Team:Calgary/Project/CpxP

From 2010.igem.org

(Difference between revisions)
 
(32 intermediate revisions not shown)
Line 63: Line 63:
<div class="container">
<div class="container">
 +
<div class="sidebar">
<div class="sidebar">
 +
<h1>Project Descriptions</h1>
<h1>Project Descriptions</h1>
 +
<ul>
<ul>
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li>
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li>
-
<li>Protein Misfolding Reporters
+
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a>
<ul>
<ul>
-
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">IbpAB Circuit</a></li>
+
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li>
-
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">CpxP Circuit</a></li>
+
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li>
</ul>
</ul>
</li>
</li>
 +
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing Our System</a></li>
 +
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li>
</ul>
</ul>
Line 82: Line 87:
<div class="mainbody">
<div class="mainbody">
-
<span id="bodytitle"><h1>Cpx Circuits</h1></span>
+
<span id="bodytitle"><h1>Periplasmic Stress Detectors</h1></span>
<br/>
<br/>
 +
<h2 style="color:#0066CC">What Causes Periplasmic Stress?</h2>
 +
<p> Periplasmic stress, also known as envelope stress, is triggered by several factors that influence the ability of the bacteria to communicate with other cells through quorum sensing, pathological pathway, pili formation, outer membrane protein formation which plays a role in adhesion of the cells allowing them to form proper colonies and survive. Many stresses in the periplasmic region are triggered by improper formation of outer protein structures such as pili and lipoprotein disruption that reduces pathogenicity of the bacteria.</p>
 +
 +
<h2 style="color:#0066CC">Team Calgary circuits for periplasmic stress detector</h2>
 +
 +
 +
<table>
 +
<tr><td>
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/cpxp-1.png"></img>
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/cpxp-1.png"></img>
<br/>
<br/>
Line 90: Line 103:
<br/>
<br/>
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/degp-1.png"></img>
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/degp-1.png"></img>
-
<p>
+
</td>
-
The Cpx pathway is an outer membrane stress response system in <i>E. coli</i> that consists of two main components: CpxA, a sensor histidine kinase, and CpxR, a cytoplasmic response regulator (Raivio, Laird, Joly, & Silhavy, 2000). CpxA is a transmembrane protein that when induced, will phosphorylate CpxR, which is a transcriptional regulator of a large number of Cpx-induced proteins including chaperones and proteases associated with relieving the toxicity of misfolded protein (Keller & Hunke, 2009). This is achieved through the presence of a CpxR binding site (5’-GTAAAN<sub>5</sub>GTAAA-3’) present within 100 bp of the transcriptional start site of these genes (Price & Raivio, 2009). In the absence of induction signals, CpxP, a periplasmic protein, inhibits the autophosphorylating activity of CpxA, stopping the capability of CpxR to bind to these sites (Keller & Hunke, 2009).
+
 +
<td>
 +
These images illustrate the promoters of choice by team Calgary for detection of periplasmic stress. The promoters of choice include CpxP, CpxR and DegP promoters. </td></tr></table>
-
There are many stresses that are known to activate the Cpx pathway including elevated pH, overproduction of outer membrane lipoproteins such as NlpE, accumulation of pilus subunits, and the presence of misfolded proteins such as variants of maltose binding protein (Keller & Hunke, 2009). Though the exact response mechanism to each of the signals of the Cpx system is unknown, it has been proposed that each of these inducing signals act by causing the accumulation of misfolded proteins (Ruiz & Silhavy, 2005). Misfolded protein can titrate CpxP away from the periplasmic domain of CpxA, resulting in Cpx pathway activation (Raivio et al., 2000). Among the main proteins that are associated with outer membrane protein folding, CpxP shows the most sensitivity to Cpx activating conditions (Price & Raivio, 2009).  It has also been shown that overexpression of CpxP will inhibit the activation of the Cpx pathway (MacRitchie et al., 2008). There are currently 50 genes that have shown experimental evidence for Cpx regulation (Price & Raivio, 2009). This year, we have constructed 3 Cpx inducible promoter regions to RFP reporter devices: CpxR, DegP, and CpxP.
+
<h2 style="color:#0066CC">How does a native <i>E. coli</i> cell combat periplasmic stress?</h2>
 +
<p>
 +
<i>E coli </i> have several native sigma factors in the genome that are upregulated during envelope stress. Some of the sigma factors include sigma E and sigma 70. These two factors act upon and induce different proteases and chaperones which act to correct the misfolding agent. There is also a new regulon that is currently studied widely, called the Cpx regulon. A well characterized periplasmic regulon found in E. coli is the Cpx regulon. Cpx pathway is involved in maintaining cellular adhesion and keeping periplasmic environment in check. It regulates proteins which are responsible for cellular adhesion and pili formation (Diguiseppe and Silhavy, 2003). Cpx also acts as a periplasmic heat shock pathway. The Cpx pathway is activated by factors which cause protein aggregation in the periplasmic space of gram negative bacteria. Extracellular stresses such as exposure to ethanol, change in pH and temperature change induce the activation of the Cpx heat shock regulon. This increases the transcription of proteins such as CpxP, DegP and CpxR.  The Cpx pathway is activated by misfolded proteins or excessive protein concentration in the periplasmic space as well.  </p>
 +
 +
<table> <tr><td><a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Cpxmechanism.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Cpxmechanism.png" border="0" alt="Cpx PATHWAY"></a></td>
 +
 +
<td>Figure illustrates Cpx regulon activation pathway. In the lack of stress, the CpxP protein is docked on CpxA transmembrane protein. In the presence of misfolded protein in the envelope, CpxP binds to the misfolded protein which allows CpxA to phosphorylate CpxR which acts as a TF for downstream proteases such as DegP.(Silhavy and Raivio,2001)</td>
 +
</tr>
 +
</table>
 +
 +
<h3>CpxP</h3>
 +
 +
<p> CpxP is a member of the Cpx regulon which docks on to CpxA, a transmembrane protein normally. But in the case of envelope stress causing protein misfolding in the periplasmic space, CpxP detaches from CpxA and attaches to the misfolded protein in the periplasmic space. </p>
 +
 +
<h3>CpxR</h3>
 +
 +
<p>
 +
CpxR is a transcription factor that is generally dephoshorylated (inactive form). However, in occasion of misfolded protein in the periplasmic space, CpxR is phosphorylated by CpxA which autophosphorylates itself and also has kinase activity towards CpxR. The phosphorylated CpxR then binds to promoter regions that code for proteases such as DegP, ppiD etc.
-
These circuits are designed with the purpose of detecting misfolding protein within the periplasm. If a gene of interest coding for a periplasmic protein is misfolded within the periplasm, it will activate the Cpx stress response pathway, and phosphorylated CpxR will be able to bind to our promoter regions, giving an RFP output.
 
-
Figure 2 shows characterization of a CpxR promoter region constructed upstream of an RFP reporter device put under stress from temperature. The results at 47&deg;C show the best example of an induction of the Cpx pathway. An increase in temperature is a known Cpx induction signal, possibly resulting from the aggregation of misfolded protein in the periplasm (Ruiz & Silhavy, 2005). Therefore, CpxP leaving the periplasmic domain of CpxA, and phosphorylated CpxR acting on the CpxR promoter, which increases transcription of RFP, can explain the spike in RFP output within the first hour of stress. It is possible that the slower rate of increase of RFP between the first and third hours is a result of lowered phosphorylated CpxR in the periplasm, resulting from the accumulation of proteases, which are being overexpressed, in response to the toxicity of aggregation of misfolded protein. Because CpxP overproduction is known to inhibit the Cpx pathway activation, a large amount of it present within the periplasm could then show the decrease in RFP levels. The accumulation of CpxP, as well as other periplasmic proteases such as DegP could be alleviating stress caused by misfolded protein in the periplasm, allowing CpxP to remain bound to the transmembrane CpxA.
 
</p>
</p>
 +
<h3>DegP</h3>
 +
 +
<p> DegP is a protease that is activated by CpxR or sigma70 in the event of envelope stress. DegP is transported into the periplasm where it binds to and degrades the misfolded protein. This allows the CpxP protein to be free resulting in binding to CpxA and shutting the Cpx regulon off.
 +
 +
The activation of the Cpx regulon is cyclic in nature and can be reversed when there is a downregulation of activating agents such as misfolded proteins in the cytoplasm.
 +
 +
 +
</p>
 +
<h2 style="color:#0066CC">Circuit usage and sensitivity</h2>
 +
 +
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/CO_cK2D4l1s?hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/CO_cK2D4l1s?hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object>
 +
 +
<p>The circuit that will be constructed to detect protein misfolding will have the CpxP promoter. This has been shown to be the most inducible member of the Cpx regulon. Two other similar circuits will be constructed containing two other Cpx inducible promoter, DegP and CpxR. All three promoters contain binding sites for phosphorylated CpxR, which will induce transcription of our circuit. This allows our promoters to be activated in presence of Cpx inducing signals. Although there is a variety of signals which will activate the cpx cascade, it has been proposed that each of these inducing signals act by creating the accumulation of misfolded proteins. Hence, if periplasmic misfolding occurs in the cell of an E.coli bacteria, the reporter gene, in this case the Red Fluroscent Protein (RFP), will be activated. This provides a simple visual output for protein misfolding within the periplasm of E.coli.
 +
</p>
 +
 +
<br/>
<br/>
 +
<table>
 +
<tr>
 +
<td>
 +
<a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Cpxresponse.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Cpxresponse.png" border="0" alt="Cpx response graph"></a></td>
 +
 +
<td> This diagram illustrated that the CpxP promoter is the most sensitive in the stress regulon. CpxP is followed by CpxR and DegP respectively. These were selected because they are some of the most sensitive in the periplasmic stress detection pathways</td>
 +
 +
</tr>
 +
</table>
 +
 +
<h2 style="color:#0066CC">References</h2>
 +
<p>
 +
DiGiuseppe, P. A., & Silhavy, T. J. (2003). Signal detection and target gene induction by the CpxRA two-component system. Journal of Bacteriology, 185(8), 2432-2440. </p>
 +
<p>
 +
 +
Otto, K., & Silhavy, T. J. (2002). Surface sensing and adhesion of escherichia coli controlled by the cpx-signaling pathway. Proceedings of the National Academy of Sciences of the United States of America, 99(4), 2287-2292. </p>
 +
 +
<p>Raivio T., and Silhavy, T. (2001). Periplasmic stress and ECF Sigma factors. Annual Review of Microbiology, 55, 591–624.
 +
 +
</p>
-
</html>[[Image:DegP Cpx Project Figure.png|thumb|400px|left|DiGuiseppe, P.A., & Silhavy, T.J. (2003). Signal detection and target gene induction by the cpxra two-component system. Journal of Bacteriology, 185(8), 2436-2436.]]<html>
 
-
</div>
 
</div>
</div>

Latest revision as of 03:53, 28 October 2010

Periplasmic Stress Detectors


What Causes Periplasmic Stress?

Periplasmic stress, also known as envelope stress, is triggered by several factors that influence the ability of the bacteria to communicate with other cells through quorum sensing, pathological pathway, pili formation, outer membrane protein formation which plays a role in adhesion of the cells allowing them to form proper colonies and survive. Many stresses in the periplasmic region are triggered by improper formation of outer protein structures such as pili and lipoprotein disruption that reduces pathogenicity of the bacteria.

Team Calgary circuits for periplasmic stress detector



These images illustrate the promoters of choice by team Calgary for detection of periplasmic stress. The promoters of choice include CpxP, CpxR and DegP promoters.

How does a native E. coli cell combat periplasmic stress?

E coli have several native sigma factors in the genome that are upregulated during envelope stress. Some of the sigma factors include sigma E and sigma 70. These two factors act upon and induce different proteases and chaperones which act to correct the misfolding agent. There is also a new regulon that is currently studied widely, called the Cpx regulon. A well characterized periplasmic regulon found in E. coli is the Cpx regulon. Cpx pathway is involved in maintaining cellular adhesion and keeping periplasmic environment in check. It regulates proteins which are responsible for cellular adhesion and pili formation (Diguiseppe and Silhavy, 2003). Cpx also acts as a periplasmic heat shock pathway. The Cpx pathway is activated by factors which cause protein aggregation in the periplasmic space of gram negative bacteria. Extracellular stresses such as exposure to ethanol, change in pH and temperature change induce the activation of the Cpx heat shock regulon. This increases the transcription of proteins such as CpxP, DegP and CpxR. The Cpx pathway is activated by misfolded proteins or excessive protein concentration in the periplasmic space as well.

Cpx PATHWAY Figure illustrates Cpx regulon activation pathway. In the lack of stress, the CpxP protein is docked on CpxA transmembrane protein. In the presence of misfolded protein in the envelope, CpxP binds to the misfolded protein which allows CpxA to phosphorylate CpxR which acts as a TF for downstream proteases such as DegP.(Silhavy and Raivio,2001)

CpxP

CpxP is a member of the Cpx regulon which docks on to CpxA, a transmembrane protein normally. But in the case of envelope stress causing protein misfolding in the periplasmic space, CpxP detaches from CpxA and attaches to the misfolded protein in the periplasmic space.

CpxR

CpxR is a transcription factor that is generally dephoshorylated (inactive form). However, in occasion of misfolded protein in the periplasmic space, CpxR is phosphorylated by CpxA which autophosphorylates itself and also has kinase activity towards CpxR. The phosphorylated CpxR then binds to promoter regions that code for proteases such as DegP, ppiD etc.

DegP

DegP is a protease that is activated by CpxR or sigma70 in the event of envelope stress. DegP is transported into the periplasm where it binds to and degrades the misfolded protein. This allows the CpxP protein to be free resulting in binding to CpxA and shutting the Cpx regulon off. The activation of the Cpx regulon is cyclic in nature and can be reversed when there is a downregulation of activating agents such as misfolded proteins in the cytoplasm.

Circuit usage and sensitivity

The circuit that will be constructed to detect protein misfolding will have the CpxP promoter. This has been shown to be the most inducible member of the Cpx regulon. Two other similar circuits will be constructed containing two other Cpx inducible promoter, DegP and CpxR. All three promoters contain binding sites for phosphorylated CpxR, which will induce transcription of our circuit. This allows our promoters to be activated in presence of Cpx inducing signals. Although there is a variety of signals which will activate the cpx cascade, it has been proposed that each of these inducing signals act by creating the accumulation of misfolded proteins. Hence, if periplasmic misfolding occurs in the cell of an E.coli bacteria, the reporter gene, in this case the Red Fluroscent Protein (RFP), will be activated. This provides a simple visual output for protein misfolding within the periplasm of E.coli.


Cpx response graph This diagram illustrated that the CpxP promoter is the most sensitive in the stress regulon. CpxP is followed by CpxR and DegP respectively. These were selected because they are some of the most sensitive in the periplasmic stress detection pathways

References

DiGiuseppe, P. A., & Silhavy, T. J. (2003). Signal detection and target gene induction by the CpxRA two-component system. Journal of Bacteriology, 185(8), 2432-2440.

Otto, K., & Silhavy, T. J. (2002). Surface sensing and adhesion of escherichia coli controlled by the cpx-signaling pathway. Proceedings of the National Academy of Sciences of the United States of America, 99(4), 2287-2292.

Raivio T., and Silhavy, T. (2001). Periplasmic stress and ECF Sigma factors. Annual Review of Microbiology, 55, 591–624.