Using Genetically Encoded Fluorescent Sensors to Accurately Observe the Physiology of Persister Cells Through Single-Cell Microscopy

Yao, Thomas

Undergraduate Honors Thesis

Using Genetically Encoded Fluorescent Sensors to Accurately Observe the Physiology of Persister Cells Through Single-Cell Microscopy Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/dr26xz04h

Abstract

Isogenic bacterial communities are capable of harboring a small subpopulation of phenotypically distinguished cells that are transiently resistant to antibiotics. Upon exposure to antibiotics, these cells - termed persisters - enter a dormant state, rendering them tolerant to the antibiotic stimulus. Once the antibiotic stimulus subsides, these cells revert to a normal state and continue dividing to repopulate the community. This feature makes it extremely difficult to eradicate persisters, consolidating their role in the recurrence of chronic infections. While multiple mechanisms can trigger entry into the persistent state, the underlying factor leading to dormancy has been determined to stem from a downregulation of metabolic activity and dissipation of the cells’ membrane potential. During my previous experiments in the Kralj Lab I have been able to isolate and inspect persisters cells under fluorescence microscopy. I discovered that in response to a single antibiotic stimulus, persister cells could exhibit different phenotypes; whilst some cells arrest their growth, others elongate, continuing to replicate once the stimulus has subsided. This project envisioned the use of three genetically encoded fluorescent sensors to allow for the probing of the persister state upon entry into dormancy at high spatio-temporal resolutions: Perceval which senses ATP, PROPS which senses membrane potential, and pHRed which senses pH. This project succeeded in creating a plasmid containing Perceval and pHRed (Perceval-pHRed), expressing said plasmid in Escherichia coli and imaging its responses to different stimuli. Perceval-pHRed was found to faithfully report differences in the pH of cells, and cellular ATP. Due to logistical reasons including an inadequate microscope setup, PROPS was unable to be incorporated together with the Perceval-pHRed plasmid during the time of this project. In future PROPS will be added to the Perceval-pHRed plasmid, allowing for the probing of the the persister physiology as a whole.

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