JOURNAL OF BACTERIOLOGY, Apr. 2008, p. 2306–2313
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 190, No. 7
Comparative Genomics and an Insect Model Rapidly Identify Novel
Virulence Genes of Burkholderia mallei?†
Mark A. Schell,1* Lyla Lipscomb,1and David DeShazer2
Department of Microbiology, University of Georgia, Athens, Georgia 30602,1and Bacteriology Division,
United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick,
Received 30 October 2007/Accepted 8 January 2008
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.
Burkholderiaceae is a family of the betaproteobacteria that
colonize a variety of ecological niches. Some species are plant
or animal pathogens, while others are important environmen-
tal bacteria, including nitrogen-fixing symbionts, plant growth-
promoting rhizobacteria, chemolithoautotrophs, and bioreme-
diation agents. Nearly all members of the Burkholderiaceae
have large (5- to 9-Mb), multireplicon genomes (http://www
.genomesonline.org/) comprised of a large core of orthologous
genes and smaller subsets of species- or isolate-specific genes
for ecological specialization. Burkholderia pseudomallei and
Burkholderia mallei are aggressive human pathogens (7–9, 13)
categorized as select agent biothreats with a high potential for
misuse (3, 55, 67). The genome sequences of several strains of
B. mallei and B. pseudomallei have revealed that B. mallei is a
deletion clone of B. pseudomallei (27) which lost ?1,000 genes.
Many of the gene deletions appear to have been caused by
insertion sequence-mediated events (49) and likely explain the
many physiological differences that led to the classification of
these two pathogens as separate species (54). Nearly all genes
retained by B. mallei share ?99.5% DNA-DNA sequence
identity with their B. pseudomallei homologs.
B. pseudomallei, which causes melioidosis, is an endemic and
opportunistic pathogen that inhabits the tropical soils and wa-
ters of Southeast Asia and Northern Australia (2, 8, 58, 70),
whereas B. mallei appears to be a zoonotic pathogen that
infects a variety of animals, including equines and humans. The
disease caused by B. mallei, glanders, is not well studied as
there have been few well-documented cases since 1950. For
both pathogens, infection usually occurs through wounds, as-
piration, and possibly inhalation. Studies of intraperitoneally
infected animals have shown that B. pseudomallei and B. mallei
can rapidly migrate to the spleen and liver, where they multiply
extensively within membrane-bound phagosomes and form ab-
scesses (25, 37). Untreated infections are often fatal.
Small-animal models are available for studying the patho-
genesis of B. pseudomallei and B. mallei (15, 34, 40, 42, 68).
These models include a sensitive (50% lethal dose, ?10 cells)
Syrian hamster model utilizing intraperitoneal injection and
two mouse models utilizing C57BL/6 or BALB/c mice and
aerosol, intranasal, or intraperitoneal infection (40, 42). How-
ever, these models are expensive, cumbersome, and hazardous
due to biosafety and biosecurity issues. A Caenorhabditis
elegans nematode infection model for B. pseudomallei has been
described (24, 50, 59), although it may have limited sensitivity
due to the extremely high doses required for killing.
Compared to other bacterial pathogens, little is known
about the virulence factors of B. mallei and B. pseudomallei or
the molecular basis of the pathogenicity of these organisms.
The only verified virulence factors for these pathogens are
those that are well known and shared by most, if not all, animal
and plant pathogens: exopolysaccharide capsule (CAP) (14,
53), type III secretion systems (T3SS) (19, 20, 23, 60, 64, 66),
and lipopolysaccharide O antigen (LPS) (12). Type II secre-
tion, type IV pili, and flagella (10, 11, 18) have been implicated
in B. pseudomallei pathogenesis. Recently, a type VI secretion
* Corresponding author. Mailing address: Department of Microbi-
ology, University of Georgia, Athens, GA 30602. Phone: (706) 542-
2815. Fax: (706) 542-2674. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jb
?Published ahead of print on 25 January 2008.
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VOL. 190, 2008NOVEL VIRULENCE GENES OF B. MALLEI 2313