# Molecular Characterization of Putative Virulence Determinants inBurkholderia pseudomallei

Authors: Suat Moi Puah; S. D. Puthucheary; Jin Town Wang; Yi Jiun Pan; Kek Heng Chua
Journal: The Scientific World Journal
 (2014)

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## Abstract

The Gram-negative saprophyteBurkholderia 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 ofB. pseudomallei and an attenuated strain of the sameB. pseudomallei isolate, 6 genesBPSL2033, 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 geneBPSL3147 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 andBURPS1710A_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 inBURPS1710A_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 ofB. pseudomallei.


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## Body

## 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 ofB. 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 severalB. pseudomallei strains has rapidly added candidate virulence genes to databases.In general, past studies have focused on genomic differences between species, that is, the virulentB. pseudomallei and a closely related but avirulent family memberB. thailandensis [5, 6]. PCR-based subtractive hybridization was recently undertaken in our laboratory using a virulent clinical isolateB. pseudomallei (v) and an attenuated strain of the sameB. pseudomallei isolate (av) [13]. PCR-based subtractive hybridization successfully demonstrated 6 subtracted DNA fragments that were unique to the virulent strain ofB. 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 aVacJ lipoprotein inShigella flexneri. The Tn10 mutant ofS. flexneri YSH6000TVacJ lipoprotein was unable to spread from cell to cell, suggestingVacJ 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, usingin vitro andin vivo assays.


## 2. Material and Methods

### 2.1. Bacterial Strains, Media, and Culture Conditions

Bacterial strains and plasmids used are listed in Table1. The clinicalB. pseudomalleistrain 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). TheEscherichia 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-BPSL2033plasmid; 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_A0094plasmid; 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_1419plasmid; 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_mcrAΔ(mrr-hsdRMS-mcrBC)ϕ80lacZΔM15ΔlacX74 recA1 endA1 araD 139Δ(ara, leu)7697 galU galKλ-rpsL nupG
Prof. Dr. Jin-Town Wang
CC118λpir
Δ(ara-leu)araDΔlacX74 galE galK phoA20 thi-1 rpsE rpoB argE(Am)recA1λpirphage lysogen
S17-1λpir
hsdR recA pro RP4-2 (Tc::Mu; Km::Tn7) (λpir)
OP50-1
A streptomycin-resistant derivative ofE.coliOP50 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 byEcoRI, 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

GenesBPSL2033,BP1026B_I2784,BP1026B_I2780,BURPS1106A_A0094,BURPS1106A_1131,BURPS1710A_1419, andBPSL3147 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 intoE. coli S17-1λpir and introduced into the wild-typeB. 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 ofBPSL2033::Km
C2033R
CATCACTCGGCGCAATGAGACTG
Complementation ofBPSL2033::Km
BP1026B_I2784
2784IF
GTCGAGAGTACGGTGTGTTC
Internal PCR
2784IR
CCTGCGAAATCCTTATCAC
Internal PCR
M2784F
CTGTTTTCTAAGCGTCAGAAG
Mutant confirmation
M2784R
AAATATGCAGGAAATAGCCCG
Mutant confirmation
C2784F
CCTTCGCGCTGATTTGGT
Complementation ofBP1026B_I2784::Km
C2784R
CTACTTCGTAGCTTGATGCGCC
Complementation ofBP1026B_I2784::Km
BP1026B_I2780
2780IF
AAACCAGAAGGGCGATTTC
Internal PCR
2780IR
GCGTTCTTTTAAGAATTGGGTAG
Internal PCR
M2780F
CAGGTACGATTCATGGAACG
Mutant confirmation
M2780R
GGTATTCCGTGACCTGAATGT
Mutant confirmation
C2780F
AGGCTCGGAGTAGTAACACTT
Complementation ofBP1026B_I2780::Km
C2780R
CTACTGCCTATGCTGGGGTAT
Complementation ofBP1026B_I2780::Km
BURPS1106A_A0094
94IF
AGTCGGGGGGTACACCTAC
Internal PCR
94IR
CGACACCGAGGAAAATTTC
Internal PCR
M94F
GTGAATGTCGATCTTGCG
Mutant confirmation
M94R
TCAATCTCCAGCGAGCTT
Mutant confirmation
C94F
TTCTACCAGCGACTTGGC
Complementation ofBURPS1106A_A0094::Km
C94R
TCAATCTCCAGCGAGCTT
Complementation ofBURPS1106A_A0094::Km
BURPS1106A_1131
1131IF
AAGGAGTTGGGTACGTCG
Internal PCR
1131R
CCCTTGTGCCATTGATAG
Internal PCR
pUT-R
TTTGAGTGACACAGGAACAC
Mutant confirmation
C1131F
GTGGTTCAGCCAGGCACG
Complementation ofBURPS1106A_1131::Km
C1131R
GTATGTGTCGTCCGCATTTG
Complementation ofBURPS1106A_1131::Km
BURPS1710A_1419
1419IF
GCGATAGCGATTGGAAAACT
Internal PCR
1419IR
GAATCCAGACCCATTCCGT
Internal PCR
KmF
ATGAGCCATATTCAACGGGA
Mutant confirmation
C1419F
TGCACAAGCTGTTCAAATG
Complementation ofBURPS1710A_1419::Km
C1419R
TTAAGGCTTGGGTGCAAG
Complementation ofBURPS1710A_1419::Km
BPSL3147
3147IF
GACCAGTACGCGCTCAAG
Internal PCR
3147IR
GAACGAATACTTGTCGATCG
Internal PCR
M3147F
GATGTACACGTTCAACGACAAG
Mutant confirmation
M3147R
CTCTTCCGGCATCTCGTA
Mutant confirmation
C3147F
TTCAGGGTTACGAAGCGAAG
Complementation ofBPSL3147::Km
C3147R
TCAGTGCAGCCGGATGCTCG
Complementation ofBPSL3147::Km


### 2.3. Construction of Complemented Plasmids

Intact genesBURPS1710A_1419, BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, andBPSL3147 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 ofB. 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 of1×105 cells/well into 24-well culture plates (Corning, USA) and incubated overnight at 37°C in 5% CO2. An overnight culture ofB. 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 withC. elegans

The wild-typeC. 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 sourceE. 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]. AllB. pseudomallei derived strains (wild-type Bp-CMS, 7 insertion mutants, and 7 complemented strains) andE. 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 ofC. 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.


## 2.1. Bacterial Strains, Media, and Culture Conditions

Bacterial strains and plasmids used are listed in Table1. The clinicalB. pseudomalleistrain 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). TheEscherichia 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-BPSL2033plasmid; 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_A0094plasmid; 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_1419plasmid; 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_mcrAΔ(mrr-hsdRMS-mcrBC)ϕ80lacZΔM15ΔlacX74 recA1 endA1 araD 139Δ(ara, leu)7697 galU galKλ-rpsL nupG
Prof. Dr. Jin-Town Wang
CC118λpir
Δ(ara-leu)araDΔlacX74 galE galK phoA20 thi-1 rpsE rpoB argE(Am)recA1λpirphage lysogen
S17-1λpir
hsdR recA pro RP4-2 (Tc::Mu; Km::Tn7) (λpir)
OP50-1
A streptomycin-resistant derivative ofE.coliOP50 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 byEcoRI, 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

GenesBPSL2033,BP1026B_I2784,BP1026B_I2780,BURPS1106A_A0094,BURPS1106A_1131,BURPS1710A_1419, andBPSL3147 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 intoE. coli S17-1λpir and introduced into the wild-typeB. 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 ofBPSL2033::Km
C2033R
CATCACTCGGCGCAATGAGACTG
Complementation ofBPSL2033::Km
BP1026B_I2784
2784IF
GTCGAGAGTACGGTGTGTTC
Internal PCR
2784IR
CCTGCGAAATCCTTATCAC
Internal PCR
M2784F
CTGTTTTCTAAGCGTCAGAAG
Mutant confirmation
M2784R
AAATATGCAGGAAATAGCCCG
Mutant confirmation
C2784F
CCTTCGCGCTGATTTGGT
Complementation ofBP1026B_I2784::Km
C2784R
CTACTTCGTAGCTTGATGCGCC
Complementation ofBP1026B_I2784::Km
BP1026B_I2780
2780IF
AAACCAGAAGGGCGATTTC
Internal PCR
2780IR
GCGTTCTTTTAAGAATTGGGTAG
Internal PCR
M2780F
CAGGTACGATTCATGGAACG
Mutant confirmation
M2780R
GGTATTCCGTGACCTGAATGT
Mutant confirmation
C2780F
AGGCTCGGAGTAGTAACACTT
Complementation ofBP1026B_I2780::Km
C2780R
CTACTGCCTATGCTGGGGTAT
Complementation ofBP1026B_I2780::Km
BURPS1106A_A0094
94IF
AGTCGGGGGGTACACCTAC
Internal PCR
94IR
CGACACCGAGGAAAATTTC
Internal PCR
M94F
GTGAATGTCGATCTTGCG
Mutant confirmation
M94R
TCAATCTCCAGCGAGCTT
Mutant confirmation
C94F
TTCTACCAGCGACTTGGC
Complementation ofBURPS1106A_A0094::Km
C94R
TCAATCTCCAGCGAGCTT
Complementation ofBURPS1106A_A0094::Km
BURPS1106A_1131
1131IF
AAGGAGTTGGGTACGTCG
Internal PCR
1131R
CCCTTGTGCCATTGATAG
Internal PCR
pUT-R
TTTGAGTGACACAGGAACAC
Mutant confirmation
C1131F
GTGGTTCAGCCAGGCACG
Complementation ofBURPS1106A_1131::Km
C1131R
GTATGTGTCGTCCGCATTTG
Complementation ofBURPS1106A_1131::Km
BURPS1710A_1419
1419IF
GCGATAGCGATTGGAAAACT
Internal PCR
1419IR
GAATCCAGACCCATTCCGT
Internal PCR
KmF
ATGAGCCATATTCAACGGGA
Mutant confirmation
C1419F
TGCACAAGCTGTTCAAATG
Complementation ofBURPS1710A_1419::Km
C1419R
TTAAGGCTTGGGTGCAAG
Complementation ofBURPS1710A_1419::Km
BPSL3147
3147IF
GACCAGTACGCGCTCAAG
Internal PCR
3147IR
GAACGAATACTTGTCGATCG
Internal PCR
M3147F
GATGTACACGTTCAACGACAAG
Mutant confirmation
M3147R
CTCTTCCGGCATCTCGTA
Mutant confirmation
C3147F
TTCAGGGTTACGAAGCGAAG
Complementation ofBPSL3147::Km
C3147R
TCAGTGCAGCCGGATGCTCG
Complementation ofBPSL3147::Km


## 2.3. Construction of Complemented Plasmids

Intact genesBURPS1710A_1419, BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, andBPSL3147 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 ofB. 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 of1×105 cells/well into 24-well culture plates (Corning, USA) and incubated overnight at 37°C in 5% CO2. An overnight culture ofB. 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 withC. elegans

The wild-typeC. 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 sourceE. 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]. AllB. pseudomallei derived strains (wild-type Bp-CMS, 7 insertion mutants, and 7 complemented strains) andE. 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 ofC. 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 Figure1. 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 ofBPSL2033::Km mutant by PCR using specific alignment primers. M and W represent mutant and wild-type strains, respectively, while L indicates 1kb 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 strainsBPSL2033::Km, BP1026B_I2780::Km, BURPS1106A_A0094::Km, BURPS1710A_1419::Km, andBPSL3147::Km showed reduced intracellular survival inside RAW264.7 cells at 4 and 8 h post infection, but onlyBPSL2033::Km reached statistical significance (P=0.049) at 8 h post infection. Another 2 mutant strainsBP1026B_I2784::Km andBURPS1106A_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 forBP1026B_I2780::Km, BURPS1106A_A0094::Km, andBURPS1106A_1131::Km. The results suggest that 3 genes,BPSL2033::Km (P=0.049),BURPS1710A_1419::Km (P=0.165), andBPSL3147::Km (P=0.076), individually had little effect on the intracellular survival ofB. pseudomallei in phagocytic cells (Figures 2(a) to 2(c)).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.
(a)
BPSL2033::Km and complemented strain
(b)
BURPS1710A_1419::Km and complemented strain
(c)
BPSL3147::Km and complemented strain


### 3.3.Caenorhabditis elegans Killing Assay

The 6 mutants (BPSL2033::Km, BP1026B_I2784::Km, BP1026B_I2780::Km, BURPS1106A_1131::Km, BURPS1710A_1419::Km, andBPSL3147::Km) exhibited low levels of attenuated virulence inC. 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 inBURPS1710A_1419::Km as 51% worms were able to survive, followed byBPSL2033::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 andBPSL2033::Km, showed at least partial restoration of virulence, thus suggesting a minor role in the nematode infection model.Kaplan-Meier survival curves forC. elegans infected with different strains ofB. pseudomallei. Values are the pooled data from triplicate of 3 separate experiments (N=270) (E. coli OP50-1 was the negative control).
(a)
BPSL2033::Km and its complemented strain
(b)
BURPS1710A_1419::Km and its complemented strain


## 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 Figure1. 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 ofBPSL2033::Km mutant by PCR using specific alignment primers. M and W represent mutant and wild-type strains, respectively, while L indicates 1kb 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 strainsBPSL2033::Km, BP1026B_I2780::Km, BURPS1106A_A0094::Km, BURPS1710A_1419::Km, andBPSL3147::Km showed reduced intracellular survival inside RAW264.7 cells at 4 and 8 h post infection, but onlyBPSL2033::Km reached statistical significance (P=0.049) at 8 h post infection. Another 2 mutant strainsBP1026B_I2784::Km andBURPS1106A_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 forBP1026B_I2780::Km, BURPS1106A_A0094::Km, andBURPS1106A_1131::Km. The results suggest that 3 genes,BPSL2033::Km (P=0.049),BURPS1710A_1419::Km (P=0.165), andBPSL3147::Km (P=0.076), individually had little effect on the intracellular survival ofB. pseudomallei in phagocytic cells (Figures 2(a) to 2(c)).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.
(a)
BPSL2033::Km and complemented strain
(b)
BURPS1710A_1419::Km and complemented strain
(c)
BPSL3147::Km and complemented strain


## 3.3.Caenorhabditis elegans Killing Assay

The 6 mutants (BPSL2033::Km, BP1026B_I2784::Km, BP1026B_I2780::Km, BURPS1106A_1131::Km, BURPS1710A_1419::Km, andBPSL3147::Km) exhibited low levels of attenuated virulence inC. 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 inBURPS1710A_1419::Km as 51% worms were able to survive, followed byBPSL2033::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 andBPSL2033::Km, showed at least partial restoration of virulence, thus suggesting a minor role in the nematode infection model.Kaplan-Meier survival curves forC. elegans infected with different strains ofB. pseudomallei. Values are the pooled data from triplicate of 3 separate experiments (N=270) (E. coli OP50-1 was the negative control).
(a)
BPSL2033::Km and its complemented strain
(b)
BURPS1710A_1419::Km and its complemented strain


## 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 geneBPSL2033 (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 withBURPS1710A_1419 (putative lipoprotein) andBPSL3147 (lipoprotein).At 8 h post infection, loss of geneBPSL2033 showed a 5-fold reduction in survival time in RAW264.7 cells while bothBURPS1710A_1419 andBPSL3147 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 byBPSL2033::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 whichB. 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 ofB. pseudomallei contributed significantly to the growth inside murine macrophage [19, 20]. Future experiments involving the use of double mutants (i.e.,BPSL2033 andBURPS1710A_1419) may prove this possibility. In the context ofBPSL3147, there may have been other unidentified gene(s) acting together for full virulence inB. pseudomallei infections. It is unclear why these genesBPSL2033,BURPS1710A_1419, andBPSL3147 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 killedC. 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 ofC. elegans occur among wild-type strains ofB. pseudomallei [23, 25, 26]. For instance, the percentage of killing of worms by variousB. 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 ofB. 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 killingC. 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 ofC. elegans killing between the mutants and wild type. Two complemented strains ofBPSL2033::Km andBURPS1710A_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.(i)
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 inB. pseudomallei. A double mutant ΔrelAΔspoT was reported to exhibit significant and severe attenuation in larva of the wax mothGalleria mellonella and C57BL/6 in mice models, which was not seen with the single mutant [20].(ii)
C. elegans possesses mechanisms to avoid or move away from pathogenic bacteria likeB. 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 onE. 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 asB. pseudomallei, especially in leaving bahaviours. Nonetheless, it is impossible to raise worms onB. 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 ofBPSL2033 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 asFrancisella tularensis [30] andLegionella 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 thatBPSL2033 forms a major facilitator superfamily fold and shares very low sequence identity (16%) to glycerol-3-phosphate transporter protein fromE. coli with known three-dimensional structure (PDB template: 1pw4A). The results imply thatBPSL2033 might exhibit new structural and/or functional characteristics and further X-ray crystallography analysis may provide valuable information. At present, we postulate thatBPSL2033 transports nutrients (probably glycerol-3-phosphate) that are essential for the replication ofB. pseudomallei. Database searches with the NCBI blastp tool identifiedBURPS1710A_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 thatBURPS1710A_1419 gene product has new or different functional characteristics.In conclusion, our results suggest thatBPSL2033 andBURPS1710A_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 inB. pseudomallei.



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*Source: 590803-2014-08-11.xml*