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Journal of Bacteriology, October 2008, p. 6580-6588, Vol. 190, No. 20
0021-9193/08/$08.00+0     doi:10.1128/JB.00761-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization of the Putative Type III Secretion ATPase CdsN (Cpn0707) of Chlamydophila pneumoniae{triangledown}

Chris B. Stone, Dustin L. Johnson, David C. Bulir, Jodi D. Gilchrist, and James B. Mahony*

M. G. DeGroote Institute for Infectious Disease Research, Faculty of Health Sciences and Department of Pathology and Molecular Medicine, McMaster University, and Father Sean O'Sullivan Research Centre, St. Joseph's Healthcare, Hamilton, Ontario, Canada

Received 28 May 2008/ Accepted 1 August 2008

Type III secretion (T3S) is utilized by a wide range of gram-negative bacterial pathogens to allow the efficient delivery of effector proteins into the host cell cytoplasm through the use of a syringe-like injectisome. Chlamydophila pneumoniae is a gram-negative, obligate intracellular pathogen that has the structural genes coding for a T3S system, but the functionality of the system has not yet been demonstrated. T3S is dependent on ATPase activity, which catalyzes the unfolding of proteins and the secretion of effector proteins through the injectisome. CdsN (Cpn0707) is predicted to be the T3S ATPase of C. pneumoniae based on sequence similarity to other T3S ATPases. Full-length CdsN and a C-terminal truncation of CdsN were cloned as glutathione S-transferase (GST)-tagged constructs and expressed in Escherichia coli. The GST-tagged C-terminal truncation of CdsN possessed ATPase activity, catalyzing the release of ADP and Pi from ATP at a rate of 0.55 ± 0.07 µmol min–1 mg–1 in a time- and dose-dependent manner. CdsN formed oligomers and high-molecular-weight multimers, as assessed by formaldehyde fixation and nondenaturing polyacrylamide gel electrophoresis. Using bacterial two-hybrid and GST pull-down assays, CdsN was shown to interact with CdsD, CdsL, CdsQ, and CopN, four putative structural components of the C. pneumoniae T3S system. CdsN also interacted with an unannotated protein, Cpn0706, a putative CdsN chaperone. Interactions between CdsN, CdsD, and CopN represent novel interactions not previously reported for other bacterial T3S systems and may be important in the localization and/or function of the ATPase at the inner membrane of C. pneumoniae.

* Corresponding author. Mailing address: Regional Virology and Chlamydiology Laboratory, St. Joseph's Hospital, 50 Charlton Avenue East, Hamilton, Ontario L8N 4A6, Canada. Phone: (905) 521-6021. Fax: (905) 521-6083. E-mail: mahonyj{at}mcmaster.ca

{triangledown} Published ahead of print on 15 August 2008.

Journal of Bacteriology, October 2008, p. 6580-6588, Vol. 190, No. 20
0021-9193/08/$08.00+0     doi:10.1128/JB.00761-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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