title: 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, pGPOmegaTp. 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 degrees 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.
authors.affiliation: Department of Microbiology and Immunology, Dental Sciences Building, Rm. 3014, University of Western Ontario, London, Ontario, Canada N6A 5C1.
authors.forename: Ronald S
authors.initials: RS
authors.lastname: Flannagan
authors.forename: Daniel
authors.initials: D
authors.lastname: Aubert
authors.forename: Cora
authors.initials: C
authors.lastname: Kooi
authors.forename: Pamela A
authors.initials: PA
authors.lastname: Sokol
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Valvano
mesh_terms: D000222:Adaptation, Physiological; D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D001665:Binding Sites; D015153:Blotting, Western; D019121:Burkholderia Infections / Q000382:microbiology; D042602:Burkholderia cepacia complex / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development* / Q000472:pathogenicity; D003087:Colicins / Q000032:analysis / Q000235:genetics; D004195:Disease Models, Animal; D017353:Gene Deletion; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006358:Hot Temperature; D050296:Microbial Viability; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009876:Operon; D009997:Osmotic Pressure; D033903:Periplasmic Proteins / Q000032:analysis; D010957:Plasmids / Q000235:genetics; D018410:Pneumonia, Bacterial / Q000382:microbiology; D012329:RNA, Bacterial / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D012697:Serine Endopeptidases / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D003087:Colicins; D033903:Periplasmic Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012697:Serine Endopeptidases