Isolation of polymyxin B-susceptible mutants of Burkholderia pseudomallei and molecular characterization of genetic loci involved in polymyxin B resistance

Abstract

Burkholderia pseudomallei is a gram-negative bacterium that causes the disease known as melioidosis. This pathogen is endemic to Southeast Asia and northern Australia and is particularly problematic in northeastern Thailand. It has been previously reported that B. pseudomallei is resistant to the killing action of cationic antimicrobial peptides, including human neutrophil peptide, protamine sulfate, poly-L-lysine, magainins, and polymyxins. Recently, we have also found that the virulent clinical isolate B. pseudomallei 1026b is capable of replicating in media containing polymyxin B at concentrations of >100 mg/ml. In order to identify genetic loci that are associated with this particular resistance phenotype, we employed a Tn5-OT182 mutagenesis system in coordination with a replica plating screen to isolate polymyxin B-susceptible mutants. Of the 17,000 Tn5-OT182 mutants screened via this approach, five polymyxin B-susceptible mutants were obtained. Three of these mutants harbored Tn5-OT182 insertions within a genetic locus demonstrating strong homology to the lytB gene present in other gram-negative bacteria. Of the remaining two mutants, one contained a transposon insertion in a locus involved in lipopolysaccharide core biosynthesis (waaF), while the other contained an insertion in an open reading frame homologous to UDP-glucose dehydrogenase genes. Isogenic mutants were also constructed via allelic exchange and used in complementation analysis studies to further characterize the relative importance of each of the various genetic loci with respect to the polymyxin B resistance phenotype exhibited by B. pseudomallei 1026b.

FIG. 1

DPX binding assay results. (a) DPX binding of 1026b and Tn5-OT182 mutants. (b) DPX binding of 1026b and allelic exchange mutants. Conc, concentration. Error bars indicate standard deviations.

FIG. 2

Map of genetic loci involved in PMB resistance. Locations of Tn5-OT182 integrations in the mutants PMB-7, PMB-20, PMB-4, PMB-14, and PMB-17 are shown. (a) waaF gene, with approximate position of the Tn5-OT182 integration. (b) udg, waaE, and gmhD genes, with approximate position of the Tn5-OT182 integration in the udg gene. The direction of transcription of these genes relative to one another is shown by arrows. (c) lytB gene, with approximate positions of the three different Tn5-OT182 integrations.

FIG. 3

Silver stain analysis of 1026b, PMB-7, and MB100. Approximately 50 μl from overnight cultures of 1026b (lane A), PMB-7 (lane B), and MB100 (lane C) was proteinase K treated, boiled, and silver stained. The power supply was shut off with the dye front approximately 2 cm from the bottom of the gel. Lane MW, protein molecular mass standards (Gibco BRL) (the apparent molecular mass of bovine serum albumin is marked).

FIG. 4

ELISA with type II O-PS-specific MAb. The values are the means and standard deviations from a single experiment performed in triplicate. O.D. 405 nm, optical density at 405 nm.

FIG. 5

(a) Immunoblot analysis of LPSs from 1026b, E264, and PMB-7. Approximately 3 μg of purified LPS from 1026b (lane A), 3 μg of purified LPS from E264 (lane B), and 30 μg of purified LPS from PMB-7 (lane C) were reacted with a 1:250 dilution of polyclonal rabbit sera containing antibodies specific for type I and II O-PS. (b) Immunoblot analysis of LPSs from 1026b and PMB-20. Approximately 50 μl from overnight cultures of 1026b (lane A) and PMB-20 (lane B) was proteinase K treated, boiled, and reacted with a 1:250 dilution of polyclonal rabbit sera containing antibodies specific for type I and II O-PS. Lanes MW, prestained protein molecular mass standards (Gibco BRL).

FIG. 6

Outer membrane protein preparations. Approximately 20 μg of protein was solubilized in sample buffer with β-mercaptoethanol and boiled for 5 min. Lane A, 1026b; lane B, PMB-4; lane C, PMB-14; lane D, PMB-17; lane E, PMB-7; lane F, PMB-20; lane MW, prestained protein molecular mass standards (Gibco BRL). Arrows indicate the positions of apparent missing protein bands.