Team:IISc-Bangalore/PAIR

PAIR


Phage Assisted Immune Recruitment

The Problem

Endotoxins

Lipopolysaccharide (LPS), commonly known as bacterial endotoxin, is a regular component of the outer membrane in all Gram-negative bacterial species, known to elicit specific immune responses by activation of macrophages. While LPS, in small amounts, is potent in signaling the body's innate immune system, its sudden release in large quantities initiates the release of dysregulated and potentially lethal set of inflammatory mediators, proving deleterious to the host. In extreme cases, endotoxin has been recognized as a major component in the pathogenesis of sepsis and septic shocks. The mortality rate of septic shocks is around 30-45%[1] - Endotoxin was detected in 51.2% of patients with severe sepsis or severe shock.[2]

Limitations of Current Therapy

Antibiotics

Conventional antibacterial therapy, by means of antibiotics, effect bactericidal activity by creating protrusions on the bacterial surface, and subsequent release of LPS through these. This results in a 5-70 fold increase of endotoxin levels in the bloodstream (after a 2 hour period), depending on the specific antibiotic used, in spite of reduced bacteremia.[2]

Phage Therapy

Conventional Phage therapy, too, meets with the same issue - bacterial lysis as a result of phage infection leads to a sudden release of physiologically high amounts of endotoxin[3], posing threats to individuals with a hyper responsive immune system. Further, lysis can also result in a spike in the amount of toxins like the ‘Shiga-like toxin’ from E. coli O157 into the bloodstream in case of an infection, proving harmful for the host.

Our Solution

Engineer a lysis deficient phage with the additional capability of immune cell recruitment.

Removal of e gene

The T4 lysis mechanism is known to mainly involve the sequential action of two genes:

  • t gene: Encodes holin, directing the degradation of the host cell membrane
  • e gene: Encodes the phage lysozyme, which degrades the peptidoglycan layer, hence mediating cell wall rupture

In an e-/t+ system, the cell membrane is ruptured, while the cell wall remains intact. Thus, by removal of the e-gene, we can engineer our phage such that bacterial metabolism is impeded due to other viral factors without effecting host cell lysis.

Replacement of e gene by mcp-1

Monocyte Chemoattractant Protein (MCP-1) is a key chemokine responsible for the migration and recruitment of macrophages, helper T cells, and NK cells to the site of infection. The wild-type protein is glycosylated, but it has been shown that the bacterial produced, non-glycosylated version has a higher chemotactic potency[13].
MCP-1, being a C-C chemokine, was chosen opposed to a C-X-C chemokine, as it induces migration of macrophages whereas overproduction of the latter was directly associated with the growth and development of certain tumors.[8]
By replacing the e-gene within the T4 genome with the gene encoding MCP-1, we can address concerns regarding the uncertainty of infection of each and every bacterial invader by our phages- the infected bacterial invaders now recruit macrophages for the clearance of both the infected and active bacteria. We thus create a bacteriostatic phage, which further enhances the rate of recruitment of macrophages to the site of infection, for timely and efficient clearance of infection.

Inclusion of Signal Peptide

In order for MCP-1 to display its chemoattractant properties, it would first need to diffuse out of the bacterial membranes. In order to facilitate the movement of the protein across the bacterial membranes, this was fused with two signal sequences:

  • HlyA signal sequence : A C-terminus signalling sequence, used for the transmembrane translocation of the E.coli haemolysin from the cytoplasm, to the extracellular medium, through Type I secretion system. It is cleaved off by the OmpT protease in the periplasmic space. Any protein with the attached signaling sequence is transported across the membranes, through membrane channels composed of the HlyB and HlyD proteins.
  • PelB signal sequence: An N terminus signalling sequence, which is responsible for transport of the attached protein from the cytoplasm to the periplasmic space, whence it is shuttled across the outer membrane due to an increased permeability of the cell membrane.

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

References

[1] Opal, S. M. (2010). Endotoxins and Other Sepsis Triggers. Endotoxemia and Endotoxin Shock, 14–24. doi:10.1159/000315915
[2] Antibiotic-induced endotoxin release and clinical sepsis: a review
[3] Dwayne R. Roach, Laurent Debarbieux. Phage therapy: awakening a sleeping giant. doi: 10.1042/ETLS20170002
[4] Samir H Moussa, Vladimir Kuznetsov, Tram Anh T Tran, James C Sacchettini, Ry Young.Protein determinants of phage T4 lysis inhibition. doi: 10.1002/pro.2042
[5] Christine Rouault Vanessa Pellegrinelli Raphaela Schilch Aurélie Cotillard Christine Poitou Joan Tordjman Henrike Sell Karine Clément Danièle Lacasa. Roles of Chemokine Ligand-2 (CXCL2) and Neutrophils in Influencing Endothelial Cell Function and Inflammation of Human Adipose Tissue. Endocrinology, Volume 154, Issue 3, 1 March 2013, Pages 1069–1079
[6] Lysis-deficient bacteriophage therapy decreases endotoxin and inflammatory mediator release and improves survival in a murine peritonitis model; Matsuda, Takeaki et al.; Surgery , Volume 137 , Issue 6 , 639 - 646
[7] Satish L. Deshmane, Sergey Kremlev, Shohreh Amini, Bassel E. Sawaya. Monocyte Chemoattractant Protein-1 (MCP-1): An Overview. J Interferon Cytokine Res. 2009 Jun; 29(6): 313–326. doi: 10.1089/jir.2008.0027
[8] Whiteside, T. L. (n.d.). Immune Cells in the Tumor Microenvironment. Cancer Immune Therapy, 95–118. doi:10.1002/3527600795.ch6
[9] GRAY, L., BAKER, K., KENNY, B., MACKMAN, N., HAIGH, R., & HOLLAND, I. B. (1989). A novel C-terminal signal sequence targets Escherichia coli haemolysin directly to the medium. Journal of Cell Science, 1989(Supplement 11), 45–57. doi:10.1242/jcs.1989.supplement_11.4
[10] Mergulhão, F. J. M., Summers, D. K., & Monteiro, G. A. (2005). Recombinant protein secretion in Escherichia coli. Biotechnology Advances, 23(3), 177–202. doi:10.1016/j.biotechadv.2004.11.003
[11] Choi, J. H., & Lee, S. Y. (2004). Secretory and extracellular production of recombinant proteins using Escherichia coli. Applied Microbiology and Biotechnology, 64(5), 625–635. doi:10.1007/s00253-004-1559-9
[12] Hanke, C., Hess, J., Schumacher, G., & Goebel, W. (1992). Processing by OmpT of fusion proteins carrying the HlyA transport signal during secretion by the Escherichia coli hemolysin transport system. MGG Molecular & General Genetics, 233(1-2), 42–48. doi:10.1007/bf00587559
[13] Yao, Yao, and Stella E. Tsirka. "Mouse monocyte chemoattractant protein 1 (MCP1) functions as a monomer." The international journal of biochemistry & cell biology 55 (2014): 51-59.