Expansion of a Salmonella Type III Secretion System Effector Protein Secretion Kinetics Assay

Beckham, Wesley

Undergraduate Honors Thesis

Expansion of a Salmonella Type III Secretion System Effector Protein Secretion Kinetics Assay Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/n009w392q

Abstract

The Type III Secretion System (T3SS) is a needle-like assembly that Gram-negative
bacteria such as Salmonella enterica use to deliver virulence effector proteins into target cells.
The inner diameter of the T3SS needle is ~2 nm, and cannot accommodate fully folded proteins.
Therefore, effectors must be unfolded prior to secretion through the T3SS. Our lab has proposed
that effector proteins have evolved to be mechanically labile while retaining typical
thermodynamic stability, and that such evolutionary pressure underlies the extreme sequence
divergence observed in T3SS effectors. This project is focused on implementing a live-cell
imaging assay to compare the secretion kinetics of effector proteins to that of non-secreted
homologs. The approach involves fusing a 3X Tetracysteine tag to the proteins of interest, which
binds a membrane-permeable fluorescent molecule called FlAsH and has been shown to not
interfere with T3SS secretion, unlike other fluorescent markers like GFP. The goal is to introduce
the fluorescently labeled proteins of interest in Salmonella typhimurium and carry out an
infection of mammalian (HeLa) cells. The tagged proteins will be highly concentrated within the
bacterial cells allowing their detection. Upon HeLa cell invasion, effector proteins are secreted
into the mammalian cells, which can be directly monitored by the decrease in the fluorescence
intensity inside the bacteria. The assay is expected to allow direct comparison of the secretion
kinetics of T3SS effector proteins and their non-secreted homologs to test the hypothesis that
effector proteins have evolved to be unfolded easier and therefore, secreted faster.

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