Team:IISc-Bangalore/Design
Design
PACMAN
Phage Antibotic Complementarity Mediated Immune Recruitment
Countering antibiotic resistance with the aid of biologically engineered phages.
PAIR
Phage Assisted Immune Recruitment
Eliminating the problem of toxin release during treatment.
PACMAN
Mutating Gp37
The first step in the execution of PACMAN was designing a sequence with an increased binding affinity for phosphoethanolamine (abbreviated pEtN hereon) - the surface molecule that is responsible for mediating resistance against Colistin[1]. To achieve this, we pulled the native Gp37 structure from RCSB PDB[2] and docked it to phosphoethanolamine (using AutoDock scoring function) to find a basal binding affinity of -3.4kcal/mol. The docking was done near the receptor binding tip to ensure that the attachment to the cell is in the same orientation as the native phage in the presence of pEtN on the cell surface.
Following this, the residues within 4Å of the docked ligand were identified as the binding pocket for pEtN on the gp37 terminal. Using PocketAnneal, a software that uses a simulated annealing algorithm for modifying residues in a specified pocket, we came up with four gp37 sequences that were predicted to have a stronger binding affinity for pEtN. All the four modified sequences were predicted to have affinities close to -6.5 kcal/mol, thus effectively compounding the affinity with which pEtN and gp37 will associate with each other. We also examined other sites on the modified protein to ensure that the highest specificity was at the modified pocket.
Modification I
Predicted binding affinity:
-6.5 kcal/mol
BBa_K2609002 and BBa_K2609012
| Amino acid number | Original residue | Modified residue |
|---|---|---|
| 933 | I | D |
| 935 | A | D |
| 944 | N | Y |
| 945 | K | L |
| 946 | M | S |
| 959 | N | D |
| 960 | T | E |
| 961 | N | E |
Modification II
Predicted binding affinity:
-6.42 kcal/mol
BBa_K2609003 and BBa_K2609013
| Amino acid number | Original residue | Modified residue |
|---|---|---|
| 933 | I | D |
| 934 | E | D |
| 936 | W | Y |
| 944 | N | W |
| 945 | K | N |
| 946 | M | E |
| 959 | N | D |
| 960 | T | E |
| 961 | N | D |
Modification III
Predicted binding affinity:
-6.35 kcal/mol
BBa_K2609004 and BBa_K2609014
| Amino acid number | Original residue | Modified residue |
|---|---|---|
| 933 | I | D |
| 934 | E | D |
| 935 | A | P |
| 944 | N | F |
| 945 | K | N |
| 946 | M | E |
| 959 | N | D |
| 960 | T | E |
| 961 | N | E |
Modification IV
Predicted binding affinity:
-6.24 kcal/mol
BBa_K2609005 and BBa_K2609015
| Amino acid number | Original residue | Modified residue |
|---|---|---|
| 933 | I | D |
| 934 | E | D |
| 935 | A | D |
| 944 | N | T |
| 945 | K | Y |
| 946 | M | E |
| 958 | S | T |
| 959 | N | D |
| 960 | T | E |
| 961 | N | E |
Noting that this whole strategy was very cumbersome and required the use of multiple tools to obtain the final results, we decided to pack the whole algorithm into a pipeline called PhageModifier that will allow fast and easy modification of proteins for increased binding to small molecules.
Optimizing the sequence
After generating the amino acid sequence for these modifications we converted them to DNA sequences and codon optimized them for expression E. coli. Any problematic restriction sites in the coding sequence were also removed to keep the sequences compatible with BioBrick standard RFC10.
Generator
We obtained three plasmids from Dr. Mark Van Raaij (CNB-CSIC) for generating the wild-type T4 phage proteins: Gp37, Gp38 and Gp57. The generators for our modified Gp37 proteins were designed keeping the sequences for these in mind. A 6xHis tag was added to the N-term of the coding sequences for easy purification. An internal Enterokinase site was also added between the 6xHis tag and gp37 to cleave off the 6xHis tag if required.
The complete coding sequence containing 6xHis-EK Site-gp37(mod) was put under a T7 expression system and cloned into the BioBrick standard vector pSB1C3. (See Assembly).
Folding
Our initial plans to express Gp37 in isolation suffered a hit when we realized that the protein might not fold on its own inside the cell[3]. The problem, however, was solved easily when Dr. Raaij agreed to send the plasmids for the Gp37 chaperones - Gp38 and Gp57 - for our use. Hereon, all expression for Gp37 was done from triple transformants containing generator plasmids for all the three proteins.
To make sure that Gp37 did fold properly, we purified it from the triple transformants and used Circular Dichroism measurements to assay the rough secondary structure. Note that Gp37 has a predominantly β-sheet structure with intervening random coils. The only α-helices are at the viral attachment region which was not expressed in our construct. As such, we expected the CD spectrum to have a significant dip at around 215nm (a characteristic of β-sheet secondary structure) and no other major features elsewhere (region of spectrum below 200nm was not analysed because of equipment limitations). We also made sure that our assumption was correct by referring to the appropriate literature where this has been done for gp37.[4]
References
[1] Beceiro, Alejandro, et al. "Phosphoethanolamine modification of lipid A in colistin-resistant variants of Acinetobacter baumannii mediated by the pmrAB two-component regulatory system." Antimicrobial agents and chemotherapy (2011): AAC-00079.
[2] Bartual, Sergio G., et al. "Structure of the bacteriophage T4 long tail fiber receptor-binding tip." Proceedings of the National Academy of Sciences 107.47 (2010): 20287-20292.
[3] Bartual, Sergio Galan, et al. "Two-chaperone assisted soluble expression and purification of the bacteriophage T4 long tail fibre protein gp37." Protein expression and purification 70.1 (2010): 116-121.
[4] Brzozowska, Ewa, et al. "Recognition of bacterial lipopolysaccharide using bacteriophage-adhesin-coated long-period gratings." Biosensors and Bioelectronics 67 (2015): 93-99.
PAIR
Generator
Our first goal with regards to PAIR was to see if Mcp-1 can be recombinantly expressed in bacteria and whether it will retain its chemotactic activity after purification. To achieve this, we created a generator for Mcp-1 with the protein coding sequence fused to a 6xHis tag and a TEV protease site on the N-terminal. The whole sequence was put under a T7 promoter and terminator for expression and was cloned into BioBrick standard vector pSB1C3.
Secretion
The next goal in PAIR was to make sure that mcp-1 will be secreted out of the cell so it could be picked up by immune cells after diffusion leading to their recruitment. To achieve this, we started looking up export signals that were well conserved among bacteria and that could be taken over by Mcp-1 to ensure its secretion.
The two signals we centered upon after a literature review were:
- PelB: A N-terminal signal sequence that directs the fused protein to the periplasmic space.[5]
- HlyA: A C-terminal signal sequence that uses the hemolysin secretion system (T1SS) for directing the fused protein directly to the extracellular space.[6]
Post secretion, PelB is cleaved by the proteases in the periplasmic space giving the mature protein. The HlyA pathway, however, requires the presence of a cell surface protease called OmpT for removal of the tag along with a specific linker sequence.
To test the activity of Mcp-1 after signal peptide-mediated secretion and linker cleavage, we designed parts that contain the chemokine fused to these tags under a T7 expression system.
Recombination
The final step in testing the viability of our project was to engineer a phage that is lysis deficient and contains the mcp-1 sequence under a viral promoter. To achieve this, we designed parts containing Mcp-1 fused to the appropriate signal sequence flanked by homologies from both sides of the T4 e-gene.
The e-gene is responsible for initiating degradation of the cell wall in the latter part of the infection. e-gene mutants have been shown to have a lysis deficient phenotype where the host bacterium ceases functioning without any leakage of the cytoplasm or its components into extracellular space[7]. Adding the e-gene upstream and downstream homology to mcp-1 will allow recombination of the sequence into the phage genome.
To increase the efficiency of recombination, we also made a generator for the Lambda red recombinases Exo, Beta and Gam. These recombinases both stabilize as well as directly participate in the recombination increased the basal frequency in the cell by about four orders of magnitude.[8]
Screening for Recombinants
After recombination, the resulting phages will be obtained as plaques on a lawn of susceptible E. coli. Unless a procedure is developed to narrow down the search, the number of plaques to be screened will be very high. To get around the issue, we developed a part that expresses the e-gene product - T4 endolysin - constitutively.
For screening, the phages will first be plated with endolysin containing bacteria. Following this, the plaques will be restreaked onto a plate of wild-type bacteria containing no plasmid. If the plaque is formed by a recombined phage lacking the e-gene on the endolysin plate, the streaking will give rise to either none or a small plaque on the wild-type bacteria because of a lack of proper lysis in the absence of endolysin.
Note that endolysin is a lysozyme but cannot act on the peptidoglycan layer (i.e the cell wall) directly from inside the cell unless the membrane is first degraded (a fate accomplished by the T4 holin protein). As such, the part by itself is not toxic to bacteria harbouring it.[9]
References
[5] Singh, Pranveer, et al. "Effect of signal peptide on stability and folding of Escherichia coli thioredoxin." PloS one 8.5 (2013): e63442.
[6] GRAY, LINDSAY, et al. "A novel C-terminal signal sequence targets Escherichia coli haemolysin directly to the medium." J Cell Sci 1989.Supplement 11 (1989): 45-57.
[7] Paul, Vivek Daniel, et al. "Lysis-deficient phages as novel therapeutic agents for controlling bacterial infection." BMC microbiology 11.1 (2011): 195.
[8] Yu, Daiguan, et al. "An efficient recombination system for chromosome engineering in Escherichia coli." Proceedings of the National Academy of Sciences 97.11 (2000): 5978-5983.
[9] Lim, Jeong-A., et al. "Exogenous lytic activity of SPN9CC endolysin against gram-negative bacteria." J. Microbiol. Biotechnol 24.6 (2014): 803-811.