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Journal of Bacteriology, February 2004, p. 829-841, Vol. 186, No. 3
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.3.829-841.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Binding of SycH Chaperone to YscM1 and YscM2 Activates Effector yop Expression in Yersinia enterocolitica

Eric D. Cambronne, Joseph A. Sorg, and Olaf Schneewind*

Committee on Microbiology, University of Chicago, Chicago, Illinois 60637

Received 20 August 2003/ Accepted 15 October 2003

Yersinia enterocolitica transports YscM1 and YscM2 via the type III pathway, a mechanism that is required for the establishment of bacterial infections. Prior to host cell contact, YscM1 and YscM2 exert posttranscriptional regulation to inhibit expression of effector yop genes, which encode virulence factors that travel the type III pathway into the cytoplasm of macrophages. Relief from repression has been predicted to occur via the type III secretion of YscM1 and YscM2 into the extracellular medium, resulting in the depletion of regulatory molecules from the bacterial cytoplasm. Using digitonin fractionation and fluorescence microscopy of FlAsH-labeled polypeptides in Yersinia-infected cells, we have localized YscM1 and YscM2 within the host cell cytoplasm. Type III injection of YscM1 and YscM2 required the SycH chaperone. Expression of C-terminal fusions of YscM1 and YscM2 to the neomycin phosphotransferase reporter revealed sequences required for regulatory activity and for secretion in the absence of SycH. Coexpression of SycH and glutathione S-transferase (GST)-YscM1 or GST-YscM2, hybrid GST variants that cannot be transported by the type III apparatus, also relieved repression of Yop synthesis. GST-SycH bound to YscM1 and YscM2 and activated effector yop expression without initiation of the bound regulatory molecules into the type III pathway. Further, regulation of yop expression by YscM1, YscM2, and SycH is shown to act independently of factors that regulate secretion, and gel filtration chromotography revealed populations of YscM1 and YscM2 that are not bound to SycH under conditions where Yop synthesis is repressed. Taken together, these results suggest that YscM1- and YscM2-mediated repression may be relieved through binding to the cytoplasmic chaperone SycH prior to their type III injection into host cells.

* Corresponding author. Mailing address: Committee on Microbiology, University of Chicago, 920 East 58th St., Chicago, IL 60637. Phone: (773) 834-9060. Fax: (773) 834-8150. E-mail: oschnee{at}delphi.bsd.uchicago.edu.

Journal of Bacteriology, February 2004, p. 829-841, Vol. 186, No. 3
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.3.829-841.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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