Discovery Park Undergraduate Research Internship Program

"Assessing Fic proteins from Helicobacter pylori as novel Virulence Factors involved in Gastric Cancer"

About the Project

Project Time & Type:
Full-Year 2018 - CPIP
Research area(s):
Biochemistry, Genetics, Molecular Biology
Project Description:
Helicobacter pylori is a spiral shaped bacterium that survives the harsh acidic environment of the human stomach and grows within the stomach’s mucus layer. Infection with H. pylori is the primary identified cause of gastric cancer (or cancer of the stomach), the second most common cause of cancer related deaths worldwide. Although our immune cells recognize and attack invading H. pylori, they are unable to clear infection because H. pylori has developed virulence strategies that interfere with the immune response. The main virulence factor from H. pylori associated with infection and risk for gastric cancer is a secreted toxin called CagA, which causes chronic inflammation and predisposes cells of the stomach lining to become cancerous. However, the H. pylori virulence factors involved in evading immune responses remain largely elusive. We have recently discovered a new family of bacterial toxins, called Fic proteins, that function to kill phagocytic immune cells. Interestingly, clinical strains of H. pylori encode anywhere from one to five different Fic proteins. We hypothesize that one or more of these Fic proteins could function as novel virulence factors to promote H. pylori infection. Our goal is to enzymatically characterize 5 Fic proteins from two H. pylori clinical strains, Puno120 and G27; assess the ability of these Fic proteins to be secreted by H. pylori; and determine whether these Fic proteins induce cytotoxicity when expressed in human macrophages and epithelial cells. Fic proteins shown to possess enzymatic activity and associated with host cell cytotoxicity will be targeted for deletion in H. pylori and the mutant strain assessed for virulence.
Expected Student Contributions:
The CPIP intern will be involved in purifying bacterially expressed H. pylori Fic proteins, subjecting them to biophysical analysis, and assessing their enzymatic activity using in vitro adenylylation assays. Biophysical characterization of proteins will be conducted in collaboration with the Bindley Biophysical Analysis Core. The intern will also use molecular biology techniques to clone these Fic proteins in mammalian expression vectors, and assess them by transfecting human epithelial cells. Efficient transfection will be assessed both by confocal fluorescent microscopy and western blotting. The intern will also carry out site directed mutagenesis to assess enzymatic mutants of Fic proteins. The techniques indicated above are routine for my laboratory; the CPIP intern will obtain training in these methodologies by working closely with my technician.
Related Website(s):
Please visit these 3 websites: https://www.bio.purdue.edu/People/faculty_dm/directory.php?refID=1079 https://www.bio.purdue.edu/news/articles/2016/summer-happenings-in-the-mattoo-lab.html http://www.purdue.edu/newsroom/releases/2015/Q2/protein-determines-life-or-death-fate-of-stressed-cells.html
Desired Qualifications:
1. GPA above 3.3 2. Coursework in Freshman Biology and Freshman Chemistry. Additional courses in Biochemistry, Cell Biology, and/or Organic Chemistry preferred but not required. 3. Prior Molecular Biology experience in a lab preferred. 4. Strong work ethic.
Estimated Weekly Hours:
12
Department awards independent research credits for this project?
Yes, 4 credit hours

Professor in Charge

Name:
Mattoo, Seema
Deptartment/College:
biological sciences

Student Supervisor

Name:
Erica Zbornik
Title:
Lab Manager/Technician

Cooperating Faculty

Name:
Jia Ma
Deptartment/College:
Bindley Biosciences - Biophysical Analysis