The bacteria used in our construct is Bifidobacterium longum - a Gram-positive, catalase-negative and rod-shaped bacteria. B. longum forms white, glossy colonies in a convex shape. It is non-pathogenic and is often added to food products. Bifidobacterium can be classified into 3 main groups - B.longum, B. infantis, B. suis. It's difficult to distinguish them phenotypically but can be distinguished via carbohydrate fermentation patterns.
B.longum has varied strains depending upon its probiotic activity; these can be distinguished through PCR reaction on subtly different 16s rRNA gene sequences.
WHY B. LONGUM?
B. longum colonizes the human gastrointestinal tract, representing upto 90% of the bacteria of an infant's gastrointestinal tract and goes upto 3% in an adult’s gastrointestinal tract. B. longum has a high tolerance to gastric acid and bile. It also produces lactic acid. This helps in the prevention of the growth of pathogenic organisms and also helps bacteria survive.
This probiotic bacteria plays a significant role in immunomodulation and displays antitumour properties.
These gut-residing lactic acid-producing bacteria have shown regression to carcinogenesis and have the ability to both increase and decrease the production of anti-inflammatory cytokines, which play an important role in preventing carcinogenesis.
They are also capable of activating phagocytes in order to eliminate early-stage cancer cells.
Application of heat-killed probiotic bacteria coupled with radiation had a positive influence on enhancing immunological recognition of cancer cells.
VECTOR FOR DELIVERY
pMB1 is a 3207 base pairs size bacterial expression vector constructed for Bifidobacterium gene delivery. pMB1 has been derived and modified from the pBR322 plasmid.
WHAT ARE BACTERIAL EXPRESSION CLONING VECTORS?
A cloning vector is a small piece of DNA that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes. It can be taken from any bacterium, plasmid, virus or higher organism. It should contain ori, reporter gene, selectable marker gene, and suitable cloning sites (these may include- Multiple Cloning Site (MCS) or unique restriction sites).
A cloning vector need not contain suitable elements for the expression of a cloned target gene, such as a promoter and ribosomal binding site (RBS), many however do, and may then work as an expression vector. Thus a cloning vector can be an expression vector when the inserted gene is under the control of promoter and ribosomal binding site.
pMB1 allows amplification with the antibiotic chloramphenicol, spectinomycin and a relaxed control i.e. it has a copy number of 100-1000 per cell. More copy numbers can be obtained if the plasmid lacks some regulatory genes. It already contains the eGFP reporter gene in its plasmid. Plasmid becomes incompatible when more than one type of plasmid with similar ori are present in the same cell. It has unique restriction sites and can be screened by Blue white screening.
Also according to literature studies, pMB1 plasmid was shown to be a stable vector in B. longum for transporting anti-cancer genes in cancer gene therapy. Researchers could successfully deliver endostatin (can help in suppressing the cell cycle and inducing cellular apoptosis) by expressing it in B. longum and targeting it to the tumour microenvironment. Previous results indicated that the plasmid when electroporated into B. longum, was maintained stably in the absence of selective antibiotics and did not significantly affect the biological characteristics of B. longum. In addition, the plasmid injected in B. longum showed a strong inhibitory effect on the growth of murine liver solid tumor in vivo.
Through this project, we wish to accomplish the induction of apoptosis in solid tumour cells by injecting our recombinant plasmid pMB1 that is expressed in B.longum, thus providing a supplement or an alternative to major therapies like radiotherapy, chemotherapy etc.
The three major components of our genetic circuit include:
- KILLER : TRAIL-SMAC FUSION CONSTRUCT
- SWITCH: KILL SWITCH
- SENSOR: AND GATE
KILLER: TRAIL-SMAC FUSION CONSTRUCT
This is a fusion construct comprising two main peptides fused together, namely, SMAC (Second Mitochondria-derived Activator of Caspase), also called DIABLO and TRAIL (TNF-Related Apoptosis-Inducing Ligand). This fusion construct induces cellular apoptosis by both, intrinsic and extrinsic pathways. In this fusion construct, we have used Smac N7, i.e. only seven residues of the SMAC polypeptide, which can be cleaved and is mainly responsible for intrinsic cellular apoptosis. This fusion construct also has a Cell-Penetrating Peptide (CPP) consisting of 8 Arginine residues for efficient membrane penetration into cells. There is a cleavage site present between the SMAC and TRAIL peptide after the polyarginine peptide. This protease cleavage site is recognised by Matrix Metalloproteinases (MMP-3 and MMP-9) and Urokinase (uPa) that will cleave the smac portion once the fusion construct reaches solid tumour cancer cells.
Moreover, the whole fusion peptide is linked to a HlyA signal peptide (C terminal signal peptide) that allows secretion of the protein from the cytoplasm to the extracellular matrix directly by the bacteria. A 6X-His Tag is incorporated for efficient purification of the protein.
Why is Killer: TRAIL-SMAC Fusion construct required?
The TRAIL-SMAC Fusion construct is the integral component of our project. It induces apoptosis in the solid tumour cancer cells, which is our ultimate goal. TRAIL handles the extrinsic apoptosis pathway, while SMAC/DIABLO monitors the intrinsic apoptosis pathway.
This fusion construct was tested against a panel of cancer cells (including lung, colorectal, pancreatic, liver, kidney and uterine) and showed a potent cytotoxic effect. It was well tolerated by animals and significantly reduced the rate of tumour growth in colon and lung adenocarcinoma animal models
- Tamer E Fandy1, Sharmila Shankar2 ,Rakesh K Srivastava et al;
- “Smac/DIABLO enhances the therapeutic potential of chemotherapeutic drugs and irradiation, and sensitizes TRAIL-resistant breast cancer cells”; Molecular Cancer; 2008 ; doi: 10.1186/1476-4598-7-60
MECHANISM OF APOPTOSIS
EXTRINSICALLY BY TRAIL:
Initially, the TRAIL ligand binds to its two death receptors, DR4 (TRAIL-R1) and DR5 (TRAIL-R2). Death receptor binds to FAS-associating protein with death domain (FADD) and TNF-related associated death domain (TRADD). FADD promotes the formation of a death-inducing signalling complex, DISC, which then binds to cysteine proteases (caspases) that cleave specific proteins.The two caspases predominantly bound to these adaptor molecules are caspase 8 and caspase 10.
Caspase-8 or caspase 10 activation initiates a cascade where other caspases, including caspase-3, are activated, ultimately resulting in an irreversible commitment of cells to undergo apoptosis. In the type I pathway, activated caspase-8 directly cleaves and activates caspase-3.
INTRINSICALLY BY SMAC/DIABLO:
It has been observed that some cancer cells gain resistance against TRAIL by overexpressing Inhibitors of APoptosis (IAP) proteins like XIAP, that block the Caspase pathway of apoptosis. Thus we have made this fusion construct in which we have a potent cytotoxic drug that can efficiently induce apoptosis.
Once the fusion protein reaches cancer cells, certain proteases over expressed in cancer cells like MMP-3 , MMP-9 and uPa will recognize the protease cleavage site and cleave the smac portion along with the CPP. The smac with the help of CPP (8 Arginine residues) internalizes into the cancer cells and starts its function to inhibit XIAP thus activating the Caspase pathway.
- Jerzy S. Pieczykolan , Konrad Kubiński , Maciej Masłyk et.al; “AD-O53.2—a novel recombinant fusion protein combining the activities of TRAIL/Apo2L and Smac/Diablo, overcomes resistance of human cancer cells to TRAIL/Apo2L”; Springer, 04 September 2014; doi :10.1007/s10637-014-0153-y
SWITCH: KILL SWITCH
The Anti Holin Holin Kill switch consists of two main cassettes: antiholin-cI repressor cassette and holin-endolysin cassette.
The antiholin-cI repressor cassette is under the control of a lactate inducible promoter (a constitutive promoter flanked by lacO operator sites for lldR product binding). The other cassette is the holin-endolysin cassette, which is responsible for the production of killer proteins against the bacterium. This cassette is under the control of cI promoter. This promoter is negatively regulated by cI repressor.
Our kill switch consists of a holin-antiholin toxin-antitoxin system. The holin protein creates holes in the bacterial cell membrane, which allows endolysin protein to pass through and degrade the cell wall, killing the cell. The antiholin protein binds to the holin protein, inactivating it. When both proteins are expressed, whether the cell dies depends on the relative concentrations of both the holin and antiholin components.
WHY ANTIHOLIN/ HOLIN KILL SWITCH?
This Antiholin/Holin kill switch ensures colonization exclusively in the tumour regions and not elsewhere. From previous literature, we know that B.longum can colonize in the human gut. Thus we have added some resources to increase the stringency of our kill switch. We have expressed antiholin and cI repressor in a single operon under a lactate inducible promoter that allows expression in the environment found in tumours (lac operon), which ensures that during culturing there are enough kill switch delaying proteins so as to allow the bacteria enough time to colonize the tumour microenvironment.
The cI repressor is degradation tagged, so its level will only be sustainable in the tumor microenvironmental conditions. If these conditions are not provided, the repressor is degraded, and holin and endolysin expression kickstarts. The holin that is initially made is neutralized by the already present antiholin, and meanwhile endolysin keeps building up in the cytosol. Once holin overwhelms antiholin, holin starts to form holes in the membrane and the endolysin now present in large concentration in the cytosol lyses the cell wall, thus effectively killing the cell.
This model ensures that our kill switch actually works in a switch-like manner with greater stringency.
SENSOR: AND GATE
The AND Gate is designed in such a way that it ensures secretion only when both conditions, i.e. hypoxia and lactate presence, are satisfied. A hypoxia inducible promoter is intercalated between two lactate operators. This promoter regulates the transcription of the TRAIL-smac fusion construct. Lactate permease and lactate repressor are constitutively synthesized. Lactate permease allows lactate to enter the cell while lactate repressor binds to the lactate operators in the absence of lactate. This repressor-operator complex interacts with the adjoining repressor-operator complex, conforming the gene circuit in such a way that the promoter is no longer accessible to the RNA polymerase 2 for transcription. Thus the TRAIL-smac fusion construct isn’t transcribed because the hypoxia inducible promoter was nestled in an inaccessible conformation. In the presence of lactate however, lactate binding supersedes repressor binding to the operator and thus the genetic circuit reverts back to an accessible conformation, thus leading to the transcription of the TRAIL-smac fusion construct exclusively in the presence of lactate and hypoxia, both.