Comparative genomics and an insect model rapidly identify novel virulence genes of Burkholderia mallei

Abstract

Burkholderia pseudomallei and its host-adapted deletion clone Burkholderia mallei cause the potentially fatal human diseases melioidosis and glanders, respectively. The antibiotic resistance profile and ability to infect via aerosol of these organisms and the absence of protective vaccines have led to their classification as major biothreats and select agents. Although documented infections by these bacteria date back over 100 years, relatively little is known about their virulence and pathogenicity mechanisms. We used in silico genomic subtraction to generate their virulome, a set of 650 putative virulence-related genes shared by B. pseudomallei and B. mallei but not present in five closely related nonpathogenic Burkholderia species. Although most of these genes are clustered in putative operons, the number of targets for mutant construction and verification of reduced virulence in animal models is formidable. Therefore, Galleria mellonella (wax moth) larvae were evaluated as a surrogate host; we found that B. pseudomallei and B. mallei, but not other phylogenetically related bacteria, were highly pathogenic for this insect. More importantly, four previously characterized B. mallei mutants with reduced virulence in hamsters or mice had similarly reduced virulence in G. mellonella larvae. Site-specific inactivation of selected genes in the computationally derived virulome identified three new potential virulence genes, each of which was required for rapid and efficient killing of larvae. Thus, this approach may provide a means to quickly identify high-probability virulence genes in B. pseudomallei, B. mallei, and other pathogens.

FIG. 1.

Killing curves for G. mellonella larvae inoculated with wild-type and mutant B. mallei cells. (A) Time course of larval killing by B. mallei wild type (WT) and mutants with defects in characterized virulence genes. (B) Time course of larval killing by wild type and mutants with defects in genes selected from the Bm-Bp virulome. Assays were performed at least three independent times by inoculating 10 larvae with each mutant. The actual number of cells (CFU) injected into the larvae was determined by plating duplicate aliquots of the injectant on LBG plates. The percentages of larvae killed are the averages of three assays; the standard deviations are <15%. Wild-type B. mallei and water-inoculated controls were included with each set of assays.

FIG. 2.

Organization and amino acid sequence identity for orthologous genes in B. mallei (BMA) and P. luminescens (PLU) that may encode an insect toxin. Orthologous genes have the same arrow pattern. The location of the B. mallei CDS (BMA0235) that was disrupted by insertion is indicated. DMT, drug/metabolite transporter.