Human Inflammasome Activation in Response to Bacterial Pathogens

Brian using a pipette

Students

2018
College

Faculty

Assistant Professor of Microbiology

Project Summary

I had the opportunity to study how the human inflammasome responds to various bacterial pathogens under the mentorship of Dr. Sunny Shin, at the Perelman School of Medicine in the Department of Microbiology.  Inflammasomes are large multi-protein complexes that assemble in the host cell in response to bacterial invasion and a vital component of human innate immunity.  These complexes are responsible for detecting bacterial ligands and initiating a signaling cascade, ultimately resulting in the release of pro-inflammatory interleukin (IL)-1-family cytokines to warn neighboring cells of imminent infection as well as pyroptosis, an inflammatory type of cell death to prevent further bacterial replication.  Given that dysregulated inflammasome activation has been implicated in causing sepsis, studying the human inflammasome and uncovering its mechanisms may provide potential targets for novel therapeutic strategies for treating bacterial septicemia.  

 

My project involved infecting THP-1 cells, an immortalized human cell line isolated from a monocytic leukemia patient, with various strains of the Gram-positive bacteria Listeria monocytogenes ectopically expressing inner rod proteins from a Type 3 Secretion System (T3SS).  The T3SS acts as a molecular syringe and is used by Salmonella Typhimurium to inject virulence factors into host cells, which can then manipulate host signaling to promote bacterial virulence.  The purpose of this was to investigate whether the T3SS inner rod protein PrgJ from Salmonella as well as homologs isolated from other bacterial species could activate the human inflammasome.

 

I first engineered Listeria to express the PrgJ T3SS homolog YscI isolated from Yersinia pseudotuberculosis.  Subsequently the genetically modified strain of Listeria expressing YscI, along with other modified strains expressing different T3SS inner rod protein homologs, was used to infect the THP-1 cells.  Namely, I tested Listeria ectopically expressing T3SS inner rod proteins isolated from S. Typhimurium, B. thailandensis, S. flexneri, and C. violaceum, in addition to the one isolated from Y. pseudotuberculosis.  ELISAs for the IL-1 family cytokines IL-1α and IL-1β, and lactate dehydrogenase (LDH) cell death assays were used to screen for inflammasome activation.

This summer was an especially rewarding one for me.  I had to overcome many obstacles and learn from my mistakes throughout the entire process.  I think the most important lesson I learned about biomedical research is that sometimes things that should work on paper do not necessarily work in practice, and that it is up to the researcher to be patient, diagnose the problem, and find ways to continually improve.  I am so grateful for all the help and encouragement from the talented members of the Shin lab who always challenge me to think scientifically and reflect on the significance of each step in the research process.  Because of all this, I feel that I have genuinely developed a deeper appreciation and passion for research and immunology.  With this newfound understanding of laboratory work, I look forward to continuing my research work in the fall semester.