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.
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)
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)
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)
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)
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