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Research Team (GutHub)
Research Team (GutHub)
Mia Young
Mia Young
Biology & Biochemistry Major
Rimi Chowdhury
Rimi Chowdhury
Department of Microbiology
Abstract/Introduction
Abstract/Introduction
Shigella flexneri is a human-adapted enteric pathogen that causes shigellosis, a severe form of infectious diarrhea. Upon ingestion, S. flexneri navigates to the colon and invades epithelial cells, triggering a strong inflammatory response by the immune system. Reactive oxygen species (ROS) are a major component of this inflammatory response, aimed at killing the invading pathogen. However, the mechanisms by which S. flexneri manages to survive in this hostile environment are poorly understood. Cysteine, an essential amino acid having a free sulfhydryl group, can neutralize ROS. Therefore, we hypothesized that the cysteine biosynthesis pathway may play a crucial role in neutralizing free radicals and ROS. To test this, we first identified the essential gene cysM involved in the cysteine biosynthetic pathway, followed by constructing deletion mutants by replacing cysM with kan and testing their growth rate relative to wildtype S. flexneri in presence of ROS. We found that the wildtype S. flexneri could survive in moderate amounts of ROS stress, but the mutant strain failed to grow at all. This confirmed our hypothesis that the cysteine biosynthesis gene cysM is essential for S. flexneri’s survival under oxidative stress. This study advances the field of host-pathogen interactions by uncovering how S. flexneri withstands oxidative stress during its infection cycle in the human host.
Research Question
Research Question
Does S. flexneri utilize the cysteine biosynthesis pathway to promote defense against oxidative stress?
Methods
Methods
Construct a targeted gene deletion of cysM, a key gene in the cysteine biosynthesis pathway, in S. flexneri.
Compare oxidative stress survival between wildtype and mutant strains by exposing them to hydrogen peroxide (H₂O₂).
PCR-amplify kan from E. coli pKD4 plasmid
Transform kan fragment into S. flexneri with ƛ Red recombinase to replace cysM
Select successful transformants (ΔcysM) on plates containing kanamycin
Perform bacterial growth curve assays with a plate reader
Measure OD600 every 30 min. for 24 h.
Compare wildtype vs. mutant with and without hydrogen peroxide
Results
Results
Wildtype
Wildtype
ΔcysM
ΔcysM
Wildtype vs. ΔcysM
Wildtype vs. ΔcysM
Survival of both strains decreases with increasing PO concentrations in the growth media, indicating oxidative stress. Both reach essentially the same saturation point by stationary phase; however, the mutant exhibits slower growth. At 750uM PO, wildtype narrowly tolerates the oxidative stress while ΔcysM does not survive at all.
Future Study
Future Study
Quantify mutant and wildtype cysteine production
Rescue survival with exogenous cysteine supplementation or a plasmid returning cysM functionality
Construct a ΔcysK strain (CysM and CysK are isoenzymes)
NACE Career Readiness Competencies
NACE Career Readiness Competencies
Critical Thinking
My research required me to analyze experimental data, interpret results, and troubleshoot unexpected outcomes. For example, when results did not align with expected trends, I had to evaluate possible sources of error, reconsider experimental design, and adjust methods accordingly. This strengthened my ability to think analytically and make evidence-based decisions.
Communication
I enhanced my ability to communicate scientific information clearly in both written and verbal formats. Writing about and presenting findings helped me translate complex concepts into understandable explanations for different audiences. I also learned the importance of clarity, organization, and precision in scientific communication.
Teamwork
Working in a research group taught me how to collaborate effectively with others. I contributed to group discussions, shared responsibilities, and supported my peers in completing experiments. This experience improved my ability to work toward shared goals while respecting diverse perspectives and approaches.
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