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Infection and Immunity, April 2007, p. 1679-1689, Vol. 75, No. 4
0019-9567/07/$08.00+0     doi:10.1128/IAI.01581-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Burkholderia cenocepacia Requires a Periplasmic HtrA Protease for Growth under Thermal and Osmotic Stress and for Survival In Vivo{triangledown} ,{dagger}

Ronald S. Flannagan,1 Daniel Aubert,1 Cora Kooi,3 Pamela A. Sokol,3 and Miguel A. Valvano1,2*

Infectious Diseases Research Group, Departments of Microbiology and Immunology,1 Medicine, Siebens-Drake Research Institute, University of Western Ontario, London, Ontario, Canada,2 Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada3

Received 29 September 2006/ Returned for modification 12 December 2006/ Accepted 31 December 2006

Burkholderia cenocepacia, a member of the B. cepacia complex, is an opportunistic pathogen that causes serious infections in patients with cystic fibrosis. We identified a six-gene cluster in chromosome 1 encoding a two-component regulatory system (BCAL2831 and BCAL2830) and an HtrA protease (BCAL2829) hypothesized to play a role in the B. cenocepacia stress response. Reverse transcriptase PCR analysis of these six genes confirmed they are cotranscribed and comprise an operon. Genes in this operon, including htrA, were insertionally inactivated by recombination with a newly created suicide plasmid, pGP{Omega}Tp. Genetic analyses and complementation studies revealed that HtrABCAL2829 was required for growth of B. cenocepacia upon exposure to osmotic stress (NaCl or KCl) and thermal stress (44°C). In addition, replacement of the serine residue in the active site with alanine (S245A) and deletion of the HtrABCAL2829 PDZ domains demonstrated that these areas are required for protein function. HtrABCAL2829 also localizes to the periplasmic compartment, as shown by Western blot analysis and a colicin V reporter assay. Using the rat agar bead model of chronic lung infection, we also demonstrated that inactivation of the htrA gene is associated with a bacterial survival defect in vivo. Together, our data demonstrate that HtrABCAL2829 is a virulence factor in B. cenocepacia.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Dental Sciences Building, Rm. 3014, University of Western Ontario, London, Ontario, Canada N6A 5C1. Phone: (519) 661-3427. Fax: (519) 661-3499. E-mail: mvalvano{at}uwo.ca.

{triangledown} Published ahead of print on 12 January 2007.

Editor: V. J. DiRita

{dagger} Supplemental material for this article may be found at http://iai.asm.org/.

Infection and Immunity, April 2007, p. 1679-1689, Vol. 75, No. 4
0019-9567/07/$08.00+0     doi:10.1128/IAI.01581-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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