Team:IISc-Bangalore/PACMAN

PACMAN


Phage-Antibiotic Complementarity Mediated Antimicrobial Network

The Problem

Antibiotic Resistance

The rapid emergence of antibiotic resistance has rendered many previously usable antibiotics obsolete. Coupled with the enormous time and cost of the introduction of newer antibacterial remedies with reducing economic returns, (and the increasingly lesser time it is taking for bacteria to develop resistance to them) we are truly staring at an uncertain future, with fears that we may soon return to pre-antibiotic days if no concrete steps are taken.[1]

Limitations of Current Therapy

Antibiotics

Conventional antibiotic therapy would have us invest in finding newer antibiotics, or use combinations of antibiotics to try and kill bacteria that are resistant to one type of antibiotic. This is exactly what has been happening ever since the introduction of antibiotics - any antibiotic resistance is taken care of by the introduction of newer, more novel antibiotics.[1] But the rate of approval of new antibiotics has plummeted.[2] Antibiotic research is considered uneconomical by pharmaceuticals[3] due to a myriad of reasons, ranging from long research durations to unknown profitability due to rampant misuse coming from overpresciption[4] and incomplete courses.

Phage Therapy

Conventional Phage therapy would have the usage of lytic phages to target the bacteria. However, bacteria can develop resistance against phages as well, and through a vast repertoire of mechanisms[5]. So, the introduction of phage therapy in its classical form risks the same issue that we currently face with antibiotics. Though in this case, it will take a lot of time for the situation to reach the state that antibiotics are in. Nevertheless, if classical phage therapy is used, it will just be a matter of time before a certain phage cocktail becomes very inefficient in dealing with the bacteria it was supposed to act against.

Our Solution

Engineer a phage that can recognize a antibiotic resistance mediating agent (preferably a small molecule) on the cell surface as a specificity marker.

Antibiotic resistance is usually mediated by the introduction of small molecules onto the cell surface - either emerging from genetic modification of the population[6] occurring due to exposure to the antibiotic in question and subsequent natural selection of the resistant bacteria, horizontal gene transfer[7], or due to some intrinsic non-genetic mechanism.[8]

For our project we have used the T4 phage and colistin as a model.

Phage Tail Modification

The T4 phage adsorbs onto the bacterial surface by the following mechanism:- [9][10]

  1. The Long Tail Fibres interact reversibly with the OmpC receptor present on the bacterial surface.
  2. Once three of the long tail fibres have interacted with the OmpC receptors, there occurs a change in the conformation of the baseplate proteins, which makes the phage "squat".
  3. This allows the Short tail Fibres to interact irreversibly with the LPS on the surface, changing the baseplate conformation.
  4. This conformation change drives the tail tube through the outer membrane, through which DNA is then inserted.

The long tail fibre's binding with OmpC thus acts as the identifier for the phage.[11] If we can change it's binding, we are one step closer to our goal. The phage tail protein that binds to the OmpC receptor is gp37.[12] (It is the terminal long tail fibre protein on the tail).

gp37, the terminal long tail fibre protein

Modifying gp37 to increase its Binding to petN

Phosphoethanolamine (pEtN) is a small molecule transferred to the surface of the cell, where it is attached to the Lipid A of Lipopolysaccharide (LPS), by the action of phosphatidylethanolamine transferase, an enzyme encoded for by the gene MCR -1 (Mobilized Colistin Resistance -1), the first identified horizontally transferable Colistin resistance gene.[13][14].

phosphoethanolamine (pEtN)

Colistin, also called polymyxin E, is an antibiotic that works against most Gram-negative bacteria. It fell out of favour due to its extreme nephrotoxicity, and is hence a last resort antibiotic, to be used for Multi-Drug Resistant cases of Pseudomonas aeroginosa, Klebsiella pneumoniae and Acinetobacter.
It is said to work on the principle of electrostatic attraction - it is positively charged - while the surface of the bacteria is negatively charged. pEtN reduces the negative charge on the bacterial surface by virtue of being positively charged itself, thereby reducing the effectiveness of Colistin.
We aim to get the phage to target bacteria with pEtN expressed on their surface by increasing the binding affinity of gp37 with pEtN to pharmaceutically significant levels. This leads to the pathogen being in a situation of being "between a rock and a hard place" - if it has pEtN to be resistant to colistin, then the phage would kill it. If it doesn't, then colistin will. We are saved from the possibility of simultaneous resistance by modifications of the molecule as it is a small molecule with no major possible sites available for genetically encoded changes to occur.

How we plan to achieve this goal can be found on the Design page.

References

1] A Ventola CL. The Antibiotic Resistance Crisis: Part 1: Causes and Threats. Pharmacy and Therapeutics. 2015;40(4):277-283
2] Gould IM, Bal AM. New antibiotic agents in the pipeline and how they can overcome microbial resistance. Virulence. 2013;4(2):185–191.
3] Bartlett JG, Gilbert DN, Spellberg B. Seven ways to preserve the miracle of antibiotics. Clin Infect Dis. 2013;56(10):1445–1450
4] The antibiotic alarm. Nature. 2013;495(7440):141.
5] Bacteriophage resistance mechanisms. Nature Reviews Microbiology volume 8, pages 317–327 (2010)
6] Grayson ML, Heymann D, Pittet D. The evolving threat of antimicrobial resistance. In: Martinez L, editor. The evolving threat of antimicrobial resistance: options for action. World Health Organization; Geneva: 2012. p. 1-10
7] Barlow M. What antimicrobial resistance has taught us about horizontal gene transfer. Methods Mol Biol 2009;532:397-411
8] El-Halfawy, O. M., & Valvano, M. A. (2012). Non-genetic mechanisms communicating antibiotic resistance: rethinking strategies for antimicrobial drug design. Expert Opinion on Drug Discovery, 7(10), 923–933. doi:10.1517/17460441.2012.712512
9] Rossmann, Michael G., et al. "The bacteriophage T4 DNA injection machine." Current opinion in structural biology 14.2 (2004): 171-180.
10]Leiman, Petr G., et al. "Three-dimensional rearrangement of proteins in the tail of bacteriophage T4 on infection of its host." Cell 118.4 (2004): 419-429.
11]Moran G. Goren, Ido Yosef, Udi Qimron, Programming Bacteriophages by Swapping Their Specificity Determinants, Trends in Microbiology, Volume 23, Issue 12, 2015, Pages 744-746.
12]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.
13]Hinchliffe, Philip; Yang, Qiu E.; Portal, Edward; Young, Tom; Li, Hui; Tooke, Catherine L.; et al. (2017). "Insights into the Mechanistic Basis of Plasmid-Mediated Colistin Resistance from Crystal Structures of the Catalytic Domain of MCR-1". Scientific Reports. 7: 39392.
14]Yi-Yun Liu, Yang Wang, Timothy R Walsh et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study, The Lancet Infectious Diseases, Volume 16, Issue 2, 2016, Pages 161-168