, strongly suggesting competition between antitoxins for degradation machinery, i.e. queueing coupling. Here we present a theoretical understanding of the dynamics of multiple TA modules that are coupled through either proteolytic queueing, a toxic effect on cell growth rate, or both. We conclude that indirect queueing coordination between multiple TA modules may be central to controlling bacterial persistence. NSF Award Number MCB-1330180.

Antibody-Mediated Inhibition of Ricin Toxin Retrograde Transport

PubMed Central

Yermakova, Anastasiya; Klokk, Tove Irene; Cole, Richard; Sandvig, Kirsten; Mantis, Nicholas J.

2014-01-01

ABSTRACT Ricin is a member of the ubiquitous family of plant and bacterial AB toxins that gain entry into the cytosol of host cells through receptor-mediated endocytosis and retrograde traffic through the trans-Golgi network (TGN) and endoplasmic reticulum (ER). While a few ricin toxin-specific neutralizing monoclonal antibodies (MAbs) have been identified, the mechanisms by which these antibodies prevent toxin-induced cell death are largely unknown. Using immunofluorescence confocal microscopy and a TGN-specific sulfation assay, we demonstrate that 24B11, a MAb against ricin’s binding subunit (RTB), associates with ricin in solution or when prebound to cell surfaces and then markedly enhances toxin uptake into host cells. Following endocytosis, however, toxin-antibody complexes failed to reach the TGN; instead, they were shunted to Rab7-positive late endosomes and LAMP-1-positive lysosomes. Monovalent 24B11 Fab fragments also interfered with toxin retrograde transport, indicating that neither cross-linking of membrane glycoproteins/glycolipids nor the recently identified intracellular Fc receptor is required to derail ricin en route to the TGN. Identification of the mechanism(s) by which antibodies like 24B11 neutralize ricin will advance our fundamental understanding of protein trafficking in mammalian cells and may lead to the discovery of new classes of toxin inhibitors and therapeutics for biodefense and emerging infectious diseases. PMID:24713323

A cell-based fluorescent assay to detect the activity of AB toxins that inhibit protein synthesis

USDA-ARS?s Scientific Manuscript database

AB-type protein toxins, produced by numerous bacterial pathogens and some plants, elicit a cytotoxic effect involving the inhibition of protein synthesis. To develop an improved method to detect the inhibition of protein synthesis by AB-type toxins, the present study characterized a Vero cell line t...

[Consumption of psychoactive drugs and exposure to bacterial toxins carried by food: a dangerous association].

PubMed

Corma-Gómez, Anaïs; López-Sepúlveda, Rocío; Capitán-Del Río, Inés; Sánchez Mariscal, María Dolores; López-Hernández, Begoña

2017-11-01

To describe and analyse from a clinical and epidemiological point of view, a food borne outbreak in a psychiatric institution in Granada, in 2015, and to examine whether treatment with psychoactive drugs constitutes a risk factor for the development of a food borne disease, analysing the degree of susceptibility according to the therapeutic group consumed. Ambispective cohort study. Residents were the unit of analysis. Our group carried out an active case search and a food survey. A search for other risks was developed as well as a food inspection. Location, time and individual variables were studied. A descriptive analysis was conducted (absolute and relative frequencies). Calculation of attack rates by building and by menu was made. Bi-variant analysis (Chi-square test, t-Student test) and relative risk were used as a measure of strength of association. For risk analysis of medication, a multivariate analysis using logistic regression was carried out. 18 cases with diarrhoea without fever were found (incubation period from 6 to 16hours). Cases were mild and self-limiting. The clinical manifestations, the temporal grouping of cases and the characteristics of the ingested foods, focussed suspicion on a bacterial toxin. Being equal in the rest of variables, the N03AF, and N03AG therapeutic groups confer greater risk of disease (odds ratio [OR]: 8.626; 95% confidence interval [95%CI]: 2.050-36.308; p=0.003; and OR: 14.516; 95%CI: 3.155-66.784; p=0.001, respectively). Decreased intestinal transit, caused by the administration of anticonvulsants, may increase exposure time of the intestinal mucosa to the toxin, increasing the risk of disease and suffering from complications. An additional hygienic effort should be made in this type of institution to prevent these pathologies. Copyright © 2017 SESPAS. Publicado por Elsevier España, S.L.U. All rights reserved.

Antibodies derived from a toxoid MEFA (multiepitope fusion antigen) show neutralizing activities against heat-labile toxin (LT), heat-stable toxins (STa, STb), and Shiga toxin 2e (Stx2e) of porcine enterotoxigenic Escherichia coli (ETEC).

PubMed

Rausch, Dana; Ruan, Xiaosai; Nandre, Rahul; Duan, Qiangde; Hashish, Emad; Casey, Thomas A; Zhang, Weiping

2017-04-01

Enterotoxigenic Escherichia coli (ETEC) strains are the main cause of diarrhea in pigs. Pig diarrhea especially post-weaning diarrhea remains one of the most important swine diseases. ETEC bacterial fimbriae including K88, F18, 987P, K99 and F41 promote bacterial attachment to intestinal epithelial cells and facilitate ETEC colonization in pig small intestine. ETEC enterotoxins including heat-labile toxin (LT) and heat-stable toxins type Ia (porcine-type STa) and type II (STb) stimulate fluid hyper-secretion, leading to watery diarrhea. Blocking bacteria colonization and/or neutralizing enterotoxicity of ETEC toxins are considered effective prevention against ETEC diarrhea. In this study, we applied the MEFA (multiepitope fusion antigen) strategy to create toxoid MEFAs that carried antigenic elements of ETEC toxins, and examined for broad antitoxin immunogenicity in a murine model. By embedding STa toxoid STa P12F (NTFYCCELCCNFACAGCY), a STb epitope (KKDLCEHY), and an epitope of Stx2e A subunit (QSYVSSLN) into the A1 peptide of a monomeric LT toxoid (LT R192G ), two toxoid MEFAs, 'LT R192G -STb-Stx2e-STa P12F ' and 'LT R192G -STb-Stx2e-3xSTa P12F ' which carried three copies of STa P12F , were constructed. Mice intraperitoneally immunized with each toxoid MEFA developed IgG antibodies to all four toxins. Induced antibodies showed in vitro neutralizing activities against LT, STa, STb and Stx2e toxins. Moreover, suckling piglets born by a gilt immunized with 'LT R192G -STb-Stx2e-3xSTa P12F ' were protected when challenged with ETEC strains, whereas piglets born by a control gilt developed diarrhea. Results from this study showed that the toxoid MEFA induced broadly antitoxin antibodies, and suggested potential application of the toxoid MEFA for developing a broad-spectrum vaccine against ETEC diarrhea in pigs. Copyright © 2016 Elsevier B.V. All rights reserved.

An Overview of Helicobacter pylori VacA Toxin Biology

PubMed Central

Foegeding, Nora J.; Caston, Rhonda R.; McClain, Mark S.; Ohi, Melanie D.; Cover, Timothy L.

2016-01-01

The VacA toxin secreted by Helicobacter pylori enhances the ability of the bacteria to colonize the stomach and contributes to the pathogenesis of gastric adenocarcinoma and peptic ulcer disease. The amino acid sequence and structure of VacA are unrelated to corresponding features of other known bacterial toxins. VacA is classified as a pore-forming toxin, and many of its effects on host cells are attributed to formation of channels in intracellular sites. The most extensively studied VacA activity is its capacity to stimulate vacuole formation, but the toxin has many additional effects on host cells. Multiple cell types are susceptible to VacA, including gastric epithelial cells, parietal cells, T cells, and other types of immune cells. This review focuses on the wide range of VacA actions that are detectable in vitro, as well as actions of VacA in vivo that are relevant for H. pylori colonization of the stomach and development of gastric disease. PMID:27271669

PemK toxin encoded by the Xylella fastidiosa IncP-1 plasmid pXF-RIV11 is a ribonuclease

USDA-ARS?s Scientific Manuscript database

Stable inheritance of the IncP-1 plasmid pXF-RIV11 in Xylella fastidiosa is conferred by the pemI/pemK plasmid addiction system. PemK serves as a toxin inhibiting bacterial growth; PemI is the corresponding antitoxin that blocks activity of PemK toxin by direct binding. Here, PemK toxin and PemI ant...

Geldanamycin Enhances Retrograde Transport of Shiga Toxin in HEp-2 Cells

PubMed Central

Simm, Roger; Torgersen, Maria Lyngaas; Sandvig, Kirsten

2015-01-01

The heat shock protein 90 (Hsp90) inhibitor geldanamycin (GA) has been shown to alter endosomal sorting, diverting cargo destined for the recycling pathway into the lysosomal pathway. Here we investigated whether GA also affects the sorting of cargo into the retrograde pathway from endosomes to the Golgi apparatus. As a model cargo we used the bacterial toxin Shiga toxin, which exploits the retrograde pathway as an entry route to the cytosol. Indeed, GA treatment of HEp-2 cells strongly increased the Shiga toxin transport to the Golgi apparatus. The enhanced Golgi transport was not due to increased endocytic uptake of the toxin or perturbed recycling, suggesting that GA selectively enhances endosomal sorting into the retrograde pathway. Moreover, GA activated p38 and both inhibitors of p38 or its substrate MK2 partially counteracted the GA-induced increase in Shiga toxin transport. Thus, our data suggest that GA-induced p38 and MK2 activation participate in the increased Shiga toxin transport to the Golgi apparatus. PMID:26017782

Cholix Toxin, a Novel ADP-ribosylating Factor from Vibrio cholerae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jorgensen, Rene; Purdy, Alexandra E.; Fieldhouse, Robert J.

2008-07-15

The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report the characterization of a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells (IC50 = 4.6 {+-} 0.4 ng/ml) and crustaceans (Artemia nauplii LD50 = 10 {+-} 2 {mu}g/ml). Here we show that this toxin is the third member of the diphthamide-specific class of ADP-ribose transferases and that it possesses specific ADP-ribose transferase activity againstmore » ribosomal eukaryotic elongation factor 2. We also describe the high resolution crystal structures of the multidomain toxin and its catalytic domain at 2.1- and 1.25-{angstrom} resolution, respectively. The new structural data show that cholix toxin possesses the necessary molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm, and inhibition of protein synthesis by specific modification of elongation factor 2. The crystal structures also provide important insight into the structural basis for activation of toxin ADP-ribosyltransferase activity. These results indicate that cholix toxin may be an important virulence factor of Vibrio cholerae that likely plays a significant role in the survival of the organism in an aquatic environment.« less

Immunization with Bacillus Spores Expressing Toxin A Peptide Repeats Protects against Infection with Clostridium difficile Strains Producing Toxins A and B ▿ †

PubMed Central

Permpoonpattana, Patima; Hong, Huynh A.; Phetcharaburanin, Jutarop; Huang, Jen-Min; Cook, Jenny; Fairweather, Neil F.; Cutting, Simon M.

2011-01-01

Clostridium difficile is a leading cause of nosocomial infection in the developed world. Two toxins, A and B, produced by most strains of C. difficile are implicated as virulence factors, yet only recently has the requirement of these for infection been investigated by genetic manipulation. Current vaccine strategies are focused mostly on parenteral delivery of toxoids. In this work, we have used bacterial spores (Bacillus subtilis) as a delivery vehicle to evaluate the carboxy-terminal repeat domains of toxins A and B as protective antigens. Our findings are important and show that oral immunization of the repeat domain of toxin A is sufficient to confer protection in a hamster model of infection designed to closely mimic the human course of infection. Importantly, neutralizing antibodies to the toxin A repeat domain were shown to be cross-reactive with the analogous domain of toxin B and, being of high avidity, provided protection against challenge with a C. difficile strain producing toxins A and B (A+B+). Thus, although many strains produce both toxins, antibodies to only toxin A can mediate protection. Animals vaccinated with recombinant spores were fully able to survive reinfection, a property that is particularly important for a disease with which patients are prone to relapse. We show that mucosal immunization, not parenteral delivery, is required to generate secretory IgA and that production of these neutralizing polymeric antibodies correlates with protection. This work demonstrates that an effective vaccine against C. difficile can be designed around two attributes, mucosal delivery and the repeat domain of toxin A. PMID:21482682

Possible mistranslation of Shiga toxin from pathogenic Escherichia coli as measured by MALDI-TOF and Orbitrap mass spectrometry

USDA-ARS?s Scientific Manuscript database

RATIONALE: Shiga toxin-producing Escherichia coli (STEC) are often subjected to DNA damaging antibiotics during culturing in order to elicit the bacterial SOS response and up-regulation of bacteriophage-encoded proteins including Shiga toxin (Stx). However, such antibiotic exposure and stress may al...

Observing the Confinement Potential of Bacterial Pore-Forming Toxin Receptors Inside Rafts with Nonblinking Eu3+-Doped Oxide Nanoparticles

PubMed Central

Türkcan, Silvan; Masson, Jean-Baptiste; Casanova, Didier; Mialon, Geneviève; Gacoin, Thierry; Boilot, Jean-Pierre; Popoff, Michel R.; Alexandrou, Antigoni

2012-01-01

We track single toxin receptors on the apical cell membrane of MDCK cells with Eu-doped oxide nanoparticles coupled to two toxins of the pore-forming toxin family: α-toxin of Clostridium septicum and ε-toxin of Clostridium perfringens. These nonblinking and photostable labels do not perturb the motion of the toxin receptors and yield long uninterrupted trajectories with mean localization precision of 30 nm for acquisition times of 51.3 ms. We were thus able to study the toxin-cell interaction at the single-molecule level. Toxins bind to receptors that are confined within zones of mean area 0.40 ± 0.05 μm2. Assuming that the receptors move according to the Langevin equation of motion and using Bayesian inference, we determined mean diffusion coefficients of 0.16 ± 0.01 μm2/s for both toxin receptors. Moreover, application of this approach revealed a force field within the domain generated by a springlike confining potential. Both toxin receptors were found to experience forces characterized by a mean spring constant of 0.30 ± 0.03 pN/μm at 37°C. Furthermore, both toxin receptors showed similar distributions of diffusion coefficient, domain area, and spring constant. Control experiments before and after incubation with cholesterol oxidase and sphingomyelinase show that these two enzymes disrupt the confinement domains and lead to quasi-free motion of the toxin receptors. Our control data showing cholesterol and sphingomyelin dependence as well as independence of actin depolymerization and microtubule disruption lead us to attribute the confinement of both receptors to lipid rafts. These toxins require oligomerization to develop their toxic activity. The confined nature of the toxin receptors leads to a local enhancement of the toxin monomer concentration and may thus explain the virulence of this toxin family. PMID:22677383

Chloroquine derivatives block the translocation pores and inhibit cellular entry of Clostridium botulinum C2 toxin and Bacillus anthracis lethal toxin.

PubMed

Kreidler, Anna-Maria; Benz, Roland; Barth, Holger

2017-03-01

The pathogenic bacteria Clostridium botulinum and Bacillus anthracis produce the binary protein toxins C2 and lethal toxin (LT), respectively. These toxins consist of a binding/transport (B 7 ) component that delivers the separate enzyme (A) component into the cytosol of target cells where it modifies its specific substrate and causes cell death. The B 7 components of C2 toxin and LT, C2IIa and PA 63 , respectively, are ring-shaped heptamers that bind to their cellular receptors and form complexes with their A components C2I and lethal factor (LF), respectively. After receptor-mediated endocytosis of the toxin complexes, C2IIa and PA 63 insert into the membranes of acidified endosomes and form trans-membrane pores through which C2I and LF translocate across endosomal membranes into the cytosol. C2IIa and PA 63 also form channels in planar bilayer membranes, and we used this approach earlier to identify chloroquine as a potent blocker of C2IIa and PA 63 pores. Here, a series of chloroquine derivatives was investigated to identify more efficient toxin inhibitors with less toxic side effects. Chloroquine, primaquine, quinacrine, and fluphenazine blocked C2IIa and PA 63 pores in planar lipid bilayers and in membranes of living epithelial cells and macrophages, thereby preventing the pH-dependent membrane transport of the A components into the cytosol and protecting cells from intoxication with C2 toxin and LT. These potent inhibitors of toxin entry underline the central role of the translocation pores for cellular uptake of binary bacterial toxins and as relevant drug targets, and might be lead compounds for novel pharmacological strategies against severe enteric diseases and anthrax.

The assembly dynamics of the cytolytic pore toxin ClyA

PubMed Central

Benke, Stephan; Roderer, Daniel; Wunderlich, Bengt; Nettels, Daniel; Glockshuber, Rudi; Schuler, Benjamin

2015-01-01

Pore-forming toxins are protein assemblies used by many organisms to disrupt the membranes of target cells. They are expressed as soluble monomers that assemble spontaneously into multimeric pores. However, owing to their complexity, the assembly processes have not been resolved in detail for any pore-forming toxin. To determine the assembly mechanism for the ring-shaped, homododecameric pore of the bacterial cytolytic toxin ClyA, we collected a diverse set of kinetic data using single-molecule spectroscopy and complementary techniques on timescales from milliseconds to hours, and from picomolar to micromolar ClyA concentrations. The entire range of experimental results can be explained quantitatively by a surprisingly simple mechanism. First, addition of the detergent n-dodecyl-β-D-maltopyranoside to the soluble monomers triggers the formation of assembly-competent toxin subunits, accompanied by the transient formation of a molten-globule-like intermediate. Then, all sterically compatible oligomers contribute to assembly, which greatly enhances the efficiency of pore formation compared with simple monomer addition. PMID:25652783

Human-Specific Bacterial Pore-Forming Toxins Induce Programmed Necrosis in Erythrocytes

PubMed Central

LaRocca, Timothy J.; Stivison, Elizabeth A.; Hod, Eldad A.; Spitalnik, Steven L.; Cowan, Peter J.; Randis, Tara M.

2014-01-01

ABSTRACT A subgroup of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins (PFTs) has an unusually narrow host range due to a requirement for binding to human CD59 (hCD59), a glycosylphosphatidylinositol (GPI)-linked complement regulatory molecule. hCD59-specific CDCs are produced by several organisms that inhabit human mucosal surfaces and can act as pathogens, including Gardnerella vaginalis and Streptococcus intermedius. The consequences and potential selective advantages of such PFT host limitation have remained unknown. Here, we demonstrate that, in addition to species restriction, PFT ligation of hCD59 triggers a previously unrecognized pathway for programmed necrosis in primary erythrocytes (red blood cells [RBCs]) from humans and transgenic mice expressing hCD59. Because they lack nuclei and mitochondria, RBCs have typically been thought to possess limited capacity to undergo programmed cell death. RBC programmed necrosis shares key molecular factors with nucleated cell necroptosis, including dependence on Fas/FasL signaling and RIP1 phosphorylation, necrosome assembly, and restriction by caspase-8. Death due to programmed necrosis in RBCs is executed by acid sphingomyelinase-dependent ceramide formation, NADPH oxidase- and iron-dependent reactive oxygen species formation, and glycolytic formation of advanced glycation end products. Bacterial PFTs that are hCD59 independent do not induce RBC programmed necrosis. RBC programmed necrosis is biochemically distinct from eryptosis, the only other known programmed cell death pathway in mature RBCs. Importantly, RBC programmed necrosis enhances the growth of PFT-producing pathogens during exposure to primary RBCs, consistent with a role for such signaling in microbial growth and pathogenesis. PMID:25161188

Crystal structure of the toxin Msmeg_6760, the structural homolog of Mycobacterium tuberculosis Rv2035, a novel type II toxin involved in the hypoxic response

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bajaj, R. Alexandra; Arbing, Mark A.; Shin, Annie

The structure of Msmeg_6760, a protein of unknown function, has been determined. Biochemical and bioinformatics analyses determined that Msmeg_6760 interacts with a protein encoded in the same operon, Msmeg_6762, and predicted that the operon is a toxin–antitoxin (TA) system. Structural comparison of Msmeg_6760 with proteins of known function suggests that Msmeg_6760 binds a hydrophobic ligand in a buried cavity lined by large hydrophobic residues. Access to this cavity could be controlled by a gate–latch mechanism. The function of the Msmeg_6760 toxin is unknown, but structure-based predictions revealed that Msmeg_6760 and Msmeg_6762 are homologous to Rv2034 and Rv2035, a predicted novelmore » TA system involved inMycobacterium tuberculosislatency during macrophage infection. The Msmeg_6760 toxin fold has not been previously described for bacterial toxins and its unique structural features suggest that toxin activation is likely to be mediated by a novel mechanism.« less

Variability of cholesterol accessibility in human red blood cells measured using a bacterial cholesterol-binding toxin

PubMed Central

Chakrabarti, Rima S; Ingham, Sally A; Kozlitina, Julia; Gay, Austin; Cohen, Jonathan C; Radhakrishnan, Arun; Hobbs, Helen H

2017-01-01

Cholesterol partitions into accessible and sequestered pools in cell membranes. Here, we describe a new assay using fluorescently-tagged anthrolysin O, a cholesterol-binding bacterial toxin, to measure accessible cholesterol in human red blood cells (RBCs). Accessible cholesterol levels were stable within individuals, but varied >10 fold among individuals. Significant variation was observed among ethnic groups (Blacks>Hispanics>Whites). Variation in accessibility of RBC cholesterol was unrelated to the cholesterol content of RBCs or plasma, but was associated with the phospholipid composition of the RBC membranes and with plasma triglyceride levels. Pronase treatment of RBCs only modestly altered cholesterol accessibility. Individuals on hemodialysis, who have an unexplained increase in atherosclerotic risk, had significantly higher RBC cholesterol accessibility. Our data indicate that RBC accessible cholesterol is a stable phenotype with significant inter-individual variability. Factors both intrinsic and extrinsic to the RBC contribute to variation in its accessibility. This assay provides a new tool to assess cholesterol homeostasis among tissues in humans. DOI: http://dx.doi.org/10.7554/eLife.23355.001 PMID:28169829

[Toxins of Clostridium perfringens as a natural and bioterroristic threats].

PubMed

Omernik, Andrzej; Płusa, Tadeusz

2015-09-01

Clostridium perfringens is absolutely anaerobic rod-shaped, sporeforming bacterium. The morbidity is connected with producing toxins. Depending on the type of toxin produced Clostridium perfringens can be divided into five serotypes:A-E. Under natural conditions, this bacterium is responsible for local outbreaks of food poisoning associated with eating contaminated food which which was improperly heat treated. Some countries with lower economic level are endemic foci of necrotizing enteritis caused by Clostridium perfringens. The bacterium is also a major cause of gas gangrene. It is a disease, associated with wound infection, with potentially fatal prognosis in the case of treatment's delays. In the absence of early radical surgery, antibiotic therapy and (if available) hyperbaric treatment leads to the spread of toxins in the body causing shock, coma and death. Due to the force of produced toxins is a pathogen that poses a substrate for the production of biological weapons. It could potentially be used to induce outbreaks of food poisoning and by missiles contamination by spore lead to increased morbidity of gas gangrene in injured soldiers. C. perfringens types B and D produce epsilon toxin considered to be the third most powerful bacterial toxin. Because of the ability to disperse the toxin as an aerosol and a lack of methods of treatment and prevention of poisoning possible factors it is a potential tool for bioterrorism It is advisable to continue research into vaccines and treatments for poisoning toxins of C. perfringens. © 2015 MEDPRESS.

Regulating Toxin-Antitoxin Expression: Controlled Detonation of Intracellular Molecular Timebombs

PubMed Central

Hayes, Finbarr; Kędzierska, Barbara

2014-01-01

Genes for toxin-antitoxin (TA) complexes are widely disseminated in bacteria, including in pathogenic and antibiotic resistant species. The toxins are liberated from association with the cognate antitoxins by certain physiological triggers to impair vital cellular functions. TAs also are implicated in antibiotic persistence, biofilm formation, and bacteriophage resistance. Among the ever increasing number of TA modules that have been identified, the most numerous are complexes in which both toxin and antitoxin are proteins. Transcriptional autoregulation of the operons encoding these complexes is key to ensuring balanced TA production and to prevent inadvertent toxin release. Control typically is exerted by binding of the antitoxin to regulatory sequences upstream of the operons. The toxin protein commonly works as a transcriptional corepressor that remodels and stabilizes the antitoxin. However, there are notable exceptions to this paradigm. Moreover, it is becoming clear that TA complexes often form one strand in an interconnected web of stress responses suggesting that their transcriptional regulation may prove to be more intricate than currently understood. Furthermore, interference with TA gene transcriptional autoregulation holds considerable promise as a novel antibacterial strategy: artificial release of the toxin factor using designer drugs is a potential approach to induce bacterial suicide from within. PMID:24434949

Antibody to ricin a chain hinders intracellular routing of toxin and protects cells even after toxin has been internalized.

PubMed

Song, Kejing; Mize, R Ranney; Marrero, Luis; Corti, Miriam; Kirk, Jason M; Pincus, Seth H

2013-01-01

Mechanisms of antibody-mediated neutralization are of much interest. For plant and bacterial A-B toxins, A chain mediates toxicity and B chain binds target cells. It is generally accepted and taught that antibody (Ab) neutralizes by preventing toxin binding to cells. Yet for some toxins, ricin included, anti-A chain Abs afford greater protection than anti-B. The mechanism(s) whereby Abs to the A chain neutralize toxins are not understood. We use quantitative confocal imaging, neutralization assays, and other techniques to study how anti-A chain Abs function to protect cells. Without Ab, ricin enters cells and penetrates to the endoplasmic reticulum within 15 min. Within 45-60 min, ricin entering and being expelled from cells reaches equilibrium. These results are consistent with previous observations, and support the validity of our novel methodology. The addition of neutralizing Ab causes ricin accumulation at the cell surface, delays internalization, and postpones retrograde transport of ricin. Ab binds ricin for >6hr as they traffic together through the cell. Ab protects cells even when administered hours after exposure. CONCLUSIONS/KEY FINDINGS: We demonstrate the dynamic nature of the interaction between the host cell and toxin, and how Ab can alter the balance in favor of the cell. Ab blocks ricin's entry into cells, hinders its intracellular routing, and can protect even after ricin is present in the target organelle, providing evidence that the major site of neutralization is intracellular. These data add toxins to the list of pathogenic agents that can be neutralized intracellularly and explain the in vivo efficacy of delayed administration of anti-toxin Abs. The results encourage the use of post-exposure passive Ab therapy, and show the importance of the A chain as a target of Abs.

Antibody to Ricin A Chain Hinders Intracellular Routing of Toxin and Protects Cells Even after Toxin Has Been Internalized

PubMed Central

Song, Kejing; Mize, R. Ranney; Marrero, Luis; Corti, Miriam; Kirk, Jason M.; Pincus, Seth H.

2013-01-01

Background Mechanisms of antibody-mediated neutralization are of much interest. For plant and bacterial A-B toxins, A chain mediates toxicity and B chain binds target cells. It is generally accepted and taught that antibody (Ab) neutralizes by preventing toxin binding to cells. Yet for some toxins, ricin included, anti-A chain Abs afford greater protection than anti-B. The mechanism(s) whereby Abs to the A chain neutralize toxins are not understood. Methodology/Principal Findings We use quantitative confocal imaging, neutralization assays, and other techniques to study how anti-A chain Abs function to protect cells. Without Ab, ricin enters cells and penetrates to the endoplasmic reticulum within 15 min. Within 45–60 min, ricin entering and being expelled from cells reaches equilibrium. These results are consistent with previous observations, and support the validity of our novel methodology. The addition of neutralizing Ab causes ricin accumulation at the cell surface, delays internalization, and postpones retrograde transport of ricin. Ab binds ricin for >6hr as they traffic together through the cell. Ab protects cells even when administered hours after exposure. Conclusions/Key Findings We demonstrate the dynamic nature of the interaction between the host cell and toxin, and how Ab can alter the balance in favor of the cell. Ab blocks ricin’s entry into cells, hinders its intracellular routing, and can protect even after ricin is present in the target organelle, providing evidence that the major site of neutralization is intracellular. These data add toxins to the list of pathogenic agents that can be neutralized intracellularly and explain the in vivo efficacy of delayed administration of anti-toxin Abs. The results encourage the use of post-exposure passive Ab therapy, and show the importance of the A chain as a target of Abs. PMID:23638075

Fungal toxins bind to the URF13 protein in maize mitochondria and Escherichia coli.

PubMed Central

Braun, C J; Siedow, J N; Levings, C S

1990-01-01

Expression of the maize mitochondrial T-urf13 gene results in a sensitivity to a family of fungal pathotoxins and to methomyl, a structurally unrelated systemic insecticide. Similar effects of pathotoxins and methomyl are observed when T-urf13 is cloned and expressed in Escherichia coli. An interaction between these compounds and the membrane-bound URF13 protein permeabilizes the inner mitochondrial and bacterial plasma membranes. To understand the toxin-URF13 effects, we have investigated whether toxin specifically binds to the URF13 protein. Our studies indicate that toxin binds to the URF13 protein in maize mitochondria and in E. coli expressing URF13. Binding analysis in E. coli reveals cooperative toxin binding. A low level of specific toxin binding is also demonstrated in cms-T and cms-T-restored mitochondria; however, binding does not appear to be cooperative in maize mitochondria. Competition and displacement studies in E. coli demonstrate that toxin binding is reversible and that the toxins and methomyl compete for the same, or for overlapping, binding sites. Two toxin-insensitive URF13 mutants display a diminished capability to bind toxin in E. coli, which identifies residues of URF13 important in toxin binding. A third toxin-insensitive URF13 mutant shows considerable toxin binding in E. coli, demonstrating that toxin binding can occur without causing membrane permeabilization. Our results indicate that toxin-mediated membrane permeabilization only occurs when toxin or methomyl is bound to URF13. PMID:2136632

CNF1-like deamidase domains: common Lego bricks among cancer-promoting immunomodulatory bacterial virulence factors.

PubMed

Ho, Mengfei; Mettouchi, Amel; Wilson, Brenda A; Lemichez, Emmanuel

2018-05-03

Alterations of the cellular proteome over time due to spontaneous or toxin-mediated enzymatic deamidation of glutamine (Gln) and asparagine (Asn) residues contribute to bacterial infection and might represent a source of aging-related diseases. Here, we put into perspective what is known about the mode of action of the CNF1 toxin from pathogenic E. coli, a paradigm of bacterial deamidases that activate Rho GTPases, to illustrate the importance of determining whether exposure to these factors are risk factors in the etiology age-related diseases, such as cancer. In particular, through in silico analysis of the distribution of the CNF1-like deamidase active site Gly-Cys-(Xaa)n-His sequence motif in bacterial genomes, we unveil the wide distribution of the super-family of CNF-like toxins and CNF-like deamidase domains among members of the enterobacteriacae and in association with a large variety of toxin delivery systems. We extent our discussion with recent findings concerning cellular systems that control activated Rac1 GTPase stability and provide protection against cancer. These findings point to the urgency for developing holistic approaches toward personalized medicine that include monitoring for asymptomatic carriage of pathogenic toxin-producing bacteria and that ultimately might lead to improved public health and increased lifespans.

Cholera toxin subunit B-mediated intracellular trafficking of mesoporous silica nanoparticles toward the endoplasmic reticulum

NASA Astrophysics Data System (ADS)

Walker, William Andrew

In recent decades, pharmaceutical research has led to the development of numerous treatments for human disease. Nanoscale delivery systems have the potential to maximize therapeutic outcomes by enabling target specific delivery of these therapeutics. The intracellular localization of many of these materials however, is poorly controlled, leading to sequestration in degradative cellular pathways and limiting the efficacy of their payloads. Numerous proteins, particularly bacterial toxins, have evolved mechanisms to subvert the degradative mechanisms of the cell. Here, we have investigated a possible strategy for shunting intracellular delivery of encapsulated cargoes from these pathways by modifying mesoporous silica nanoparticles (MSNs) with the well-characterized bacterial toxin Cholera toxin subunit B (CTxB). Using established optical imaging methods we investigated the internalization, trafficking, and subcellular localization of our modified MSNs in an in vitro animal cell model. We then attempted to demonstrate the practical utility of this approach by using CTxB-modified mesoporous silica nanoparticles to deliver propidium iodide, a membrane-impermeant fluorophore.

Immunological and functional comparison between Clostridium perfringens iota toxin, C. spiroforme toxin, and anthrax toxins.

PubMed

Perelle, S; Scalzo, S; Kochi, S; Mock, M; Popoff, M R

1997-01-01

Clostridium perfringens iota and C. spiroforme toxins consist of two separate proteins. One is the binding component and the other the enzymatic component. The two toxins secreted by Bacillus anthracis are composed of binary combinations of three proteins: protective antigen, lethal factor, and edema factor. As shown by Western blotting and ELISA, the binding component of anthrax toxin shares common epitopes with that of iota toxin and C. spiroforme toxin which are closely related immunologically. However, no functional complementation was observed between iota toxin and anthrax toxin components. The binding components can form toxins active on macrophages only in combination with their respective enzymatic components. Agents which prevent acidification of endosomes do not have the same effects on anthrax toxin activity as they do on iota and C. spiroforme toxins. Therefore, the mechanisms of entry into the cells are presumably different. Since the binding components of anthrax toxins and iota toxin share a conserved putative translocation domain, these binding components could have a common mode of insertion into the cell membranes.

Identification of RNA species in the RNA-toxin complex and structure of the complex in Clostridium botulinum type E.

PubMed

Kitamura, Masaru

2002-02-15

Clostridium botulinum type E toxin was isolated in the form of a complex with RNA(s) from bacterial cells. Characterization of the complexed RNA remains to be elucidated. The RNA is identified here as ribosomal RNA (rRNA) having 23S and 16S components. The RNA-toxin complexes were found to be made up of three types with different molecular sizes. The three types of RNA-toxin complex are toxin bound to both the 23S and 16S rRNA, toxin bound to the 16S rRNA and a small amount of 23S rRNA, and toxin bound only to the 16S rRNA. ©2002 Elsevier Science (USA).

Contact-dependent growth inhibition induces high levels of antibiotic-tolerant persister cells in clonal bacterial populations.

PubMed

Ghosh, Anirban; Baltekin, Özden; Wäneskog, Marcus; Elkhalifa, Dina; Hammarlöf, Disa L; Elf, Johan; Koskiniemi, Sanna

2018-05-02

Bacterial populations can use bet-hedging strategies to cope with rapidly changing environments. One example is non-growing cells in clonal bacterial populations that are able to persist antibiotic treatment. Previous studies suggest that persisters arise in bacterial populations either stochastically through variation in levels of global signalling molecules between individual cells, or in response to various stresses. Here, we show that toxins used in contact-dependent growth inhibition (CDI) create persisters upon direct contact with cells lacking sufficient levels of CdiI immunity protein, which would otherwise bind to and neutralize toxin activity. CDI-mediated persisters form through a feedforward cycle where the toxic activity of the CdiA toxin increases cellular (p)ppGpp levels, which results in Lon-mediated degradation of the immunity protein and more free toxin. Thus, CDI systems mediate a population density-dependent bet-hedging strategy, where the fraction of non-growing cells is increased only when there are many cells of the same genotype. This may be one of the mechanisms of how CDI systems increase the fitness of their hosts. © 2018 The Authors.

Removal of Hepatitis C Virus-Infected Cells by a Zymogenized Bacterial Toxin

PubMed Central

Shapira, Assaf; Shapira, Shiran; Gal-Tanamy, Meital; Zemel, Romy; Tur-Kaspa, Ran; Benhar, Itai

2012-01-01

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and has become a global health threat. No HCV vaccine is currently available and treatment with antiviral therapy is associated with adverse side effects. Moreover, there is no preventive therapy for recurrent hepatitis C post liver transplantation. The NS3 serine protease is necessary for HCV replication and represents a prime target for developing anti HCV therapies. Recently we described a therapeutic approach for eradication of HCV infected cells that is based on protein delivery of two NS3 protease-activatable recombinant toxins we named “zymoxins”. These toxins were inactivated by fusion to rationally designed inhibitory peptides via NS3-cleavable linkers. Once delivered to cells where NS3 protease is present, the inhibitory peptide is removed resulting in re-activation of cytotoxic activity. The zymoxins we described suffered from two limitations: they required high levels of protease for activation and had basal activities in the un-activated form that resulted in a narrow potential therapeutic window. Here, we present a solution that overcame the major limitations of the “first generation zymoxins” by converting MazF ribonuclease, the toxic component of the E. coli chromosomal MazEF toxin-antitoxin system, into an NS3-activated zymoxin that is introduced to cells by means of gene delivery. We constructed an expression cassette that encodes for a single polypeptide that incorporates both the toxin and a fragment of its potent natural antidote, MazE, linked via an NS3-cleavable linker. While covalently paired to its inhibitor, the ribonuclease is well tolerated when expressed in naïve, healthy cells. In contrast, activating proteolysis that is induced by even low levels of NS3, results in an eradication of NS3 expressing model cells and HCV infected cells. Zymoxins may thus become a valuable tool in eradicating cells infected by intracellular pathogens that express

Hybrid microarray based on double biomolecular markers of DNA and carbohydrate for simultaneous genotypic and phenotypic detection of cholera toxin-producing Vibrio cholerae.

PubMed

Shin, Hwa Hui; Seo, Jeong Hyun; Kim, Chang Sup; Hwang, Byeong Hee; Cha, Hyung Joon

2016-05-15

Life-threatening diarrheal cholera is usually caused by water or food contaminated with cholera toxin-producing Vibrio cholerae. For the prevention and surveillance of cholera, it is crucial to rapidly and precisely detect and identify the etiological causes, such as V. cholerae and/or its toxin. In the present work, we propose the use of a hybrid double biomolecular marker (DBM) microarray containing 16S rRNA-based DNA capture probe to genotypically identify V. cholerae and GM1 pentasaccharide capture probe to phenotypically detect cholera toxin. We employed a simple sample preparation method to directly obtain genomic DNA and secreted cholera toxin as target materials from bacterial cells. By utilizing the constructed DBM microarray and prepared samples, V. cholerae and cholera toxin were detected successfully, selectively, and simultaneously; the DBM microarray was able to analyze the pathogenicity of the identified V. cholerae regardless of whether the bacteria produces toxin. Therefore, our proposed DBM microarray is a new effective platform for identifying bacteria and analyzing bacterial pathogenicity simultaneously. Copyright © 2015 Elsevier B.V. All rights reserved.

Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins.

PubMed

Barth, Holger; Aktories, Klaus; Popoff, Michel R; Stiles, Bradley G

2004-09-01

Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic "A-B" paradigm for protein toxins. The binary toxins produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects. The "B" components are synthesized as precursors that are subsequently activated by serine-type proteases on the targeted cell surface and/or in solution. Following release of a 20-kDa N-terminal peptide, the activated "B" components form homoheptameric rings that subsequently dock with an "A" component(s) on the cell surface. By following an acidified endosomal route and translocation into the cytosol, "A" molecules disable a cell (and host organism) via disruption of the actin cytoskeleton, increasing intracellular levels of cyclic AMP, or inactivation of signaling pathways linked to mitogen-activated protein kinase kinases. Recently, B. anthracis has gleaned much notoriety as a biowarfare/bioterrorism agent, and of primary interest has been the edema and lethal toxins, their role in anthrax, as well as the development of efficacious vaccines and therapeutics targeting these virulence factors and ultimately B. anthracis. This review comprehensively surveys the literature and discusses the similarities, as well as distinct differences, between each Clostridium and Bacillus binary toxin in terms of their biochemistry, biology, genetics, structure, and applications in science and medicine. The information may foster future studies that aid novel vaccine and drug development, as well as a better understanding of a conserved intoxication process utilized by various gram-positive, spore-forming bacteria.

Binary Bacterial Toxins: Biochemistry, Biology, and Applications of Common Clostridium and Bacillus Proteins

PubMed Central

Barth, Holger; Aktories, Klaus; Popoff, Michel R.; Stiles, Bradley G.

2004-01-01

Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic “A-B” paradigm for protein toxins. The binary toxins produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects. The “B” components are synthesized as precursors that are subsequently activated by serine-type proteases on the targeted cell surface and/or in solution. Following release of a 20-kDa N-terminal peptide, the activated “B” components form homoheptameric rings that subsequently dock with an “A” component(s) on the cell surface. By following an acidified endosomal route and translocation into the cytosol, “A” molecules disable a cell (and host organism) via disruption of the actin cytoskeleton, increasing intracellular levels of cyclic AMP, or inactivation of signaling pathways linked to mitogen-activated protein kinase kinases. Recently, B. anthracis has gleaned much notoriety as a biowarfare/bioterrorism agent, and of primary interest has been the edema and lethal toxins, their role in anthrax, as well as the development of efficacious vaccines and therapeutics targeting these virulence factors and ultimately B. anthracis. This review comprehensively surveys the literature and discusses the similarities, as well as distinct differences, between each Clostridium and Bacillus binary toxin in terms of their biochemistry, biology, genetics, structure, and applications in science and medicine. The information may foster future studies that aid novel vaccine and drug development, as well as a better understanding of a conserved intoxication process utilized by various gram-positive, spore-forming bacteria. PMID:15353562

Mixed biofilm formation by Shiga toxin-producing Escherichia coli and Salmonella enterica serovar Typhimurium enhanced bacterial resistance to sanitization due to extracellular polymeric substances.

PubMed

Wang, Rong; Kalchayanand, Norasak; Schmidt, John W; Harhay, Dayna M

2013-09-01

Shiga toxin-producing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium are important foodborne pathogens capable of forming single-species biofilms or coexisting in multispecies biofilm communities. Bacterial biofilm cells are usually more resistant to sanitization than their planktonic counterparts, so these foodborne pathogens in biofilms pose a serious food safety concern. We investigated how the coexistence of E. coli O157:H7 and Salmonella Typhimurium strains would affect bacterial planktonic growth competition and mixed biofilm composition. Furthermore, we also investigated how mixed biofilm formation would affect bacterial resistance to common sanitizers. Salmonella Typhimurium strains were able to outcompete E. coli strains in the planktonic growth phase; however, mixed biofilm development was highly dependent upon companion strain properties in terms of the expression of bacterial extracellular polymeric substances (EPS), including curli fimbriae and exopolysaccharide cellulose. The EPS-producing strains with higher biofilm-forming abilities were able to establish themselves in mixed biofilms more efficiently. In comparison to single-strain biofilms, Salmonella or E. coli strains with negative EPS expression obtained significantly enhanced resistance to sanitization by forming mixed biofilms with an EPS-producing companion strain of the other species. These observations indicate that the bacterial EPS components not only enhance the sanitizer resistance of the EPS-producing strains but also render protections to their companion strains, regardless of species, in mixed biofilms. Our study highlights the potential risk of cross-contamination by multispecies biofilms in food safety and the need for increased attention to proper sanitization practices in food processing facilities.

A bioanalytical platform for simultaneous detection and quantification of biological toxins.

PubMed

Weingart, Oliver G; Gao, Hui; Crevoisier, François; Heitger, Friedrich; Avondet, Marc-André; Sigrist, Hans

2012-01-01

Prevalent incidents support the notion that toxins, produced by bacteria, fungi, plants or animals are increasingly responsible for food poisoning or intoxication. Owing to their high toxicity some toxins are also regarded as potential biological warfare agents. Accordingly, control, detection and neutralization of toxic substances are a considerable economic burden to food safety, health care and military biodefense. The present contribution describes a new versatile instrument and related procedures for array-based simultaneous detection of bacterial and plant toxins using a bioanalytical platform which combines the specificity of covalently immobilized capture probes with a dedicated instrumentation and immuno-based microarray analytics. The bioanalytical platform consists of a microstructured polymer slide serving both as support of printed arrays and as incubation chamber. The platform further includes an easy-to-operate instrument for simultaneous slide processing at selectable assay temperature. Cy5 coupled streptavidin is used as unifying fluorescent tracer. Fluorescence image analysis and signal quantitation allow determination of the toxin's identity and concentration. The system's performance has been investigated by immunological detection of Botulinum Neurotoxin type A (BoNT/A), Staphylococcal enterotoxin B (SEB), and the plant toxin ricin. Toxins were detectable at levels as low as 0.5-1 ng · mL(-1) in buffer or in raw milk.

Crystal Structures of Phd-Doc, HigA, and YeeU Establish Multiple Evolutionary Links between Microbial Growth-Regulating Toxin-Antitoxin Systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arbing, Mark A.; Handelman, Samuel K.; Kuzin, Alexandre P.

2010-09-27

Bacterial toxin-antitoxin (TA) systems serve a variety of physiological functions including regulation of cell growth and maintenance of foreign genetic elements. Sequence analyses suggest that TA families are linked by complex evolutionary relationships reflecting likely swapping of functional domains between different TA families. Our crystal structures of Phd-Doc from bacteriophage P1, the HigA antitoxin from Escherichia coli CFT073, and YeeU of the YeeUWV systems from E. coli K12 and Shigella flexneri confirm this inference and reveal additional, unanticipated structural relationships. The growth-regulating Doc toxin exhibits structural similarity to secreted virulence factors that are toxic for eukaryotic target cells. The Phdmore » antitoxin possesses the same fold as both the YefM and NE2111 antitoxins that inhibit structurally unrelated toxins. YeeU, which has an antitoxin-like activity that represses toxin expression, is structurally similar to the ribosome-interacting toxins YoeB and RelE. These observations suggest extensive functional exchanges have occurred between TA systems during bacterial evolution.« less

Structural constraints-based evaluation of immunogenic avirulent toxins from Clostridium botulinum C2 and C3 toxins as subunit vaccines.

PubMed

Prisilla, A; Prathiviraj, R; Sasikala, R; Chellapandi, P

2016-10-01

Clostridium botulinum (group-III) is an anaerobic bacterium producing C2 and C3 toxins in addition to botulinum neurotoxins in avian and mammalian cells. C2 and C3 toxins are members of bacterial ADP-ribosyltransferase superfamily, which modify the eukaryotic cell surface proteins by ADP-ribosylation reaction. Herein, the mutant proteins with lack of catalytic and pore forming function derived from C2 (C2I and C2II) and C3 toxins were computationally evaluated to understand their structure-function integrity. We have chosen many structural constraints including local structural environment, folding process, backbone conformation, conformational dynamic sub-space, NAD-binding specificity and antigenic determinants for screening of suitable avirulent toxins. A total of 20 avirulent mutants were identified out of 23 mutants, which were experimentally produced by site-directed mutagenesis. No changes in secondary structural elements in particular to α-helices and β-sheets and also in fold rate of all-β classes. Structural stability was maintained by reordered hydrophobic and hydrogen bonding patterns. Molecular dynamic studies suggested that coupled mutations may restrain the binding affinity to NAD(+) or protein substrate upon structural destabilization. Avirulent toxins of this study have stable energetic backbone conformation with a common blue print of folding process. Molecular docking studies revealed that avirulent mutants formed more favorable hydrogen bonding with the side-chain of amino acids near to conserved NAD-binding core, despite of restraining NAD-binding specificity. Thus, structural constraints in the avirulent toxins would determine their immunogenic nature for the prioritization of protein-based subunit vaccine/immunogens to avian and veterinary animals infected with C. botulinum. Copyright © 2016 Elsevier B.V. All rights reserved.

Mycobacterium tuberculosis toxin Rv2872 is an RNase involved in vancomycin stress response and biofilm development.

PubMed

Wang, Xiaoyu; Zhao, Xiaokang; Wang, Hao; Huang, Xue; Duan, Xiangke; Gu, Yinzhong; Lambert, Nzungize; Zhang, Ke; Kou, Zhenhao; Xie, Jianping

2018-06-11

Bacterial toxin-antitoxin (TA) systems are emerging important regulators of multiple cellular physiological events and candidates for novel antibiotic targets. To explore the role of Mycobacterium tuberculosis function, unknown toxin gene Rv2872 was heterologously expressed in Mycobacterium smegmatis (MS_Rv2872). Upon induction, MS_Rv2872 phenotype differed significantly from the control, such as increased vancomycin resistance, retarded growth, cell wall, and biofilm structure. This phenotype change might result from the RNase activity of Rv2872 as purified Rv2872 toxin protein can cleave the products of several key genes involved in abovementioned phenotypes. In summary, toxin Rv2872 was firstly reported to be a endonuclease involved in antibiotic stress responses, cell wall structure, and biofilm development.

[Malaria in the Republic of Djibouti. Strategy for control using a biological antilarval campaign: indigenous larvivorous fishes (Aphanius dispar) and bacterial toxins].

PubMed

Louis, J P; Albert, J P

1988-01-01

The authors take stock of the present situation of malaria in the Republic of Djibouti which, after several decades of silence, seems to have been reintroduced at the beginning of the seventies. Actually it is hypo-endemic malaria with Plasmodium falciparum of which the only vector seems to be Anopheles arabiensis, gambiae complex. The specificity of the larvae nests allows a control strategy based on the only treatment of larvae sites by biological control: larvivorous fishes (Aphanius Dispar) and in addition the pin-point use of bacterial toxins as a complementary measure. The first results obtained in the rural zones around the capital are encouraging and permit to envisage the extensions of such a strategy to the whole of the territory of the Republic.

Yersinia enterocolitica YopT and Clostridium difficile Toxin B Induce Expression of GILZ in Epithelial Cells

PubMed Central

Köberle, Martin; Göppel, David; Grandl, Tanja; Gaentzsch, Peer; Manncke, Birgit; Berchtold, Susanne; Müller, Steffen; Lüscher, Bernhard; Asselin-Labat, Marie-Liesse; Pallardy, Marc; Sorg, Isabel; Langer, Simon; Barth, Holger; Zumbihl, Robert; Autenrieth, Ingo B.; Bohn, Erwin

2012-01-01

Glucocorticoid induced-leucine zipper (GILZ) has been shown to be induced in cells by different stimuli such as glucocorticoids, IL-10 or deprivation of IL-2. GILZ has anti-inflammatory properties and may be involved in signalling modulating apoptosis. Herein we demonstrate that wildtype Yersinia enterocolitica which carry the pYV plasmid upregulated GILZ mRNA levels and protein expression in epithelial cells. Infection of HeLa cells with different Yersinia mutant strains revealed that the protease activity of YopT, which cleaves the membrane-bound form of Rho GTPases was sufficient to induce GILZ expression. Similarly, Clostridium difficile toxin B, another bacterial inhibitor of Rho GTPases induced GILZ expression. YopT and toxin B both increased transcriptional activity of the GILZ promoter in HeLa cells. GILZ expression could not be linked to the inactivation of an individual Rho GTPase by these toxins. However, forced expression of RhoA and RhoB decreased basal GILZ promoter activity. Furthermore, MAPK activation proved necessary for profound GILZ induction by toxin B. Promoter studies and gel shift analyses defined binding of upstream stimulatory factor (USF) 1 and 2 to a canonical c-Myc binding site (E-box) in the GILZ promoter as a crucial step of its trans-activation. In addition we could show that USF-1 and USF-2 are essential for basal as well as toxin B induced GILZ expression. These findings define a novel way of GILZ promoter trans-activation mediated by bacterial toxins and differentiate it from those mediated by dexamethasone or deprivation of IL-2. PMID:22792400

A novel mechanism of programmed cell death in bacteria by toxin-antitoxin systems corrupts peptidoglycan synthesis.

PubMed

Mutschler, Hannes; Gebhardt, Maike; Shoeman, Robert L; Meinhart, Anton

2011-03-01

Most genomes of bacteria contain toxin-antitoxin (TA) systems. These gene systems encode a toxic protein and its cognate antitoxin. Upon antitoxin degradation, the toxin induces cell stasis or death. TA systems have been linked with numerous functions, including growth modulation, genome maintenance, and stress response. Members of the epsilon/zeta TA family are found throughout the genomes of pathogenic bacteria and were shown not only to stabilize resistance plasmids but also to promote virulence. The broad distribution of epsilon/zeta systems implies that zeta toxins utilize a ubiquitous bacteriotoxic mechanism. However, whereas all other TA families known to date poison macromolecules involved in translation or replication, the target of zeta toxins remained inscrutable. We used in vivo techniques such as microscropy and permeability assays to show that pneumococcal zeta toxin PezT impairs cell wall synthesis and triggers autolysis in Escherichia coli. Subsequently, we demonstrated in vitro that zeta toxins in general phosphorylate the ubiquitous peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) and that this activity is counteracted by binding of antitoxin. After identification of the product we verified the kinase activity in vivo by analyzing metabolite extracts of cells poisoned by PezT using high pressure liquid chromatograpy (HPLC). We further show that phosphorylated UNAG inhibitis MurA, the enzyme catalyzing the initial step in bacterial peptidoglycan biosynthesis. Additionally, we provide what is to our knowledge the first crystal structure of a zeta toxin bound to its substrate. We show that zeta toxins are novel kinases that poison bacteria through global inhibition of peptidoglycan synthesis. This provides a fundamental understanding of how epsilon/zeta TA systems stabilize mobile genetic elements. Additionally, our results imply a mechanism that connects activity of zeta toxin PezT to virulence of pneumococcal infections

A novel Hsp70 inhibitor prevents cell intoxication with the actin ADP-ribosylating Clostridium perfringens iota toxin

PubMed Central

Ernst, Katharina; Liebscher, Markus; Mathea, Sebastian; Granzhan, Anton; Schmid, Johannes; Popoff, Michel R.; Ihmels, Heiko; Barth, Holger; Schiene-Fischer, Cordelia

2016-01-01

Hsp70 family proteins are folding helper proteins involved in a wide variety of cellular pathways. Members of this family interact with key factors in signal transduction, transcription, cell-cycle control, and stress response. Here, we developed the first Hsp70 low molecular weight inhibitor specifically targeting the peptide binding site of human Hsp70. After demonstrating that the inhibitor modulates the Hsp70 function in the cell, we used the inhibitor to show for the first time that the stress-inducible chaperone Hsp70 functions as molecular component for entry of a bacterial protein toxin into mammalian cells. Pharmacological inhibition of Hsp70 protected cells from intoxication with the binary actin ADP-ribosylating iota toxin from Clostridium perfringens, the prototype of a family of enterotoxins from pathogenic Clostridia and inhibited translocation of its enzyme component across cell membranes into the cytosol. This finding offers a starting point for novel therapeutic strategies against certain bacterial toxins. PMID:26839186

Antibody-Based Sensors: Principles, Problems and Potential for Detection of Pathogens and Associated Toxins

PubMed Central

Byrne, Barry; Stack, Edwina; Gilmartin, Niamh; O'Kennedy, Richard

2009-01-01

Antibody-based sensors permit the rapid and sensitive analysis of a range of pathogens and associated toxins. A critical assessment of the implementation of such formats is provided, with reference to their principles, problems and potential for ‘on-site’ analysis. Particular emphasis is placed on the detection of foodborne bacterial pathogens, such as Escherichia coli and Listeria monocytogenes, and additional examples relating to the monitoring of fungal pathogens, viruses, mycotoxins, marine toxins and parasites are also provided. PMID:22408533

Ultrasensitive detection of protein translocated through toxin pores in droplet-interface bilayers

PubMed Central

Fischer, Audrey; Holden, Matthew A.; Pentelute, Brad L.; Collier, R. John

2011-01-01

Many bacterial toxins form proteinaceous pores that facilitate the translocation of soluble effector proteins across cellular membranes. With anthrax toxin this process may be monitored in real time by electrophysiology, where fluctuations in ionic current through these pores inserted in model membranes are used to infer the translocation of individual protein molecules. However, detecting the minute quantities of translocated proteins has been a challenge. Here, we describe use of the droplet-interface bilayer system to follow the movement of proteins across a model membrane separating two submicroliter aqueous droplets. We report the capture and subsequent direct detection of as few as 100 protein molecules that have translocated through anthrax toxin pores. The droplet-interface bilayer system offers new avenues of approach to the study of protein translocation. PMID:21949363

Noninvasive Imaging Technologies Reveal Edema Toxin as a Key Virulence Factor in Anthrax

PubMed Central

Dumetz, Fabien; Jouvion, Grégory; Khun, Huot; Glomski, Ian Justin; Corre, Jean-Philippe; Rougeaux, Clémence; Tang, Wei-Jen; Mock, Michèle; Huerre, Michel; Goossens, Pierre Louis

2011-01-01

Powerful noninvasive imaging technologies enable real-time tracking of pathogen-host interactions in vivo, giving access to previously elusive events. We visualized the interactions between wild-type Bacillus anthracis and its host during a spore infection through bioluminescence imaging coupled with histology. We show that edema toxin plays a central role in virulence in guinea pigs and during inhalational infection in mice. Edema toxin (ET), but not lethal toxin (LT), markedly modified the patterns of bacterial dissemination leading, to apparent direct dissemination to the spleen and provoking apoptosis of lymphoid cells. Each toxin alone provoked particular histological lesions in the spleen. When ET and LT are produced together during infection, a specific temporal pattern of lesion developed, with early lesions typical of LT, followed at a later stage by lesions typical of ET. Our study provides new insights into the complex spatial and temporal effects of B. anthracis toxins in the infected host, suggesting a greater role than previously suspected for ET in anthrax and suggesting that therapeutic targeting of ET contributes to protection. PMID:21641378

Messing with Bacterial Quorum Sensing

PubMed Central

González, Juan E.; Keshavan, Neela D.

2006-01-01

Quorum sensing is widely recognized as an efficient mechanism to regulate expression of specific genes responsible for communal behavior in bacteria. Several bacterial phenotypes essential for the successful establishment of symbiotic, pathogenic, or commensal relationships with eukaryotic hosts, including motility, exopolysaccharide production, biofilm formation, and toxin production, are often regulated by quorum sensing. Interestingly, eukaryotes produce quorum-sensing-interfering (QSI) compounds that have a positive or negative influence on the bacterial signaling network. This eukaryotic interference could result in further fine-tuning of bacterial quorum sensing. Furthermore, recent work involving the synthesis of structural homologs to the various quorum-sensing signal molecules has resulted in the development of additional QSI compounds that could be used to control pathogenic bacteria. The creation of transgenic plants that express bacterial quorum-sensing genes is yet another strategy to interfere with bacterial behavior. Further investigation on the manipulation of quorum-sensing systems could provide us with powerful tools against harmful bacteria. PMID:17158701

The Trojan Horse of the microbiological arms race: phage-encoded toxins as a defence against eukaryotic predators.

PubMed

Arnold, Jason W; Koudelka, Gerald B

2014-02-01

Phage-encoded Shiga toxin (Stx) acts as a bacterial defence against the eukaryotic predator Tetrahymena. To function as an effective bacterial anti-predator defence, Stx must kill a broad spectrum of predators. Consistent with that assertion, we show here that bacterially encoded Stx efficiently kills the bacteriovore Acanthamoeba castellanii in co-culture. We also show that, in addition to Stx, the phage-encoded exotoxin, diphtheria toxin (Dtx) expressed by Corynebacterium diphtheriae also can function as part of an anti-predator strategy; it kills Acanthamoeba in co-culture. Interestingly, only exotoxins produced by bacteria internalized by the Acanthamoeba predator are cytolethal; the presence of purified Dtx or Stx in culture medium has no effect on predator viability. This finding is consistent with our results indicating that intoxication of Acanthamoeba by these exotoxins does not require a receptor. Thus bacteria, in the disguise of a food source, function as a 'Trojan Horse', carrying genes encoding an exotoxin into target organisms. This 'Trojan Horse' mechanism of exotoxin delivery into predator cells allows intoxication of predators that lack a cell surface receptor for the particular toxin, allowing bacteria-bearing exotoxins to kill a broader spectrum of predators, increasing the fitness of the otherwise 'defenceless' prey bacteria. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Antimicrobial Nanoparticle for the Treatment of Bacterial Infection

NASA Astrophysics Data System (ADS)

Pornpattananangkul, Dissaya

Liposomes are spherical lipid vesicles with bilayered membrane structure, which have been recognized as one of the most widely used carriers for delivering a myriad of pharmaceuticals. Liposomes can carry both hydrophilic and hydrophobic agents with high efficiency and protect them from undesired effects of external conditions. However, the applications of liposomes are usually limited by their instability during storage. They are inclined to fuse with one another immediately after preparation, resulting in undesired mixing, increase in size, and payload loss. To overcome this limitation, this dissertation will focus on the technology to stabilize liposomes during storage and destabilize at specific conditions in order to allow controllable therapeutic release, as well as demonstrate their application to treat one of the bacterial infection diseases, acne vulgaris. The first area of this research is stimuli-responsive liposomes development, where the liposomes are stabilized by introducing gold nanoparticles to adsorb to their surface. As a result, the liposomes are prevented from fusing with one another and undesirable payload release during storage or physiological environments. Moreover, therapeutic is controllably released depending on environment conditions, such as acidic pH and bacterial virulence factor. In case of acid-responsive liposomes, the bound gold nanoparticles can effectively prevent liposomes from fusing with one another at neutral pH value, while at acidic environment (e.g. pH<5), the gold particle stabilizers will fall off from the liposomes, thereby reinstalling the fusion activity of liposomes. The fusion activity of the stabilized liposomes is found to be 25% at pH=7, in contrast to 80% at pH=4. Another stimulus that can activate drug release from liposomes is virulence factor released from bacteria themselves, such as bacterial toxin. When nanoparticle-stabilized liposomes encounter with bacteria that secrete toxin, the toxin will insert

New monoclonal antibodies against a novel subtype of Shiga toxin 1 produced by Enterobacter cloacae and their use in analysis of human serum

USDA-ARS?s Scientific Manuscript database

Shiga toxin (Stx) is a major virulence factor for several bacterial pathogens that cause potentially fatal illness, including Escherichia coli and Shigella spp. The continual emergence of new subtypes of Stxs presents challenges in clinical diagnosis of infections caused by Shiga toxin-producing org...

An interbacterial NAD(P) + glycohydrolase toxin requires elongation factor Tu for delivery to target cells

DOE PAGES

Whitney, John C.; Quentin, Dennis; Sawai, Shin; ...

2015-10-08

Type VI secretion (T6S) influences the composition of microbial communities by catalyzing the delivery of toxins between adjacent bacterial cells. Here, we demonstrate that a T6S integral membrane toxin from Pseudomonas aeruginosa, Tse6, acts on target cells by degrading the universally essential dinucleotides NAD + and NADP +. Structural analyses of Tse6 show that it resembles mono-ADP-ribosyltransferase proteins, such as diphtheria toxin, with the exception of a unique loop that both excludes proteinaceous ADP-ribose acceptors and contributes to hydrolysis. We find that entry of Tse6 into target cells requires its binding to an essential housekeeping protein, translation elongation factor Tumore » (EF-Tu). These proteins participate in a larger assembly that additionally directs toxin export and provides chaperone activity. Lastly, visualization of this complex by electron microscopy defines the architecture of a toxin-loaded T6S apparatus and provides mechanistic insight into intercellular membrane protein delivery between bacteria.« less

An Interbacterial NAD(P)+ Glycohydrolase Toxin Requires Elongation Factor Tu for Delivery to Target Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whitney, John C.; Quentin, Dennis; Sawai, Shin

2015-10-08

Type VI secretion (T6S) influences the composition of microbial communities by catalyzing the delivery of toxins between adjacent bacterial cells. Here, we demonstrate that a T6S integral membrane toxin from Pseudomonas aeruginosa, Tse6, acts on target cells by degrading the universally essential dinucleotides NAD + and NADP +. Structural analyses of Tse6 show that it resembles mono-ADP-ribosyltransferase proteins, such as diphtheria toxin, with the exception of a unique loop that both excludes proteinaceous ADP-ribose acceptors and contributes to hydrolysis. We find that entry of Tse6 into target cells requires its binding to an essential housekeeping protein, translation elongation factor Tumore » (EF-Tu). These proteins participate in a larger assembly that additionally directs toxin export and provides chaperone activity. Visualization of this complex by electron microscopy defines the architecture of a toxin-loaded T6S apparatus and provides mechanistic insight into intercellular membrane protein delivery between bacteria.« less

Alpha-Toxin Promotes Staphylococcus aureus Mucosal Biofilm Formation

PubMed Central

Anderson, Michele J.; Lin, Ying-Chi; Gillman, Aaron N.; Parks, Patrick J.; Schlievert, Patrick M.; Peterson, Marnie L.

2012-01-01

Staphylococcus aureus causes many diseases in humans, ranging from mild skin infections to serious, life-threatening, superantigen-mediated Toxic Shock Syndrome (TSS). S. aureus may be asymptomatically carried in the anterior nares or vagina or on the skin, serving as a reservoir for infection. Pulsed-field gel electrophoresis clonal type USA200 is the most widely disseminated colonizer and the leading cause of TSS. The cytolysin α-toxin (also known as α-hemolysin or Hla) is the major epithelial proinflammatory exotoxin produced by TSS S. aureus USA200 isolates. The current study aims to characterize the differences between TSS USA200 strains [high (hla+) and low (hla−) α-toxin producers] in their ability to disrupt vaginal mucosal tissue and to characterize the subsequent infection. Tissue viability post-infection and biofilm formation of TSS USA200 isolates CDC587 and MN8, which contain the α-toxin pseudogene (hla−), MNPE (hla+), and MNPE isogenic hla knockout (hlaKO), were observed via LIVE/DEAD® staining and confocal microscopy. All TSS strains grew to similar bacterial densities (1–5 × 108 CFU) on the mucosa and were proinflammatory over 3 days. However, MNPE formed biofilms with significant reductions in the mucosal viability whereas neither CDC587 (hla−), MN8 (hla−), nor MNPE hlaKO formed biofilms. The latter strains were also less cytotoxic than wild-type MNPE. The addition of exogenous, purified α-toxin to MNPE hlaKO restored the biofilm phenotype. We speculate that α-toxin affects S. aureus phenotypic growth on vaginal mucosa by promoting tissue disruption and biofilm formation. Further, α-toxin mutants (hla−) are not benign colonizers, but rather form a different type of infection, which we have termed high density pathogenic variants (HDPV). PMID:22919655

A Bioanalytical Platform for Simultaneous Detection and Quantification of Biological Toxins

PubMed Central

Weingart, Oliver G.; Gao, Hui; Crevoisier, François; Heitger, Friedrich; Avondet, Marc-André; Sigrist, Hans

2012-01-01

Prevalent incidents support the notion that toxins, produced by bacteria, fungi, plants or animals are increasingly responsible for food poisoning or intoxication. Owing to their high toxicity some toxins are also regarded as potential biological warfare agents. Accordingly, control, detection and neutralization of toxic substances are a considerable economic burden to food safety, health care and military biodefense. The present contribution describes a new versatile instrument and related procedures for array-based simultaneous detection of bacterial and plant toxins using a bioanalytical platform which combines the specificity of covalently immobilized capture probes with a dedicated instrumentation and immuno-based microarray analytics. The bioanalytical platform consists of a microstructured polymer slide serving both as support of printed arrays and as incubation chamber. The platform further includes an easy-to-operate instrument for simultaneous slide processing at selectable assay temperature. Cy5 coupled streptavidin is used as unifying fluorescent tracer. Fluorescence image analysis and signal quantitation allow determination of the toxin’s identity and concentration. The system’s performance has been investigated by immunological detection of Botulinum Neurotoxin type A (BoNT/A), Staphylococcal enterotoxin B (SEB), and the plant toxin ricin. Toxins were detectable at levels as low as 0.5–1 ng·mL−1 in buffer or in raw milk. PMID:22438766

Targeted Silencing of Anthrax Toxin Receptors Protects against Anthrax Toxins*

PubMed Central

Arévalo, Maria T.; Navarro, Ashley; Arico, Chenoa D.; Li, Junwei; Alkhatib, Omar; Chen, Shan; Diaz-Arévalo, Diana; Zeng, Mingtao

2014-01-01

Anthrax spores can be aerosolized and dispersed as a bioweapon. Current postexposure treatments are inadequate at later stages of infection, when high levels of anthrax toxins are present. Anthrax toxins enter cells via two identified anthrax toxin receptors: tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2). We hypothesized that host cells would be protected from anthrax toxins if anthrax toxin receptor expression was effectively silenced using RNA interference (RNAi) technology. Thus, anthrax toxin receptors in mouse and human macrophages were silenced using targeted siRNAs or blocked with specific antibody prior to challenge with anthrax lethal toxin. Viability assays were used to assess protection in macrophages treated with specific siRNA or antibody as compared with untreated cells. Silencing CMG2 using targeted siRNAs provided almost complete protection against anthrax lethal toxin-induced cytotoxicity and death in murine and human macrophages. The same results were obtained by prebinding cells with specific antibody prior to treatment with anthrax lethal toxin. In addition, TEM8-targeted siRNAs also offered significant protection against lethal toxin in human macrophage-like cells. Furthermore, silencing CMG2, TEM8, or both receptors in combination was also protective against MEK2 cleavage by lethal toxin or adenylyl cyclase activity by edema toxin in human kidney cells. Thus, anthrax toxin receptor-targeted RNAi has the potential to be developed as a life-saving, postexposure therapy against anthrax. PMID:24742682

Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates.

PubMed

Baums, Christoph G; Schotte, Ulrich; Amtsberg, Gunter; Goethe, Ralph

2004-05-20

In this study we provide a protocol for genotyping Clostridium perfringens with a new multiplex PCR. This PCR enables reliable and specific detection of the toxin genes cpa, cpb, etx, iap, cpe and cpb2 from heat lysed bacterial suspensions. The efficiency of the protocol was demonstrated by typing C. perfringens reference strains and isolates from veterinary bacteriological routine diagnostic specimens.

Identification of a lambda toxin-negative Clostridium perfringens strain that processes and activates epsilon prototoxin intracellularly.

PubMed

Harkness, Justine M; Li, Jihong; McClane, Bruce A

2012-10-01

Clostridium perfringens type B and D strains produce epsilon toxin (ETX), which is one of the most potent clostridial toxins and is involved in enteritis and enterotoxemias of domestic animals. ETX is produced initially as an inactive prototoxin that is typically then secreted and processed by intestinal proteases or possibly, for some strains, lambda toxin. During the current work a unique C. perfringens strain was identified that intracellularly processes epsilon prototoxin to an active form capable of killing MDCK cells. This activated toxin is not secreted but instead is apparently released upon lysis of bacterial cells entering stationary phase. These findings broaden understanding of the pathogenesis of type B and D infections by identifying a new mechanism of ETX activation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Keeping the Wolves at Bay: Antitoxins of Prokaryotic Type II Toxin-Antitoxin Systems.

PubMed

Chan, Wai Ting; Espinosa, Manuel; Yeo, Chew Chieng

2016-01-01

In their initial stages of discovery, prokaryotic toxin-antitoxin (TA) systems were confined to bacterial plasmids where they function to mediate the maintenance and stability of usually low- to medium-copy number plasmids through the post-segregational killing of any plasmid-free daughter cells that developed. Their eventual discovery as nearly ubiquitous and repetitive elements in bacterial chromosomes led to a wealth of knowledge and scientific debate as to their diversity and functionality in the prokaryotic lifestyle. Currently categorized into six different types designated types I-VI, type II TA systems are the best characterized. These generally comprised of two genes encoding a proteic toxin and its corresponding proteic antitoxin, respectively. Under normal growth conditions, the stable toxin is prevented from exerting its lethal effect through tight binding with the less stable antitoxin partner, forming a non-lethal TA protein complex. Besides binding with its cognate toxin, the antitoxin also plays a role in regulating the expression of the type II TA operon by binding to the operator site, thereby repressing transcription from the TA promoter. In most cases, full repression is observed in the presence of the TA complex as binding of the toxin enhances the DNA binding capability of the antitoxin. TA systems have been implicated in a gamut of prokaryotic cellular functions such as being mediators of programmed cell death as well as persistence or dormancy, biofilm formation, as defensive weapons against bacteriophage infections and as virulence factors in pathogenic bacteria. It is thus apparent that these antitoxins, as DNA-binding proteins, play an essential role in modulating the prokaryotic lifestyle whilst at the same time preventing the lethal action of the toxins under normal growth conditions, i.e., keeping the proverbial wolves at bay. In this review, we will cover the diversity and characteristics of various type II TA antitoxins. We shall

Keeping the Wolves at Bay: Antitoxins of Prokaryotic Type II Toxin-Antitoxin Systems

PubMed Central

Chan, Wai Ting; Espinosa, Manuel; Yeo, Chew Chieng

2016-01-01

In their initial stages of discovery, prokaryotic toxin-antitoxin (TA) systems were confined to bacterial plasmids where they function to mediate the maintenance and stability of usually low- to medium-copy number plasmids through the post-segregational killing of any plasmid-free daughter cells that developed. Their eventual discovery as nearly ubiquitous and repetitive elements in bacterial chromosomes led to a wealth of knowledge and scientific debate as to their diversity and functionality in the prokaryotic lifestyle. Currently categorized into six different types designated types I–VI, type II TA systems are the best characterized. These generally comprised of two genes encoding a proteic toxin and its corresponding proteic antitoxin, respectively. Under normal growth conditions, the stable toxin is prevented from exerting its lethal effect through tight binding with the less stable antitoxin partner, forming a non-lethal TA protein complex. Besides binding with its cognate toxin, the antitoxin also plays a role in regulating the expression of the type II TA operon by binding to the operator site, thereby repressing transcription from the TA promoter. In most cases, full repression is observed in the presence of the TA complex as binding of the toxin enhances the DNA binding capability of the antitoxin. TA systems have been implicated in a gamut of prokaryotic cellular functions such as being mediators of programmed cell death as well as persistence or dormancy, biofilm formation, as defensive weapons against bacteriophage infections and as virulence factors in pathogenic bacteria. It is thus apparent that these antitoxins, as DNA-binding proteins, play an essential role in modulating the prokaryotic lifestyle whilst at the same time preventing the lethal action of the toxins under normal growth conditions, i.e., keeping the proverbial wolves at bay. In this review, we will cover the diversity and characteristics of various type II TA antitoxins. We shall

Virulence and the Environment: a Novel Role for Vibrio cholerae Toxin-Coregulated Pili in Biofilm Formation on Chitin

PubMed Central

Reguera, Gemma; Kolter, Roberto

2005-01-01

The toxin-coregulated pilus (TCP) of Vibrio cholerae is required for intestinal colonization and cholera toxin acquisition. Here we report that TCP mediates bacterial interactions required for biofilm differentiation on chitinaceous surfaces. We also show that undifferentiated TCP− biofilms have reduced ecological fitness and, thus, that chitin colonization may represent an ecological setting outside the host in which selection for a host colonization factor may take place. PMID:15866944

Epsilon toxin: a fascinating pore-forming toxin.

PubMed

Popoff, Michel R

2011-12-01

Epsilon toxin (ETX) is produced by strains of Clostridium perfringens classified as type B or type D. ETX belongs to the heptameric β-pore-forming toxins including aerolysin and Clostridium septicum alpha toxin, which are characterized by the formation of a pore through the plasma membrane of eukaryotic cells consisting in a β-barrel of 14 amphipatic β strands. By contrast to aerolysin and C. septicum alpha toxin, ETX is a much more potent toxin and is responsible for enterotoxemia in animals, mainly sheep. ETX induces perivascular edema in various tissues and accumulates in particular in the kidneys and brain, where it causes edema and necrotic lesions. ETX is able to pass through the blood-brain barrier and stimulate the release of glutamate, which accounts for the symptoms of nervous excitation observed in animal enterotoxemia. At the cellular level, ETX causes rapid swelling followed by cell death involving necrosis. The precise mode of action of ETX remains to be determined. ETX is a powerful toxin, however, it also represents a unique tool with which to vehicle drugs into the central nervous system or target glutamatergic neurons. © 2011 The Author Journal compilation © 2011 FEBS.

Noninvasive imaging technologies reveal edema toxin as a key virulence factor in anthrax.

PubMed

Dumetz, Fabien; Jouvion, Grégory; Khun, Huot; Glomski, Ian Justin; Corre, Jean-Philippe; Rougeaux, Clémence; Tang, Wei-Jen; Mock, Michèle; Huerre, Michel; Goossens, Pierre Louis

2011-06-01

Powerful noninvasive imaging technologies enable real-time tracking of pathogen-host interactions in vivo, giving access to previously elusive events. We visualized the interactions between wild-type Bacillus anthracis and its host during a spore infection through bioluminescence imaging coupled with histology. We show that edema toxin plays a central role in virulence in guinea pigs and during inhalational infection in mice. Edema toxin (ET), but not lethal toxin (LT), markedly modified the patterns of bacterial dissemination leading, to apparent direct dissemination to the spleen and provoking apoptosis of lymphoid cells. Each toxin alone provoked particular histological lesions in the spleen. When ET and LT are produced together during infection, a specific temporal pattern of lesion developed, with early lesions typical of LT, followed at a later stage by lesions typical of ET. Our study provides new insights into the complex spatial and temporal effects of B. anthracis toxins in the infected host, suggesting a greater role than previously suspected for ET in anthrax and suggesting that therapeutic targeting of ET contributes to protection. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Simultaneous Microcystis Algicidal and Microcystin Degrading Capability by a Single Acinetobacter Bacterial Strain.

PubMed

Li, Hong; Ai, Hainan; Kang, Li; Sun, Xingfu; He, Qiang

2016-11-01

Measures for removal of toxic harmful algal blooms often cause lysis of algal cells and release of microcystins (MCs). In this study, Acinetobacter sp. CMDB-2 that exhibits distinct algal lysing activity and MCs degradation capability was isolated. The physiological response and morphological characteristics of toxin-producing Microcystis aeruginosa, the dynamics of intra- and extracellular MC-LR concentration were studied in an algal/bacterial cocultured system. The results demonstrated that Acinetobacter sp. CMDB-2 caused thorough decomposition of algal cells and impairment of photosynthesis within 24 h. Enhanced algal lysis and MC-LR release appeared with increasing bacterial density from 1 × 10 3 to 1 × 10 7 cells/mL; however, the MC-LR was reduced by nearly 94% within 14 h irrespective of bacterial density. Measurement of extracellular and intracellular MC-LR revealed that the toxin was decreased by 92% in bacterial cell incubated systems relative to control and bacterial cell-free filtrate systems. The results confirmed that the bacterial metabolite caused 92% lysis of Microcystis aeruginosa cells, whereas the bacterial cells were responsible for approximately 91% reduction of MC-LR. The joint efforts of the bacterium and its metabolite accomplished the sustainable removal of algae and MC-LR. This is the first report of a single bacterial strain that achieves these dual actions.

Stool C difficile toxin

MedlinePlus

... toxin; Colitis - toxin; Pseudomembranous - toxin; Necrotizing colitis - toxin; C difficile - toxin ... be analyzed. There are several ways to detect C difficile toxin in the stool sample. Enzyme immunoassay ( ...

Enhanced detection and identification of Shiga toxin 1 and 2 from pathogenic bacteria by MALDI-TOF-TOF-MS/MS-PSD and top-down proteomic analysis

USDA-ARS?s Scientific Manuscript database

Shiga toxin producing Escherichia coli (STEC) represent a continuing threat to the Nation’s food supply and public health. Shiga toxin genes (stx) are encoded in lambda-like bacteriophages whose genome is inserted into the bacterial DNA. Environmental stress can trigger bacteriophage replication a...

Structure-Based Design and Synthesis of a Small Molecule that Exhibits Anti-inflammatory Activity by Inhibition of MyD88-mediated Signaling to Bacterial Toxin Exposure.

PubMed

Alam, Shahabuddin; Javor, Sacha; Degardin, Melissa; Ajami, Dariush; Rebek, Mitra; Kissner, Teri L; Waag, David M; Rebek, Julius; Saikh, Kamal U

2015-08-01

Both Gram-positive and Gram-negative pathogens or pathogen-derived components, such as staphylococcal enterotoxins (SEs) and endotoxin (LPS) exposure, activate MyD88-mediated pro-inflammatory cellular immunity for host defense. However, dysregulated MyD88-mediated signaling triggers exaggerated immune response that often leads to toxic shock and death. Previously, we reported a small molecule compound 1 mimicking BB-loop structure of MyD88 was capable of inhibiting pro-inflammatory response to SEB exposure in mice. In this study, we designed a dimeric structure compound 4210 covalently linked with compound 1 by a non-polar cyclohexane linker which strongly inhibited the production of pro-inflammatory cytokines in human primary cells to SEB (IC50 1-50 μm) or LPS extracted from Francisella tularensis, Escherichia coli, or Burkholderia mallei (IC50 10-200 μm). Consistent with cytokine inhibition, in a ligand-induced cell-based reporter assay, compound 4210 inhibited Burkholderia mallei or LPS-induced MyD88-mediated NF-kB-dependent expression of reporter activity (IC50 10-30 μm). Furthermore, results from a newly expressed MyD88 revealed that 4210 inhibited MyD88 dimer formation which is critical for pro-inflammatory signaling. Importantly, a single administration of compound 4210 in mice showed complete protection from lethal toxin challenge. Collectively, these results demonstrated that compound 4210 inhibits toxin-induced inflated pro-inflammatory immune signaling, thus displays a potential bacterial toxin therapeutic. © 2014 John Wiley & Sons A/S.

An Acoustic Plate Mode Sensor for Biowarfare Toxins, Phase II

DTIC Science & Technology

1997-10-01

Biological agents -- such as bacteria , bacterial toxins and viruses -- must be detected rapidly to allow their neutralization or the quick treatment of...Results were comparable. 16 * r Cyclic voltammetry (CV) studies indicate that the monolayers made with the thiodialkyne, D1, which have been photolyzed...Microprocessor system development is ahead of schedule. Preliminary biosensor data is offered using Y. Pestis. The data indi- cates marginal detection

Review of the inhibition of biological activities of food-related selected toxins by natural compounds.

PubMed

Friedman, Mendel; Rasooly, Reuven

2013-04-23

There is a need to develop food-compatible conditions to alter the structures of fungal, bacterial, and plant toxins, thus transforming toxins to nontoxic molecules. The term 'chemical genetics' has been used to describe this approach. This overview attempts to survey and consolidate the widely scattered literature on the inhibition by natural compounds and plant extracts of the biological (toxicological) activity of the following food-related toxins: aflatoxin B1, fumonisins, and ochratoxin A produced by fungi; cholera toxin produced by Vibrio cholerae bacteria; Shiga toxins produced by E. coli bacteria; staphylococcal enterotoxins produced by Staphylococcus aureus bacteria; ricin produced by seeds of the castor plant Ricinus communis; and the glycoalkaloid α-chaconine synthesized in potato tubers and leaves. The reduction of biological activity has been achieved by one or more of the following approaches: inhibition of the release of the toxin into the environment, especially food; an alteration of the structural integrity of the toxin molecules; changes in the optimum microenvironment, especially pH, for toxin activity; and protection against adverse effects of the toxins in cells, animals, and humans (chemoprevention). The results show that food-compatible and safe compounds with anti-toxin properties can be used to reduce the toxic potential of these toxins. Practical applications and research needs are suggested that may further facilitate reducing the toxic burden of the diet. Researchers are challenged to (a) apply the available methods without adversely affecting the nutritional quality, safety, and sensory attributes of animal feed and human food and (b) educate food producers and processors and the public about available approaches to mitigating the undesirable effects of natural toxins that may present in the diet.

Review of the Inhibition of Biological Activities of Food-Related Selected Toxins by Natural Compounds

PubMed Central

Friedman, Mendel; Rasooly, Reuven

2013-01-01

There is a need to develop food-compatible conditions to alter the structures of fungal, bacterial, and plant toxins, thus transforming toxins to nontoxic molecules. The term ‘chemical genetics’ has been used to describe this approach. This overview attempts to survey and consolidate the widely scattered literature on the inhibition by natural compounds and plant extracts of the biological (toxicological) activity of the following food-related toxins: aflatoxin B1, fumonisins, and ochratoxin A produced by fungi; cholera toxin produced by Vibrio cholerae bacteria; Shiga toxins produced by E. coli bacteria; staphylococcal enterotoxins produced by Staphylococcus aureus bacteria; ricin produced by seeds of the castor plant Ricinus communis; and the glycoalkaloid α-chaconine synthesized in potato tubers and leaves. The reduction of biological activity has been achieved by one or more of the following approaches: inhibition of the release of the toxin into the environment, especially food; an alteration of the structural integrity of the toxin molecules; changes in the optimum microenvironment, especially pH, for toxin activity; and protection against adverse effects of the toxins in cells, animals, and humans (chemoprevention). The results show that food-compatible and safe compounds with anti-toxin properties can be used to reduce the toxic potential of these toxins. Practical applications and research needs are suggested that may further facilitate reducing the toxic burden of the diet. Researchers are challenged to (a) apply the available methods without adversely affecting the nutritional quality, safety, and sensory attributes of animal feed and human food and (b) educate food producers and processors and the public about available approaches to mitigating the undesirable effects of natural toxins that may present in the diet. PMID:23612750

Characterizing RecA-Independent Induction of Shiga toxin2-Encoding Phages by EDTA Treatment

PubMed Central

Imamovic, Lejla; Muniesa, Maite

2012-01-01

Background The bacteriophage life cycle has an important role in Shiga toxin (Stx) expression. The induction of Shiga toxin-encoding phages (Stx phages) increases toxin production as a result of replication of the phage genome, and phage lysis of the host cell also provides a means of Stx toxin to exit the cell. Previous studies suggested that prophage induction might also occur in the absence of SOS response, independently of RecA. Methodology/Principal Findings The influence of EDTA on RecA-independent Stx2 phage induction was assessed, in laboratory lysogens and in EHEC strains carrying Stx2 phages in their genome, by Real-Time PCR. RecA-independent mechanisms described for phage λ induction (RcsA and DsrA) were not involved in Stx2 phage induction. In addition, mutations in the pathway for the stress response of the bacterial envelope to EDTA did not contribute to Stx2 phage induction. The effect of EDTA on Stx phage induction is due to its chelating properties, which was also confirmed by the use of citrate, another chelating agent. Our results indicate that EDTA affects Stx2 phage induction by disruption of the bacterial outer membrane due to chelation of Mg2+. In all the conditions evaluated, the pH value had a decisive role in Stx2 phage induction. Conclusions/Significance Chelating agents, such as EDTA and citrate, induce Stx phages, which raises concerns due to their frequent use in food and pharmaceutical products. This study contributes to our understanding of the phenomenon of induction and release of Stx phages as an important factor in the pathogenicity of Shiga toxin-producing Escherichia coli (STEC) and in the emergence of new pathogenic strains. PMID:22393404

Toxin-Antitoxin Systems Are Important for Niche-Specific Colonization and Stress Resistance of Uropathogenic Escherichia coli

PubMed Central

Norton, J. Paul; Mulvey, Matthew A.

2012-01-01

Toxin-antitoxin (TA) systems are prevalent in many bacterial genomes and have been implicated in biofilm and persister cell formation, but the contribution of individual chromosomally encoded TA systems during bacterial pathogenesis is not well understood. Of the known TA systems encoded by Escherichia coli, only a subset is associated with strains of extraintestinal pathogenic E. coli (ExPEC). These pathogens colonize diverse niches and are a major cause of sepsis, meningitis, and urinary tract infections. Using a murine infection model, we show that two TA systems (YefM-YoeB and YbaJ-Hha) independently promote colonization of the bladder by the reference uropathogenic ExPEC isolate CFT073, while a third TA system comprised of the toxin PasT and the antitoxin PasI is critical to ExPEC survival within the kidneys. The PasTI TA system also enhances ExPEC persister cell formation in the presence of antibiotics and markedly increases pathogen resistance to nutrient limitation as well as oxidative and nitrosative stresses. On its own, low-level expression of PasT protects ExPEC from these stresses, whereas overexpression of PasT is toxic and causes bacterial stasis. PasT-induced stasis can be rescued by overexpression of PasI, indicating that PasTI is a bona fide TA system. By mutagenesis, we find that the stress resistance and toxic effects of PasT can be uncoupled and mapped to distinct domains. Toxicity was specifically linked to sequences within the N-terminus of PasT, a region that also promotes the development of persister cells. These results indicate discrete, multipurpose functions for a TA-associated toxin and demonstrate that individual TA systems can provide bacteria with pronounced fitness advantages dependent on toxin expression levels and the specific environmental niche occupied. PMID:23055930

Intimin, tir, and shiga toxin 1 do not influence enteropathogenic responses to shiga toxin-producing Escherichia coli in bovine ligated intestinal loops.

PubMed

Stevens, Mark P; Marchès, Olivier; Campbell, June; Huter, Veronika; Frankel, Gad; Phillips, Alan D; Oswald, Eric; Wallis, Timothy S

2002-02-01

Shiga toxin-producing Escherchia coli (STEC) comprises a group of attaching and effacing (A/E) enteric pathogens of animals and humans. Natural and experimental infection of calves with STEC may result in acute enteritis or subclinical infection, depending on serotype- and host-specific factors. To quantify intestinal secretory and inflammatory responses to STEC in the bovine intestine, serotypes that are associated with human disease (O103:H2 and O157:H7) were introduced into ligated mid-ileal loops in gnotobiotic and conventional calves, and fluid accumulation and recruitment of radiolabeled neutrophils were measured after 12 h. STEC serotype O103:H2, but not serotype O157:H7, elicited strong enteropathogenic responses. To determine if the inflammatory response to STEC O103:H2 in calves requires Shiga toxin 1 or intimate bacterial attachment to the intestinal epithelium, defined mutations were made in the stx1, eae, and tir genes. Our data indicate that some STEC induce intestinal inflammatory responses in calves by a mechanism that is independent of A/E-lesion formation, intimin, or Shiga toxin 1. This may have implications for strategies to reduce STEC carriage in cattle.

Intimin, Tir, and Shiga Toxin 1 Do Not Influence Enteropathogenic Responses to Shiga Toxin-Producing Escherichia coli in Bovine Ligated Intestinal Loops

PubMed Central

Stevens, Mark P.; Marchès, Olivier; Campbell, June; Huter, Veronika; Frankel, Gad; Phillips, Alan D.; Oswald, Eric; Wallis, Timothy S.

2002-01-01

Shiga toxin-producing Escherchia coli (STEC) comprises a group of attaching and effacing (A/E) enteric pathogens of animals and humans. Natural and experimental infection of calves with STEC may result in acute enteritis or subclinical infection, depending on serotype- and host-specific factors. To quantify intestinal secretory and inflammatory responses to STEC in the bovine intestine, serotypes that are associated with human disease (O103:H2 and O157:H7) were introduced into ligated mid-ileal loops in gnotobiotic and conventional calves, and fluid accumulation and recruitment of radiolabeled neutrophils were measured after 12 h. STEC serotype O103:H2, but not serotype O157:H7, elicited strong enteropathogenic responses. To determine if the inflammatory response to STEC O103:H2 in calves requires Shiga toxin 1 or intimate bacterial attachment to the intestinal epithelium, defined mutations were made in the stx1, eae, and tir genes. Our data indicate that some STEC induce intestinal inflammatory responses in calves by a mechanism that is independent of A/E-lesion formation, intimin, or Shiga toxin 1. This may have implications for strategies to reduce STEC carriage in cattle. PMID:11796630

New insights into the biological effects of anthrax toxins: linking cellular to organismal responses

PubMed Central

Guichard, Annabel; Nizet, Victor; Bier, Ethan

2013-01-01

The anthrax toxins lethal toxin (LT) and edema toxin (ET), are essential virulence factors produced by B. anthracis. These toxins act during two distinct phases of anthrax infection. During the first, prodromal phase, which is often asymptomatic, anthrax toxins act on cells of the immune system to help the pathogen establish infection. Then, during the rapidly progressing (or fulminant) stage of the disease bacteria disseminate via a hematological route to various target tissues and organs, which are typically highly vascularized. As bacteria proliferate in the bloodstream LT and ET begin to accumulate rapidly reaching a critical threshold level that will cause death even when the bacterial proliferation is curtailed by antibiotics. During this final phase of infection the toxins cause an increase in vascular permeability and a decrease in function of target organs including the heart, spleen, kidney, adrenal gland, and brain. In this review, we examine the various biological effects of anthrax toxins, focusing on the fulminant stage of the disease and on mechanisms by which the two toxins may collaborate to cause cardiovascular collapse. We discuss normal mechanisms involved in maintaining vascular integrity and based on recent studies indicating that LT and ET cooperatively inhibit membrane trafficking to cell-cell junctions we explore several potential mechanisms by which the toxins may achieve their lethal effects. We also summarize the effects of other potential virulence factors secreted by B. anthracis and consider the role of toxic factors in the evolutionarily recent emergence of this devastating disease. PMID:21930233

Phages and the Evolution of Bacterial Pathogens: from Genomic Rearrangements to Lysogenic Conversion

PubMed Central

Brüssow, Harald; Canchaya, Carlos; Hardt, Wolf-Dietrich

2004-01-01

Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like “swarms” of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework. PMID:15353570

Neutralizing Monoclonal Antibodies against Disparate Epitopes on Ricin Toxin’s Enzymatic Subunit Interfere with Intracellular Toxin Transport

PubMed Central

Yermakova, Anastasiya; Klokk, Tove Irene; O’Hara, Joanne M.; Cole, Richard; Sandvig, Kirsten; Mantis, Nicholas J.

2016-01-01

Ricin is a member of the A-B family of bacterial and plant toxins that exploit retrograde trafficking to the Golgi apparatus and endoplasmic reticulum (ER) as a means to deliver their cytotoxic enzymatic subunits into the cytoplasm of mammalian cells. In this study we demonstrate that R70 and SyH7, two well-characterized monoclonal antibodies (mAbs) directed against distinct epitopes on the surface of ricin’s enzymatic subunit (RTA), interfere with toxin transport from the plasma membrane to the trans Golgi network. Toxin-mAb complexes formed on the cell surface delayed ricin’s egress from EEA-1+ and Rab7+ vesicles and enhanced toxin accumulation in LAMP-1+ vesicles, suggesting the complexes were destined for degradation in lysosomes. Three other RTA-specific neutralizing mAbs against different epitopes were similar to R70 and SyH7 in terms of their effects on ricin retrograde transport. We conclude that interference with toxin retrograde transport may be a hallmark of toxin-neutralizing antibodies directed against disparate epitopes on RTA. PMID:26949061

Thermal stability and structural changes in bacterial toxins responsible for food poisoning

PubMed Central

Regenthal, Paulina; Hansen, Jesper S.; André, Ingemar

2017-01-01

The staphylococcal enterotoxins (SEs) are secreted by the bacteria Staphylococcus aureus and are the most common causative agent in staphylococcal food poisoning. The staphylococcal enterotoxin A (SEA) has been associated with large staphylococcal food poisoning outbreaks, but newer identified SEs, like staphylococcal enterotoxin H (SEH) has recently been shown to be present at similar levels as SEA in food poisoning outbreaks. Thus, we set out to investigate the thermo-stability of the three-dimensional structures of SEA, SEH and staphylococcal enterotoxin E (SEE), since heat inactivation is a common method to inactivate toxins during food processing. Interestingly, the investigated toxins behaved distinctly different upon heating. SEA and SEE were more stable at slightly acidic pH values, while SEH adopted an extremely stable structure at neutral pH, with almost no effects on secondary structural elements upon heating to 95°C, and with reversible formation of tertiary structure upon subsequent cooling to room temperature. Taken together, the data suggests that the family of staphylococcal enterotoxins have different ability to withstand heat, and thus the exact profile of heat inactivation for all SEs causing food poisoning needs to be considered to improve food safety. PMID:28207867

Augmenting the Efficacy of Immunotoxins and Other Targeted Protein Toxins by Endosomal Escape Enhancers.

PubMed

Fuchs, Hendrik; Weng, Alexander; Gilabert-Oriol, Roger

2016-07-01

The toxic moiety of almost all protein-based targeted toxins must enter the cytosol of the target cell to mediate its fatal effect. Although more than 500 targeted toxins have been investigated in the past decades, no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date. Missing efficacy can be attributed in many cases to insufficient endosomal escape and therefore subsequent lysosomal degradation of the endocytosed toxins. To overcome this drawback, many strategies have been described to weaken the membrane integrity of endosomes. This comprises the use of lysosomotropic amines, carboxylic ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light-induced techniques. Although the efficacy of the targeted toxins was typically augmented in cell culture hundred or thousand fold, in exceptional cases more than million fold, the combination of several substances harbors new problems including additional side effects, loss of target specificity, difficulties to determine the therapeutic window and cell type-dependent variations. This review critically scrutinizes the chances and challenges of endosomal escape enhancers and their potential role in future developments.

Augmenting the Efficacy of Immunotoxins and Other Targeted Protein Toxins by Endosomal Escape Enhancers

PubMed Central

Fuchs, Hendrik; Weng, Alexander; Gilabert-Oriol, Roger

2016-01-01

The toxic moiety of almost all protein-based targeted toxins must enter the cytosol of the target cell to mediate its fatal effect. Although more than 500 targeted toxins have been investigated in the past decades, no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date. Missing efficacy can be attributed in many cases to insufficient endosomal escape and therefore subsequent lysosomal degradation of the endocytosed toxins. To overcome this drawback, many strategies have been described to weaken the membrane integrity of endosomes. This comprises the use of lysosomotropic amines, carboxylic ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light-induced techniques. Although the efficacy of the targeted toxins was typically augmented in cell culture hundred or thousand fold, in exceptional cases more than million fold, the combination of several substances harbors new problems including additional side effects, loss of target specificity, difficulties to determine the therapeutic window and cell type-dependent variations. This review critically scrutinizes the chances and challenges of endosomal escape enhancers and their potential role in future developments. PMID:27376327

Repurposing a Prokaryotic Toxin-Antitoxin System for the Selective Killing of Oncogenically Stressed Human Cells.

PubMed

Preston, Mark A; Pimentel, Belén; Bermejo-Rodríguez, Camino; Dionne, Isabelle; Turnbull, Alice; de la Cueva-Méndez, Guillermo

2016-07-15

Prokaryotes express intracellular toxins that pass unnoticed to carrying cells until coexpressed antitoxin partners are degraded in response to stress. Although not evolved to function in eukaryotes, one of these toxins, Kid, induces apoptosis in mammalian cells, an effect that is neutralized by its cognate antitoxin, Kis. Here we engineered this toxin-antitoxin pair to create a synthetic system that becomes active in human cells suffering a specific oncogenic stress. Inspired by the way Kid becomes active in bacterial cells, we produced a Kis variant that is selectively degraded in human cells expressing oncoprotein E6. The resulting toxin-antitoxin system functions autonomously in human cells, distinguishing those that suffer the oncogenic insult, which are killed by Kid, from those that do not, which remain protected by Kis. Our results provide a framework for developing personalized anticancer strategies avoiding off-target effects, a challenge that has been hardly tractable by other means thus far.

Addressing the Immunogenicity of the Cargo and of the Targeting Antibodies with a Focus on Deimmunized Bacterial Toxins and on Antibody-Targeted Human Effector Proteins

PubMed Central

Grinberg, Yehudit; Benhar, Itai

2017-01-01

Third-generation immunotoxins are composed of a human, or humanized, targeting moiety, usually a monoclonal antibody or an antibody fragment, and a non-human effector molecule. Due to the non-human origin of the cytotoxic domain, these molecules stimulate potent anti-drug immune responses, which limit treatment options. Efforts are made to deimmunize such immunotoxins or to combine treatment with immunosuppression. An alternative approach is using the so-called “human cytotoxic fusion proteins”, in which antibodies are used to target human effector proteins. Here, we present three relevant approaches for reducing the immunogenicity of antibody-targeted protein therapeutics: (1) reducing the immunogenicity of the bacterial toxin, (2) fusing human cytokines to antibodies to generate immunocytokines and (3) addressing the immunogenicity of the targeting antibodies. PMID:28574434

Inhibition of cholera toxin and other AB toxins by polyphenolic compounds

USDA-ARS?s Scientific Manuscript database

All AB-type protein toxins have intracellular targets despite an initial extracellular location. These toxins use different methods to reach the cytosol and have different effects on the target cell. Broad-spectrum inhibitors against AB toxins are therefore hard to develop because the toxins use dif...

Structure, Biology, and Therapeutic Application of Toxin-Antitoxin Systems in Pathogenic Bacteria.

PubMed

Lee, Ki-Young; Lee, Bong-Jin

2016-10-22

Bacterial toxin-antitoxin (TA) systems have received increasing attention for their diverse identities, structures, and functional implications in cell cycle arrest and survival against environmental stresses such as nutrient deficiency, antibiotic treatments, and immune system attacks. In this review, we describe the biological functions and the auto-regulatory mechanisms of six different types of TA systems, among which the type II TA system has been most extensively studied. The functions of type II toxins include mRNA/tRNA cleavage, gyrase/ribosome poison, and protein phosphorylation, which can be neutralized by their cognate antitoxins. We mainly explore the similar but divergent structures of type II TA proteins from 12 important pathogenic bacteria, including various aspects of protein-protein interactions. Accumulating knowledge about the structure-function correlation of TA systems from pathogenic bacteria has facilitated a novel strategy to develop antibiotic drugs that target specific pathogens. These molecules could increase the intrinsic activity of the toxin by artificially interfering with the intermolecular network of the TA systems.

Pseudomonas aeruginosa Type III Secretory Toxin ExoU and Its Predicted Homologs.

PubMed

Sawa, Teiji; Hamaoka, Saeko; Kinoshita, Mao; Kainuma, Atsushi; Naito, Yoshifumi; Akiyama, Koichi; Kato, Hideya

2016-10-26

Pseudomonas aeruginosa ExoU, a type III secretory toxin and major virulence factor with patatin-like phospholipase activity, is responsible for acute lung injury and sepsis in immunocompromised patients. Through use of a recently updated bacterial genome database, protein sequences predicted to be homologous to Ps. aeruginosa ExoU were identified in 17 other Pseudomonas species ( Ps. fluorescens , Ps. lundensis , Ps. weihenstephanensis , Ps. marginalis, Ps. rhodesiae, Ps. synxantha , Ps. libanensis , Ps. extremaustralis , Ps. veronii , Ps. simiae , Ps. trivialis , Ps. tolaasii , Ps. orientalis , Ps. taetrolens , Ps. syringae , Ps. viridiflava , and Ps. cannabina ) and 8 Gram-negative bacteria from three other genera ( Photorhabdus , Aeromonas , and Paludibacterium ). In the alignment of the predicted primary amino acid sequences used for the phylogenetic analyses, both highly conserved and nonconserved parts of the toxin were discovered among the various species. Further comparative studies of the predicted ExoU homologs should provide us with more detailed information about the unique characteristics of the Ps. aeruginosa ExoU toxin.

Poisons, ruffles and rockets: bacterial pathogens and the host cell cytoskeleton.

PubMed

Steele-Mortimer, O; Knodler, L A; Finlay, B B

2000-02-01

The cytoskeleton of eukaryotic cells is affected by a number of bacterial and viral pathogens. In this review we consider three recurring themes of cytoskeletal involvement in bacterial pathogenesis: 1) the effect of bacterial toxins on actin-regulating small GTP-binding proteins; 2) the invasion of non-phagocytic cells by the bacterial induction of ruffles at the plasma membrane; 3) the formation of actin tails and pedestals by intracellular and extracellular bacteria, respectively. Considerable progress has been made recently in the characterization of these processes. It is becoming clear that bacterial pathogens have developed a variety of sophisticated mechanisms for utilizing the complex cytoskeletal system of host cells. These bacterially-induced processes are now providing unique insights into the regulation of fundamental eukaryotic mechanisms.

Approaches to treatment of emerging Shiga toxin-producing Escherichia coli infections highlighting the O104:H4 serotype

PubMed Central

Rahal, Elias A.; Fadlallah, Sukayna M.; Nassar, Farah J.; Kazzi, Natalie; Matar, Ghassan M.

2015-01-01

Shiga toxin-producing Escherichia coli (STEC) are a group of diarrheagenic bacteria associated with foodborne outbreaks. Infection with these agents may result in grave sequelae that include fatality. A large number of STEC serotypes has been identified to date. E. coli serotype O104:H4 is an emerging pathogen responsible for a 2011 outbreak in Europe that resulted in over 4000 infections and 50 deaths. STEC pathogenicity is highly reliant on the production of one or more Shiga toxins that can inhibit protein synthesis in host cells resulting in a cytotoxicity that may affect various organ systems. Antimicrobials are usually avoided in the treatment of STEC infections since they are believed to induce bacterial cell lysis and the release of stored toxins. Some antimicrobials have also been reported to enhance toxin synthesis and production from these organisms. Various groups have attempted alternative treatment approaches including the administration of toxin-directed antibodies, toxin-adsorbing polymers, probiotic agents and natural remedies. The utility of antibiotics in treating STEC infections has also been reconsidered in recent years with certain modalities showing promise. PMID:25853096

Effect of environmental conditions on production of toxic shock syndrome toxin 1 by Staphylococcus aureus.

PubMed Central

Wong, A C; Bergdoll, M S

1990-01-01

The kinetics of toxic shock syndrome toxin 1 (TSST-1) production by Staphylococcus aureus was studied in a fermentor in which aeration rate, atmospheric composition, pH, and temperature were controlled. The toxin was synthesized at a maximal rate during the exponential phase. High bacterial populations were not necessarily accompanied by high TSST-1 yields. Aerobiosis increased TSST-1 production, but excessive aeration had an adverse effect. Addition of CO2 enhanced TSST-1 yield by increasing toxin production rate and efficiency. Cultures with no pH control made more TSST-1 than those maintained at pH 5.5 to 7.5. Maximum TSST-1 yields were obtained when cultures were supplied with air (20 cm3/min) and CO2 (5 cm3/min) via a sintered glass sparger. PMID:2108084

Identification of four type II toxin-antitoxin systems in Actinobacillus pleuropneumoniae.

PubMed

Zheng, Chengkun; Zhao, Xigong; Zeng, Ting; Cao, Manman; Xu, Jiali; Shi, Guolin; Li, Jinquan; Chen, Huanchun; Bei, Weicheng

2017-07-03

Toxin-antitoxin (TA) systems are small genetic elements that are widely prevalent in the genomes of bacteria and archaea. These modules have been identified in various bacteria and proposed to play an important role in bacterial physiology and virulence. However, their presence in the genomes of Actinobacillus species has received no attention. In this study, we describe the identification of four type II TA systems in Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. Reverse transcription PCR analysis revealed that the genes encoding the toxin and antitoxin are co-transcribed. Overexpression of each toxin inhibited the growth of Escherichia coli, and the toxic effect could be counteracted by its cognate antitoxin. The pull-down experiments demonstrated that each toxin interacts with its cognate antitoxin in vivo. The promoter activity assays showed that each antitoxin could autoregulate either positively or negatively the TA operon transcription. In addition, the APJL_0660/0659 TA system is present in half of the detected serovars of A. pleuropneumoniae, while the others are present in all. Collectively, we identified four type II TA systems in A. pleuropneumoniae, and this study has laid the foundation for further functional study of these TA systems. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Binding of ATP by pertussis toxin and isolated toxin subunits

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hausman, S.Z.; Manclark, C.R.; Burns, D.L.

1990-07-03

The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner;more » however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.« less

A Bacterial Toxin with Analgesic Properties: Hyperpolarization of DRG Neurons by Mycolactone.

PubMed

Song, Ok-Ryul; Kim, Han-Byul; Jouny, Samuel; Ricard, Isabelle; Vandeputte, Alexandre; Deboosere, Nathalie; Marion, Estelle; Queval, Christophe J; Lesport, Pierre; Bourinet, Emmanuel; Henrion, Daniel; Oh, Seog Bae; Lebon, Guillaume; Sandoz, Guillaume; Yeramian, Edouard; Marsollier, Laurent; Brodin, Priscille

2017-07-18

Mycolactone, a polyketide molecule produced by Mycobacterium ulcerans , is the etiological agent of Buruli ulcer. This lipid toxin is endowed with pleiotropic effects, presents cytotoxic effects at high doses, and notably plays a pivotal role in host response upon colonization by the bacillus. Most remarkably, mycolactone displays intriguing analgesic capabilities: the toxin suppresses or alleviates the pain of the skin lesions it inflicts. We demonstrated that the analgesic capability of mycolactone was not attributable to nerve damage, but instead resulted from the triggering of a cellular pathway targeting AT₂ receptors (angiotensin II type 2 receptors; AT₂R), and leading to potassium-dependent hyperpolarization. This demonstration paves the way to new nature-inspired analgesic protocols. In this direction, we assess here the hyperpolarizing properties of mycolactone on nociceptive neurons. We developed a dedicated medium-throughput assay based on membrane potential changes, and visualized by confocal microscopy of bis-oxonol-loaded Dorsal Root Ganglion (DRG) neurons. We demonstrate that mycolactone at non-cytotoxic doses triggers the hyperpolarization of DRG neurons through AT₂R, with this action being not affected by known ligands of AT₂R. This result points towards novel AT₂R-dependent signaling pathways in DRG neurons underlying the analgesic effect of mycolactone, with the perspective for the development of new types of nature-inspired analgesics.

Serogroup-Specific Bacterial Engineered Glycoproteins as Novel Antigenic Targets for Diagnosis of Shiga Toxin-Producing-Escherichia coli-Associated Hemolytic-Uremic Syndrome

PubMed Central

Melli, Luciano J.; Ciocchini, Andrés E.; Caillava, Ana J.; Vozza, Nicolás; Chinen, Isabel; Rivas, Marta; Feldman, Mario F.

2014-01-01

Human infection with Shiga toxin-producing Escherichia coli (STEC) is a major cause of postdiarrheal hemolytic-uremic syndrome (HUS), a life-threatening condition characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. E. coli O157:H7 is the dominant STEC serotype associated with HUS worldwide, although non-O157 STEC serogroups can cause a similar disease. The detection of anti-O157 E. coli lipopolysaccharide (LPS) antibodies in combination with stool culture and detection of free fecal Shiga toxin considerably improves the diagnosis of STEC infections. In the present study, we exploited a bacterial glycoengineering technology to develop recombinant glycoproteins consisting of the O157, O145, or O121 polysaccharide attached to a carrier protein as serogroup-specific antigens for the serological diagnosis of STEC-associated HUS. Our results demonstrate that using these antigens in indirect ELISAs (glyco-iELISAs), it is possible to clearly discriminate between STEC O157-, O145-, and O121-infected patients and healthy children, as well as to confirm the diagnosis in HUS patients for whom the classical diagnostic procedures failed. Interestingly, a specific IgM response was detected in almost all the analyzed samples, indicating that it is possible to detect the infection in the early stages of the disease. Additionally, in all the culture-positive HUS patients, the serotype identified by glyco-iELISAs was in accordance with the serotype of the isolated strain, indicating that these antigens are valuable not only for diagnosing HUS caused by the O157, O145, and O121 serogroups but also for serotyping and guiding the subsequent steps to confirm diagnosis. PMID:25472487

Plasma bacterial and mitochondrial DNA distinguish bacterial sepsis from sterile systemic inflammatory response syndrome and quantify inflammatory tissue injury in nonhuman primates.

PubMed

Sursal, Tolga; Stearns-Kurosawa, Deborah J; Itagaki, Kiyoshi; Oh, Sun-Young; Sun, Shiqin; Kurosawa, Shinichiro; Hauser, Carl J

2013-01-01

Systemic inflammatory response syndrome (SIRS) is a fundamental host response common to bacterial infection and sterile tissue injury. Systemic inflammatory response syndrome can cause organ dysfunction and death, but its mechanisms are incompletely understood. Moreover, SIRS can progress to organ failure or death despite being sterile or after control of the inciting infection. Biomarkers discriminating between sepsis, sterile SIRS, and postinfective SIRS would therefore help direct care. Circulating mitochondrial DNA (mtDNA) is a damage-associated molecular pattern reflecting cellular injury. Circulating bacterial 16S DNA (bDNA) is a pathogen-associated pattern (PAMP) reflecting ongoing infection. We developed quantitative polymerase chain reaction assays to quantify these markers, and predicting their plasma levels might help distinguish sterile injury from infection. To study these events in primates, we assayed banked serum from Papio baboons that had undergone a brief challenge of intravenous Bacillus anthracis delta Sterne (modified to remove toxins) followed by antibiotics (anthrax) that causes organ failure and death. To investigate the progression of sepsis to "severe" sepsis and death, we studied animals where anthrax was pretreated with drotrecogin alfa (activated protein C), which attenuates sepsis in baboons. We also contrasted lethal anthrax bacteremia against nonlethal E. coli bacteremia and against sterile tissue injury from Shiga-like toxin 1. Bacterial DNA and mtDNA levels in timed samples were correlated with blood culture results and assays of organ function. Sterile injury by Shiga-like toxin 1 increased mtDNA, but bDNA was undetectable: consistent with the absence of infection. The bacterial challenges caused parallel early bDNA and mtDNA increases, but bDNA detected pathogens even after bacteria were undetectable by culture. Sublethal E. coli challenge only caused transient rises in mtDNA consistent with a self-limited injury. In lethal

Contact-dependent growth inhibition toxins exploit multiple independent cell-entry pathways

PubMed Central

Willett, Julia L. E.; Gucinski, Grant C.; Fatherree, Jackson P.; Low, David A.; Hayes, Christopher S.

2015-01-01

Contact-dependent growth inhibition (CDI) systems function to deliver toxins into neighboring bacterial cells. CDI+ bacteria export filamentous CdiA effector proteins, which extend from the inhibitor-cell surface to interact with receptors on neighboring target bacteria. Upon binding its receptor, CdiA delivers a toxin derived from its C-terminal region. CdiA C-terminal (CdiA-CT) sequences are highly variable between bacteria, reflecting the multitude of CDI toxin activities. Here, we show that several CdiA-CT regions are composed of two domains, each with a distinct function during CDI. The C-terminal domain typically possesses toxic nuclease activity, whereas the N-terminal domain appears to control toxin transport into target bacteria. Using genetic approaches, we identified ptsG, metI, rbsC, gltK/gltJ, yciB, and ftsH mutations that confer resistance to specific CdiA-CTs. The resistance mutations all disrupt expression of inner-membrane proteins, suggesting that these proteins are exploited for toxin entry into target cells. Moreover, each mutation only protects against inhibition by a subset of CdiA-CTs that share similar N-terminal domains. We propose that, following delivery of CdiA-CTs into the periplasm, the N-terminal domains bind specific inner-membrane receptors for subsequent translocation into the cytoplasm. In accord with this model, we find that CDI nuclease domains are modular payloads that can be redirected through different import pathways when fused to heterologous N-terminal “translocation domains.” These results highlight the plasticity of CDI toxin delivery and suggest that the underlying translocation mechanisms could be harnessed to deliver other antimicrobial agents into Gram-negative bacteria. PMID:26305955

Surface-enhanced Raman for monitoring toxins in water

NASA Astrophysics Data System (ADS)

Spencer, Kevin M.; Sylvia, James M.; Clauson, Susan L.; Bertone, Jane F.; Christesen, Steven D.

2004-02-01

Protection of the drinking water supply from a terrorist attack is of critical importance. Since the water supply is vast, contamination prevention is difficult. Therefore, rapid detection of contaminants, whether a military chemical/biological threat, a hazardous chemical spill, naturally occurring toxins, or bacterial build-up is a priority. The development of rapid environmentally portable and stable monitors that allow continuous monitoring of the water supply is ideal. EIC Laboratories has been developing Surface-Enhanced Raman Spectroscopy (SERS) to detect chemical agents, toxic industrial chemicals (TICs), viruses, cyanotoxins and bacterial agents. SERS is an ideal technique for the Joint Service Agent Water Monitor (JSAWM). SERS uses the enhanced Raman signals observed when an analyte adsorbs to a roughened metal substrate to enable trace detection. Proper development of the metal substrate will optimize the sensitivity and selectivity towards the analytes of interest.

Isolation, purification and spectrometric analysis of PSP toxins from moraxella sp., a bacterium associated with a toxic dinoflagellate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boyce, S.D.; Doucette, G.J.

1994-12-31

Paralytic shellfish poisoning (PSP) is a seafood intoxication syndrome caused by the injestion of shellfish contaminated with toxins produced by algae known as dinoflagellates. The PSP toxins, saxitoxin and its derivatives, act to block voltage-dependent sodium channels and can cause paralysis and even death at higher doses. It is well documented that bacteria coexist with many harmful or toxic algal species, though the exact nature of the association in relation to toxin production is unknown. Recently, the bacterium Moraxella sp. was isolated from the PSP toxin producing dinoflagellate Alexandrium tamarense. Through HPLC analysis and saxitoxin receptor binding assays performed onmore » crude bacterial extracts, it appears that Moraxella sp. is capable of producing saxitoxin and several of its derivatives. However, physical confirmation (e.g. mass spectrometry) of these results is still needed.« less

Failure of botulinum toxin injection for neurogenic detrusor overactivity: Switch of toxin versus second injection of the same toxin.

PubMed

Peyronnet, Benoit; Castel-Lacanal, Evelyne; Manunta, Andréa; Roumiguié, Mathieu; Marque, Philippe; Rischmann, Pascal; Gamé, Xavier

2015-12-01

To evaluate the efficacy of a second injection of the same toxin versus switching to a different botulinum toxin A after failure of a first detrusor injection in patients with neurogenic detrusor overactivity. The charts of all patients who underwent detrusor injections of botulinum toxin A (either abobotulinumtoxinA or onabotulinumtoxinA) for the management of neurogenic detrusor overactivity at a single institution were retrospectively reviewed. Patients in whom a first detrusor injection had failed were included in the present study. They were managed by a second injection of the same toxin at the same dosage or by a new detrusor injection using a different botulinum toxin A. Success was defined as a resolution of urgency, urinary incontinence and detrusor overactivity in a patient self-catheterizing seven times or less per 24 h. A total of 58 patients were included for analysis. A toxin switch was carried out in 29 patients, whereas the other 29 patients received a reinjection of the same toxin at the same dose. The success rate was higher in patients who received a toxin switch (51.7% vs. 24.1%, P = 0.03). Patients treated with a switch from abobotulinumtoxinA to onabotulinumtoxinA and those treated with a switch from onabotulinumtoxinA to abobotulinumtoxinA had similar success rates (52.9% vs. 50%, P = 0.88). After failure of a first detrusor injection of botulinum toxin for neurogenic detrusor overactivity, a switch to a different toxin seems to be more effective than a second injection of the same toxin. The replacement of onabotulinumtoxin by abobotulinumtoxin or the reverse provides similar results. © 2015 The Japanese Urological Association.

Dissecting the role of ADAM10 as a mediator of Staphylococcus aureus α-toxin action.

PubMed

von Hoven, Gisela; Rivas, Amable J; Neukirch, Claudia; Klein, Stefan; Hamm, Christian; Qin, Qianqian; Meyenburg, Martina; Füser, Sabine; Saftig, Paul; Hellmann, Nadja; Postina, Rolf; Husmann, Matthias

2016-07-01

Staphylococcus aureus is a leading cause of bacterial infections in humans, including life-threatening diseases such as pneumonia and sepsis. Its small membrane-pore-forming α-toxin is considered an important virulence factor. By destroying cell-cell contacts through cleavage of cadherins, the metalloproteinase ADAM10 (a disintegrin and metalloproteinase 10) critically contributes to α-toxin-dependent pathology of experimental S. aureus infections in mice. Moreover, ADAM10 was proposed to be a receptor for α-toxin. However, it is unclear whether the catalytic activity or specific domains of ADAM10 are involved in mediating binding and/or subsequent cytotoxicity of α-toxin. Also, it is not known how α-toxin triggers ADAM10's enzymatic activity, and whether ADAM10 is invariably required for all α-toxin action on cells. In the present study, we show that efficient cleavage of the ADAM10 substrate epithelial cadherin (E-cadherin) requires supra-cytotoxic concentrations of α-toxin, leading to significant increases in intracellular [Ca(2+)]; the fall in cellular ATP levels, typically following membrane perforation, became observable at far lower concentrations. Surprisingly, ADAM10 was dispensable for α-toxin-dependent xenophagic targeting of S. aureus, whereas a role for α-toxin attack on the plasma membrane was confirmed. The catalytic site of ADAM10, furin cleavage site, cysteine switch and intracellular domain of ADAM10 were not required for α-toxin binding and subsequent cytotoxicity. In contrast, an essential role for the disintegrin domain and the prodomain emerged. Thus, co-expression of the prodomain with prodomain-deficient ADAM10 reconstituted binding of α-toxin and susceptibility of ADAM10-deficient cells. The results of the present study may help to inform structural analyses of α-toxin-ADAM10 interactions and to design novel strategies to counteract S. aureus α-toxin action. © 2016 The Author(s). published by Portland Press Limited on behalf

Toxic shock syndrome toxin-1, not α-toxin, mediated Bundaberg fatalities.

PubMed

Mueller, Elizabeth A; Merriman, Joseph A; Schlievert, Patrick M

2015-12-01

The 1928 Bundaberg disaster is one of the greatest vaccine tragedies in history. Of 21 children immunized with a diphtheria toxin-antitoxin preparation contaminated with Staphylococcus aureus, 18 developed life-threatening disease and 12 died within 48  h. Historically, the deaths have been attributed to α-toxin, a secreted cytotoxin produced by most S. aureus strains, yet the ability of the Bundaberg contaminant microbe to produce the toxin has never been verified. For the first time, the ability of the original strain to produce α-toxin and other virulence factors is investigated. The study investigates the genetic and regulatory loci mediating α-toxin expression by PCR and assesses production of the cytotoxin in vitro using an erythrocyte haemolysis assay. This analysis is extended to other secreted virulence factors produced by the strain, and their sufficiency to cause lethality in New Zealand white rabbits is determined. Although the strain possesses a wild-type allele for α-toxin, it must have a defective regulatory system, which is responsible for the strain's minimal α-toxin production. The strain encodes and produces staphylococcal superantigens, including toxic shock syndrome toxin-1 (TSST-1), which is sufficient to cause lethality in patients. The findings cast doubt on the belief that α-toxin is the major virulence factor responsible for the Bundaberg fatalities and point to the superantigen TSST-1 as the cause of the disaster.

Three Dimensional Structure of the MqsR:MqsA Complex: A Novel TA Pair Comprised of a Toxin Homologous to RelE and an Antitoxin with Unique Properties

PubMed Central

Kim, Younghoon; Arruda, Jennifer M.; Davenport, Andrew; Wood, Thomas K.; Peti, Wolfgang; Page, Rebecca

2009-01-01

One mechanism by which bacteria survive environmental stress is through the formation of bacterial persisters, a sub-population of genetically identical quiescent cells that exhibit multidrug tolerance and are highly enriched in bacterial toxins. Recently, the Escherichia coli gene mqsR (b3022) was identified as the gene most highly upregulated in persisters. Here, we report multiple individual and complex three-dimensional structures of MqsR and its antitoxin MqsA (B3021), which reveal that MqsR:MqsA form a novel toxin:antitoxin (TA) pair. MqsR adopts an α/β fold that is homologous with the RelE/YoeB family of bacterial ribonuclease toxins. MqsA is an elongated dimer that neutralizes MqsR toxicity. As expected for a TA pair, MqsA binds its own promoter. Unexpectedly, it also binds the promoters of genes important for E. coli physiology (e.g., mcbR, spy). Unlike canonical antitoxins, MqsA is also structured throughout its entire sequence, binds zinc and coordinates DNA via its C- and not N-terminal domain. These studies reveal that TA systems, especially the antitoxins, are significantly more diverse than previously recognized and provide new insights into the role of toxins in maintaining the persister state. PMID:20041169

Superantigens Modulate Bacterial Density during Staphylococcus aureus Nasal Colonization

PubMed Central

Xu, Stacey X.; Kasper, Katherine J.; Zeppa, Joseph J.; McCormick, John K.

2015-01-01

Superantigens (SAgs) are potent microbial toxins that function to activate large numbers of T cells in a T cell receptor (TCR) Vβ-specific manner, resulting in excessive immune system activation. Staphylococcus aureus possesses a large repertoire of distinct SAgs, and in the context of host-pathogen interactions, staphylococcal SAg research has focused primarily on the role of these toxins in severe and invasive diseases. However, the contribution of SAgs to colonization by S. aureus remains unclear. We developed a two-week nasal colonization model using SAg-sensitive transgenic mice expressing HLA-DR4, and evaluated the role of SAgs using two well-studied stains of S. aureus. S. aureus Newman produces relatively low levels of staphylococcal enterotoxin A (SEA), and although we did not detect significant TCR-Vβ specific changes during wild-type S. aureus Newman colonization, S. aureus Newman Δsea established transiently higher bacterial loads in the nose. S. aureus COL produces relatively high levels of staphylococcal enterotoxin B (SEB), and colonization with wild-type S. aureus COL resulted in clear Vβ8-specific T cell skewing responses. S. aureus COL Δseb established consistently higher bacterial loads in the nose. These data suggest that staphylococcal SAgs may be involved in regulating bacterial densities during nasal colonization. PMID:26008236

Molecular Evolutionary Constraints that Determine the Avirulence State of Clostridium botulinum C2 Toxin.

PubMed

Prisilla, A; Prathiviraj, R; Chellapandi, P

2017-04-01

Clostridium botulinum (group-III) is an anaerobic bacterium producing C2 toxin along with botulinum neurotoxins. C2 toxin is belonged to binary toxin A family in bacterial ADP-ribosylation superfamily. A structural and functional diversity of binary toxin A family was inferred from different evolutionary constraints to determine the avirulence state of C2 toxin. Evolutionary genetic analyses revealed evidence of C2 toxin cluster evolution through horizontal gene transfer from the phage or plasmid origins, site-specific insertion by gene divergence, and homologous recombination event. It has also described that residue in conserved NAD-binding core, family-specific domain structure, and functional motifs found to predetermine its virulence state. Any mutational changes in these residues destabilized its structure-function relationship. Avirulent mutants of C2 toxin were screened and selected from a crucial site required for catalytic function of C2I and pore-forming function of C2II. We found coevolved amino acid pairs contributing an essential role in stabilization of its local structural environment. Avirulent toxins selected in this study were evaluated by detecting evolutionary constraints in stability of protein backbone structure, folding and conformational dynamic space, and antigenic peptides. We found 4 avirulent mutants of C2I and 5 mutants of C2II showing more stability in their local structural environment and backbone structure with rapid fold rate, and low conformational flexibility at mutated sites. Since, evolutionary constraints-free mutants with lack of catalytic and pore-forming function suggested as potential immunogenic candidates for treating C. botulinum infected poultry and veterinary animals. Single amino acid substitution in C2 toxin thus provides a major importance to understand its structure-function link, not only of a molecule but also of the pathogenesis.

In silico analysis of protein toxin and bacteriocins from Lactobacillus paracasei SD1 genome and available online databases

PubMed Central

Surachat, Komwit; Sangket, Unitsa; Deachamag, Panchalika; Chotigeat, Wilaiwan

2017-01-01

Lactobacillus paracasei SD1 is a potential probiotic strain due to its ability to survive several conditions in human dental cavities. To ascertain its safety for human use, we therefore performed a comprehensive bioinformatics analysis and characterization of the bacterial protein toxins produced by this strain. We report the complete genome of Lactobacillus paracasei SD1 and its comparison to other Lactobacillus genomes. Additionally, we identify and analyze its protein toxins and antimicrobial proteins using reliable online database resources and establish its phylogenetic relationship with other bacterial genomes. Our investigation suggests that this strain is safe for human use and contains several bacteriocins that confer health benefits to the host. An in silico analysis of protein-protein interactions between the target bacteriocins and the microbial proteins gtfB and luxS of Streptococcus mutans was performed and is discussed here. PMID:28837656

Binary actin-ADP-ribosylating toxins and their use as molecular Trojan horses for drug delivery into eukaryotic cells.

PubMed

Barth, Holger; Stiles, Bradley G

2008-01-01

Binary bacterial toxins are unique AB-type toxins, composed of two non-linked proteins that act as a binding/translocation component and an enzyme component. All known actin-ADP-ribosylating toxins from clostridia possess this binary structure. This toxin family is comprised of the prototypical Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, Clostridium difficile CDT, and Clostridium spiroforme toxin. Once in the cytosol of host cells, these toxins transfer an ADP-ribose moiety from nicotinamide-adenosine-dinucleotide onto G-actin that then leads to depolymerization of actin filaments. In recent years much progress has been made towards understanding the cellular uptake mechanism of binary actin-ADP-ribosylating toxins, and in particular that of C2 toxin. Both components act in a precisely concerted manner to intoxicate eukaryotic cells. The binding/translocation (B-) component forms a complex with the enzyme (A-) component and mediates toxin binding to a cell-surface receptor. Following receptor-mediated endocytosis, the enzyme component escapes from acidic endosomes into the cytosol. Acidification of endosomes triggers pore formation by the binding/translocation component in endosomal membranes and the enzyme component subsequently translocates through the pore. This step requires a host cell chaperone, Hsp90. Due to their unique structure, binary toxins are naturally "tailor made" for transporting foreign proteins into the cytosol of host cells. Several highly specific and cell-permeable recombinant fusion proteins have been designed and successfully used in experimental cell research. This review will focus on the recent progress in studying binary actin ADP-ribosylating toxins as highly effective virulence factors and innovative tools for cell physiology as well as pharmacology.

Toxin-antitoxin systems and regulatory mechanisms in Mycobacterium tuberculosis.

PubMed

Slayden, Richard A; Dawson, Clinton C; Cummings, Jason E

2018-06-01

There has been a significant reduction in annual tuberculosis incidence since the World Health Organization declared tuberculosis a global health threat. However, treatment of M. tuberculosis infections requires lengthy multidrug therapeutic regimens to achieve a durable cure. The development of new drugs that are active against resistant strains and phenotypically diverse organisms continues to present the greatest challenge in the future. Numerous phylogenomic analyses have revealed that the Mtb genome encodes a significantly expanded repertoire of toxin-antitoxin (TA) loci that makes up the Mtb TA system. A TA loci is a two-gene operon encoding a 'toxin' protein that inhibits bacterial growth and an interacting 'antitoxin' partner that neutralizes the inhibitory activity of the toxin. The presence of multiple chromosomally encoded TA loci in Mtb raises important questions in regard to expansion, regulation and function. Thus, the functional roles of TA loci in Mtb pathogenesis have received considerable attention over the last decade. The cumulative results indicate that they are involved in regulating adaptive responses to stresses associated with the host environment and drug treatment. Here we review the TA families encoded in Mtb, discuss the duplication of TA loci in Mtb, regulatory mechanism of TA loci, and phenotypic heterogeneity and pathogenesis.

Tetanus toxin interaction with human erythrocytes. II. Kinetic properties of toxin association and evidence for a ganglioside-toxin macromolecular complex formation.

PubMed

Lazarovici, P; Yavin, E

1985-01-25

The properties of tetanus toxin interaction with human erythrocytes supplemented with disialo- and trisialo-gangliosides have been investigated. Binding of toxin is linear with time for 1 h and is 3-4-fold higher at 37 degrees C than at 4 degrees C during incubation of long duration. It exhibits saturation at toxin concentrations between 0.1 and 1 microgram/ml; however, it is nonsaturable between 1 and up to 50 micrograms/ml. It is effectively prevented by free gangliosides and antibodies or by pretreatment with sialidase but is unaffected by a number of closely related ligands including toxoid and toxin fragments. NaCl (1 M) removes a great portion (86%) of cell-associated toxin while Triton X-100 extracts an additional fraction (30%) of the salt-resistant cell-bound toxin. The residual sequestred toxin after detergent extraction is sensitive to proteolytic degradation. The trypsin-stable fraction (1.5%) is biotoxic and may be indicative of internalization of toxin. A macromolecular complex of about 700 kDa containing toxin and gangliosides has been isolated and characterized by Sephacryl S-300 gel permeation chromatography, SDS-gel electrophoresis, immunoprecipitability and biotoxicity. This complex is obtained only in ganglioside-supplemented cells and not when free 3H-labeled GD1b is reacted with 125I-labeled toxin in solution in the absence of cells. The hydrophobicity properties acquired as a result of ganglioside-toxin interaction, presumably at the cell surface, suggest a conformational change of the toxin which may enable its penetration into the bilayer.

A Bacterial Toxin with Analgesic Properties: Hyperpolarization of DRG Neurons by Mycolactone

PubMed Central

Song, Ok-Ryul; Kim, Han-Byul; Jouny, Samuel; Ricard, Isabelle; Vandeputte, Alexandre; Deboosere, Nathalie; Marion, Estelle; Queval, Christophe J.; Lesport, Pierre; Henrion, Daniel; Oh, Seog Bae; Lebon, Guillaume; Sandoz, Guillaume; Yeramian, Edouard; Marsollier, Laurent; Brodin, Priscille

2017-01-01

Mycolactone, a polyketide molecule produced by Mycobacterium ulcerans, is the etiological agent of Buruli ulcer. This lipid toxin is endowed with pleiotropic effects, presents cytotoxic effects at high doses, and notably plays a pivotal role in host response upon colonization by the bacillus. Most remarkably, mycolactone displays intriguing analgesic capabilities: the toxin suppresses or alleviates the pain of the skin lesions it inflicts. We demonstrated that the analgesic capability of mycolactone was not attributable to nerve damage, but instead resulted from the triggering of a cellular pathway targeting AT2 receptors (angiotensin II type 2 receptors; AT2R), and leading to potassium-dependent hyperpolarization. This demonstration paves the way to new nature-inspired analgesic protocols. In this direction, we assess here the hyperpolarizing properties of mycolactone on nociceptive neurons. We developed a dedicated medium-throughput assay based on membrane potential changes, and visualized by confocal microscopy of bis-oxonol-loaded Dorsal Root Ganglion (DRG) neurons. We demonstrate that mycolactone at non-cytotoxic doses triggers the hyperpolarization of DRG neurons through AT2R, with this action being not affected by known ligands of AT2R. This result points towards novel AT2R-dependent signaling pathways in DRG neurons underlying the analgesic effect of mycolactone, with the perspective for the development of new types of nature-inspired analgesics. PMID:28718822

Selective inhibitor of endosomal trafficking pathways exploited by multiple toxins and viruses

PubMed Central

Gillespie, Eugene J.; Ho, Chi-Lee C.; Balaji, Kavitha; Clemens, Daniel L.; Deng, Gang; Wang, Yao E.; Elsaesser, Heidi J.; Tamilselvam, Batcha; Gargi, Amandeep; Dixon, Shandee D.; France, Bryan; Chamberlain, Brian T.; Blanke, Steven R.; Cheng, Genhong; de la Torre, Juan Carlos; Brooks, David G.; Jung, Michael E.; Colicelli, John; Damoiseaux, Robert; Bradley, Kenneth A.

2013-01-01

Pathogenic microorganisms and toxins have evolved a variety of mechanisms to gain access to the host-cell cytosol and thereby exert virulent effects upon the host. One common mechanism of cellular entry requires trafficking to an acidified endosome, which promotes translocation across the host membrane. To identify small-molecule inhibitors that block this process, a library of 30,000 small molecules was screened for inhibitors of anthrax lethal toxin. Here we report that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone, the most active compound identified in the screen, inhibits intoxication by lethal toxin and blocks the entry of multiple other acid-dependent bacterial toxins and viruses into mammalian cells. This compound, which we named EGA, also delays lysosomal targeting and degradation of the EGF receptor, indicating that it targets host-membrane trafficking. In contrast, EGA does not block endosomal recycling of transferrin, retrograde trafficking of ricin, phagolysosomal trafficking, or phagosome permeabilization by Franciscella tularensis. Furthermore, EGA does not neutralize acidic organelles, demonstrating that its mechanism of action is distinct from pH-raising agents such as ammonium chloride and bafilomycin A1. EGA is a powerful tool for the study of membrane trafficking and represents a class of host-targeted compounds for therapeutic development to treat infectious disease. PMID:24191014

Inhibitory effects of anthocyanins on secretion of Helicobacter pylori CagA and VacA toxins.

PubMed

Kim, Sa-Hyun; Park, Min; Woo, Hyunjun; Tharmalingam, Nagendran; Lee, Gyusang; Rhee, Ki-Jong; Eom, Yong Bin; Han, Sang Ik; Seo, Woo Duck; Kim, Jong Bae

2012-01-01

Anthocyanins have been studied as potential antimicrobial agents against Helicobacter pylori. We investigated whether the biosynthesis and secretion of cytotoxin-associated protein A (CagA) and vacuolating cytotoxin A (VacA) could be suppressed by anthocyanin treatment in vitro. H. pylori reference strain 60190 (CagA(+)/VacA(+)) was used in this study to investigate the inhibitory effects of anthocyanins; cyanidin 3-O-glucoside (C3G), peonidin 3-O-glucoside (Peo3G), pelargonidin 3-O-glucoside (Pel3G), and malvidin 3-O-glucoside (M3G) on expression and secretion of H. pylori toxins. Anthocyanins were added to bacterial cultures and Western blotting was used to determine secretion of CagA and VacA. Among them, we found that C3G inhibited secretion of CagA and VacA resulting in intracellular accumulation of CagA and VacA. C3G had no effect on cagA and vacA expression but suppressed secA transcription. As SecA is involved in translocation of bacterial proteins, the down-regulation of secA expression by C3G offers a mechanistic explanation for the inhibition of toxin secretion. To our knowledge, this is the first report suggesting that C3G inhibits secretion of the H. pylori toxins CagA and VacA via suppression of secA transcription.

Inhibitory Effects of Anthocyanins on Secretion of Helicobacter pylori CagA and VacA Toxins

PubMed Central

Kim, Sa-Hyun; Park, Min; Woo, Hyunjun; Tharmalingam, Nagendran; Lee, Gyusang; Rhee, Ki-Jong; Eom, Yong Bin; Han, Sang Ik; Seo, Woo Duck; Kim, Jong Bae

2012-01-01

Anthocyanins have been studied as potential antimicrobial agents against Helicobacter pylori. We investigated whether the biosynthesis and secretion of cytotoxin-associated protein A (CagA) and vacuolating cytotoxin A (VacA) could be suppressed by anthocyanin treatment in vitro. H. pylori reference strain 60190 (CagA+/VacA+) was used in this study to investigate the inhibitory effects of anthocyanins; cyanidin 3-O-glucoside (C3G), peonidin 3-O-glucoside (Peo3G), pelargonidin 3-O-glucoside (Pel3G), and malvidin 3-O-glucoside (M3G) on expression and secretion of H. pylori toxins. Anthocyanins were added to bacterial cultures and Western blotting was used to determine secretion of CagA and VacA. Among them, we found that C3G inhibited secretion of CagA and VacA resulting in intracellular accumulation of CagA and VacA. C3G had no effect on cagA and vacA expression but suppressed secA transcription. As SecA is involved in translocation of bacterial proteins, the down-regulation of secA expression by C3G offers a mechanistic explanation for the inhibition of toxin secretion. To our knowledge, this is the first report suggesting that C3G inhibits secretion of the H. pylori toxins CagA and VacA via suppression of secA transcription. PMID:23155357

Crystal structures of ricin toxin's enzymatic subunit (RTA) in complex with neutralizing and non-neutralizing single-chain antibodies.

PubMed

Rudolph, Michael J; Vance, David J; Cheung, Jonah; Franklin, Matthew C; Burshteyn, Fiana; Cassidy, Michael S; Gary, Ebony N; Herrera, Cristina; Shoemaker, Charles B; Mantis, Nicholas J

2014-08-26

Ricin is a select agent toxin and a member of the RNA N-glycosidase family of medically important plant and bacterial ribosome-inactivating proteins. In this study, we determined X-ray crystal structures of the enzymatic subunit of ricin (RTA) in complex with the antigen binding domains (VHH) of five unique single-chain monoclonal antibodies that differ in their respective toxin-neutralizing activities. None of the VHHs made direct contact with residues involved in RTA's RNA N-glycosidase activity or induced notable allosteric changes in the toxin's subunit. Rather, the five VHHs had overlapping structural epitopes on the surface of the toxin and differed in the degree to which they made contact with prominent structural elements in two folding domains of the RTA. In general, RTA interactions were influenced most by the VHH CDR3 (CDR, complementarity-determining region) elements, with the most potent neutralizing antibody having the shortest and most conformationally constrained CDR3. These structures provide unique insights into the mechanisms underlying toxin neutralization and provide critically important information required for the rational design of ricin toxin subunit vaccines. Copyright © 2014 Elsevier Ltd. All rights reserved.

Clostridial Binary Toxins: Basic Understandings that Include Cell Surface Binding and an Internal "Coup de Grâce".

PubMed

Stiles, Bradley G

2017-01-01

Clostridium species can make a remarkable number of different protein toxins, causing many diverse diseases in humans and animals. The binary toxins of Clostridium botulinum, C. difficile, C. perfringens, and C. spiroforme are one group of enteric-acting toxins that attack the actin cytoskeleton of various cell types. These enterotoxins consist of A (enzymatic) and B (cell binding/membrane translocation) components that assemble on the targeted cell surface or in solution, forming a multimeric complex. Once translocated into the cytosol via endosomal trafficking and acidification, the A component dismantles the filamentous actin-based cytoskeleton via mono-ADP-ribosylation of globular actin. Knowledge of cell surface receptors and how these usurped, host-derived molecules facilitate intoxication can lead to novel ways of defending against these clostridial binary toxins. A molecular-based understanding of the various steps involved in toxin internalization can also unveil therapeutic intervention points that stop the intoxication process. Furthermore, using these bacterial proteins as medicinal shuttle systems into cells provides intriguing possibilities in the future. The pertinent past and state-of-the-art present, regarding clostridial binary toxins, will be evident in this chapter.

The Protective Antigen Component of Anthrax Toxin Forms Functional Octameric Complexes

PubMed Central

Kintzer, Alexander F.; Thoren, Katie L.; Sterling, Harry J.; Dong, Ken C.; Feld, Geoffrey K.; Tang, Iok I.; Zhang, Teri T.; Williams, Evan R.; Berger, James M.; Krantz, Bryan A.

2009-01-01

The assembly of bacterial toxins and virulence factors is critical to their function, but the regulation of assembly during infection has not been studied. We begin to address this question using anthrax toxin as a model. The protective antigen (PA) component of the toxin assembles into ring-shaped homooligomers that bind the two other enzyme components of the toxin, lethal factor (LF) and edema factor (EF), to form toxic complexes. To disrupt the host, these toxic complexes are endocytosed, such that the PA oligomer forms a membrane-spanning channel that LF and EF translocate through to enter the cytosol. We show using single-channel electrophysiology that PA channels contain two populations of conductance states, which correspond with two different PA pre-channel oligomers observed by electron microscopy—the well-described heptamer and a novel octamer. Mass spectrometry demonstrates that the PA octamer binds four LFs, and assembly routes leading to the octamer are populated with even-numbered, dimeric and tetrameric, PA intermediates. Both heptameric and octameric PA complexes can translocate LF and EF with similar rates and efficiencies. Here we also report a 3.2-Å crystal structure of the PA octamer. The octamer comprises ∼20−30% of the oligomers on cells, but outside of the cell, the octamer is more stable than the heptamer under physiological pH. Thus the PA octamer is a physiological, stable, and active assembly state capable of forming lethal toxins that may withstand the hostile conditions encountered in the bloodstream. This assembly mechanism may provide a novel means to control cytotoxicity. PMID:19627991

Anthrax Toxin

DTIC Science & Technology

1984-10-26

focused initially on EF because it seemed possible that this component, like cholera toxin, might cause edema in skin through elevation of cellular cAMP...behavior differed from that seen in cells exposed to cholera toxin, where cellular cAMP levels remain elevated upon toxin removal. Studies in CHO cell...LF, the rat bioassay is not likely to be an appropriate system for studying the cellular and molecular mechanisms of action of LF. Therefore, a survey

Larvicidal Activities of Indigenous Bacillus thuringiensis Isolates and Nematode Symbiotic Bacterial Toxins against the Mosquito Vector, Culex pipiens (Diptera: Culicidae).

PubMed

Ahmed, Ashraf M; Hussein, Hamdy I; El-Kersh, Talat A; Al-Sheikh, Yazeed A; Ayaad, Tahany H; El-Sadawy, Hanan A; Al-Mekhlafi, Fahd A; Ibrahim, Mohamed S; Al-Tamimi, Jameel; Nasr, Fahd A

2017-06-01

The incidence of mosquito-borne diseases and the resistance of mosquitoes to conventional pesticides have recently caused a panic to the authorities in the endemic countries. This study was conducted to identify native larvicidal biopesticides against Culex pipiens for utilization in the battle against mosquito-borne diseases. Larvicidal activities of new indigenous Bacillus thuringiensis isolates and crude toxin complexes (TCs) of two nematode bacterial-symbionts, Photorhabdus luminescens akhurstii (HRM1) and Ph. luminescens akhurstii (HS1) that tested against Cx. pipiens . B. thuringiensis isolates were recovered from different environmental samples in Saudi Arabia, and the entomopathogenic nematodes, Heterorhabditis indica (HRM1) and He. sp (HS1) were isolated from Egypt. Larvicidal activities (LC 50 and LC 95 ) of the potentially active B. thuringiensis strains or TCs were then evaluated at 24 and 48h post-treatment. Three B. thuringiensis isolates were almost as active as the reference B. thuringiensis israelensis (Bti-H14), and seven isolates were 1.6-5.4 times more toxic than Bti-H14. On the other hand, the TCs of the bacterial symbionts, HRM1 and HS1, showed promising larvicidal activities. HS1 showed LC 50 of 2.54 folds that of HRM1 at 24h post-treatment. Moreover, histopathological examinations of the HS1-treated larvae showed deformations in midgut epithelial cells at 24h post-treatment. Synergistic activity and molecular characterization of these potentially active biocontrol agents are currently being investigated. These results may lead to the identification of eco-friend mosquito larvicidal product(s) that could contribute to the battle against mosquito-borne diseases.

Larvicidal Activities of Indigenous Bacillus thuringiensis Isolates and Nematode Symbiotic Bacterial Toxins against the Mosquito Vector, Culex pipiens (Diptera: Culicidae)

PubMed Central

Ahmed, Ashraf M; Hussein, Hamdy I; El-Kersh, Talat A; Al-Sheikh, Yazeed A; Ayaad, Tahany H; El-Sadawy, Hanan A; Al-Mekhlafi, Fahd A; Ibrahim, Mohamed S; Al-Tamimi, Jameel; Nasr, Fahd A

2017-01-01

Background: The incidence of mosquito-borne diseases and the resistance of mosquitoes to conventional pesticides have recently caused a panic to the authorities in the endemic countries. This study was conducted to identify native larvicidal biopesticides against Culex pipiens for utilization in the battle against mosquito-borne diseases. Methods: Larvicidal activities of new indigenous Bacillus thuringiensis isolates and crude toxin complexes (TCs) of two nematode bacterial-symbionts, Photorhabdus luminescens akhurstii (HRM1) and Ph. luminescens akhurstii (HS1) that tested against Cx. pipiens. B. thuringiensis isolates were recovered from different environmental samples in Saudi Arabia, and the entomopathogenic nematodes, Heterorhabditis indica (HRM1) and He. sp (HS1) were isolated from Egypt. Larvicidal activities (LC50 and LC95) of the potentially active B. thuringiensis strains or TCs were then evaluated at 24 and 48h post-treatment. Results: Three B. thuringiensis isolates were almost as active as the reference B. thuringiensis israelensis (Bti-H14), and seven isolates were 1.6–5.4 times more toxic than Bti-H14. On the other hand, the TCs of the bacterial symbionts, HRM1 and HS1, showed promising larvicidal activities. HS1 showed LC50 of 2.54 folds that of HRM1 at 24h post-treatment. Moreover, histopathological examinations of the HS1-treated larvae showed deformations in midgut epithelial cells at 24h post-treatment. Conclusion: Synergistic activity and molecular characterization of these potentially active biocontrol agents are currently being investigated. These results may lead to the identification of eco-friend mosquito larvicidal product(s) that could contribute to the battle against mosquito-borne diseases. PMID:29062851

Bioterrorism: toxins as weapons.

PubMed

Anderson, Peter D

2012-04-01

The potential for biological weapons to be used in terrorism is a real possibility. Biological weapons include infectious agents and toxins. Toxins are poisons produced by living organisms. Toxins relevant to bioterrorism include ricin, botulinum, Clostridium perfrigens epsilson toxin, conotoxins, shigatoxins, saxitoxins, tetrodotoxins, mycotoxins, and nicotine. Toxins have properties of biological and chemical weapons. Unlike pathogens, toxins do not produce an infection. Ricin causes multiorgan toxicity by blocking protein synthesis. Botulinum blocks acetylcholine in the peripheral nervous system leading to muscle paralysis. Epsilon toxin damages cell membranes. Conotoxins block potassium and sodium channels in neurons. Shigatoxins inhibit protein synthesis and induce apoptosis. Saxitoxin and tetrodotoxin inhibit sodium channels in neurons. Mycotoxins include aflatoxins and trichothecenes. Aflatoxins are carcinogens. Trichothecenes inhibit protein and nucleic acid synthesis. Nicotine produces numerous nicotinic effects in the nervous system.

Metabolism of HT-2 Toxin and T-2 Toxin in Oats

PubMed Central

Meng-Reiterer, Jacqueline; Bueschl, Christoph; Rechthaler, Justyna; Berthiller, Franz; Lemmens, Marc; Schuhmacher, Rainer

2016-01-01

The Fusarium mycotoxins HT-2 toxin (HT2) and T-2 toxin (T2) are frequent contaminants in oats. These toxins, but also their plant metabolites, may contribute to toxicological effects. This work describes the use of 13C-assisted liquid chromatography–high-resolution mass spectrometry for the first comprehensive study on the biotransformation of HT2 and T2 in oats. Using this approach, 16 HT2 and 17 T2 metabolites were annotated including novel glycosylated and hydroxylated forms of the toxins, hydrolysis products, and conjugates with acetic acid, putative malic acid, malonic acid, and ferulic acid. Further targeted quantitative analysis was performed to study toxin metabolism over time, as well as toxin and conjugate mobility within non-treated plant tissues. As a result, HT2-3-O-β-d-glucoside was identified as the major detoxification product of both parent toxins, which was rapidly formed (to an extent of 74% in HT2-treated and 48% in T2-treated oats within one day after treatment) and further metabolised. Mobility of the parent toxins appeared to be negligible, while HT2-3-O-β-d-glucoside was partly transported (up to approximately 4%) through panicle side branches and stem. Our findings demonstrate that the presented combination of untargeted and targeted analysis is well suited for the comprehensive elucidation of mycotoxin metabolism in plants. PMID:27929394

Biotinylation of environmentally isolated Shiga toxin-producing Escherichia coli (STEC) – specific bacteriophages for biosensor and biocontrol applications

USDA-ARS?s Scientific Manuscript database

Like common bacteriophages, Shiga toxin-producing Escherichia coli (STEC) bacteriophages are viruses that recognize and bind to specific bacterial host (STEC) for propagation. They co-exist with STEC hosts, which cause epidemic food and waterborne illnesses, but may act as host populations limiting ...

Structure, Function and Evolution of Clostridium botulinum C2 and C3 Toxins: Insight to Poultry and Veterinary Vaccines.

PubMed

Chellapandi, Paulchamy; Prisilla, Arokiyasamy

2017-01-01

Clostridium botulinum group III strains are able to produce cytotoxins, C2 toxin and C3 exotoxin, along with botulinum neurotoxin types C and D. C2 toxin and C3 exotoxin produced by this organism are the most important members of bacterial ADP-ribosyltransferase superfamily. Both toxins have distinct pathophysiological functions in the avian and mammalian hosts. The members of this superfamily transfer an ADP-ribose moiety of NAD+ to specific eukaryotic target proteins. The present review describes the structure, function and evolution aspects of these toxins with a special emphasis to the development of veterinary vaccines. C2 toxin is a binary toxin that consists of a catalytic subunit (C2I) and a translocation subunit (C2II). C2I component is structurally and functionally similar to the VIP2 and iota A toxin whereas C2II component shows a significant homology with the protective antigen from anthrax toxin and iota B. Unlike C2 toxin, C3 toxin is devoid of translocation/binding subunit. Extensive studies on their sequence-structure-function link spawn additional efforts to understand the catalytic mechanisms and target recognition. Structural and functional relationships with them are often determined by using evolutionary constraints as valuable biological measures. Enzyme-deficient mutants derived from these toxins have been used as drug/protein delivery systems in eukaryotic cells. Thus, current knowledge on their molecular diversity is a well-known perspective to design immunotoxin or subunit vaccine for C. botulinum infection. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Revisiting the Concept of Targeting Only Bacillus anthracis Toxins as a Treatment for Anthrax.

PubMed

Glinert, Itai; Bar-David, Elad; Sittner, Assa; Weiss, Shay; Schlomovitz, Josef; Ben-Shmuel, Amir; Mechaly, Adva; Altboum, Zeev; Kobiler, David; Levy, Haim

2016-08-01

Protective antigen (PA)-based vaccines are effective in preventing the development of fatal anthrax disease both in humans and in relevant animal models. The Bacillus anthracis toxins lethal toxin (lethal factor [LF] plus PA) and edema toxin (edema factor [EF] plus PA) are essential for the establishment of the infection, as inactivation of these toxins results in attenuation of the pathogen. Since the toxins reach high toxemia levels at the bacteremic stages of the disease, the CDC's recommendations include combining antibiotic treatment with antitoxin (anti-PA) immunotherapy. We demonstrate here that while treatment with a highly potent neutralizing monoclonal antibody was highly efficient as postexposure prophylaxis treatment, it failed to protect rabbits with any detectable bacteremia (≥10 CFU/ml). In addition, we show that while PA vaccination was effective against a subcutaneous spore challenge, it failed to protect rabbits against systemic challenges (intravenous injection of vegetative bacteria) with the wild-type Vollum strain or a toxin-deficient mutant. To test the possibility that additional proteins, which are secreted by the bacteria under pathogenicity-stimulating conditions in vitro, may contribute to the vaccine's potency, we immunized rabbits with a secreted protein fraction from a toxin-null mutant. The antiserum raised against the secreted fraction reacts with the bacteria in an immunofluorescence assay. Immunization with the secreted protein fraction did not protect the rabbits against a systemic challenge with the fully pathogenic bacteria. Full protection was obtained only by a combined vaccination with PA and the secreted protein fraction. Therefore, these results indicate that an effective antiserum treatment in advanced stages of anthrax must include toxin-neutralizing antibodies in combination with antibodies against bacterial cell targets. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Millipede toxin

MedlinePlus

... The toxin that millipedes release keeps away most predators. Some large millipede species can spray these toxins ... Your local poison control center can be reached directly by calling the national toll-free Poison Help hotline (1-800-222-1222) from ...

Mass spectrometry-based method of detecting and distinguishing type 1 and type 2 Shiga-like toxins in human serum

USDA-ARS?s Scientific Manuscript database

Shiga-like toxins (verotoxins) are a class of AB5 holotoxins that are responsible for the virulence associated with bacterial pathogens such as Shigella dysenteriae, shigatoxigenic and enterohemorrhagic strains of Escherichia coli (STEC and EHEC), and some Enterobacter strains. The actual expression...

Ingested Shiga Toxin 2 (Stx2) Causes Histopathological Changes in Kidney, Spleen and Thymus Tissues and Mortality in Mice

USDA-ARS?s Scientific Manuscript database

The Shiga toxin (Stxs) producing bacterial strain, Escherichia coli O157:H7, colonizes the distal small intestine and the colon, initiating a very broad spectrum of illnesses such as hemolytic-uremic syndrome (HUS) characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal ...

[Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

PubMed

Inoue, Masaharu; Kikuchi, Maho; Komoriya, Tomoe; Watanabe, Kunitomo; Kouno, Hideki

2007-01-01

Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.

Cytolethal Distending Toxin From Campylobacter jejuni Requires the Cytoskeleton for Toxic Activity.

PubMed

Méndez-Olvera, Estela T; Bustos-Martínez, Jaime A; López-Vidal, Yolanda; Verdugo-Rodríguez, Antonio; Martínez-Gómez, Daniel

2016-10-01

Campylobacter jejuni is one of the major causes of infectious diarrhea worldwide. The distending cytolethal toxin (CDT) of Campylobacter spp. interferes with normal cell cycle progression. This toxic effect is considered a result of DNase activity that produces chromosomal DNA damage. To perform this event, the toxin must be endocytosed and translocated to the nucleus. The aim of this study was to evaluate the role of the cytoskeleton in the translocation of CDT to the nucleus. Campylobacter jejuni ATCC 33291 and seven isolates donated from Instituto de Biotecnologia were used in this study. The presence of CDT genes in C. jejuni strains was determined by PCR. To evaluate the effect of CDT, HeLa cells were treated with bacterial lysate, and the damage and morphological changes were analyzed by microscopy, immunofluorescence staining, and flow cytometry. To evaluate the role of the cytoskeleton, HeLa cells were treated with either latrunculin A or by nocodazole and analyzed by microscopy, flow cytometry, and immunoquantification (ELISA). The results obtained showed that the eight strains of C. jejuni , including the reference strain, had the ability to produce the toxin. Usage of latrunculin A and nocodazole, two cytoskeletal inhibitors, blocked the toxic effect in cells treated with the toxin. This phenomenon was evident in flow cytometry analysis and immunoquantification of Cdc2-phosphorylated. This work showed that the cytotoxic activity of the C. jejuni CDT is dependent on its endocytosis. The alteration in the microtubules and actin filaments caused a blockage transit of the toxin, preventing it from reaching the nucleus of the cell, as well as preventing DNA fragmentation and alteration of the cell cycle. The CDT toxin appears to be an important element for the pathogenesis of campylobacteriosis, since all clinical isolates showed the presence of cdtA , cdtB and cdtC genes.

Cytolethal Distending Toxin From Campylobacter jejuni Requires the Cytoskeleton for Toxic Activity

PubMed Central

Méndez-Olvera, Estela T.; Bustos-Martínez, Jaime A.; López-Vidal, Yolanda; Verdugo-Rodríguez, Antonio; Martínez-Gómez, Daniel

2016-01-01

Background Campylobacter jejuni is one of the major causes of infectious diarrhea worldwide. The distending cytolethal toxin (CDT) of Campylobacter spp. interferes with normal cell cycle progression. This toxic effect is considered a result of DNase activity that produces chromosomal DNA damage. To perform this event, the toxin must be endocytosed and translocated to the nucleus. Objectives The aim of this study was to evaluate the role of the cytoskeleton in the translocation of CDT to the nucleus. Methods Campylobacter jejuni ATCC 33291 and seven isolates donated from Instituto de Biotecnologia were used in this study. The presence of CDT genes in C. jejuni strains was determined by PCR. To evaluate the effect of CDT, HeLa cells were treated with bacterial lysate, and the damage and morphological changes were analyzed by microscopy, immunofluorescence staining, and flow cytometry. To evaluate the role of the cytoskeleton, HeLa cells were treated with either latrunculin A or by nocodazole and analyzed by microscopy, flow cytometry, and immunoquantification (ELISA). Results The results obtained showed that the eight strains of C. jejuni, including the reference strain, had the ability to produce the toxin. Usage of latrunculin A and nocodazole, two cytoskeletal inhibitors, blocked the toxic effect in cells treated with the toxin. This phenomenon was evident in flow cytometry analysis and immunoquantification of Cdc2-phosphorylated. Conclusions This work showed that the cytotoxic activity of the C. jejuni CDT is dependent on its endocytosis. The alteration in the microtubules and actin filaments caused a blockage transit of the toxin, preventing it from reaching the nucleus of the cell, as well as preventing DNA fragmentation and alteration of the cell cycle. The CDT toxin appears to be an important element for the pathogenesis of campylobacteriosis, since all clinical isolates showed the presence of cdtA, cdtB and cdtC genes. PMID:27942359

Contribution of pertussis toxin to the pathogenesis of pertussis disease

PubMed Central

Carbonetti, Nicholas H.

2015-01-01

Pertussis toxin (PT) is a multisubunit protein toxin secreted by Bordetella pertussis, the bacterial agent of the disease pertussis or whooping cough. PT in detoxified form is a component of all licensed acellular pertussis vaccines, since it is considered to be an important virulence factor for this pathogen. PT inhibits G protein-coupled receptor signaling through Gi proteins in mammalian cells, an activity that has led to its widespread use as a cell biology tool. But how does this activity of PT contribute to pertussis, including the severe respiratory symptoms of this disease? In this minireview, the contribution of PT to the pathogenesis of pertussis disease will be considered based on evidence from both human infections and animal model studies. Although definitive proof of the role of PT in humans is lacking, substantial evidence supports the idea that PT is a major contributor to pertussis pathology, including the severe respiratory symptoms associated with this disease. PMID:26394801

Botulinum toxin in parkinsonism: The when, how, and which for botulinum toxin injections.

PubMed

Cardoso, Francisco

2018-06-01

The aim of this article is to provide a review of the use of injections of botulinum toxin in the management of selected symptoms and signs of Parkinson's disease and other forms of parkinsonism. Sialorrhea is defined as inability to control oral secretions, resulting in excessive saliva in the oropharynx. There is a high level of evidence for the treatment of sialorrhea in parkinsonism with injections of different forms of botulinum toxin type A as well as botulinum toxin type B. Tremor can be improved by the use of botulinum toxin injections but improved tremor control often leads to concomitant motor weakness, limiting its use. Levodopa induced dyskinesias are difficult to treat with botulinum toxin injections because of their variable frequency and direction. Apraxia of eyelid opening, a sign more commonly seen in progressive supranuclear palsy and other tauopathies, often improves after botulinum toxin injections. Recent data suggest that regardless of the underlying mechanism, pain in parkinsonism can be alleviated by botulinum toxin injections. Finally, freezing of gait, camptocormia and Pisa syndrome in parkinsonism almost invariably fail to respond to botulinum toxin injections. Copyright © 2017 Elsevier Ltd. All rights reserved.

Particle size fractionation of paralytic shellfish toxins (PSTs): seasonal distribution and bacterial production in the St Lawrence estuary, Canada.

PubMed

Michaud, S; Levasseur, M; Doucette, G; Cantin, G

2002-10-01

We determined the seasonal distribution of paralytic shellfish toxins (PSTs) and PST producing bacteria in > 15, 5-15, and 0.22-5 microm size fractions in the St Lawrence. We also measured PSTs in a local population of Mytilus edulis. PST concentrations were determined in each size fraction and in laboratory incubations of sub-samples by high performance liquid chromatography (HPLC), including the rigorous elimination of suspected toxin 'imposter' peaks. Mussel toxin levels were determined by mouse bioassay and HPLC. PSTs were detected in all size fractions during the summer sampling season, with 47% of the water column toxin levels associated with particles smaller than Alexandrium tamarense (< 15 microm). Even in the > 15 microm size fraction, we estimated that as much as 92% of PSTs could be associated with particles other than A. tamarense. Our results stress the importance of taking into account the potential presence of PSTs in size fractions other than that containing the known algal producer when attempting to model shellfish intoxication, especially during years of low cell abundance. Finally, our HPLC results confirmed the presence of bacteria capable of autonomous PST production in the St Lawrence as well as demonstrating their regular presence and apparent diversity in the plankton. Copyright 2002 Elsevier Science Ltd.

A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough.

PubMed

Nguyen, Annalee W; Wagner, Ellen K; Laber, Joshua R; Goodfield, Laura L; Smallridge, William E; Harvill, Eric T; Papin, James F; Wolf, Roman F; Padlan, Eduardo A; Bristol, Andy; Kaleko, Michael; Maynard, Jennifer A

2015-12-02

Despite widespread vaccination, pertussis rates are rising in industrialized countries and remain high worldwide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. We humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human immunoglobulin G1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the Bordetella pertussis-induced rise in white blood cell counts and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated, but not untreated control animals, experienced a blunted rise in white blood cell counts and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care. Copyright © 2015, American Association for the Advancement of Science.

A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough

PubMed Central

Nguyen, Annalee W.; Wagner, Ellen K.; Laber, Joshua R.; Goodfield, Laura L.; Smallridge, William E.; Harvill, Eric T.; Papin, James F.; Wolf, Roman F.; Padlan, Eduardo A.; Bristol, Andy; Kaleko, Michael; Maynard, Jennifer A.

2016-01-01

In spite of wide-spread vaccination, pertussis rates are rising in industrialized countries and remain high world-wide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. Here, we humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human IgG1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the B. pertussis-induced rise in white blood cell count and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated but not control animals experienced a blunted rise in white blood cell count and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care. PMID:26631634

Mathematical modeling of growth of non-O157 Shiga Toxin-producing Escherichia coli in raw ground beef

USDA-ARS?s Scientific Manuscript database

The objective of this study was to investigate the growth of Shiga toxin-producing Escherichia coli (STEC, including serogroups O45, O103, O111, O121, and O145) in raw ground beef and to develop mathematical models to describe the bacterial growth under different temperature conditions. Three prima...

Antibacterial activity of plant extracts on foodborne bacterial pathogens and food spoilage bacteria

USDA-ARS?s Scientific Manuscript database

Bacterial foodborne diseases are caused by consumption of foods contaminated with bacteria and/or their toxins. In this study, we evaluated antibacterial properties of twelve different extracts including turmeric, lemon and different kinds of teas against four major pathogenic foodborne bacteria inc...

Pertussis toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sekura, R.D.; Moss, J.; Vaughan, M.

1985-01-01

This book contains 13 selections. Some of the titles are: Genetic and Functional Studies of Pertussis Toxin Substrates; Effect of Pertussis Toxin on the Hormonal Responsiveness of Different Tissues; Extracellular Adenylate Cyclase of Bordetella pertussis; and GTP-Regulatory Proteins are Introcellular Messagers: A Model for Hormone Action.

The investigation of epsilon toxin effects on different cancerous cell lines and its synergism effect with methotrexate.

PubMed

Shekarsaraei, Azin Gholami; Hasannia, Sadegh; Pirooznia, Nazanin; Ataiee, Fariba

2014-01-01

The overall goal of this study is to use a bacterial toxin as drug delivery agents for chemotherapy drugs and overcome the development of resistance to these medicines. COR-L105 and MDA-MB 231 which are epithelial-like were used in this study. Cytotoxicity assays were performed by 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) as metabolic indicator. The toxin was essential to kill 50% (CT50) and IC 50 value (inhibition growth value) for methotrexate were determined as optical density at 540 nm. Epsilon toxin-loaded PLGA nanoparticles were prepared using non-aqueous technique. Surface morphology, in vitro drug release, and encapsulation efficiency of the nanoparticles was determined. Results confirmed that using non-toxic concentration of epsilon toxin, resistance to cancerous cell decreased significantly, which could be an important result in cancer therapy. The synergistic effect of MTX and epsilon toxin showed that bio toxins can be used as supplement with chemical drugs and increase the effect of chemotherapy. The results illustrated that application of PLGA as drug delivery system due to its controlled release properties was beneficial. These finding proposed that due to the ease of local accessibility of lung tumors with aerosol drug delivery, biotoxins can directly be used with chemotherapy drugs in aerosol form.

Higher cytotoxicity of divalent antibody-toxins than monovalent antibody-toxins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Won, JaeSeon; Nam, PilWon; Lee, YongChan

2009-04-24

Recombinant antibody-toxins are constructed via the fusion of a 'carcinoma-specific' antibody fragment to a toxin. Due to the high affinity and high selectivity of the antibody fragments, antibody-toxins can bind to surface antigens on cancer cells and kill them without harming normal cells [L.H. Pai, J.K. Batra, D.J. FitzGerald, M.C. Willingham, I. Pastan, Anti-tumor activities of immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin, Proc. Natl. Acad. Sci. USA 88 (1991) 3358-3362]. In this study, we constructed the antibody-toxin, Fab-SWn-PE38, with SWn (n = 3, 6, 9) sequences containing n-time repeated (G{sub 4}S) between the Fabmore » fragment and PE38 (38 kDa truncated form of Pseudomonas exotoxin A). The SWn sequence also harbored one cysteine residue that could form a disulfide bridge between two Fab-SWn-PE38 monomers. We assessed the cytotoxicity of the monovalent (Fab-SWn-PE38), and divalent ([Fab-SWn-PE38]{sub 2}) antibody-toxins. The cytotoxicity of the dimer against the CRL1739 cell line was approximately 18.8-fold higher than that of the monomer on the ng/ml scale, which was approximately 37.6-fold higher on the pM scale. These results strongly indicate that divalency provides higher cytotoxicity for an antibody-toxin.« less

Inhibition of Shiga toxin 2 (Stx2) in apple juices and its resistance to pasteurization.

PubMed

Rasooly, Reuven; Do, Paula M; Levin, Carol E; Friedman, Mendel

2010-06-01

In the present study, we evaluated Shiga toxin (Stx2) activity in apple juices by measuring a decrease in dehydrogenase activity of Vero cells with the microculture tetrazolium (MTT) assay. Freshly prepared juice from Red Delicious apples and Golden Delicious apples inhibited the biological activity of the bacterial toxin Stx2 produced by E. coli O157:H7 strains. Studies with immunomagnetic beads bearing specific antibodies against the toxin revealed that Stx2 activity was restored when removed from the apple juice. SDS gel electrophoresis revealed no difference (P < 0.05) in the densities or molecular weights between Stx2 in either PBS or apple juices. These results suggest that Stx2 may be reversibly bound to small molecular weight constituents in the juice. The Stx2 toxin was not inactivated on exposure to heat programs (63 degrees C for 30 min, 72 degrees C for 15 s, 89 degrees C for 1 s) commonly used to pasteurize apple juice, but lost all activity when exposed to 100 degrees C for 5 min. The results suggest that pasteurization of apple juice used to inactivate E. coli O157:H7 has no effect on Stx2, and that food-compatible and safe antitoxin compounds can be used to inhibit the biological activity of the Shiga toxin.

Genetic Fusions of a CFA/I/II/IV MEFA (Multiepitope Fusion Antigen) and a Toxoid Fusion of Heat-Stable Toxin (STa) and Heat-Labile Toxin (LT) of Enterotoxigenic Escherichia coli (ETEC) Retain Broad Anti-CFA and Antitoxin Antigenicity

PubMed Central

Ruan, Xiaosai; Sack, David A.; Zhang, Weiping

2015-01-01

Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC) strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs) and two distinct enterotoxins [heat-labile toxin (LT) and heat-stable toxin type Ib (STa or hSTa)]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA) strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2):243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTaA14Q-dmLT or 3xSTaN12S-dmLT [IAI 2014, 82(5):1823-32] for CFA/I/II/IV-STaA14Q-dmLT and CFA/I/II/IV-STaN12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTaN12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTaN12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in multivalent

Genetic fusions of a CFA/I/II/IV MEFA (multiepitope fusion antigen) and a toxoid fusion of heat-stable toxin (STa) and heat-labile toxin (LT) of enterotoxigenic Escherichia coli (ETEC) retain broad anti-CFA and antitoxin antigenicity.

PubMed

Ruan, Xiaosai; Sack, David A; Zhang, Weiping

2015-01-01

Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC) strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs) and two distinct enterotoxins [heat-labile toxin (LT) and heat-stable toxin type Ib (STa or hSTa)]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA) strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2):243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTaA14Q-dmLT or 3xSTaN12S-dmLT [IAI 2014, 82(5):1823-32] for CFA/I/II/IV-STaA14Q-dmLT and CFA/I/II/IV-STaN12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTaN12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTaN12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in multivalent

Toxin Plasmids of Clostridium perfringens

PubMed Central

Li, Jihong; Adams, Vicki; Bannam, Trudi L.; Miyamoto, Kazuaki; Garcia, Jorge P.; Uzal, Francisco A.; Rood, Julian I.

2013-01-01

SUMMARY In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1.

PubMed

Ivie, Susan E; McClain, Mark S

2012-09-25

Clostridium perfringens epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1.

Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1

PubMed Central

Ivie, Susan E.; McClain, Mark S.

2012-01-01

Clostridium perfringens epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1. PMID:22938730

Comparative toxicity and efficacy of engineered anthrax lethal toxin variants with broad anti-tumor activities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peters, Diane E.; Program of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, MA; Hoover, Benjamin

2014-09-01

We have previously designed and characterized versions of anthrax lethal toxin that are selectively cytotoxic in the tumor microenvironment and which display broad and potent anti-tumor activities in vivo. Here, we have performed the first direct comparison of the safety and efficacy of three engineered anthrax lethal toxin variants requiring activation by either matrix-metalloproteinases (MMPs), urokinase plasminogen activator (uPA) or co-localized MMP/uPA activities. C57BL/6J mice were challenged with six doses of engineered toxins via intraperitoneal (I.P.) or intravenous (I.V.) dose routes to determine the maximum tolerated dose for six administrations (MTD6) and dose-limiting toxicities. Efficacy was evaluated using the B16-BL6more » syngraft model of melanoma; mice bearing established tumors were treated with six I.P. doses of toxin and tumor measurements and immunohistochemistry, paired with terminal blood work, were used to elaborate upon the anti-tumor mechanism and relative efficacy of each variant. We found that MMP-, uPA- and dual MMP/uPA-activated anthrax lethal toxins exhibited the same dose-limiting toxicity; dose-dependent GI toxicity. In terms of efficacy, all three toxins significantly reduced primary B16-BL6 tumor burden, ranging from 32% to 87% reduction, and they also delayed disease progression as evidenced by dose-dependent normalization of blood work values. While target organ toxicity and effective doses were similar amongst the variants, the dual MMP/uPA-activated anthrax lethal toxin exhibited the highest I.P. MTD6 and was 1.5–3-fold better tolerated than the single MMP- and uPA-activated toxins. Overall, we demonstrate that this dual MMP/uPA-activated anthrax lethal toxin can be administered safely and is highly effective in a preclinical model of melanoma. This modified bacterial cytotoxin is thus a promising candidate for further clinical development and evaluation for use in treating human cancers. - Highlights: • Toxicity and

Comparative toxicity and efficacy of engineered anthrax lethal toxin variants with broad anti-tumor activities.

PubMed

Peters, Diane E; Hoover, Benjamin; Cloud, Loretta Grey; Liu, Shihui; Molinolo, Alfredo A; Leppla, Stephen H; Bugge, Thomas H

2014-09-01

We have previously designed and characterized versions of anthrax lethal toxin that are selectively cytotoxic in the tumor microenvironment and which display broad and potent anti-tumor activities in vivo. Here, we have performed the first direct comparison of the safety and efficacy of three engineered anthrax lethal toxin variants requiring activation by either matrix-metalloproteinases (MMPs), urokinase plasminogen activator (uPA) or co-localized MMP/uPA activities. C57BL/6J mice were challenged with six doses of engineered toxins via intraperitoneal (I.P.) or intravenous (I.V.) dose routes to determine the maximum tolerated dose for six administrations (MTD6) and dose-limiting toxicities. Efficacy was evaluated using the B16-BL6 syngraft model of melanoma; mice bearing established tumors were treated with six I.P. doses of toxin and tumor measurements and immunohistochemistry, paired with terminal blood work, were used to elaborate upon the anti-tumor mechanism and relative efficacy of each variant. We found that MMP-, uPA- and dual MMP/uPA-activated anthrax lethal toxins exhibited the same dose-limiting toxicity; dose-dependent GI toxicity. In terms of efficacy, all three toxins significantly reduced primary B16-BL6 tumor burden, ranging from 32% to 87% reduction, and they also delayed disease progression as evidenced by dose-dependent normalization of blood work values. While target organ toxicity and effective doses were similar amongst the variants, the dual MMP/uPA-activated anthrax lethal toxin exhibited the highest I.P. MTD6 and was 1.5-3-fold better tolerated than the single MMP- and uPA-activated toxins. Overall, we demonstrate that this dual MMP/uPA-activated anthrax lethal toxin can be administered safely and is highly effective in a preclinical model of melanoma. This modified bacterial cytotoxin is thus a promising candidate for further clinical development and evaluation for use in treating human cancers. Published by Elsevier Inc.

Comparative toxicity and efficacy of engineered anthrax lethal toxin variants with broad anti-tumor activities

PubMed Central

Peters, Diane E.; Hoover, Benjamin; Cloud, Loretta Grey; Liu, Shihui; Molinolo, Alfredo A.; Leppla, Stephen H.; Bugge, Thomas H.

2014-01-01

We have previously designed and characterized versions of anthrax lethal toxin that are selectively cytotoxic in the tumor microenvironment and which display broad and potent anti-tumor activities in vivo. Here, we have performed the first direct comparison of the safety and efficacy of three engineered anthrax lethal toxin variants requiring activation by either matrix-metalloproteinases (MMPs), urokinase plasminogen activator (uPA) or co-localized MMP/uPA activities. C57BL/6J mice were challenged with six doses of engineered toxins via intraperitoneal (I.P.) or intravenous (I.V.) dose routes to determine the maximum tolerated dose for six administrations (MTD6) and dose-limiting toxicities. Efficacy was evaluated using the B16-BL6 syngraft model of melanoma; Mice bearing established tumors were treated with six I.P. doses of toxin and tumor measurements and immunohistochemistry, paired with terminal blood work, were used to elaborate upon the anti-tumor mechanism and relative efficacy of each variant. We found that MMP-, uPA- and dual MMP/uPA- activated anthrax lethal toxins exhibited the same dose-limiting toxicity; dose-dependent GI toxicity. In terms of efficacy, all three toxins significantly reduced primary B16-BL6 tumor burden, ranging from 32%–87% reduction, and they also delayed disease progression as evidenced by dose-dependent normalization of blood work values. While target organ toxicity and effective doses were similar amongst the variants, the dual MMP/uPA-activated anthrax lethal toxin exhibited the highest I.P. MTD6 and was 1.5–3-fold better tolerated than the single MMP- and uPA-activated toxins. Overall, we demonstrate that this dual MMP/uPA-activated anthrax lethal toxin can be administered safely and is highly effective in a preclinical model of melanoma. This modified bacterial cytotoxin is thus a promising candidate for further clinical development and evaluation for use in treating human cancers. PMID:24971906

The Adenylate Cyclase Toxins of Bacillus anthracis and Bordetella pertussis Promote Th2 Cell Development by Shaping T Cell Antigen Receptor Signaling

PubMed Central

Rossi Paccani, Silvia; Benagiano, Marisa; Capitani, Nagaja; Zornetta, Irene; Ladant, Daniel; Montecucco, Cesare; D'Elios, Mario M.; Baldari, Cosima T.

2009-01-01

The adjuvanticity of bacterial adenylate cyclase toxins has been ascribed to their capacity, largely mediated by cAMP, to modulate APC activation, resulting in the expression of Th2–driving cytokines. On the other hand, cAMP has been demonstrated to induce a Th2 bias when present during T cell priming, suggesting that bacterial cAMP elevating toxins may directly affect the Th1/Th2 balance. Here we have investigated the effects on human CD4+ T cell differentiation of two adenylate cyclase toxins, Bacillus anthracis edema toxin (ET) and Bordetella pertussis CyaA, which differ in structure, mode of cell entry, and subcellular localization. We show that low concentrations of ET and CyaA, but not of their genetically detoxified adenylate cyclase defective counterparts, potently promote Th2 cell differentiation by inducing expression of the master Th2 transcription factors, c-maf and GATA-3. We also present evidence that the Th2–polarizing concentrations of ET and CyaA selectively inhibit TCR–dependent activation of Akt1, which is required for Th1 cell differentiation, while enhancing the activation of two TCR–signaling mediators, Vav1 and p38, implicated in Th2 cell differentiation. This is at variance from the immunosuppressive toxin concentrations, which interfere with the earliest step in TCR signaling, activation of the tyrosine kinase Lck, resulting in impaired CD3ζ phosphorylation and inhibition of TCR coupling to ZAP-70 and Erk activation. These results demonstrate that, notwithstanding their differences in their intracellular localization, which result in focalized cAMP production, both toxins directly affect the Th1/Th2 balance by interfering with the same steps in TCR signaling, and suggest that their adjuvanticity is likely to result from their combined effects on APC and CD4+ T cells. Furthermore, our results strongly support the key role of cAMP in the adjuvanticity of these toxins. PMID:19266022

T3SS-Independent Uptake of the Short-Trip Toxin-Related Recombinant NleC Effector of Enteropathogenic Escherichia coli Leads to NF-κB p65 Cleavage.

PubMed

Stolle, Anne-Sophie; Norkowski, Stefanie; Körner, Britta; Schmitz, Jürgen; Lüken, Lena; Frankenberg, Maj; Rüter, Christian; Schmidt, M Alexander

2017-01-01

Effector proteins secreted by the type 3 secretion system (T3SS) of pathogenic bacteria have been shown to precisely modulate important signaling cascades of the host for the benefit of the pathogens. Among others, the non-LEE encoded T3SS effector protein NleC of enteropathogenic Escherichia coli (EPEC) is a Zn-dependent metalloprotease and suppresses innate immune responses by directly targeting the NF-κB signaling pathway. Many pathogenic bacteria release potent bacterial toxins of the A-B type, which-in contrast to the direct cytoplasmic injection of T3SS effector proteins-are released first into the environment. In this study, we found that NleC displays characteristics of bacterial A-B toxins, when applied to eukaryotic cells as a recombinant protein. Although lacking a B subunit, that typically mediates the uptake of toxins, recombinant NleC (rNleC) induces endocytosis via lipid rafts and follows the endosomal-lysosomal pathway. The conformation of rNleC is altered by low pH to facilitate its escape from acidified endosomes. This is reminiscent of the homologous A-B toxin AIP56 of the fish pathogen Photobacterium damselae piscicida ( Phdp ). The recombinant protease NleC is functional inside eukaryotic cells and cleaves p65 of the NF-κB pathway. Here, we describe the endocytic uptake mechanism of rNleC, characterize its intracellular trafficking and demonstrate that its specific activity of cleaving p65 requires activation of host cells e.g., by IL1β. Further, we propose an evolutionary link between some T3SS effector proteins and bacterial toxins from apparently unrelated bacteria. In summary, these properties might suggest rNleC as an interesting candidate for future applications as a potential therapeutic against immune disorders.

Delivery of CdiA Nuclease Toxins into Target Cells during Contact-Dependent Growth Inhibition

PubMed Central

Webb, Julia S.; Nikolakakis, Kiel C.; Willett, Julia L. E.; Aoki, Stephanie K.

2013-01-01

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CDI systems deploy a variety of distinct toxins, which are contained within the polymorphic C-terminal region (CdiA-CT) of CdiA proteins. Several CdiA-CTs are nucleases, suggesting that the toxins are transported into the target cell cytoplasm to interact with their substrates. To analyze CdiA transfer to target bacteria, we used the CDI system of uropathogenic Escherichia coli 536 (UPEC536) as a model. Antibodies recognizing the amino- and carboxyl-termini of CdiAUPEC536 were used to visualize transfer of CdiA from CDIUPEC536+ inhibitor cells to target cells using fluorescence microscopy. The results indicate that the entire CdiAUPEC536 protein is deposited onto the surface of target bacteria. CdiAUPEC536 transfer to bamA101 mutants is reduced, consistent with low expression of the CDI receptor BamA on these cells. Notably, our results indicate that the C-terminal CdiA-CT toxin region of CdiAUPEC536 is translocated into target cells, but the N-terminal region remains at the cell surface based on protease sensitivity. These results suggest that the CdiA-CT toxin domain is cleaved from CdiAUPEC536 prior to translocation. Delivery of a heterologous Dickeya dadantii CdiA-CT toxin, which has DNase activity, was also visualized. Following incubation with CDI+ inhibitor cells targets became anucleate, showing that the D.dadantii CdiA-CT was delivered intracellularly. Together, these results demonstrate that diverse CDI toxins are efficiently translocated across target cell envelopes. PMID:23469034

Toxin-Based Therapeutic Approaches

PubMed Central

Shapira, Assaf; Benhar, Itai

2010-01-01

Protein toxins confer a defense against predation/grazing or a superior pathogenic competence upon the producing organism. Such toxins have been perfected through evolution in poisonous animals/plants and pathogenic bacteria. Over the past five decades, a lot of effort has been invested in studying their mechanism of action, the way they contribute to pathogenicity and in the development of antidotes that neutralize their action. In parallel, many research groups turned to explore the pharmaceutical potential of such toxins when they are used to efficiently impair essential cellular processes and/or damage the integrity of their target cells. The following review summarizes major advances in the field of toxin based therapeutics and offers a comprehensive description of the mode of action of each applied toxin. PMID:22069564

Yersinia pestis endowed with increased cytotoxicity is avirulent in a bubonic plague model and induces rapid protection against pneumonic plague.

PubMed

Zauberman, Ayelet; Tidhar, Avital; Levy, Yinon; Bar-Haim, Erez; Halperin, Gideon; Flashner, Yehuda; Cohen, Sara; Shafferman, Avigdor; Mamroud, Emanuelle

2009-06-16

An important virulence strategy evolved by bacterial pathogens to overcome host defenses is the modulation of host cell death. Previous observations have indicated that Yersinia pestis, the causative agent of plague disease, exhibits restricted capacity to induce cell death in macrophages due to ineffective translocation of the type III secretion effector YopJ, as opposed to the readily translocated YopP, the YopJ homologue of the enteropathogen Yersinia enterocolitica Oratio8. This led us to suggest that reduced cytotoxic potency may allow pathogen propagation within a shielded niche, leading to increased virulence. To test the relationship between cytotoxic potential and virulence, we replaced Y. pestis YopJ with YopP. The YopP-expressing Y. pestis strain exhibited high cytotoxic activity against macrophages in vitro. Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 10(7)-fold reduction in virulence. Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain. The subcutaneous administration of the cytotoxic Y. pestis strain appears to activate a rapid and potent systemic, CTL-independent, immunoprotective response, allowing the organism to overcome simultaneous coinfection with 10,000 LD(50) of virulent Y. pestis. Moreover, three days after subcutaneous administration of this strain, animals were also protected against septicemic or primary pneumonic plague. Our findings indicate that an inverse relationship exists between the cytotoxic potential of Y. pestis and its virulence following subcutaneous infection. This appears to be associated with the ability of the engineered cytotoxic Y. pestis strain to induce very rapid, effective and long-lasting protection against bubonic and pneumonic plague. These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed new light on the

Challenging the roles of CD44 and lipolysis stimulated lipoprotein receptor in conveying Clostridium perfringens iota toxin cytotoxicity in breast cancer.

PubMed

Fagan-Solis, Katerina D; Reaves, Denise K; Rangel, M Cristina; Popoff, Michel R; Stiles, Bradley G; Fleming, Jodie M

2014-07-02

Translational exploration of bacterial toxins has come to the forefront of research given their potential as a chemotherapeutic tool. Studies in select tissues have demonstrated that Clostridium perfringens iota toxin binds to CD44 and lipolysis stimulated lipoprotein receptor (LSR) cell-surface proteins. We recently demonstrated that LSR expression correlates with estrogen receptor positive breast cancers and that LSR signaling directs aggressive, tumor-initiating cell behaviors. Herein, we identify the mechanisms of iota toxin cytotoxicity in a tissue-specific, breast cancer model with the ultimate goal of laying the foundation for using iota toxin as a targeted breast cancer therapy. In vitro model systems were used to determine the cytotoxic effect of iota toxin on breast cancer intrinsic subtypes. The use of overexpression and knockdown technologies confirmed the roles of LSR and CD44 in regulating iota toxin endocytosis and induction of cell death. Lastly, cytotoxicity assays were used to demonstrate the effect of iota toxin on a validated set of tamoxifen resistant breast cancer cell lines. Treatment of 14 breast cancer cell lines revealed that LSR+/CD44- lines were highly sensitive, LSR+/CD44+ lines were slightly sensitive, and LSR-/CD44+ lines were resistant to iota cytotoxicity. Reduction in LSR expression resulted in a significant decrease in toxin sensitivity; however, overexpression of CD44 conveyed toxin resistance. CD44 overexpression was correlated with decreased toxin-stimulated lysosome formation and decreased cytosolic levels of iota toxin. These findings indicated that expression of CD44 drives iota toxin resistance through inhibition of endocytosis in breast cancer cells, a role not previously defined for CD44. Moreover, tamoxifen-resistant breast cancer cells exhibited robust expression of LSR and were highly sensitive to iota-induced cytotoxicity. Collectively, these data are the first to show that iota toxin has the potential to be an

Toxins of Prokaryotic Toxin-Antitoxin Systems with Sequence-Specific Endoribonuclease Activity

PubMed Central

Masuda, Hisako; Inouye, Masayori

2017-01-01

Protein translation is the most common target of toxin-antitoxin system (TA) toxins. Sequence-specific endoribonucleases digest RNA in a sequence-specific manner, thereby blocking translation. While past studies mainly focused on the digestion of mRNA, recent analysis revealed that toxins can also digest tRNA, rRNA and tmRNA. Purified toxins can digest single-stranded portions of RNA containing recognition sequences in the absence of ribosome in vitro. However, increasing evidence suggests that in vivo digestion may occur in association with ribosomes. Despite the prevalence of recognition sequences in many mRNA, preferential digestion seems to occur at specific positions within mRNA and also in certain reading frames. In this review, a variety of tools utilized to study the nuclease activities of toxins over the past 15 years will be reviewed. A recent adaptation of an RNA-seq-based technique to analyze entire sets of cellular RNA will be introduced with an emphasis on its strength in identifying novel targets and redefining recognition sequences. The differences in biochemical properties and postulated physiological roles will also be discussed. PMID:28420090

A single-step purification and molecular characterization of functional Shiga toxin 2 variants from pathogenic Escherichia coli

USDA-ARS?s Scientific Manuscript database

Shiga toxin (Stx) 2 variants, Stx2a, Stx2c, Stx2d and Stx2g were purified to homogeneity from bacterial culture supernatants by a one-step monoclonal anti-Stx affinity chromatography method. The method was based on the binding affinity of these Stxs for a monoclonal antibody against the Stx2 A-subun...

Identification and characterization of two novel toxins expressed by the lethal honey bee pathogen Paenibacillus larvae, the causative agent of American foulbrood.

PubMed

Fünfhaus, Anne; Poppinga, Lena; Genersch, Elke

2013-11-01

Paenibacillus larvae is a Gram-positive bacterial pathogen causing the epizootic American foulbrood in honey bee larvae. Four so-called enterobacterial repetitive intergenic consensus (ERIC) genotypes of P. larvae exist with P. larvae genotypes ERIC I and ERIC II being responsible for disease outbreaks all over the world. Very few molecular data on the pathogen, on pathogenesis or on virulence factors exist. We now identified two genomic loci in P. larvae ERIC I coding for two binary AB toxins, Plx1 and Plx2. In silico analyses revealed that Plx1 is the third member of an enigmatic family of AB toxins so far only comprising MTX1 of Lysinibacillus sphaericus and pierisin-like toxins expressed by several butterflies. Plx2 is also remarkable because the A-domain is highly similar to C3 exoenzymes, which normally are single domain proteins, while the B-domain is homologous to B-domains of C2-toxins. We constructed P. larvae mutants lacking expression of Plx1, Plx2 or both toxins and demonstrated that these toxins are important virulence factors for P. larvae ERIC I. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

The role of TcdB and TccC subunits in secretion of the Photorhabdus Tcd toxin complex.

PubMed

Yang, Guowei; Waterfield, Nicholas R

2013-01-01

The Toxin Complex (TC) is a large multi-subunit toxin encoded by a range of bacterial pathogens. The best-characterized examples are from the insect pathogens Photorhabdus, Xenorhabdus and Yersinia. They consist of three large protein subunits, designated A, B and C that assemble in a 5∶1∶1 stoichiometry. Oral toxicity to a range of insects means that some have the potential to be developed as pest control technology. The three subunit proteins do not encode any recognisable export sequences and as such little progress has been made in understanding their secretion. We have developed heterologous TC production and secretion models in E. coli and used them to ascribe functions to different domains of the crucial B+C sub-complex. We have determined that the B and C subunits use a secretion mechanism that is either encoded by the proteins themselves or employ an as yet undefined system common to laboratory strains of E. coli. We demonstrate that both the N-terminal domains of the B and C subunits are required for secretion of the whole complex. We propose a model whereby the N-terminus of the C-subunit toxin exports the B+C sub-complex across the inner membrane while that of the B-subunit allows passage across the outer membrane. We also demonstrate that even in the absence of the B-subunit, that the C-subunit can also facilitate secretion of the larger A-subunit. The recognition of this novel export system is likely to be of importance to future protein secretion studies. Finally, the identification of homologues of B and C subunits in diverse bacterial pathogens, including Burkholderia and Pseudomonas, suggests that these toxins are likely to be important in a range of different hosts, including man.

A portable cell-based optical detection device for rapid detection of Listeria and Bacillus toxins

NASA Astrophysics Data System (ADS)

Banerjee, Pratik; Banada, Padmapriya P.; Rickus, Jenna L.; Morgan, Mark T.; Bhunia, Arun K.

2005-11-01

A mammalian cell-based optical biosensor was built to detect pathogenic Listeria and Bacillus species. This sensor measures the ability of the pathogens to infect and induce cytotoxicity on hybrid lymphocyte cell line (Ped-2E9) resulting in the release of alkaline phosphatase (ALP) that can be detected optically using a portable spectrophotometer. The Ped-2E9 cells were encapsulated in collagen gel matrices and grown in 48-well plates or in specially designed filtration tube units. Toxin preparations or bacterial cells were introduced and ALP release was assayed after 3-5 h. Pathogenic L. monocytogenes strains or the listeriolysin toxins preparation showed cytotoxicity ranging from 55% - 92%. Toxin preparations (~20 μg/ml) from B. cereus strains showed 24 - 98% cytotoxicity. In contrast, a non-pathogenic L. innocua (F4247) and a B. substilis induced only 2% and 8% cytotoxicity, respectively. This cell-based detection device demonstrates its ability to detect the presence of pathogenic Listeria and Bacillus species and can potentially be used onsite for food safety or in biosecurity application.

Evaluation of in vitro properties of di-tri-octahedral smectite on clostridial toxins and growth.

PubMed

Weese, J S; Cote, N M; deGannes, R V G

2003-11-01

Clostridial colitis and endotoxaemia of intestinal origin are significant causes of morbidity and mortality in horses. Intestinal adsorbents are available for treatment of these conditions; however, little information exists supporting their use. To evaluate the ability of di-tri-octahedral smectite to bind to Clostridium difficile toxins A and B, C. perfringens enterotoxin and endotoxin, inhibit clostridial growth and the actions of metronidazole in vitro. Clostridium difficile toxins, C. perfringens enterotoxin and endotoxin were mixed with serial dilutions of di-tri-octahedral smectite, then tested for the presence of clostridial toxins or endotoxin using commercial tests. Serial dilutions of smectite were tested for the ability to inhibit growth of C. perfringens in culture broth, and to interfere with the effect of metronidazole on growth of C. perfringens in culture broth. Clostridium difficile toxins A and B, and C. perfringens enterotoxin were completely bound at dilutions of 1:2 to 1:16. Partial binding of C. difficile toxins occurred at dilutions up to 1:256 while partial binding of C. perfringens enterotoxin occurred up to a dilution of 1:128. Greater than 99% binding of endotoxin occurred with dilutions 1:2 to 1:32. No inhibition of growth of C. difficile or C. perfringens was present at any dilution, and there was no effect on the action of metronidazole. Di-tri-octahedral smectite possesses the ability to bind C. difficile toxins A and B, C. perfringens enterotoxin and endotoxin in vivo while having no effect on bacterial growth or the action of metronidazole. In vivo studies are required to determine whether di-tri-octahedral smectite might be a useful adjunctive treatment of clostridial colitis and endotoxaemia in horses.

A Nanocoaxial-Based Electrochemical Sensor for the Detection of Cholera Toxin

NASA Astrophysics Data System (ADS)

Archibald, Michelle M.; Rizal, Binod; Connolly, Timothy; Burns, Michael J.; Naughton, Michael J.; Chiles, Thomas C.

2015-03-01

Sensitive, real-time detection of biomarkers is of critical importance for rapid and accurate diagnosis of disease for point of care (POC) technologies. Current methods do not allow for POC applications due to several limitations, including sophisticated instrumentation, high reagent consumption, limited multiplexing capability, and cost. Here, we report a nanocoaxial-based electrochemical sensor for the detection of bacterial toxins using an electrochemical enzyme-linked immunosorbent assay (ELISA) and differential pulse voltammetry (DPV). Proof-of-concept was demonstrated for the detection of cholera toxin (CT). The linear dynamic range of detection was 10 ng/ml - 1 μg/ml, and the limit of detection (LOD) was found to be 2 ng/ml. This level of sensitivity is comparable to the standard optical ELISA used widely in clinical applications. In addition to matching the detection profile of the standard ELISA, the nanocoaxial array provides a simple electrochemical readout and a miniaturized platform with multiplexing capabilities for the simultaneous detection of multiple biomarkers, giving the nanocoax a desirable advantage over the standard method towards POC applications. Sensitive, real-time detection of biomarkers is of critical importance for rapid and accurate diagnosis of disease for point of care (POC) technologies. Current methods do not allow for POC applications due to several limitations, including sophisticated instrumentation, high reagent consumption, limited multiplexing capability, and cost. Here, we report a nanocoaxial-based electrochemical sensor for the detection of bacterial toxins using an electrochemical enzyme-linked immunosorbent assay (ELISA) and differential pulse voltammetry (DPV). Proof-of-concept was demonstrated for the detection of cholera toxin (CT). The linear dynamic range of detection was 10 ng/ml - 1 μg/ml, and the limit of detection (LOD) was found to be 2 ng/ml. This level of sensitivity is comparable to the standard optical

Characterization of Alpha-Toxin hla Gene Variants, Alpha-Toxin Expression Levels, and Levels of Antibody to Alpha-Toxin in Hemodialysis and Postsurgical Patients with Staphylococcus aureus Bacteremia

PubMed Central

Wu, Yuling; Tabor, David E.; Mok, Hoyin; Sellman, Bret R.; Jenkins, Amy; Yu, Li; Jafri, Hasan S.; Rude, Thomas H.; Ruffin, Felicia; Schell, Wiley A.; Park, Lawrence P.; Yan, Qin; Thaden, Joshua T.; Messina, Julia A.; Esser, Mark T.

2014-01-01

Alpha-toxin is a major Staphylococcus aureus virulence factor. This study evaluated potential relationships between in vitro alpha-toxin expression of S. aureus bloodstream isolates, anti-alpha-toxin antibody in serum of patients with S. aureus bacteremia (SAB), and clinical outcomes in 100 hemodialysis and 100 postsurgical SAB patients. Isolates underwent spa typing and hla sequencing. Serum anti-alpha-toxin IgG and neutralizing antibody levels were measured by using an enzyme-linked immunosorbent assay and a red blood cell (RBC)-based hemolysis neutralization assay. Neutralization of alpha-toxin by an anti-alpha-toxin monoclonal antibody (MAb MEDI4893) was tested in an RBC-based lysis assay. Most isolates encoded hla (197/200; 98.5%) and expressed alpha-toxin (173/200; 86.5%). In vitro alpha-toxin levels were inversely associated with survival (cure, 2.19 μg/ml, versus failure, 1.09 μg/ml; P < 0.01). Both neutralizing (hemodialysis, 1.26 IU/ml, versus postsurgical, 0.95; P < 0.05) and IgG (hemodialysis, 1.94 IU/ml, versus postsurgical, 1.27; P < 0.05) antibody levels were higher in the hemodialysis population. Antibody levels were also significantly higher in patients infected with alpha-toxin-expressing S. aureus isolates (P < 0.05). Levels of both neutralizing antibodies and IgG were similar among patients who were cured and those not cured (failures). Sequence analysis of hla revealed 12 distinct hla genotypes, and all genotypic variants were susceptible to a neutralizing monoclonal antibody in clinical development (MEDI4893). These data demonstrate that alpha-toxin is highly conserved in clinical S. aureus isolates. Higher in vitro alpha-toxin levels were associated with a positive clinical outcome. Although patients infected with alpha-toxin-producing S. aureus exhibited higher anti-alpha-toxin antibody levels, these levels were not associated with a better clinical outcome in this study. PMID:25392350

Lymphocyte receptors for pertussis toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clark, C.G.; Armstrong, G.D.

1990-12-01

We have investigated human T-lymphocyte receptors for pertussis toxin by affinity isolation and photoaffinity labeling procedures. T lymphocytes were obtained from peripheral human blood, surface iodinated, and solubilized in Triton X-100. The iodinated mixture was then passed through pertussis toxin-agarose, and the fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of the fixed, dried gels revealed several bands in the pertussis toxin-bound fraction that were not observed in fractions obtained from histone or fetuin-agarose. Further investigations employed a photoaffinity labeling reagent, sulfosuccinimidyl 2-(p-azido-salicylamido)-1,3'-dithiopropionate, to identify pertussis toxin receptors in freshly isolated peripheral blood monocytic cells, T lymphocytes, andmore » Jurkat cells. In all three cell systems, the pertussis toxin affinity probe specifically labeled a single protein species with an apparent molecular weight of 70,000 that was not observed when the procedure was performed in the presence of excess unmodified pertussis toxin. A protein comparable in molecular weight to the one detected by the photoaffinity labeling technique was also observed among the species that bound to pertussis toxin-agarose. The results suggest that pertussis toxin may bind to a 70,000-Da receptor in human T lymphocytes.« less

Botulinum toxin: Bioweapon & magic drug

PubMed Central

Dhaked, Ram Kumar; Singh, Manglesh Kumar; Singh, Padma; Gupta, Pallavi

2010-01-01

Botulinum neurotoxins, causative agents of botulism in humans, are produced by Clostridium botulinum, an anaerobic spore-former Gram positive bacillus. Botulinum neurotoxin poses a major bioweapon threat because of its extreme potency and lethality; its ease of production, transport, and misuse; and the need for prolonged intensive care among affected persons. A single gram of crystalline toxin, evenly dispersed and inhaled, can kill more than one million people. The basis of the phenomenal potency of botulinum toxin is enzymatic; the toxin is a zinc proteinase that cleaves neuronal vesicle associated proteins responsible for acetylcholine release into the neuromuscular junction. As a military or terrorist weapon, botulinum toxin could be disseminated via aerosol or by contamination of water or food supplies, causing widespread casualties. A fascinating aspect of botulinum toxin research in recent years has been development of the most potent toxin into a molecule of significant therapeutic utility. It is the first biological toxin which is licensed for treatment of human diseases. In the late 1980s, Canada approved use of the toxin to treat strabismus, in 2001 in the removal of facial wrinkles and in 2002, the FDA in the United States followed suit. The present review focuses on both warfare potential and medical uses of botulinum neurotoxin. PMID:21149997

Botulinum toxin: bioweapon & magic drug.

PubMed

Dhaked, Ram Kumar; Singh, Manglesh Kumar; Singh, Padma; Gupta, Pallavi

2010-11-01

Botulinum neurotoxins, causative agents of botulism in humans, are produced by Clostridium botulinum, an anaerobic spore-former Gram positive bacillus. Botulinum neurotoxin poses a major bioweapon threat because of its extreme potency and lethality; its ease of production, transport, and misuse; and the need for prolonged intensive care among affected persons. A single gram of crystalline toxin, evenly dispersed and inhaled, can kill more than one million people. The basis of the phenomenal potency of botulinum toxin is enzymatic; the toxin is a zinc proteinase that cleaves neuronal vesicle associated proteins responsible for acetylcholine release into the neuromuscular junction. As a military or terrorist weapon, botulinum toxin could be disseminated via aerosol or by contamination of water or food supplies, causing widespread casualties. A fascinating aspect of botulinum toxin research in recent years has been development of the most potent toxin into a molecule of significant therapeutic utility . It is the first biological toxin which is licensed for treatment of human diseases. In the late 1980s, Canada approved use of the toxin to treat strabismus, in 2001 in the removal of facial wrinkles and in 2002, the FDA in the United States followed suit. The present review focuses on both warfare potential and medical uses of botulinum neurotoxin.

Topical botulinum toxin.

PubMed

Collins, Ashley; Nasir, Adnan

2010-03-01

Nanotechnology is a rapidly growing discipline that capitalizes on the unique properties of matter engineered on the nanoscale. Vehicles incorporating nanotechnology have led to great strides in drug delivery, allowing for increased active ingredient stability, bioavailability, and site-specific targeting. Botulinum toxin has historically been used for the correction of neurological and neuromuscular disorders, such as torticollis, blepharospasm, and strabismus. Recent dermatological indications have been for the management of axillary hyperhydrosis and facial rhytides. Traditional methods of botulinum toxin delivery have been needle-based. These have been associated with increased pain and cost. Newer methods of botulinum toxin formulation have yielded topical preparations that are bioactive in small pilot clinical studies. While there are some risks associated with topical delivery, the refinement and standardization of delivery systems and techniques for the topical administration of botulinum toxin using nanotechnology is anticipated in the near future.

Down-regulation of annexin A1 in the urothelium decreases cell survival after bacterial toxin exposure.

PubMed

Monastyrskaya, Katia; Babiychuk, Eduard B; Draeger, Annette; Burkhard, Fiona C

2013-07-01

We examined the role of annexins in bladder urothelium. We characterized expression and distribution in normal bladders, biopsies from patients with bladder pain syndrome, cultured human urothelium and urothelial TEU-2 cells. Annexin expression in bladder layers was analyzed by quantitative reverse transcriptase-polymerase chain reaction and immunofluorescence. We assessed cell survival after exposure to the pore forming bacterial toxin streptolysin O by microscopy and alamarBlue® assay. Bladder dome biopsies were obtained from 8 asymptomatic controls and 28 patients with symptoms of bladder pain syndrome. Annexin A1, A2, A5 and A6 were differentially distributed in bladder layers. Annexin A6 was abundant in detrusor smooth muscle and low in urothelium, while annexin A1 was the highest in urothelium. Annexin A2 was localized to the lateral membrane of umbrella cells but excluded from tight junctions. TEU-2 cell differentiation caused up-regulation of annexin A1 and A2 and down-regulation of annexin A6 mRNA. Mature urothelium dedifferentiation during culture caused the opposite effect, decreasing annexin A1 and increasing annexin A6. Annexin A2 influenced TEU-2 cell epithelial permeability. siRNA mediated knockdown of annexin A1 in TEU-2 cells caused significantly decreased cell survival after streptolysin O exposure. Annexin A1 was significantly reduced in biopsies from patients with bladder pain syndrome. Several annexins are expressed in human bladder and TEU-2 cells, in which levels are regulated during urothelial differentiation. Annexin A1 down-regulation in patients with bladder pain syndrome might decrease cell survival and contribute to compromised urothelial function. Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Detecting and discriminating among Shiga toxins

USDA-ARS?s Scientific Manuscript database

The virulence associated with Shiga toxin producing Escherichia coli (STEC) infections is from the Shiga toxins produced by the E. coli strain. Although Shiga toxins are associated with E. coli, the expression of the toxins is actually controlled by a temperate lambdoid phage that infects the host. ...

Botulinum Toxin and Gastrointestinal Tract Disorders

PubMed Central

Weiser, Kirsten; Kennedy, Abigail

2008-01-01

The history of botulinum toxin is fascinating. First recognized as the cause of botulism nearly 200 years ago, it was originally feared as a deadly poison. Over the last 30 years, however, botulinum toxin has been transformed into a readily available medication used to treat a variety of medical disorders. Interest in the use of botulinum toxin has been particularly strong for patients with spastic smooth muscle disorders of the gastrointestinal tract. Patients with achalasia, diffuse esophageal spasm, gastroparesis, sphincter of Oddi dysfunction, and anal fissures have all been treated with botulinum toxin injections, often with impressive results. However, not all patients respond to botulinum toxin therapy, and large randomized controlled trials are lacking for many conditions commonly treated with botulinum toxin. This paper reviews the history, microbiology, and pharmacology of botulinum toxin, discusses its mechanism of action, and then presents recent evidence from the literature regarding the use of botulinum toxin for the treatment of a variety of gastrointestinal tract disorders. PMID:21960915

Structure-based receptor MIMICS targeted against bacterial superantigen toxins

DOEpatents

Gupta, Goutam [Santa Fe, NM; Hong-Geller, Elizabeth [Los Alamos, NM; Shiflett, Patrick R [Los Alamos, NM; Lehnert, Nancy M [Albuquerque, NM

2009-08-18

The invention provides therapeutic compositions useful in the treatment of bacterial superantigen mediated conditions, such as Toxic Shock Syndrome. The compositions comprise genetically engineered bifunctional polypeptides containing a specific T-cell receptor binding domain and a specific MHC class II receptor binding domain, each targeting non-overlapping epitopes on a superantigen molecule against which they are designed. The anti-superantigen "receptor mimetics" or "chimeras" are rationally designed to recreate the modality of superantigen binding directly to both the TCR and the MHC-II receptor, and are capable of acting as decoys for superantigen binding, effectively out-competing the host T-cell and MHC-II receptors, the natural host receptors.

Pore-forming activity of clostridial binary toxins.

PubMed

Knapp, O; Benz, R; Popoff, M R

2016-03-01

Clostridial binary toxins (Clostridium perfringens Iota toxin, Clostridium difficile transferase, Clostridium spiroforme toxin, Clostridium botulinum C2 toxin) as Bacillus binary toxins, including Bacillus anthracis toxins consist of two independent proteins, one being the binding component which mediates the internalization into cell of the intracellularly active component. Clostridial binary toxins induce actin cytoskeleton disorganization through mono-ADP-ribosylation of globular actin and are responsible for enteric diseases. Clostridial and Bacillus binary toxins share structurally and functionally related binding components which recognize specific cell receptors, oligomerize, form pores in endocytic vesicle membrane, and mediate the transport of the enzymatic component into the cytosol. Binding components retain the global structure of pore-forming toxins (PFTs) from the cholesterol-dependent cytotoxin family such as perfringolysin. However, their pore-forming activity notably that of clostridial binding components is more related to that of heptameric PFT family including aerolysin and C. perfringens epsilon toxin. This review focuses upon pore-forming activity of clostridial binary toxins compared to other related PFTs. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale. Copyright © 2015 Elsevier B.V. All rights reserved.

Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control

PubMed Central

Bravo, Alejandra; Gill, Sarjeet S.; Soberón, Mario

2007-01-01

Bacillus thuringiensis Cry and Cyt protein families are a diverse group of proteins with activity against insects of different orders - Lepidoptera, Coleoptera, Diptera and also against other invertebrates such as nematodes. Their primary action is to lyse midgut epithelial cells by inserting into the target membrane and forming pores. Among this group of proteins, members of the 3-Domain Cry family are used worldwide for insect control, and their mode of action has been characterized in some detail. Phylogenetic analyses established that the diversity of the 3-Domain Cry family evolved by the independent evolution of the three domains and by swapping of domain III among toxins. Like other pore-forming toxins (PFT) that affect mammals, Cry toxins interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in the formation of a pre-pore oligomeric structure that is insertion competent. In contrast, Cyt toxins directly interact with membrane lipids and insert into the membrane. Recent evidence suggests that Cyt synergize or overcome resistance to mosquitocidal-Cry proteins by functioning as a Cry-membrane bound receptor. In this review we summarize recent findings on the mode of action of Cry and Cyt toxins, and compare them to the mode of action of other bacterial PFT. Also, we discuss their use in the control of agricultural insect pests and insect vectors of human diseases. PMID:17198720

Identification of a functional toxin-antitoxin system located in the genomic island PYG1 of piezophilic hyperthermophilic archaeon Pyrococcus yayanosii.

PubMed

Li, Zhen; Song, Qinghao; Wang, Yinzhao; Xiao, Xiang; Xu, Jun

2018-05-01

Toxin-antitoxin (TA) system is bacterial or archaeal genetic module consisting of toxin and antitoxin gene that be organized as a bicistronic operon. TA system could elicit programmed cell death, which is supposed to play important roles for the survival of prokaryotic population under various physiological stress conditions. The phage abortive infection system (AbiE family) belongs to bacterial type IV TA system. However, no archaeal AbiE family TA system has been reported so far. In this study, a putative AbiE TA system (PygAT), which is located in a genomic island PYG1 in the chromosome of Pyrococcus yayanosii CH1, was identified and characterized. In Escherichia coli, overexpression of the toxin gene pygT inhibited its growth while the toxic effect can be suppressed by introducing the antitoxin gene pygA in the same cell. PygAT also enhances the stability of shuttle plasmids with archaeal plasmid replication protein Rep75 in E. coli. In P. yayanosii, disruption of antitoxin gene pygA cause a significantly growth delayed under high hydrostatic pressure (HHP). The antitoxin protein PygA can specifically bind to the PygAT promoter region and regulate the transcription of pygT gene in vivo. These results show that PygAT is a functional TA system in P. yayanosii, and also may play a role in the adaptation to HHP environment.

Botulinum toxin injection - larynx

MedlinePlus

Injection laryngoplasty; Botox - larynx: spasmodic dysphonia-BTX; Essential voice tremor (EVT)-btx; Glottic insufficiency; Percutaneous electromyography - guided botulinum toxin treatment; Percutaneous indirect laryngoscopy - guided botulinum toxin treatment; ...

Detection of Clostridium difficile toxins from the small intestine and cecum of rabbits with naturally acquired enterotoxemia.

PubMed

Perkins, S E; Fox, J G; Taylor, N S; Green, D L; Lipman, N S

1995-08-01

Four specific-pathogen-free rabbits with anorexia died peracutely; decreased fecal output, nasal exudate, and labored breathing were the only other clinical abnormalities observed in two of the rabbits before death. The animals, three juveniles and one adult, were on a standard polyclonal antibody production regimen and had received immunizations approximately 2 weeks before presentation. External examination revealed distended abdomen and perineal fecal staining. At necropsy the small intestine was distended with fluid, and the cecum was distended with chyme. The small intestines and cecum had marked serosal hyperemia. Anaerobic bacterial culture techniques were used to isolate Clostridium difficile from the small intestine (3/4) and cecum (2/4). In all cases C. difficile toxin B was detected at high titers (10(2) to > 10(5)) in the small intestine by cytotoxicity assay with HeLa 229 cell culture. In two of the four rabbits C. difficile was isolated, and cytotoxin titers were detected at 10(1) and 10(4) in the cecum of affected rabbits. Toxin B was neutralized with C. sordellii antiserum but not C. spiroforme antiserum. In addition, toxin A was detected in each of the cytotoxin B-positive samples by a commercial toxin A enzyme immunosorbent assay. In vitro production of toxins A and B was detected from each culture isolate after incubation in chopped meat broth. These cases are noteworthy because spontaneous (nonantibiotic-associated) C. difficile enterotoxemia has not been previously reported in rabbits. Also the toxins of clostridial organisms are usually documented in the cecum, not the small intestine, of rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)

The MqsRA Toxin-Antitoxin System from Xylella fastidiosa Plays a Key Role in Bacterial Fitness, Pathogenicity, and Persister Cell Formation.

PubMed

Merfa, Marcus V; Niza, Bárbara; Takita, Marco A; De Souza, Alessandra A

2016-01-01

Through the formation of persister cells, bacteria exhibit tolerance to multidrug and other environmental stresses without undergoing genetic changes. The toxin-antitoxin (TA) systems are involved in the formation of persister cells because they are able to induce cell dormancy. Among the TA systems, the MqsRA system has been observed to be highly induced in persister cells of Xylella fastidiosa (causal agent of citrus variegated chlorosis-CVC) activated by copper stress, and has been described in Escherichia coli as related to the formation of persister cells and biofilms. Thus, we evaluated the role of this TA system in X. fastidiosa by overexpressing the MqsR toxin, and verified that the toxin positively regulated biofilm formation and negatively cell movement, resulting in reduced pathogenicity in citrus plants. The overexpression of MqsR also increased the formation of persister cells under copper stress. Analysis of the gene and protein expression showed that this system likely has an autoregulation mechanism to express the toxin and antitoxin in the most beneficial ratio for the cell to oppose stress. Our results suggest that this TA system plays a key role in the adaptation and survival of X. fastidiosa and reveal new insights into the physiology of phytopathogen-host interactions.

The MqsRA Toxin-Antitoxin System from Xylella fastidiosa Plays a Key Role in Bacterial Fitness, Pathogenicity, and Persister Cell Formation

PubMed Central

Merfa, Marcus V.; Niza, Bárbara; Takita, Marco A.; De Souza, Alessandra A.

2016-01-01

Through the formation of persister cells, bacteria exhibit tolerance to multidrug and other environmental stresses without undergoing genetic changes. The toxin-antitoxin (TA) systems are involved in the formation of persister cells because they are able to induce cell dormancy. Among the TA systems, the MqsRA system has been observed to be highly induced in persister cells of Xylella fastidiosa (causal agent of citrus variegated chlorosis—CVC) activated by copper stress, and has been described in Escherichia coli as related to the formation of persister cells and biofilms. Thus, we evaluated the role of this TA system in X. fastidiosa by overexpressing the MqsR toxin, and verified that the toxin positively regulated biofilm formation and negatively cell movement, resulting in reduced pathogenicity in citrus plants. The overexpression of MqsR also increased the formation of persister cells under copper stress. Analysis of the gene and protein expression showed that this system likely has an autoregulation mechanism to express the toxin and antitoxin in the most beneficial ratio for the cell to oppose stress. Our results suggest that this TA system plays a key role in the adaptation and survival of X. fastidiosa and reveal new insights into the physiology of phytopathogen-host interactions. PMID:27375608

A polyclonal antibody based immunoassay detects seven subtypes of Shiga toxin 2 produced by escherichia coli in human and environmental samples

USDA-ARS?s Scientific Manuscript database

The increase of outbreaks and illnesses linked to Shiga toxin-producing Escherichia coli (STEC) has necessitated the development of effective detection methods for these pathogens in various matrices. The best way to determine if a bacterial strain is a STEC is to examine the production of Shiga tox...

Influence of the Vaginal Microbiota on Toxic Shock Syndrome Toxin 1 Production by Staphylococcus aureus

PubMed Central

MacPhee, Roderick A.; Miller, Wayne L.; Gloor, Gregory B.; McCormick, John K.; Hammond, Jo-Anne; Burton, Jeremy P.

2013-01-01

Menstrual toxic shock syndrome (TSS) is a serious illness that afflicts women of premenopausal age worldwide and arises from vaginal infection by Staphylococcus aureus and concurrent production of toxic shock syndrome toxin-1 (TSST-1). Studies have illustrated the capacity of lactobacilli to reduce S. aureus virulence, including the capacity to suppress TSST-1. We hypothesized that an aberrant microbiota characteristic of pathogenic bacteria would induce the increased production of TSST-1 and that this might represent a risk factor for the development of TSS. A S. aureus TSST-1 reporter strain was grown in the presence of vaginal swab contents collected from women with a clinically healthy vaginal status, women with an intermediate status, and those diagnosed with bacterial vaginosis (BV). Bacterial supernatant challenge assays were also performed to test the effects of aerobic vaginitis (AV)-associated pathogens toward TSST-1 production. While clinical samples from healthy and BV women suppressed toxin production, in vitro studies demonstrated that Streptococcus agalactiae and Enterococcus spp. significantly induced TSST-1 production, while some Lactobacillus spp. suppressed it. The findings suggest that women colonized by S. aureus and with AV, but not BV, may be more susceptible to menstrual TSS and would most benefit from prophylactic treatment. PMID:23315732

Influence of the vaginal microbiota on toxic shock syndrome toxin 1 production by Staphylococcus aureus.

PubMed

MacPhee, Roderick A; Miller, Wayne L; Gloor, Gregory B; McCormick, John K; Hammond, Jo-Anne; Burton, Jeremy P; Reid, Gregor

2013-03-01

Menstrual toxic shock syndrome (TSS) is a serious illness that afflicts women of premenopausal age worldwide and arises from vaginal infection by Staphylococcus aureus and concurrent production of toxic shock syndrome toxin-1 (TSST-1). Studies have illustrated the capacity of lactobacilli to reduce S. aureus virulence, including the capacity to suppress TSST-1. We hypothesized that an aberrant microbiota characteristic of pathogenic bacteria would induce the increased production of TSST-1 and that this might represent a risk factor for the development of TSS. A S. aureus TSST-1 reporter strain was grown in the presence of vaginal swab contents collected from women with a clinically healthy vaginal status, women with an intermediate status, and those diagnosed with bacterial vaginosis (BV). Bacterial supernatant challenge assays were also performed to test the effects of aerobic vaginitis (AV)-associated pathogens toward TSST-1 production. While clinical samples from healthy and BV women suppressed toxin production, in vitro studies demonstrated that Streptococcus agalactiae and Enterococcus spp. significantly induced TSST-1 production, while some Lactobacillus spp. suppressed it. The findings suggest that women colonized by S. aureus and with AV, but not BV, may be more susceptible to menstrual TSS and would most benefit from prophylactic treatment.

[Today's threat of ricin toxin].

PubMed

From, Sławomir; Płusa, Tadeusz

2015-09-01

Since the late 70s of the last century there were more than 700 incidents related to the use of the ricin toxin. For this reason, CDC (Center of Disease Control and Prevention) recognized toxin as a biological weapon category B. The lethal dose of ricin toxin after parenteral administration is 0.0001 mg/kg and after oral administration 0.2 mg. The first symptoms of poisoning occur within a few hours after application of toxin as a nausea, vomiting and abdominal pain. In the final stage there are observed: cardiac arrhythmia, collapse and symptoms suggestive of involvement of the central nervous system. Stage immediately preceding death is a state of coma. The ricin toxin is still the substance against which action has no optimal antidote. Developed a vaccine called RiVax is waiting for its registration. It should be pointed out that the availability of a ricin toxin makes it possible to use it for real bioterrorists. © 2015 MEDPRESS.

Purification of the Clostridium spiroforme binary toxin and activity of the toxin on HEp-2 cells.

PubMed

Popoff, M R; Milward, F W; Bancillon, B; Boquet, P

1989-08-01

The two components Sa (Mr, 44,000) and Sb (Mr, 92,000) of Clostridium spiroforme toxin were identified and characterized. Serological data permitted the identification of two groups of actin ADP-ribosylating clostridial toxins. The first consists of only C. botulinum C2. The second group includes spiroforme toxin, iota toxin of C. perfringens E, and an enzyme called CDT found in one strain of C. difficile, antibodies against which cross-react with all of the members of both groups. C. spiroforme toxin acted on cells by disrupting microfilaments by ADP-ribosylation of G actin. Toxicity was not blocked by 10 or 20 mM ammonium chloride and was only moderately inhibited by 30 mM NH4Cl. Inhibition of coated-pit formation in HEp-2 cells by potassium depletion strongly protected against the effect of C. spiroforme toxin. Toxicity was not blocked by incubation of HEp-2 cells and spiroforme toxin at 15 degrees C. These results suggest that this new binary toxin enters cells via the coated-pit-coated-vesicle pathway and might reach the cytoplasm at the same time as or before transfer to early endosomes.

Purification of the Clostridium spiroforme binary toxin and activity of the toxin on HEp-2 cells.

PubMed Central

Popoff, M R; Milward, F W; Bancillon, B; Boquet, P

1989-01-01

The two components Sa (Mr, 44,000) and Sb (Mr, 92,000) of Clostridium spiroforme toxin were identified and characterized. Serological data permitted the identification of two groups of actin ADP-ribosylating clostridial toxins. The first consists of only C. botulinum C2. The second group includes spiroforme toxin, iota toxin of C. perfringens E, and an enzyme called CDT found in one strain of C. difficile, antibodies against which cross-react with all of the members of both groups. C. spiroforme toxin acted on cells by disrupting microfilaments by ADP-ribosylation of G actin. Toxicity was not blocked by 10 or 20 mM ammonium chloride and was only moderately inhibited by 30 mM NH4Cl. Inhibition of coated-pit formation in HEp-2 cells by potassium depletion strongly protected against the effect of C. spiroforme toxin. Toxicity was not blocked by incubation of HEp-2 cells and spiroforme toxin at 15 degrees C. These results suggest that this new binary toxin enters cells via the coated-pit-coated-vesicle pathway and might reach the cytoplasm at the same time as or before transfer to early endosomes. Images PMID:2545625

Challenging the roles of CD44 and lipolysis stimulated lipoprotein receptor in conveying Clostridium perfringens iota toxin cytotoxicity in breast cancer

PubMed Central

2014-01-01

Background Translational exploration of bacterial toxins has come to the forefront of research given their potential as a chemotherapeutic tool. Studies in select tissues have demonstrated that Clostridium perfringens iota toxin binds to CD44 and lipolysis stimulated lipoprotein receptor (LSR) cell-surface proteins. We recently demonstrated that LSR expression correlates with estrogen receptor positive breast cancers and that LSR signaling directs aggressive, tumor-initiating cell behaviors. Herein, we identify the mechanisms of iota toxin cytotoxicity in a tissue-specific, breast cancer model with the ultimate goal of laying the foundation for using iota toxin as a targeted breast cancer therapy. Methods In vitro model systems were used to determine the cytotoxic effect of iota toxin on breast cancer intrinsic subtypes. The use of overexpression and knockdown technologies confirmed the roles of LSR and CD44 in regulating iota toxin endocytosis and induction of cell death. Lastly, cytotoxicity assays were used to demonstrate the effect of iota toxin on a validated set of tamoxifen resistant breast cancer cell lines. Results Treatment of 14 breast cancer cell lines revealed that LSR+/CD44- lines were highly sensitive, LSR+/CD44+ lines were slightly sensitive, and LSR-/CD44+ lines were resistant to iota cytotoxicity. Reduction in LSR expression resulted in a significant decrease in toxin sensitivity; however, overexpression of CD44 conveyed toxin resistance. CD44 overexpression was correlated with decreased toxin-stimulated lysosome formation and decreased cytosolic levels of iota toxin. These findings indicated that expression of CD44 drives iota toxin resistance through inhibition of endocytosis in breast cancer cells, a role not previously defined for CD44. Moreover, tamoxifen-resistant breast cancer cells exhibited robust expression of LSR and were highly sensitive to iota-induced cytotoxicity. Conclusions Collectively, these data are the first to show that iota

Structure–function analyses of a pertussis-like toxin from pathogenic Escherichia coli reveal a distinct mechanism of inhibition of trimeric G-proteins

PubMed Central

Littler, Dene R.; Ang, Sheng Y.; Moriel, Danilo G.; Kocan, Martina; Kleifeld, Oded; Johnson, Matthew D.; Tran, Mai T.; Paton, Adrienne W.; Paton, James C.; Summers, Roger J.; Schembri, Mark A.; Rossjohn, Jamie; Beddoe, Travis

2017-01-01

Pertussis-like toxins are secreted by several bacterial pathogens during infection. They belong to the AB5 virulence factors, which bind to glycans on host cell membranes for internalization. Host cell recognition and internalization are mediated by toxin B subunits sharing a unique pentameric ring-like assembly. Although the role of pertussis toxin in whooping cough is well-established, pertussis-like toxins produced by other bacteria are less studied, and their mechanisms of action are unclear. Here, we report that some extra-intestinal Escherichia coli pathogens (i.e. those that reside in the gut but can spread to other bodily locations) encode a pertussis-like toxin that inhibits mammalian cell growth in vitro. We found that this protein, EcPlt, is related to toxins produced by both nontyphoidal and typhoidal Salmonella serovars. Pertussis-like toxins are secreted as disulfide-bonded heterohexamers in which the catalytic ADP-ribosyltransferase subunit is activated when exposed to the reducing environment in mammalian cells. We found here that the reduced EcPlt exhibits large structural rearrangements associated with its activation. We noted that inhibitory residues tethered within the NAD+-binding site by an intramolecular disulfide in the oxidized state dissociate upon the reduction and enable loop restructuring to form the nucleotide-binding site. Surprisingly, although pertussis toxin targets a cysteine residue within the α subunit of inhibitory trimeric G-proteins, we observed that activated EcPlt toxin modifies a proximal lysine/asparagine residue instead. In conclusion, our results reveal the molecular mechanism underpinning activation of pertussis-like toxins, and we also identified differences in host target specificity. PMID:28663369

The SXT conjugative element and linear prophage N15 encode toxin-antitoxin-stabilizing systems homologous to the tad-ata module of the Paracoccus aminophilus plasmid pAMI2.

PubMed

Dziewit, Lukasz; Jazurek, Magdalena; Drewniak, Lukasz; Baj, Jadwiga; Bartosik, Dariusz

2007-03-01

A group of proteic toxin-antitoxin (TA) cassettes whose representatives are widely distributed among bacterial genomes has been identified. These cassettes occur in chromosomes, plasmids, bacteriophages, and noncomposite transposons, as well as in the SXT conjugative element of Vibrio cholerae. The following four homologous loci were subjected to detailed comparative studies: (i) tad-ata from plasmid pAMI2 of Paracoccus aminophilus (the prototype of this group), (ii) gp49-gp48 from the linear bacteriophage N15 of Escherichia coli, (iii) s045-s044 from SXT, and (iv) Z3230-Z3231 from the genomic island of enterohemorrhagic Escherichia coli O157:H7 strain EDL933. Functional analysis revealed that all but one of these loci (Z3230-Z3231) are able to stabilize heterologous replicons, although the host ranges varied. The TA cassettes analyzed have the following common features: (i) the toxins are encoded by the first gene of each operon; (ii) the antitoxins contain a predicted helix-turn-helix motif of the XRE family; and (iii) the cassettes have two promoters that are different strengths, one which is located upstream of the toxin gene and one which is located upstream of the antitoxin gene. All four toxins tested are functional in E. coli; overexpression of the toxins (in the absence of antitoxin) results in a bacteriostatic effect manifested by elongation of bacterial cells and growth arrest. The toxins have various effects on cell viability, which suggests that they may recognize different intracellular targets. Preliminary data suggest that different cellular proteases are involved in degradation of antitoxins encoded by the loci analyzed.

Clostridium perfringens type A–E toxin plasmids

PubMed Central

Freedman, John C.; Theoret, James R.; Wisniewski, Jessica A.; Uzal, Francisco A.; Rood, Julian I.; McClane, Bruce A.

2014-01-01

Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell. PMID:25283728

THE PRODUCTION OF DIPHTHERIA TOXIN

PubMed Central

Park, W. H.; Williams, A. W.

1896-01-01

Toxin of sufficient strength to kill a 400-gramme guinea-pig in three days and a half in a dose of 0·cubic centimetre developed in suitable bouillon, contained in ordinary Erlenmeyer flasks, within a period of twenty-four hours. In such boullon the toxin reached its greatest strength in from four to seven days (0·005 cubic centimetre killing a 500-gramme guinea-pig in three days). This period of time covered that of the greatest growth of the bacilli, as shown both by the appearance of the culture and by the number of colonies developing an agar plates. The bodies of the diphtheria bacili did not at any time contain toxin in cosiderable amounts. The type of growth of the bacili and the rapidity and extent of the production of toxin depended more on the reaction of the bouillon than upon any other single factor. The best results were obtained in bouillon which, after being neutralized to litmus, had about seven cubic centimetres of normal soda solution added to each litre. An excessive amount of either acid or alkali prevented the development of toxin. Strong toxin was produced in bouillon containing peptone ranging from one to ten per cent. The strength of toxin averaged greater in the two and four-per-cent peptone solutions than in the one-percent. When the stage of acid reaction was brief and the degree of acidity probably slight, strong toxin developed while the culture bouillon was still acid; but when the stage of acid reaction was prolonged, little if any toxin was produced until just before the fluid became alkaline. Glucose is deleterious to the growth of the diphtheria bacillus and to the production of toxin when it is present in sufficient amounts to cause by its disintegration too great a degree of acidity in the fluid culture. When the acid resulting from decomposition of glucose is neutralized by the addition of alkali the diphtheria bacilus again grows abundantly. Glucose is not present, at least as a rule, in sufficient amounts in the meat as

[Intoxication of botulinum toxin].

PubMed

Chudzicka, Aleksandra

2015-09-01

Botulinum toxin is an egzotoxin produced by Gram positive bacteria Clostridium botulinum. It is among the most potent toxins known. The 3 main clinical presentations of botulism are as follows: foodborne botulism, infant botulism and wound botulism. The main symptom of intoxication is flat muscles paralysis. The treatment is supportive care and administration of antitoxin. In prevention the correct preparing of canned food is most important. Botulinum toxin is accepted as a biological weapon. © 2015 MEDPRESS.

The YopJ superfamily of type III efforts in plant-associated bacteria

USDA-ARS?s Scientific Manuscript database

Bacterial pathogens employ the type III secretion system to secrete and translocate effector proteins into their hosts. The primary function of these effector proteins is believed to be the suppression of host defense responses or innate immunity. However, some effector proteins may be recognized by...

Active Shiga-Like Toxin Produced by Some Aeromonas spp., Isolated in Mexico City.

PubMed

Palma-Martínez, Ingrid; Guerrero-Mandujano, Andrea; Ruiz-Ruiz, Manuel J; Hernández-Cortez, Cecilia; Molina-López, José; Bocanegra-García, Virgilio; Castro-Escarpulli, Graciela

2016-01-01

Shiga-like toxins (Stx) represent a group of bacterial toxins involved in human and animal diseases. Stx is produced by enterohemorrhagic Escherichia coli, Shigella dysenteriae type 1, Citrobacter freundii , and Aeromonas spp.; Stx is an important cause of bloody diarrhea and hemolytic uremic syndrome (HUS). The aim of this study was to identify the stx 1 /stx 2 genes in clinical strains and outer membrane vesicles (OMVs) of Aeromonas spp., 66 strains were isolated from children who live in Mexico City, and Stx effects were evaluated in Vero cell cultures. The capacity to express active Stx1 and Stx2 toxins was determined in Vero cell cultures and the concentration of Stx was evaluated by 50% lethal dose (LD 50 ) assays, observing inhibition of damaged cells by specific monoclonal antibodies. The results obtained in this study support the hypothesis that the stx gene is another putative virulence factor of Aeromonas , and since this gene can be transferred horizontally through OMVs this genus should be included as a possible causal agents of gastroenteritis and it should be reported as part of standard health surveillance procedures. Furthermore, these results indicate that the Aeromonas genus might be a potential causative agent of HUS.

Active Shiga-Like Toxin Produced by Some Aeromonas spp., Isolated in Mexico City

PubMed Central

Palma-Martínez, Ingrid; Guerrero-Mandujano, Andrea; Ruiz-Ruiz, Manuel J.; Hernández-Cortez, Cecilia; Molina-López, José; Bocanegra-García, Virgilio; Castro-Escarpulli, Graciela

2016-01-01

Shiga-like toxins (Stx) represent a group of bacterial toxins involved in human and animal diseases. Stx is produced by enterohemorrhagic Escherichia coli, Shigella dysenteriae type 1, Citrobacter freundii, and Aeromonas spp.; Stx is an important cause of bloody diarrhea and hemolytic uremic syndrome (HUS). The aim of this study was to identify the stx1/stx2 genes in clinical strains and outer membrane vesicles (OMVs) of Aeromonas spp., 66 strains were isolated from children who live in Mexico City, and Stx effects were evaluated in Vero cell cultures. The capacity to express active Stx1 and Stx2 toxins was determined in Vero cell cultures and the concentration of Stx was evaluated by 50% lethal dose (LD50) assays, observing inhibition of damaged cells by specific monoclonal antibodies. The results obtained in this study support the hypothesis that the stx gene is another putative virulence factor of Aeromonas, and since this gene can be transferred horizontally through OMVs this genus should be included as a possible causal agents of gastroenteritis and it should be reported as part of standard health surveillance procedures. Furthermore, these results indicate that the Aeromonas genus might be a potential causative agent of HUS. PMID:27725813

Why do we study animal toxins?

PubMed Central

ZHANG, Yun

2015-01-01

Venom (toxins) is an important trait evolved along the evolutionary tree of animals. Our knowledges on venoms, such as their origins and loss, the biological relevance and the coevolutionary patterns with other organisms are greatly helpful in understanding many fundamental biological questions, i.e., the environmental adaptation and survival competition, the evolution shaped development and balance of venoms, and the sophisticated correlations among venom, immunity, body power, intelligence, their genetic basis, inherent association, as well as the cost-benefit and trade-offs of biological economy. Lethal animal envenomation can be found worldwide. However, from foe to friend, toxin studies have led lots of important discoveries and exciting avenues in deciphering and fighting human diseases, including the works awarded the Nobel Prize and lots of key clinic therapeutics. According to our survey, so far, only less than 0.1% of the toxins of the venomous animals in China have been explored. We emphasize on the similarities shared by venom and immune systems, as well as the studies of toxin knowledge-based physiological toxin-like proteins/peptides (TLPs). We propose the natural pairing hypothesis. Evolution links toxins with humans. Our mission is to find out the right natural pairings and interactions of our body elements with toxins, and with endogenous toxin-like molecules. Although, in nature, toxins may endanger human lives, but from a philosophical point of view, knowing them well is an effective way to better understand ourselves. So, this is why we study toxins. PMID:26228472

Bordetella Adenylate Cyclase-Hemolysin Toxins

PubMed Central

Guiso, Nicole

2017-01-01

Adenylate cyclase-hemolysin toxin is secreted and produced by three classical species of the genus Bordetella: Bordetella pertussis, B. parapertussis and B. bronchiseptica. This toxin has several properties such as: (i) adenylate cyclase activity, enhanced after interaction with the eukaryotic protein, calmodulin; (ii) a pore-forming activity; (iii) an invasive activity. It plays an important role in the pathogenesis of these Bordetella species responsible for whooping cough in humans or persistent respiratory infections in mammals, by modulating host immune responses. In contrast with other Bordetella toxins or adhesins, lack of (or very low polymorphism) is observed in the structural gene encoding this toxin, supporting its importance as well as a potential role as a vaccine antigen against whooping cough. In this article, an overview of the investigations undertaken on this toxin is presented. PMID:28892012

Pore-forming toxins in Cnidaria.

PubMed

Podobnik, Marjetka; Anderluh, Gregor

2017-12-01

The ancient phylum of Cnidaria contains many aquatic species with peculiar lifestyle. In order to survive, these organisms have evolved attack and defense mechanisms that are enabled by specialized cells and highly developed venoms. Pore-forming toxins are an important part of their venomous arsenal. Along some other types, the most representative are examples of four protein families that are commonly found in other kingdoms of life: actinoporins, Cry-like proteins, aerolysin-like toxins and MACPF/CDC toxins. Some of the homologues of pore-forming toxins may serve other functions, such as in food digestion, development and response against pathogenic organisms. Due to their interesting physico-chemical properties, the cnidarian pore-forming toxins may also serve as tools in medical research and nanobiotechnological applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Temperate bacterial viruses as double-edged swords in bacterial warfare.

PubMed

Gama, João Alves; Reis, Ana Maria; Domingues, Iolanda; Mendes-Soares, Helena; Matos, Ana Margarida; Dionisio, Francisco

2013-01-01

It has been argued that bacterial cells may use their temperate viruses as biological weapons. For instance, a few bacterial cells among a population of lysogenic cells could release the virus and kill susceptible non-lysogenic competitors, while their clone mates would be immune. Because viruses replicate inside their victims upon infection, this process would amplify their number in the arena. Sometimes, however, temperate viruses spare recipient cells from death by establishing themselves in a dormant state inside cells. This phenomenon is called lysogenization and, for some viruses such as the λ virus, the probability of lysogenization increases with the multiplicity of infection. Therefore, the amplification of viruses leads to conflicting predictions about the efficacy of temperate viruses as biological weapons: amplification can increase the relative advantage of clone mates of lysogens but also the likelihood of saving susceptible cells from death, because the probability of lysogenization is higher. To test the usefulness of viruses as biological weapons, we performed competition experiments between lysogenic Escherichia coli cells carrying the λ virus and susceptible λ-free E. coli cells, either in a structured or unstructured habitat. In structured and sometimes in unstructured habitats, the λ virus qualitatively behaved as a "replicating toxin". However, such toxic effect of λ viruses ceased after a few days of competition. This was due to the fact that many of initially susceptible cells became lysogenic. Massive lysogenization of susceptible cells occurred precisely under the conditions where the amplification of the virus was substantial. From then on, these cells and their descendants became immune to the λ virus. In conclusion, if at short term bacterial cells may use temperate viruses as biological weapons, after a few days only the classical view of temperate bacterial viruses as parasitic agents prevails.

Plant Insecticidal Toxins in Ecological Networks

PubMed Central

Ibanez, Sébastien; Gallet, Christiane; Després, Laurence

2012-01-01

Plant secondary metabolites play a key role in plant-insect interactions, whether constitutive or induced, C- or N-based. Anti-herbivore defences against insects can act as repellents, deterrents, growth inhibitors or cause direct mortality. In turn, insects have evolved a variety of strategies to act against plant toxins, e.g., avoidance, excretion, sequestration and degradation of the toxin, eventually leading to a co-evolutionary arms race between insects and plants and to co-diversification. Anti-herbivore defences also negatively impact mutualistic partners, possibly leading to an ecological cost of toxin production. However, in other cases toxins can also be used by plants involved in mutualistic interactions to exclude inadequate partners and to modify the cost/benefit ratio of mutualism to their advantage. When considering the whole community, toxins have an effect at many trophic levels. Aposematic insects sequester toxins to defend themselves against predators. Depending on the ecological context, toxins can either increase insects’ vulnerability to parasitoids and entomopathogens or protect them, eventually leading to self-medication. We conclude that studying the community-level impacts of plant toxins can provide new insights into the synthesis between community and evolutionary ecology. PMID:22606374

*CYANOBACTERIA AND THEIR TOXINS

EPA Science Inventory

Cyanobacteria, or blue-green algae, are naturally-occurring contaminants of surface waters worldwide. These photosynthesizing prokaryotes thrive in warm, shallow, nutrient-rich waters. Many produce potent toxins as secondary metabolites. Cyanobacteria toxins have been document...

A Versatile Family 3 Glycoside Hydrolase from Bifidobacterium adolescentis Hydrolyzes β-Glucosides of the Fusarium Mycotoxins Deoxynivalenol, Nivalenol, and HT-2 Toxin in Cereal Matrices

PubMed Central

Varga, Elisabeth; Malachova, Alexandra; Nguyen, Nhung Thi; Lorenz, Cindy; Haltrich, Dietmar; Berthiller, Franz; Adam, Gerhard

2015-01-01

Glycosylation plays a central role in plant defense against xenobiotics, including mycotoxins. Glucoconjugates of Fusarium toxins, such as deoxynivalenol-3-O-β-d-glucoside (DON-3G), often cooccur with their parental toxins in cereal-based food and feed. To date, only limited information exists on the occurrence of glucosylated mycotoxins and their toxicological relevance. Due to a lack of analytical standards and the requirement of high-end analytical instrumentation for their direct determination, hydrolytic cleavage of β-glucosides followed by analysis of the released parental toxins has been proposed as an indirect determination approach. This study compares the abilities of several fungal and recombinant bacterial β-glucosidases to hydrolyze the model analyte DON-3G. Furthermore, substrate specificities of two fungal and two bacterial (Lactobacillus brevis and Bifidobacterium adolescentis) glycoside hydrolase family 3 β-glucosidases were evaluated on a broader range of substrates. The purified recombinant enzyme from B. adolescentis (BaBgl) displayed high flexibility in substrate specificity and exerted the highest hydrolytic activity toward 3-O-β-d-glucosides of the trichothecenes deoxynivalenol (DON), nivalenol, and HT-2 toxin. A Km of 5.4 mM and a Vmax of 16 μmol min−1 mg−1 were determined with DON-3G. Due to low product inhibition (DON and glucose) and sufficient activity in several extracts of cereal matrices, this enzyme has the potential to be used for indirect analyses of trichothecene-β-glucosides in cereal samples. PMID:25979885

A functional antigen in a practical crop: LT-B producing maize protects mice against Escherichia coli heat labile enterotoxin (LT) and cholera toxin (CT).

PubMed

Chikwamba, Rachel; Cunnick, Joan; Hathaway, Diane; McMurray, Jennifer; Mason, Hugh; Wang, Kan

2002-10-01

We have produced a functional heat labile enterotoxin (LT-) B subunit of Escherichia coli in maize. LT-B is a multimeric protein that presents an ideal model for an edible vaccine, displaying stability in the gut and inducing mucosal and systemic immune responses. Transgenic maize was engineered to synthesize the LT-B polypeptides, which assembled into oligomeric structures with affinity for G(M1) gangliosides. We orally immunized BALB/c mice by feeding transgenic maize meal expressing LT-B or non-transgenic maize meal spiked with bacterial LT-B. Both treatments stimulated elevated IgA and IgG antibodies against LT-B and the closely related cholera toxin B subunit (CT-B) in serum, and elevated IgA in fecal pellets. The transgenic maize induced a higher anti-LT-B and anti-CT-B mucosal and serum IgA response compared to the equivalent amount of bacterial LT-B spiked into maize. Following challenge by oral administration of the diarrhea inducing toxins LT and CT, transgenic maize-fed mice displayed reduced fluid accumulation in the gut compared to non-immunized mice. Moreover, the gut to carcass ratio of immunized mice was not significantly different from the PBS (non-toxin) challenged control group. We concluded that maize-synthesized LT-B had features of the native bacterial LT-B such as molecular weight, G(M1) binding ability, and induction of serum and mucosal immunity. We have demonstrated that maize, a major food and feed ingredient, can be efficiently transformed to produce, accumulate, and store a fully assembled and functional candidate vaccine antigen.

Implications of free Shiga toxin-converting bacteriophages occurring outside bacteria for the evolution and the detection of Shiga toxin-producing Escherichia coli.

PubMed

Martínez-Castillo, Alexandre; Muniesa, Maite

2014-01-01

In this review we highlight recent work that has increased our understanding of the distribution of Shiga toxin-converting phages that can be detected as free phage particles, independently of Shiga toxin-producing bacteria (STEC). Stx phages are a quite diverse group of temperate phages that can be found in their prophage state inserted within the STEC chromosome, but can also be found as phages released from the cell after activation of their lytic cycle. They have been detected in extraintestinal environments such as water polluted with feces from humans or animals, food samples or even in stool samples of healthy individuals. The high persistence of phages to several inactivation conditions makes them suitable candidates for the successful mobilization of stx genes, possibly resulting in the genes reaching a new bacterial genomic background by means of transduction, where ultimately they may be expressed, leading to Stx production. Besides the obvious fact that Stx phages circulating between bacteria can be, and probably are, involved in the emergence of new STEC strains, we review here other possible ways in which free Stx phages could interfere with the detection of STEC in a given sample by current laboratory methods and how to avoid such interference.

Quorum Sensing Extracellular Death Peptides Enhance the Endoribonucleolytic Activities of Mycobacterium tuberculosis MazF Toxins

PubMed Central

Nigam, Akanksha; Kumar, Sathish

2018-01-01

ABSTRACT mazEF is a toxin-antitoxin module located on chromosomes of most bacteria. MazF toxins are endoribonucleases antagonized by MazE antitoxins. Previously, we characterized several quorum sensing peptides called "extracellular death factors" (EDFs). When secreted from bacterial cultures, EDFs induce interspecies cell death. EDFs also enhance the endoribonucleolytic activity of Escherichia coli MazF. Mycobacterium tuberculosis carries several mazEF modules. Among them, the endoribonucleolytic activities of MazF proteins mt-1, mt-3, and mt-6 were identified. MazF-mt6 and MazF-mt-3 cleave M. tuberculosis rRNAs. Here we report the in vitro effects of EDFs on the endoribonucleolytic activities of M. tuberculosis MazFs. Escherichia coli EDF (EcEDF) and the three Pseudomonas aeruginosa EDFs (PaEDFs) individually enhance the endoribonucleolytic activities of MazF-mt6 and MazF-mt3 and overcome the inhibitory effect of MazE-mt3 or MazE-mt6 on the endoribonucleolytic activities of the respective toxins. We propose that these EDFs can serve as a basis for a novel class of antibiotics against M. tuberculosis. PMID:29717013

Synthesis and biology of cyclic imine toxins, an emerging class of potent, globally distributed marine toxins.

PubMed

Stivala, Craig E; Benoit, Evelyne; Aráoz, Rómulo; Servent, Denis; Novikov, Alexei; Molgó, Jordi; Zakarian, Armen

2015-03-01

From a small group of exotic compounds isolated only two decades ago, Cyclic Imine (CI) toxins have become a major class of marine toxins with global distribution. Their distinct chemical structure, biological mechanism of action, and intricate chemistry ensures that CI toxins will continue to be the subject of fascinating fundamental studies in the broad fields of chemistry, chemical biology, and toxicology. The worldwide occurrence of potent CI toxins in marine environments, their accumulation in shellfish, and chemical stability are important considerations in assessing risk factors for human health. This review article aims to provide an account of chemistry, biology, and toxicology of CI toxins from their discovery to the present day.

Giardia duodenalis Infection Reduces Granulocyte Infiltration in an In Vivo Model of Bacterial Toxin-Induced Colitis and Attenuates Inflammation in Human Intestinal Tissue

PubMed Central

Cotton, James A.; Motta, Jean-Paul; Schenck, L. Patrick; Hirota, Simon A.; Beck, Paul L.; Buret, Andre G.

2014-01-01

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a predominant cause of waterborne diarrheal disease that may lead to post-infectious functional gastrointestinal disorders. Although Giardia-infected individuals could carry as much as 106 trophozoites per centimetre of gut, their intestinal mucosa is devoid of overt signs of inflammation. Recent studies have shown that in endemic countries where bacterial infectious diseases are common, Giardia infections can protect against the development of diarrheal disease and fever. Conversely, separate observations have indicated Giardia infections may enhance the severity of diarrheal disease from a co-infecting pathogen. Polymorphonuclear leukocytes or neutrophils (PMNs) are granulocytic, innate immune cells characteristic of acute intestinal inflammatory responses against bacterial pathogens that contribute to the development of diarrheal disease following recruitment into intestinal tissues. Giardia cathepsin B cysteine proteases have been shown to attenuate PMN chemotaxis towards IL-8/CXCL8, suggesting Giardia targets PMN accumulation. However, the ability of Giardia infections to attenuate PMN accumulation in vivo and how in turn this effect may alter the host inflammatory response in the intestine has yet to be demonstrated. Herein, we report that Giardia infection attenuates granulocyte tissue infiltration induced by intra-rectal instillation of Clostridium difficile toxin A and B in an isolate-dependent manner. This attenuation of granulocyte infiltration into colonic tissues paralled decreased expression of several cytokines associated with the recruitment of PMNs. Giardia trophozoite isolates that attenuated granulocyte infiltration in vivo also decreased protein expression of cytokines released from inflamed mucosal biopsy tissues collected from patients with active Crohn’s disease, including several cytokines associated with PMN recruitment. These results demonstrate for the first time that certain

Giardia duodenalis infection reduces granulocyte infiltration in an in vivo model of bacterial toxin-induced colitis and attenuates inflammation in human intestinal tissue.

PubMed

Cotton, James A; Motta, Jean-Paul; Schenck, L Patrick; Hirota, Simon A; Beck, Paul L; Buret, Andre G

2014-01-01

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a predominant cause of waterborne diarrheal disease that may lead to post-infectious functional gastrointestinal disorders. Although Giardia-infected individuals could carry as much as 106 trophozoites per centimetre of gut, their intestinal mucosa is devoid of overt signs of inflammation. Recent studies have shown that in endemic countries where bacterial infectious diseases are common, Giardia infections can protect against the development of diarrheal disease and fever. Conversely, separate observations have indicated Giardia infections may enhance the severity of diarrheal disease from a co-infecting pathogen. Polymorphonuclear leukocytes or neutrophils (PMNs) are granulocytic, innate immune cells characteristic of acute intestinal inflammatory responses against bacterial pathogens that contribute to the development of diarrheal disease following recruitment into intestinal tissues. Giardia cathepsin B cysteine proteases have been shown to attenuate PMN chemotaxis towards IL-8/CXCL8, suggesting Giardia targets PMN accumulation. However, the ability of Giardia infections to attenuate PMN accumulation in vivo and how in turn this effect may alter the host inflammatory response in the intestine has yet to be demonstrated. Herein, we report that Giardia infection attenuates granulocyte tissue infiltration induced by intra-rectal instillation of Clostridium difficile toxin A and B in an isolate-dependent manner. This attenuation of granulocyte infiltration into colonic tissues paralled decreased expression of several cytokines associated with the recruitment of PMNs. Giardia trophozoite isolates that attenuated granulocyte infiltration in vivo also decreased protein expression of cytokines released from inflamed mucosal biopsy tissues collected from patients with active Crohn's disease, including several cytokines associated with PMN recruitment. These results demonstrate for the first time that certain

Mathematical modeling and numerical analysis of the growth of Non-O157 shiga toxin-producing Escherichia coli in spinach leaves

USDA-ARS?s Scientific Manuscript database

This study was conducted to investigate the growth of non-O157 Shiga toxin-producing Escherichia coli (STEC) in spinach leaves and to develop kinetic models to describe the bacterial growth. Six serogroups of non-O157 STEC, including O26, O45, O103, O111, O121, and O145, were used in the growth stu...

Food toxin detection with atomic force microscope

USDA-ARS?s Scientific Manuscript database

Externally introduced toxins or internal spoilage correlated pathogens and their metabolites are all potential sources of food toxins. To prevent and protect unsafe food, many food toxin detection techniques have been developed to detect various toxins for quality control. Although several routine m...

The Interactions of Human Neutrophils with Shiga Toxins and Related Plant Toxins: Danger or Safety?

PubMed Central

Brigotti, Maurizio

2012-01-01

Shiga toxins and ricin are well characterized similar toxins belonging to quite different biological kingdoms. Plant and bacteria have evolved the ability to produce these powerful toxins in parallel, while humans have evolved a defense system that recognizes molecular patterns common to foreign molecules through specific receptors expressed on the surface of the main actors of innate immunity, namely monocytes and neutrophils. The interactions between these toxins and neutrophils have been widely described and have stimulated intense debate. This paper is aimed at reviewing the topic, focusing particularly on implications for the pathogenesis and diagnosis of hemolytic uremic syndrome. PMID:22741061

Cellular Entry of Clostridium perfringens Iota-Toxin and Clostridium botulinum C2 Toxin.

PubMed

Takehara, Masaya; Takagishi, Teruhisa; Seike, Soshi; Oda, Masataka; Sakaguchi, Yoshihiko; Hisatsune, Junzo; Ochi, Sadayuki; Kobayashi, Keiko; Nagahama, Masahiro

2017-08-11

Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin are composed of two non-linked proteins, one being the enzymatic component and the other being the binding/translocation component. These latter components recognize specific receptors and oligomerize in plasma membrane lipid-rafts, mediating the uptake of the enzymatic component into the cytosol. Enzymatic components induce actin cytoskeleton disorganization through the ADP-ribosylation of actin and are responsible for cell rounding and death. This review focuses upon the recent advances in cellular internalization of clostridial binary toxins.

Protein- and DNA-based anthrax toxin vaccines confer protection in guinea pigs against inhalational challenge with Bacillus cereus G9241.

PubMed

Palmer, John; Bell, Matt; Darko, Christian; Barnewall, Roy; Keane-Myers, Andrea

2014-11-01

In the past decade, several Bacillus cereus strains have been isolated from otherwise healthy individuals who succumbed to bacterial pneumonia presenting symptoms resembling inhalational anthrax. One strain was indistinguishable from B. cereus G9241, previously cultured from an individual who survived a similar pneumonia-like illness and which was shown to possess a complete set of plasmid-borne anthrax toxin-encoding homologs. The finding that B. cereus G9241 pathogenesis in mice is dependent on pagA1-derived protective antigen (PA) synthesis suggests that an anthrax toxin-based vaccine may be effective against this toxin-encoding B. cereus strain. Dunkin Hartley guinea pigs were immunized with protein- and DNA-based anthrax toxin-based vaccines, immune responses were evaluated and survival rates were calculated after lethal aerosol exposure with B. cereus G9241 spores. Each vaccine induced seroconversion with the protein immunization regimen eliciting significantly higher serum levels of antigen-specific antibodies at the prechallenge time-point compared with the DNA-protein prime-boost immunization schedule. Complete protection against lethal challenge was observed in all groups with a detectable prechallenge serum titer of toxin neutralizing antibodies. For the first time, we demonstrated that the efficacy of fully defined anthrax toxin-based vaccines was protective against lethal B. cereus G9241 aerosol challenge in the guinea pig animal model. Published 2014. This article is a US Government work and is in the public domain in the USA.

Conditional Toxin Splicing Using a Split Intein System.

PubMed

Alford, Spencer C; O'Sullivan, Connor; Howard, Perry L

2017-01-01

Protein toxin splicing mediated by split inteins can be used as a strategy for conditional cell ablation. The approach requires artificial fragmentation of a potent protein toxin and tethering each toxin fragment to a split intein fragment. The toxin-intein fragments are, in turn, fused to dimerization domains, such that addition of a dimerizing agent reconstitutes the split intein. These chimeric toxin-intein fusions remain nontoxic until the dimerizer is added, resulting in activation of intein splicing and ligation of toxin fragments to form an active toxin. Considerations for the engineering and implementation of conditional toxin splicing (CTS) systems include: choice of toxin split site, split site (extein) chemistry, and temperature sensitivity. The following method outlines design criteria and implementation notes for CTS using a previously engineered system for splicing a toxin called sarcin, as well as for developing alternative CTS systems.

Mutant with diphtheria toxin receptor and acidification function but defective in entry of toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kohno, Kenji; Hayes, H.; Mekada, Eisuke

1987-09-01

A mutant of Chinese hamster ovary cells, GE1, that is highly resistant to diphtheria toxin was isolated. The mutant contains 50% ADP-ribosylatable elongation factor 2, but its protein synthesis was not inhibited by the toxin even at concentrations above 100 {mu}g/ml. {sup 125}I-labeled diphtheria toxin was associated with GE1 cells as well as with the parent cells but did not block protein synthesis of GE1 cells even when the cells were exposed to low pH in the presence or absence of NH{sub 4}Cl. The infections of GE1 cells and the parent cells by vesicular stomatitis virus were similar. GE1 cellsmore » were cross-resistant to Pseudomonas aeruginosa exotoxin A and so were about 1,000 times more resistant to this toxin than the parent cells. Hybrids of GE1 cells and the parent cells or mutant cells lacking a functional receptor were more sensitive to diphtheria toxin than GE1 cells. These results suggest that entry of diphtheria toxin into cells requires a cellular factor(s) in addition to those involved in receptor function and acidification of endosomes and that GE1 cells do not express this cellular factor. This character is recessive in GE1 cells.« less

Plasma Bacterial and Mitochondrial DNA Distinguish Bacterial Sepsis from Sterile SIRS and Quantify Inflammatory Tissue Injury in Nonhuman Primates

PubMed Central

Sursal, Tolga; Stearns-Kurosawa, Deborah J; Itagaki, Kiyoshi; Oh, Sun-Young; Sun, Shiqin; Kurosawa, Shinichiro; Hauser, Carl J

2012-01-01

Systemic inflammatory response syndrome (SIRS) is a fundamental host response common to bacterial infection and sterile tissue injury. SIRS can cause organ dysfunction and death but its mechanisms are incompletely understood. Moreover, SIRS can progress to organ failure or death despite being sterile or after control of the inciting infection. Biomarkers discriminating between sepsis, sterile SIRS and post-infective SIRS would therefore help direct care. Circulating mitochondrial DNA (mtDNA) is a damage-associated molecular pattern (DAMP) reflecting cellular injury. Circulating bacterial 16S-DNA (bDNA) is a pathogen-associated pattern (PAMP) reflecting ongoing infection. We developed qPCR assays to quantify these markers and predicted their plasma levels might help distinguish sterile injury from infection. To study these events in primates we assayed banked serum from papio baboons that had undergone a brief challenge of intravenous Bacillus anthracis deltaSterne (modified to remove toxins) followed by antibiotics (anthrax) that causes organ failure and death. To investigate the progression of sepsis to “severe” sepsis and death we studied animals where anthrax was pretreated with drotrecogin alfa (aPC), which attenuates sepsis in baboons. We also contrasted lethal anthrax bacteremia against non-lethal E.coli bacteremia and against sterile tissue injury from Shiga-like toxin-1 (Stx1). bDNA and mtDNA levels in timed samples were correlated with blood culture results and assays of organ function. Sterile injury by Stx1 increased mtDNA but bDNA was undetectable: consistent with the absence of infection. The bacterial challenges caused parallel early bDNA and mtDNA increases, but bDNA detected pathogens even after bacteria were undetectable by culture. Sub-lethal E.coli challenge only caused transient rises in mtDNA consistent with a self-limited injury. In lethal anthrax challenge (n=4) bDNA increased transiently but mtDNA levels remained elevated until death

Computational Studies of Snake Venom Toxins

PubMed Central

Ojeda, Paola G.; Caballero, Julio; Kaas, Quentin; González, Wendy

2017-01-01

Most snake venom toxins are proteins, and participate to envenomation through a diverse array of bioactivities, such as bleeding, inflammation, and pain, cytotoxic, cardiotoxic or neurotoxic effects. The venom of a single snake species contains hundreds of toxins, and the venoms of the 725 species of venomous snakes represent a large pool of potentially bioactive proteins. Despite considerable discovery efforts, most of the snake venom toxins are still uncharacterized. Modern bioinformatics tools have been recently developed to mine snake venoms, helping focus experimental research on the most potentially interesting toxins. Some computational techniques predict toxin molecular targets, and the binding mode to these targets. This review gives an overview of current knowledge on the ~2200 sequences, and more than 400 three-dimensional structures of snake toxins deposited in public repositories, as well as of molecular modeling studies of the interaction between these toxins and their molecular targets. We also describe how modern bioinformatics have been used to study the snake venom protein phospholipase A2, the small basic myotoxin Crotamine, and the three-finger peptide Mambalgin. PMID:29271884

Polymorphic toxin systems: Comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics

PubMed Central

2012-01-01

Background Proteinaceous toxins are observed across all levels of inter-organismal and intra-genomic conflicts. These include recently discovered prokaryotic polymorphic toxin systems implicated in intra-specific conflicts. They are characterized by a remarkable diversity of C-terminal toxin domains generated by recombination with standalone toxin-coding cassettes. Prior analysis revealed a striking diversity of nuclease and deaminase domains among the toxin modules. We systematically investigated polymorphic toxin systems using comparative genomics, sequence and structure analysis. Results Polymorphic toxin systems are distributed across all major bacterial lineages and are delivered by at least eight distinct secretory systems. In addition to type-II, these include type-V, VI, VII (ESX), and the poorly characterized “Photorhabdus virulence cassettes (PVC)”, PrsW-dependent and MuF phage-capsid-like systems. We present evidence that trafficking of these toxins is often accompanied by autoproteolytic processing catalyzed by HINT, ZU5, PrsW, caspase-like, papain-like, and a novel metallopeptidase associated with the PVC system. We identified over 150 distinct toxin domains in these systems. These span an extraordinary catalytic spectrum to include 23 distinct clades of peptidases, numerous previously unrecognized versions of nucleases and deaminases, ADP-ribosyltransferases, ADP ribosyl cyclases, RelA/SpoT-like nucleotidyltransferases, glycosyltranferases and other enzymes predicted to modify lipids and carbohydrates, and a pore-forming toxin domain. Several of these toxin domains are shared with host-directed effectors of pathogenic bacteria. Over 90 families of immunity proteins might neutralize anywhere between a single to at least 27 distinct types of toxin domains. In some organisms multiple tandem immunity genes or immunity protein domains are organized into polyimmunity loci or polyimmunity proteins. Gene-neighborhood-analysis of polymorphic toxin systems

Clostridial toxins active locally in the gastrointestinal tract.

PubMed

Wilkins, T; Krivan, H; Stiles, B; Carman, R; Lyerly, D

1985-01-01

Clostridium difficile and Clostridium spiroforme have only in recent years been recognized as intestinal pathogens. They both produce toxins that are also produced by other clostridia. C. difficile toxins A and B are produced by C. sordellii and a few strains of C. perfringens whereas C. spiroforme produces the same toxins as C. perfringens Type E (iota toxin). Iota toxin activity may be the product of two proteins. Toxigenic strains of C. spiroforme and Type E produce two antigens which possess much more biological activity when administered together than when given alone. C. difficile was thought for some time to produce only a single toxin, but then the enterotoxic activity was shown to be due to a separate toxin (toxin A). This toxin increases the oral toxicity of toxin B (the main cytotoxin) and may increase the permeability of the colon. Toxin A binds to a specific receptor in hamster brush border membranes and in the membranes of rabbit erythrocytes. This receptor appears to be a glycoprotein. The receptor can be extracted from the membrane with Triton and binds to immobilized toxin A. The receptor can be extracted and used to coat plastic plates as a first phase in an ELISA assay. Another assay has been developed in which the toxin A binds to the red cells and then the erythrocytes are agglutinated with antitoxin. An even more sensitive assay consists of using rabbit erythrocyte ghosts to bind the toxin and then precipitating the ghosts with antibody to toxin A attached to latex beads. Monoclonal antibodies to toxin A also have been developed and are used in these and other assays.

pH-dependent conformational changes of diphtheria toxin adsorbed to lipid monolayers by neutron and X-ray reflection

NASA Astrophysics Data System (ADS)

Kent, Michael; Yim, Hyun; Satija, Sushil; Kuzmenko, Ivan

2006-03-01

Several important bacterial toxins, such as diphtheria, tetanus, and botulinum, invade cells through a process of high affinity binding, internalization via endosome formation, and subsequent membrane penetration of the catalytic domain activated by a pH drop in the endosome. These toxins are composed of three domains: a binding domain, a translocation domain, and an enzyme. The translocation process is not well understood with regard to the detailed conformational changes that occur at each step, To address this, we performed neutron reflectivity measurements for diphtheria toxin bound to lipid monolayers as a function of pH. While the final membrane inserted conformation will not be reproduced with the present monolayer system, important insights can still be gained into several intermediate stages. In particular, we show that no adsorption occurs at pH = 7.6, but strong adsorption occurs over at a pH range from 6.5 to 6.0. Following binding, at least two stages of conformational change occur, as the thickness increases from pH 6.3 to 5.3 and then decreases from pH 5.3 to 4.5. In addition, the dimension of the adsorbed layer substantially exceeds that of the largest dimension in the crystal structure of monomeric diphtheria, suggesting that the toxin may be present as multimers.

Autoregulation and Virulence Control by the Toxin-Antitoxin System SavRS in Staphylococcus aureus

PubMed Central

Wen, Wen; Liu, Banghui; Xue, Lu; Zhu, Zhongliang; Niu, Liwen

2018-01-01

ABSTRACT Toxin-antitoxin (TA) systems play diverse physiological roles, such as plasmid maintenance, growth control, and persister cell formation, but their involvement in bacterial pathogenicity remains largely unknown. Here, we have identified a novel type II toxin-antitoxin system, SavRS, and revealed the molecular mechanisms of its autoregulation and virulence control in Staphylococcus aureus. Electrophoretic mobility shift assay and isothermal titration calorimetry data indicated that the antitoxin SavR acted as the primary repressor bound to its own promoter, while the toxin SavS formed a complex with SavR to enhance the ability to bind to the operator site. DNase I footprinting assay identified the SavRS-binding site containing a short and long palindrome in the promoter region. Further, mutation and DNase I footprinting assay demonstrated that the two palindromes were crucial for DNA binding and transcriptional repression. More interestingly, genetic deletion of the savRS system led to the increased hemolytic activity and pathogenicity in a mouse subcutaneous abscess model. We further identified two virulence genes, hla and efb, by real-time quantitative reverse transcription-PCR and demonstrated that SavR and SavRS could directly bind to their promoter regions to repress virulence gene expression. PMID:29440365

Brown spider dermonecrotic toxin directly induces nephrotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chaim, Olga Meiri; Sade, Youssef Bacila; Bertoni da Silveira, Rafael

2006-02-15

Brown spider (Loxosceles genus) venom can induce dermonecrotic lesions at the bite site and systemic manifestations including fever, vomiting, convulsions, disseminated intravascular coagulation, hemolytic anemia and acute renal failure. The venom is composed of a mixture of proteins with several molecules biochemically and biologically well characterized. The mechanism by which the venom induces renal damage is unknown. By using mice exposed to Loxosceles intermedia recombinant dermonecrotic toxin (LiRecDT), we showed direct induction of renal injuries. Microscopic analysis of renal biopsies from dermonecrotic toxin-treated mice showed histological alterations including glomerular edema and tubular necrosis. Hyalinization of tubules with deposition of proteinaceousmore » material in the tubule lumen, tubule epithelial cell vacuoles, tubular edema and epithelial cell lysis was also observed. Leukocytic infiltration was neither observed in the glomerulus nor the tubules. Renal vessels showed no sign of inflammatory response. Additionally, biochemical analyses showed such toxin-induced changes in renal function as urine alkalinization, hematuria and azotemia with elevation of blood urea nitrogen levels. Immunofluorescence with dermonecrotic toxin antibodies and confocal microscopy analysis showed deposition and direct binding of this toxin to renal intrinsic structures. By immunoblotting with a hyperimmune dermonecrotic toxin antiserum on renal lysates from toxin-treated mice, we detected a positive signal at the region of 33-35 kDa, which strengthens the idea that renal failure is directly induced by dermonecrotic toxin. Immunofluorescence reaction with dermonecrotic toxin antibodies revealed deposition and binding of this toxin directly in MDCK epithelial cells in culture. Similarly, dermonecrotic toxin treatment caused morphological alterations of MDCK cells including cytoplasmic vacuoles, blebs, evoked impaired spreading and detached cells from each other and

Cellular Entry of Clostridium perfringens Iota-Toxin and Clostridium botulinum C2 Toxin

PubMed Central

Takehara, Masaya; Takagishi, Teruhisa; Seike, Soshi; Oda, Masataka; Sakaguchi, Yoshihiko; Hisatsune, Junzo; Ochi, Sadayuki; Kobayashi, Keiko; Nagahama, Masahiro

2017-01-01

Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin are composed of two non-linked proteins, one being the enzymatic component and the other being the binding/translocation component. These latter components recognize specific receptors and oligomerize in plasma membrane lipid-rafts, mediating the uptake of the enzymatic component into the cytosol. Enzymatic components induce actin cytoskeleton disorganization through the ADP-ribosylation of actin and are responsible for cell rounding and death. This review focuses upon the recent advances in cellular internalization of clostridial binary toxins. PMID:28800062

EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin

PubMed Central

Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R.; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Tehran, Domenico Azarnia; Montecucco, Cesare; Barth, Holger

2016-01-01

The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins. PMID:27043629

EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin.

PubMed

Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Azarnia Tehran, Domenico; Montecucco, Cesare; Barth, Holger

2016-04-01

The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins.

Anthrax toxin receptor 1 is the cellular receptor for Seneca Valley virus

PubMed Central

Miles, Linde A.; Burga, Laura N.; Gardner, Eric E.; Bostina, Mihnea; Poirier, John T.; Rudin, Charles M.

2017-01-01

Seneca Valley virus (SVV) is an oncolytic picornavirus with selective tropism for neuroendocrine cancers. It has shown promise as a cancer therapeutic in preclinical studies and early-phase clinical trials. Here, we have identified anthrax toxin receptor 1 (ANTXR1) as the receptor for SVV using genome-wide loss-of-function screens. ANTXR1 is necessary for permissivity in vitro and in vivo. However, robust SVV replication requires an additional innate immune defect. We found that SVV interacts directly and specifically with ANTXR1, that this interaction is required for SVV binding to permissive cells, and that ANTXR1 expression is necessary and sufficient for infection in cell lines with decreased expression of antiviral IFN genes at baseline. Finally, we identified the region of the SVV capsid that is responsible for receptor recognition using cryoelectron microscopy of the SVV-ANTXR1-Fc complex. These studies identify ANTXR1, a class of receptor that is shared by a mammalian virus and a bacterial toxin, as the cellular receptor for SVV. PMID:28650343

[Biological and toxin terrorism weapons].

PubMed

Bokan, Slavko

2003-03-01

The use of biological agents and toxins in warfare and terrorism has a long history. Human, animal and plant pathogens and toxins can cause disease and can be used as a threat to humans, animals and staple crops. The same is true for biological agents. Although the use of biological agents and toxins in military conflicts has been a concern of military communities for many years, several recent events have increased the awareness of terrorist use of these weapons against civilian population. A Mass Casualty Biological (Toxin) Weapon (MCBTW) is any biological and toxin weapon capable of causing death or disease on a large scale, such that the military or civilian infrastructure of the state or organization being attacked is overwhelmed. A militarily significant (or terrorist) weapon is any weapon capable of affecting, directly or indirectly, that is physically or psychologically, the outcome of a military operation. Although many biological agents such as toxins and bioregulators can be used to cause diseases, there are only a few that can truly threaten civilian populations on a large scale. Bioregulators or modulators are biochemical compounds, such as peptides, that occur naturally in organisms. They are new class of weapons that can damage nervous system, alter moods, trigger psychological changes and kill. The potential military or terrorist use of bioregulators is similar to that of toxins. Some of these compounds are several hundred times more potent than traditional chemical warfare agents. Important features and military advantages of new bioregulators are novel sites of toxic action; rapid and specific effects; penetration of protective filters and equipment, and militarily effective physical incapacitation. This overview of biological agents and toxins is largely intended to help healthcare providers on all levels to make decisions in protecting general population from these agents.

Naturally Occurring Food Toxins

PubMed Central

Dolan, Laurie C.; Matulka, Ray A.; Burdock, George A.

2010-01-01

Although many foods contain toxins as a naturally-occurring constituent or, are formed as the result of handling or processing, the incidence of adverse reactions to food is relatively low. The low incidence of adverse effects is the result of some pragmatic solutions by the US Food and Drug Administration (FDA) and other regulatory agencies through the creative use of specifications, action levels, tolerances, warning labels and prohibitions. Manufacturers have also played a role by setting limits on certain substances and developing mitigation procedures for process-induced toxins. Regardless of measures taken by regulators and food producers to protect consumers from natural food toxins, consumption of small levels of these materials is unavoidable. Although the risk for toxicity due to consumption of food toxins is fairly low, there is always the possibility of toxicity due to contamination, overconsumption, allergy or an unpredictable idiosyncratic response. The purpose of this review is to provide a toxicological and regulatory overview of some of the toxins present in some commonly consumed foods, and where possible, discuss the steps that have been taken to reduce consumer exposure, many of which are possible because of the unique process of food regulation in the United States. PMID:22069686

Effect of endosomal acidification on small ion transport through the anthrax toxin PA63 channel.

PubMed

Kalu, Nnanya; Alcaraz, Antonio; Yamini, Goli; Momben Abolfath, Sanaz; Lucas, Laura; Kenney, Clare; Aguilella, Vicente M; Nestorovich, Ekaterina M

2017-11-01

Tight regulation of pH is critical for the structure and function of cells and organelles. The pH environment changes dramatically along the endocytic pathway, an internalization transport process that is 'hijacked' by many intracellularly active bacterial exotoxins, including the anthrax toxin. Here, we investigate the role of pH on single-channel properties of the anthrax toxin protective antigen (PA 63 ). Using conductance and current noise analysis, blocker binding, ion selectivity, and poly(ethylene glycol) partitioning measurements, we show that the channel exists in two different open states ('maximum' and 'main') at pH ≥ 5.5, while only a maximum conductance state is detected at pH < 5.5. We describe two substantially distinct patterns of PA 63 conductance dependence on KCl concentration uncovered at pH 6.5 and 4.5. © 2017 Federation of European Biochemical Societies.

Antimicrobial inflammasomes: unified signalling against diverse bacterial pathogens.

PubMed

Eldridge, Matthew J G; Shenoy, Avinash R

2015-02-01

Inflammasomes - molecular platforms for caspase-1 activation - have emerged as common hubs for a number of pathways that detect and respond to bacterial pathogens. Caspase-1 activation results in the secretion of bioactive IL-1β and IL-18 and pyroptosis, and thus launches a systemic immune and inflammatory response. In this review we discuss signal transduction leading to 'canonical' and 'non-canonical' activation of caspase-1 through the involvement of upstream caspases. Recent studies have identified a growing number of regulatory networks involving guanylate binding proteins, protein kinases, ubiquitylation and necroptosis related pathways that modulate inflammasome responses and immunity to bacterial infection. By being able to respond to extracellular, vacuolar and cytosolic bacteria, their cytosolic toxins or ligands for cell surface receptors, inflammasomes have emerged as important sentinels of infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

SVM-Based Prediction of Propeptide Cleavage Sites in Spider Toxins Identifies Toxin Innovation in an Australian Tarantula

PubMed Central

Wong, Emily S. W.; Hardy, Margaret C.; Wood, David; Bailey, Timothy; King, Glenn F.

2013-01-01

Spider neurotoxins are commonly used as pharmacological tools and are a popular source of novel compounds with therapeutic and agrochemical potential. Since venom peptides are inherently toxic, the host spider must employ strategies to avoid adverse effects prior to venom use. It is partly for this reason that most spider toxins encode a protective proregion that upon enzymatic cleavage is excised from the mature peptide. In order to identify the mature toxin sequence directly from toxin transcripts, without resorting to protein sequencing, the propeptide cleavage site in the toxin precursor must be predicted bioinformatically. We evaluated different machine learning strategies (support vector machines, hidden Markov model and decision tree) and developed an algorithm (SpiderP) for prediction of propeptide cleavage sites in spider toxins. Our strategy uses a support vector machine (SVM) framework that combines both local and global sequence information. Our method is superior or comparable to current tools for prediction of propeptide sequences in spider toxins. Evaluation of the SVM method on an independent test set of known toxin sequences yielded 96% sensitivity and 100% specificity. Furthermore, we sequenced five novel peptides (not used to train the final predictor) from the venom of the Australian tarantula Selenotypus plumipes to test the accuracy of the predictor and found 80% sensitivity and 99.6% 8-mer specificity. Finally, we used the predictor together with homology information to predict and characterize seven groups of novel toxins from the deeply sequenced venom gland transcriptome of S. plumipes, which revealed structural complexity and innovations in the evolution of the toxins. The precursor prediction tool (SpiderP) is freely available on ArachnoServer (http://www.arachnoserver.org/spiderP.html), a web portal to a comprehensive relational database of spider toxins. All training data, test data, and scripts used are available from the Spider

A novel membrane-bound toxin for cell division, CptA (YgfX), inhibits polymerization of cytoskeleton proteins, FtsZ and MreB, in Escherichia coli.

PubMed

Masuda, Hisako; Tan, Qian; Awano, Naoki; Yamaguchi, Yoshihiro; Inouye, Masayori

2012-03-01

Nearly all free-living bacteria carry toxin-antitoxin (TA) systems on their genomes, through which cell growth and death are regulated. Toxins target a variety of essential cellular functions, including DNA replication, translation, and cell division. Here, we identified a novel toxin, YgfX, on the Escherichia coli genome. The toxin, consisting of 135 residues, is composed of the N-terminal membrane domain, which encompasses two transmembrane segments, and the C-terminal cytoplasmic domain. Upon YgfX expression, the cells were initially elongated and then the middle portion of the cells became inflated to form a lemon shape. YgfX was found to interact with MreB and FtsZ, two essential cytoskeletal proteins in E. coli. The cytoplasmic domain [YgfX(C)] was found to be responsible for the YgfX toxicity, as purified YgfX(C) was found to block the polymerization of FtsZ and MreB in vitro. YgfY, located immediately upstream of YgfX, was shown to be the cognate antitoxin; notably, YgfX is the first membrane-associating toxin in bacterial TA systems. We propose to rename the toxin and the antitoxin as CptA and CptB (for Cytoskeleton Polymerization inhibiting Toxin), respectively. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Screening for the presence of mcr-1/mcr-2 genes in Shiga toxin-producing Escherichia coli recovered from a major produce-production region in California

USDA-ARS?s Scientific Manuscript database

The rapid spreading of polymyxin E (colistin) resistance among bacterial strains through the horizontally transmissible mcr-1 and mcr-2 plasmids has become a serious concern. The emergence of these genes in Shiga toxin-producing Escherichia coli (STEC), a group of human pathogenic bacteria was even ...

The novel acidophilic structure of the killer toxin from halotolerant yeast demonstrates remarkable folding similarity with a fungal killer toxin.

PubMed

Kashiwagi, T; Kunishima, N; Suzuki, C; Tsuchiya, F; Nikkuni, S; Arata, Y; Morikawa, K

1997-01-15

Several strains of yeasts and fungi produce proteinous substances, termed killer toxins, which kill sensitive strains. The SMK toxin, secreted by the halotolerant yeast Pichia farinosa KK1 strain, uniquely exhibits its maximum killer activity under conditions of acidic pH and high salt concentration. The toxin is composed of two distinct subunits, alpha and beta, which tightly interact with each other under acidic conditions. However, they are easily dissociated under neutral conditions and lose the killer activity. The three-dimensional structure of the SMK toxin will provide a better understanding of the mechanism of toxicity of this protein and the cause of its unique pH-dependent stability. Two crystal structures of the SMK toxin have been determined at 1.8 A resolution in different ionic strength conditions. The two subunits, alpha and beta, are jointly folded into an ellipsoidal, single domain structure belonging to the alpha/beta-sandwich family. The folding topology of the SMK toxin is essentially the same as that of the fungal killer toxin, KP4. This shared topology contains two left-handed split betaalphabeta motifs, which are rare in the other proteins. Many acidic residues are clustered at the bottom of the SMK toxin molecule. Some of the carboxyl sidechains interact with each other through hydrogen bonds. The ionic strength difference induces no evident structural change of the SMK toxin except that, in the high ionic strength crystal, a number of sulfate ions are electrostatically bound near the basic residues which are also locally distributed at the bottom of the toxin molecule. The two killer toxins, SMK and KP4, share a unique folding topology which contains a rare structural motif. This observation may suggest that these toxins are evolutionally and/or functionally related. The pH-dependent stability of the SMK toxin is a result of the intensive interactions between the carboxyl groups. This finding is important for protein engineering, for

Miscellaneous chemical toxins

USGS Publications Warehouse

Friend, M.

1999-01-01

The previous chapters provide information about some of the chemical toxins that have lethal effects on wild birds. The material presented in Section 7, Chemical Toxins, is far from comprehensive because wild birds are poisoned by a wide variety of toxic substances. Also, monitoring of wild bird mortality is not yet organized so that diagnostic findings can be extended to reflect the relative impacts among the types of toxins, within populations, or among species, geographic areas, and time. The data that are available are not collectively based on random sampling, nor do specimen collection and submission follow methodical assessment methods. Instead, most data simply document individual bird poisoning events. The inherent biases in this information include the species of birds observed dead (large birds in open areas are more likely to be observed dead than small forest birds); the species of birds likely to be submitted for analysis (bald eagles are more likely to be submitted than house sparrows); collection sites (agricultural fields are more likely to be observed than urban environments); geographic area of the country; season; reasons for submissions; and other variables. Nevertheless, findings from individual events reflect the causes of mortality associated with those events and collectively identify chemical toxins that repeatedly cause bird mortalities which result in carcass collection and sub

A Nanocoaxial-Based Electrochemical Sensor for the Detection of Cholera Toxin

NASA Astrophysics Data System (ADS)

Archibald, Michelle; Rizal, Binod; Connolly, Timothy; Burns, Michael J.; Naughton, Michael J.; Chiles, Thomas C.; Biology; Physics Collaboration

We report a nanocoax-based electrochemical sensor for the detection of bacterial toxins using an electrochemical enzyme-linked immunosorbent assay (ELISA) and differential pulse voltammetry (DPV). The device architecture is composed of vertically-oriented, nanoscale coaxial electrodes, with coax cores and shields serving as integrated working and counter electrodes, respectively. Proof-of-concept was demonstrated for the detection of cholera toxin (CT), with a linear dynamic range of detection was 10 ng/ml - 1 µg/ml, and a limit of detection (LOD) of 2 ng/ml. This level of sensitivity is comparable to the standard optical ELISA used widely in clinical applications. The nanocoax array thus matches the detection profile of the standard ELISA while providing a simple electrochemical readout and a miniaturized platform with multiplexing capabilities, toward point-of-care (POC) implementation. In addition, next generation nanocoax devices with extended cores are currently under development, which would provide a POC platform amenable for biofunctionalization of ELISA receptor proteins directly onto the device. This work was supported by the National Institutes of Health (National Cancer Institute Award No. CA137681 and National Institute of Allergy and Infectious Diseases Award No. AI100216).

Sea Anemone (Cnidaria, Anthozoa, Actiniaria) Toxins: An Overview

PubMed Central

Frazão, Bárbara; Vasconcelos, Vitor; Antunes, Agostinho

2012-01-01

The Cnidaria phylum includes organisms that are among the most venomous animals. The Anthozoa class includes sea anemones, hard corals, soft corals and sea pens. The composition of cnidarian venoms is not known in detail, but they appear to contain a variety of compounds. Currently around 250 of those compounds have been identified (peptides, proteins, enzymes and proteinase inhibitors) and non-proteinaceous substances (purines, quaternary ammonium compounds, biogenic amines and betaines), but very few genes encoding toxins were described and only a few related protein three-dimensional structures are available. Toxins are used for prey acquisition, but also to deter potential predators (with neurotoxicity and cardiotoxicity effects) and even to fight territorial disputes. Cnidaria toxins have been identified on the nematocysts located on the tentacles, acrorhagi and acontia, and in the mucous coat that covers the animal body. Sea anemone toxins comprise mainly proteins and peptides that are cytolytic or neurotoxic with its potency varying with the structure and site of action and are efficient in targeting different animals, such as insects, crustaceans and vertebrates. Sea anemones toxins include voltage-gated Na+ and K+ channels toxins, acid-sensing ion channel toxins, Cytolysins, toxins with Kunitz-type protease inhibitors activity and toxins with Phospholipase A2 activity. In this review we assessed the phylogentic relationships of sea anemone toxins, characterized such toxins, the genes encoding them and the toxins three-dimensional structures, further providing a state-of-the-art description of the procedures involved in the isolation and purification of bioactive toxins. PMID:23015776

Sea anemone (Cnidaria, Anthozoa, Actiniaria) toxins: an overview.

PubMed

Frazão, Bárbara; Vasconcelos, Vitor; Antunes, Agostinho

2012-08-01

The Cnidaria phylum includes organisms that are among the most venomous animals. The Anthozoa class includes sea anemones, hard corals, soft corals and sea pens. The composition of cnidarian venoms is not known in detail, but they appear to contain a variety of compounds. Currently around 250 of those compounds have been identified (peptides, proteins, enzymes and proteinase inhibitors) and non-proteinaceous substances (purines, quaternary ammonium compounds, biogenic amines and betaines), but very few genes encoding toxins were described and only a few related protein three-dimensional structures are available. Toxins are used for prey acquisition, but also to deter potential predators (with neurotoxicity and cardiotoxicity effects) and even to fight territorial disputes. Cnidaria toxins have been identified on the nematocysts located on the tentacles, acrorhagi and acontia, and in the mucous coat that covers the animal body. Sea anemone toxins comprise mainly proteins and peptides that are cytolytic or neurotoxic with its potency varying with the structure and site of action and are efficient in targeting different animals, such as insects, crustaceans and vertebrates. Sea anemones toxins include voltage-gated Na⁺ and K⁺ channels toxins, acid-sensing ion channel toxins, Cytolysins, toxins with Kunitz-type protease inhibitors activity and toxins with Phospholipase A2 activity. In this review we assessed the phylogentic relationships of sea anemone toxins, characterized such toxins, the genes encoding them and the toxins three-dimensional structures, further providing a state-of-the-art description of the procedures involved in the isolation and purification of bioactive toxins.

Ricin detection: tracking active toxin.

PubMed

Bozza, William P; Tolleson, William H; Rivera Rosado, Leslie A; Zhang, Baolin

2015-01-01

Ricin is a plant toxin with high bioterrorism potential due to its natural abundance and potency in inducing cell death. Early detection of the active toxin is essential for developing appropriate countermeasures. Here we review concepts for designing ricin detection methods, including mechanism of action of the toxin, advantages and disadvantages of current detection assays, and perspectives on the future development of rapid and reliable methods for detecting ricin in environmental samples. Published by Elsevier Inc.

Multiplex detection of protein toxins using MALDI-TOF-TOF tandem mass spectrometry: application in unambiguous toxin detection from bioaerosol.

PubMed

Alam, Syed Imteyaz; Kumar, Bhoj; Kamboj, Dev Vrat

2012-12-04

Protein toxins, such as botulinum neurotoxins (BoNTs), Clostridium perfringens epsilon toxin (ETX), staphylococcal enterotoxin B (SEB), shiga toxin (STX), and plant toxin ricin, are involved in a number of diseases and are considered as potential agents for bioterrorism and warfare. From a bioterrorism and warfare perspective, these agents are likely to cause maximum damage to a civilian or military population through an inhalational route of exposure and aerosol is considered the envisaged mode of delivery. Unambiguous detection of toxin from aerosol is of paramount importance, both for bringing mitigation protocols into operation and for implementation of effective medical countermeasures, in case a "biological cloud" is seen over a population. A multiplex, unambiguous, and qualitative detection of protein toxins is reported here using tandem mass spectrometry with MALDI-TOF-TOF. The methodology involving simple sample processing steps was demonstrated to identify toxins (ETX, Clostridium perfringes phospholipase C, and SEB) from blind spiked samples. The novel directed search approach using a list of unique peptides was used to identify toxins from a complex protein mixture. The bioinformatic analysis of seven protein toxins for elucidation of unique peptides with conservation status across all known sequences provides a high confidence for detecting toxins originating from any geographical location and source organism. Use of tandem MS data with peptide sequence information increases the specificity of the method. A prototype for generation of aerosol using a nebulizer and collection using a cyclone collector was used to provide a proof of concept for unambiguous detection of toxin from aerosol using precursor directed tandem mass spectrometry combined with protein database searching. ETX prototoxin could be detected from aerosol at 0.2 ppb concentration in aerosol.

Marine and freshwater toxins.

PubMed

Hungerford, James M

2006-01-01

In a very busy and exciting year, 2005 included First Action approval of a much needed official method for paralytic shellfish toxins and multiple international toxin symposia highlighted by groundbreaking research. These are the first-year milestones and activities of the Marine and Freshwater Toxins Task Force and Analytical Community. Inaugurated in 2004 and described in detail in last year's General Referee Report (1) this international toxins group has grown to 150 members from many regions and countries. Perhaps most important they are now making important and global contributions to food safety and to providing alternatives to animal-based assays. Official Method 2005.06 was first approved in late 2004 by the Task Force and subsequently Official First Action in 2005 (2) by the Methods Committee on Natural Toxins and Food Allergens and the Official Methods Board. This nonproprietary method (3) is a precolumn oxidation, liquid chromatographic method that makes good use of fluorescence detection to provide high sensitivity detection of the saxitoxins. It has also proven to be rugged enough for regulatory use and the highest level of validation. As pointed out in the report of method principle investigator and Study Director James Lawrence, approval of 2005.06 now provides the first official alternative to the mouse bioassay after many decades of shellfish monitoring. This past year in April 2005 the group also held their first international conference, "Marine and Freshwater Toxins Analysis: Ist Joint Symposium and AOAC Task Force Meeting," in Baiona, Spain. The 4-day conference consisted of research and stakeholder presentations and symposium-integrated subgroup sessions on ciguatoxins, saxitoxin assays and liquid chromatography (LC) methods for saxitoxins and domoic acids, okadaiates and azaspiracids, and yessotoxins. Many of these subgroups were recently formed in 2005 and are working towards their goals of producing officially validated analytical methods

Role of Botulinum Toxin in Depression.

PubMed

Parsaik, Ajay K; Mascarenhas, Sonia S; Hashmi, Aqeel; Prokop, Larry J; John, Vineeth; Okusaga, Olaoluwa; Singh, Balwinder

2016-03-01

The goal of this review was to consolidate the evidence concerning the efficacy of botulinum toxin type A (onabotulinumtoxinA) in depression. We searched MEDLINE, EMBASE, Cochrane, and Scopus through May 5, 2014, for studies evaluating the efficacy of botulinum toxin A in depression. Only randomized controlled trials were included in the meta-analysis. A pooled mean difference in primary depression score, and pooled odds ratio for response and remission rate with 95% confidence interval (CI) were estimated using the random-effects model. Heterogeneity was assessed using Cochran Q test and χ statistic. Of the 639 articles that were initially retrieved, 5 studies enrolling 194 subjects (age 49±9.6 y) were included in the systematic review, and 3 randomized controlled trials enrolling 134 subjects were included in the meta-analysis. The meta-analysis showed a significant decrease in mean primary depression scores among patients who received botulinum toxin A compared with placebo (-9.80; 95% CI, -12.90 to -6.69) with modest heterogeneity between the studies (Cochran Q test, χ=70). Response and remission rates were 8.3 and 4.6 times higher, respectively, among patients receiving botulinum toxin A compared with placebo, with no heterogeneity between the studies. The 2 studies excluded from the meta-analysis also found a significant decrease in primary depression scores in patients after receiving botulinum toxin A. A few subjects had minor side effects, which were similar between the groups receiving botulinum toxin and those receiving placebo. This study suggests that botulinum toxin A can produce significant improvement in depressive symptoms and is a safe adjunctive treatment for patients receiving pharmacotherapy for depression. Future trials are needed to evaluate the antidepressant effect per se of botulinum toxin A and to further elucidate the underlying antidepressant mechanism of botulinum toxin A.

Emerging insights into the biology of typhoid toxin

PubMed Central

Fowler, Casey C.; Chang, Shu-Jung; Gao, Xiang; Geiger, Tobias; Stack, Gabrielle; Galán, Jorge E.

2017-01-01

Typhoid toxin is a unique A2B5 exotoxin and an important virulence factor for Salmonella Typhi, the cause of typhoid fever. In the decade since its initial discovery, great strides have been made in deciphering the unusual biological program of this toxin, which is fundamentally different from related toxins in many ways. Purified typhoid toxin administered to laboratory animals causes many of the symptoms of typhoid fever, suggesting that typhoid toxin is a central factor in this disease. Further advances in understanding the biology of this toxin will help guide the development of badly needed diagnostics and therapeutic interventions that target this toxin to detect, prevent or treat typhoid fever. PMID:28213043

Botulinum toxin therapy for limb dystonias.

PubMed

Yoshimura, D M; Aminoff, M J; Olney, R K

1992-03-01

We investigated the effectiveness of botulinum toxin in 17 patients with limb dystonias (10 with occupational cramps, three with idiopathic dystonia unrelated to activity, and two each with post-stroke and parkinsonian dystonia) in a placebo-controlled, blinded study. We identified affected muscles clinically and by recording the EMG from implanted wire electrodes at rest and during performance of tasks that precipitated abnormal postures. There were three injections given with graded doses of toxin (average doses, 5 to 10, 10 to 20, and 20 to 40 units per muscle) and one with placebo, in random order. Subjective improvement occurred after 53% of injections of botulinum toxin, and this was substantial in 24%. Only one patient (7%) improved after placebo injection. Subjective improvement occurred in 82% of patients with at least one dose of toxin, lasting for 1 to 4 months. Response rates were similar between clinical groups. Objective evaluation failed to demonstrate significant improvement following treatment with toxin compared with placebo. The major side effect was transient focal weakness after 53% of injections of toxin.

Anthrax toxin.

PubMed

Bhatnagar, R; Batra, S

2001-01-01

Anthrax is primarily a disease of herbivores caused by gram-positive, aerobic, spore-forming Bacillus anthracis. Humans are accidental hosts through the food of animal origin and animal products. Anthrax is prevelant in most parts of the globe, and cases of anthrax have been reported from almost every country. Three forms of the disease have been recognized: cutaneous (through skin), gastrointestinal (through alimentary tract), and pulmonary (by inhalation of spores). The major virulence factors of Bacillus anthracis are a poly-D glutamic acid capsule and a three-component protein exotoxin. The genes coding for the toxin and the enzymes responsible for capsule production are carried on plasmid pXO1 and pXO2, respectively. The three proteins of the exotoxin are protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa). The toxins follow the A-B model with PA being the B moeity and LF/EF, the alternative A moeities. LF and EF are individually nontoxic, but in combination with PA form two toxins causing different pathogenic responses in animals and cultured cells. PA + LF forms the lethal toxin and PA + EF forms the edema toxin. During the process of intoxication, PA binds to the cell surface receptor and is cleaved at the sequence RKKR (167) by cell surface proteases such as furin generating a cell-bound, C-terminal 63 kDa protein (PA63). PA63 possesses a binding site to which LF or EF bind with high affinity. The complex is then internalized by receptor-mediated endocytosis. Acidification of the vesicle leads to instertion of PA63 into the endosomal membrane and translocation of LF/EF across the bilayer into the cytosol where they exert their toxic effects. EF has a calcium- and calmodulin-dependent adenylate cyclase activity. Recent reports indicate that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and 2), and this cleavage inactivates MAPKK1 and thus inhibits the

Partial purification of a toxin found in hamsters with antibiotic-associated colitis. Reversible binding of the toxin by cholestyramine.

PubMed

Humphrey, C D; Condon, C W; Cantey, J R; Pittman, F E

1979-03-01

A toxin with cytotoxic and enterotoxic activities was isolated from cecal contents of hamsters receiving lincomycin. The toxin was partially purified by ultracentrifugation, ultrafiltration, (NH4)2SO4 precipitation, and gel filtration. Cytotoxic activity, assayed on monolayers of HeLa cells, was restricted to material that eluted in the molecular weight range of 107,000 +/- 6,000 daltons. Cytotoxicity of crude AAC toxin could be demonstrated at concentrations as low as 0.04 microgram/ml. The toxin was heat labile (55 degrees-60 degrees C for 0.5 hr) and sensitive to trypsinization, acidification at pH 3, or alkalinization at pH 9. Cytotoxic activity was inhibited by Clostridium sordellii antitoxin. Enterotoxic activity of the crude toxin and the cytotoxic fraction from gel filtration was demonstrated by fluid secretion in ligated rabbit ileal loops. Studies were done in vitro with cholestyramine resin, vancomycin, or gentamicin to determine if the toxin was bound or denatured by these drugs. It was demonstrated that cholestyramine bound the toxin, significantly reducing its cytotoxicity. Reversible binding of the cytotoxic material was demonstrated by salt gradient elution. Neither vancomycin nor gentamicin had any effect on the in vitro cytotoxic activity of the toxin.

Identification of novel mazEF/pemIK family toxin-antitoxin loci and their distribution in the Staphylococcus genus.

PubMed

Bukowski, Michal; Hyz, Karolina; Janczak, Monika; Hydzik, Marcin; Dubin, Grzegorz; Wladyka, Benedykt

2017-10-18

The versatile roles of toxin-antitoxin (TA) systems in bacterial physiology and pathogenesis have been investigated for more than three decades. Diverse TA loci in Bacteria and Archaea have been identified in genome-wide studies. The advent of massive parallel sequencing has substantially expanded the number of known bacterial genomic sequences over the last 5 years. In staphylococci, this has translated into an impressive increase from a few tens to a several thousands of available genomes, which has allowed us for the re-evalution of prior conclusions. In this study, we analysed the distribution of mazEF/pemIK family TA system operons in available staphylococcal genomes and their prevalence in mobile genetic elements. 10 novel m azEF/pemIK homologues were identified, each with a corresponding toxin that plays a potentially different and undetermined physiological role. A detailed characterisation of these TA systems would be exceptionally useful. Of particular interest are those associated with an SCCmec mobile genetic element (responsible for multidrug resistance transmission) or representing the joint horizontal transfer of TA systems and determinants of vancomycin resistance from enterococci. The involvement of TA systems in maintaining mobile genetic elements and the associations between novel mazEF/pemIK loci and those which carry drug resistance genes highlight their potential medical importance.

Botulinum Toxin for the Management of Sjögren Syndrome-Associated Recurrent Parotitis.

PubMed

O'Neil, Luke M; Palme, Carsten E; Riffat, Faruque; Mahant, Neil

2016-12-01

Recurrent parotitis is a rare manifestation of Sjögren syndrome. The management of recurrent parotitis is challenging because conservative methods may be of limited efficacy and invasive approaches carry the risk of complications. Botulinum toxin has been shown to reduce salivary flow, and consequently, the results of its use in the management of recurrent parotitis have been encouraging. A 65-year-old female patient with recurrent parotitis due to Sjögren syndrome was referred to us, complaining of weekly bouts of inflammation. She required a course of antibiotics monthly to control bacterial superinfections. We treated her with onabotulinumtoxinA injections into both parotid glands at regular intervals. After her second injection cycle, she denied further inflammatory bouts, has not required antibiotics in more than 36 months, and denied any side effects. Botulinum toxin may be a safe and effective method of treating Sjögren syndrome-associated recurrent parotitis. Copyright © 2016 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

Marine Neurotoxins: Ingestible Toxins.

PubMed

Stommel, Elijah W.; Watters, Michael R.

2004-03-01

Fish and shellfish account for a significant portion of food-borne illnesses throughout the world. In general, three classes of diseases result from seafood consumption--intoxication, allergies, and infections. In this review, the authors discuss several seafood-borne toxins, including domoic acid, which acts on the central nervous system. In addition, the authors discuss ciguatoxin-, brevetoxin-, saxitoxin-, tetrodotoxin-, and scombroid-related histamine toxicity, all of which act primarily on the peripheral nervous system. Fish has become a very popular food in the US mostly related to its potential health benefits. Fish is consumed to such a degree that fishing stocks are reportedly at an all time low from what seemed like an endless supply even 30 years ago. One of the most significant threats to human intoxication is the recreational harvest of shellfish, often times located in remote locations where the harvesters are subsistent on fishery resources and have no monitoring in place. The hazard to intoxication is not as common in purchased seafood, which is more stringently regulated, yet still is a serious problem. Most ingestible toxins are thermo-stable and therefore unaffected by cooking, freezing, or salting. Air transport of consumable products throughout the world makes it easy to obtain exotic edibles from far away countries. A seemingly unusual toxin can be more commonly encountered than previously thought and it is important to consider this when evaluating patients. Recognition and treatment of various neurologic symptoms related to seafood ingestion is paramount in today's mobile, gastronomic world. Specific treatments vary with each individual toxin and with the individual's specific reaction to the toxin. Generally, some degree of medical care is required with all ingestible toxin exposure, ranging from simple administration of medication and hydration to ventilatory and cardiovascular support.

Engineered Toxins “Zymoxins” Are Activated by the HCV NS3 Protease by Removal of an Inhibitory Protein Domain

PubMed Central

Shapira, Assaf; Gal-Tanamy, Meital; Nahary, Limor; Litvak-Greenfeld, Dana; Zemel, Romy; Tur-Kaspa, Ran; Benhar, Itai

2011-01-01

The synthesis of inactive enzyme precursors, also known as “zymogens,” serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated “zymogenized” chimeric toxins (which we denote “zymoxins”). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the “zymoxin” approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected. PMID:21264238

Military Importance of Natural Toxins and Their Analogs.

PubMed

Pitschmann, Vladimír; Hon, Zdeněk

2016-04-28

Toxin weapon research, development, production and the ban on its uses is an integral part of international law, with particular attention paid to the protection against these weapons. In spite of this, hazards associated with toxins cannot be completely excluded. Some of these hazards are also pointed out in the present review. The article deals with the characteristics and properties of natural toxins and synthetic analogs potentially constituting the basis of toxin weapons. It briefly describes the history of military research and the use of toxins from distant history up to the present age. With respect to effective disarmament conventions, it mentions certain contemporary concepts of possible toxin applications for military purposes and the protection of public order (suppression of riots); it also briefly refers to the question of terrorism. In addition, it deals with certain traditional as well as modern technologies of the research, synthesis, and use of toxins, which can affect the continuing development of toxin weapons. These are, for example, cases of new toxins from natural sources, their chemical synthesis, production of synthetic analogs, the possibility of using methods of genetic engineering and modern biotechnologies or the possible applications of nanotechnology and certain pharmaceutical methods for the effective transfer of toxins into the organism. The authors evaluate the military importance of toxins based on their comparison with traditional chemical warfare agents. They appeal to the ethics of the scientific work as a principal condition for the prevention of toxin abuse in wars, military conflicts, as well as in non-military attacks.

Multimechanistic Monoclonal Antibodies (MAbs) Targeting Staphylococcus aureus Alpha-Toxin and Clumping Factor A: Activity and Efficacy Comparisons of a MAb Combination and an Engineered Bispecific Antibody Approach.

PubMed

Tkaczyk, C; Kasturirangan, S; Minola, A; Jones-Nelson, O; Gunter, V; Shi, Y Y; Rosenthal, K; Aleti, V; Semenova, E; Warrener, P; Tabor, D; Stover, C K; Corti, D; Rainey, G; Sellman, B R

2017-08-01

Secreted alpha-toxin and surface-localized clumping factor A (ClfA) are key virulence determinants in Staphylococcus aureus bloodstream infections. We previously demonstrated that prophylaxis with a multimechanistic monoclonal antibody (MAb) combination against alpha-toxin (MEDI4893*) and ClfA (11H10) provided greater strain coverage and improved efficacy in an S. aureus lethal bacteremia model. Subsequently, 11H10 was found to exhibit reduced affinity and impaired inhibition of fibrinogen binding to ClfA002 expressed by members of a predominant hospital-associated methicillin-resistant S. aureus (MRSA) clone, ST5. Consequently, we identified another anti-ClfA MAb (SAR114) from human tonsillar B cells with >100-fold increased affinity for three prominent ClfA variants, including ClfA002, and potent inhibition of bacterial agglutination by 112 diverse clinical isolates. We next constructed bispecific Abs (BiSAbs) comprised of 11H10 or SAR114 as IgG scaffolds and grafted anti-alpha-toxin (MEDI4893*) single-chain variable fragment to the amino or carboxy terminus of the anti-ClfA heavy chains. Although the BiSAbs exhibited in vitro potencies similar to those of the parental MAbs, only 11H10-BiSAb, but not SAR114-BiSAb, showed protective activity in murine infection models comparable to the respective MAb combination. In vivo activity with SAR114-BiSAb was observed in infection models with S. aureus lacking ClfA. Our data suggest that high-affinity binding to ClfA sequesters the SAR114-BiSAb to the bacterial surface, thereby reducing both alpha-toxin neutralization and protection in vivo These results indicate that a MAb combination targeting ClfA and alpha-toxin is more promising for future development than the corresponding BiSAb. Copyright © 2017 Tkaczyk et al.

Immunochromatographic Strip Test for Rapid Detection of Diphtheria Toxin: Description and Multicenter Evaluation in Areas of Low and High Prevalence of Diphtheria

PubMed Central

Engler, K. H.; Efstratiou, A.; Norn, D.; Kozlov, R. S.; Selga, I.; Glushkevich, T. G.; Tam, M.; Melnikov, V. G.; Mazurova, I. K.; Kim, V. E.; Tseneva, G. Y.; Titov, L. P.; George, R. C.

2002-01-01

An immunochromatographic strip (ICS) test was developed for the detection of diphtheria toxin by using an equine polyclonal antibody as the capture antibody and colloidal gold-labeled monoclonal antibodies specific for fragment A of the diphtheria toxin molecule as the detection antibody. The ICS test has been fully optimized for the detection of toxin from bacterial cultures; the limit of detection was approximately 0.5 ng of diphtheria toxin per ml within 10 min. In a comparative study with 915 pure clinical isolates of Corynebacterium spp., the results of the ICS test were in complete agreement with those of the conventional Elek test. The ICS test was also evaluated for its ability to detect toxigenicity from clinical specimens (throat swabs) in two field studies conducted within areas of the former USSR where diphtheria is epidemic. Eight hundred fifty throat swabs were examined by conventional culture and by use of directly inoculated broth cultures for the ICS test. The results showed 99% concordance (848 of 850 specimens), and the sensitivity and specificity of the ICS test were 98% (95% confidence interval, 91 to 99%) and 99% (95% confidence interval, 99 to 100%), respectively. PMID:11773096

A New Type of Toxin A-Negative, Toxin B-Positive Clostridium difficile Strain Lacking a Complete tcdA Gene

PubMed Central

Marín, Mercedes; Martín, Adoración; Rupnik, Maja

2014-01-01

Toxins A and B are the main virulence factors of Clostridium difficile and are the targets for molecular diagnostic tests. Here, we describe a new toxin A-negative, toxin B-positive, binary toxin CDT (Clostridium difficile transferase)-negative (A− B+ CDT−) toxinotype (XXXII) characterized by a variant type of pathogenicity locus (PaLoc) without tcdA and with atypical organization of the PaLoc integration site. PMID:25428159

Urea, a true uremic toxin: the empire strikes back.

PubMed

Lau, Wei Ling; Vaziri, Nosratola D

2017-01-01

Blood levels of urea rise with progressive decline in kidney function. Older studies examining acute urea infusion suggested that urea was well-tolerated at levels 8-10× above normal values. More recent in vitro and in vivo work argue the opposite and demonstrate both direct and indirect toxicities of urea, which probably promote the premature aging phenotype that is pervasive in chronic kidney disease (CKD). Elevated urea at concentrations typically encountered in uremic patients induces disintegration of the gut epithelial barrier, leading to translocation of bacterial toxins into the bloodstream and systemic inflammation. Urea induces apoptosis of vascular smooth muscle cells as well as endothelial dysfunction, thus directly promoting cardiovascular disease. Further, urea stimulates oxidative stress and dysfunction in adipocytes, leading to insulin resistance. Finally, there are widespread indirect effects of elevated urea as a result of the carbamylation reaction, where isocyanic acid (a product of urea catabolism) alters the structure and function of proteins in the body. Carbamylation has been linked with renal fibrosis, atherosclerosis and anaemia. In summary, urea is a re-emerging Dark Force in CKD pathophysiology. Trials examining low protein diet to minimize accumulation of urea and other toxins suggest a clinical benefit in terms of slowing progression of CKD. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

VapC from the Leptospiral VapBC Toxin-Antitoxin Module Displays Ribonuclease Activity on the Initiator tRNA

PubMed Central

Lopes, Alexandre P. Y.; Lopes, Luana M.; Fraga, Tatiana R.; Chura-Chambi, Rosa M.; Sanson, André L.; Cheng, Elisabeth; Nakajima, Erika; Morganti, Ligia; Martins, Elizabeth A. L.

2014-01-01

The prokaryotic ubiquitous Toxin-Antitoxin (TA) operons encode a stable toxin and an unstable antitoxin. The most accepted hypothesis of the physiological function of the TA system is the reversible cessation of cellular growth under stress conditions. The major TA family, VapBC is present in the spirochaete Leptospira interrogans. VapBC modules are classified based on the presence of a predicted ribonucleasic PIN domain in the VapC toxin. The expression of the leptospiral VapC in E. coli promotes a strong bacterial growth arrestment, making it difficult to express the recombinant protein. Nevertheless, we showed that long term induction of expression in E. coli enabled the recovery of VapC in inclusion bodies. The recombinant protein was successfully refolded by high hydrostatic pressure, providing a new method to obtain the toxin in a soluble and active form. The structural integrity of the recombinant VapB and VapC proteins was assessed by circular dichroism spectroscopy. Physical interaction between the VapC toxin and the VapB antitoxin was demonstrated in vivo and in vitro by pull down and ligand affinity blotting assays, respectively, thereby indicating the ultimate mechanism by which the activity of the toxin is regulated in bacteria. The predicted model of the leptospiral VapC structure closely matches the Shigella's VapC X-ray structure. In agreement, the ribonuclease activity of the leptospiral VapC was similar to the activity described for Shigella's VapC, as demonstrated by the cleavage of tRNAfMet and by the absence of unspecific activity towards E. coli rRNA. This finding suggests that the cleavage of the initiator transfer RNA may represent a common mechanism to a larger group of bacteria and potentially configures a mechanism of post-transcriptional regulation leading to the inhibition of global translation. PMID:25047537

VapC from the leptospiral VapBC toxin-antitoxin module displays ribonuclease activity on the initiator tRNA.

PubMed

Lopes, Alexandre P Y; Lopes, Luana M; Fraga, Tatiana R; Chura-Chambi, Rosa M; Sanson, André L; Cheng, Elisabeth; Nakajima, Erika; Morganti, Ligia; Martins, Elizabeth A L

2014-01-01

The prokaryotic ubiquitous Toxin-Antitoxin (TA) operons encode a stable toxin and an unstable antitoxin. The most accepted hypothesis of the physiological function of the TA system is the reversible cessation of cellular growth under stress conditions. The major TA family, VapBC is present in the spirochaete Leptospira interrogans. VapBC modules are classified based on the presence of a predicted ribonucleasic PIN domain in the VapC toxin. The expression of the leptospiral VapC in E. coli promotes a strong bacterial growth arrestment, making it difficult to express the recombinant protein. Nevertheless, we showed that long term induction of expression in E. coli enabled the recovery of VapC in inclusion bodies. The recombinant protein was successfully refolded by high hydrostatic pressure, providing a new method to obtain the toxin in a soluble and active form. The structural integrity of the recombinant VapB and VapC proteins was assessed by circular dichroism spectroscopy. Physical interaction between the VapC toxin and the VapB antitoxin was demonstrated in vivo and in vitro by pull down and ligand affinity blotting assays, respectively, thereby indicating the ultimate mechanism by which the activity of the toxin is regulated in bacteria. The predicted model of the leptospiral VapC structure closely matches the Shigella's VapC X-ray structure. In agreement, the ribonuclease activity of the leptospiral VapC was similar to the activity described for Shigella's VapC, as demonstrated by the cleavage of tRNAfMet and by the absence of unspecific activity towards E. coli rRNA. This finding suggests that the cleavage of the initiator transfer RNA may represent a common mechanism to a larger group of bacteria and potentially configures a mechanism of post-transcriptional regulation leading to the inhibition of global translation.

Rapid Bacterial Detection via an All-Electronic CMOS Biosensor

PubMed Central

Nikkhoo, Nasim; Cumby, Nichole; Gulak, P. Glenn; Maxwell, Karen L.

2016-01-01

The timely and accurate diagnosis of infectious diseases is one of the greatest challenges currently facing modern medicine. The development of innovative techniques for the rapid and accurate identification of bacterial pathogens in point-of-care facilities using low-cost, portable instruments is essential. We have developed a novel all-electronic biosensor that is able to identify bacteria in less than ten minutes. This technology exploits bacteriocins, protein toxins naturally produced by bacteria, as the selective biological detection element. The bacteriocins are integrated with an array of potassium-selective sensors in Complementary Metal Oxide Semiconductor technology to provide an inexpensive bacterial biosensor. An electronic platform connects the CMOS sensor to a computer for processing and real-time visualization. We have used this technology to successfully identify both Gram-positive and Gram-negative bacteria commonly found in human infections. PMID:27618185

Cationic antimicrobial peptides inactivate Shiga toxin-encoding bacteriophages

NASA Astrophysics Data System (ADS)

Del Cogliano, Manuel E.; Hollmann, Axel; Martinez, Melina; Semorile, Liliana; Ghiringhelli, Pablo D.; Maffía, Paulo C.; Bentancor, Leticia V.

2017-12-01

Shiga toxin (Stx) is the principal virulence factor during Shiga toxin-producing Escherichia coli (STEC) infections. We have previously reported the inactivation of bacteriophage encoding Stx after treatment with chitosan, a linear polysaccharide polymer with cationic properties. Cationic antimicrobial peptides (cAMPs) are short linear aminoacidic sequences, with a positive net charge, which display bactericidal or bacteriostatic activity against a wide range of bacterial species. They are promising novel antibiotics since they have shown bactericidal effects against multiresistant bacteria. To evaluate whether cationic properties are responsible for bacteriophage inactivation, we tested seven cationic peptides with proven antimicrobial activity as anti-bacteriophage agents, and one random sequence cationic peptide with no antimicrobial activity as a control. We observed bacteriophage inactivation after incubation with five cAMPs, but no inactivating activity was observed with the random sequence cationic peptide or with the non alpha helical cAMP Omiganan. Finally, to confirm peptide-bacteriophage interaction, zeta potential was analyzed by following changes on bacteriophage surface charges after peptide incubation. According to our results we could propose that: 1) direct interaction of peptides with phage is a necessary step for bacteriophage inactivation, 2) cationic properties are necessary but not sufficient for bacteriophage inactivation, and 3) inactivation by cationic peptides could be sequence (or structure) specific. Overall our data suggest that these peptides could be considered a new family of molecules potentially useful to decrease bacteriophage replication and Stx expression.

Toxins of filamentous fungi.

PubMed

Bhatnagar, Deepak; Yu, Jiujiang; Ehrlich, Kenneth C

2002-01-01

Mycotoxins are low-molecular-weight secondary metabolites of fungi. The most significant mycotoxins are contaminants of agricultural commodities, foods and feeds. Fungi that produce these toxins do so both prior to harvest and during storage. Although contamination of commodities by toxigenic fungi occurs frequently in areas with a hot and humid climate (i.e. conditions favorable for fungal growth), they can also be found in temperate conditions. Production of mycotoxins is dependent upon the type of producing fungus and environmental conditions such as the substrate, water activity (moisture and relative humidity), duration of exposure to stress conditions and microbial, insect or other animal interactions. Although outbreaks of mycotoxicoses in humans have been documented, several of these have not been well characterized, neither has a direct correlation between the mycotoxin and resulting toxic effect been well established in vivo. Even though the specific modes of action of most of the toxins are not well established, acute and chronic effects in prokaryotic and eukaryotic systems, including humans have been reported. The toxicity of the mycotoxins varies considerably with the toxin, the animal species exposed to it, and the extent of exposure, age and nutritional status. Most of the toxic effects of mycotoxins are limited to specific organs, but several mycotoxins affect many organs. Induction of cancer by some mycotoxins is a major concern as a chronic effect of these toxins. It is nearly impossible to eliminate mycotoxins from the foods and feed in spite of the regulatory efforts at the national and international levels to remove the contaminated commodities. This is because mycotoxins are highly stable compounds, the producing fungi are ubiquitous, and food contamination can occur both before and after harvest. Nevertheless, good farm management practices and adequate storage facilities minimize the toxin contamination problems. Current research is

Activation of Bacteroides fragilis toxin by a novel bacterial protease contributes to anaerobic sepsis in mice.

PubMed

Choi, Vivian M; Herrou, Julien; Hecht, Aaron L; Teoh, Wei Ping; Turner, Jerrold R; Crosson, Sean; Bubeck Wardenburg, Juliane

2016-05-01

Bacteroides fragilis is the leading cause of anaerobic bacteremia and sepsis. Enterotoxigenic strains that produce B. fragilis toxin (BFT, fragilysin) contribute to colitis and intestinal malignancy, yet are also isolated in bloodstream infection. It is not known whether these strains harbor unique genetic determinants that confer virulence in extra-intestinal disease. We demonstrate that BFT contributes to sepsis in mice, and we identify a B. fragilis protease called fragipain (Fpn) that is required for the endogenous activation of BFT through the removal of its auto-inhibitory prodomain. Structural analysis of Fpn reveals a His-Cys catalytic dyad that is characteristic of C11-family cysteine proteases that are conserved in multiple pathogenic Bacteroides spp. and Clostridium spp. Fpn-deficient, enterotoxigenic B. fragilis has an attenuated ability to induce sepsis in mice; however, Fpn is dispensable in B. fragilis colitis, wherein host proteases mediate BFT activation. Our findings define a role for B. fragilis enterotoxin and its activating protease in the pathogenesis of bloodstream infection, which indicates a greater complexity of cellular targeting and activity of BFT than previously recognized. The expression of fpn by both toxigenic and nontoxigenic strains suggests that this protease may contribute to anaerobic sepsis in ways that extend beyond its role in toxin activation. It could thus potentially serve as a target for disease modification.

Differential Requirement for the Translocation of Clostridial Binary Toxins: Iota Toxin Requires a Membrane Potential Gradient

DTIC Science & Technology

2007-02-28

be inter- changed to form biologically-active, hybrid toxins. Thereby, Ib can internalize the enzymatic components from either C. spiroforme or C...since chloro- quine, monensin, nigericin, or ammonium chloride did not in- hibit its activity (Fig. 2A). C. spiroforme toxin, which is very highly...ER [50]. These results are in agreement with a recent report [15], and an earlier finding [45] that there was no effect of C. spiroforme toxin when

Toxin Detection by Surface Plasmon Resonance

PubMed Central

Hodnik, Vesna; Anderluh, Gregor

2009-01-01

Significant efforts have been invested in the past years for the development of analytical methods for fast toxin detection in food and water. Immunochemical methods like ELISA, spectroscopy and chromatography are the most used in toxin detection. Different methods have been linked, e.g. liquid chromatography and mass spectrometry (LC-MS), in order to detect as low concentrations as possible. Surface plasmon resonance (SPR) is one of the new biophysical methods which enables rapid toxin detection. Moreover, this method was already included in portable sensors for on-site determinations. In this paper we describe some of the most common methods for toxin detection, with an emphasis on SPR. PMID:22573957

Identification of the cellular receptor of Clostridium spiroforme toxin.

PubMed

Papatheodorou, Panagiotis; Wilczek, Claudia; Nölke, Thilo; Guttenberg, Gregor; Hornuss, Daniel; Schwan, Carsten; Aktories, Klaus

2012-04-01

Clostridium spiroforme produces the binary actin-ADP-ribosylating toxin CST (C. spiroforme toxin), which has been proposed to be responsible for diarrhea, enterocolitis, and eventually death, especially in rabbits. Here we report on the recombinant production of the enzyme component (CSTa) and the binding component (CSTb) of C. spiroforme toxin in Bacillus megaterium. By using the recombinant toxin components, we show that CST enters target cells via the lipolysis-stimulated lipoprotein receptor (LSR), which has been recently identified as the host cell receptor of the binary toxins Clostridium difficile transferase (CDT) and Clostridium perfringens iota toxin. Microscopic studies revealed that CST, but not the related Clostridium botulinum C2 toxin, colocalized with LSR during toxin uptake and traffic to endosomal compartments. Our findings indicate that CST shares LSR with C. difficile CDT and C. perfringens iota toxin as a host cell surface receptor.

Identification of the Cellular Receptor of Clostridium spiroforme Toxin

PubMed Central

Papatheodorou, Panagiotis; Wilczek, Claudia; Nölke, Thilo; Guttenberg, Gregor; Hornuss, Daniel; Schwan, Carsten

2012-01-01

Clostridium spiroforme produces the binary actin-ADP-ribosylating toxin CST (C. spiroforme toxin), which has been proposed to be responsible for diarrhea, enterocolitis, and eventually death, especially in rabbits. Here we report on the recombinant production of the enzyme component (CSTa) and the binding component (CSTb) of C. spiroforme toxin in Bacillus megaterium. By using the recombinant toxin components, we show that CST enters target cells via the lipolysis-stimulated lipoprotein receptor (LSR), which has been recently identified as the host cell receptor of the binary toxins Clostridium difficile transferase (CDT) and Clostridium perfringens iota toxin. Microscopic studies revealed that CST, but not the related Clostridium botulinum C2 toxin, colocalized with LSR during toxin uptake and traffic to endosomal compartments. Our findings indicate that CST shares LSR with C. difficile CDT and C. perfringens iota toxin as a host cell surface receptor. PMID:22252869

Update on bacterial pathogenesis in BRD.

PubMed

Confer, Anthony W

2009-12-01

Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycoplasma bovis and Arcanobacterium pyogenes are all frequently implicated in bovine respiratory disease (BRD). M. haemolytica is considered the most important of the group. These bacteria are commensals in the nasopharynx and establish infection in the lungs of cattle that are subjected to a variety of stresses. Factors that permit adherence to and proliferation in the lungs and factors that cause tissue destruction and inflammation have been identified as having major roles in pathogenesis. These virulence factors include protein adhesins, capsular polysaccharide, outer membrane proteins, iron-binding proteins, lipopolysacharide or lipooligosaccharide, enzymes and toxins. These bacterial products function to evade the immune system, damage the immune system and induce a severe inflammatory response.

Proteomic Methods of Detection and Quantification of Protein Toxins.

PubMed

Duracova, Miloslava; Klimentova, Jana; Fucikova, Alena; Dresler, Jiri

2018-02-28

Biological toxins are a heterogeneous group of compounds that share commonalities with biological and chemical agents. Among them, protein toxins represent a considerable, diverse set. They cover a broad range of molecular weights from less than 1000 Da to more than 150 kDa. This review aims to compare conventional detection methods of protein toxins such as in vitro bioassays with proteomic methods, including immunoassays and mass spectrometry-based techniques and their combination. Special emphasis is given to toxins falling into a group of selected agents, according to the Centers for Disease Control and Prevention, such as Staphylococcal enterotoxins , Bacillus anthracis toxins, Clostridium botulinum toxins, Clostridium perfringens epsilon toxin, ricin from Ricinus communis , Abrin from Abrus precatorius or control of trade in dual-use items in the European Union, including lesser known protein toxins such as Viscumin from Viscum album . The analysis of protein toxins and monitoring for biological threats, i.e., the deliberate spread of infectious microorganisms or toxins through water, food, or the air, requires rapid and reliable methods for the early identification of these agents.

Proteomic Methods of Detection and Quantification of Protein Toxins

PubMed Central

Klimentova, Jana; Fucikova, Alena

2018-01-01

Biological toxins are a heterogeneous group of compounds that share commonalities with biological and chemical agents. Among them, protein toxins represent a considerable, diverse set. They cover a broad range of molecular weights from less than 1000 Da to more than 150 kDa. This review aims to compare conventional detection methods of protein toxins such as in vitro bioassays with proteomic methods, including immunoassays and mass spectrometry-based techniques and their combination. Special emphasis is given to toxins falling into a group of selected agents, according to the Centers for Disease Control and Prevention, such as Staphylococcal enterotoxins, Bacillus anthracis toxins, Clostridium botulinum toxins, Clostridium perfringens epsilon toxin, ricin from Ricinus communis, Abrin from Abrus precatorius or control of trade in dual-use items in the European Union, including lesser known protein toxins such as Viscumin from Viscum album. The analysis of protein toxins and monitoring for biological threats, i.e., the deliberate spread of infectious microorganisms or toxins through water, food, or the air, requires rapid and reliable methods for the early identification of these agents. PMID:29495560

Lipoproteins/peptides are sepsis-inducing toxins from bacteria that can be neutralized by synthetic anti-endotoxin peptides

PubMed Central

de Tejada, Guillermo Martinez; Heinbockel, Lena; Ferrer-Espada, Raquel; Heine, Holger; Alexander, Christian; Bárcena-Varela, Sergio; Goldmann, Torsten; Correa, Wilmar; Wiesmüller, Karl-Heinz; Gisch, Nicolas; Sánchez-Gómez, Susana; Fukuoka, Satoshi; Schürholz, Tobias; Gutsmann, Thomas; Brandenburg, Klaus

2015-01-01

Sepsis, a life-threatening syndrome with increasing incidence worldwide, is triggered by an overwhelming inflammation induced by microbial toxins released into the bloodstream during infection. A well-known sepsis-inducing factor is the membrane constituent of Gram-negative bacteria, lipopolysaccharide (LPS), signalling via Toll-like receptor-4. Although sepsis is caused in more than 50% cases by Gram-positive and mycoplasma cells, the causative compounds are still poorly described. In contradicting investigations lipoproteins/-peptides (LP), lipoteichoic acids (LTA), and peptidoglycans (PGN), were made responsible for eliciting this pathology. Here, we used human mononuclear cells from healthy donors to determine the cytokine-inducing activity of various LPs from different bacterial origin, synthetic and natural, and compared their activity with that of natural LTA and PGN. We demonstrate that LP are the most potent non-LPS pro-inflammatory toxins of the bacterial cell walls, signalling via Toll-like receptor-2, not only in vitro, but also when inoculated into mice: A synthetic LP caused sepsis-related pathological symptoms in a dose-response manner. Additionally, these mice produced pro-inflammatory cytokines characteristic of a septic reaction. Importantly, the recently designed polypeptide Aspidasept® which has been proven to efficiently neutralize LPS in vivo, inhibited cytokines induced by the various non-LPS compounds protecting animals from the pro-inflammatory activity of synthetic LP. PMID:26390973

Crystallization of isoelectrically homogeneous cholera toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spangler, B.D.; Westbrook, E.M.

1989-02-07

Past difficulty in growing good crystals of cholera toxin has prevented the study of the crystal structure of this important protein. The authors have determined that failure of cholera toxin to crystallize well has been due to its heterogeneity. They have now succeeded in overcoming the problem by isolating a single isoelectric variant of this oligomeric protein (one A subunit and five B subunits). Cholera toxin purified by their procedure readily forms large single crystals. The crystal form has been described previously. They have recorded data from native crystals of cholera toxin to 3.0-{angstrom} resolution with our electronic area detectors.more » With these data, they have found the orientation of a 5-fold symmetry axis within these crystals, perpendicular to the screw dyad of the crystal. They are now determining the crystal structure of cholera toxin by a combination of multiple heavy-atom isomorphous replacement and density modification techniques, making use of rotational 5-fold averaging of the B subunits.« less

Botulinum toxin in trigeminal neuralgia.

PubMed

Castillo-Álvarez, Federico; Hernando de la Bárcena, Ignacio; Marzo-Sola, María Eugenia

2017-01-06

Trigeminal neuralgia is one of the most disabling facial pain syndromes, with a significant impact on patients' quality of life. Pharmacotherapy is the first choice for treatment but cases of drug resistance often require new strategies, among which various interventional treatments have been used. In recent years a new therapeutic strategy consisting of botulinum toxin has emerged, with promising results. We reviewed clinical cases and case series, open-label studies and randomized clinical trials examining the use of botulinum toxin for drug-refractory trigeminal neuralgia published in the literature. The administration of botulinum toxin has proven to be a safe and effective therapeutic strategy in patients with drug-refractory idiopathic trigeminal neuralgia, but many questions remain unanswered as to the precise role of botulinum toxin in the treatment of this disease. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

Real-time microfluidic recombinase polymerase amplification for the toxin B gene of Clostridium difficile on a SlipChip platform.

PubMed

Tsaloglou, M-N; Watson, R J; Rushworth, C M; Zhao, Y; Niu, X; Sutton, J M; Morgan, H

2015-01-07

Clostridium difficile is one of the key bacterial pathogens that cause infectious diarrhoea both in the developed and developing world. Isothermal nucleic acid amplification methods are increasingly used for identification of toxinogenic infection by clinical labs. For this purpose, we developed a low-cost microfluidic platform based on the SlipChip concept and implemented real-time isothermal recombinase polymerase amplification (RPA). The on-chip RPA assay targets the Clostridium difficile toxin B gene (tcdB) coding for toxin B, one of the proteins responsible for bacterial toxicity. The device was fabricated in clear acrylic using rapid prototyping methods. It has six replicate 500 nL reaction wells as well as two sets of 500 nL control wells. The reaction can be monitored in real-time using exonuclease fluorescent probes with an initial sample volume of as little as 6.4 μL. We demonstrated a limit of detection of 1000 DNA copies, corresponding to 1 fg, at a time-to-result of <20 minutes. This miniaturised platform for pathogen detection has potential for use in resource-limited environments or at the point-of-care because of its ease of use and low cost, particularly if combined with preserved reagents.

Collaborative Research Program on Seafood Toxins

DTIC Science & Technology

1988-08-14

Crystallographic Structures of Saxitoxins Cl and C2 Appendix C: Collaborative Research Program an Seafcod Toxins Progress Report on Ciguatera and Related...radioimmunoassay for PSP were also evalumted. The Hokama stick test for ciguatera toxin was also evaluated. 4. initiate Studies on the Accumulation...tco•d which caie a form of b-mnn poisoning referred to as ciguatera . The respcnsible toxins originate from ll1ular rine algae of the division

Legionella pneumophila Toxin, Isolation and Purification

DTIC Science & Technology

1981-01-01

which dis- plays an in vivo lethality. The purification procedures involve acid precipitation, gel chromatography, and preparative isotachophoresis. The...Chymotrypsinogen A, Ribonuclease A, and Apoprotinin as markers. Preparation of antiserum One milliqram amounts of protein from Le jonella acid ...RESULTS Toxin isolation Step 1: Acid precipitation of crude toxin. 1.0 N HCl acid was slowly added to rapidly stirred crude toxin until pH 3.5 was

Algal toxins

USGS Publications Warehouse

Creekmore, Lynn H.

1999-01-01

Periodic blooms of algae, including true algae, dinoflagellates, and cyanobacteria or blue-green algae have been reported in marine and freshwater bodies throughout the world. Although many blooms are merely an aesthetic nuisance, some species of algae produce toxins that kill fish, shellfish, humans, livestock and wildlife. Pigmented blooms of toxinproducing marine algae are often referred to as “red tides” (Fig. 36.1). Proliferations of freshwater toxin-producing cyanobacteria are simply called “cyanobacterial blooms” or “toxic algal blooms.” Cyanobacterial blooms initially appear green and may later turn blue, sometimes forming a “scum” in the water (Fig. 36.2).Although algal blooms historically have been considered a natural phenomenon, the frequency of occurrence of harmful algae appears to have increased in recent years. Agricultural runoff and other pollutants of freshwater and marine wetlands and water bodies have resulted in increased nutrient loading of phosphorus and nitrogen, thus providing conditions favorable to the growth of potentially toxic algae. The detrimental impact of red tides and cyanobacterial blooms on wetland, shore, and pelagic species has long been suspected but not often been substantiated because information on the effects of these toxins in fish and wildlife species is lacking and diagnostic tools are limited.

Botulinum Toxin: Pharmacology and Therapeutic Roles in Pain States.

PubMed

Patil, Shilpadevi; Willett, Olga; Thompkins, Terin; Hermann, Robert; Ramanathan, Sathish; Cornett, Elyse M; Fox, Charles J; Kaye, Alan David

2016-03-01

Botulinum toxin, also known as Botox, is produced by Clostridium botulinum, a gram-positive anaerobic bacterium, and botulinum toxin injections are among the most commonly practiced cosmetic procedures in the USA. Although botulinum toxin is typically associated with cosmetic procedures, it can be used to treat a variety of other conditions, including pain. Botulinum toxin blocks the release of acetylcholine from nerve endings to paralyze muscles and to decrease the pain response. Botulinum toxin has a long duration of action, lasting up to 5 months after initial treatment which makes it an excellent treatment for chronic pain patients. This manuscript will outline in detail why botulinum toxin is used as a successful treatment for pain in multiple conditions as well as outline the risks associated with using botulinum toxin in certain individuals. As of today, the only FDA-approved chronic condition that botulinum toxin can be used to treat is migraines and this is related to its ability to decrease muscle tension and increase muscle relaxation. Contraindications to botulinum toxin treatments are limited to a hypersensitivity to the toxin or an infection at the site of injection, and there are no known drug interactions with botulinum toxin. Botulinum toxin is an advantageous and effective alternative pain treatment and a therapy to consider for those that do not respond to opioid treatment. In summary, botulinum toxin is a relatively safe and effective treatment for individuals with certain pain conditions, including migraines. More research is warranted to elucidate chronic and long-term implications of botulinum toxin treatment as well as effects in pregnant, elderly, and adolescent patients.

Tumor Targeting and Drug Delivery by Anthrax Toxin.

PubMed

Bachran, Christopher; Leppla, Stephen H

2016-07-01

Anthrax toxin is a potent tripartite protein toxin from Bacillus anthracis. It is one of the two virulence factors and causes the disease anthrax. The receptor-binding component of the toxin, protective antigen, needs to be cleaved by furin-like proteases to be activated and to deliver the enzymatic moieties lethal factor and edema factor to the cytosol of cells. Alteration of the protease cleavage site allows the activation of the toxin selectively in response to the presence of tumor-associated proteases. This initial idea of re-targeting anthrax toxin to tumor cells was further elaborated in recent years and resulted in the design of many modifications of anthrax toxin, which resulted in successful tumor therapy in animal models. These modifications include the combination of different toxin variants that require activation by two different tumor-associated proteases for increased specificity of toxin activation. The anthrax toxin system has proved to be a versatile system for drug delivery of several enzymatic moieties into cells. This highly efficient delivery system has recently been further modified by introducing ubiquitin as a cytosolic cleavage site into lethal factor fusion proteins. This review article describes the latest developments in this field of tumor targeting and drug delivery.

Staphylococcus aureus Biofilms Induce Macrophage Dysfunction Through Leukocidin AB and Alpha-Toxin

PubMed Central

Scherr, Tyler D.; Hanke, Mark L.; Huang, Ouwen; James, David B. A.; Horswill, Alexander R.; Bayles, Kenneth W.; Fey, Paul D.; Torres, Victor J.

2015-01-01

ABSTRACT The macrophage response to planktonic Staphylococcus aureus involves the induction of proinflammatory microbicidal activity. However, S. aureus biofilms can interfere with these responses in part by polarizing macrophages toward an anti-inflammatory profibrotic phenotype. Here we demonstrate that conditioned medium from mature S. aureus biofilms inhibited macrophage phagocytosis and induced cytotoxicity, suggesting the involvement of a secreted factor(s). Iterative testing found the active factor(s) to be proteinaceous and partially agr-dependent. Quantitative mass spectrometry identified alpha-toxin (Hla) and leukocidin AB (LukAB) as critical molecules secreted by S. aureus biofilms that inhibit murine macrophage phagocytosis and promote cytotoxicity. A role for Hla and LukAB was confirmed by using hla and lukAB mutants, and synergy between the two toxins was demonstrated with a lukAB hla double mutant and verified by complementation. Independent confirmation of the effects of Hla and LukAB on macrophage dysfunction was demonstrated by using an isogenic strain in which Hla was constitutively expressed, an Hla antibody to block toxin activity, and purified LukAB peptide. The importance of Hla and LukAB during S. aureus biofilm formation in vivo was assessed by using a murine orthopedic implant biofilm infection model in which the lukAB hla double mutant displayed significantly lower bacterial burdens and more macrophage infiltrates than each single mutant. Collectively, these findings reveal a critical synergistic role for Hla and LukAB in promoting macrophage dysfunction and facilitating S. aureus biofilm development in vivo. PMID:26307164

TOXINS FROM CYANOBACTERIA IN WATER

EPA Science Inventory

This project is part of a larger U. S. Environmental Protection Agency (EPA) effort, which includes the Office of Water, to investigate algal toxins in surface water supplies and drinking water. Toxins produced by cyanobacteria (blue-green algae) are among the most potent known ...

Reassessing the Role of Type II Toxin-Antitoxin Systems in Formation of Escherichia coli Type II Persister Cells.

PubMed

Goormaghtigh, Frédéric; Fraikin, Nathan; Putrinš, Marta; Hallaert, Thibaut; Hauryliuk, Vasili; Garcia-Pino, Abel; Sjödin, Andreas; Kasvandik, Sergo; Udekwu, Klas; Tenson, Tanel; Kaldalu, Niilo; Van Melderen, Laurence

2018-06-12

Persistence is a reversible and low-frequency phenomenon allowing a subpopulation of a clonal bacterial population to survive antibiotic treatments. Upon removal of the antibiotic, persister cells resume growth and give rise to viable progeny. Type II toxin-antitoxin (TA) systems were assumed to play a key role in the formation of persister cells in Escherichia coli based on the observation that successive deletions of TA systems decreased persistence frequency. In addition, the model proposed that stochastic fluctuations of (p)ppGpp levels are the basis for triggering activation of TA systems. Cells in which TA systems are activated are thought to enter a dormancy state and therefore survive the antibiotic treatment. Using independently constructed strains and newly designed fluorescent reporters, we reassessed the roles of TA modules in persistence both at the population and single-cell levels. Our data confirm that the deletion of 10 TA systems does not affect persistence to ofloxacin or ampicillin. Moreover, microfluidic experiments performed with a strain reporting the induction of the yefM-yoeB TA system allowed the observation of a small number of type II persister cells that resume growth after removal of ampicillin. However, we were unable to establish a correlation between high fluorescence and persistence, since the fluorescence of persister cells was comparable to that of the bulk of the population and none of the cells showing high fluorescence were able to resume growth upon removal of the antibiotic. Altogether, these data show that there is no direct link between induction of TA systems and persistence to antibiotics. IMPORTANCE Within a growing bacterial population, a small subpopulation of cells is able to survive antibiotic treatment by entering a transient state of dormancy referred to as persistence. Persistence is thought to be the cause of relapsing bacterial infections and is a major public health concern. Type II toxin-antitoxin systems are

Can a toxin gene NAAT be used to predict toxin EIA and the severity of Clostridium difficile infection?

PubMed

Garvey, Mark I; Bradley, Craig W; Wilkinson, Martyn A C; Holden, Elisabeth

2017-01-01

Diagnosis of C. difficile infection (CDI) is controversial because of the many laboratory methods available and their lack of ability to distinguish between carriage, mild or severe disease. Here we describe whether a low C. difficile toxin B nucleic acid amplification test (NAAT) cycle threshold (CT) can predict toxin EIA, CDI severity and mortality. A three-stage algorithm was employed for CDI testing, comprising a screening test for glutamate dehydrogenase (GDH), followed by a NAAT, then a toxin enzyme immunoassay (EIA). All diarrhoeal samples positive for GDH and NAAT between 2012 and 2016 were analysed. The performance of the NAAT CT value as a classifier of toxin EIA outcome was analysed using a ROC curve; patient mortality was compared to CTs and toxin EIA via linear regression models. A CT value ≤26 was associated with ≥72% toxin EIA positivity; applying a logistic regression model we demonstrated an association between low CT values and toxin EIA positivity. A CT value of ≤26 was significantly associated ( p  = 0.0262) with increased one month mortality, severe cases of CDI or failure of first line treatment. The ROC curve probabilities demonstrated a CT cut off value of 26.6. Here we demonstrate that a CT ≤26 indicates more severe CDI and is associated with higher mortality. Samples with a low CT value are often toxin EIA positive, questioning the need for this additional EIA test. A CT ≤26 could be used to assess the potential for severity of CDI and guide patient treatment.

New Developments in Vaccines, Inhibitors of Anthrax Toxins, and Antibiotic Therapeutics for Bacillus anthracis

PubMed Central

Beierlein, J.M.; Anderson, A.C.

2013-01-01

Bacillus anthracis, the causative agent responsible for anthrax infections, poses a significant biodefense threat. There is a high mortality rate associated with untreated anthrax infections; specifically, inhalation anthrax is a particularly virulent form of infection with mortality rates close to 100%, even with aggressive treatment. Currently, a vaccine is not available to the general public and few antibiotics have been approved by the FDA for the treatment of inhalation anthrax. With the threat of natural or engineered bacterial resistance to antibiotics and the limited population for whom the current drugs are approved, there is a clear need for more effective treatments against this deadly infection. A comprehensive review of current research in drug discovery is presented in this article, including efforts to improve the purity and stability of vaccines, design inhibitors targeting the anthrax toxins, and identify inhibitors of novel enzyme targets. High resolution structural information for the anthrax toxins and several essential metabolic enzymes has played a significant role in aiding the structure-based design of potent and selective antibiotics. PMID:22050756

Monoclonal antibodies and toxins--a perspective on function and isotype.

PubMed

Chow, Siu-Kei; Casadevall, Arturo

2012-06-01

Antibody therapy remains the only effective treatment for toxin-mediated diseases. The development of hybridoma technology has allowed the isolation of monoclonal antibodies (mAbs) with high specificity and defined properties, and numerous mAbs have been purified and characterized for their protective efficacy against different toxins. This review summarizes the mAb studies for 6 toxins--Shiga toxin, pertussis toxin, anthrax toxin, ricin toxin, botulinum toxin, and Staphylococcal enterotoxin B (SEB)--and analyzes the prevalence of mAb functions and their isotypes. Here we show that most toxin-binding mAbs resulted from immunization are non-protective and that mAbs with potential therapeutic use are preferably characterized. Various common practices and caveats of protection studies are discussed, with the goal of providing insights for the design of future research on antibody-toxin interactions.

Anticancer Activity of Bacterial Proteins and Peptides.

PubMed

Karpiński, Tomasz M; Adamczak, Artur

2018-04-30

Despite much progress in the diagnosis and treatment of cancer, tumour diseases constitute one of the main reasons of deaths worldwide. The side effects of chemotherapy and drug resistance of some cancer types belong to the significant current therapeutic problems. Hence, searching for new anticancer substances and medicines are very important. Among them, bacterial proteins and peptides are a promising group of bioactive compounds and potential anticancer drugs. Some of them, including anticancer antibiotics (actinomycin D, bleomycin, doxorubicin, mitomycin C) and diphtheria toxin, are already used in the cancer treatment, while other substances are in clinical trials (e.g., p28, arginine deiminase ADI) or tested in in vitro research. This review shows the current literature data regarding the anticancer activity of proteins and peptides originated from bacteria: antibiotics, bacteriocins, enzymes, nonribosomal peptides (NRPs), toxins and others such as azurin, p28, Entap and Pep27anal2. The special attention was paid to the still poorly understood active substances obtained from the marine sediment bacteria. In total, 37 chemical compounds or groups of compounds with antitumor properties have been described in the present article.

Anti-leukemia activity of a bacterial toxin with natural specificity for LFA-1 on white blood cells

PubMed Central

Kachlany, Scott C.; Schwartz, Amy B.; Balashova, Nataliya V.; Hioe, Catarina E.; Tuen, Michael; Le, Amy; Kaur, Manpreet; Mei, Yongyi; Rao, Jia

2009-01-01

The oral bacterium, Aggregatibacter actinomycetemcomitans, produces a leukotoxin (LtxA) that is specific for white blood cells (WBCs) from humans and Old World primates by interacting with lymphocyte function antigen-1 (LFA-1) on susceptible cells. To determine if LtxA could be used as a therapeutic agent for the treatment of WBC diseases, we tested the in vitro and in vivo anti-leukemia activity of the toxin. LtxA kills human malignant WBC lines and primary leukemia cells from acute myeloid leukemia patients, but healthy peripheral blood mononuclear cells (PBMCs) are relatively resistant to LtxA-mediated cytotoxicity. Levels of LFA-1 on cell lines correlated with killing by LtxA and the toxin preferentially killed cells expressing the activated form of LFA-1. In a SCID mouse model for human leukemia, LtxA had potent therapeutic value resulting in long-term survival in LtxA-treated mice. Intravenous infusion of LtxA into a rhesus macaque resulted in a drop in WBC counts at early times post-infusion; however, red blood cells, platelets, hemoglobin and blood chemistry values remained unaffected. Thus, LtxA may be an effective and safe novel therapeutic agent for the treatment of hematologic malignancies. PMID:19747730

Clostridium botulinum C2 toxin--new insights into the cellular up-take of the actin-ADP-ribosylating toxin.

PubMed

Aktories, Klaus; Barth, Holger

2004-04-01

Clostridium botulinum C2 toxin is a member of the family of binary actin-ADP-ribosylating toxins. It consists of the enzyme component C2I, and the separated binding/translocation component C2II. Proteolytically activated C2II forms heptamers and binds to a carbohydrate cell surface receptor. After attachment of C2I, the toxin complex is endocytosed to reach early endosomes. At low pH of endosomes, C2II-heptamers insert into the membrane, form pores and deliver C2I into the cytosol. Here, C2I ADP-ribosylates actin at Arg177 to block actin polymerization and to induce depolymerization of actin filaments. The mini-review describes main properties of C2 toxin and discusses new findings on the involvement of chaperones in the up-take process of the toxin.

Defense islands in bacterial and archaeal genomes and prediction of novel defense systems.

PubMed

Makarova, Kira S; Wolf, Yuri I; Snir, Sagi; Koonin, Eugene V

2011-11-01

The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous overrepresented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for the experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show nonrandom clustering in defense islands. It remains unclear to what extent these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic "sinks" that accumulate diverse nonessential genes, particularly those acquired via horizontal gene transfer. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are nonrandomly associated in islands, suggesting nonadaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands.

A mutagenic analysis of the RNase mechanism of the bacterial Kid toxin by mass spectrometry.

PubMed

Diago-Navarro, Elizabeth; Kamphuis, Monique B; Boelens, Rolf; Barendregt, Arjan; Heck, Albert J; van den Heuvel, Robert H; Díaz-Orejas, Ramón

2009-09-01

Kid, the toxin of the parD (kis, kid) maintenance system of plasmid R1, is an endoribonuclease that preferentially cleaves RNA at the 5' of A in the core sequence 5'-UA(A/C)-3'. A model of the Kid toxin interacting with the uncleavable mimetic 5'-AdUACA-3' is available. To evaluate this model, a significant collection of mutants in some of the key residues proposed to be involved in RNA binding (T46, A55, T69 and R85) or RNA cleavage (R73, D75 and H17) were analysed by mass spectrometry in RNA binding and cleavage assays. A pair of substrates, 5'-AUACA-3', and its uncleavable mimetic 5'-AdUACA-3', used to establish the model and structure of the Kid-RNA complex, were used in both the RNA cleavage and binding assays. A second RNA substrate, 5'-UUACU-3' efficiently cleaved by Kid both in vivo and in vitro, was also used in the cleavage assays. Compared with the wild-type protein, mutations in the residues of the catalytic site abolished RNA cleavage without substantially altering RNA binding. Mutations in residues proposed to be involved in RNA binding show reduced binding efficiency and a corresponding decrease in RNA cleavage efficiency. The cleavage profiles of the different mutants were similar with the two substrates used, but RNA cleavage required much lower protein concentrations when the 5'-UUACU-3' substrate was used. Protein synthesis and growth assays are consistent with there being a correlation between the RNase activity of Kid and its inhibitory potential. These results give important support to the available models of Kid RNase and the Kid-RNA complex.

Toxins That Affect Voltage-Gated Sodium Channels.

PubMed

Ji, Yonghua

2017-10-26

Voltage-gated sodium channels (VGSCs) are critical in generation and conduction of electrical signals in multiple excitable tissues. Natural toxins, produced by animal, plant, and microorganisms, target VGSCs through diverse strategies developed over millions of years of evolutions. Studying of the diverse interaction between VGSC and VGSC-targeting toxins has been contributing to the increasing understanding of molecular structure and function, pharmacology, and drug development potential of VGSCs. This chapter aims to summarize some of the current views on the VGSC-toxin interaction based on the established receptor sites of VGSC for natural toxins.

Botulinum toxin for the treatment of bruxism.

PubMed

Tinastepe, Neslihan; Küçük, Burcu Bal; Oral, Koray

2015-10-01

Botulinum toxin, the most potent biological toxin, has been shown to be effective for a variety of disorders in several medical conditions, when used both therapeutically and cosmetically. In recent years, there has been a rising trend in the use of this pharmacological agent to control bruxing activity, despite its reported adverse effects. The aim of this review was to provide a brief overview to clarify the underlying essential ideas for the use of botulinum toxin in bruxism based on available scientific papers. An electronic literature search was performed to identify publications related to botulinum toxin and its use for bruxism in PubMed. Hand searching of relevant articles was also made to identify additional studies. Of the eleven identified studies, only two were randomized controlled trials, compared with the effectiveness of botulinum toxins on the reduction in the frequency of bruxism events and myofascial pain after injection. The authors of these studies concluded that botulinum toxin could be used as an effective treatment for reducing nocturnal bruxism and myofascial pain in patients with bruxism. Evidence-based research was limited on this topic. More randomized controlled studies are needed to confirm that botulinum toxin is safe and reliable for routine clinical use in bruxism.

Botulinum toxin for the treatment of bruxism.

PubMed

Tinastepe, Neslihan; Küçük, Burcu Bal; Oral, Koray

2014-08-14

Aims: Botulinum toxin, the most potent biological toxin, has been shown to be effective for a variety of disorders in several medical conditions, when used both therapeutically and cosmetically. In recent years, there has been a rising trend in the use of this pharmacological agent to control bruxing activity, despite its reported adverse effects. The aim of this review was to provide a brief overview to clarify the underlying essential ideas for the use of botulinum toxin in bruxism based on available scientific papers. Methodology: An electronic literature search was performed to identify publications related to botulinum toxin and its use for bruxism in PubMed. Hand searching of relevant articles was also made to identify additional studies. Results: Of the eleven identified studies, only two were randomized controlled trials, compared with the effectiveness of botulinum toxins on the reduction in the frequency of bruxism events and myofascial pain after injection. The authors of these studies concluded that botulinum toxin could be used as an effective treatment for reducing nocturnal bruxism and myofascial pain in patients with bruxism. Conclusion: Evidence-based research was limited on this topic. More randomized controlled studies are needed to confirm that botulinum toxin is safe and reliable for routine clinical use in bruxism.

Cyanobacterial toxins: risk management for health protection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Codd, Geoffrey A.; Morrison, Louise F.; Metcalf, James S

2005-03-15

This paper reviews the occurrence and properties of cyanobacterial toxins, with reference to the recognition and management of the human health risks which they may present. Mass populations of toxin-producing cyanobacteria in natural and controlled waterbodies include blooms and scums of planktonic species, and mats and biofilms of benthic species. Toxic cyanobacterial populations have been reported in freshwaters in over 45 countries, and in numerous brackish, coastal, and marine environments. The principal toxigenic genera are listed. Known sources of the families of cyanobacterial toxins (hepato-, neuro-, and cytotoxins, irritants, and gastrointestinal toxins) are briefly discussed. Key procedures in the riskmore » management of cyanobacterial toxins and cells are reviewed, including derivations (where sufficient data are available) of tolerable daily intakes (TDIs) and guideline values (GVs) with reference to the toxins in drinking water, and guideline levels for toxigenic cyanobacteria in bathing waters. Uncertainties and some gaps in knowledge are also discussed, including the importance of exposure media (animal and plant foods), in addition to potable and recreational waters. Finally, we present an outline of steps to develop and implement risk management strategies for cyanobacterial cells and toxins in waterbodies, with recent applications and the integration of Hazard Assessment Critical Control Point (HACCP) principles.« less

Investigation of prevalence of free Shiga toxin-producing Escherichia coli (STEC)-specific bacteriophages and its correlation with STEC bacterial hosts in a produce-growing area in Salinas, California.

PubMed

Liao, Yen-Te; Quintela, Irwin A; Nguyen, Kimberly; Salvador, Alexandra; Cooley, Michael B; Wu, Vivian C H

2018-01-01

Shiga toxin-producing E. coli (STEC) causes approximately 265,000 illnesses and 3,600 hospitalizations annually and is highly associated with animal contamination due to the natural reservoir of ruminant gastrointestinal tracts. Free STEC-specific bacteriophages against STEC strains are also commonly isolated from fecal-contaminated environment. Previous studies have evaluated the correlation between the prevalence of STEC-specific bacteriophages and STEC strains to improve animal-associated environment. However, the similar information regarding free STEC-specific bacteriophages prevalence in produce growing area is lacking. Thus, the objectives of this research were to determine the prevalence of STEC-specific phages, analyze potential effects of environmental factors on the prevalence of the phages, and study correlations between STEC-specific bacteriophages and the bacterial hosts in pre-harvest produce environment. Surface water from 20 samples sites was subjected to free bacteriophage isolation using host strains of both generic E. coli and STEC (O157, six non-O157 and one O179 strains) cocktails, and isolation of O157 and non-O157 STEC strains by use of culture methods combined with PCR-based confirmation. The weather data were obtained from weather station website. Free O145- and O179-specific bacteriophages were the two most frequently isolated bacteriophages among all (O45, O145, O157 and O179) in this study. The results showed June and July had relatively high prevalence of overall STEC-specific bacteriophages with minimum isolation of STEC strains. In addition, the bacteriophages were likely isolated in the area-around or within city-with predominant human impact, whereas the STEC bacterial isolates were commonly found in agriculture impact environment. Furthermore, there was a trend that the sample sites with positive of free STEC bacteriophage did not have the specific STEC bacterial hosts. The findings of the study enable us to understand the ecology

Binding of Diphtheria Toxin to Phospholipids in Liposomes

NASA Astrophysics Data System (ADS)

Alving, Carl R.; Iglewski, Barbara H.; Urban, Katharine A.; Moss, Joel; Richards, Roberta L.; Sadoff, Jerald C.

1980-04-01

Diphtheria toxin bound to the phosphate portion of some, but not all, phospholipids in liposomes. Liposomes consisting of dimyristoyl phosphatidylcholine and cholesterol did not bind toxin. Addition of 20 mol% (compared to dimyristoyl phosphatidylcholine) of dipalmitoyl phosphatidic acid, dicetyl phosphate, phosphatidylinositol phosphate, cardiolipin, or phosphatidylserine in the liposomes resulted in substantial binding of toxin. Inclusion of phosphatidylinositol in dimyristol phosphatidylcholine / cholesterol liposomes did not result in toxin binding. The calcium salt of dipalmitoyl phosphatidic acid was more effective than the sodium salt, and the highest level of binding occurred with liposomes consisting only of dipalmitoyl phosphatidic acid (calcium salt) and cholesterol. Binding of toxin to liposomes was dependent on pH, and the pattern of pH dependence varied with liposomes having different compositions. Incubation of diphtheria toxin with liposomes containing dicetyl phosphate resulted in maximal binding at pH 3.6, whereas binding to liposomes containing phosphatidylinositol phosphate was maximal above pH 7. Toxin did not bind to liposomes containing 20 mol% of a free fatty acid (palmitic acid) or a sulfated lipid (3-sulfogalactosylceramide). Toxin binding to dicetyl phosphate or phosphatidylinositol phosphate was inhibited by UTP, ATP, phosphocholine, or p-nitrophenyl phosphate, but not by uracil. We conclude that (a) diphtheria toxin binds specifically to the phosphate portion of certain phospholipids, (b) binding to phospholipids in liposomes is dependent on pH, but is not due only to electrostatic interaction, and (c) binding may be strongly influenced by the composition of adjacent phospholipids that do not bind toxin. We propose that a minor membrane phospholipid (such as phosphatidylinositol phosphate or phosphatidic acid), or that some other phosphorylated membrane molecule (such as a phosphoprotein) may be important in the initial binding of

Drooling in Parkinson's disease: A randomized controlled trial of incobotulinum toxin A and meta-analysis of Botulinum toxins.

PubMed

Narayanaswami, Pushpa; Geisbush, Thomas; Tarulli, Andrew; Raynor, Elizabeth; Gautam, Shiva; Tarsy, Daniel; Gronseth, Gary

2016-09-01

Botulinum toxins are a therapeutic option for drooling in Parkinson's Disease (PD). The aims of this study were to: 1. evaluate the efficacy of incobotulinum toxin A for drooling in PD. 2. Perform a meta-analysis of studies of Botulinum toxins for drooling in PD. 1. Primary study: Randomized, double blind, placebo controlled, cross over trial. Incobotulinum toxin (100 units) or saline was injected into the parotid (20 units) and submandibular (30 units) glands. Subjects returned monthly for three evaluations after each injection. Outcome measures were saliva weight and Drooling Frequency and Severity Scale. 2. Systematic review of literature, followed by inverse variance meta-analyses using random effects models. 1. Primary Study: Nine of 10 subjects completed both arms. There was no significant change in the primary outcome of saliva weight one month after injection in the treatment period compared to placebo period (mean difference, gm ± SD: -0.194 ± 0.61, range: -1.28 to 0.97, 95% CI -0.71 to 0.32). Secondary outcomes also did not change. 2. Meta-analysis of six studies demonstrated significant benefit of Botulinum toxin on functional outcomes (effect size, Cohen's d: -1.32, CI -1.86 to -0.78). The other studies used a higher dose of Botulinum toxin A into the parotid glands. This study did not demonstrate efficacy of incobotulinum toxin A for drooling in PD, but lacked precision to exclude moderate benefit. The parotid/submandibular dose-ratio may have influenced results. Studies evaluating higher doses of incobotulinum toxin A into the parotid glands may be useful. Copyright © 2016 Elsevier Ltd. All rights reserved.

Array biosensor for detection of toxins

NASA Technical Reports Server (NTRS)

Ligler, Frances S.; Taitt, Chris Rowe; Shriver-Lake, Lisa C.; Sapsford, Kim E.; Shubin, Yura; Golden, Joel P.

2003-01-01

The array biosensor is capable of detecting multiple targets rapidly and simultaneously on the surface of a single waveguide. Sandwich and competitive fluoroimmunoassays have been developed to detect high and low molecular weight toxins, respectively, in complex samples. Recognition molecules (usually antibodies) were first immobilized in specific locations on the waveguide and the resultant patterned array was used to interrogate up to 12 different samples for the presence of multiple different analytes. Upon binding of a fluorescent analyte or fluorescent immunocomplex, the pattern of fluorescent spots was detected using a CCD camera. Automated image analysis was used to determine a mean fluorescence value for each assay spot and to subtract the local background signal. The location of the spot and its mean fluorescence value were used to determine the toxin identity and concentration. Toxins were measured in clinical fluids, environmental samples and foods, with minimal sample preparation. Results are shown for rapid analyses of staphylococcal enterotoxin B, ricin, cholera toxin, botulinum toxoids, trinitrotoluene, and the mycotoxin fumonisin. Toxins were detected at levels as low as 0.5 ng mL(-1).

Botulinum toxin for the treatment of strabismus.

PubMed

Rowe, Fiona J; Noonan, Carmel P

2017-03-02

The use of botulinum toxin as an investigative and treatment modality for strabismus is well reported in the medical literature. However, it is unclear how effective it is in comparison to other treatment options for strabismus. The primary objective was to examine the efficacy of botulinum toxin therapy in the treatment of strabismus compared with alternative conservative or surgical treatment options. This review sought to ascertain those types of strabismus that particularly benefit from the use of botulinum toxin as a treatment option (such as small angle strabismus or strabismus with binocular potential, i.e. the potential to use both eyes together as a pair). The secondary objectives were to investigate the dose effect and complication rates associated with botulinum toxin. We searched CENTRAL (which contains the Cochrane Eyes and Vision Trials Register) (2016, Issue 6), Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid MEDLINE Daily, Ovid OLDMEDLINE (January 1946 to July 2016), Embase (January 1980 to July 2016), Latin American and Caribbean Literature on Health Sciences (LILACS) (January 1982 to July 2016), the ISRCTN registry (www.isrctn.com/editAdvancedSearch), ClinicalTrials.gov (www.clinicaltrials.gov), and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 11 July 2016. We handsearched the British and Irish Orthoptic Journal, Australian Orthoptic Journal, proceedings of the European Strabismological Association (ESA), International Strabismological Association (ISA) and International Orthoptic Association (IOA) (www.liv.ac.uk/orthoptics/research/search.htm) and American Academy of Paediatric Ophthalmology and Strabismus meetings (AAPOS). We contacted researchers who are active in this field for information about further

Mechanisms of the cytopathic action of actin-ADP-ribosylating toxins.

PubMed

Aktories, K; Wegner, A

1992-10-01

Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, and Clostridium spiroforme toxin ADP-ribosylate actin monomers. Toxin-induced ADP-ribosylation disturbs the cellular equilibrium between monomeric and polymeric actin and traps monomeric actin in its unpolymerized form, thereby depolymerizing actin filaments and destroying the microfilament network. Furthermore, the toxins ADP-ribosylate gelsolin actin complexes. These modifications may contribute to the cytopathic action of the toxins.

Ingestion of transgenic carrots expressing the Escherichia coli heat-labile enterotoxin B subunit protects mice against cholera toxin challenge.

PubMed

Rosales-Mendoza, Sergio; Soria-Guerra, Ruth Elena; López-Revilla, Rubén; Moreno-Fierros, Leticia; Alpuche-Solís, Angel Gabriel

2008-01-01

Diarrheal diseases caused by Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) are worldwide health problems that might be prevented with vaccines based on edible plants expressing the B subunit from either the cholera toxin (CTB) or the E. coli heat labile toxin (LTB). In this work we analyzed the immunity induced in Balb/c mice by ingestion of three weekly doses of 10 mug of LTB derived from transgenic carrot material. Although the anti-LTB serum immunoglobulin G (IgG) and intestinal IgA antibody responses were higher with 10 mug-doses of pure bacterial recombinant LTB (rLTB), the transgenic carrot material also elicited significant serum and intestinal antibody responses. Serum anti-LTB IgG1 antibodies predominated over IgG2a antibodies, suggesting that mainly Th2 responses were induced. A decrease of intestinal fluid accumulation after cholera toxin challenge was observed in mice immunized with either rLTB or LTB-containing carrot material. These results demonstrate that ingestion of carrot-derived LTB induces antitoxin systemic and intestinal immunity in mice and suggest that transgenic carrots expressing LTB may be used as an effective edible vaccine against cholera and ETEC diarrhea in humans.

Global functional analyses of cellular responses to pore-forming toxins.

PubMed

Kao, Cheng-Yuan; Los, Ferdinand C O; Huffman, Danielle L; Wachi, Shinichiro; Kloft, Nicole; Husmann, Matthias; Karabrahimi, Valbona; Schwartz, Jean-Louis; Bellier, Audrey; Ha, Christine; Sagong, Youn; Fan, Hui; Ghosh, Partho; Hsieh, Mindy; Hsu, Chih-Shen; Chen, Li; Aroian, Raffi V

2011-03-01

Here we present the first global functional analysis of cellular responses to pore-forming toxins (PFTs). PFTs are uniquely important bacterial virulence factors, comprising the single largest class of bacterial protein toxins and being important for the pathogenesis in humans of many Gram positive and Gram negative bacteria. Their mode of action is deceptively simple, poking holes in the plasma membrane of cells. The scattered studies to date of PFT-host cell interactions indicate a handful of genes are involved in cellular defenses to PFTs. How many genes are involved in cellular defenses against PFTs and how cellular defenses are coordinated are unknown. To address these questions, we performed the first genome-wide RNA interference (RNAi) screen for genes that, when knocked down, result in hypersensitivity to a PFT. This screen identifies 106 genes (∼0.5% of genome) in seven functional groups that protect Caenorhabditis elegans from PFT attack. Interactome analyses of these 106 genes suggest that two previously identified mitogen-activated protein kinase (MAPK) pathways, one (p38) studied in detail and the other (JNK) not, form a core PFT defense network. Additional microarray, real-time PCR, and functional studies reveal that the JNK MAPK pathway, but not the p38 MAPK pathway, is a key central regulator of PFT-induced transcriptional and functional responses. We find C. elegans activator protein 1 (AP-1; c-jun, c-fos) is a downstream target of the JNK-mediated PFT protection pathway, protects C. elegans against both small-pore and large-pore PFTs and protects human cells against a large-pore PFT. This in vivo RNAi genomic study of PFT responses proves that cellular commitment to PFT defenses is enormous, demonstrates the JNK MAPK pathway as a key regulator of transcriptionally-induced PFT defenses, and identifies AP-1 as the first cellular component broadly important for defense against large- and small-pore PFTs.

Botulinum Toxin Use in Pediatric Plastic Surgery.

PubMed

Fu, Katherine J; Teichgraeber, John F; Greives, Matthew R

2016-11-01

Botulinum toxin has increasingly become a prevalent treatment option for a wide range of conditions, many of which have their roots in plastic surgery and have been well studied. In adults, chronic headache, hyperhidrosis, and facial muscular hypertrophy have been effectively treated with botulinum toxin, and emerging studies have begun looking at its efficacy in children, as well. Successful treatment of spasticity and muscular contraction has allowed for the creation of safety profiles and dosage guidelines for botulinum toxin usage in children. The expanded indications for its use have since flourished in all arenas of pediatric care, including plastic surgery. Recent studies have described the use of botulinum toxin as an adjunct to the treatment of congenital torticollis and cleft lip. This review discusses the various applications of botulinum toxin for pediatric patients in the field of plastic surgery.

Short toxin-like proteins abound in Cnidaria genomes.

PubMed

Tirosh, Yitshak; Linial, Itai; Askenazi, Manor; Linial, Michal

2012-11-16

Cnidaria is a rich phylum that includes thousands of marine species. In this study, we focused on Anthozoa and Hydrozoa that are represented by the Nematostella vectensis (Sea anemone) and Hydra magnipapillata genomes. We present a method for ranking the toxin-like candidates from complete proteomes of Cnidaria. Toxin-like functions were revealed using ClanTox, a statistical machine-learning predictor trained on ion channel inhibitors from venomous animals. Fundamental features that were emphasized in training ClanTox include cysteines and their spacing along the sequences. Among the 83,000 proteins derived from Cnidaria representatives, we found 170 candidates that fulfill the properties of toxin-like-proteins, the vast majority of which were previously unrecognized as toxins. An additional 394 short proteins exhibit characteristics of toxin-like proteins at a moderate degree of confidence. Remarkably, only 11% of the predicted toxin-like proteins were previously classified as toxins. Based on our prediction methodology and manual annotation, we inferred functions for over 400 of these proteins. Such functions include protease inhibitors, membrane pore formation, ion channel blockers and metal binding proteins. Many of the proteins belong to small families of paralogs. We conclude that the evolutionary expansion of toxin-like proteins in Cnidaria contributes to their fitness in the complex environment of the aquatic ecosystem.

Phospholipase C produced by Clostridium botulinum types C and D: comparison of gene, enzymatic, and biological activities with those of Clostridium perfringens alpha-toxin.

PubMed

Fatmawati, Ni Nengah Dwi; Sakaguchi, Yoshihiko; Suzuki, Tomonori; Oda, Masataka; Shimizu, Kenta; Yamamoto, Yumiko; Sakurai, Jun; Matsushita, Osamu; Oguma, Keiji

2013-01-01

Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival.

Design and expression of recombinant toxins from Mexican scorpions of the genus Centruroides for production of antivenoms.

PubMed

Jiménez-Vargas, J M; Quintero-Hernández, V; González-Morales, L; Ortiz, E; Possani, L D

2017-03-15

This manuscript describes the design of plasmids containing the genes coding for four main mammalian toxins of scorpions from the genus Centruroides (C.) of Mexico. The genes that code for toxin 2 of C. noxius (Cn2), toxin 2 from C. suffusus (Css2) and toxins 1 and 2 from C. limpidus (Cll1 and Cll2) were included into individual plasmids carrying the genetic construction for expression of fusion proteins containing a leader peptide (pelB) that directs the expressed protein to the bacterial periplasm, a carrier protein (thioredoxin), the cleavage site for enterokinase, the chosen toxin and a poly-histidine tag (6xHis-tag) for purification of the hybrid protein by immobilized metal ion affinity chromatography after expression in Escherichia coli strain BL21 (DE3). The purified hybrid proteins containing the recombinant toxins (abbreviated Thio-EK-Toxin) were used for immunization of three independent groups of ten mice and four rabbits. Challenging the first group of mice, immunized with recombinant Thio-EK-Css2, with three median lethal doses (LD 50 ) of C. suffusus soluble venom resulted in the survival of all the test animals without showing intoxication symptoms. All control mice (none immunized) died. Similar results were obtained with mice previously immunized with Thio-EK-Cn2 and challenged with C. noxius venom. The third group of mice immunized with both Thio-EK-Cll1 and Thio-EK-Cll2 showed an 80% survival ratio when challenged with only one LD 50 of C. limpidus venom, all showing symptoms of intoxication. The sera from rabbits immunized with a combination of the four recombinant toxins were collected separately and used to assess their neutralization capacity in vitro (pre-incubating the serum with the respective scorpion venom and injecting the mixture into mice), using six mice for each serum/venom combination tested. The venoms from the six most dangerous scorpion species of Mexico were assayed: C. noxius, C. suffusus, C. limpidus, C. elegans, C

Clostridium perfringens epsilon toxin inhibits the gastrointestinal transit in mice.

PubMed

Losada-Eaton, D M; Fernandez-Miyakawa, M E

2010-12-01

Epsilon toxin produced by Clostridium perfringens type B and D is a potent toxin that is responsible for a highly fatal enterotoxemia in sheep and goats. In vitro, epsilon toxin produces contraction of the rat ileum as the result of an indirect action, presumably mediated through the autonomic nervous system. To examine the impact of epsilon toxin in the intestinal transit, gastric emptying (GE) and gastrointestinal transit (GIT) were evaluated after intravenous and oral administration of epsilon toxin in mice. Orally administered epsilon toxin produced a delay on the GIT. Inhibition of the small intestinal transit was observed as early as 1 h after the toxin was administered orally but the effects were not observed after 1 week. Epsilon toxin also produced an inhibition in GE and a delay on the GIT when relatively high toxin concentrations were given intravenously. These results indicate that epsilon toxin administered orally or intravenously to mice transitorily inhibits the GIT. The delay in the GIT induced by epsilon toxin could be relevant in the pathogenesis of C. perfringens type B and D enterotoxemia. Copyright © 2010 Elsevier Ltd. All rights reserved.

Molecular basis of toxicity of Clostridium perfringens epsilon toxin.

PubMed

Bokori-Brown, Monika; Savva, Christos G; Fernandes da Costa, Sérgio P; Naylor, Claire E; Basak, Ajit K; Titball, Richard W

2011-12-01

Clostridium perfringens ε-toxin is produced by toxinotypes B and D strains. The toxin is the aetiological agent of dysentery in newborn lambs but is also associated with enteritis and enterotoxaemia in goats, calves and foals. It is considered to be a potential biowarfare or bioterrorism agent by the US Government Centers for Disease Control and Prevention. The relatively inactive 32.9 kDa prototoxin is converted to active mature toxin by proteolytic cleavage, either by digestive proteases of the host, such as trypsin and chymotrypsin, or by C. perfringens λ-protease. In vivo, the toxin appears to target the brain and kidneys, but relatively few cell lines are susceptible to the toxin, and most work has been carried out using Madin-Darby canine kidney (MDCK) cells. The binding of ε-toxin to MDCK cells and rat synaptosomal membranes is associated with the formation of a stable, high molecular weight complex. The crystal structure of ε-toxin reveals similarity to aerolysin from Aeromonas hydrophila, parasporin-2 from Bacillus thuringiensis and a lectin from Laetiporus sulphureus. Like these toxins, ε-toxin appears to form heptameric pores in target cell membranes. The exquisite specificity of the toxin for specific cell types suggests that it binds to a receptor found only on these cells. © 2011 The Authors Journal compilation © 2011 FEBS.

Model for RNA binding and the catalytic site of the RNase Kid of the bacterial parD toxin-antitoxin system.

PubMed

Kamphuis, Monique B; Bonvin, Alexandre M J J; Monti, Maria Chiara; Lemonnier, Marc; Muñoz-Gómez, Ana; van den Heuvel, Robert H H; Díaz-Orejas, Ramón; Boelens, Rolf

2006-03-17

The toxin Kid and antitoxin Kis are encoded by the parD operon of Escherichia coli plasmid R1. Kid and its chromosomal homologues MazF and ChpBK have been shown to inhibit protein synthesis in cell extracts and to act as ribosome-independent endoribonucleases in vitro. Kid cleaves RNA preferentially at the 5' side of the A residue in the nucleotide sequence 5'-UA(A/C)-3' of single-stranded regions. Here, we show that RNA cleavage by Kid yields two fragments with a 2':3'-cyclic phosphate group and a free 5'-OH group, respectively. The cleavage mechanism is similar to that of RNases A and T1, involving the uracil 2'-OH group. Via NMR titration studies with an uncleavable RNA mimic, we demonstrate that residues of both monomers of the Kid dimer together form a concatenated RNA-binding surface. Docking calculations based on the NMR chemical shifts, the cleavage mechanism and previously reported mutagenesis data provide a detailed picture of the position of the AUACA fragment within the binding pocket. We propose that residues D75, R73 and H17 form the active site of the Kid toxin, where D75 and R73 are the catalytic base and acid, respectively. The RNA sequence specificity is defined by residues T46, S47, A55, F57, T69, V71 and R73. Our data show the importance of these residues for Kid function, and the implications of our results for related toxins, such as MazF, CcdB and RelE, are discussed.

Botulinum toxin for vaginismus treatment.

PubMed

Ferreira, Juliana Rocha; Souza, Renan Pedra

2012-01-01

Vaginismus is characterized by recurrent or persistent involuntary contraction of the perineal muscles surrounding the outer third of the vagina when penile, finger, tampon, or speculum penetration is attempted. Recent results have suggested the use of botulinum toxin for the treatment of vaginismus. Here, we assessed previously published data to evaluate the therapeutic effectiveness of botulinum toxin for vaginismus. We have carried out a systematic review followed by a meta-analysis. Our results indicate that botulinum toxin is an effective therapeutic option for patients with vaginismus (pooled odds ratio of 8.723 with 95% confidence interval limits of 1.942 and 39.162, p = 0.005). This may hold particularly true in treatment-refractory patients because most of the studies included in this meta-analysis have enrolled these subjects in their primary analysis. Botulinum toxin appears to bea reasonable intervention for vaginismus. However, this conclusion should be read carefully because of the deficiency of placebo-controlled randomized clinical trials and the quality issues presented in the existing ones.

Clostridium Perfringens Toxins Involved in Mammalian Veterinary Diseases

PubMed Central

Uzal, F. A.; Vidal, J. E.; McClane, B. A.; Gurjar, A. A.

2013-01-01

Clostridium perfringens is a gram-positive anaerobic rod that is classified into 5 toxinotypes (A, B, C, D, and E) according to the production of 4 major toxins, namely alpha (CPA), beta (CPB), epsilon (ETX) and iota (ITX). However, this microorganism can produce up to 16 toxins in various combinations, including lethal toxins such as perfringolysin O (PFO), enterotoxin (CPE), and beta2 toxin (CPB2). Most diseases caused by this microorganism are mediated by one or more of these toxins. The role of CPA in intestinal disease of mammals is controversial and poorly documented, but there is no doubt that this toxin is essential in the production of gas gangrene of humans and several animal species. CPB produced by C. perfringens types B and C is responsible for necrotizing enteritis and enterotoxemia mainly in neonatal individuals of several animal species. ETX produced by C. perfringens type D is responsible for clinical signs and lesions of enterotoxemia, a predominantly neurological disease of sheep and goats. The role of ITX in disease of animals is poorly understood, although it is usually assumed that the pathogenesis of intestinal diseases produced by C. perfringens type E is mediated by this toxin. CPB2, a necrotizing and lethal toxin that can be produced by all types of C. perfringens, has been blamed for disease in many animal species, but little information is currently available to sustain or rule out this claim. CPE is an important virulence factor for C. perfringens type A gastrointestinal disease in humans and dogs; however, the data implicating CPE in other animal diseases remains ambiguous. PFO does not seem to play a direct role as the main virulence factor for animal diseases, but it may have a synergistic role with CPA-mediated gangrene and ETX-mediated enterotoxemia. The recent improvement of animal models for C. perfringens infection and the use of toxin gene knock-out mutants have demonstrated the specific pathogenic role of several toxins of C

Helicobacter pylori VacA Toxin Promotes Bacterial Intracellular Survival in Gastric Epithelial Cells▿ †

PubMed Central

Terebiznik, M. R.; Vazquez, C. L.; Torbicki, K.; Banks, D.; Wang, T.; Hong, W.; Blanke, S. R.; Colombo, M. I.; Jones, N. L.

2006-01-01

Helicobacter pylori colonizes the gastric epithelium of at least 50% of the world's human population, playing a causative role in the development of chronic gastritis, peptic ulcers, and gastric adenocarcinoma. Current evidence indicates that H. pylori can invade epithelial cells in the gastric mucosa. However, relatively little is known about the biology of H. pylori invasion and survival in host cells. Here, we analyze both the nature of and the mechanisms responsible for the formation of H. pylori's intracellular niche. We show that in AGS cells infected with H. pylori, bacterium-containing vacuoles originate through the fusion of late endocytic organelles. This process is mediated by the VacA-dependent retention of the small GTPase Rab7. In addition, functional interactions between Rab7 and its downstream effector, Rab-interacting lysosomal protein (RILP), are necessary for the formation of the bacterial compartment since expression of mutant forms of RILP or Rab7 that fail to bind each other impaired the formation of this unique bacterial niche. Moreover, the VacA-mediated sequestration of active Rab7 disrupts the full maturation of vacuoles as assessed by the lack of both colocalization with cathepsin D and degradation of internalized cargo in the H. pylori-containing vacuole. Based on these findings, we propose that the VacA-dependent isolation of the H. pylori-containing vacuole from bactericidal components of the lysosomal pathway promotes bacterial survival and contributes to the persistence of infection. PMID:17000720

Single toxin dose-response models revisited

DOE Office of Scientific and Technical Information (OSTI.GOV)

Demidenko, Eugene, E-mail: eugened@dartmouth.edu

The goal of this paper is to offer a rigorous analysis of the sigmoid shape single toxin dose-response relationship. The toxin efficacy function is introduced and four special points, including maximum toxin efficacy and inflection points, on the dose-response curve are defined. The special points define three phases of the toxin effect on mortality: (1) toxin concentrations smaller than the first inflection point or (2) larger then the second inflection point imply low mortality rate, and (3) concentrations between the first and the second inflection points imply high mortality rate. Probabilistic interpretation and mathematical analysis for each of the fourmore » models, Hill, logit, probit, and Weibull is provided. Two general model extensions are introduced: (1) the multi-target hit model that accounts for the existence of several vital receptors affected by the toxin, and (2) model with a nonzero mortality at zero concentration to account for natural mortality. Special attention is given to statistical estimation in the framework of the generalized linear model with the binomial dependent variable as the mortality count in each experiment, contrary to the widespread nonlinear regression treating the mortality rate as continuous variable. The models are illustrated using standard EPA Daphnia acute (48 h) toxicity tests with mortality as a function of NiCl or CuSO{sub 4} toxin. - Highlights: • The paper offers a rigorous study of a sigmoid dose-response relationship. • The concentration with highest mortality rate is rigorously defined. • A table with four special points for five morality curves is presented. • Two new sigmoid dose-response models have been introduced. • The generalized linear model is advocated for estimation of sigmoid dose-response relationship.« less

Dinoflagellate Toxins Responsible for Ciguatera Food Poisoning

DTIC Science & Technology

1988-12-10

0 AD DINOFLAGELLATE TOXINS RESPONSIBLE FOR CIGUATERA FOOD POISONING Annual Summary Report Donald M. Miller December 10, 1988 Supported by US ARMY...Maryland 21701-5012 62770A 62770A871 AA 377 11. TITLE (Include Security Classification) Dinoflagellate Toxins Responsible for Ciguatera Poisoning 12...block number) FIELD GROUP SUB-GROUP Ciguatera ; Toxins; Inhibitor; RA I; Dinoflagellates; 06 03 Mass Culture; Purification󈧊 j 01 19 ABSTRACT

Hemocyte–hemocyte adhesion and nodulation reactions of the greater wax moth, Galleria mellonella are influenced by cholera toxin and its B-subunit

PubMed Central

Lapointe, Jason F.; Dunphy, Gary B.; Mandato, Craig A.

2012-01-01

Nodulation, the lepidopteran insect immune response to large numbers of microbes in the blood (hemolymph) consists of the coordination of the blood cell (hemocyte) types the granular cells and plasmatocytes in terms of granular cell–bacteria adhesion and hemocyte–hemocyte adhesion (microaggregation). Hemocyte–microbe adhesion is influenced by the secondary messenger, cAMP, and cAMP-dependent protein kinase A. In the present study, cholera toxin, an AB5 protein known to indirectly stimulate adenylate cyclase, is used to examine the hemocyte responses to glass, bacteria and hemocyte–hemocyte microaggregates. In vitro, this toxin induces a bimodal hemocyte adhesion response that varies with the holotoxin concentration in terms of the individual and aggregated hemocyte adhesion responses: the lower CTX concentration (1.2 nM) increases microaggregate adhesion and decreases individual hemocyte binding to glass, as does higher concentrations (6–120 nM), however microaggregates induced by lower concentrations do not adhere to glass. Cholera toxin-induced microaggregation is inhibited by RGDS, suggestive of integrin involvement. In vivo, cholera toxin (1.2–120 nM) injected into larvae induces also a bimodal hemocytic response: low levels (1.2–6 nM) cause reduced hemocyte adhesion, while high levels (12–120 nM) increase hemocyte release or mobilization of adhesive hemocyte counts in the hemolymph. Increasing levels of cholera toxin concomitantly injected with the non-pathogenic bacterium, Bacillus subtilis produces a bimodal pattern in bacterial removal from the hemolymph which correlates with nodule frequency in larvae injected with cholera toxin only. The effects of higher concentrations of cholera toxin in vitro (6–120 nM) and in vivo (12–120 nM) are mediated by the B-subunit, whereas the isolated A-subunit has no effect on hemocyte activity. Cholera toxin and its individual subunits did not detectably alter levels of intracellular cAMP in the

Short Toxin-like Proteins Abound in Cnidaria Genomes

PubMed Central

Tirosh, Yitshak; Linial, Itai; Askenazi, Manor; Linial, Michal

2012-01-01

Cnidaria is a rich phylum that includes thousands of marine species. In this study, we focused on Anthozoa and Hydrozoa that are represented by the Nematostella vectensis (Sea anemone) and Hydra magnipapillata genomes. We present a method for ranking the toxin-like candidates from complete proteomes of Cnidaria. Toxin-like functions were revealed using ClanTox, a statistical machine-learning predictor trained on ion channel inhibitors from venomous animals. Fundamental features that were emphasized in training ClanTox include cysteines and their spacing along the sequences. Among the 83,000 proteins derived from Cnidaria representatives, we found 170 candidates that fulfill the properties of toxin-like-proteins, the vast majority of which were previously unrecognized as toxins. An additional 394 short proteins exhibit characteristics of toxin-like proteins at a moderate degree of confidence. Remarkably, only 11% of the predicted toxin-like proteins were previously classified as toxins. Based on our prediction methodology and manual annotation, we inferred functions for over 400 of these proteins. Such functions include protease inhibitors, membrane pore formation, ion channel blockers and metal binding proteins. Many of the proteins belong to small families of paralogs. We conclude that the evolutionary expansion of toxin-like proteins in Cnidaria contributes to their fitness in the complex environment of the aquatic ecosystem. PMID:23202321

In vitro reconstitution of the Clostridium botulinum type D progenitor toxin.

PubMed

Kouguchi, Hirokazu; Watanabe, Toshihiro; Sagane, Yoshimasa; Sunagawa, Hiroyuki; Ohyama, Tohru

2002-01-25

Clostridium botulinum type D strain 4947 produces two different sizes of progenitor toxins (M and L) as intact forms without proteolytic processing. The M toxin is composed of neurotoxin (NT) and nontoxic-nonhemagglutinin (NTNHA), whereas the L toxin is composed of the M toxin and hemagglutinin (HA) subcomponents (HA-70, HA-17, and HA-33). The HA-70 subcomponent and the HA-33/17 complex were isolated from the L toxin to near homogeneity by chromatography in the presence of denaturing agents. We were able to demonstrate, for the first time, in vitro reconstitution of the L toxin formed by mixing purified M toxin, HA-70, and HA-33/17. The properties of reconstituted and native L toxins are indistinguishable with respect to their gel filtration profiles, native-PAGE profiles, hemagglutination activity, binding activity to erythrocytes, and oral toxicity to mice. M toxin, which contained nicked NTNHA prepared by treatment with trypsin, could no longer be reconstituted to the L toxin with HA subcomponents, whereas the L toxin treated with proteases was not degraded into M toxin and HA subcomponents. We conclude that the M toxin forms first by assembly of NT with NTNHA and is subsequently converted to the L toxin by assembly with HA-70 and HA-33/17.

Botulinum Toxin for Rhinitis.

PubMed

Ozcan, Cengiz; Ismi, Onur

2016-08-01

Rhinitis is a common clinical entity. Besides nasal obstruction, itching, and sneezing, one of the most important symptoms of rhinitis is nasal hypersecretion produced by nasal glands and exudate from the nasal vascular bed. Allergic rhinitis is an IgE-mediated inflammatory reaction of nasal mucosa after exposure to environmental allergens. Idiopathic rhinitis describes rhinitis symptoms that occur after non-allergic, noninfectious irritants. Specific allergen avoidance, topical nasal decongestants, nasal corticosteroids, immunotherapy, and sinonasal surgery are the main treatment options. Because the current treatment modalities are not enough for reducing rhinorrhea in some patients, novel treatment options are required to solve this problem. Botulinum toxin is an exotoxin generated by Clostridium botulinum. It disturbs the signal transmission at the neuromuscular and neuroglandular junction by inhibiting the acetylcholine release from the presynaptic nerve terminal. It has been widely used in neuromuscular, hypersecretory, and autonomic nerve system disorders. There have been a lot of published articles concerning the effect of this toxin on rhinitis symptoms. Based on the results of these reports, intranasal botulinum toxin A administration appears to be a safe and effective treatment method for decreasing rhinitis symptoms in rhinitis patients with a long-lasting effect. Botulinum toxin type A will be a good treatment option for the chronic rhinitis patients who are resistant to other treatment methods.

Interactions of cnidarian toxins with the immune system.

PubMed

Suput, Dusan

2011-10-01

Cnidarians comprise four classes of toxic marine animals: Anthozoa, Cubozoa, Scyphozoa and Hydrozoa. They are the largest and probably the oldest phylum of toxic marine animals. Any contact with a cnidarian, especially the box jellyfish (Chironex fleckeri), can be fatal, but most cnidarians do not possess sufficiently strong venomous apparatus to penetrate the human skin, whereas others rarely come into contact with human beings. Only a small, almost negligible percentage of the vast wealth of cnidarian toxins has been studied in detail. Many polypeptide cnidarian toxins are immunogenic, and cross-reactivity between several jellyfish venoms has been reported. Cnidarians also possess components of innate immunity, and some of those components have been preserved in evolution. On the other hand, cnidarian toxins have already been used for the design of immunotoxins to treat cancer, whereas other cnidarian toxins can modulate the immune system in mammals, including man. This review will focus on a short overview of cnidarian toxins, on the innate immunity of cnidarians, and on the mode of action of cnidarian toxins which can modulate the immune system in mammals. Emphasis is palced on those toxins which block voltage activated potassium channels in the cells of the immune system.

Bacterial superantigens bypass Lck-dependent T cell receptor signaling by activating a Galpha11-dependent, PLC-beta-mediated pathway.

PubMed

Bueno, Clara; Lemke, Caitlin D; Criado, Gabriel; Baroja, Miren L; Ferguson, Stephen S G; Rahman, A K M Nur-Ur; Tsoukas, Constantine D; McCormick, John K; Madrenas, Joaquin

2006-07-01

The paradigm to explain antigen-dependent T cell receptor (TCR) signaling is based on the activation of the CD4 or CD8 coreceptor-associated kinase Lck. It is widely assumed that this paradigm is also applicable to signaling by bacterial superantigens. However, these bacterial toxins can activate human T cells lacking Lck, suggesting the existence of an additional pathway of TCR signaling. Here we showed that this alternative pathway operates in the absence of Lck-dependent tyrosine-phosphorylation events and was initiated by the TCR-dependent activation of raft-enriched heterotrimeric Galpha11 proteins. This event, in turn, activated a phospholipase C-beta and protein kinase C-mediated cascade that turned on the mitogen-activated protein kinases ERK-1 and ERK-2, triggered Ca(2+) influx, and translocated the transcription factors NF-AT and NF-kappaB to the nucleus, ultimately inducing the production of interleukin-2 in Lck-deficient T cells. The triggering of this alternative pathway by superantigens suggests that these toxins use a G protein-coupled receptor as a coreceptor on T cells.

Temperature Effects Explain Continental Scale Distribution of Cyanobacterial Toxins

PubMed Central

Mantzouki, Evanthia; Fastner, Jutta; de Senerpont Domis, Lisette; Wilk-Woźniak, Elżbieta; Koreivienė, Judita; Verstijnen, Yvon; Krztoń, Wojciech; Walusiak, Edward; Karosienė, Jūratė; Kasperovičienė, Jūratė; Savadova, Ksenija; Vitonytė, Irma; Budzyńska, Agnieszka; Szeląg-Wasielewska, Elżbieta; Domek, Piotr; Messyasz, Beata; Pełechata, Aleksandra; Pełechaty, Mariusz; Kokocinski, Mikolaj; García-Murcia, Ana; Real, Monserrat; Romans, Elvira; Noguero-Ribes, Jordi; Duque, David Parreño; Karakaya, Nusret; Häggqvist, Kerstin; Beklioğlu, Meryem; Filiz, Nur; Iskin, Uğur; Bezirci, Gizem; Tavşanoğlu, Ülkü Nihan; Panou, Manthos; Fakioglu, Özden; Avagianos, Christos; Çelik, Kemal; Yilmaz, Mete; Marcé, Rafael; Buck, Moritz; Colom-Montero, William; Mustonen, Kristiina; Pierson, Don; Yang, Yang; Raposeiro, Pedro M.; Antoniou, Maria G.; Tsiarta, Nikoletta; McCarthy, Valerie; Perello, Victor C.; Feldmann, Tõnu; Panksep, Kristel; Tuvikene, Lea; Gagala, Ilona; Çınar, Şakir; Çapkın, Kadir; Yağcı, Abdulkadir; Cesur, Mehmet; Bilgin, Fuat; Bulut, Cafer; Uysal, Rahmi; Boscaini, Adriano; Cerasino, Leonardo; Richardson, Jessica; Visser, Petra M.; Verspagen, Jolanda M. H.; Karan, Tünay; Ochocka, Agnieszka; Pasztaleniec, Agnieszka; Köker, Latife; Albay, Meriç; Maronić, Dubravka Špoljarić; Stević, Filip; Pfeiffer, Tanja Žuna; Fonvielle, Jeremy; Rothhaupt, Karl-Otto; Hansson, Lars-Anders; Bláha, Luděk; Geriš, Rodan; Fránková, Markéta; Koçer, Mehmet Ali Turan; Alp, Mehmet Tahir; Remec-Rekar, Spela; Elersek, Tina; Hiskia, Anastasia; Haande, Sigrid; Skjelbred, Birger; Madrecka, Beata; Nemova, Hana; Drastichova, Iveta; Chomova, Lucia; Edwards, Christine; Sevindik, Tuğba Ongun; Tunca, Hatice; Önem, Burçin; Aleksovski, Boris; Krstić, Svetislav; Vucelić, Itana Bokan; Nawrocka, Lidia; Salmi, Pauliina; Machado-Vieira, Danielle; de Oliveira, Alinne Gurjão; Delgado-Martín, Jordi; García, David; Cereijo, Jose Luís; Trapote, Mari Carmen; Obrador, Biel; Grabowska, Magdalena; Chmura, Damian; Úbeda, Bárbara; Warming, Trine Perlt; Kobos, Justyna; Mazur-Marzec, Hanna; Arvola, Lauri; Alcaraz-Párraga, Pablo; Toporowska, Magdalena; Pawlik-Skowronska, Barbara; Niedźwiecki, Michał; Pęczuła, Wojciech; Moreno-Ostos, Enrique; Blanco, José María; Rodríguez, Valeriano; Montes-Pérez, Jorge Juan; Palomino, Roberto L.; Rodríguez-Pérez, Estela; Carballeira, Rafael; Picazo, Antonio; Santamans, Anna C.; Ferriol, Carmen; Romo, Susana; Dunalska, Julita; Sieńska, Justyna; Szymański, Daniel; Kostrzewska-Szlakowska, Iwona; Jasser, Iwona; Žutinić, Petar; Udovič, Marija Gligora; Plenković-Moraj, Anđelka; Frąk, Magdalena; Bańkowska-Sobczak, Agnieszka; Wasilewicz, Michał; Özkan, Korhan; Kangro, Kersti; Ibelings, Bas W.

2018-01-01

Insight into how environmental change determines the production and distribution of cyanobacterial toxins is necessary for risk assessment. Management guidelines currently focus on hepatotoxins (microcystins). Increasing attention is given to other classes, such as neurotoxins (e.g., anatoxin-a) and cytotoxins (e.g., cylindrospermopsin) due to their potency. Most studies examine the relationship between individual toxin variants and environmental factors, such as nutrients, temperature and light. In summer 2015, we collected samples across Europe to investigate the effect of nutrient and temperature gradients on the variability of toxin production at a continental scale. Direct and indirect effects of temperature were the main drivers of the spatial distribution in the toxins produced by the cyanobacterial community, the toxin concentrations and toxin quota. Generalized linear models showed that a Toxin Diversity Index (TDI) increased with latitude, while it decreased with water stability. Increases in TDI were explained through a significant increase in toxin variants such as MC-YR, anatoxin and cylindrospermopsin, accompanied by a decreasing presence of MC-LR. While global warming continues, the direct and indirect effects of increased lake temperatures will drive changes in the distribution of cyanobacterial toxins in Europe, potentially promoting selection of a few highly toxic species or strains. PMID:29652856

Temperature Effects Explain Continental Scale Distribution of Cyanobacterial Toxins.

PubMed

Mantzouki, Evanthia; Lürling, Miquel; Fastner, Jutta; de Senerpont Domis, Lisette; Wilk-Woźniak, Elżbieta; Koreivienė, Judita; Seelen, Laura; Teurlincx, Sven; Verstijnen, Yvon; Krztoń, Wojciech; Walusiak, Edward; Karosienė, Jūratė; Kasperovičienė, Jūratė; Savadova, Ksenija; Vitonytė, Irma; Cillero-Castro, Carmen; Budzyńska, Agnieszka; Goldyn, Ryszard; Kozak, Anna; Rosińska, Joanna; Szeląg-Wasielewska, Elżbieta; Domek, Piotr; Jakubowska-Krepska, Natalia; Kwasizur, Kinga; Messyasz, Beata; Pełechaty, Aleksandra; Pełechaty, Mariusz; Kokocinski, Mikolaj; García-Murcia, Ana; Real, Monserrat; Romans, Elvira; Noguero-Ribes, Jordi; Duque, David Parreño; Fernández-Morán, Elísabeth; Karakaya, Nusret; Häggqvist, Kerstin; Demir, Nilsun; Beklioğlu, Meryem; Filiz, Nur; Levi, Eti E.; Iskin, Uğur; Bezirci, Gizem; Tavşanoğlu, Ülkü Nihan; Özhan, Koray; Gkelis, Spyros; Panou, Manthos; Fakioglu, Özden; Avagianos, Christos; Kaloudis, Triantafyllos; Çelik, Kemal; Yilmaz, Mete; Marcé, Rafael; Catalán, Nuria; Bravo, Andrea G.; Buck, Moritz; Colom-Montero, William; Mustonen, Kristiina; Pierson, Don; Yang, Yang; Raposeiro, Pedro M.; Gonçalves, Vítor; Antoniou, Maria G.; Tsiarta, Nikoletta; McCarthy, Valerie; Perello, Victor C.; Feldmann, Tõnu; Laas, Alo; Panksep, Kristel; Tuvikene, Lea; Gagala, Ilona; Mankiewicz-Boczek, Joana; Yağcı, Meral Apaydın; Çınar, Şakir; Çapkın, Kadir; Yağcı, Abdulkadir; Cesur, Mehmet; Bilgin, Fuat; Bulut, Cafer; Uysal, Rahmi; Obertegger, Ulrike; Boscaini, Adriano; Flaim, Giovanna; Salmaso, Nico; Cerasino, Leonardo; Richardson, Jessica; Visser, Petra M.; Verspagen, Jolanda M. H.; Karan, Tünay; Soylu, Elif Neyran; Maraşlıoğlu, Faruk; Napiórkowska-Krzebietke, Agnieszka; Ochocka, Agnieszka; Pasztaleniec, Agnieszka; Antão-Geraldes, Ana M.; Vasconcelos, Vitor; Morais, João; Vale, Micaela; Köker, Latife; Akçaalan, Reyhan; Albay, Meriç; Špoljarić Maronić, Dubravka; Stević, Filip; Žuna Pfeiffer, Tanja; Fonvielle, Jeremy; Straile, Dietmar; Rothhaupt, Karl-Otto; Hansson, Lars-Anders; Urrutia-Cordero, Pablo; Bláha, Luděk; Geriš, Rodan; Fránková, Markéta; Koçer, Mehmet Ali Turan; Alp, Mehmet Tahir; Remec-Rekar, Spela; Elersek, Tina; Triantis, Theodoros; Zervou, Sevasti-Kiriaki; Hiskia, Anastasia; Haande, Sigrid; Skjelbred, Birger; Madrecka, Beata; Nemova, Hana; Drastichova, Iveta; Chomova, Lucia; Edwards, Christine; Sevindik, Tuğba Ongun; Tunca, Hatice; Önem, Burçin; Aleksovski, Boris; Krstić, Svetislav; Vucelić, Itana Bokan; Nawrocka, Lidia; Salmi, Pauliina; Machado-Vieira, Danielle; de Oliveira, Alinne Gurjão; Delgado-Martín, Jordi; García, David; Cereijo, Jose Luís; Gomà, Joan; Trapote, Mari Carmen; Vegas-Vilarrúbia, Teresa; Obrador, Biel; Grabowska, Magdalena; Karpowicz, Maciej; Chmura, Damian; Úbeda, Bárbara; Gálvez, José Ángel; Özen, Arda; Christoffersen, Kirsten Seestern; Warming, Trine Perlt; Kobos, Justyna; Mazur-Marzec, Hanna; Pérez-Martínez, Carmen; Ramos-Rodríguez, Eloísa; Arvola, Lauri; Alcaraz-Párraga, Pablo; Toporowska, Magdalena; Pawlik-Skowronska, Barbara; Niedźwiecki, Michał; Pęczuła, Wojciech; Leira, Manel; Hernández, Armand; Moreno-Ostos, Enrique; Blanco, José María; Rodríguez, Valeriano; Montes-Pérez, Jorge Juan; Palomino, Roberto L.; Rodríguez-Pérez, Estela; Carballeira, Rafael; Camacho, Antonio; Picazo, Antonio; Rochera, Carlos; Santamans, Anna C.; Ferriol, Carmen; Romo, Susana; Soria, Juan Miguel; Dunalska, Julita; Sieńska, Justyna; Szymański, Daniel; Kruk, Marek; Kostrzewska-Szlakowska, Iwona; Jasser, Iwona; Žutinić, Petar; Gligora Udovič, Marija; Plenković-Moraj, Anđelka; Frąk, Magdalena; Bańkowska-Sobczak, Agnieszka; Wasilewicz, Michał; Özkan, Korhan; Maliaka, Valentini; Kangro, Kersti; Grossart, Hans-Peter; Paerl, Hans W.; Carey, Cayelan C.; Ibelings, Bas W.

2018-04-13

Insight into how environmental change determines the production and distribution of cyanobacterial toxins is necessary for risk assessment. Management guidelines currently focus on hepatotoxins (microcystins). Increasing attention is given to other classes, such as neurotoxins (e.g., anatoxin-a) and cytotoxins (e.g., cylindrospermopsin) due to their potency. Most studies examine the relationship between individual toxin variants and environmental factors, such as nutrients, temperature and light. In summer 2015, we collected samples across Europe to investigate the effect of nutrient and temperature gradients on the variability of toxin production at a continental scale. Direct and indirect effects of temperature were the main drivers of the spatial distribution in the toxins produced by the cyanobacterial community, the toxin concentrations and toxin quota. Generalized linear models showed that a Toxin Diversity Index (TDI) increased with latitude, while it decreased with water stability. Increases in TDI were explained through a significant increase in toxin variants such as MC-YR, anatoxin and cylindrospermopsin, accompanied by a decreasing presence of MC-LR. While global warming continues, the direct and indirect effects of increased lake temperatures will drive changes in the distribution of cyanobacterial toxins in Europe, potentially promoting selection of a few highly toxic species or strains.

Endosymbiont-dependent host reproduction maintains bacterial-fungal mutualism.

PubMed

Partida-Martinez, Laila P; Monajembashi, Shamci; Greulich, Karl-Otto; Hertweck, Christian

2007-05-01

Bacterial endosymbionts play essential roles for many organisms, and thus specialized mechanisms have evolved during evolution that guarantee the persistence of the symbiosis during or after host reproduction. The rice seedling blight fungus Rhizopus microsporus represents a unique example of a mutualistic life form in which a fungus harbors endobacteria (Burkholderia sp.) for the production of a phytotoxin. Here we report the unexpected observation that in the absence of endosymbionts, the host is not capable of vegetative reproduction. Formation of sporangia and spores is restored only upon reintroduction of endobacteria. To monitor this process, we succeeded in GFP labeling cultured endosymbionts. We also established a laserbeam transformation technique for the first controlled introduction of bacteria into fungi to observe their migration to the tips of the aseptate hyphae. The persistence of this fungal-bacterial mutualism through symbiont-dependent sporulation is intriguing from an evolutionary point of view and implies that the symbiont produces factors that are essential for the fungal life cycle. Reproduction of the host has become totally dependent on endofungal bacteria, which in return provide a highly potent toxin for defending the habitat and accessing nutrients from decaying plants. This scenario clearly highlights the significance for a controlled maintenance of this fungal-bacterial symbiotic relationship.

Calcium-dependent disorder-to-order transitions are central to the secretion and folding of the CyaA toxin of Bordetella pertussis, the causative agent of whooping cough.

PubMed

O'Brien, Darragh P; Perez, Ana Cristina Sotomayor; Karst, Johanna; Cannella, Sara E; Enguéné, Véronique Yvette Ntsogo; Hessel, Audrey; Raoux-Barbot, Dorothée; Voegele, Alexis; Subrini, Orso; Davi, Marilyne; Guijarro, J Inaki; Raynal, Bertrand; Baron, Bruno; England, Patrick; Hernandez, Belen; Ghomi, Mahmoud; Hourdel, Véronique; Malosse, Christian; Chamot-Rooke, Julia; Vachette, Patrice; Durand, Dominique; Brier, Sébastien; Ladant, Daniel; Chenal, Alexandre

2018-07-01

The adenylate cyclase toxin (CyaA) plays an essential role in the early stages of respiratory tract colonization by Bordetella pertussis, the causative agent of whooping cough. Once secreted, CyaA invades eukaryotic cells, leading to cell death. The cell intoxication process involves a unique mechanism of translocation of the CyaA catalytic domain directly across the plasma membrane of the target cell. Herein, we review our recent results describing how calcium is involved in several steps of this intoxication process. In conditions mimicking the low calcium environment of the crowded bacterial cytosol, we show that the C-terminal, calcium-binding Repeat-in-ToXin (RTX) domain of CyaA, RD, is an extended, intrinsically disordered polypeptide chain with a significant level of local, secondary structure elements, appropriately sized for transport through the narrow channel of the secretion system. Upon secretion, the high calcium concentration in the extracellular milieu induces the refolding of RD, which likely acts as a scaffold to favor the refolding of the upstream domains of the full-length protein. Due to the presence of hydrophobic regions, CyaA is prone to aggregate into multimeric forms in vitro, in the absence of a chaotropic agent. We have recently defined the experimental conditions required for CyaA folding, comprising both calcium binding and molecular confinement. These parameters are critical for CyaA folding into a stable, monomeric and functional form. The monomeric, calcium-loaded (holo) toxin exhibits efficient liposome permeabilization and hemolytic activities in vitro, even in a fully calcium-free environment. By contrast, the toxin requires sub-millimolar calcium concentrations in solution to translocate its catalytic domain across the plasma membrane, indicating that free calcium in solution is actively involved in the CyaA toxin translocation process. Overall, this data demonstrates the remarkable adaptation of bacterial RTX toxins to the

ALTERATIONS OF BACTERIAL TOXINS BY THE CONSTITUENTS OF CHAMOMILE AND HORSERADISH. VI. STUDY ON THE QUESTION OF THE CONVERSION OF LETHALLY ACTING STAPHYLOCOCCI TOXIN INTO TOXOID UNDER THE ACTION OF CHAMOMILES AND HORSERADISH CONSTITUENS

DTIC Science & Technology

On the basis of the investigations it is concluded that the chamomile and horseradish constituents tested, in addition to their hitherto known effects, very probably also have a detoxicating effect upon bacteria toxins.

Induction of rapid and selective cell necrosis in Drosophila using Bacillus thuringiensis Cry toxin and its silkworm receptor.

PubMed

Obata, Fumiaki; Tanaka, Shiho; Kashio, Soshiro; Tsujimura, Hidenobu; Sato, Ryoichi; Miura, Masayuki

2015-07-08

Genetic ablation of target cells is a powerful tool to study the origins and functions of cells, tissue regeneration, or pathophysiology in a human disease model in vivo. Several methods for selective cell ablation by inducing apoptosis have been established, using exogenous toxins or endogenous proapoptotic genes. However, their application is limited to cells with intact apoptotic machinery. Herein, we established a method for inducing rapid and selective cell necrosis by the pore-forming bacterial toxin Cry1Aa, which is specifically active in cells expressing the Cry1Aa receptor (CryR) derived from the silkworm Bombyx mori. We demonstrated that overexpressing CryR in Drosophila melanogaster tissues induced rapid cell death of CryR-expressing cells only, in the presence of Cry1Aa toxin. Cry/CryR system was effective against both proliferating cells in imaginal discs and polyploid postmitotic cells in the fat body. Live imaging analysis of cell ablation revealed swelling and subsequent osmotic lysis of CryR-positive cells after 30 min of incubation with Cry1Aa toxin. Osmotic cell lysis was still triggered when apoptosis, JNK activation, or autophagy was inhibited, suggesting that Cry1Aa-induced necrotic cell death occurred independently of these cellular signaling pathways. Injection of Cry1Aa into the body cavity resulted in specific ablation of CryR-expressing cells, indicating the usefulness of this method for in vivo cell ablation. With Cry toxins from Bacillus thuringiensis, we developed a novel method for genetic induction of cell necrosis. Our system provides a "proteinous drill" for killing target cells through physical injury of the cell membrane, which can potentially be used to ablate any cell type in any organisms, even those that are resistant to apoptosis or JNK-dependent programmed cell death.

Proteomic Identification of Novel Secreted Antibacterial Toxins of the Serratia marcescens Type VI Secretion System*

PubMed Central

Fritsch, Maximilian J.; Trunk, Katharina; Diniz, Juliana Alcoforado; Guo, Manman; Trost, Matthias; Coulthurst, Sarah J.

2013-01-01

It has recently become apparent that the Type VI secretion system (T6SS) is a complex macromolecular machine used by many bacterial species to inject effector proteins into eukaryotic or bacterial cells, with significant implications for virulence and interbacterial competition. “Antibacterial” T6SSs, such as the one elaborated by the opportunistic human pathogen, Serratia marcescens, confer on the secreting bacterium the ability to rapidly and efficiently kill rival bacteria. Identification of secreted substrates of the T6SS is critical to understanding its role and ability to kill other cells, but only a limited number of effectors have been reported so far. Here we report the successful use of label-free quantitative mass spectrometry to identify at least eleven substrates of the S. marcescens T6SS, including four novel effector proteins which are distinct from other T6SS-secreted proteins reported to date. These new effectors were confirmed as antibacterial toxins and self-protecting immunity proteins able to neutralize their cognate toxins were identified. The global secretomic study also unexpectedly revealed that protein phosphorylation-based post-translational regulation of the S. marcescens T6SS differs from that of the paradigm, H1-T6SS of Pseudomonas aeruginosa. Combined phosphoproteomic and genetic analyses demonstrated that conserved PpkA-dependent threonine phosphorylation of the T6SS structural component Fha is required for T6SS activation in S. marcescens and that the phosphatase PppA can reverse this modification. However, the signal and mechanism of PpkA activation is distinct from that observed previously and does not appear to require cell–cell contact. Hence this study has not only demonstrated that new and species-specific portfolios of antibacterial effectors are secreted by the T6SS, but also shown for the first time that PpkA-dependent post-translational regulation of the T6SS is tailored to fit the needs of different bacterial

Dinoflagellate Toxins Responsible for Ciguatera Food Poisoning

DTIC Science & Technology

1987-12-10

the occurrence and importance of secondary toxins (12, 13, 14, 15, 16 and 17). Hutner and McLaughlin (18) suggested that the zooxanthellae ...argued that zooxanthellae were unlikely prospects because very few fishes eat coral and saxitoxin (STX = a paralytic shellfish poison which originates...of the toxins was not based on tests of zooxanthellae species but rather on the assumption that all dinoflagellate toxins would be expected to be

Cwp19 Is a Novel Lytic Transglycosylase Involved in Stationary-Phase Autolysis Resulting in Toxin Release in Clostridium difficile.

PubMed

Wydau-Dematteis, Sandra; El Meouche, Imane; Courtin, Pascal; Hamiot, Audrey; Lai-Kuen, René; Saubaméa, Bruno; Fenaille, François; Butel, Marie-José; Pons, Jean-Louis; Dupuy, Bruno; Chapot-Chartier, Marie-Pierre; Peltier, Johann

2018-06-12

. difficile generally produce two toxins that have been identified as the major virulence factors. The mechanism for release of these toxins from bacterial cells is not yet fully understood but is thought to be partly mediated by bacteriolysis. Here we identify a novel peptidoglycan hydrolase in C. difficile , Cwp19, exhibiting lytic transglycosylase activity. We show that Cwp19 contributes to C. difficile cell autolysis in the stationary phase and, consequently, to toxin release, most probably as a response to environmental conditions such as nutritional signals. These data highlight that Cwp19 constitutes a promising target for the development of new preventive and curative strategies. Copyright © 2018 Wydau-Dematteis et al.

Diphtheria toxin translocation across cellular membranes is regulated by sphingolipids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spilsberg, Bjorn; Hanada, Kentaro; Sandvig, Kirsten

2005-04-08

Diphtheria toxin is translocated across cellular membranes when receptor-bound toxin is exposed to low pH. To study the role of sphingolipids for toxin translocation, both a mutant cell line lacking the first enzyme in de novo sphingolipid synthesis, serine palmitoyltransferase, and a specific inhibitor of the same enzyme, myriocin, were used. The serine palmitoyltransferase-deficient cell line (LY-B) was found to be 10-15 times more sensitive to diphtheria toxin than the genetically complemented cell line (LY-B/cLCB1) and the wild-type cell line (CHO-K1), both when toxin translocation directly across the plasma membrane was induced by exposing cells with surface-bound toxin to lowmore » pH, and when the toxin followed its normal route via acidified endosomes into the cytosol. Toxin binding was similar in these three cell lines. Furthermore, inhibition of serine palmitoyltransferase activity by addition of myriocin sensitized the two control cell lines (LY-B/cLCB1 and CHO-K1) to diphtheria toxin, whereas, as expected, no effect was observed in cells lacking serine palmitoyltransferase (LY-B). In conclusion, diphtheria toxin translocation is facilitated by depletion of membrane sphingolipids.« less

Formation and Control of Cyanobacterial Toxins

EPA Science Inventory

This presentation will cover the formation of harmful algal blooms and the control of their toxins. Data will be presented from current ORD projects on the treatment of cyanobacterial toxins through drinking water treatment facilities. The results will demonstrate that current c...

Natural toxins of plant origin (phytotoxins)

USDA-ARS?s Scientific Manuscript database

Over 100,000 secondary compounds have been identified in plants including alkaloids, glycosides, proteins, polypeptides, amines and non-protein amino acids, organic acids, alcohols, polyacetylenes, resinous toxins and mineral toxins. For thousands of years man has used some of these compounds as fl...

Resistance to Bacillus thuringiensis Mediated by an ABC Transporter Mutation Increases Susceptibility to Toxins from Other Bacteria in an Invasive Insect

PubMed Central

Zhang, Dandan; Gong, Lingling; He, Fei; Soberón, Mario; Bravo, Alejandra; Tabashnik, Bruce E.; Wu, Kongming

2016-01-01

Evolution of pest resistance reduces the efficacy of insecticidal proteins from the gram-positive bacterium Bacillus thuringiensis (Bt) used widely in sprays and transgenic crops. Recent efforts to delay pest adaptation to Bt crops focus primarily on combinations of two or more Bt toxins that kill the same pest, but this approach is often compromised because resistance to one Bt toxin causes cross-resistance to others. Thus, integration of Bt toxins with alternative controls that do not exhibit such cross-resistance is urgently needed. The ideal scenario of negative cross-resistance, where selection for resistance to a Bt toxin increases susceptibility to alternative controls, has been elusive. Here we discovered that selection of the global crop pest, Helicoverpa armigera, for >1000-fold resistance to Bt toxin Cry1Ac increased susceptibility to abamectin and spineotram, insecticides derived from the soil bacteria Streptomyces avermitilis and Saccharopolyspora spinosa, respectively. Resistance to Cry1Ac did not affect susceptibility to the cyclodiene, organophospate, or pyrethroid insecticides tested. Whereas previous work demonstrated that the resistance to Cry1Ac in the strain analyzed here is conferred by a mutation disrupting an ATP-binding cassette protein named ABCC2, the new results show that increased susceptibility to abamectin is genetically linked with the same mutation. Moreover, RNAi silencing of HaABCC2 not only decreased susceptibility to Cry1Ac, it also increased susceptibility to abamectin. The mutation disrupting ABCC2 reduced removal of abamectin in live larvae and in transfected Hi5 cells. The results imply that negative cross-resistance occurs because the wild type ABCC2 protein plays a key role in conferring susceptibility to Cry1Ac and in decreasing susceptibility to abamectin. The negative cross-resistance between a Bt toxin and other bacterial insecticides reported here may facilitate more sustainable pest control. PMID:26872031

The life history of a botulinum toxin molecule.

PubMed

Simpson, Lance

2013-06-01

There is an emerging literature describing the absorption, distribution, metabolism and elimination of botulinum toxin. This work reveals that the toxin can be absorbed by both the oral and inhalation routes. The primary mechanism for absorption is binding and transport across epithelial cells. Toxin that enters the body undergoes a distribution phase, which is quite short, and an elimination phase, which is comparatively long. During the distribution phase, botulinum toxin migrates to the peri-neuronal microcompartment in the vicinity of vulnerable cells, such as cholinergic nerve endings. Only these cells have the ability to selectively accumulate the molecule. When the toxin moves from the cell membrane to the cell interior, it undergoes programmed death. This is coincident with release of the catalytically active light chain that paralyzes transmission. Intraneuronal metabolism of light chain is via the ubiquitination-proteasome pathway. Systemic metabolism and elimination is assumed to be via the liver. The analysis of absorption, distribution, metabolism and elimination of the toxin helps to create a life history of the molecule in the body. This has many benefits, including: a) clarifying the mechanisms that underlie the disease botulism, b) providing insights for development of medical countermeasures against the toxin, and c) helping to explain the meaning of a lethal dose of toxin. It is likely that work intended to enhance understanding of the fate of botulinum toxin in the body will intensify. These efforts will include new and powerful analytic tools, such as single molecule-single cell analyses in vitro and real time, 3-dimensional pharmacokinetic studies in vivo. Copyright © 2013 Elsevier Ltd. All rights reserved.

The Bordetella Adenylate Cyclase Repeat-in-Toxin (RTX) Domain Is Immunodominant and Elicits Neutralizing Antibodies*

PubMed Central

Wang, Xianzhe; Maynard, Jennifer A.

2015-01-01

The adenylate cyclase toxin (ACT) is a multifunctional virulence factor secreted by Bordetella species. Upon interaction of its C-terminal hemolysin moiety with the cell surface receptor αMβ2 integrin, the N-terminal cyclase domain translocates into the host cell cytosol where it rapidly generates supraphysiological cAMP concentrations, which inhibit host cell anti-bacterial activities. Although ACT has been shown to induce protective immunity in mice, it is not included in any current acellular pertussis vaccines due to protein stability issues and a poor understanding of its role as a protective antigen. Here, we aimed to determine whether any single domain could recapitulate the antibody responses induced by the holo-toxin and to characterize the dominant neutralizing antibody response. We first immunized mice with ACT and screened antibody phage display libraries for binding to purified ACT. The vast majority of unique antibodies identified bound the C-terminal repeat-in-toxin (RTX) domain. Representative antibodies binding two nonoverlapping, neutralizing epitopes in the RTX domain prevented ACT association with J774A.1 macrophages and soluble αMβ2 integrin, suggesting that these antibodies inhibit the ACT-receptor interaction. Sera from mice immunized with the RTX domain showed similar neutralizing activity as ACT-immunized mice, indicating that this domain induced an antibody response similar to that induced by ACT. These data demonstrate that RTX can elicit neutralizing antibodies and suggest it may present an alternative to ACT. PMID:25505186

Risk Assessment of Shellfish Toxins

PubMed Central

Munday, Rex; Reeve, John

2013-01-01

Complex secondary metabolites, some of which are highly toxic to mammals, are produced by many marine organisms. Some of these organisms are important food sources for marine animals and, when ingested, the toxins that they produce may be absorbed and stored in the tissues of the predators, which then become toxic to animals higher up the food chain. This is a particular problem with shellfish, and many cases of poisoning are reported in shellfish consumers each year. At present, there is no practicable means of preventing uptake of the toxins by shellfish or of removing them after harvesting. Assessment of the risk posed by such toxins is therefore required in order to determine levels that are unlikely to cause adverse effects in humans and to permit the establishment of regulatory limits in shellfish for human consumption. In the present review, the basic principles of risk assessment are described, and the progress made toward robust risk assessment of seafood toxins is discussed. While good progress has been made, it is clear that further toxicological studies are required before this goal is fully achieved. PMID:24226039

CD44 Promotes intoxication by the clostridial iota-family toxins.

PubMed

Wigelsworth, Darran J; Ruthel, Gordon; Schnell, Leonie; Herrlich, Peter; Blonder, Josip; Veenstra, Timothy D; Carman, Robert J; Wilkins, Tracy D; Van Nhieu, Guy Tran; Pauillac, Serge; Gibert, Maryse; Sauvonnet, Nathalie; Stiles, Bradley G; Popoff, Michel R; Barth, Holger

2012-01-01

Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44(+) melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins.

CD44 Promotes Intoxication by the Clostridial Iota-Family Toxins

PubMed Central

Wigelsworth, Darran J.; Ruthel, Gordon; Schnell, Leonie; Herrlich, Peter; Blonder, Josip; Veenstra, Timothy D.; Carman, Robert J.; Wilkins, Tracy D.; Van Nhieu, Guy Tran; Pauillac, Serge; Gibert, Maryse; Sauvonnet, Nathalie; Stiles, Bradley G.; Popoff, Michel R.; Barth, Holger

2012-01-01

Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44+ melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins. PMID:23236484

Structural Insights into Clostridium perfringens Delta Toxin Pore Formation

PubMed Central

Huyet, Jessica; Naylor, Claire E.; Savva, Christos G.; Gibert, Maryse; Popoff, Michel R.; Basak, Ajit K.

2013-01-01

Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present GM2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus β-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 Å crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal α-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins. PMID:23805259

[Staphylococcal toxin of toxic shock syndrome].

PubMed

Fluer, F S

2007-01-01

Literature data on toxic shock syndrome staphylococcal toxin (TSST-1) are summarized; properties of Staphylococcus aureus strains producing TSST-1, nutrient media, and factors influencing on production of TSST-1 are reviewed. Physical and chemical properties of the toxin, its molecular characteristics, genetic regulation of its production, mechanism of action, and diseases which it causes are also discussed. Clinical and histologic signs of toxic shock syndrome (TSS), its diagnostic criteria, susceptibility of people to TSS, antigenic and serologic properties of the toxin, epidemiology of the infection caused by TSST-1-producing strains of staphylococci, methods of TSST-1 extraction and identification are described.

Structural Basis of Clostridium perfringens Toxin Complex Formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adams,J.; Gregg, K.; Bayer, E.

2008-01-01

The virulent properties of the common human and livestock pathogen Clostridium perfringens are attributable to a formidable battery of toxins. Among these are a number of large and highly modular carbohydrate-active enzymes, including the {mu}-toxin and sialidases, whose catalytic properties are consistent with degradation of the mucosal layer of the human gut, glycosaminoglycans, and other cellular glycans found throughout the body. The conservation of noncatalytic ancillary modules among these enzymes suggests they make significant contributions to the overall functionality of the toxins. Here, we describe the structural basis of an ultra-tight interaction (Ka = 1.44 x 1011 M-1) between themore » X82 and dockerin modules, which are found throughout numerous C. perfringens carbohydrate-active enzymes. Extensive hydrogen-bonding and van der Waals contacts between the X82 and dockerin modules give rise to the observed high affinity. The {mu}-toxin dockerin module in this complex is positioned {approx}180 relative to the orientation of the dockerin modules on the cohesin module surface within cellulolytic complexes. These observations represent a unique property of these clostridial toxins whereby they can associate into large, noncovalent multitoxin complexes that allow potentiation of the activities of the individual toxins by combining complementary toxin specificities.« less

Voltage-dependent blockade of muscle Na+ channels by guanidinium toxins

PubMed Central

1984-01-01

Na+ channels from rat muscle plasma membrane vesicles were inserted into neutral planar phospholipid bilayers and were activated by batrachotoxin. Single channel blocking events induced by the addition of various guanidinium toxins were analyzed to derive the rates of channel-toxin association and dissociation. Blocking by tetrodotoxin, saxitoxin, and six natural saxitoxin derivatives containing sulfate or hydroxyl groups were studied. Although the binding affinities vary over 2,000-fold, all of the toxins exhibit identical voltage dependence of the blocking reactions, regardless of the toxin's net charge. The results suggest that the voltage dependence of toxin binding is due to a voltage-dependent conformational equilibrium of the toxin receptor, rather than to direct entry of the charged toxin molecule into the applied transmembrane electric field. PMID:6096479

The Microcosm Mediates the Persistence of Shiga Toxin-Producing Escherichia coli in Freshwater Ecosystems

PubMed Central

Mauro, Steven A.; Opalko, Hannah; Lindsay, Kyle; Colon, Michael P.

2013-01-01

Water is a major route for infection of humans by exotoxin-producing bacteria, including Shiga toxin-producing Escherichia coli (STEC). While STEC has the potential to be present in nearly every type of water source, its distribution is sporadic, and an understanding of factors that govern its emergence and persistence within water is lacking. In this study, we examined the influence of microbe content on STEC persistence in freshwater. We found that depletion of microbes in the water leads to a considerable increase in the persistence of STEC, an effect that can be mitigated by adding grazing protists to the water. STEC strains appear to be more resistant to the impact of grazing protists than E. coli strains that lack the Shiga toxin (stx) gene. Our results demonstrate that the microcosm can dramatically influence the persistence of STEC in aquatic ecosystems and that the overall impact by microbes on STEC strains is fundamentally different from that of non-STEC strains of bacteria. Overall, these results provide insight into why STEC and possibly other exotoxin-producing bacterial pathogens display such variability in abundance, distribution, and persistence in aquatic ecosystems. PMID:23747699

Structure of CARDS toxin, a unique ADP-ribosylating and vacuolating cytotoxin from Mycoplasma pneumoniae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Becker, Argentina; Kannan, T. R.; Taylor, Alexander B.

Mycoplasma pneumoniae (Mp) infections cause tracheobronchitis and “walking” pneumonia, and are linked to asthma and other reactive airway diseases. As part of the infectious process, the bacterium expresses a 591-aa virulence factor with both mono-ADP ribosyltransferase (mART) and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). CARDS TX binds to human surfactant protein A and annexin A2 on airway epithelial cells and is internalized, leading to a range of pathogenetic events. In this paper, we present the structure of CARDS TX, a triangular molecule in which N-terminal mART and C-terminal tandem β-trefoil domains associate to form anmore » overall architecture distinct from other well-recognized ADP-ribosylating bacterial toxins. We demonstrate that CARDS TX binds phosphatidylcholine and sphingomyelin specifically over other membrane lipids, and that cell surface binding and internalization activities are housed within the C-terminal β-trefoil domain. Finally, the results enhance our understanding of Mp pathogenicity and suggest a novel avenue for the development of therapies to treat Mp-associated asthma and other acute and chronic airway diseases.« less

Structure of CARDS toxin, a unique ADP-ribosylating and vacuolating cytotoxin from Mycoplasma pneumoniae

DOE PAGES

Becker, Argentina; Kannan, T. R.; Taylor, Alexander B.; ...

2015-04-06

Mycoplasma pneumoniae (Mp) infections cause tracheobronchitis and “walking” pneumonia, and are linked to asthma and other reactive airway diseases. As part of the infectious process, the bacterium expresses a 591-aa virulence factor with both mono-ADP ribosyltransferase (mART) and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). CARDS TX binds to human surfactant protein A and annexin A2 on airway epithelial cells and is internalized, leading to a range of pathogenetic events. In this paper, we present the structure of CARDS TX, a triangular molecule in which N-terminal mART and C-terminal tandem β-trefoil domains associate to form anmore » overall architecture distinct from other well-recognized ADP-ribosylating bacterial toxins. We demonstrate that CARDS TX binds phosphatidylcholine and sphingomyelin specifically over other membrane lipids, and that cell surface binding and internalization activities are housed within the C-terminal β-trefoil domain. Finally, the results enhance our understanding of Mp pathogenicity and suggest a novel avenue for the development of therapies to treat Mp-associated asthma and other acute and chronic airway diseases.« less

Comparison of T-2 Toxin and HT-2 Toxin Distributed in the Skeletal System with That in Other Tissues of Rats by Acute Toxicity Test.