2026-09-21T18:46:48Zhttps://ecommons.cornell.edu/server/oai/requestoai:ecommons.cornell.edu:1813/335512026-05-14T13:55:41Zcom_1813_35col_1813_47
Seckute, Jolita
Nicholson, Linda K
Ealick, Steven Edward
Oswald, Robert Edward
2013-07-23T18:23:43Z
2016-06-01T06:15:47Z
2011-01-31
https://hdl.handle.net/1813/33551
8213846
Bacteria have evolved several secretion systems as direct conduits into host cell cytoplasm for bacterial effector protein trafficking to further their symbiotic or pathogenic goals, while avoiding detection and host immune responses. Gram-negative bacterium Pseudomonas syringae uses the type III secretion system that spans bacterial membranes and extends with a needlelike protrusion to open up through the host cell membrane. There are structural constraints to passage through this conduit, where the inner channel diameter of 2-3 nm prevents effector protein export in the folded form. The requirement for transient unfolding places unique restrictions on the stability thresholds for effector structure and function, essential for bacterial pathogenicity. Effector tertiary structure is typically needed for function in the host, as has been shown for one of the most widely studied effectors, AvrPto. AvrPto is an 18-kDa protein with a helical core of low stability and disordered N- and C-termini, secreted by the plant pathogen Pseudomonas syringae. AvrPto features a single amino acid folding switch governed by histidine 87 that acts in the moderately acidic pH range expected in the biological environment of the bacterium. At neutral plant host cytosol of pH ~7.4 AvrPto is predominantly in the folded form. We disabled the His 87encoded pH switch by the H87Y mutation and observed a slight increase in stability and a dramatic shift in the acid denaturation into the more acidic pH. This mutation significantly affected the translocation efficiency of AvrPto during the initial hours of Pseudomonas syringae infection at different temperatures. Through direct comparison of in vivo data with in vitro AvrPto stability trends we conclude that the amount of available cytoplasmic unfolded AvrPto is a factor regulating AvrPto translocation, and favors an acidic bacterial cytosol. Preliminary ratiometric GFP fluorescence data predicts bacterial intracellular pH of < 6, in agreement with this prediction. Taken together, a mechanism for effector transport emerges that depends on destabilized effector pools for the T3SS as a significant factor in effector translocation hierarchy and the overall infection process.
en_US
Histidine
protein folding switch
type III secretion system
Histidine Folding Switch In Effector Protein Avrpto Regulates Its Transport Through The Bacterial Type Iii Secretion System, As Determined By Nmr, Fluorescence, Circular Dichroism Spectroscopies And Translocation Assays
dissertation or thesis
application/pdf
Biophysics
Cornell University
Doctor of Philosophy
Ph. D., Biophysics
Publication
Cornell Theses and Dissertations
Seckute, Jolita
5893a6ea-7af3-41d7-abc6-04bcd26ab5df
open.access
open.access
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<Title>Histidine Folding Switch In Effector Protein Avrpto Regulates Its Transport Through The Bacterial Type Iii Secretion System, As Determined By Nmr, Fluorescence, Circular Dichroism Spectroscopies And Translocation Assays</Title>
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<Abstract>Bacteria have evolved several secretion systems as direct conduits into host cell cytoplasm for bacterial effector protein trafficking to further their symbiotic or pathogenic goals, while avoiding detection and host immune responses. Gram-negative bacterium Pseudomonas syringae uses the type III secretion system that spans bacterial membranes and extends with a needlelike protrusion to open up through the host cell membrane. There are structural constraints to passage through this conduit, where the inner channel diameter of 2-3 nm prevents effector protein export in the folded form. The requirement for transient unfolding places unique restrictions on the stability thresholds for effector structure and function, essential for bacterial pathogenicity. Effector tertiary structure is typically needed for function in the host, as has been shown for one of the most widely studied effectors, AvrPto. AvrPto is an 18-kDa protein with a helical core of low stability and disordered N- and C-termini, secreted by the plant pathogen Pseudomonas syringae. AvrPto features a single amino acid folding switch governed by histidine 87 that acts in the moderately acidic pH range expected in the biological environment of the bacterium. At neutral plant host cytosol of pH ~7.4 AvrPto is predominantly in the folded form. We disabled the His 87encoded pH switch by the H87Y mutation and observed a slight increase in stability and a dramatic shift in the acid denaturation into the more acidic pH. This mutation significantly affected the translocation efficiency of AvrPto during the initial hours of Pseudomonas syringae infection at different temperatures. Through direct comparison of in vivo data with in vitro AvrPto stability trends we conclude that the amount of available cytoplasmic unfolded AvrPto is a factor regulating AvrPto translocation, and favors an acidic bacterial cytosol. Preliminary ratiometric GFP fluorescence data predicts bacterial intracellular pH of < 6, in agreement with this prediction. Taken together, a mechanism for effector transport emerges that depends on destabilized effector pools for the T3SS as a significant factor in effector translocation hierarchy and the overall infection process.</Abstract>
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