Subhada Pattanayak

Useful proteins from Bacillus thuringiensis for

non-agricultural applications

OUTLINE

Bt for cancer treatment

Bt against protozoan diseases

Mosquito control

Endoparasite control

Bacteriocins

Enzyme production

Introduction

Applications

Conclusion

Discussion

IntroductionIntroduction

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 protein world.A conceptual diagram of the present and previous B. thuringiensis crystal protein world.

(Kitada et al., 2006)

Applications of Bt

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.

Bt proteins for cancer treatment

Applications…

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.

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 AB186914 Yamagiwa et al.2004 TK-E6

PS3Aa1

Cry41Aa1 AB116649 Yamashita et

al.2005

A1462

PS3Ab1

Cry41Ab1 AB116651 Yamashita et

al.2005

A1462

PS4Aa1 Cry45Aa1 AB180980 Okumura & Saitoh

2004

A1470

PS5Aa1 submitting AB555650 Ekino & Shin 2009 A1100

PS6Aa1 submitting AB375063 Nagamatsu et al.

2010

CP84, M019

List of parasporins ( till March 2012)

(http://parasporin.fitc.pref.fukuoka.jp/index.html)

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)

CharacteristicsCharacteristics

Cytotoxicity spectra of parasporinsCytotoxicity 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)

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

Cytocidal action and cell specificities

Cytocidal action and cell specificities

(Wong et al., 2010)

Morphological changes caused by PSsMorphological changes caused by PSs

PS-1: Blebbing of the cells

PS-2: Balloon shaped

PS-3: Fragmenting

PS-4: Detachment

HeLa

HepG2

HepG2

MOLT-4

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 coloncancer 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 interferencemicroscope. 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

Crystal proteins could protect erythrocytes from Plasmodium attack.

This study suggested a novel way to control plasmodial infections and even

malaria.

Survival days of mice infected with Plasmodium bergheiafter 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

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 plate assay after feeding nematodespecies E. coli transformed with empty vector (Left), vector plus nontoxic Cry protein insert (Center), or vector plus toxic Cry protein insert

Anterior intestine of nematodes fed with four toxic crystal proteins in E. coli. showing reduction of width of intestine at one position near theanterior.

(Wei et al., 2003)

Parasitic roundworms. a A. ceylanicum b. Heligmosomoides polygyrus bakeri

Efficacy of Cry proteins against roundwormsEfficacy of Cry proteins against roundworms

(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 ?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.

Source Bacteriocin Mol wt(kDa) Activity

(Abriouel et al., 2011)

Bacteriocins produced by Bacillus thuringiensis

Bt for enzyme productionBt for enzyme production

Proteases 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)

CellulasesCellulases

Bacillus thuringiensis strains produced novel cellulases which could

liberate glucose from soluble cellulose, carboxymethyl cellulose

(CMC), and insoluble crystalline cellulose.(Bisht, 2010)

Chitinases: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 as a major or unique

carbon source

(Bisht, 2010).

Autolysins 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)

ConclusionConclusion

DiscussionDiscussion