Undergraduate Honors Thesis

 

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

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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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Date Awarded
  • 2024-04-18
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  • 2024-04-22
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