Molecular characterization of putative virulence determinants in Burkholderia pseudomallei.

The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033::Km demonstrated weak statistical significance (P = 0.049) at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei.

1. Introduction

Burkholderia pseudomallei is a Gram-negative motile bacillus, that is, a facultative anaerobe and an environmental saprophyte. It is readily recovered from the soil and surface waters in endemic areas, that is, Southeast Asia and Northern Australia [1]. This bacterium possesses a remarkable capacity to infect humans and animals, causing melioidosis which is an important cause of sepsis in the tropics [2]. It has been considered a potential bioweapon and so virulence factors that correspond to its pathogenesis are being intensively studied at an increasing rate.

The availability of complete genome sequence database of organisms allows researchers to discover many molecules and mechanisms that may be involved in the virulence of B. pseudomallei [3, 4]. Several approaches, including subtractive hybridization [5], comparative genomics [6], signature-tagged mutagenesis [7], transposon mutagenesis [8], in vivo expression technology [9], microarray [10], and computational methods [11, 12], have accelerated the discovery of virulence factors over the past decades. The availability of the genomic sequences of several B. pseudomallei strains has rapidly added candidate virulence genes to databases.

In general, past studies have focused on genomic differences between species, that is, the virulent B. pseudomallei and a closely related but avirulent family member B. thailandensis [5, 6]. PCR-based subtractive hybridization was recently undertaken in our laboratory using a virulent clinical isolate B. pseudomallei (v) and an attenuated strain of the same B. pseudomallei isolate (av) [13]. PCR-based subtractive hybridization successfully demonstrated 6 subtracted DNA fragments that were unique to the virulent strain of B. pseudomallei, whereas these DNA fragments were not seen in its “attenuated” strain [13]. Sequencing of the subtracted DNA fragments revealed 6 unique genes with unknown functions as follows: BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419.

Besides the 6 putative “virulence” determinants, BPSL3147 is another potential virulence determinant identified by Cuccui et al. [7]. This gene encodes a putative lipoprotein and is reported to be a putative lipoprotein containing 39.16% amino acid that is identical to a VacJ lipoprotein in Shigella flexneri. The Tn10 mutant of S. flexneri YSH6000T VacJ lipoprotein was unable to spread from cell to cell, suggesting VacJ is important for intercellular spread of the organism [14].

The aim of this study was to extensively characterize the 6 putative virulence determinants which were absent in the “attenuated” strain (av) that is believed to have reduced virulence that occurred after several subcultures and long-term storage in the laboratory, together with an additional candidate, BPSL3147, using the same methodology, that is, gene knockout approach, using in vitro and in vivo assays.

2. Material and Methods

2.1. Bacterial Strains, Media, and Culture Conditions

Bacterial strains and plasmids used are listed in Table 1. The clinical B. pseudomallei strain was isolated from the blood of a patient CMS, at the University Hospital, University of Malaya, Kuala Lumpur, who died from melioidosis as described in previous report [11] and this strain was used throughout the study (henceforth referred to as Bp-CMS). The Escherichia coli strains DH10B, CC118λpir, S17-1λpir, vector pUT-Km, and chloramphenicol acetyltransferase (CAT) cassette were obtained from Prof. Dr. Wang Jin-Town (National Taiwan University, Taiwan). All strains were grown in Luria-Bertani (LB) medium at 37°C and, when appropriate, antibiotics were used at the following final concentrations: ampicillin 100 μg/mL, kanamycin 50 μg/mL, chloramphenicol 100 μg/mL, and streptomycin 100 μg/mL. The mouse leukaemic monocyte macrophage cell line RAW264.7 was obtained from American Type Culture Collection (ATCC, USA). It was cultured and maintained in flasks (Corning, USA) with DMEM (Gifco, USA) supplemented with 10% (v/v) fetal bovine serum (Gifco, USA), 4 mM L-glutamine, and an antibiotic mixture containing 100 U/mL penicillin and 0.1 mg/mL streptomycin at 37°C in 5% CO2.

Table 1

Bacterial strains and plasmids used.

Strains or plasmids	Relevant characteristic(s)	Reference
Strains
B. pseudomallei
Bp-CMS	Clinical isolate from blood; wild type parental strain for generation of insertion mutants	Current study
BPSL2033::Km	BPSL2033::Km derivative of CMS; KanR
BPSL2033::Km (pC-BPSL2033)	BPSL2033::Km complemented with pC-BPSL2033 plasmid; KanR CmR
BP1026B_I2784::Km	BP1026B_I2784::Km derivative of CMS; KanR
BP1026B_I2784::Km (pC-BP1026B_I2784)	BP1026B_I2784::Km complemented with pC-BP1026B_I2784 plasmid; KanR CmR
BP1026B_I2780::Km	BP1026B_I2780::Km derivative of CMS; KanR
BP1026B_I2780::Km (pC-BP1026B_I2780)	BP1026B_I2780::Km complemented with pC-BP1026B_I2780 plasmid; KanR CmR
BURPS1106A_A0094::Km	BURPS1106A_A0094::Km derivative of CMS; KanR
BURPS1106A_A0094::Km   (pC-BURPS1106A_A0094)	BURPS1106A_A0094 ::Km complemented with pC-BURPS1106A_A0094 plasmid; KanR CmR
BURPS1106A_1131::Km	BURPS 1106A_1131::Km derivative of CMS; KanR
BURPS1106A_1131::Km   (pC-BURPS1106A_1131)	BURPS 1106A_1131::Km complemented with pC-BURPS1106A_1131 plasmid; KanR CmR
BURPS1710A_1419::Km	BURPS1710A_1419::Km derivative of CMS; KanR
BURPS1710A_1419::Km   (pC-BURPS1710A_1419)	BURPS1710A_1419::Km complemented in trans with pC-BURPS1710A_1419 plasmid; KanR CmR
BPSL3147::Km	BPSL3147::Km derivative of CMS; KanR
BPSL3147::Km (pC-BPSL3147)	BPSL3147::Km complemented with pC-BPSL3147 plasmid; KanR CmR
E. coli
DH10B	F_ mcrA Δ(mrr-hsdRMS-mcrBC)ϕ80lacZ ΔM15 ΔlacX74 recA1 endA1 araD 139 Δ(ara, leu)7697 galU galK λ − rpsL nupG	Prof. Dr. Jin-Town Wang
CC118λpir	Δ(ara-leu) araD Δ lacX74 galE galK phoA20 thi-1 rpsE rpoB argE(Am) recA1 λpir phage lysogen
S17-1λpir	hsdR recA pro RP4-2 (Tc::Mu; Km::Tn7) (λpir)
OP50-1	A streptomycin-resistant derivative of E.coli OP50 used as food source in C. elegans cultivation and uninfected control	Kind gifts from Prof. Dr. Sheila Nathan
Plasmid
pUT-Km	pUT-Km1 derived plasmid, with miniTn5 excised by EcoRI, tnp excised by SalI, and bla removed by ApaLI and then with an insertion of Km resistance cassette from pUC4K into PstI site; oriR6K mobRP4 KanR AmpR	Chuang et al., 2006 [15]
pGEM-T easy	Cloning vector for PCR cloning; AmpR	Promega, USA

2.2. Construction of Mutants

Genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, BURPS1710A_1419, and BPSL3147 were disrupted by homologous recombination using a suicide vector pUT-Km [15, 16]. Briefly, a partial region of the gene to be inactivated (serving as a significant site of gene exchange through conjugation and recombinant events) was amplified by polymerase chain reaction (PCR) using primer pairs as listed in Table 2. The amplicon was subcloned into pGEM-T easy vector, digested with EcoRI, and subcloned into the suicide vector pUT-Km [15]. Selection of transformants by plating onto LB agar containing kanamycin and rapid screening for desired inserts by colony PCR was performed using primers KmF and KmR. The construct was then transformed into E. coli S17-1λpir and introduced into the wild-type B. pseudomallei (Bp-CMS) by conjugation. An insertion mutant was selected using LB agar supplemented with kanamycin and site-specific chromosomal integration was verified by PCR using vector- and corresponding gene-specific primer pairs.

Table 2

Primers used for PCR and construction of mutants and complemented plasmids.

Gene	Primer name	Nucleotide sequence (5′ to 3′)	Purpose
BPSL2033	2033IF	AGAACTTCGAGCAATTGCTG	Internal PCR
	2033IR	GAGAGATGACGTTCGGTCTT	Internal PCR
	M2033F	GTGAACTGGTACAAAGAAATATCG	Mutant confirmation
	M2033R	CACGTTTCTCGGATAGAGC	Mutant confirmation
	C2033F	CTAGAGCGCGGCCTCGCG	Complementation of BPSL2033::Km
	C2033R	CATCACTCGGCGCAATGAGACTG	Complementation of BPSL2033::Km
BP1026B_I2784	2784IF	GTCGAGAGTACGGTGTGTTC	Internal PCR
	2784IR	CCTGCGAAATCCTTATCAC	Internal PCR
	M2784F	CTGTTTTCTAAGCGTCAGAAG	Mutant confirmation
	M2784R	AAATATGCAGGAAATAGCCCG	Mutant confirmation
	C2784F	CCTTCGCGCTGATTTGGT	Complementation of BP1026B_I2784::Km
	C2784R	CTACTTCGTAGCTTGATGCGCC	Complementation of BP1026B_I2784::Km
BP1026B_I2780	2780IF	AAACCAGAAGGGCGATTTC	Internal PCR
	2780IR	GCGTTCTTTTAAGAATTGGGTAG	Internal PCR
	M2780F	CAGGTACGATTCATGGAACG	Mutant confirmation
	M2780R	GGTATTCCGTGACCTGAATGT	Mutant confirmation
	C2780F	AGGCTCGGAGTAGTAACACTT	Complementation of BP1026B_I2780::Km
	C2780R	CTACTGCCTATGCTGGGGTAT	Complementation of BP1026B_I2780::Km
BURPS1106A_A0094	94IF	AGTCGGGGGGTACACCTAC	Internal PCR
	94IR	CGACACCGAGGAAAATTTC	Internal PCR
	M94F	GTGAATGTCGATCTTGCG	Mutant confirmation
	M94R	TCAATCTCCAGCGAGCTT	Mutant confirmation
	C94F	TTCTACCAGCGACTTGGC	Complementation of BURPS1106A_A0094::Km
	C94R	TCAATCTCCAGCGAGCTT	Complementation of BURPS1106A_A0094::Km
BURPS1106A_1131	1131IF	AAGGAGTTGGGTACGTCG	Internal PCR
	1131R	CCCTTGTGCCATTGATAG	Internal PCR
	pUT-R	TTTGAGTGACACAGGAACAC	Mutant confirmation
	C1131F	GTGGTTCAGCCAGGCACG	Complementation of BURPS1106A_1131::Km
	C1131R	GTATGTGTCGTCCGCATTTG	Complementation of BURPS1106A_1131::Km
BURPS1710A_1419	1419IF	GCGATAGCGATTGGAAAACT	Internal PCR
	1419IR	GAATCCAGACCCATTCCGT	Internal PCR
	KmF	ATGAGCCATATTCAACGGGA	Mutant confirmation
	C1419F	TGCACAAGCTGTTCAAATG	Complementation of BURPS1710A_1419::Km
	C1419R	TTAAGGCTTGGGTGCAAG	Complementation of BURPS1710A_1419::Km
BPSL3147	3147IF	GACCAGTACGCGCTCAAG	Internal PCR
	3147IR	GAACGAATACTTGTCGATCG	Internal PCR
	M3147F	GATGTACACGTTCAACGACAAG	Mutant confirmation
	M3147R	CTCTTCCGGCATCTCGTA	Mutant confirmation
	C3147F	TTCAGGGTTACGAAGCGAAG	Complementation of BPSL3147::Km
	C3147R	TCAGTGCAGCCGGATGCTCG	Complementation of BPSL3147::Km

2.3. Construction of Complemented Plasmids

Intact genes BURPS1710A_1419, BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BPSL3147 and their promoters were amplified by PCR and cloned into a pGEM-T-CAT plasmid. These complemented plasmids were then reintroduced into the corresponding insertion mutants by transformation. The resulting complemented strains were selected using LB agar containing chloramphenicol.

2.4. Bacterial Growth Curve

The growth of B. pseudomallei strains in LB broth was monitored over 8 h by taking 1 mL of culture broth every hour to perform OD600 readings. The wild-type Bp-CMS was used as a positive control for growth in LB.

2.5. Bacterial Replication Assays

Intracellular bacterial survival and replication was assayed in the mouse macrophage-like cell line RAW 264.7. Cells were seeded at a density of 1 × 105 cells/well into 24-well culture plates (Corning, USA) and incubated overnight at 37°C in 5% CO2. An overnight culture of B. pseudomallei strain was diluted 1 : 100 and grown at 37°C for 3 h with shaking to reach mid-log phase. Cell monolayers were then washed twice with PBS and incubated in fresh DMEM without antibiotic for 1 h prior to infection with bacteria. The bacterial suspension was added at a MOI of 100 : 1 and the coculture was immediately centrifuged at room temperature at 170 ×g for 5 min to bring the bacteria in direct contact with the host cells. After 1 h, the cells were washed twice with PBS and incubated in fresh DMEM containing 300 μg/mL of tetracycline to suppress the growth of residual extracellular bacteria. Tetracycline was used instead of gentamicin, as Bp-CMS is resistant to gentamicin. At 2, 4, and 8 h after infection, infected monolayers were washed twice with PBS and lysed with 0.1% (v/v) Triton X-100 for 15 min. Serial dilutions of the released bacteria (expressed as colony forming units CFU) were plated on tryptic soy agar (TSA) plates to enumerate bacterial loads by direct colony counts. The number of internalized bacteria obtained at 2 h after infection represented the initial entry of bacteria, whereas at 4 and 8 h after infection represented intracellular bacterial replication. Bacterial survival was normalized to counts obtained at 2 h after infection and the relative survival rate presented as a percentage.

2.6. Virulence Testing with C. elegans

The wild-type C. elegans N2 was obtained from Carolina Biological Supply Company (USA). The nematode was propagated on nematode growth medium (NGM) plate and fed on the normal food source E. coli OP50-1, which was a kind gift from Prof. Dr. Sheila Nathan (National University of Malaysia, Malaysia). Killing assays were performed as previously described by Tan et al. [17] with minor modifications. All nematodes were age-synchronized by a bleaching procedure prior to the killing assay [18]. All B. pseudomallei derived strains (wild-type Bp-CMS, 7 insertion mutants, and 7 complemented strains) and E. coli OP50-1 were grown overnight at 37°C and 40 μL of each culture was spread on NGM plates containing 50 μg/mL 5-fluorodeoxyuridine (Merck, USA) to inhibit the eggs of C. elegans from hatching. Plates were incubated at 37°C for 24 h and then allowed to equilibrate for 24 h at room temperature before coculturing with the host worms. Thirty age-matched hermaphrodites were individually transferred to freshly lawned plates by using the flattened tip of a worm pick (platinum wire). The plates were incubated at room temperature and virulence was tracked by counting the number of live and dead worms every 24 h for 3 days. Three independent experiments were carried out for each strain and each test was performed in triplicate (with a total of 270 worms). A worm was considered dead on failure to respond to gentle touch by the worm pick. E. coli OP50-1 was used as a negative control. The resulting data was analyzed with GraphPad Prism 5 software and plotted using the Kaplan-Meier survival plot.

3. Results

3.1. Insertion Mutant Construction and Growth

Positive chromosomal integration mutants were successfully constructed for the 7 candidate genes, on the first or second attempt, and a representative of construct is shown in Figure 1. All 7 mutant strains demonstrated similar growth rates to the parental strain in liquid media over 8 h.

Figure 1

Representative of mutant construct. Verification of the construction of BPSL2033::Km mutant by PCR using specific alignment primers. M and W represent mutant and wild-type strains, respectively, while L indicates 1 kb DNA ladder from Fermentas (USA).

3.2. Bacterial Replication and Survival Assay

Overall, wild-type bacteria were able to survive and replicate in macrophage cells over the course of the experiments. In contrast, the 5 mutant strains BPSL2033::Km, BP1026B_I2780::Km, BURPS1106A_A0094::Km, BURPS1710A_1419::Km, and BPSL3147::Km showed reduced intracellular survival inside RAW264.7 cells at 4 and 8 h post infection, but only BPSL2033::Km reached statistical significance (P = 0.049) at 8 h post infection. Another 2 mutant strains BP1026B_I2784::Km and BURPS1106A_1131::Km demonstrated no difference in survival in RAW264.7 cells at 4 and 8 h post infection.

Most of these plasmid-complemented strains partially restored intracellular survival and replication except for BP1026B_I2780::Km, BURPS1106A_A0094::Km, and BURPS1106A_1131::Km. The results suggest that 3 genes, BPSL2033::Km (P = 0.049), BURPS1710A_1419::Km (P = 0.165), and BPSL3147::Km (P = 0.076), individually had little effect on the intracellular survival of B. pseudomallei in phagocytic cells (Figures 2(a) to 2(c)).

Figure 2

Bacterial survival and replication within RAW264.7 macrophage-like cells infected at MOI of 100 using wild-type strain and the insertion mutants as well as their complemented strains. Data represents means and standard errors of 3 separate experiments; each experiment was carried out in 3 technical replicates for each time point. Asterisk indicates significant differences (P < 0.05) relative to the wild-type strain Bp-CMS at each time point.

3.3. Caenorhabditis elegans Killing Assay

The 6 mutants (BPSL2033::Km, BP1026B_I2784::Km, BP1026B_I2780::Km, BURPS1106A_1131::Km, BURPS1710A_1419::Km, and BPSL3147::Km) exhibited low levels of attenuated virulence in C. elegans, where survival rates of worms were only 2-fold higher than that of the wild type (data not shown). Among them, the most attenuation of virulence was observed in BURPS1710A_1419::Km as 51% worms were able to survive, followed by BPSL2033::Km (47%) compared to the wild-type parental strain (23%) after 3 days (Figures 3(a) and 3(b)). The complemented strains of both mutants, BURPS1710A_1419::Km and BPSL2033::Km, showed at least partial restoration of virulence, thus suggesting a minor role in the nematode infection model.

Figure 3

Kaplan-Meier survival curves for C. elegans infected with different strains of B. pseudomallei. Values are the pooled data from triplicate of 3 separate experiments (N = 270) (E. coli OP50-1 was the negative control).

4. Discussion

There was no difference in the growth rates of all 7 mutants compared to that of the wild type when assayed in rich media (data not shown), ruling out the possibility that these genes are not affecting the growth but involved in other aspects such as pathogenicity. The infection assay on phagocytic cells showed that, without the presence of gene BPSL2033 (putative transport-related membrane protein), the mutants demonstrated reduced ability to replicate and survive over 8 h. This result was supported by a plasmid-encoded complemented strain, which demonstrated partial restoration of intracellular survival when compared to wild-type Bp-CMS. Similar outcomes were seen with BURPS1710A_1419 (putative lipoprotein) and BPSL3147 (lipoprotein).

At 8 h post infection, loss of gene BPSL2033 showed a 5-fold reduction in survival time in RAW264.7 cells while both BURPS1710A_1419 and BPSL3147 resulted in a 3-fold reduction in survival, indicating that these genes may be involved in intracellular survival. There was a weak but still significant difference (P = 0.049) exhibited by BPSL2033::Km compared to Bp-CMS. Thus, BPSL2033 may act in concert with other genes and play an essential role in virulence. Subtractive hybridization, as reported in our earlier study, demonstrated that Bp-CMS contained 6 DNA sequences that were not found in the attenuated strain, indicating that maximal virulence probably requires multiple genes acting together in concert [13]. This hypothesis is further supported by the present study in which B. pseudomallei virulence in the phagocytic cell line model was not critically dependent on any single putative gene tested.

Several studies have suggested that a double mutant and not a single mutant of B. pseudomallei contributed significantly to the growth inside murine macrophage [19, 20]. Future experiments involving the use of double mutants (i.e., BPSL2033 and BURPS1710A_1419) may prove this possibility. In the context of BPSL3147, there may have been other unidentified gene(s) acting together for full virulence in B. pseudomallei infections. It is unclear why these genes BPSL2033, BURPS1710A_1419, and BPSL3147 with their corresponding complemented plasmids only restored intracellular survival and replication to approximately 50% of the wild-type level. One possible explanation for this may be due to the gene being present on multiple copies of the plasmid.

C. elegans has been used as a simple surrogate host for modeling bacterial diseases [21]. It has been shown that on a low nutrient nematode growth medium (NGM) B. pseudomallei killed C. elegans strain N2 within 3 days and this type of killing is referred to as “slow killing” [22-24]. In our study, wild-type strain Bp-CMS killed 74% of the nematode population at 72 h time point in NGM. Our results are in agreement with published reports that differences in killing efficiency of C. elegans occur among wild-type strains of B. pseudomallei [23, 25, 26]. For instance, the percentage of killing of worms by various B. pseudomallei strains, that is, ATCC23343, EY4, number #40, and KHW, was approximately 50, 60, 75, and 90%, respectively, at 72 h time points [23]. Virulence of B. pseudomallei for the nematode is likely to be variable due to the different genetic determinants in the strains.

In the present study, all the constructed mutants were less effective in killing C. elegans under slow-killing conditions; that is, twice as many worms survived when fed on mutant strains compared to worms fed on wild-type Bp-CMS after 72 h of coculture. However, there was no significant difference of C. elegans killing between the mutants and wild type. Two complemented strains of BPSL2033::Km and BURPS1710A_1419::Km achieved at least partial restoration of virulence at 72 h coculture, thus suggesting that both genes are most probably involved in bacterial virulence.

The low level attenuation of virulence in the mutants, compared to the wild type, may possibly be due to the following.

A single gene was insufficient to mediate full killing in the animal model. At least 2 genes are probably required to act together for achieving virulence in B. pseudomallei. A double mutant ΔrelAΔspoT was reported to exhibit significant and severe attenuation in larva of the wax moth Galleria mellonella and C57BL/6 in mice models, which was not seen with the single mutant [20].

C. elegans possesses mechanisms to avoid or move away from pathogenic bacteria like B. pseudomallei. It has a simple nervous system that consists of 302 neurons that facilitate the identification of molecules, neurons, and circuits involved in their behavior [27]. It uses chemotaxis to find food on the plate and is able to discriminate food both physically based on size and chemically based on taste and olfaction [28]. Worms can modify their olfactory preference via the neurotransmitter serotonin to avoid odours from pathogens and this learning occurs with exposure as short as 4 h [29]. In our study, C. elegans was cultivated on E. coli OP50-1 that best supports growth, so the worms had already experienced good food, which might have increased their exploratory bahaviour when switching to very bad food such as B. pseudomallei, especially in leaving bahaviours. Nonetheless, it is impossible to raise worms on B. pseudomallei alone because of the virulence of the organism.

BPSL2033 is a 428-amino-acid protein with a molecular mass of 46 kDa as a transport-related membrane protein; BURPS1710A_1419 is a 74-amino-acid protein with a calculated molecular mass of 8 kDa which is a putative lipoprotein. Further bioinformatics analysis suggests that amino acids 23-324 of BPSL2033 encode a domain belonging to major facilitator superfamily (MFS). MFS transporters are ubiquitous and found in all classes of organisms and in several pathogens such as Francisella tularensis [30] and Legionella pneumophila [31]. Chatfiled and colleagues [32] have shown that MFS protein plays a role in virulence by promoting bacterial iron-siderophore import.

The Phyre 2 model [33] predicts that BPSL2033 forms a major facilitator superfamily fold and shares very low sequence identity (16%) to glycerol-3-phosphate transporter protein from E. coli with known three-dimensional structure (PDB template: 1pw4A). The results imply that BPSL2033 might exhibit new structural and/or functional characteristics and further X-ray crystallography analysis may provide valuable information. At present, we postulate that BPSL2033 transports nutrients (probably glycerol-3-phosphate) that are essential for the replication of B. pseudomallei. Database searches with the NCBI blastp tool identified BURPS1710A_1419, as a putative lipoprotein within genus level, is diverged from 37 to 82% with no conserved domain identified, as well as a lack of three-dimensional structural information, possibly suggesting that BURPS1710A_1419 gene product has new or different functional characteristics.

In conclusion, our results suggest that BPSL2033 and BURPS1710A_1419 individually are likely to contribute to a minor role in virulence and provide a basis for further characterization of their role in pathogenesis. We hypothesize that the combination effect of both genes can provide a clear virulence role in B. pseudomallei.