Team:Paris Bettencourt/Project/TB-ception
From 2013.igem.org
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<b>Killing assay inside the macrophages: microscopy and video movie</b><br> | <b>Killing assay inside the macrophages: microscopy and video movie</b><br> | ||
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+ | <h3>Discussion</h3> | ||
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+ | Our results show an indeniable role of the protein TDMH in the lysis of M.smegmatis. This target is independant of the Mycobacteria’s growth making it a molecule of choice in the war against tuberculosis.<br> | ||
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+ | Using E.coli as a vector allows us to target the macrophages using a natural process: the phagocytosis. We showed that E.coli can enter a macrophage where there is already M.smegmatis. They didn’t seem to be in the same phagosome but the TDMH produced by E.coli can reach M.smegmatis. This is possible thanks to the lysteriolysin that lysed E.coli’s phagosome. <br> | ||
+ | <br> | ||
+ | Lysteriolysin can’t lyse a phagosome from outside. However, M.smegmatis alters the phagosome membrane and the protein delivery is possible. <br> | ||
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+ | <br> | ||
+ | <h3>Perspectives</h3> | ||
+ | <br> | ||
+ | |||
+ | It will be interesting to study the stability of TDMH at the phagosome’s pH and to imagine a protein delivery system that can target the macrophages without using a living organism (for example like they do with imiglucerase). The problem is our protein is not glycosylated so it will not be that easy. | ||
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Revision as of 15:29, 2 October 2013
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![](https://static.igem.org/mediawiki/2013/6/6c/PB_infiltrate.gif)
Bacterial vectors offer a biological route to gene and protein delivery to cells such as macrophages. We want to investigate the potential use of Escherichia coli as a bacterial vector to kill Mycobacerium tuberculosis. We will introduce an expression system containing a gene for a cutinase-like serine esterase that triggers rapid lysis of the mycobacterial cell wall and a gene encoding a protein which forms large pores in the phagosomal membrane where mycobacteria are located, thus releasing the target protein into the cytosol.
Tuberculosis is a disease on the rise in recent years. Despite numerous technological advances, this disease is difficult to treat and diagnose. We are interested in for this project only to the difficulties inherent to the treatment.
When we think about bacteria and health we always think about problems such as antibiotic resistances. Imagine that this time the bacteria will not be against you but with you.
Problematic
Mycobacterium tuberculosis subverts the immune system. It stays latent in the macrophages and evades destruction by preventing the phagosome maturation. We need to find a new way to reach it.
Aim
Use bactofection to target the immune system (especially the macrophages) and deliver a protein that will kill the Mycobacteria.
Background
Why in the 21st century is it so difficult to treat tuberculosis?
This is due to several parameters linked to the nature of the pathogen, Mycobacterium tuberculosis:
- This bacterium has the power to replicate or remain dormant for years inside the macrophages. It happens by many complex mechanisms including prevention of the phagosomal maturation. This phenomenon involves coronin-1.
- The cell wall of M.tuberculosis is very complex and it's difficult for drugs to get inside.
- The growth of the bacteria is very slow and majority of drugs can reach only bacteria which are dividing.
Approach
We wanted to overcome these issues using 2 approaches:
In the first one we want to introduce an expression system containing a gene for a cutinase-like serine esterase that triggers rapid lysis of the mycobacterial cell wall and a gene encoding a protein which forms large pores in the phagosomal membrane where mycobacteria are located, thus releasing the target protein into the cytosol. In the second one we want to develop a bacteria-based iRNA delivery system for silencing the coronin-1 gene of the macrophages. Coronin-1 is a protein which surrounds the mycobacterial phagosome allowing mycobacteria to survive within them. Silencing of this gene will result in the enhancement of the lysosome/phagosome fusion.
Only the first approach was developped during iGEM.
Model
We used Mycobacterium smegmatis as a model. This model was chosen because it is non-pathogenic and it grows quickly (comparing to Mycobacterium tuberculosis)
Main results
Killing assay outside the macrophages
We performed a killing assay using different ratios of bacteria:
1 E.coli for 1 M.smegmatis
1 E.coli for 10 M.smegmatis
1 E.coli for 100 M.smegmatis
2 ratios were successful regarding the killing of M.smegmatis:
1 to 10 and 1 to 1.
In the 1 to 100 ratio we observed a recovery of M.smegmatis.
We decided that we will use the 1/1 ratio for the rest of our experiments.
Killing assay inside the macrophages: microscopy and video movie
Discussion
Our results show an indeniable role of the protein TDMH in the lysis of M.smegmatis. This target is independant of the Mycobacteria’s growth making it a molecule of choice in the war against tuberculosis.Using E.coli as a vector allows us to target the macrophages using a natural process: the phagocytosis. We showed that E.coli can enter a macrophage where there is already M.smegmatis. They didn’t seem to be in the same phagosome but the TDMH produced by E.coli can reach M.smegmatis. This is possible thanks to the lysteriolysin that lysed E.coli’s phagosome.
Lysteriolysin can’t lyse a phagosome from outside. However, M.smegmatis alters the phagosome membrane and the protein delivery is possible.
Perspectives
It will be interesting to study the stability of TDMH at the phagosome’s pH and to imagine a protein delivery system that can target the macrophages without using a living organism (for example like they do with imiglucerase). The problem is our protein is not glycosylated so it will not be that easy.
Bibliography
1. Yang Y, Bhatti A, Ke D, Gonzalez-Juarrero M, Lenaerts A, Kremer L, Guerardel Y, Zhang P, Ojha AK (2012) : Exposure to a cutinase-like serine esterase triggers rapid lysis of multiple mycobacterial species. J Biol Chem. 2013 Jan 4;288(1):382-92.
2. Rajesh Jayachandran, 4, Varadharajan Sundaramurthy, Benoit Combaluzier , Philipp Mueller, Hannelie Korf, Kris Huygen, Toru Miyazaki, Imke Albrecht, Jan Massner, Jean Pieters (2007) : Survival of Mycobacteria in Macrophages Is Mediated by Coronin 1-Dependent Activation of Calcineurin. Cell, Volume 130, Issue 1, 13 July 2007, Pages 12-14
![](https://static.igem.org/mediawiki/2013/0/0f/TB_Ception_Figure.png)
![](https://static.igem.org/mediawiki/2013/e/e5/Figure_1_M.smeg.png)
Killing assay