Mapping, Tracking and Modeling the Movements of Single Membrane-Bound Transcription Activator Proteins in Live Vibrio cholerae.

Abstract

Cholera has been known since ancient times, yet there is still no cure. Vibrio cholerae bacteria cause cholera by producing cholera toxin. Cholera toxin production is regulated by the protein ToxT, which itself is regulated by two membrane-bound proteins, TcpP and ToxR, that work together to bind DNA and activate transcription of toxT. The molecular-scale details of this unusual membrane-bound transcription activation mechanism are unclear and cannot be observed using traditional light microscopy due to the diffraction limit of light.

In this thesis, we use single-molecule tracking and super-resolution localization microscopy to overcome the diffraction limit and directly observe the motions and interactions of TcpP labeled with the fluorescent proteins Dendra2, mCherry and PAmCherry in live V. cholerae cells. We describe methods developed–and obstacles encountered–in the course of our studies of these protein fusions, and find, of the three fluorescent proteins examined, PAmCherry is the best choice for tracking TcpP motion.

By using mean squared displacement and cumulative probability distribution analyses of single-molecule trajectories, we compare TcpP-PAmCherry motions across three V. cholerae strains. In each strain, the native copy of TcpP is removed and replaced with TcpP-PAmCherry expressed ectopically. We find TcpP motion can be categorized into three populations: fast, slow and immobile; and TcpP-PAmCherry moves faster when both binding partners (ToxR and the toxT promoter) are present than when either is lacking. Our findings support a mechanism for TcpP--ToxR--toxT promoter interaction in which ToxR recruits TcpP to the toxT promoter.

Although PAmCherry is adequate for our single-molecule microscopy experiments, it is not an ideal fluorophore. A brighter fluorescent label that resists photobleaching would enable faster imaging and longer measured trajectories. We present a protocol for enhancing PAmCherry fluorescence by coupling TcpP-PAmCherry in the membrane of live V. cholerae cells to extracellular gold nanoisland films and nanotriangle arrays to achieve plasmon-enhanced fluorescence. We can detect single-molecule fluorescence above background scatter on both nanostructured gold surfaces, which is promising for further live cell studies.

Greater understanding of the ToxR regulon may lead to novel therapeutics to combat cholera, and enhanced fluorescence will help us observe such interactions with greater detail.