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Endospores and Bioweapons

05 December 2005 M.S. Peppler Dept of MMI mark.peppler@ualberta.ca 1-69 Medical Sciences Building. Endospores and Bioweapons. Endospores and Bioweapons. Objectives. After today’s session you will understand: 1. Which organisms produce endopores and how they are formed.

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Endospores and Bioweapons

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  1. 05 December 2005 M.S. Peppler Dept of MMI mark.peppler@ualberta.ca 1-69 Medical Sciences Building Endospores and Bioweapons

  2. Endospores and Bioweapons Objectives. After today’s session you will understand: 1. Which organisms produce endopores and how they are formed. 2. How durable and resilient endopores are. 3. Why developers of bioweapons have focused on anthrax.

  3. Objective 1a. The Nature and Source of Endospores. ENDOSPORES (meaning “within” + “seed”) (often just called “spores”, but this imprecise term does not help differentiate them from other, less elaborate structures. So, we will call them by the proper term, endospores). What they are: Survival structures formed by ca. 20 Gram +ve genera (no Gram –ve genera or Archaea form endospores). e.g. Clostridium spp. (strict anaerobes) Bacillus spp. (aerobes and facultative anaerobes)

  4. Objective 1b. The Nature and Source of Endospores. Refractile structures visible under phase contrast, or by special stain under light microscope. Placement in the cell is characteristic of a species e.g. a) terminal, b) subterminal, c) central. a. b. c.

  5. Objective 1c. The Nature and Source of Endospores. “Vegetative” cells (i.e., cells that are metabolically active) produce the metabolically inactive endospore as a survival response, NOT a reproductive response: 1 vegetative cell  1 endospore  1 vegetative cell (sporulates) (germinates) i.e., There is NO net increase in the # of cells.

  6. * J Bacteriol. 2005.187:6832-6840. Objective 1d. The Nature and Source of Endospores. Sporulation and endospore structure. Takes ~ 8 hours a. An environmental trigger such as C or N starvation, or low temperature (i.e. stationary phase or death phase cells), etc. causes expression of large number of genes under control of a specific sigma factor*. These genes are expressed in the presence of specific sigma factors. After this point, the cell is committed to sporulation and cannot “change its mind”. b. One copy of the chromosome condenses at one end of the cell, and is surrounded twice by an invagination of the cell membrane (two membranes surrounding DNA). Meanwhile, the other copy of the chromosome directs the synthesis of spore-specific proteins. c. Many layers of peptidoglycan are laid down between the two layers of membrane  very thick cell wall called CORTEX, sandwiched between the two membranes. d. The interior (CORE) produces calcium dipicolinate (unique to endospores) and SASPs (small acid-soluble spore proteins believed to stabilize the DNA and provide a C+N source when the endospore eventually germinates), and becomes dehydrated, and metabolically inert (no protein synthesis occurs). e. A thick, keratin-like protein layer is synthesized (by the mother cell) outside the cortex, called the SPORE COAT. f. A thin, loose protein layer is sometimes present outside of the coat, called an EXOSPORIUM. g.Vegetative cell dies, releasing the MATURE ENDOSPORE.

  7. Objective 1e. The Nature and Source of Endospores. a. g. e., f. d. b. c.

  8. Objective 1f. The Nature and Source of Endospores.

  9. Objective 2a. Endospore Durability. The vegetative cells are usually not extremophiles (many are common soil and water organisms). But, because of their dehydration and thick wall and coat, their MATURE endospores ARE very resistant to environmental insults: e.g. chemicals (toxins, metals, free radicals, antibiotics etc.) UV light, gamma irradiation pH desiccation temperature (boiling, freezing) TIME Examples of demonstrated survival: decades in the lab (see text) centuries, millennia in pharaoh’s tombs perhaps millions of years?

  10. Objective 2b. Endospore Durability. Controversies surround how long endospores can survive. e.g., (Cano RJ, Borucki MK. 1995. Revival and identification of bacterial-spores in 25-million-year-old to 40-million-year-old dominican amber; Science 268:1060-1064; Commentary p. 977). More recently, (Vreeland RH, Rosenzweig WD, Powers DW.2000. Isolation of a 250 million-year-old halotolerant bacterium from a primary salt crystal.Nature 407:897-900.See Commentary p. 844), resuscitated bacterial endospores entombed in salt crystals with liquid inclusions, 250 million years ago. The cultures were halotolerant (tolerated 20% NaCl; 10X seawater), so are not likely lab contaminants, may be real. They resemble Bacillus marismortuii literally, “Dead Sea rods”. Next time you sprinkle salt on your food, wonder whether you are eating 100-million-year-old bacteria!

  11. Objective 2c. Endospore Durability. Could endospores survive in outer space? Concept of panspermia, with microbes travelling between planets for millions of years before landing somewhere hospitable and germinating. WHAT IS THE UPPER TIME LIMIT TO SURVIVAL? They may sound indestructible, but they CAN be killed (recall moist heat death). Some species are more resistant than others.

  12. Objective 2c. Endospore Durability. • Germination: Calcium dipicolinate is lost from the cortex and the SASPs are used as immediate energy source to begin synthesis of macromolecules. • Outgrowth: rapidly, the coat and cortex loosen and take up water to re-hydrate the cortex and permit active metabolism • The cell is again sensitive to environmental stresses. • Becomes a vegetative cell identical to the original cell. Full activation, germination and outgrowth takes 60 – 90 minutes, even after decades of inactivity. AMAZING!

  13. Objective 3a. Endospores, Anthrax and Bioweapons. What is the importance of endospores? Some endospore-formers are facultative pathogens when the endospores are introduced into wounds (e.g., puncture wounds like stepping on a nail) or ingested (e.g., in contaminated food) or inhaled (e.g., anthrax). Many of these pathogens cause disease by surviving in the environment as endospores, then germinating in the host (or in food). The newly germinated vegetative cells then produce very potent exotoxins. e.g. botulinum toxin (Botox – most lethal toxin known). anthrax toxins perfringens toxin (food poisoning) tetanus toxin (lockjaw)

  14. Objective 3b. Endospores, Anthrax and Bioweapons. Some are biowarfare agents: either the endospores (e.g., anthrax), or the toxins (e.g., botulinum toxin). (Note: biowarfare is not new: it has been practiced in different forms for millennia, from smallpox to plague to cholera). see website: www.bioterry.com/HistoryBioTerr.html

  15. Objective 3c. Endospores, Anthrax and Bioweapons. Bacillus anthracis Gram positive rod. Facultative anaerobe, but only sporulates when growing aerobically. Natural flora of soils in both vegetative and endospore forms. All cells can produce endospores, but only certain strains can cause anthrax. These are strains that carry two large virulence plasmids: Plasmid pXO1 (110 MDa) is required for synthesis of the three anthrax toxin proteins; Plasmid pXO2 (60 MDa) carries the genes required for synthesis of an antiphagocytic capsule facilitating host immune system evasion. BOTH plasmids are required for maximum virulence and strains lacking either the pX01 or pX02 plasmid are considered either avirulent or significantly attenuated.

  16. Objective 3d. Endospores, Anthrax and Bioweapons. Disease begins with contact between endospore and host, but the route of infection dictates the type of disease caused and severity of disease: scratches on skin: “cutaneous anthrax” (woolsorters’ disease), “low” mortality (10- 50%). (name “anthracis”, like anthracite coal, refers to the black color of skin infected with cutaneous anthrax). ingestion: “intestinal anthrax” type of food poisoning, also “low” mortality (10-50%). inhalation: “pulmonary anthrax” high mortality (up to 90%, even with antibiotic treatment). The endospores germinate in the lungs, the new vegetative cells multiply rapidly, and release exotoxin that causes clotting in pulmonary capillaries, lymph nodes: death from respiratory failure.

  17. Objective 3e. Endospores, Anthrax and Bioweapons. Theoretically B. anthracis is a good bioterrorism agent because of long shelf life, stability, rapid death once administered through respiratory route. In practice, there are problems “weaponizing” the endospores because they tend to clump together and either clog spray nozzles (Tokyo attempts), or become too heavy to stay airborne to cause pulmonary disease. In Oct. 2001 US cases, victims had to handle the contaminated letters to get sick. Based on data from studies of primates, the estimated infectious dose by the respiratory route required to cause inhalation anthrax in humans is 8,000--50,000 spores. The influence of the bacterium strain or host factors on this infectious dose is not completely understood.

  18. Objective 3f. Endospores, Anthrax and Bioweapons. Still, the World Health Organization has estimated that 50 kg of B. anthracis released upwind of a population center of 500,000 could result in 95,000 deaths and 125,000 hospitalizations. Again, this is nothing new - In British biowarfare trial on Gruinard Island in 1941, all sheep on the island died of anthrax, and it still was not habitable by 1987. There is a veterinary vaccine prepared from the plasmid-free [avirulent, non-encapsulated] strain; i.e., vaccine is not against the toxin or capsule, but rather against the bacterium itself) available to protect grazing animals from endospores encountered in contaminated soils (endospores can lay dormant for years, then be ingested or inhaled during feeding). A vaccine for humans is available and is made from isolated “protective antigen” (the cell-binding part of anthrax toxin).

  19. Objective 3g. Endospores, Anthrax and Bioweapons. Close to home! Recurrent outbreaks in buffalo herds (e.g., Wood Bison Nat’l Park) are “treated” by killing all bison in the area and burning their carcasses then burying the ashes. A recent PhD thesis in Dept of MMI, U of A, studied these outbreaks. The student had to remove some details on the geographical location of the outbreaks from his thesis so that it could not be used to guide bioterrorists to the exact site to collect soil for growing virulent anthrax!

  20. Objective 3h. Endospores, Anthrax and Bioweapons. Prevention and treatment: Besides vaccine (an ounce of prevention), antibiotic treatment (e.g., ciprofloxacin) can be effective, but must begin immediately (within 12 h of inhalation). It still may not have enough effect to stop rapid progression of the disease (a pound of cure). The antibiotic is inactive against mature endospores, only works against vegetative cells.

  21. Objective 3i. Endospores, Anthrax and Bioweapons. What else do we know? The most deadly anthrax epidemic known, which occurred at a Soviet biological weapons facility located in Sverdlovsk (now Ekaterinberg, Russia) in 1979, resulted in at least 68 human deaths. See the following for the full story: http://www.gwu.edu/~nsarchiv/NSAEBB/NSAEBB61/ In 1992, Dr. Kanatjan Alibekov fled to the U.S. He carried with him intimate knowledge of the Soviet Union's biological weapons program had served as deputy chief of Biopreparat, the agency at the heart of the Soviet program. Alibekov, who later changed his name to Ken Alibek, wrote about his experience.

  22. Objective 3j. Endospores, Anthrax and Bioweapons. What else do we know? To learn Soviet secrets, the U.S. government turned to longtime bioweaponeer Bill Patrick to debrief the Alibek in a series of clandestine meetings. You can read reminisces about their first meetings together and their insiders' views of the threat of bioterrorism today at the PBS Nova site: http://www.pbs.org/wgbh/nova/bioterror/biow_alibek.html

  23. Next time (Last class, Dec 07) Exam review. Bring your questions (and answers!) Dr. Foght and Dr. Peppler will both be there.

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