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J Bacteriol. Apr 2008; 190(7): 2306–2313.
Published online Jan 25, 2008. doi:  10.1128/JB.01735-07
PMCID: PMC2293206

Comparative Genomics and an Insect Model Rapidly Identify Novel Virulence Genes of Burkholderia mallei[down-pointing small open triangle]

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.

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 environmental bacteria, including nitrogen-fixing symbionts, plant growth-promoting rhizobacteria, chemolithoautotrophs, and bioremediation 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 waters 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, aspiration, 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 abscesses (25, 37). Untreated infections are often fatal.

Small-animal models are available for studying the pathogenesis 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). However, 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 secretion, type IV pili, and flagella (10, 11, 18) have been implicated in B. pseudomallei pathogenesis. Recently, a type VI secretion (T6S) gene cluster was shown to be required for the virulence of B. mallei (51, 56). Several putative virulence factors, including two proteases (10, 41), a hemolysin (1), a rhamnolipid (28), predicted transporter proteins, and three hypothetical proteins, have been identified using either nematode killing assays (24) or biochemical analysis. However, these factors are yet to be verified by animal experiments. Recently, Tuanyok et al. (62) identified a phospholipase C and a two-component regulator which are required for full virulence of B. pseudomallei.

The genome sequences of >25 Burkholderiaceae isolates representing >12 species and four genera are available (http://www.genomesonline.org/). These isolates occupy a wide diversity of ecological niches, yet they share a common core genome comprised of long syntenic regions containing >2,500 shared “housekeeping” genes whose products share >60% amino acid sequence identity. Of greater interest are the species- or isolate-specific genes, such as those found only in human-pathogenic Burkholderia spp. Therefore, we used in silico genomic subtraction (22) to derive a set of 650 genes that are present in both B. pseudomallei and B. mallei but are not present in the sequenced nonpathogenic members of the Burkholderiaceae. This set is predicted to be highly enriched in novel virulence genes and has been designated the Bm-Bp virulome. We investigated this virulome by selecting genes likely to encode novel virulence factors, inactivating them, and testing the corresponding mutants to determine whether they exhibited reduced virulence in wax moth larvae, a surrogate model that we adapted and verified as correlating with the hamster model commonly used to test B. mallei virulence. Our results show that this is a powerful and easily applicable approach that can accelerate research on these important pathogens and serve as an adjunct to much more expensive and problematic animal testing.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

Strains and plasmids used are listed in Table Table1.1. Bacteria were grown at 37°C in LB (45) medium supplemented with 2% glycerol (LBG) or in brain heart infusion (BHI) medium. For some experiments, B. mallei was also grown on minimal medium plates (4) with 5% sucrose as the sole carbon source. When necessary, antibiotics were used at the following concentrations: kanamycin, 50 μg/ml; polymyxin B, 20 μg/ml; and gentamicin, 20 μg/ml.

TABLE 1.
Bacterial strains and plasmids

Preparation of electrocompetent B. mallei.

An overnight culture was diluted to obtain an A600 of 0.1 in BHI medium and shaken for ~3 h until the A600 was 0.8. Cells were harvested by centrifugation at 6°C at 4,000 × g and washed with 0.5 culture volume of ice-cold sterile water and then with 0.25 volume of ice-cold sterile 10% glycerol. Cells were finally resuspended in 0.01 volume of 10% glycerol and frozen at −80°C.

Construction of a sacB deletion strain of B. mallei ATCC 23344.

Four PCR primers (see Table S1 in the supplemental material) were used to generate DNA fragments of the 5′ and 3′ ends of the sacB gene (BMAA0466) from B. mallei ATCC 23344, and the fragments were cloned into separate pCR2.1-TOPO vectors. The Sac7/Sac8 product was cut out of pCR2.1-TOPO with EcoRI, and the Sac5/Sac6 product was cut out with XbaI and BamHI. Both fragments were cloned stepwise into the corresponding sites of pGRV2 (63), resulting in pDD159, which was confirmed by sequencing. Subsequently, pDD159 was transformed into Escherichia coli S17-1 (56) and mobilized into B. mallei ATCC 23344. Transconjugants were inoculated into LBG broth without antibiotics, and 10-fold dilutions of the overnight cultures were spread onto LBG plates with 5% sucrose. Sucrose-resistant colonies were screened for the sacB deletion mutation by PCR, and one confirmed isolate was designated B. mallei GRS 23344.

Construction of putative virulence mutant.

Internal fragments (400 to 900 bp; lacking both 5′ and 3′ ends) of target coding sequences (CDSs) were PCR amplified with appropriate primers (see Table S1 in the supplemental material), directly cloned into pCR-XL-TOPO (Invitrogen), and transformed into chemically competent E. coli TOP10. Approximately 0.5 μg of each of the resultant plasmids was electroporated into 70 μl of ice-cold electrocompetent B. mallei cells with an Eppendorf w-150 electroporator at 2.3 kV, 600 Ω, and 10 μF for 5 ms. Cells were immediately diluted 10-fold with LBG and shaken for 4 h at 37°C, after which aliquots were spread onto LBG plates containing kanamycin. Colonies that grew after 3 to 4 days were picked, the genomic DNA was isolated, and the presence of the appropriate cis-merodiploid mutant allele was verified by PCR. Typically, 25% of the colonies harbored the expected mutant allele.

BMAA1517 and BMAA1621 were refractory to insertional inactivation, so we used splice overlap extension PCR (32) with a modified sacB-based deletion method (43, 63). Briefly, four PCR primers (see Table S1 in the supplemental material) were used to amplify two ~500-bp fragments of DNA flanking each target gene. Gel-purified amplicons were mixed in a second PCR mixture with the forward primer of the upstream amplicon and the reverse primer of the downstream amplicon. The ~1,000-bp chimeric fragments were cloned into pCR-XL-TOPO, cut out with restriction enzymes (Table (Table1),1), and ligated into the appropriate sites in pGRV2 (63). The resulting plasmids were transformed into E. coli S17-1 (57) and mobilized into the B. mallei sacB deletion mutant GRS 23344. Transconjugants were spread onto minimal medium sucrose plates. Colonies of putative deletion mutants that grew after 5 to 7 days of incubation were screened by PCR to verify deletion of the target sequence. Approximately 10% of the colonies tested harbored the deletion allele.

Infection of Galleria mellonella larvae.

B. mallei strains were grown at 37°C for 24 to 48 h on LBG plates, resuspended at an A600 of 0.2 in BHI medium, and shaken at 37°C until the A600 was between 0.4 and 0.6. Culture aliquots were diluted in sterile water, and ~10 or 250 cells in 5 μl were injected under the carapace into the hemocoel of 10 fifth-instar G. mellonella larvae (Vanderhorst Wholesale, Marysville OH) using a 50-μl gas-tight Hamilton syringe equipped with a 30.5-gauge needle (Becton Dickinson 305106). Inoculated larvae were sealed in 2-oz hinged-lid polypropylene cups (Fisher 03-405-39) with five 2-mm ventilation holes in the lid. Larval death was scored daily for 1 week; dead larvae no longer moved when the cup was tapped and/or had turned black.

Bioinformatic methods.

In silico genomic subtraction was performed as previously described (22). Briefly, using BLASTP, the amino acid sequences encoded by all ~6,000 Glimmer-predicted CDSs of B. mallei ATCC 23344 were individually aligned with the best hits for predicted CDSs of five closely related nonpathogenic strains: Burkholderia sp. strain 383 (= ATCC 17660 = R-18194), Ralstonia eutropha JMP134, Burkholderia ambifaria AMMD, Burkholderia xenovorans LB400, and Burkholderia vietnamiensis G4. The Burkholderia strains were chosen because they are nonclinical (environmental) isolates with a long history of use and no reports of human infection. Sequences of their CDS products and contigs were obtained from JGI/ORNL (http://compbio.ornl.gov/channel). For each CDS alignment, an “orthology score” was calculated by using amino acid sequence identity, the ratio of alignment and query lengths, and the ratio of query and hit lengths. Utilizing an orthology score cutoff value based on the average amino acid sequence identities of the 100 most similar ortholog pairs of each, a set of B. mallei CDS products lacking orthologs in any of the closely related nonpathogens was derived. TBLASTN was then used to compare each CDS in this set to the genomic DNA sequence of the same five bacteria to find and remove orthologous CDSs of B. mallei present in the DNA sequence but not in the called CDS sets. This method provided results similar to those obtained with the reciprocal best BLAST hit method but, in contrast, can be used with raw unfinished genome sequences (22).

Select agent handling.

Gmr B. mallei strains constructed during this work were generated before the Centers for Disease Control Select Agent Program (CDC-SAP) informed us that, despite the fact that Gmr had been widely used in B. mallei before 2006, introduction of Gmr into B. mallei is now considered a “restricted experiment” that requires prior CDC-SAP approval. Thus, all Gmr B. mallei strains that we constructed have been destroyed. Otherwise, all regulations and procedures mandated by CDC-SAP were followed during this work.

RESULTS AND DISCUSSION

Derivation of a virulome for B. mallei and B. pseudomallei.

Despite the extensive ecological diversity of the organisms, genome comparisons of members of the Burkholderiaceae have revealed a core genome comprised of long syntenic regions containing thousands of shared “housekeeping” genes. Of greater interest are the hundreds of species- or isolate-specific genes that confer on each isolate its ability to colonize and survive in a specific niche or host. To find genes that are specific to B. mallei and B. pseudomallei and also have increased probability of major involvement in virulence, we used an in silico genomic subtraction method in which the amino acid sequences encoded by all B. mallei CDSs were individually compared to the amino acid sequences encoded by all the CDSs of four closely related nonpathogenic Burkholderia strains, and all CDS products found in “orthologous” pairs were removed. This yielded a set of 650 CDSs designated the Bm-Bp virulome (see Table S2 in the supplemental material), all of which are present in both B. mallei and B. pseudomallei but whose products have <45% amino acid sequence identity with the product of any other CDS in the genomes of the nonpathogens. In contrast, the average amino acid identity observed for most orthologous CDS pairs is >65%. This virulome comprises ~12% of the genome of B. mallei and appears to be highly enriched in CDSs/genes encoding probable virulence and pathogenicity factors since it contains most of the genes in the animal pathogen-like T3SS (T3SSAP), CAP, T6S, and LPS biosynthetic gene clusters, the only published loci experimentally proven to be critical for causing disease in the hamster model (14, 56, 64). When we attempted to use the total CDSs of Burkholderia thailandensis E264, a strain previously considered a nonpathogen (26, 47), in a subsequent in silico subtraction experiment to further reduce and refine the virulome, we found orthologs for 70% of the virulome CDSs in the nonpathogen, including the majority of the CDSs in the three large gene clusters (T3SS, T6S, and CAP) that are essential for B. mallei virulence. In contrast, orthologs for <10% of the virulome CDSs were found in the genome of Burkholderia cenocepacia J2315 or B. cenocepacia AU1054, two pathogenic strains isolated from cystic fibrosis patients (5, 44, 65). This is perhaps not surprising considering that B. thailandensis E264 is very closely related to B. pseudomallei and that at relatively high doses it has the ability to kill nematodes (50) and hamsters (2) and has the ability to survive in macrophages (F. Gherardini, personal communication).

Most of the 650 virulome CDSs occur in ~40 operonlike clusters of four or more contiguous genes (only 100 genes are singletons). Not surprisingly, most clusters are on the smaller replicon, which carries the majority of nonhousekeeping genes (31, 49). Particularly high concentrations of the CDSs are found between BMAA0345 and BMAA0410, between BMAA0729 and BMAA0758, and between BMAA1517 and BMAA1565. Very few of the CDSs in clusters have Karlin score deviations (38), G+C contents, or GC skews that significantly differ from those of the flanking regions or the genome averages. Nearly 130 CDSs are predicted by SignalP V.3.0 (48) to encode signal peptides for secretion, a value which is about four times higher than the predicted genome-wide average. In the clusters with >10 genes are two complete sets of T3SS genes and several predicted secondary metabolite-producing clusters encoding nonribosomal peptide or polyketide synthetases possibly involved in the cytotoxic activities ascribed to B. pseudomallei (6, 29, 30). Also present are CDSs encoding major parts of both the CAP and LPS biosynthesis clusters and two fimbria/pilus biogenesis pathways, in addition to >100 CDSs encoding predicted membrane proteins. The presence of CDSs encoding proteins with predicted enzymatic functions in other clusters suggests that these CDSs may encode parts of metabolic pathways. However, many of the clustered genes have no predicted function; >200 CDSs lack any functional description, and ~100 do not have PFAM domains.

G. mellonella larvae are a valid surrogate host for B. mallei.

The ultimate goal of deriving the virulome was to extract a comprehensive set of potentially novel virulence genes that contained few enough genes that the majority could be individually knocked out and mutants evaluated to determine whether the knockouts reduced virulence in animal models. Uncertainty about the pathogenic potential of B. thailandensis precluded using in silico subtraction to reduce the virulome to a size at which we could realistically test a large proportion of the genes in mammalian models under cumbersome BSL3 conditions. In contrast, higher-throughput surrogate host systems can be very simple and inexpensive, yet quite accurate for study of human pathogens (61). G. mellonella (wax moth) larvae have served as an insect host and simple pathogenesis model for several mammalian pathogens (39); they are easy to acquire and contain without special facilities. Most importantly, G. mellonella has a circulatory system and a complex innate and mammal-like immune response that effectively deals with a wide range of microbial pathogens (39). In some cases the virulence of a pathogen and attenuated mutants in animal hosts has been accurately reflected in G. mellonella (33, 46). Therefore, we tested G. mellonella as a pathogenesis model for verification of B. mallei virulence genes in the Bm-Bp virulome.

When ~10 wild-type B. mallei ATCC 23344 (or B. mallei GRS 23344) cells were injected into the hemocoel of larvae, the majority of the larvae were killed within 4 days. In ~20 experiments we inoculated more than 300 larvae with anywhere from 3 to 200 cells of wild-type B. mallei and always observed >90% killing within 6 days. Injecting 10 B. pseudomallei K96243 cells into the larvae killed them nearly twice as fast (>80% mortality by 2 days). Similar to hamsters infected with B. mallei or B. pseudomallei, infected larvae showed extensive paralysis 12 h before death. At the onset of paralysis, typically >104 B. mallei cells/ml were present in the hemolymph; just prior to death >106 B. mallei or B. pseudomallei cells/ml were found. These results indicate that B. mallei and B. pseudomallei can multiply to very high numbers in the hemocoel of wax moth larvae and are highly pathogenic for this insect. Injection of up to 105 cells of the pathogenic cystic fibrosis epidemic strain B. cenocepacia J2315, Pseudomonas putida, B. xenovorans, or E. coli W3110 killed <15% of the larvae by day 6; using an inoculum containing 104 cells reduced the percentage of dead larvae at day 6 to <10%, which is identical to the level observed if water was injected. Injection of >105 B. thailandensis cells killed ~35% of the larvae after 7 days. Although this is somewhat inconsistent with the “nonpathogenic” label for B. thailandensis, as mentioned above, this species harbors ~70% of the genes in the Bm-Bp virulome and at relatively high doses (>105 cells) it also can kill hamsters and nematodes. Moreover, at least one opportunistic infection of a human by B. thailandensis has been reported (26).

To show correspondence between the wax moth larva model and mammalian models, we first tested B. mallei RD01, which has a polar insertion in the T3SSAP gene cluster (64). This T3SS is essential for the virulence of B. mallei and B. pseudomallei in mice and hamsters, respectively (64, 66). Compared to the wild type, the ability of B. mallei RD01 to kill wax moth larvae was dramatically reduced (Fig. (Fig.1A,1A, compare WT and T3SS); at lower infection levels (<20 cells/larva), RD01 was largely avirulent (data not shown). When B. mallei strain DD3008, a CPS-deficient mutant with dramatically reduced virulence in hamsters (10), was used, the killing of wax moth larvae was delayed and reduced (Fig. (Fig.1A,1A, compare WT and CAP). Next, we constructed an isoleucine-valine auxotroph of B. mallei by insertional inactivation of ilvI (BMA1848) encoding a subunit of acetolactate synthase III. The resultant mutant was not able to grow on minimal glucose medium and, similar to what has been observed with mice (63), showed attenuated virulence in larvae (Fig. (Fig.1A,1A, compare WT and IlvI). Thus, the behavior of the three mutants whose virulence in animal models is attenuated correlated well with their virulence in larvae. Inactivation of type II secretion affects the virulence of P. aeruginosa (17, 69), some plant pathogens (36, 43), and B. pseudomallei (10). Therefore, we insertionally inactivated gspD (BMA2786), a key component of type II secretion, and found that the killing of wax moth larvae by the resultant GspD mutant was reduced and delayed (Fig. (Fig.1A),1A), but not to the extent caused by the T3SS mutation. These data suggest that the wax moth model of infection is an efficient predictor of potential virulence genes in B. mallei.

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 ...

To verify the stability of the mutant genotype in the wax moth larvae, bacterial cells were recovered from the hemolymph at days 2 and 5 by plating on nonselective LBG agar. All of the 250 resultant colonies tested retained the antibiotic resistance encoded by the plasmid insertion. When auxotrophic ilvI mutant cells were recovered from larval hemolymph and plated on both LBG and minimal media, we found that <1 in 105 of the ex vivo cells had reverted to prototrophy, further demonstrating the high level of stability of the cis-merodiploid mutants.

Use of the wax moth larva model to screen putative virulence mutants.

Based on a manual review of bioinformatic data for virulome CDSs, 10 CDSs were evaluated to determine whether they have a role in virulence (Table (Table2).2). Some of these CDSs were chosen because their best BLAST hits were in phylogenetically distant pathogens or in a eukaryote, some were chosen because they appeared to be potential virulence gene regulators, and some were chosen because they appeared to encode polyketide or peptide synthetases that may be involved in cytotoxin production. Knockout mutants for each CDS were constructed and verified by PCR, and their virulence in wax moth larvae was tested as described above. B. mallei strains with mutations in BMA0847, BMAA1111, BMA1123, BMAA1013, and BMAA1621 had marginal or no differences in virulence compared with the wild type (Fig. (Fig.1A1A and data not shown), whereas with strains harboring insertions in BMA0235, BMAA1204, BMAA1517, BMAA1785, and BMAA1900 there was reduced and/or delayed killing of the larvae (Fig. (Fig.1B1B).

TABLE 2.
Potential virulence genes targeted for mutagenesis

Of all the mutants tested, inactivation of BMAA1517, encoding an AraC-type transcriptional regulator, resulted in the most dramatic reduction in virulence in the larvae. At 7 days postinfection 80% of the larvae infected with ~200 CFU of the BMAA1517 mutant remained asymptomatic, similar to the behavior of the T3SSAP mutant. Whole-genome expression profiling of a B. mallei strain overexpressing BMAA1517 has shown that a cluster of genes found in the Bm-Bp virulome (BMAA0727 to BMAA0744) is exclusively upexpressed on average 4-fold; the levels of some genes are elevated >10-fold (56). This previous study also showed that these genes encode components of a T6S system which, when inactivated, rendered B. mallei avirulent in hamsters. When we tested a mutant lacking a critical component of the T6S system (BMAA0739), its virulence in larvae was reduced to the same extent as that of BMAA1517 mutants (not shown). These results further support the hypothesis that the wax moth larva model is an accurate predictor of B. mallei virulence genes.

Inactivation of BMA0235 in B. mallei dramatically delayed and reduced killing of larvae in the wax moth infection model. BMA0235 is in an operonlike cluster of six genes that is conserved in all sequenced B. pseudomallei and B. mallei strains but is not present in any other Burkholderia genome or in the 600 microbial genomes at Integrated Microbial Genomes (http://img.jgi.doe.gov/cgi-bin/pub/main.cgi), with the exception of the genome of Photorhabdus luminescens. In fact, P. luminescens, which can be an insect pathogen or nematode symbiont (21), has similarly organized orthologs of the five genes flanking BMA0235 (Fig. (Fig.2).2). The average amino acid identity between the products of the six CDS pairs from these phylogenetically distant bacteria is 66%, which is quite remarkable considering that the G+C content of the P. luminescens CDSs is ~38%, while the average G+C content of the B. mallei orthologs is 68%. The BMA0237 protein has a domain similar to a domain in chorismate mutase (PFAM1042), while the BMA0236a and BMA0235 proteins are both assigned to the enoylpyruvate carboxytransferase family (PFAM 00275) and are related to 3-phosphoshikimate 1-carboxyvinyl transferases. Both of the latter proteins were originally annotated as AroA (EC 2.5.1.19), which is involved in aromatic amino acid biosynthesis. However, these proteins show <25% identity to each other and to E. coli AroA. The AroA function of B. mallei more likely resides in the BMA0430 protein, annotated as a bifunctional prephenate dehydrogenase/3-phosphoshikimate 1-carboxyvinyl transferase enzyme whose C-terminal and N-terminal halves show ~50% amino acid identity to full-length E. coli AroA and TyrA, respectively. Thus, the BMA0235 and BMA0236a proteins are more likely novel carboxyvinyl transferases that transfer a vinyl group from phosphoenolpyruvate to an unknown substrate. The BMA0234 protein is annotated as a putative asparagine synthase B (EC 6.3.5.4), as are the products of two other CDSs in the B. mallei genome (BMAA1158 and BMAA1921). The products of these three CDSs do not show >30% amino acid identity to E. coli AsnB. The BMA0234 protein is likely involved in an AsnB-like reaction, transfer of the amido group of glutamine to an aspartatelike substrate. The BMA0233a protein may be a carboxylase, while the BMA0232 protein is a putative drug/metabolite transporter (PFAM 05297). It is plausible that the gene clusters shown in Fig. Fig.22 synthesize a secondary metabolite or toxin that plays a role in insect pathogenesis in P. luminescens and perhaps in B. mallei. This metabolite could be derived from carboxyvinylation of 3,4-dihydroxy-2-butanone 4-phosphate made by the adjacent ribA gene, followed by amidation of the carboxyvinyl group. P. luminescens and its close relatives are well known for production of insect toxins.

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 ...

BMAA1204 mutants took twice as long as the wild type to start killing larvae; after 7 days only 60% of the mutant-infected larvae were dead, compared to the 100% mortality caused by the wild type. BMAA1204 is a ~4,200-residue CDS annotated as encoding a putative polyketide synthase (PKS) in COG family 0332. PKSs are giant multidomain proteins ubiquitous in the prokaryotes. In a manner analogous to fatty acid biosynthesis, they condense or “polymerize” organic acids (e.g., acetate and malonate) into linear polyketides. The resultant polyketides are often cyclized and modified, yielding 300- to 1,500-Da molecules that exhibit diverse antibiotic, antitumor, or even immunosuppressive activities. BMAA1204 is in a large gene cluster along with genes encoding two other putative PKSs and several CDSs whose products are in protein families associated with polyketide synthesis and/or modification. In fact, most CDSs in this region (BMAA1201 to BMAA1222) are in the virulome. It is possible that BMAA1204 and/or the adjacent PKS genes encode production of one of the “cytotoxin” activities found previously in culture supernatants of B. pseudomallei (16, 41, 42), whose characteristics (e.g., heat stability and molecular weight) are consistent with a polyketide origin. PKSs have been implicated in pathogenesis by several other bacteria (e.g., Mycobacterium ulcerans [35]). B. mallei has seven putative PKS genes; three of these genes (BMAA1021, BMAA1204, and BMAA1451) are present in the virulome but are not present in B. thailandensis.

Inactivation of BMAA1785, whose product is annotated as a “chitin binding domain protein,” reduced virulence quantitatively to an extent very similar to the extent observed after inactivation of the PKS gene BMAA1204. Although nonpathogenic Burkholderia spp. lack orthologs of the BMAA1785 protein, Yersinia pestis and Yersinia pseudotuberculosis have orthologs with 55% amino acid sequence identity. The actual function of the BMAA1785 protein is not clear, but its so-called chitin binding domain 3 (PFAM03067) is related to baculoviral spheroidin and spindolin capsid proteins. It has a predicted signal sequence and is present at moderately high levels in culture supernatants of B. pseudomallei and B. mallei (52; data not shown). We speculate that it binds to some type of chitin-like oligosaccharide moiety and has an as-yet-unknown function.

Inactivation of BMAA1900 caused only a moderate delay (1 day) in the onset of killing and a minor reduction in B. mallei-related mortality of wax moth larvae. This gene is in a cluster (BMAA1897 to BMAA1915) that is predicted to encode one of the four predicted T6S systems in B. mallei (56). BMAA1900 encodes a putative pentapeptide repeat protein (COG family 1357) that is 38% identical to a putative exported protein of Bordetella bronchiseptica, which is located in an analogous T6S system cluster. While one can speculate that BMAA1900 is involved in T6S system-mediated secretion of a virulence factor(s), it seems that this T6S system plays a relatively minor role in virulence for wax moth larvae.

Conclusion.

To date, relatively few virulence factors of B. mallei have been identified, and most of the factors that have been found are widespread in diverse pathogens (e.g., T3SS and capsule). Some of the methods used to identify these virulence factors, such as subtractive hybridization and transposon mutagenesis, are time-consuming and especially cumbersome with BSL3-requiring select agent pathogens. With the availability of hundreds of bacterial genome sequences, it makes sense to exploit these databases to use new bioinformatic methods to direct functional genomic studies. In silico genomic subtraction provided a set of B. mallei genes that contained genes encoding all known virulence factors and therefore was very valuable as a starting point for functional analyses to discover new genes with a role in pathogenesis.

G. mellonella larvae have successfully been used as a model for infection by several pathogenic bacteria (39), as well as in screening for bacterial mutants with reduced virulence and/or altered host colonization ability (33, 46). We have shown that G. mellonella also is a good model for B. mallei pathogenesis, because four B. mallei strains with mutations in different characterized virulence gene clusters (T3SS, CAP, type II secretion system, and T6S system) showed reduced virulence that correlated well with their behavior in the hamster model of infection. The larva model is a facile and inexpensive method that can be used to test potential virulence mutants of B. mallei before hamster or mouse models are used. The value of the larva model is enhanced by the observation that G. mellonella has many sophisticated defenses analogous to those found in mammals, such as circulating hemocytes utilizing surface receptors similar to those found in mammalian phagocytes to help encapsulate and destroy microbial invaders (39). Other antimicrobial responses of these insects to pathogens are also similar to those of mammals, including oxidative burst and inducible production of lysozyme and other antibacterial proteins, such as cecropins, defensins, and proline-rich peptides. Besides demonstrating the utility and validity of the G. mellonella insect model for screening mutants for virulence phenotypes, some of our results (i.e., the killing of larvae by very low infectious doses and the presence of multiple insect pathogen-associated genes in the virulome [Fig. [Fig.2;2; see Table S2 in the supplemental material]) suggest that some soil-dwelling insects may be natural hosts and reservoirs of B. pseudomallei (and perhaps B. mallei). However, if this is true, the use of the larva model may result in a bias toward discovery of genes for virulence in insects that may or may not play as significant a role in mammalian pathogenesis.

Supplementary Material

[Supplemental material]

Acknowledgments

This work was supported by National Institutes of Health grant R21-AAI69081 to M.A.S.

We thank Michele Sturgeon and Erin Raybon for their technical assistance and Benjamin Hasselbring and Joanne Rue for critiquing the manuscript.

Footnotes

[down-pointing small open triangle]Published ahead of print on 25 January 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

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