Brittany Kartchner and Dr. Eric Wilson, Microbiology and Molecular Biology
Introduction
For this project, I hypothesized that the amino acid composition of the C terminus of chemokine proteins allows some chemokines to bind to specific proteins on the cell membrane of bacteria. Previous research has shown that the C terminal region of the chemokine CCL28 is largely responsible for the antimicrobial activity (Liu, 2010). However, it has not been shown what the C terminus region of the chemokine binds to. The C terminus region could bind to the negatively charged LPS on the bacterial membrane, or have a specific protein target on the cell membrane. Polymyxin B, another antimicrobial protein that has a similar structure to CCL28, has been shown to bind directly to the LPS of bacteria, while CXCL10, another antimicrobial chemokine, has been shown to directly bind to the membrane protein FtsX in Bacillus anthracis (Applemelk, 1992; Crawford, 2011). During the course of this experiment, I used a variety of methods to try and determine if a protein was the target of chemokine binding, and if so, which protein was the specific protein target for CCL28.
Methods
To determine if a protein was the chemokine target, about 105 colony-forming units of exponentially growing bacteria are incubated at 37 degress C with sodium periodate or with proteinase K. Sodium periodate works by opening saccharide rings between vicinal diols leaving two aldehyde groups, effectively destroying the O-antigen and core structures of the LPS. Proteinase K works by digesting any protein that has hydrophobic amino acids, which is includes membrane bound proteins. If a protein is the molecular target of CCL28, then when proteinase K is added to cells, the amount of chemokine that can bind to the bacteria will be significantly reduced. If the cell’s LPS is the molecular target for CCL28, then after sodium periodate is added, the chemokine will not be able to bind to the bacteria as well as a bacteria that has not been exposed to sodium periodate. The amount of chemokine binding to Yersinia pseudotuberculosis was analyzed through flow cytometry.
The specific protein target of CCL28 was initially attempted through a pull-down assay in which the antibody to the chemokine was stuck to a bead. This would allow anything that stuck to the bead to be pulled out of solution. The chemokine and antibody bound to the bead was added to bacterial lysate, which was created through sonication, and allowed to incubate and bind for about two hours. The proteins bound to the bead were then eluted from the beads by incubating the solution in a 90˚ C water bath for 10 minutes. The resulting proteins were then run on an SDS-PAGE gel and analyzed through a blue dye to expose where the protein bands were.
The next method used lysate generated through a pressure homogenizer. This allowed for the cell to be lysed more completely and generated a higher number of usable proteins. A pull-down assay and colorimetric analysis was used as before.
The assay used now involves biotinylating the outer membrane proteins prior to lysis by the pressure homogenizer. This allows only the outer membrane proteins to be detected when the SDS-PAGE gel is analyzed through western blot techniques. This method allows for high sensitivity and quick results.
Results
My initial results were confirmed when it shown that proteinase K resulted in significantly decreased chemokine binding. Sodium periodate also slightly increased chemokine binding, but not to the same degree that proteinase K did. This indicates that the LPS hinders the CCL28 from reaching its protein target.
The first attempt to identify the specific protein target resulted in only a few bands showing up where the lysate was run on the gel. This indicated that our lysis methods were not efficient as hardly any proteins were able to be detected.
The pressure homogenizer generated more usable proteins for our analysis, but I was still not able to detect the proteins in the right concentrations. My chemokine band did not show the amount of chemokine added to the initial assay. This led me to believe that the colorimetric assay was not sensitive enough.
The assay that I am currently using uses biotinylation to tag the outer membrane proteins. Since I am fairly certain that the protein that CCL28 binds to is an outer membrane protein, this makes sense. The pull down assay is run as before, but instead of using a colorimetric analysis, the gel is then run through a western blot protocol which uses streptavidin to bind to the biotinylated proteins and fluoresce them. This fluorescence is then detected through film and the development of that film. There have been films which shown a protein of interest, but there is too much background to be confident in those results.
Conclusion
Progress has been made on detecting the target of the antimicrobial protein CCL28, but more analysis and experimentation is needed. CCL28 has been confirmed as having a protein target, but the specific protein is yet unknown. Other chemokines and antimicrobial proteins could be tested in this same way to determine their binding targets. This could be useful in the development of new and effective antibiotics.
References
- Liu, Bin, and Eric Wilson. “The Antimicrobial Activity of CCL28 Is Dependent on C-terminal Positively charge Amino Acids.” European Journal of immunology 40.1 (2010): 186-96. Print.
- Appelmelk, B., D. Su, A. A. Verqij-van Vught, B. G. Thihs, and D. M. MacLaren. “Polymyxin B-horseradish Peroxidase Conjugates as Tools in Edotoxin Research.” Analytical Biochemistry 207.2 (1992): 311-16. Print.