BT proteins for non agricultural purposes

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Bacillus thuringiensis, an aerobic, Gram positive, spore forming bacterium produces unique proteinaceous crystalline parasporal inclusions during sporulation which have insecticidal properties. Besides being widely used as an insecticide in agriculture, Bt has been found to be useful in several fields like medicine, endoparasite control, bacteriocin production as well as enzyme production. Parasporin, a new category of bacterial parasporal protein capable of discriminately killing the cancer cells have been discovered. There are six classes of parasporins having different mode of action and cell specificities against cancer and tumor cells (Ohba et al., 2009).Bt proteins have also been used successfully to suppress the population levels of medically important Dipteran pests like mosquitoes by use of mosquitocidal strains that produce Cry proteins (Zhang et al., 2012) as well as potential therapeutic agent against protozoan disease Leishmaniases (El-Sadawy et al., 2008). Crystal proteins, like Cry5B from Bacillus thuringiensis are found to be safe to vertebrates and have been shown to have efficacy against intestinal hookworm parasites (Capello et al., 2006). Thus the multifarious applications of Bacillus thuringiensis have made it a microbe to reckon with and further study its genome for future developments.

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  • Erythrocytes of mice infected with Plasmodium berghei. A, B Three days after inoculation with P. berghei. C, D Five days after inoculation. E, F Seven days after inoculation. A, C and E are samples from a control mouse; B, D and F show samples from a mouse treated with crystal proteins extracted from Bacillus thuringiensis strain 037. Arrows in A show examples of regular, infected, and lysed cells
  • BT proteins for non agricultural purposes

    1. 1. Useful proteins from Bacillus thuringiensis for non-agricultural applications Subhada Pattanayak
    2. 2. OUTLINE Bt for cancer treatmentIntroduction Bt against protozoan diseasesApplications Mosquito control Endoparasite controlConclusion BacteriocinsDiscussion Enzyme production
    3. 3. IntroductionBacillus thuringiensis- a gram positive,spore forming bacteria producescrystalline parasporal inclusionsToxic to agriculturally and medicallyimportant pestsNon-insecticidal Bt strains havecytocidal activity
    4. 4. A conceptual diagram of the present and previous B. thuringiensis crystalA conceptual diagram of the present and previous B. thuringiensis crystalprotein world.protein world. (Kitada et al., 2006)
    5. 5. Applications of Bt
    6. 6. Applications… Bt proteins for cancer treatment Scanning electron micrograph of HTLV-I virus (green) infecting a human T-lymphocyte (yellow). Infection with this virus can stimulate the T-cells to proliferate at an increased rate, causing a risk of developing leukemia.
    7. 7. Brief history….In 1970s, Prasad and Shethna had carried out research on the anti-tumoureffects of Bt.Parasporal proteins of Bacillus thuringiensis serovar shandongiensis foundcytotoxic to human leukaemic T cells (Lee et al., 2000). A soil isolate belonging to Bacillus thuringiensis serovar dakota (H15) wasfound to produce non-cyt inclusion proteins that were highly cytotoxic againsthuman leukaemic T cells (MOLT-4) and moderately cytotoxic to humancervical cancer cells (HeLa) (Kim et al., 2000).
    8. 8. Parasporins“Parasporin” was first used by Mizuki et al to describe anovel protein with a unique cytotoxicity in 2000.Defined as Bt and related bacterial parasporal proteinsthat are non-haemolytic but capable of preferentiallykilling cancer cells
    9. 9. Classification of Parasporins• In 2006, the Committee of Parasporin Classification and Nomenclature was organized to construct a taxonomically sound classification system based on the amino acid identity• In the nomenclature scheme, the number and letter system B. thuringiensis was adopted so that a novel parasporin protein is assigned to a new class incorporating four ranks• Currently, approximately 95, 78, and 45% sequence identities are the borders of the four ranks.
    10. 10. The parasporins are broadly divided into 6 main classes• Till date 19 parasporins discovered and placed on the list of parasporins• Mechanism of action of PS1, PS2, PS3 and PS4 has been well studied and much less is known about PS5 and PS6.
    11. 11. List of parasporins ( till March 2012)Name Cry No. Acc No. Authors & YearPS1Aa1 Cry31Aa1 AB031065 Mizuki et al. 2000 A1190PS1Aa2 Cry31Aa2 AY081052 Jung & Côté 2002 M15PS1Aa3 Cry31Aa3 AB250922 Uemori et al. 2006 B195PS1Aa4 Cry31Aa4 AB274826 Yasutake et al. 2006 Bt 79-25PS1Aa5 Cry31Aa5 AB274827 Yasutake et al.2006 Bt 92-10PS1Aa6 submitting AB375062 Nagamatsu et al.2010 CP78A, M019PS1Ab1 Cry31Ab1 AB250923 Uemori et al. 2006 B195PS1Ab2 Cry31Ab2 AB274825 Yasutake et al.2006 Bt 31-5PS1Ac1 Cry31Ac1 AB276125 Yasutake et al.2006 Bt 87-29PS1Ac2 Cry31Ac2 AB731600 Kuroda et al. 2012 B0462PS1Ad1 submitting AB375062 Nagamatsu et al.2010 CP78B, M019PS2Aa1 Cry46Aa1 AB099515 Ito & Kitada 2004 A1547PS2Aa2 Cry46Aa2 AB454419 Ishikawa et al.2008 A1470PS2Ab1 Cry46Ab1 (http://parasporin.fitc.pref.fukuoka.jp/index.html) AB186914 Yamagiwa et al.2004 TK-E6
    12. 12. CharacteristicsDiffer in molecular weight and compositionEach PS has different cell specificity due to the presenceof different receptor, genome sequence, mode of actionand targeting specificities (Wong,2010)
    13. 13. Cytotoxicity spectra of parasporins Cytotoxicity spectra of parasporinsThe levels of cytotoxicity based on the EC50 values in cell proliferation assay graded as follows:extremely high (++++), high (+++), moderate (++), low (+), and very low / non-toxic (–). NT: Not tested (Ohba et al., 2009)
    14. 14. Cytocidal action and cell specificitiesPS-1 has high toxicity against cancer cell lines like HeLa cells (cell line fromcervical cancer cells)PS-2 shows toxicity against cell like MOLT-4 (Leukemic T cells), Jurkat (cellline from T lymphocyte cells) and Hep G2 (Hepato cellular carcinoma-livertissue)PS-3 has toxicity against Hep G2 and HL-60 (myeloid leukemia cells)PS-4 shows toxicity against CACO-2 (epithelial colorectal adenocarcinomacells)PS-5 – UnknownPS-6 shows anticancer activity against human hepatocyte cancer cells andcervical cancer cells (Wong et al., 2010)
    15. 15. Morphological changes caused by PSs HeLa PS-1: Blebbing of the cells HepG2 PS-2: Balloon shaped HepG2 PS-3: Fragmenting MOLT-4 PS-4: Detachment 2006
    16. 16. Parasporin 1 Exists as 81 kDa protein, Pro-PS-1 Upon activation, PS-1 exists as a 15 kDa and 56 kDa heterodimer. Affects membrane permeability, calcium homeostasis Several conclusions were drawn from the studies on PS1:1) PS-1 cytotoxic against HeLa cells2) PS-1 causes an increase in Ca2+ influx but the influx not related to Ca2+ channels and due to extracellular Ca2+ 3) Heterotrimetric G-proteins or G-protein coupled receptors involved in parasporin-1 induced Ca2+ influx4) PS-1 not a pore forming toxin5) Mode of cell death most likely apoptotic. (Katayama et al., 2007)
    17. 17. (Akiba et al., 2009)
    18. 18. Parasporin- 2• 30kDa protein found to be a pore forming toxin which caused increased permeability to the susceptible cells.• PS-2 distributed at the cell periphery• PS-2 oligomerised at the cell surface via binding to lipid rafts which led to cell lysis and that glycosyl phosphatidyl inositol (GPI)- anchored proteins involved in such cytocidal activity (Kitada et al, 2009)
    19. 19. Detection of parasporin-2 in hepatocellularcarcinoma.• Cancer and non-neoplastic tissuesincubated with parasporin-2, and analyzedusing an anti-parasporin-2 antibody.• Extensive presence of parasporin-2 inhepatocellular carcinoma cells but not innon-neoplastic liver cells• Parasporin-2 binds specifically to thecancer cells. Detection of parasporin-2 in colon cancer cells. • Sections of colon cancer tissues treated with parasporin-2 • Columnar cancer and fibroblastic cells indicated by arrows and arrowheads, respectively. • The toxins efficiently bound to the cancer cells but not to the peripheral fibroblastic cells. (Kitada et al., 2006)
    20. 20. Parasporin-3• PS-3 is a typical three-domain-type Cry protein• PS-3 acts as a pore-forming toxin on the plasma membrane of cancer cells and increases plasma membrane permeability of target cells (Wong et al., 2010)
    21. 21. Parasporin-4Parasporin-4 (PS4) is a cytotoxic protein produced by Bacillusthuringiensis strain A1470.It exhibits high cytotoxicity against human cancer cell lines, CACO-2, Sawano, and MOLT-4 cellsExhibits strong cytotoxicity against several human tumor cell lineswhen activated by protease treatment but does not exhibitinsecticidal or hemolytic activitiesPS4 binds non-specifically to the plasma membrane andoligomerizes to form pores only in target cells, inducing cell death. (Okumura et al., 2011)
    22. 22. Cytopathic effect of parasporin-4 (PS4) against CACO-2 , HeLa , MOLT-4Cells were observed by phase contrast microscopy before and 4 h after treatment with4 μg/ml PS4. (Okumura et al., 2011)
    23. 23. Cytopathic effects in MOLT-4 cells observed with a differential interferencemicroscope. PS4 was added at a final concentration of 2 μg/ml (Okumura et al., 2011)
    24. 24. Bt against protozoan diseases
    25. 25. Erythrocytes of mice infected withPlasmodium bergheiB. thuringiensis protein samples of 0.3 mleach were injected into infected micethrough the tail vein on the 1st and 2nd dayafter infection
    26. 26. Survival days of mice infected with Plasmodium bergheiafter treatment with Bacillus thuringiensis crystalproteins Bt crystal proteins injection prolonged the survival of the infected mice. The mean life for plasmodium- infected mice was about 8.5 days. For those injected with crystal proteins from strains 007, 020, 021, 030, or 032, the mean life was extended to 13.5–15 days Crystal proteins could protect erythrocytes from Plasmodium attack. This study suggested a novel way to control plasmodial infections and even malaria.
    27. 27. Leishmaniasis are caused by haemoflagellate protozoan which belongs togenus Leishmania that infect vertebrate hosts through the bites of sand flyfemales Phlebotomus spp.
    28. 28. Active non truncated core toxin of B.t. serovar thuringiensis (H14) 43 kDafor their activity against Leishmania major promastigotes used. The active protein fraction was bioassayed against L. major promastigotessuspension (7×107 promastigotes /ml) in final concentrations ranging from100 - 0.78 μg /ml aseptically in replicates. The bioassay of protein fraction showed its LC50 is 4.95 μg/ml
    29. 29. Cytopathological changes in Leishmania major promastigotes started withswelling (A) followed by changing from spindle shape to spheroid (B) berryshaped cells (C) cytoplasmic proteins with a giant increase in size (D).While the untreated promastigotes keep motile and alive in pairs (E&F)
    30. 30. Bt for mosquito control
    31. 31. Mosquitoes act worldwide as vectors transmitting disease causing viruses andparasites such as malaria, yellow fever, dengue fever, filariasis, St. Louisencephalitis and the West Nile virus between humans and animals (Tolle, 2009).B. thuringiensis subsp. israelensis (Bti) has been extensively studied forits specific and high toxicity to mosquito and black fly larvae since its discoveryin 1976.The parasporal inclusion body of Bti consists of four major insecticidal crystalproteins (Cry4Aa,Cry4Ba, Cry11Aa and Cyt1Aa) (Stein et al., 2006).
    32. 32. Although Bti and its toxins have been successfully commercialized formosquito control, screening programs have continued worldwide to identifyand characterize new mosquitocidal Bt isolates and toxin genesThe Bt S2160-1 strain was isolated from soil samples collected fromSouthern China and found to have a comparable mosquitocidal activity toBti.PCR-restriction fragment length polymorphism identification system wasdeveloped and used in order to identify novel cry-type genes cry30Ea,cry30Ga, cry50Ba and cry54Ba (Zhang et al., 2012)
    33. 33. Bt for endoparasite control
    34. 34. • Hookworms, whipworms (Ancylostoma duodenale, Necator americanus, and Ancylostoma ceylanicum ) are major soil-transmitted helminths (nematodes, roundworms) that parasitize humans, infecting 576– 740 million people globally and are the leading source of iron-deficient anemia in endemic areas(Bethony et al., 2006).• For mass drug administration against soil-transmitted helminths like hookworms, the current drug of choice is albendazole.• One promising group of alternative anthelmintics is roundworm- active crystal proteins, in particular Cry5B, made by Bacillus thuringiensis (Hu & Aroian, 2012)
    35. 35. • Cry5B and Cry21A have therapeutic activities against infections of the roundworm Heligmosomoides polygyrus bakeri in mice.• Cry5B shows highly therapeutic activity against Ancylostoma ceylanicum infection in hamsters, a minor hookworm parasite of humans• Cry proteins show excellent combinatorial therapeutic properties with nicotinic acetylcholine receptor (nAChR) agonists, one of the two classes of compounds approved by the World Health Organization for the treatment for intestinal roundworms in humans. (Hu & Aroian, 2012)
    36. 36. Typical morphology (at 360 magnification) seen in the L4 Anterior intestine of nematodes fed with four toxicplate assay after feeding nematode crystal proteins in E. coli. showing reduction ofspecies E. coli transformed with empty vector (Left), width of intestine at one position near thevector plus nontoxic Cry protein insert (Center), or vector anterior.plus toxic Cry protein insert (Wei et al., 2003)
    37. 37. Efficacy of Cry proteins against roundworms Efficacy of Cry proteins against roundwormsParasitic roundworms. a A. ceylanicum b. Heligmosomoides polygyrusbakeri (Hu & Aroian, 2012)
    38. 38. Glycolipids that bind Cry5B known as arthroseries glycolipidsare specific to roundworms (nematodes) but lacking inmammals and vertebrates (Griffitts and Aroian , 2005).Thus, Cry proteins like Cry5B are non-toxic to vertebrates dueto lack of the Cry5B arthroseries glycolipid receptorsGiven their non-toxicity to humans and their broad spectrumof nematicidal action, Cry proteins show great potential asnext-generation anthelmintics.
    39. 39. • Anthelminthic activities of purified recombinant Cry5B against the hookworm parasite Ancylostoma ceylanicum, a bloodfeeding gastrointestinal nematode for which humans are permissive hosts.• Cry5B was found to be highly toxic to early stage hookworm larvae.• Exposure of adult A. ceylanicum to Cry5B was also associated with significant toxicity including a substantial reduction in egg excretion by adult female worms.
    40. 40. Exposure to Cry5B impairs motility ofadult hookworms in culture.Cry5B toxin reduces A. ceylanicum eggexcretion.
    41. 41. Cry5B treatment reduceshookworm infection as measuredby weight gain and bloodhaemoglobin
    42. 42. Bt for bacteriocins
    43. 43. Bacteriocins ?• Inhibitory peptides or proteins, produced by different groups of bacteria, which have bactericidal effects on micro-organisms closely related to the producer(Jack et al. 1995).• Produced by bacteria as a defense mechanism in complex environments.
    44. 44. Bacteriocins produced by Bacillus thuringiensis Source Bacteriocin Mol wt(kDa) Activity (Abriouel et al., 2011)
    45. 45. Bt for enzyme productionBt for enzyme production
    46. 46. Proteases• Proteases are essential for biological processes like cell cycle regulation, cell growth and differentiation and sporulation.• Bt is an excellent source of protease enzymes. (Brar et al., 2007) Cellulases Cellulases Bacillus thuringiensis strains produced novel cellulases which could liberate glucose from soluble cellulose, carboxymethyl cellulose (CMC), and insoluble crystalline cellulose. (Bisht, 2010)
    47. 47. Chitinases:• Bacillus thuringiensis produces chitinases• The presence of endochitinase and exochitinase genes was detected via PCR screening of 16 B. thuringiensis isolates which showed also an important chitinolytic activity on plates containing colloidal chitin (Bisht, 2010). as a major or unique carbon source
    48. 48. AutolysinsEndogenous peptidoglycan hydrolases that digest cell wallpeptidoglycans of the producer bacterium and of other bacteriaThe characterisation of the autolytic phenotype of 112 B. thuringiensisstrains showed seven major proteins of molecular weights rangingbetween 25 and 90 kDa which exhibited peptidoglycan hydrolase activity,particularly at alkaline pH.Several of these proteins retained lytic activity against other bacterialspecies such as Micrococcus lysodeikticus, Listeria monocytogenes andStaphylococcus aureus.These are of great interest in field application of B. thuringiensis forimproving bacterial or insect biocontrol by coupling with otherantagonistic factors such as bacteriocins or chitinases (Bisht, 2010)
    49. 49. Conclusion
    50. 50. Discussion

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