Identification of a novel virulence factor in Burkholderia cenocepacia H111 required for efficient slow killing of Caenorhabditis elegans

Abstract

Burkholderia cenocepacia H111, which was isolated from a cystic fibrosis patient, employs a quorum-sensing (QS) system, encoded by cep, to control the expression of virulence factors as well as the formation of biofilms. The QS system is thought to ensure that pathogenic traits are expressed only when the bacterial population density is high enough to overwhelm the host before it is able to mount an efficient response. While the wild-type strain effectively kills the nematode Caenorhabditis elegans, the pathogenicity of mutants with defective quorum sensing is attenuated. To date, very little is known about the cep-regulated virulence factors required for nematode killing. Here we report the identification of a cep-regulated gene, whose predicted amino acid sequence is highly similar to the QS-regulated protein AidA of the plant pathogen Ralstonia solanacearum. By use of polyclonal antibodies directed against AidA, it is demonstrated that the protein is expressed in the late-exponential phase and accumulates during growth arrest. We show that B. cenocepacia H111 AidA is essential for slow killing of C. elegans but has little effect on fast killing, suggesting that the protein plays a role in the accumulation of the strain in the nematode gut. Thus, AidA appears to be required for establishing an infection-like process rather than acting as a toxin. Furthermore, evidence is provided that AidA is produced not only by B. cenocepacia but also by many other strains of the Burkholderia cepacia complex.

FIG. 1.

Surface proteins of B. cenocepacia H111 and the cepI mutant H111-I. Surface proteins were extracted by treatment of cells with 0.25% SDS, and samples were separated on an SDS-15% PAGE gel. The band missing in H111-I is marked with an arrow. Lane M, protein markers (in kilodaltons).

FIG. 2.

Comparison of the AidA homologues of B. cenocepacia H111 (AidA and AidA′) and R. solanacearum AW1 (AidA-R). Shown on a black background are identical amino acids; shown on a grey background are similar amino acids.

FIG. 3.

(a) Genetic organization of the aidA locus of B. cenocepacia. Genes are represented by open arrows pointing in the direction of transcription. Shown below is the nucleotide sequence of the aidA upstream region of B. cenocepacia H111. Putative −35 and −10 promoter sequences are boxed, and an inverted repeat that exhibits similarity to lux box sequences is underlined. The solid arrowhead above the diagram marks the location of the npt cassette in the aidA mutant H111-A. m.p., predicted membrane protein; ABC, putative ABC transporter component, substrate binding protein. (b) Nucleotide sequence comparison of the lux box-like sequence upstream of aidA and the operator region of cepI (36). Boldfaced nucleotides, identical bases. Boxed sequences represent the minimal consensus defined for operators of QS-regulated genes in P. aeruginosa (58).

FIG. 4.

Expression of AidA is controlled by the cep QS system. The B. cenocepacia wild-type strain H111 and various isogenic mutants were grown in LB medium overnight. Total-cell proteins were separated by SDS-PAGE, and AidA was visualized by immunoblotting with antisera directed against the protein. (a) Lanes: 1, H111; 2, H111-I (cepI); 3, H111-I (cepI) plus 200 nM C8-HSL; 4, H111-R (cepR); 5, H111-R(pBAH27). (b) Lanes: 1, H111; 2, H111-A (aidA); 3, H111-A(pBAH44); 4, H111-A(pBAH62).

FIG. 5.

Expression of AidA is growth phase dependent. The B. cenocepacia wild-type strain H111 was grown in LB medium, and samples were taken along the growth curve. AHL concentrations were quantified by mixing 10 μl of cell-free culture supernatant with 100 μl of an exponentially growing culture of the GFP-based AHL biosensor P. putida(pAS-C8). Following incubation for 4 h, relative fluorescence units (rfu) were measured with a fluorimeter (a). Expression of AidA was assessed by normalizing samples to an OD₆₀₀ of 1 and using 10 μl for Western blotting with anti-AidA antibodies (b).

FIG. 6.

Killing of nematodes by B. cenocepacia H111 (solid bars), the aidA mutant H111-A (open bars), and the complemented aidA mutants H111-A(pBAH44) (aidA⁺) (light shaded bars) and H111-A(pBAH62) (aidA⁺ aidA′⁺) (dark shaded bars) under conditions of fast killing (a) and slow killing (b). Each data point represents the mean and standard deviation from eight (fast killing) or five (slow killing) independent trials.

FIG. 7.

The ability of B. cenocepacia H111 to accumulate in the C. elegans intestine is dependent on AidA. Nematodes were fed on GFP-tagged derivatives of the B. cenocepacia wild-type strain H111 (a) or the aidA mutant H111-A (b). Worms were inspected with a confocal laser scanning microscope, and transmission images were merged with epifluorescence micrographs showing bacterial GFP fluorescence. The entire lumen of a nematode fed on GFP-tagged wild-type H111 was filled with green fluorescent cells (a). In contrast, only a few GFP-tagged cells were observed in the nematode gut when the aidA mutant H111-A (b) was used as a food source.