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A method for identification of receptors for YopE in the type III secretion (T3S) system of Yersinia pseudotuberculosis


Numerous Gram-negative bacterial pathogens have virulence mechanisms that modulate host targets. One such mechanism is the type III secretion (T3S) system. YopE is one of the six virulence proteins delivered by the T3S system of the bacterial pathogen Yersinia into host cells. The targeting signals that direct virulence factors into the host have been mapped, but the receptors for these signals are unknown. The Y. pseudotuberculosis effector YopE requires both an N-terminal region, which is termed the signal sequence (SS) and consists of amino acids 1-15, and a region just downstream, which is termed the chaperone- binding (Cb) region, for translocation into host cells. The focus of this project is to set up a system to identify the receptor, or receptors, that recognize the YopE SS. Prior work has shown that interactions in the T3S system tend to be transient. Thus, I have taken an approach that utilizes formaldehyde as a crosslinking reagent. Formaldehyde is highly reactive, cell permeable, and forms short crosslinks (2.3-2.7 Å). Furthermore, its crosslinks may be reversed at boiling temperatures. Two additional crosslinking reagents, glutaraldehyde and disuccinimidyl tartrate (DST), are presented as alternatives for optimization of crosslinking efficiency. Glutaraldehyde forms a 5 Å irreversible crosslink and DST forms a 6.4 Å reversible crosslink. Reversibility is not strictly necessary, but allows for ease in isolation and identification of potential receptors through mass spectrometry. Recommendations are made for future experiments using the crosslinking parameters proposed for identification of receptors for YopE's signal sequence region

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