Genome-wide analysis of DNA repeats in Burkholderia cenocepacia J2315 identifies a novel adhesin-like gene unique to epidemic-associated strains of the ET-12 lineage

Abstract

Members of the Burkholderia cepacia complex (Bcc) are respiratory pathogens in patients with cystic fibrosis (CF). Close repetitive DNA sequences often associate with surface antigens to promote genetic variability in pathogenic bacteria. The genome of Burkholderia cenocepacia J2315, a CF isolate belonging to the epidemic lineage Edinburgh-Toronto (ET-12), was analysed for the presence of close repetitive DNA sequences. Among the 422 DNA close repeats, 45 genes potentially involved in virulence were identified and grouped into 12 classes; of these, 13 genes were included in the antigens class. Two trimeric autotransporter adhesins (TAA) among the 13 putative antigens are absent from the other Burkholderia genomes and are clustered downstream of the cci island that is a marker for transmissible B. cenocepacia strains. This cluster contains four adhesins, one outer-membrane protein, one sensor histidine kinase and two transcriptional regulators. By using PCR, we analysed three genes among 47 Bcc isolates to determine whether the cluster was conserved. These three genes were present in the isolates of the ET-12 lineage but absent in all the other members. Furthermore, the BCAM0224 gene was exclusively detected in this epidemic lineage and may serve as a valuable new addition to the field of Bcc diagnostics. The BCAM0224 gene encodes a putative TAA that demonstrates adhesive properties to the extracellular matrix protein collagen type I. Quantitative real-time PCR analysis indicated that BCAM0224 gene expression occurred preferentially for cells grown under high osmolarity, oxygen-limited conditions and oxidative stress. Inactivation of BCAM0224 in B. cenocepacia attenuates the ability of the mutant to promote cell adherence in vitro and impairs the overall bacterial virulence against Galleria mellonella as a model of infection. Together, our data show that BCAM0224 from B. cenocepacia J2315 represents a new collagen-binding TAA with no bacterial orthologues which has an important role in cellular adhesion and virulence.

Characterization of BCAM0224, a Multifunctional Trimeric Autotransporter from the Human Pathogen Burkholderia cenocepacia

“…In a previous study, we reported that the BCAM0224 gene was detected exclusively in epidemic B. cenocepacia strain J2315. We also found that a B. cenocepacia BCAM0224 mutant exhibited attenuated virulence in a Galleria mellonella infection model (22).…”

“…In a previous work (22), we identified the BCAM0224 gene, encoding a protein with similarity to the prototype member of the TAA family, the Yersinia adhesin YadA. In an attempt to gain insight into its structure, we have built 3D models for BCAM0224 domains using the Webbased I-TASSER server (http://zhanglab.ccmb.med.umich.edu /I-TASSER/) (26).…”

“…In a previous study, we identified a gene, BCAM0224, encoding a trimeric autotransporter adhesin (TAA), which appears to be restricted to epidemic-associated B. cenocepacia strains of the ET-12 lineage (22). Transcriptional analysis revealed that BCAM0224 is located within a bicistronic operon composed of 9 genes, which comprises, among others, two additional TAA-encoding genes (BCAM0219 and BCAM0223) (21).…”

“…The complete coding sequence of the BCAM0224 gene was obtained from plasmid pDM3 (22) by digestion with NdeI and XbaI restriction enzymes. This DNA fragment was ligated into plasmid pIN177 (tac promoter; Cm r ) (A. Vergunst and D. O'Callagan, unpublished data) before transformation into E. coli DH5␣ and selection with 100 mg/liter chloramphenicol.…”

“…B. cenocepacia clinical isolate K56-2, an ET-12 isolate, was kindly provided by J. J. LiPuma (University of Michigan). The B. cenocepacia K56-2 BCAM0224::Tp mutant and Escherichia coli strain BL21(DE3) expressing the BCAM0224 gene were described in a previous study (22). Bacteria were cultured in Luria-Bertani (LB) broth (Conda, Pronadisa) at 37°C with orbital agitation at 250 rpm.…”