1、Biotoxins,Toxins,Poisonous substances produced by microorganisms (and Others)toxins - primary factor - pathogenicity 220 known bacterial toxins 40% cause disease by damaging the Eukaryotic cell membrane Toxemia Toxins in the bloodstream,Poisonous mushrooms The deathcap one bite can prove fatal,Cyano
2、bacterial Toxins:Cyanobacteria can produce a wide array of neurotoxins, liver toxins, cell toxins and skin irritants. In addition, many genera, such as Anabaena, can produce multiple toxins.,Toxin-Producing Organism Classes,Viruses (e.g. stx phage, cytotoxins, lysins) Bacteria (e.g. endotoxins, exot
3、oxins) Fungi (e.g. tricothecenes) Protozoa (endotoxin, phospholipase, protease) Algae (microcystins, ASP, PSP) Plants (alkaloids, tannins, cyanogenic glycosides) Higher Animals (fish, insects, snakes, frogs),Biological Agents of Concern,e.g.: Clostridium botulinum toxin Staphylococcal enterotoxin B
4、Ricin toxin Trichothecene Mycotoxins Others (marine neurotoxins, venoms),Classes of Toxins by Site of Action,e.g.: Neurotoxins Nerve Tissue Enterotoxins Enteric System Cytotoxins Cells Hemotoxins Blood Cells Dermatotoxins Skin Cells Hepatotoxins Liver tissue,Water Treatment & Biological Toxins,Water
5、 Treatment & Biological Toxins,Weaponization and No Observed Adverse Effect Level (NOAEL) for Biotoxins,Weaponization and No Observed Adverse Effect Level (NOAEL) for Biotoxins,Relative Toxicity of Some Poisons in Water,Exotoxins,Products of bacterial metabolism that are elaborated and excreted into
6、 growth media as bacteria grow Virulence Factors; Waste products Chromosomal or on mobile elements One or Two subunit toxins One subunit (e.g. pore forming cytolysins),Subunit Toxins,2 subunits A subunit: enzymatic subunit conferring toxicity B subunit: cell recognition, toxin entry subunit Protoxin
7、 from single gene with post-translation proteolytic cleavage eg, C diphtheriae diphtheria toxin (AB) A & B subunits are products of different genes eg, Vibrio cholerae cholera toxin (A1B5) Bipartite A & B subunits are products of different genes & do not associate until modified by the host target c
8、ell eg, Bacillus anthracis lethal factor (A) and protective antigen (B) Pseudo Subunit Toxins: Bifunctional toxins with single polypeptide having distinct toxic and entry functions eg, Bordatella pertussis adenylate cyclase-hemolysin,E coli heat liable enterotoxin (LT): a) subunit B pentamer, b) sub
9、unit A. (Nature 355:561-564;1992),Acquisition of virulence genes,Bacteria have three ways of exchanging DNA Transformation cells take up naked DNA Transduction phages carry DNA Conjugation cells mate through specialised appendages,Mobile genetic elements,Transposons ST enterotoxin genes Virulence Pl
10、asmids e.g. TTSSs in Shigella, Yersinia; toxins in Salmonella, E. coli, anthrax Phage-encoded virulence e.g. botulinum toxins, diphtheria toxin, shiga-like toxin (linked to lysis), staphylococcal toxins, TTSS substrates in Salmonella.,Exotoxin Classification,Site and features of intoxication eg, neu
11、rotoxin, enterotoxin Structure eg, AB subunit toxins Heat-liable vs heat-stable eg, EC heat-sensitive & heat-liable enterotoxins Mechanism of action eg, pore-forming cytolysin Host cell target eg, plasma membrane integrity,Host Cell Toxin Targets,Host Cell Membrane Integrity Pore-forming cytolysins
12、Phospholipases Surfactant-like Host Cell Macromolecular Synthesis/Stability Protein Synthesis & modification DNA lysis Aberrant Host Cell Regulation Altered adenylate cyclase activity Altered GTPase activity,Types of Exotoxins,1. Cytotoxins kill cells 2. Neurotoxins interfere with normal nerve impul
13、ses 3. Enterotoxins effect cells lining the G.I. Tract,Most genes that code for exotoxins - plasmids or phages,Lysogenic convergence Diphtheria Cytotoxin inhibits protein synthesis - resulting in cell death Pseudomembrane fibrin, dead tissue, bacterial cells,Lysogenic Convergence,Scarlet Fever Strep
14、tococcus pyogenes lysogenic convergence prophage cytotoxin - damages blood capillaries and results in a skin rash Strep Thoat with a rash,Diseases caused by Neurotoxins,Botulism Clostridium botulinum Gram (+), anaerobic, spore-forming rod, found in soil works at the neuromuscular junction prevents i
15、mpulse from nerve cell to muscle cell results in muscle paralysis,Clostridium General Information,Anaerobic Rods Form spores Produce toxins Species of note: C. botulinum C. tetani C. perfringens,Clostridium botulinum,Gram positive spore forming rods Spores are heat resistant Widely found in nature T
16、oxins produced are most potent Seven types of botulism (A, B, C, D, E, F, G) Toxin not heat stable Pathologies Paralysis Infantile botulism,Action of Botulinum Toxin,0.1-1 nanograms (ng) needed to cause illness Toxin is absorbed in intestine, transported to neuromuscular junctions via blood stream C
17、leaves proteins which enable synaptic vesicle fusion in neurons Neuromuscular neurotransmitter blocked Motor dysfunction,Sources: Types of Food Incriminated,Home canned foodsFish preserved by salting or smokingPrepared meats eaten uncookedHoney can be problematic for infants,Tetanus (Lock Jaw),Clost
18、ridium tetani Gram (+), spore-forming, anaerobic rod neurotoxin acts on nerves, resulting in the inhibition of muscle relaxation tetanospasmin - “spasms” or “Lock Jaw”,Clostridium perfringens,Previously named C. welchii Anaerobic, gram-positive, sporulating Widely distributed Soil and sediments Inte
19、stinal tracts Areas with fecal contamination Toxins can cause problems for humans At least 12 identified,http:/medinfo.ufl.edu/year2/mmid/bms5300/bugs/closper.html,C. perfringens Food Poisoning,Type A strain Symptoms Abdominal cramps, diarrhea Death rare Symptoms appear 8-22 hours after bacteria con
20、sumed Usually over in 24 hours Unreported cases,Gas Gangrene and Necrotic Enteritis,Wound invasion causes gas gangrene Type A strain Ingestion causes necrosis of intestines Type C strain Results in septicemia Rare in the united states Often fatal C. perfringens one of 6 species causing necrotic ente
21、ritis Present in 80-90% of all cases.,Diseases caused by Enterotoxins,Cholera Vibrio cholerae Gram (-) comma shaped rods,Cholera toxin,Converts ATP into cAMP causes cells to excrete Cl- ions and inhibits absorption of Na+ ions Electrolyte imbalance H2O leaves by osmosis H2O Loss (Diarrhea),Severe ca
22、ses, 12 - 20 liters of liquid lost in a day,Untreated cases - Mortality Rate about 50%Mortality may be reduced to about 1% administering fluids and electrolytes,EHEC (Enterohemorrhagic E. coli),E. coli (0157:H7) enterotoxin causes a hemolytic inflammation of the intestines results in bloody diarrhea
23、 Toxin alters the 60S ribosomal subunit inhibits Protein Synthesis Results in cell death lining of intestine is “shed” Bloody Diarrhea (Dysentary),Staphylococcus Enterotoxin B,Exotoxin produced by Staphylococcus aureus Food poisoning Moderately stable Exposure Inhalation (incubation 3-12 hours) Inge
24、stion (incubation 4-10 hours) Skin contact,Staphylococcus Enterotoxin B Signs & Symptoms,Inhalation specific Non-productive cough, chest pain, dyspnea Pulmonary edema & respiratory failure (severe cases) Gastrointestinal,Ingestion specific Nausea Vomiting Diarrhea,Non-specific flu-like symptoms- Fev
25、er Chills Headache Myalgia Prostration,Staphylococcus Enterotoxin B,Toxins No person to person transmission Not dermally active Secondary aerosols not a hazard Significant morbidity Inhalation 50-80% (untreated),Endotoxins,Structural component in cell wall; Majority found in gram negative bacteria T
26、hree components: Core polysaccharide Side chain sugars (antigenic; highly variable) Lipid A molecule (conserved) LPS; O antigen Not secreted (externally); may be released by destruction of cell (phagocytic host cells); targets receptive site,Endotoxin of Gram-negatives,ricinus communis,Ricin, a pote
27、nt cytotoxin (toxic at cellular level) concentrated in the castor bean also contains highly toxic glycoproteins that block the synthesis of other good proteins causing cell death.,Ricin,Ricinus communis or Castor Bean plant is mildly toxic including the stalk, leaf and the bean is very toxic. One be
28、an chewed up is sufficient to kill a small child. Plants can grow to about 12 feet and has large five to seven inch long finger like leaves and is green to reddish purple in color. Seed pods are spinney and green to red in color. Castor Bean oil is used for lubricants and the left over pulp or cake
29、is boiled and pressed for use as animal feed or discarded. Boiling makes it non-toxic.,Castor Beans “ricinus communis”,Ricin Routes of Entry,There are three methods of ricin poisoning ; Intravenous-Introduced into a puncture or cut. Inhalation- Aerosolized liquid or powder inhaled directly into the
30、lungs. Ingestion- Ricin enters through contaminated food or water into the stomach. Symptoms range from fever, cough, nausea, chest tightness, sweating, cyanosis, hypo tension (very low blood pressure), dyspnea (labored breathing). Circulatory and respiratory collapse occur within 36-72 hrs leading
31、to death.,Ricin Signs & Symptoms,Inhalation Coughing, chest tightness, nausea, difficulty breathing, muscle aches (1st few hours) Inflammed airways, excess fluid in lungs, blue skin, breathing more difficult (next few hours),Ricin Signs & Symptoms,Ingestion Internal bleeding (stomach, intestines) Bl
32、ood diarrhea and vomiting Liver, spleen and kidney failure Low/no urine output,Ingestion Pupil dilation Fever Thirst Sore throat Headache Vascular collapse Shock,Ricin Signs & Symptoms,Injection Muscle and lymph node death (injection site) Liver, kidney and spleen failure Massive bleeding from stoma
33、ch & intestines Death multiple organ failure 36-48 hours after exposure,Toxicity,Ricin is one of the deadliest toxins known to man. Ricin is 400 times more toxic than cobra venom. Ricin is 1200 times more toxic than cyanide. Ricin is 4000 times more toxic than arsenic. An amount in size between a ha
34、lf and a full grain of salt is sufficient to cause death in most persons. Mortality rate is about 85% . There is no-antitoxin available currently. NOTE: Ricin poisoning is not contagious. It does not produce a communicable disease. Caution should be taken to ensure victims are decontaminated prior t
35、o clothing or skin contact with health care givers.,Ricin,Castor bean cake or meal can be fed to animals without being toxic due to boiling, then ricin shares the same heat instability towards toxicity. Exposure to high or very warm temperatures and or high humidity will diminish the strength of the
36、 toxin (denaturing effect). Currently, there is no antidote for ricin but one is being worked on by USAMRID and the Nat. Inst. Of Health. The only treatment is large amounts of intestinal protectives via stomach tube and lots of intravenous fluids may possibly help.,Ricin History as a Weapon,Ricin i
37、s a choice toxin of terrorists/assassins, because without motive or witnesses, death appears to be from pneumonia. Death can be caused by a very small amount (.015 milligram / size of a grain of salt). 1978 Ricin was the toxin used to assassinate Georgy Markov (Bulgarian Defector) in London. Markov
38、was stuck with an umbrella gun that shot a Platinum ball containing Ricin. Markov died three days later. 1991 Members of a Minnesota Patriot organization manufactured ricin in an attempt to kill a US Marshal. Their plans and the amount of toxin made could have killed more than a hundred people. They
39、 were unsuccessful.,Ricin History of Use,1993 Thomas Lavy (Neo Nazi) arrested in Canada en-route to the US, was found to have enough ricin to kill 30,000 people, four guns and 20,00 rounds of ammo. 1995 Disneyland gets threat letter and a video of someone mixing chemicals possibly Ricin or Sarin. 19
40、97 Thomas Leahy arrested in a shooting. Raid on home finds Ricin lab in basement along with nicotine sulfate and he was trying to grow botulism.,Ricin - Continued,2003 Seven men (4 Algerians) were arrested in a London apartment where they had manufactured ricin, a quantity of which could not be acco
41、unted for. Authorities refused to announce the nationality of the three other suspects.,Recombinant DNA,http:/www4.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm,Recombinant DNA,Recombinant DNA molecules are either: 1) molecules which are constructed outside living cells by joining natur
42、al or synthetic DNA segments to DNA molecules that can replicate in a living cell; or 2) DNA molecules that result from the replication of those described in 1). Synthetic DNA segments which are likely to yield a potentially harmful polynucleotide or polypeptide (e.g., a toxin or a pharmacologically
43、 active agent) are considered as equivalent to their natural DNA counterpart. However,if the synthetic DNA segment is not expressed in vivo as a biologically active polynucleotide or polypeptide product, it is exempt from the NIH Guidelines.,IBC Approval,Principal Investigator must submit a registra
44、tion document to the Institutional Biosafety Committee which contains the following information: (i) the source(s) of DNA; (ii) the nature of the inserted DNA sequences; (iii) the host(s) and vector(s) to be used; (iv) if an attempt will be made to obtain expression of a foreign gene, and if so, ind
45、icate the protein that will be produced; and (v) the containment conditions that will be implemented as specified in the NIH Guidelines,Required Approval,Experiments that Require Institutional Biosafety Committee Approval, RAC Review, and NIH Director Approval Before Initiation The deliberate transf
46、er of a drug resistance trait to microorganisms that are not known to acquire the trait naturally Experiments That Require NIH/OBA and Institutional Biosafety Committee Approval Before Initiation Deliberate formation of recombinant DNA containing genes for the biosynthesis of toxin molecules lethal
47、for vertebrates at an LD50 of less than 100 nanograms per kilogram body weight (e.g., microbial toxins such as the botulinum toxins, tetanus toxin, diphtheria toxin, and Shigella dysenteriae neurotoxin) Experiments that Require Institutional Biosafety Committee and Institutional Review Board Approva
48、ls and RAC Review Before Research Participant Enrollment The deliberate transfer of recombinant DNA, or DNA or RNA derived from recombinant DNA, into human research participants (human gene transfer),Require IBC Approval Prior to Initiation,Experiments Using Risk Group 2, Risk Group 3, Risk Group 4,
49、 or Restricted Agents as Host-Vector Systems Experiments in Which DNA From Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents is Cloned into Nonpathogenic Prokaryotic or Lower Eukaryotic Host-Vector Systems Experiments Involving the Use of Infectious DNA or RNA Viruses or Defective DNA or RNA Viruses in the Presence of Helper Virus in Tissue Culture Systems Experiments Involving Whole Animals Experiments Involving Whole Plants (BL 2P+ and above) Experiments Involving More than 10 Liters of Culture,Require IBC Approval Simultaneous with Initiation,
