Undergraduate Honors Thesis

 

Effector Stability as a Determinant for Secretion by the Type Three Secretion System Public Deposited

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https://scholar.colorado.edu/concern/undergraduate_honors_theses/gq67jr68g
Abstract
  • The Type III Secretion System (TTSS) is present in many Gram-negative bacteria. The TTSS is an important protein complex which allows the bacteria to inject effector proteins from the bacterial cytosol into the host cell. This process is crucial for virulence of bacteria. The injectisome, the needle-like assembly of the TTSS, has a narrow inner diameter of 1.3-2.5 nm. Folded effector proteins are too large to fit through the needle so they must be mechanically unfolded by the TTSS unfoldase. The TTSS unfoldase has a weak motor. Evidence for this is seen in experiments where SptP, an effector protein, is fused with GFP, the protein becomes trapped in the injectisome. This has led to the current model that thermodynamically stable proteins are unable to be unfolded by the TTSS. To test this hypothesis, the thermodynamic stability of SopE2 and SptP, two Salmonella effectors, were tested using urea denaturation monitored by Circular Dichroism spectroscopy. They were found to have average thermodynamic stability, providing evidence against this hypothesis. This led to an alternative hypothesis, that effector proteins have evolved to be mechanically unstable to allow for unfolding by the TTSS but they have maintained thermodynamically stable so that they can refold in the host cell. To test this hypothesis, the TTSS effector protein NleC was characterized by Atomic Force Microscopy-based Single Molecular force Spectroscopy (AFM-SMFS) to determine the mechanical stability. NleC was found to be compliant and unfold at low force, providing evidence for this hypothesis.

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  • 2019-01-01
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  • 2023-12-03
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