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Useful proteins from Bacillus thuringiensis
     for non-agricultural applications




                            Subhada Pattanayak
OUTLINE


                 Bt for cancer treatment
Introduction     Bt against protozoan diseases

Applications     Mosquito control
                 Endoparasite control
Conclusion
                 Bacteriocins
Discussion
                 Enzyme production
Introduction

Bacillus thuringiensis- a gram positive,
spore    forming     bacteria   produces
crystalline parasporal inclusions


Toxic to agriculturally and medically
important pests


Non-insecticidal     Bt   strains   have
cytocidal activity
A conceptual diagram of the present and previous B. thuringiensis crystal
A conceptual diagram of the present and previous B. thuringiensis crystal
protein world.
protein world.
                                             (Kitada et al., 2006)
Applications of Bt
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.
Brief history….

In 1970s, Prasad and Shethna had carried out research on the anti-tumour
effects of Bt.


Parasporal proteins of Bacillus thuringiensis serovar shandongiensis found
cytotoxic to human leukaemic T cells (Lee et al., 2000).


 A soil isolate belonging to Bacillus thuringiensis serovar dakota (H15) was
found to produce non-cyt inclusion proteins that were highly cytotoxic against
human leukaemic T cells (MOLT-4) and moderately cytotoxic to human
cervical cancer cells (HeLa) (Kim et al., 2000).
Parasporins

“Parasporin” was first used by Mizuki et al to describe a
novel protein with a unique cytotoxicity in 2000.



Defined as Bt and related bacterial parasporal proteins
that are non-haemolytic but capable of preferentially
killing cancer cells
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.
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.
List of parasporins ( till March 2012)
Name       Cry No.       Acc No.       Authors & Year
PS1Aa1     Cry31Aa1      AB031065      Mizuki et al. 2000     A1190
PS1Aa2     Cry31Aa2      AY081052      Jung & Côté 2002       M15
PS1Aa3     Cry31Aa3      AB250922      Uemori et al. 2006     B195
PS1Aa4     Cry31Aa4      AB274826      Yasutake et al. 2006   Bt 79-25


PS1Aa5     Cry31Aa5      AB274827      Yasutake et al.2006    Bt 92-10
PS1Aa6     submitting    AB375062      Nagamatsu et al.2010   CP78A, M019
PS1Ab1     Cry31Ab1      AB250923      Uemori et al. 2006     B195


PS1Ab2     Cry31Ab2      AB274825      Yasutake et al.2006    Bt 31-5


PS1Ac1     Cry31Ac1      AB276125      Yasutake et al.2006    Bt 87-29


PS1Ac2     Cry31Ac2      AB731600      Kuroda et al. 2012     B0462


PS1Ad1     submitting    AB375062      Nagamatsu et al.2010   CP78B, M019


PS2Aa1     Cry46Aa1      AB099515      Ito & Kitada 2004      A1547


PS2Aa2     Cry46Aa2      AB454419      Ishikawa et al.2008    A1470
PS2Ab1     Cry46Ab1     (http://parasporin.fitc.pref.fukuoka.jp/index.html)
                         AB186914      Yamagiwa et al.2004 TK-E6
Characteristics

Differ in molecular weight and composition


Each PS has different cell specificity due to the presence
of different receptor, genome sequence, mode of action
and targeting specificities



                                              (Wong,
2010)
Cytotoxicity spectra of parasporins
            Cytotoxicity spectra of parasporins




The 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)
Cytocidal action and cell specificities
PS-1 has high toxicity against cancer cell lines like HeLa cells (cell line from
cervical cancer cells)

PS-2 shows toxicity against cell like MOLT-4 (Leukemic T cells), Jurkat (cell
line from T lymphocyte cells) and Hep G2 (Hepato cellular carcinoma-liver
tissue)

PS-3 has toxicity against Hep G2 and HL-60 (myeloid leukemia cells)

PS-4 shows toxicity against CACO-2 (epithelial colorectal adenocarcinoma
cells)

PS-5 – Unknown

PS-6 shows anticancer activity against human hepatocyte cancer cells and
cervical cancer cells                                    (Wong et al., 2010)
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
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 cells

2) 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+ influx

4) PS-1 not a pore forming toxin

5) Mode of cell death most likely apoptotic.

                                               (Katayama et al., 2007)
(Akiba et al., 2009)
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)
Detection of parasporin-2 in hepatocellular
carcinoma.
•    Cancer and non-neoplastic tissues
incubated with parasporin-2, and analyzed
using an anti-parasporin-2 antibody.
• Extensive presence of parasporin-2 in
hepatocellular carcinoma cells but not in
non-neoplastic liver cells
• Parasporin-2 binds specifically to the
cancer 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)
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)
Parasporin-4
Parasporin-4 (PS4) is a cytotoxic protein produced by Bacillus
thuringiensis strain A1470.
It exhibits high cytotoxicity against human cancer cell lines, CACO-
2, Sawano, and MOLT-4 cells

Exhibits strong cytotoxicity against several human tumor cell lines
when activated by protease treatment but does not exhibit
insecticidal or hemolytic activities

PS4    binds   non-specifically   to   the   plasma   membrane     and
oligomerizes to form pores only in target cells, inducing cell death.


                                                  (Okumura et al., 2011)
Cytopathic effect of parasporin-4 (PS4) against CACO-2 , HeLa , MOLT-4
Cells were observed by phase contrast microscopy before and 4 h after treatment with
4 μg/ml PS4.
                                                         (Okumura et al., 2011)
Cytopathic effects in MOLT-4 cells observed with a differential interference
microscope. PS4 was added at a final concentration of 2 μg/ml
                                                          (Okumura et al., 2011)
Bt against protozoan
      diseases
Erythrocytes of mice infected with
Plasmodium berghei


B. thuringiensis protein samples of 0.3 ml
each were injected into infected mice
through the tail vein on the 1st and 2nd day
after infection
Survival days of mice infected with Plasmodium berghei
after treatment with Bacillus thuringiensis crystal
proteins


                                                           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.
Leishmaniasis are caused by haemoflagellate protozoan which belongs to
genus Leishmania that infect vertebrate hosts through the bites of sand fly
females Phlebotomus spp.
Active non truncated core toxin of B.t. serovar thuringiensis (H14) 43 kDa
for their activity against Leishmania major promastigotes used.



  The active protein fraction was bioassayed against L. major promastigotes
suspension (7×107 promastigotes /ml) in final concentrations ranging from
100 - 0.78 μg /ml aseptically in replicates.


  The bioassay of protein fraction showed its LC50 is 4.95 μg/ml
Cytopathological changes in Leishmania major promastigotes started with
swelling (A) followed by changing from spindle shape to spheroid (B) berry
shaped cells (C) cytoplasmic proteins with a giant increase in size (D).
While the untreated promastigotes keep motile and alive in pairs (E&F)
Bt for mosquito control
Mosquitoes act worldwide as vectors transmitting disease causing viruses and
parasites such as malaria, yellow fever, dengue fever, filariasis, St. Louis
encephalitis and the West Nile virus between humans and animals (Tolle, 2009).



B. thuringiensis subsp. israelensis (Bti) has been extensively studied for
its specific and high toxicity to mosquito and black fly larvae since its discovery
in 1976.



The parasporal inclusion body of Bti consists of four major insecticidal crystal
proteins (Cry4Aa,Cry4Ba, Cry11Aa and Cyt1Aa) (Stein et al., 2006).
Although Bti and its toxins have been successfully commercialized for
mosquito control, screening programs have continued worldwide to identify
and characterize new mosquitocidal Bt isolates and toxin genes

The Bt S2160-1 strain was isolated from soil samples collected from
Southern China and found to have a comparable mosquitocidal activity to
Bti.
PCR-restriction fragment length polymorphism identification system was
developed and used in order to identify novel cry-type genes cry30Ea,
cry30Ga, cry50Ba and cry54Ba




                                                     (Zhang et al., 2012)
Bt for endoparasite
      control
•   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)
•   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)
Typical morphology (at 360 magnification) seen in the L4      Anterior intestine of nematodes fed with four toxic
plate assay after feeding nematode                            crystal proteins in E. coli. showing reduction of
species E. coli transformed with empty vector (Left),         width of intestine at one position near the
vector plus nontoxic Cry protein insert (Center), or vector   anterior.
plus toxic Cry protein insert
                                                                                     (Wei et al., 2003)
Efficacy of Cry proteins against roundworms
             Efficacy of Cry proteins against roundworms




Parasitic roundworms. a A. ceylanicum b. Heligmosomoides polygyrus
bakeri                                                (Hu & Aroian, 2012)
Glycolipids that bind Cry5B known as arthroseries glycolipids
are specific to roundworms (nematodes) but lacking in
mammals and vertebrates (Griffitts and Aroian , 2005).

Thus, Cry proteins like Cry5B are non-toxic to vertebrates due
to lack of the Cry5B arthroseries glycolipid receptors

Given their non-toxicity to humans and their broad spectrum
of nematicidal action, Cry proteins show great potential as
next-generation anthelmintics.
•   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.
Exposure to Cry5B impairs motility of
adult hookworms in culture.




Cry5B toxin reduces A. ceylanicum egg
excretion.
Cry5B         treatment         reduces
hookworm infection as measured
by   weight      gain     and     blood
haemoglobin
Bt for bacteriocins
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.
Bacteriocins produced by Bacillus thuringiensis
    Source    Bacteriocin   Mol wt(kDa)   Activity




                                              (Abriouel et al., 2011)
Bt for enzyme production
Bt for enzyme production
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)
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
Autolysins
Endogenous      peptidoglycan    hydrolases    that       digest   cell   wall
peptidoglycans of the producer bacterium and of other bacteria
The characterisation of the autolytic phenotype of 112 B. thuringiensis
strains showed seven major proteins of molecular weights ranging
between 25 and 90 kDa which exhibited peptidoglycan hydrolase activity,
particularly at alkaline pH.
Several of these proteins retained lytic activity against other bacterial
species such as Micrococcus lysodeikticus, Listeria monocytogenes and
Staphylococcus aureus.
These are of great interest in field application of B. thuringiensis for
improving bacterial or insect biocontrol by coupling with other
antagonistic factors such as bacteriocins or chitinases

                                                            (Bisht, 2010)
Conclusion
Discussion

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BT proteins for non agricultural purposes

  • 1. Useful proteins from Bacillus thuringiensis for non-agricultural applications Subhada Pattanayak
  • 2. OUTLINE Bt for cancer treatment Introduction Bt against protozoan diseases Applications Mosquito control Endoparasite control Conclusion Bacteriocins Discussion Enzyme production
  • 3. Introduction Bacillus thuringiensis- a gram positive, spore forming bacteria produces crystalline parasporal inclusions Toxic to agriculturally and medically important pests Non-insecticidal Bt strains have cytocidal activity
  • 4. A conceptual diagram of the present and previous B. thuringiensis crystal A conceptual diagram of the present and previous B. thuringiensis crystal protein world. protein world. (Kitada et al., 2006)
  • 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. Brief history…. In 1970s, Prasad and Shethna had carried out research on the anti-tumour effects of Bt. Parasporal proteins of Bacillus thuringiensis serovar shandongiensis found cytotoxic to human leukaemic T cells (Lee et al., 2000). A soil isolate belonging to Bacillus thuringiensis serovar dakota (H15) was found to produce non-cyt inclusion proteins that were highly cytotoxic against human leukaemic T cells (MOLT-4) and moderately cytotoxic to human cervical cancer cells (HeLa) (Kim et al., 2000).
  • 8. Parasporins “Parasporin” was first used by Mizuki et al to describe a novel protein with a unique cytotoxicity in 2000. Defined as Bt and related bacterial parasporal proteins that are non-haemolytic but capable of preferentially killing cancer cells
  • 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. 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. List of parasporins ( till March 2012) Name Cry No. Acc No. Authors & Year PS1Aa1 Cry31Aa1 AB031065 Mizuki et al. 2000 A1190 PS1Aa2 Cry31Aa2 AY081052 Jung & Côté 2002 M15 PS1Aa3 Cry31Aa3 AB250922 Uemori et al. 2006 B195 PS1Aa4 Cry31Aa4 AB274826 Yasutake et al. 2006 Bt 79-25 PS1Aa5 Cry31Aa5 AB274827 Yasutake et al.2006 Bt 92-10 PS1Aa6 submitting AB375062 Nagamatsu et al.2010 CP78A, M019 PS1Ab1 Cry31Ab1 AB250923 Uemori et al. 2006 B195 PS1Ab2 Cry31Ab2 AB274825 Yasutake et al.2006 Bt 31-5 PS1Ac1 Cry31Ac1 AB276125 Yasutake et al.2006 Bt 87-29 PS1Ac2 Cry31Ac2 AB731600 Kuroda et al. 2012 B0462 PS1Ad1 submitting AB375062 Nagamatsu et al.2010 CP78B, M019 PS2Aa1 Cry46Aa1 AB099515 Ito & Kitada 2004 A1547 PS2Aa2 Cry46Aa2 AB454419 Ishikawa et al.2008 A1470 PS2Ab1 Cry46Ab1 (http://parasporin.fitc.pref.fukuoka.jp/index.html) AB186914 Yamagiwa et al.2004 TK-E6
  • 12. Characteristics Differ in molecular weight and composition Each PS has different cell specificity due to the presence of different receptor, genome sequence, mode of action and targeting specificities (Wong, 2010)
  • 13. Cytotoxicity spectra of parasporins Cytotoxicity spectra of parasporins The 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. Cytocidal action and cell specificities PS-1 has high toxicity against cancer cell lines like HeLa cells (cell line from cervical cancer cells) PS-2 shows toxicity against cell like MOLT-4 (Leukemic T cells), Jurkat (cell line from T lymphocyte cells) and Hep G2 (Hepato cellular carcinoma-liver tissue) PS-3 has toxicity against Hep G2 and HL-60 (myeloid leukemia cells) PS-4 shows toxicity against CACO-2 (epithelial colorectal adenocarcinoma cells) PS-5 – Unknown PS-6 shows anticancer activity against human hepatocyte cancer cells and cervical cancer cells (Wong et al., 2010)
  • 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. 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 cells 2) 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+ influx 4) PS-1 not a pore forming toxin 5) Mode of cell death most likely apoptotic. (Katayama et al., 2007)
  • 17. (Akiba et al., 2009)
  • 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. Detection of parasporin-2 in hepatocellular carcinoma. • Cancer and non-neoplastic tissues incubated with parasporin-2, and analyzed using an anti-parasporin-2 antibody. • Extensive presence of parasporin-2 in hepatocellular carcinoma cells but not in non-neoplastic liver cells • Parasporin-2 binds specifically to the cancer 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. 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. Parasporin-4 Parasporin-4 (PS4) is a cytotoxic protein produced by Bacillus thuringiensis strain A1470. It exhibits high cytotoxicity against human cancer cell lines, CACO- 2, Sawano, and MOLT-4 cells Exhibits strong cytotoxicity against several human tumor cell lines when activated by protease treatment but does not exhibit insecticidal or hemolytic activities PS4 binds non-specifically to the plasma membrane and oligomerizes to form pores only in target cells, inducing cell death. (Okumura et al., 2011)
  • 22. Cytopathic effect of parasporin-4 (PS4) against CACO-2 , HeLa , MOLT-4 Cells were observed by phase contrast microscopy before and 4 h after treatment with 4 μg/ml PS4. (Okumura et al., 2011)
  • 23. Cytopathic effects in MOLT-4 cells observed with a differential interference microscope. PS4 was added at a final concentration of 2 μg/ml (Okumura et al., 2011)
  • 25. Erythrocytes of mice infected with Plasmodium berghei B. thuringiensis protein samples of 0.3 ml each were injected into infected mice through the tail vein on the 1st and 2nd day after infection
  • 26. Survival days of mice infected with Plasmodium berghei after treatment with Bacillus thuringiensis crystal proteins 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. Leishmaniasis are caused by haemoflagellate protozoan which belongs to genus Leishmania that infect vertebrate hosts through the bites of sand fly females Phlebotomus spp.
  • 28. Active non truncated core toxin of B.t. serovar thuringiensis (H14) 43 kDa for their activity against Leishmania major promastigotes used. The active protein fraction was bioassayed against L. major promastigotes suspension (7×107 promastigotes /ml) in final concentrations ranging from 100 - 0.78 μg /ml aseptically in replicates. The bioassay of protein fraction showed its LC50 is 4.95 μg/ml
  • 29. Cytopathological changes in Leishmania major promastigotes started with swelling (A) followed by changing from spindle shape to spheroid (B) berry shaped cells (C) cytoplasmic proteins with a giant increase in size (D). While the untreated promastigotes keep motile and alive in pairs (E&F)
  • 30. Bt for mosquito control
  • 31. Mosquitoes act worldwide as vectors transmitting disease causing viruses and parasites such as malaria, yellow fever, dengue fever, filariasis, St. Louis encephalitis and the West Nile virus between humans and animals (Tolle, 2009). B. thuringiensis subsp. israelensis (Bti) has been extensively studied for its specific and high toxicity to mosquito and black fly larvae since its discovery in 1976. The parasporal inclusion body of Bti consists of four major insecticidal crystal proteins (Cry4Aa,Cry4Ba, Cry11Aa and Cyt1Aa) (Stein et al., 2006).
  • 32. Although Bti and its toxins have been successfully commercialized for mosquito control, screening programs have continued worldwide to identify and characterize new mosquitocidal Bt isolates and toxin genes The Bt S2160-1 strain was isolated from soil samples collected from Southern China and found to have a comparable mosquitocidal activity to Bti. PCR-restriction fragment length polymorphism identification system was developed and used in order to identify novel cry-type genes cry30Ea, cry30Ga, cry50Ba and cry54Ba (Zhang et al., 2012)
  • 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. 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. Typical morphology (at 360 magnification) seen in the L4 Anterior intestine of nematodes fed with four toxic plate assay after feeding nematode crystal proteins in E. coli. showing reduction of species E. coli transformed with empty vector (Left), width of intestine at one position near the vector plus nontoxic Cry protein insert (Center), or vector anterior. plus toxic Cry protein insert (Wei et al., 2003)
  • 37. Efficacy of Cry proteins against roundworms Efficacy of Cry proteins against roundworms Parasitic roundworms. a A. ceylanicum b. Heligmosomoides polygyrus bakeri (Hu & Aroian, 2012)
  • 38. Glycolipids that bind Cry5B known as arthroseries glycolipids are specific to roundworms (nematodes) but lacking in mammals and vertebrates (Griffitts and Aroian , 2005). Thus, Cry proteins like Cry5B are non-toxic to vertebrates due to lack of the Cry5B arthroseries glycolipid receptors Given their non-toxicity to humans and their broad spectrum of nematicidal action, Cry proteins show great potential as next-generation anthelmintics.
  • 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. Exposure to Cry5B impairs motility of adult hookworms in culture. Cry5B toxin reduces A. ceylanicum egg excretion.
  • 41. Cry5B treatment reduces hookworm infection as measured by weight gain and blood haemoglobin
  • 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. Bacteriocins produced by Bacillus thuringiensis Source Bacteriocin Mol wt(kDa) Activity (Abriouel et al., 2011)
  • 45. Bt for enzyme production Bt for enzyme production
  • 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. 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. Autolysins Endogenous peptidoglycan hydrolases that digest cell wall peptidoglycans of the producer bacterium and of other bacteria The characterisation of the autolytic phenotype of 112 B. thuringiensis strains showed seven major proteins of molecular weights ranging between 25 and 90 kDa which exhibited peptidoglycan hydrolase activity, particularly at alkaline pH. Several of these proteins retained lytic activity against other bacterial species such as Micrococcus lysodeikticus, Listeria monocytogenes and Staphylococcus aureus. These are of great interest in field application of B. thuringiensis for improving bacterial or insect biocontrol by coupling with other antagonistic factors such as bacteriocins or chitinases (Bisht, 2010)

Notes de l'éditeur

  1. 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