A Broad-Host-Range Tailocin from Burkholderia cenocepacia

Abstract

The Burkholderia cepacia complex (Bcc) consists of 20 closely related Gram-negative bacterial species that are significant pathogens for persons with cystic fibrosis (CF). Some Bcc strains are highly transmissible and resistant to multiple antibiotics, making infection difficult to treat. A tailocin (phage tail-like bacteriocin), designated BceTMilo, with a broad host range against members of the Bcc, was identified in B. cenocepacia strain BC0425. Sixty-eight percent of Bcc representing 10 species and 90% of non-Bcc Burkholderia strains tested were sensitive to BceTMilo. BceTMilo also showed killing activity against Pseudomonas aeruginosa PAO1 and derivatives. Liquid chromatography-mass spectrometry analysis of the major BceTMilo proteins was used to identify a 23-kb tailocin locus in a draft BC0425 genome. The BceTMilo locus was syntenic and highly similar to a 24.6-kb region on chromosome 1 of B. cenocepacia J2315 (BCAL0081 to BCAL0107). A close relationship and synteny were observed between BceTMilo and Burkholderia phage KL3 and, by extension, with paradigm temperate myophage P2. Deletion mutants in the gene cluster encoding enzymes for biosynthesis of lipopolysaccharide (LPS) in the indicator strain B. cenocepacia K56-2 conferred resistance to BceTMilo. Analysis of the defined mutants in LPS biosynthetic genes indicated that an α-d-glucose residue in the core oligosaccharide is the receptor for BceTMilo.IMPORTANCE BceTMilo, presented in this study, is a broad-host-range tailocin active against Burkholderia spp. As such, BceTMilo and related or modified tailocins have potential as bactericidal therapeutic agents against plant- and human-pathogenic Burkholderia.

Keywords: Burkholderia cenocepacia; LPS; broad host range; receptor; tailocin.

FIG 1

Transmission electron micrographs of IEF-purified tailocin BceTMilo. Samples were negatively stained with 2% (wt/vol) aqueous uranyl acetate. Bar, 100 nm. (Left panel) Magnification, ×25,000. Contracted tailocins are shown. (Right panel) Magnification, ×100,000. Arrows indicate tail fibers.

FIG 2

SDS-PAGE of IEF-purified tailocin BceTMilo. Protein subunits of tailocin BceTMilo were dissociated as described in Materials and Methods and loaded on a (A) 10 to 20% Tris-HCl SDS gel or (B) 7.5% Tris-HCl SDS gel. Lane 1, molecular mass standards (Precision Plus protein standard from Bio-Rad; 10 to 250 kDa); lane 2, subunits of tailocin BceTMilo. Proteins were visualized using Coomassie blue staining. Please note that the white lines between lanes 1 and 2 for both panel A and panel B indicate that a lane was removed from each of the original gels, since the data were not presented in Results.

FIG 3

Genomic map of BceTMilo and syntenic comparison. (A) Syntenic relationship of tailocin BceTMilo to tailocin locus on B. cenocepacia J2315. (B) Syntenic comparison of tailocin BceTMilo, Burkholderia phage KL3, and Enterobacteria phage P2. The genomic maps were drawn to scale. Genes are color coded according to Dice amino acid similarity compared to Burkholderia phage KL3, and the related genes are indicated with dotted lines.

FIG 4

Effect of Burkholderia LPS on BceTMilo. Increasing amounts of purified LPS from (A) B. cenocepacia strain PC184 (sensitive) and (B) B. cenocepacia strain AU10487 (resistant) were mixed with tailocin BceTMilo and allowed to incubate for 30 min. A 1:2 dilution series (see Materials and Methods) of mixtures from the experiments represented in panel A and panel B were spotted onto overlays seeded with strain PC184 to determine residual BceTMilo activity. Results are representative of triplicate experiments.

FIG 5

Sugar inhibition assay. Data represent the effects of d-galactose, α-d-glucose, d-fructose, d-mannoheptose, l-rhamnose, d-cellobiose, lactose, sucrose, and d-raffinose on tailocin adsorption to B. cenocepacia strain PC184. The titer was expressed as the reciprocal of the highest dilution showing inhibition. Asterisks (*) indicate sugars that inhibited tailocin BceTMilo adsorption. Results are representative of triplicate experiments. Each bar represents the standard deviation.

FIG 6

Sensitivity of P. aeruginosa LPS mutants to tailocin BceTMilo. (A) Activity of serially diluted (1:10) tailocin BceTMilo against PAO1 (i), PAO1wbpM (ii), PAO1wbpL (iii), PAO1rmlC (iv), or PAO1algC (v). WT, wild type. (B) Numbers in parentheses indicated in panel A correspond to depicted LPS structures with the same numbers as those indicated in panel B. Putative residues involved in BceTMilo adsorption are circled. Abbreviations: Man, mannose; Fuc, fucose; GalN, N-galactosamine; HEP, mannoheptose; NAG, N-acetylglucosamine; KDO, 2-keto-3 deoxyoctulosonic acid. (Adapted from reference with permission of the publisher.)

FIG 7

Electrophoretic profiles of LPS from B. cenocepacia K56-2, J2315, K56-2, and K56-2 deletion mutants and complements. LPS was prepared as described in Materials and Methods. A total of 1.0 μg LPS was loaded in each lane of a 16.5% polyacrylamide gel in a Tricine-SDS system and developed by silver staining as described in Materials and Methods. Please note that the white line between lanes 3 and 4 indicates that a lane was removed from the original gel, since the data were not presented for the strain in Results.

FIG 8

Sensitivity of B. cenocepacia strains K56-2 and J2315 and K56-2 LPS mutants to tailocin BceTMilo. (A) Activity of serially diluted (1:10) tailocin BceTMilo against strain K56-2 (i), J2315 (ii), K56-2 ΔwaaL (iii), K56-2 ΔwaaC (iv), K56-2 ΔwaaL complement (v), or K56-2 ΔwaaC complement (vi). (B) Numbers in parentheses indicated in panel A correspond to depicted LPS structure with the same numbers as those indicated in panel B. Residues involved in BceTMilo adsorption are circled. Abbreviations: OS, oligosaccharide; HEP, l-glycero-d-manno-heptose; KO, d-glycero-α-d-talo-oct-2-ulopyranolsylonic acid; KDO, 2-keto-3 deoxyoctulosonic acid; QuiNAc, β-d-QuiNAc. The structure shown in panel B was modified from Ortega et al. (30).