Title of Abstract

Characterization of the Putative Foldase XopAZ from Xanthomonas cynarae

Submitting Student(s)

Alyssa PettyFollow

Faculty Mentor

One WU mentor: Jason Hurlbert, Ph.D.; hurlbertj@winthrop.edu

College

College of Arts and Sciences

Department

Chemistry, Physics & Geology

Faculty Mentor

Jason Hurlbert, Ph.D.

Abstract

Xanthomonas cynarae is a bacterium that infects and elicits a hypersensitive response in artichokes. This response is due to bacterial avirulence (Avr) proteins interacting with the host cell’s resistance proteins. The Avr proteins enter the plant cell cytosol through the Type III Secretion System (T3SS), which uses a modified flagellum to puncture the plant cell wall and deliver proteins into the cytosol. The diameter of the T3SS “needle” is too narrow for folded proteins to be transported, so bacterial Avr proteins are unfolded prior to passing through the system. Once in the plant cytosol, the Avr proteins refold into their active state. Our collaborators have identified a protein from X. cynarae called XopAZ that, based upon sequence identity, may be involved in the refolding of Avr proteins as they emerge from the T3SS system. XopAZ shows sequence similarities to multiple peptidyl prolyl isomerases (PPIases) and sensitive to lysis chaperonins. Homology modeling of XopAZ also suggests that it has peptidyl-prolyl isomerase activity and chaperonin activity. In this study, recombinant, hexahistidine-tagged XopAZ was expressed in Escherichia coli and purified using Ni2+ chelating affinity chromatography. Aggregation assays were conducted to determine the chaperonin activity of XopAZ using absorbance monitoring of lysozyme aggregation in addition to monitoring the fluorescent scattering of thermally denatured citrate synthase; however, XopAZ was unable to prevent the aggregation of lysozyme or citrate synthase. Remaining work involves quantitating the peptidyl-prolyl isomerase activity and performing crystallization screening.

Additional Fields About Your Abstract

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Honors Thesis Committee

Jason Hurlbert, Ph.D.; Michael Lipscomb, Ph.D.; Victoria Frost, Ph.D.; Timea Fernandez, Ph.D.

Honors Thesis Committee

Jason Hurlbert, Ph.D.; Michael Lipscomb, Ph.D.; Victoria Frost, Ph.D.; Timea Fernandez, Ph.D.

Course Assignment

CHEM 552 - Hurlbert and HONR 451H - Lipscomb

Other Presentations/Performances

2019 Summer Undergraduate Research Experience Poster Session, Rock Hill, SC, October 2019.

Grant Support

This work was supported by grants from the National Center for Research Resources (5 P20 RR16461) and the National Institute of General Medical Sciences (8 P20 GM103499) from the National Institutes of Health and the National Science Foundation EPSCoR Program under NSF Award # OIA-1655740

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Characterization of the Putative Foldase XopAZ from Xanthomonas cynarae

Xanthomonas cynarae is a bacterium that infects and elicits a hypersensitive response in artichokes. This response is due to bacterial avirulence (Avr) proteins interacting with the host cell’s resistance proteins. The Avr proteins enter the plant cell cytosol through the Type III Secretion System (T3SS), which uses a modified flagellum to puncture the plant cell wall and deliver proteins into the cytosol. The diameter of the T3SS “needle” is too narrow for folded proteins to be transported, so bacterial Avr proteins are unfolded prior to passing through the system. Once in the plant cytosol, the Avr proteins refold into their active state. Our collaborators have identified a protein from X. cynarae called XopAZ that, based upon sequence identity, may be involved in the refolding of Avr proteins as they emerge from the T3SS system. XopAZ shows sequence similarities to multiple peptidyl prolyl isomerases (PPIases) and sensitive to lysis chaperonins. Homology modeling of XopAZ also suggests that it has peptidyl-prolyl isomerase activity and chaperonin activity. In this study, recombinant, hexahistidine-tagged XopAZ was expressed in Escherichia coli and purified using Ni2+ chelating affinity chromatography. Aggregation assays were conducted to determine the chaperonin activity of XopAZ using absorbance monitoring of lysozyme aggregation in addition to monitoring the fluorescent scattering of thermally denatured citrate synthase; however, XopAZ was unable to prevent the aggregation of lysozyme or citrate synthase. Remaining work involves quantitating the peptidyl-prolyl isomerase activity and performing crystallization screening.