Comparative Genomics and an Insect Model Rapidly Identify Novel Virulence Genes of Burkholderia mallei

Burkholderia pseudomallei and its host-adapted deletion clone Burkholderia mallei, cause the potentially fatal human diseases melioidosis and glanders, respectively. Their antibiotic resistance profile, ability to infect via aerosol, and the absence of protective vaccines has 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 absent from 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 related bacteria, were highly pathogenic in this insect. More importantly, four previously characterized B. mallei mutants with reduced virulence in hamsters or mice were similarly reduced in virulence on G. mellonella larvae. Site-specific inactivation of select genes from the computationally derived virulome identified three potentially new virulence genes, each of which were 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. Published in Journal of Bacteriology, v190 n7 p2306-2313, Apr 2008.