Contribution of Burkholderia cenocepacia flagella to infectivity and inflammation

Abstract

Burkholderia cenocepacia is an opportunistic pathogen that can cause severe lung infections in cystic fibrosis patients. To understand the contribution of B. cenocepacia flagella to infection, a strain mutated in the major flagellin subunit, fliCII, was constructed in B. cenocepacia K56-2 and tested in a murine agar bead model of lung infection. C57/BL6 mice infected with approximately 10(8) wild-type K56-2 bacteria exhibited 40% mortality after 3 days, whereas no mortality was noted in mice infected with the fliCII mutant. Among the mice surviving the infection with either strain, there was no significant difference in the bacterial loads in the lungs and spleen, bacteremia, weight loss, or infiltration of immune effector cells at 3 days postinfection. Similar results were observed at 24 h, prior to expression of the lethality phenotype. KC, a murine interleukin-8 (IL-8) homolog, was elevated in both the bronchoalveolar lavage fluid and serum of mice infected with the wild type compared to the fliCII mutant at 24 h, suggesting that flagella stimulated host cells. To demonstrate that flagella contributed to these responses, the interaction between B. cenocepacia and Toll-like receptor 5 (TLR5) was investigated. Infection of HEK293 cells with heat-killed wild-type K56-2, but not infection with the fliCII mutant, resulted in both NF-kappaB activation and IL-8 secretion that was dependent upon expression of TLR5. Together, these results demonstrate that B. cenocepacia flagella contribute to virulence in an in vivo infection model, and that induction of host immune responses through interaction with TLR5 may contribute to its overall pathogenic potential.

FIG. 1.

Physical map of the B. cenocepacia fliCII gene with insertion of a Tp cassette (Tp^r). The fliCII gene was interrupted at the AgeI site at 583 bp with a Tp cassette in the B. cenocepacia K56-2 chromosome. The large arrow indicates the direction of fliCII transcription. Primers generated for gene replacement (fliCII-1 and fliCII-2) and complementation (fliCIIcompF and fliCIIcompR) are represented by directional arrows. Restriction endonuclease sites for PstI used for Southern blot analysis are shown. The numbers refer to nucleotide positions with respect to the fliCII start site.

FIG. 2.

Confirmation of fliCII::Tp by PCR and Southern blot hybridization. (A) PCR was performed on single-colony resuspensions in LB with primers fliCII-1 and fliCII-2, as indicated in Fig. 1. Lanes: 1, K56-2; 2, fliCII::Tp; 3, pTOPO-fliCII-Tp; MW, molecular weight markers (sizes in base pairs are shown at the left). (B) PstI-digested genomic DNA was used for Southern hybridization. DNA was probed with an 848-bp fragment of fliCII generated with primers fliCII-1 and fliCII-2, indicated in Fig. 1. Lanes: 1, K56-2; 2, fliCII::Tp. Sizes of molecular weight markers in base pairs are shown at the left.

FIG. 3.

fliCII::Tp lacks expression of FliCII, as detected by Coomassie staining and immunoblot analysis. Flagella were isolated and separated by SDS-10% PAGE. (A) Coomassie blue staining after SDS-PAGE. Lanes: 1, K56-2; 2, fliCII::Tp; MW, molecular weight markers (sizes in kilodaltons are shown at the left). (B) After SDS-PAGE, proteins were transferred to a nitrocellulose membrane. The membrane was probed with Salmonella H antiserum poly a-z antibody, and reactivity was detected by BCIP/NBT precipitation. Lanes: 1, K56-2; 2, fliCII::Tp. Sizes of molecular weight markers in kilodaltons are shown at the left.

FIG. 4.

fliCII is necessary for swimming motility. Colonies were stabbed into 0.4% LA, incubated overnight at 37°C, and observed for growth around the point of the stab as a sign of swimming motility. Quadrants: A, K56-2; B, fliCII::Tp; C, fliCII::Tp(pUCP18Tc); D, fliCII::Tp(pUCP18Tc-fliCII).

FIG. 5.

KC is elevated in BAL fluid and serum of wild-type-infected mice. The concentration of KC was determined by enzyme-linked immunosorbent assay. (A) BAL fluid was harvested from mice infected with K56-2 (grey bars) or fliCII::Tp (black bars) at 24 h. The results are representative of two independent experiments. (B) Blood was taken from tails of mice infected with K56-2 (grey bars) or fliCII::Tp (black bars) at 24 h.

FIG. 6.

Verification of V5-TLR5 expression in HEK293 cells. Transfected HEK293 cells were lysed and separated by SDS-8% PAGE. Separated proteins were transferred to nitrocellulose and probed with anti-V5-horseradish peroxidase antibody or loading control p44/42 MAP kinase antibody. Reactivity of p44/42 MAP kinase was detected by BCIP/NBT precipitation. Lanes: 1, pcDNA3.1/Zeo; 2, pEF6::hTLR5.

FIG. 7.

Flagellum-expressing B. cenocepacia activates NF-κB through TLR5. HEK293 cells were transiently transfected with pcDNA3.1/Zeo (grey bars) or pEF6::hTLR5 (black bars). Cells were treated with a PBS control or heat-killed bacteria. NF-κB activation was determined by luciferase production (measured in relative light units) 18 h postinfection. These results are representative of three independent experiments. Error bars indicate standard deviations within the experiment.

FIG. 8.

Flagellum-expressing B. cenocepacia induces IL-8 secretion through TLR5. HEK293 cells were transiently transfected with pcDNA3.1/Zeo (grey bars) or pEF6::hTLR5 (black bars). Cells were treated with a PBS control or heat-killed bacteria. Supernatants were harvested, and IL-8 secretion was measured by enzyme-linked immunosorbent assay. The results are representative of three independent experiments. Error bars indicate standard deviations within the experiment.