The Cpx Pathway Of Epec Biology Essay


Along with this, I have also performed some 96-well plate assays to determine the minimum inhibitory concentration of the PA0757/757 mutant thought to be encode for a two-component regulatory system in both planktonic and biofilm cells, using tobramycin as the antibiotic.

Dr. Tracy Raivio is a researcher, associate professor and AHFMR senior scholar at the University of Alberta, under the Department of Biological Sciences.

She completed her BSc at the University of Alberta, her PhD at the University of Calgary, and her post-doc fellowship at Princeton University. She is funded by the Alberta Heritage Foundation for Medical Research, CIHR-IRSC, and NSERC-CRSNG. The current members of her lab are Nicole Acosta, Shannon Leblanc, Stefanie Vogt, and Julia Wong.

Her lab is currently working with the Cpx stress response system in enteropathogenic Escherichia coli (EPEC) as a model system for understanding how bacteria respond to their environment. As observed by the lab, under conditions of stress, the Cpx pathway is able to downregulate the production of unessential proteins in the envelope, including virulence factors. This could potentially lead to the use of the Cpx pathway as a target for future antibiotic therapeutic agents.

Lady using a tablet
Lady using a tablet


Essay Writers

Lady Using Tablet

Get your grade
or your money back

using our Essay Writing Service!

Essay Writing Service

More information can be found on:

The lab of Dr. Tracy Raivio works almost exclusively with enteropathogenic Escherichia coli or more commonly known as EPEC. EPEC is a gram-negative, rod-shaped bacterium. It plays a large role in the field of medicine as it is one of the most common causes of infantile diarrhoea in the world [1]. As with other pathogens that infect cells of the human gastrointestinal tract, EPEC infects the host by attaching to the epithelial cells of the intestines. First, the pathogen adheres to the cell using bundle forming pili (or BFP) which are formed from type IV fimbriae [5]. The second step of EPEC pathogenesis is the secretion of virulent proteins from the bacterial cell into the host epithelial cells. This is done via the type 3 secretion system [2,5], whose proteins, along with other virulent secreted proteins, are encoded on the locus of enterocyte effacement (LEE), a pathogenecity island in the genome of about 35 kB in size [2]. One of the proteins that are secreted is Tir, which binds to the host cell and serves as a ligand for the bacteria's adhesion molecule, intimin [2,5]. This forms a pedestal [5]. It is through this way that the bacteria forms a close attachment to the intestinal epithelial cells, effacing the microvili of the epithelial cells [5] and thus destroying them. The destruction of cells and the consequent electrolyte imbalance is what leads to the characteristic diarrhoea of EPEC infections [1].

When looking at EPEC, one cannot look at the Type 3 secretion system of EPEC as a separate entity to the rest of the cell but rather as a system that works in conjunction with and is influenced by other systems. This fact is part of the basis of Dr. Raivio's research. While her lab focuses primarily on the Cpx pathway of EPEC, as we shall see in some of her research papers, the Cpx pathway has an influence on other pathways and systems and vice verse [2].

The Cpx pathway of EPEC is a two component regulatory system that is able to detect stress to the cell envelope and responds accordingly in order to maintain integrity of the cell envelope. Stress could include an alkali pH, the build-up of misfolded proteins in the envelope [2] or the accumulation of overexpressed envelope proteins (like NlpE or P pilus subunits) [4]. Stress is detected by the inner membrane-bound histidine kinase CpxA and CpxA releases CpxP. CpxP goes on to phosphorylate the cytoplasmic response regulator, CpxR [3]. The CpxR-P complex serves as a transcriptional activator [3], binding to promoters of genes that encode for periplasmic protein folding and degradation factors [2,4]. There are potentially around 100 promoters that are thought to be regulated by the CpxR-P transcriptional activator complex [3]. When there is a lack of stress, CpxA will dephosphorylate the CpxR-P complex and the Cpx pathway is thus downregulated [3].

Along with responding to stress to the cell envelope, the Cpx pathway, as alluded to earlier, is believed to be involved in a number of other pathways and systems. These include the attachment process [2], chemoreception, motility [3], biofilm formation, aromatic amino acid biosynthesis, etc [4]. This has then become the goal of the Raivio lab: to see how Cpx pathway, in response to stress, affects other pathways and systems in the cell and how this knowledge can help understand how the cell responds to its environment as well as working toward developing new therapeutic agents against EPEC and other similar systems.

Lady using a tablet
Lady using a tablet


Writing Services

Lady Using Tablet

Always on Time

Marked to Standard

Order Now

The relationship between the Cpx pathway and the type 3 secretion systems was explored in the Dr. Raivio lab's paper "Activation of the Cpx envelope stress response down-regulates expression of severl locus of enterocytes effacement-encoded genes in enteropathogenic Escherichia coli" [2]. The expression of type 3 secretion system components was observed during activation of the Cpx pathway. The study was done using the EPEC strain E2348/69, a strain that has been used as a prototype to study the biology, virulence and genetics of EPEC [6]. As explained in this paper by Dr. Raivio's lab, activating the Cpx pathway decreased the intracellular levels of substrates in the type 3 secretion system. More specifically, there is a decrease in transcription on the LEE operons LEE4 and LEE5. These operons encodes for various effector and translocator proteins involved in the secretion, including: Tir, the translocated receptor; EspA, monomers that polymerize and are thought to form the needle extension that attaches and then forms into a pore; EspB, forms a pore once inserted into the membrane (alone with EspD). With a diminishing of the transcription of these proteins, the type 3 secretion system would not be able to assemble properly.

These observations are important because they show us that when the envelope of EPEC is under stress and the Cpx pathway is activated to respond to that stress, the Cpx pathway is able to inhibit traffic of proteins in the envelope that are not needed at the critical time of stress response. It would be quite inconvenient for the cell if it began to construct a secretion system and secrete virulence factors while it was under some sort of stress. From this, a future objective was made in the paper: determining if the Cpx pathway does indeed reduce the ability of EPEC to become virulent and attach and efface intestinal epithelial cells and if this applies for other enteropathogenic microbes.

If this indeed is true, then research could be directed to establishing novel therapeutic agents that work in such a way that they activate the Cpx pathway, thus diminishing the ability of EPEC to infect intestinal cells.

In the 2008 paper, "Activation of the Cpx regulon destabilizes the F plasmid transfer activator, TraJ, via the HslVU protease in Escherichia coli", the Raivio lab presents F plasmid conjugation as another bacterial system that is under control of the Cpx pathway [3]. F plasmid conjugation involves the transfer of an F plasmid from a donor bacterial cell to a recipient bacterial cell via the conjugative pilus. Synthesis of the pilus and conjugation are both highly regulated processes. The promoter responsible for F plasmid conjugation is the Pγ promoter, which contains all the tra genes. Transcription of the promoter is under the control of TraJ and ArcA. Without these genes, there is a reduction in mating efficiency. The product of the F transfer operon is a Type 4 secretion system that crosses the bacterial cell's envelope. Since this system crosses the cell envelope and the Cpx system is responsible for stress related to the envelope, it was hypothesized that the Cpx system could have some effect on F plasmid conjugation.

As it was observed in Dr. Raivio's research, TraJ, the activating factor responsible for transcription of the Pγ promoter was degraded in the presence of envelope stress, such as overexpression of NlpE. As explained previously, stress caused by NlpE expression is dealt by the Cpx pathway. Therefore, it was concluded that the Cpx pathway does indeed have an effect on F plasmid conjugation, via degradation of TraJ. More specifically, TraJ is degraded as a substrate of HslVU, a heat shock protease-chaperone protein. However, TraJ degradation under the influence of the Cpx pathway does not always happen under any circumstance. In the absence of stress, the presence of the F plasmid seemed to protect TraJ from being degraded by HslVU. More significantly, TraJ could no longer be degraded on expression and synthesis of the F plasmid apparatus had been initiated. Again, this is significant for the purposes of creating novel therapeutic agents that work through the Cpx pathway.

The research that I found the most interesting by the Raivio Lab is explained in their 2009 paper "The contribution of small cryptic plasmids to the antibiotic resistance of enteropathogenic Escherichia coli E2348/69", which did not relate to the Cpx pathway but rather on another interesting system of EPEC bacteria [1]. The lab researched small cryptic plasmids (SCP), which are found in EPEC as well as other bacterial species such as Pseudonomas and Aeronomas. The exact purpose of these small plasmids is still relatively unknown but they are thought to contribute to antibiotic resistance. The Raivio lab proposed to isolate and characterize two such plasmids taken from EPEC E2348/69 and determine their functions. One SCP was approximately 5.2 kb in size and was named p5217 and the other SCP was approximately 6.2 kb in size and named p6148. P5217 was found to not directly influence bacterial phenotype but it did contain genes that are responsible for mobilizing proteins for transfer between hosts, thus potentially aiding other plasmids in their transfer functions.

Lady using a tablet
Lady using a tablet

This Essay is

a Student's Work

Lady Using Tablet

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

Examples of our work

The p6148 plasmid was found to contain the strA-strB-sul2 gene clusters, which are common in several bacterial species' plasmids (like Salmonella, etc.), encoding resistance to streptomycin (strA and strB) and sulphonamide antibiotics (sul2). The sul2 gene of p6148 was actually 382 bp smaller than sul2 genes found in other bacterial strains and therefore it did not contribute to the level of sulphonamide resistance of E2348/69. The strA and strB genes of the gene cluster found on p6148 did confer streptomycin resistance for E2348/69, encoding for a phosphotransferase that would inactivate streptomycin activity.

Therefore, Raivio's research is significant because it shows that SCP's do play an important role in EPEC, either aiding in mobilization of transfer products or in antibiotic resistance. While more research is needed to fully understand their purpose, it is significant to note that the presence of antibiotic resistance genes on these SCP's are probably one of the reasons why these genes have not diminished among bacteria, even with the decreased use of antibiotics such as sulphonamides.

One of the reasons why I wish to work with the Raivio lab is because I am very interested in the subject matter. Of the various science courses I've taken over the last four years in university, microbiology has consistently interested me the most. Bacteria play a huge role in both the medical field as well as other aspects of everyday human life. Dr. Raivio works with EPEC, a bacteria that is considered one of the leading causes of infantile diarrhoea in the world and is a component of food poisoning.

Another portion of Dr. Raivio's research that interests me is the work related to antibiotic resistance. Again, the issue of antibiotic resistance is very relevant to modern medicine; the number of bacteria resistant to antibiotics and the number of antibiotics that bacteria are resistant to have increased over the years. Personally, I feel that it is very important to understand the various mechanisms that aid bacteria to be resistant to antibiotics as well as find possible ways to overcome these mechanisms. I find it interesting that from her research on Cpx pathway, Dr. Raivio was able to observe that the Cpx pathway had an effect on virulence factors, like the important type 3 secretion system. I would like to further explore this part of her research: to see what other virulence factors of EPEC the Cpx pathway may have an effect on and further on, to see how the Cpx pathway could be manipulated as a way of decreasing the function of virulence factors in EPEC, and by this extension, other bacteria.

I would also like to work in the lab of Dr. Raivio because I wish to work with the methods and materials that she uses for her research. I already have some experience with some of the experimental procedures undertaken in the Raivio lab and I feel that for this reason I could also be an important addition to the lab. For example, the majority of the growth conditions used for EPEC E2348/69 are similar to the ones used for the strains of Pseudonomas aeruginosa that I am currently working with (PA14 and PAO1); both bacterial species are grown in LB (Luria-Bertani) or M63 minimal media. In the paper about small cryptic plasmids [1], the lab used 96-well plates to determine the minimum inhibitory concentration (MIC) for streptomycin and sulphonamides. At the very beginning of my research, under the supervision of Dr. Mah, I also used 96-well plates to determine the MIC of three strains of P. aeruginosa to tobramycin (the wild-type, the negative control ΔndvB, the mutant ΔPA0756/757). Thus, I feel that the fact that I already have some knowledge and practical familiarity with some of the methods used in the Raivio lab and this would help me to work in her lab.

However, the Raivio lab also uses a variety of methods that I have had no exposure to but would like to learn as a way of expanding my practical repertoire to whatever I may pursue after graduate studies. Some of these include Western blots, microarray analysis, immunoblot analysis, β-galactosidase, pH sensitivity assay. Other methods include extensions of the prototypical 96-well plate assay, such as the bioluminescence assay, the copper sensitivity assay and the biofilm assay. Having some experience with the 96-well plate, I am interested to see how these forms of assay function and how they could be used to further research for the Raivio lab.

Along with my experience with relevant materials and methods, I feel that I could be an asset to the Raivio lab and contribute to their research because my own research here at the University of Ottawa has given me general knowledge in antibiotic resistance and various ways to measure antibiotic resistance. I would like to join the Raivio lab by further exploring antibiotic resistance in the context of virulence factors under the influence of the Cpx pathway. For example, if the Cpx pathway was activated in such a way that there was a decrease in the function of the type 3 secretion system, how virulent would the EPEC cell be? Would another virulence determinant arise to replace the type 3 secretion system?

Although it does not deal with the Cpx pathway, which is the main focus of the Raivio lab, I was really fascinated by the research on SCP's and wish to expand on this research. Using the resources available, I would like to see just how much the SCP's have an effect on EPEC function, virulence and antibiotic resistance. For example, if one were to remove the p6148 plasmid from EPEC, could the cell still be resistant to streptomycin? What other mechanisms could arise to compensate for the loss of the strA and strB genes found on the plasmid? Such a question could be answered using a 96-well plate and comparing these hypothetical results with those of the previous paper [1].

Thus, I am very interested in the work of Dr. Tracy Raivio and feel that I could contribute to her lab and the research that has already been done by the lab.