Burkholderia cenocepacia Requires a Periplasmic HtrA Protease for Growth under Thermal and Osmotic Stress and for Survival In Vivo

Abstract

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, pGPΩTp. Genetic analyses and complementation studies revealed that HtrA _BCAL2829 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 HtrA _BCAL2829 PDZ domains demonstrated that these areas are required for protein function. HtrA _BCAL2829 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 HtrA _BCAL2829 is a virulence factor in B. cenocepacia .