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Helicobacter pylori, Cell Shape, & Mutant CcmA Alleles:
How a Summer of Lab Research Leads to High School Cancer Curricula
August 2024
Summer vacation, something school aged children and education professionals look forward to all year. No bells, no meetings, just time off. Well, that's true for most of us at least. This summer, I was selected as a Hutch Fellowship for Excellence in STEM Teaching participant, the first of a two-summer long program. This means I spent my summer vacation working in a lab at Fred Hutchinson Cancer Center in Seattle, Washington. I completed real cancer research while developing curricula for my high school physics and chemistry students at Lincoln High School in Tacoma, WA. While it may not be everyone’s idea of relaxing, it was an exceptionally rewarding and productive summer.
Elainea Kesler-Horan using a blue loop to spread H. pylori bacteria onto a horse blood agar plate. Photo credit: Caren Brinkema Photography/ Fred Hutch Cancer Center.
Why HTF?
You may be asking yourself why a teacher would give up their summer vacation to work a second full time job. Well, on a selfish and superficial level, it is incredible to work on cancer research to play a small role in helping cure cancer! But for me, the reason goes much deeper. You see, I had first heard about the Hutch Teacher Fellowship (HTF) program while at an appointment at Fred Hutchinson Cancer Center (“Fred Hutch”) for my father, Mark Kesler. Mark was a patient at Fred Hutch (formerly Seattle Cancer Care Alliance, SCCA) and University of Washington Medical Center. He was treated by his doctors for Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML) until his passing in October 2023.
My dad taught me to use our existence on earth for the betterment of others, thus that is what I try to do. So when I received an email forwarded to me by my principal on behalf of the coordinator of the HTF program I had learned of a year prior, I knew I had to apply. Truthfully, I did not think I would be selected, as I don't teach life science, nor do I have a background in biology. But what I perceived to lack in bio knowledge, I made up for through my passion for cancer research, my overall commitment to learning and growing personally and professionally, and my expertise in physics and chemistry. Thus, I found a perfect placement with the biochemical and biophysical Salama Lab. There I could effectively use my lab research to develop high school physics and chemistry curricula.
Mark Kesler (left) with Elainea Kesler-Horan (right) while Mark was in the intensive care unit at University of Washington Medical Center shortly before his passing in October 2023. Photo credit: Dylan Kesler, brother of Elainea Kesler-Horan.
The Lab
The Salama Lab at Fred Hutch is run by principal investigator Dr. Nina Salama. While working in the lab, I had two mentors, Niyati Rodricks and Kayleen Lederman, both PhD candidates in the University of Washington Microbiology department. They were integral to my success in this program, as they selected the methods and protocols to follow to complete our experiments and taught necessary lab skills I was lacking. They also would explain each experimental step, its purpose in our overarching lab goals, and its function within the human body system. The lab focus is on a Group 1 carcinogenic bacteria called Helicobacter pylori. H. pylori infects over half of the world's population and is responsible for over 80% of gastric (stomach) cancers. Stomach cancer is the fourth leading cancer killer worldwide.
Elainea Kesler-Horan (center) with mentors Kayleen Lederman (left) and Niyati Rodricks (right). Photo credit: Caren Brinkema Photography/Fred Hutch Cancer Center.
My Research
H. pylori is a gram negative bacteria that uses its helical shape to burrow into the mucus lining in the stomach, similar to a screw into drywall. My individual research project leverages the work of former HTF & Science Education Partnership (SEP) teacher Maggie Lewis, as well as the work of Dr. Sophie Sichel, who completed her PhD while in the Salama Lab. Together, they created four mutations of H. pylori. Dr. Sichel did work to understand CcmA, a cytoskeletal protein that polymerizes and self-stabilizes to form higher order structures. Dr. Sichel’s work found that CcmA has an N-terminal and C-terminal domain, while the central bactofilin part of the protein is conserved. She tried to understand what the N and C termini are doing; when just the N-terminus is deleted, CcmA and Csd7 interact, which causes Csd1 and Csd2 to destabilize and modify the peptidoglycan. She also found three different amino acid residues which are potentially involved with regulating CcmA interaction with Csd7. Maggie Lewis generated four mutations based upon Dr. Sichel’s work, three from the amino acid residues and one that is a combination of two different mutations.
Figure 1. Image above shows the methods and tools used to confirm CcmA expression in wild type LHS100 H. pylori and the Maggie Lewis H. pylori mutants. Image created using BioRender.
My research project uses the mutants that Maggie created. We’re interested in characterizing the interaction between CcmA and other proteins to determine what the phenotype of the different mutations are. To do so, I grew H. pylori cultures using the wild type (unmutated) LHS100, a CcmA knockout control, and the four mutants Maggie Lewis made (MLH1, MLH2, MLH3, and MLH4). I suspended the H. pylori samples in brucella broth, spun down to pellet cells in the centrifuge, resuspended the pellet in a buffer solution, boiled and ran cell lysate on SDS-PAGE before washing in milk protein and TBST (See Figure 1 for reference of methods).
We completed a western blot of the four mutations Maggie generated that confirmed that CcmA is still being expressed (Figure 2). While the western blot in figure 2A could suggest that there are lower levels of CcmA in MLH 2, MLH3, and MLH4, based on the control blot of Cag3, there may have been less total protein loaded in those lanes of the gel. We also completed a western blot of Csd1, which showed preliminary results that Csd1 is expressed in 3 of the four mutants, MLH1, MLH2, and MLH4. The goal with these experiments is to understand the role Csd1 and CcmA play in building and maintaining H. pylori’s helical shape. Next summer, we plan on running new western blots to verify Csd1 and CcmA expression levels in the mutant strains.
Figure 2A
Figure 2B
Figure 2A (left) shows western blot of CcmA, with the load control Cag3, kilodalton ladder, wild type LHS100 H. pylori, a CcmA knockout, a Csd1 knockout, and the four Maggie Lewis H. pylori mutants. Figure 2B (right) shows a higher contrast image at the 17kDa line from the same western blot.
Figure 3A
Figure 3B
Figure 3A (left) shows PCR of the wild type LHS100 H. pylori and the four Maggie Lewis H. pylori mutants. Figure 3B (right) shows the second PCR of MLH 2 and MLH4.
Elainea Kesler-Horan using a micropipette onto horse blood agar while mentor Niyati Rodricks observes. Photo credit: Caren Brinkema Photography/Fred Hutch Cancer Center.
The other component of our research is to see what the actual structure of CcmA is with these mutations. To do so, we can purify the mutant ccmA alleles after expressing the mutant proteins in E. coli, putting them on a grid, then image them using transmission electron microscopy. This would allow us to see if they form polymers differently than un-mutated CcmA. The first step was to extract the genomic DNA of the mutants and use a polymerase chain reaction (PCR) to amplify the DNA. When we did this, we were able to extract and PCR all four mutants. We then purified the PCR in order to clone the mutant gene into a plasmid optimized for expressing the protein in E. coli.
Unfortunately, our clones did not grow well onto the E. coli selection plates, indicating unsuccessful transformation. We eventually want to take the plasmids expressing the mutant proteins, purify out the CcmA, and see how it changes how the proteins behave when visualized by transmission electron microscopy. This will be a focus next summer after we attempt cloning transformation into E. coli again.
My Curricula
The final component of the HTF program is to create and pilot curriculum based on my summer cancer research project. As I teach physics and chemistry, I am creating two curricula. For physics, I plan on using H. pylori as a grounding phenomenon for circular and rotational motion. Students will investigate the physical constraints of H. pylori and how its shape uses physics to cause cancer. For my chemistry classes, students will get an introduction to biochemistry. Students will investigate how to neutralize acids using bases just like how H. pylori produces urease to survive in stomach acid. The purpose of this curricula is to engage students in hands-on activities that model real biological systems. It helps to show students that science is not something that happens solely in a classroom, nor does the learning only come from a textbook. The final component which will be integrated into both curricula is a focus on inequities in healthcare and cancer care. Historically, women and people of color have had lesser quality of care and concerns regarding their health compared to their white male counterparts. Students will investigate these inequities and what they could mean for themselves and their families. They will use this investigation and what they learned about H. pylori for their culminating project. The best culminating projects across all classes will be submitted to the Fred Hutch STEAM for Justice, a symposium for high school science students to use science to promote community service, social justice, and social change. The hope is for students to see their education as part of a larger role within society. As the next generation, students have an inherent and integral part in creating our society’s future which does not need to wait until tomorrow–they can make change today.
Elainea Kesler-Horan snowshoeing on Mt. Rainier while chaperoning with the Lincoln High School Science Outdoors program at the Mount Rainier Institute.
Photo credit: Elainea Kesler-Horan.
Overall, this was a summer where I pushed myself further than I thought I was capable. I learned far more lab skills, routines, and content knowledge than I had imagined. You may be asking if it is worth it only having a few days off before returning to teacher meetings and school year prep. To answer, I would say teaching is not something I do from 7 to 3, Monday through Friday, September through June. It is not simply informing content from a textbook into a large set of brains. Teachers are the bedrock of core memories, helping grow students to become better humans. Teachers often wear different hats: caretaker, counselor, cheerleader. This summer, this teacher got to wear a new hat: Cancer Researcher. That is a hat no amount of time off could replace.
Acknowledgements
I would like to acknowledge my principal investigator Dr. Nina Salama for taking in another Hutch Teacher Fellow and providing the guidance and resources of her lab for our program. I also greatly appreciate my mentors, Niyati and Kayleen, without whom I would be unable to attempt my research. Their patience and in-depth explanations have allowed me to learn more than I had imagined in this program, and we’re only one summer in. I want to acknowledge Dr. Kristen Bergsman with the HTF program, as her guidance is essential to success. My fellow HTF teachers and Partners in Science 2.0 @ Fred Hutch teachers are excellent resources, companions, and collaborators. I want to acknowledge the support I have from my principal, Mr. Karl Hoseth, as well as my department chairs, Natalie Reszka and Jennifer Roe, whose support has made my work at LHS feel like home. Finally, I would like to acknowledge my friends and family; without your support this program would not be possible for me.
I live, work, and teach on the ceded and unceded lands and waterways of the Puyallup, Nisqually, and Snoqualmie tribes. I acknowledge that these Indigenous Peoples were, and are, the original stewards of these places.
Elainea Kesler-Horan teaches physics and chemistry classes at Lincoln High School in Tacoma, WA. They are also a partner in the LHS Science Outdoors Program, which hopes to gain equitable access to the outdoors for all students at Lincoln and beyond. LHS Science Outdoors hosts a public symposium in June to celebrate the successes of the school year. Elainea is also a proud disabled educator and disability advocate.
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