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Infection and Immunity, October 2005, p. 6260-6271, Vol. 73, No. 10
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.10.6260-6271.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification of New Secreted Effectors in Salmonella enterica Serovar Typhimurium

Kaoru Geddes,* Micah Worley, George Niemann, and Fred Heffron

Department of Microbiology and Immunology, Oregon Health and Sciences University, Portland, Oregon

Received 5 May 2005/ Returned for modification 7 June 2005/ Accepted 16 June 2005

A common theme in bacterial pathogenesis is the secretion of bacterial products that modify cellular functions to overcome host defenses. Gram-negative bacterial pathogens use type III secretion systems (TTSSs) to inject effector proteins into host cells. The genes encoding the structural components of the type III secretion apparatus are conserved among bacterial species and can be identified by sequence homology. In contrast, the sequences of secreted effector proteins are less conserved and are therefore difficult to identify. A strategy was developed to identify virulence factors secreted by Salmonella enterica serovar Typhimurium into the host cell cytoplasm. We constructed a transposon, which we refer to as mini-Tn5-cycler, to generate translational fusions between Salmonella chromosomal genes and a fragment of the calmodulin-dependent adenylate cyclase gene derived from Bordetella pertussis (cyaA'). In-frame fusions to bacterial proteins that are secreted into the eukaryotic cell cytoplasm were identified by high levels of cyclic AMP in infected cells. The assay was sufficiently sensitive that a single secreted fusion could be identified among several hundred that were not secreted. This approach identified three new effectors as well as seven that have been previously characterized. A deletion of one of the new effectors, steA (Salmonella translocated effector A), attenuated virulence. In addition, SteA localizes to the trans-Golgi network in both transfected and infected cells. This approach has identified new secreted effector proteins in Salmonella and will likely be useful for other organisms, even those in which genetic manipulation is more difficult.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Oregon Health and Sciences University, Portland, OR 97201. Phone: (503) 494-6841. Fax: (503) 494-6862. E-mail: geddesk{at}ohsu.edu.

Editor: F. C. Fang

Infection and Immunity, October 2005, p. 6260-6271, Vol. 73, No. 10
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.10.6260-6271.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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