Research Article Molecular Characterization of Putative Virulence …downloads.hindawi.com/journals/tswj/2014/590803.pdf · 2019-07-31 · Research Article Molecular Characterization

Research ArticleMolecular Characterization of Putative Virulence Determinantsin Burkholderia pseudomallei

Suat Moi Puah1 S D Puthucheary2 Jin Town Wang34 Yi Jiun Pan3 and Kek Heng Chua1

1 Department of Biomedical Science Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia2 Department of Medical Education Research and Evaluation Duke-NUS Graduate Medical School Singapore8 College Road Singapore 169857

3Department of Microbiology National Taiwan University College of Medicine Section 1 Jen-Ai Road Taipei 10051 Taiwan4Department of Internal Medicine National Taiwan University Hospital Taipei 10051 Taiwan

Correspondence should be addressed to Kek Heng Chua khchuaumedumy

Received 24 May 2014 Accepted 9 July 2014 Published 11 August 2014

Academic Editor Adhar C Manna

Copyright copy 2014 Suat Moi Puah et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis an infectious disease whichis endemic in Southeast Asia and northern Australia This bacterium possesses many virulence factors which are thought tocontribute to its survival and pathogenicity Using a virulent clinical isolate of B pseudomallei and an attenuated strain of thesame B pseudomallei isolate 6 genes BPSL2033 BP1026B I2784 BP1026B I2780 BURPS1106A A0094 BURPS1106A 1131 andBURPS1710A 1419 were identified earlier by PCR-based subtractive hybridization These genes were extensively characterized atthe molecular level together with an additional gene BPSL3147 that had been identified by other investigators Through a reversegenetic approach single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis andwere confirmed by PCR BPSL2033Km and BURPS1710A 1419Kmmutants showed reduced rates of survival inside macrophageRAW 2647 cells and also low levels of virulence in the nematode infection model BPSL2033Km demonstrated weak statisticalsignificance (119875 = 0049) at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A 1419KmNevertheless complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studiesthus suggesting that they individually play a minor role in the virulence of B pseudomallei

1 Introduction

Burkholderia pseudomallei is a Gram-negative motile bacil-lus that is a facultative anaerobe and an environmentalsaprophyte It is readily recovered from the soil and surfacewaters in endemic areas that is Southeast Asia and NorthernAustralia [1] This bacterium possesses a remarkable capacityto infect humans and animals causing melioidosis which isan important cause of sepsis in the tropics [2] It has beenconsidered a potential bioweapon and so virulence factorsthat correspond to its pathogenesis are being intensivelystudied at an increasing rate

The availability of complete genome sequence databaseof organisms allows researchers to discover many moleculesand mechanisms that may be involved in the virulence of

B pseudomallei [3 4] Several approaches including subtrac-tive hybridization [5] comparative genomics [6] signature-tagged mutagenesis [7] transposon mutagenesis [8] in vivoexpression technology [9] microarray [10] and computa-tional methods [11 12] have accelerated the discovery ofvirulence factors over the past decades The availability ofthe genomic sequences of several B pseudomallei strains hasrapidly added candidate virulence genes to databases

In general past studies have focused on genomic dif-ferences between species that is the virulent B pseudo-mallei and a closely related but avirulent family memberB thailandensis [5 6] PCR-based subtractive hybridizationwas recently undertaken in our laboratory using a virulentclinical isolate B pseudomallei (v) and an attenuated strainof the same B pseudomallei isolate (av) [13] PCR-based

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 590803 9 pageshttpdxdoiorg1011552014590803

2 The Scientific World Journal

subtractive hybridization successfully demonstrated 6 sub-tracted DNA fragments that were unique to the virulentstrain of B pseudomallei whereas these DNA fragments werenot seen in its ldquoattenuatedrdquo strain [13] Sequencing of thesubtracted DNA fragments revealed 6 unique genes withunknown functions as follows BPSL2033 BP1026B I2784BP1026B I2780 BURPS1106A A0094 BURPS1106A 1131 andBURPS1710A 1419

Besides the 6 putative ldquovirulencerdquo determinantsBPSL3147 is another potential virulence determinant iden-tified by Cuccui et al [7] This gene encodes a putativelipoprotein and is reported to be a putative lipoproteincontaining 3916 amino acid that is identical to a VacJlipoprotein in Shigella flexneri The Tn10 mutant of S flexneriYSH6000T VacJ lipoprotein was unable to spread from cellto cell suggesting VacJ is important for intercellular spreadof the organism [14]

The aim of this study was to extensively characterizethe 6 putative virulence determinants which were absent inthe ldquoattenuatedrdquo strain (av) that is believed to have reducedvirulence that occurred after several subcultures and long-term storage in the laboratory together with an additionalcandidate BPSL3147 using the same methodology that isgene knockout approach using in vitro and in vivo assays

2 Material and Methods

21 Bacterial Strains Media and Culture Conditions Bacter-ial strains and plasmids used are listed in Table 1 Theclinical B pseudomallei strain was isolated from the bloodof a patient CMS at the University Hospital Universityof Malaya Kuala Lumpur who died from melioidosis asdescribed in previous report [11] and this strain was usedthroughout the study (henceforth referred to as Bp-CMS)The Escherichia coli strains DH10B CC118120582pir S17-1120582pir vec-tor pUT-Km and chloramphenicol acetyltransferase (CAT)cassette were obtained from Prof Dr Wang Jin-Town(National Taiwan University Taiwan) All strains were grownin Luria-Bertani (LB)mediumat 37∘Candwhen appropriateantibiotics were used at the following final concentrationsampicillin 100 120583gmL kanamycin 50 120583gmL chlorampheni-col 100 120583gmL and streptomycin 100 120583gmL The mouseleukaemic monocyte macrophage cell line RAW2647 wasobtained from American Type Culture Collection (ATCCUSA) It was cultured and maintained in flasks (CorningUSA) with DMEM (Gifco USA) supplemented with 10(vv) fetal bovine serum (Gifco USA) 4mM L-glutamineand an antibioticmixture containing 100UmLpenicillin and01mgmL streptomycin at 37∘C in 5 CO

2

22 Construction of Mutants Genes BPSL2033 BP1026BI2784 BP1026B I2780 BURPS1106A A0094 BURPS1106A1131 BURPS1710A 1419 and BPSL3147 were disrupted byhomologous 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 conju-gation and recombinant events) was amplified by polymerasechain reaction (PCR) using primer pairs as listed in Table 2

The amplicon was subcloned into pGEM-T easy vectordigested with EcoRI and subcloned into the suicide vectorpUT-Km [15] Selection of transformants by plating onto LBagar containing kanamycin and rapid screening for desiredinserts by colony PCR was performed using primers KmFand KmR The construct was then transformed into E coliS17-1120582pir and introduced into the wild-type B pseudomallei(Bp-CMS) by conjugation An insertion mutant was selectedusing LB agar supplementedwith kanamycin and site-specificchromosomal integration was verified by PCR using vector-and corresponding gene-specific primer pairs

23 Construction of Complemented Plasmids Intact genesBURPS1710A 1419 BPSL2033 BP1026B I2784 BP1026BI2780 BURPS1106A A0094 BURPS1106A 1131 andBPSL3147and their promoters were amplified by PCR and cloned into apGEM-T-CAT plasmid These complemented plasmids werethen reintroduced into the corresponding insertion mutantsby transformation The resulting complemented strains wereselected using LB agar containing chloramphenicol

24 Bacterial Growth Curve The growth of B pseudomalleistrains in LB broth was monitored over 8 h by taking 1mLof culture broth every hour to perform OD600 readings Thewild-type Bp-CMS was used as a positive control for growthin LB

25 Bacterial Replication Assays Intracellular bacterial sur-vival and replication was assayed in the mouse macrophage-like cell line RAW 2647 Cells were seeded at a density of1 times 10

5 cellswell into 24-well culture plates (Corning USA)and incubated overnight at 37∘C in 5 CO

2 An overnight

culture of B pseudomallei strain was diluted 1 100 and grownat 37∘C for 3 h with shaking to reach mid-log phase Cellmonolayers were then washed twice with PBS and incubatedin fresh DMEM without antibiotic for 1 h prior to infectionwith bacteria The bacterial suspension was added at a MOIof 100 1 and the coculture was immediately centrifuged atroom temperature at 170timesg for 5min to bring the bacteriain direct contact with the host cells After 1 h the cells werewashed twice with PBS and incubated in fresh DMEM con-taining 300 120583gmL of tetracycline to suppress the growth ofresidual extracellular bacteria Tetracycline was used insteadof gentamicin as Bp-CMS is resistant to gentamicin At 24 and 8 h after infection infected monolayers were washedtwice with PBS and lysed with 01 (vv) Triton X-100 for15min Serial dilutions of the released bacteria (expressedas colony forming units CFU) were plated on tryptic soyagar (TSA) plates to enumerate bacterial loads by directcolony counts The number of internalized bacteria obtainedat 2 h after infection represented the initial entry of bacteriawhereas at 4 and 8 h after infection represented intracellularbacterial replication Bacterial survival was normalized tocounts obtained at 2 h after infection and the relative survivalrate presented as a percentage

26 Virulence Testing with C elegans The wild-type C ele-gansN2 was obtained from Carolina Biological Supply Com-pany (USA) The nematode was propagated on nematode

The Scientific World Journal 3

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 ofinsertion mutants

Current study

BPSL2033Km BPSL2033Km derivative of CMS KanR

BPSL2033Km (pC-BPSL2033) BPSL2033Km complemented with pC-BPSL2033 plasmid KanR

CmR

BP1026B I2784Km BP1026B I2784Km derivative of CMS KanR

BP1026B I2784Km (pC-BP1026B I2784) BP1026B I2784Km complemented with pC-BP1026B I2784 plasmidKanR CmR



BURPS1106A A0094Km BURPS1106A A0094Km derivative of CMS KanR

BURPS1106A A0094Km(pC-BURPS1106A A0094)

BURPS1106A A0094 Km complemented withpC-BURPS1106A A0094 plasmid KanR CmR

BURPS1106A 1131Km BURPS 1106A 1131Km derivative of CMS KanR

BURPS1106A 1131Km(pC-BURPS1106A 1131)

BURPS 1106A 1131Km complemented with pC-BURPS1106A 1131plasmid KanR CmR

BURPS1710A 1419Km BURPS1710A 1419Km derivative of CMS KanR


BURPS1710A 1419Km complemented in trans withpC-BURPS1710A 1419 plasmid KanR CmR


BPSL3147Km (pC-BPSL3147) BPSL3147Km complemented with pC-BPSL3147 plasmid KanR CmR

E coli

DH10B F mcrA Δ(mrr-hsdRMS-mcrBC)12060180lacZ ΔM15 ΔlacX74 recA1 endA1araD 139 Δ(ara leu)7697 galU galK 120582-rpsL nupG

Prof DrJin-TownWang

CC118120582pir Δ(ara-leu) araD Δ lacX74 galE galK phoA20 thi-1 rpsE rpoB argE(Am)recA1 120582pir phage lysogen

S17-1120582pir hsdR recA pro RP4-2 (TcMu KmTn7) (120582pir)

OP50-1 A streptomycin-resistant derivative of Ecoli OP50 used as foodsource in C elegans cultivation and uninfected control

Kind giftsfrom ProfDr SheilaNathan

Plasmid

pUT-Km

pUT-Km1 derived plasmid with miniTn5 excised by EcoRI tnpexcised by SalI and bla removed by ApaLI and then with an insertionof Km resistance cassette from pUC4K into PstI site oriR6KmobRP4KanR AmpR

Chuang et al2006 [15]

pGEM-T easy Cloning vector for PCR cloning AmpR PromegaUSA

growth medium (NGM) plate and fed on the normal foodsource E coli OP50-1 which was a kind gift from Prof DrSheila Nathan (National University of Malaysia Malaysia)Killing assays were performed as previously described by Tanet 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) andE coli OP50-1 were grown overnight at 37∘C and 40 120583L ofeach culture was spread on NGMplates containing 50120583gmL5-fluorodeoxyuridine (Merck USA) to inhibit the eggs ofC elegans from hatching Plates were incubated at 37∘C


Table 2 Primers used for PCR and construction of mutants and complemented plasmids

Gene Primer name Nucleotide sequence (51015840 to 31015840) Purpose

BPSL2033

2033IF AGAACTTCGAGCAATTGCTG Internal PCR2033IR GAGAGATGACGTTCGGTCTT Internal PCRM2033F GTGAACTGGTACAAAGAAATATCG Mutant confirmationM2033R CACGTTTCTCGGATAGAGC Mutant confirmationC2033F CTAGAGCGCGGCCTCGCG Complementation of BPSL2033KmC2033R CATCACTCGGCGCAATGAGACTG Complementation of BPSL2033Km

BP1026B I2784

2784IF GTCGAGAGTACGGTGTGTTC Internal PCR2784IR CCTGCGAAATCCTTATCAC Internal PCRM2784F CTGTTTTCTAAGCGTCAGAAG Mutant confirmationM2784R AAATATGCAGGAAATAGCCCG Mutant confirmationC2784F CCTTCGCGCTGATTTGGT Complementation of BP1026B I2784KmC2784R CTACTTCGTAGCTTGATGCGCC Complementation of BP1026B I2784Km

BP1026B I2780

2780IF AAACCAGAAGGGCGATTTC Internal PCR2780IR GCGTTCTTTTAAGAATTGGGTAG Internal PCRM2780F CAGGTACGATTCATGGAACG Mutant confirmationM2780R GGTATTCCGTGACCTGAATGT Mutant confirmationC2780F AGGCTCGGAGTAGTAACACTT Complementation of BP1026B I2780KmC2780R CTACTGCCTATGCTGGGGTAT Complementation of BP1026B I2780Km

BURPS1106A A0094

94IF AGTCGGGGGGTACACCTAC Internal PCR94IR CGACACCGAGGAAAATTTC Internal PCRM94F GTGAATGTCGATCTTGCG Mutant confirmationM94R TCAATCTCCAGCGAGCTT Mutant confirmationC94F TTCTACCAGCGACTTGGC Complementation of BURPS1106A A0094KmC94R TCAATCTCCAGCGAGCTT Complementation of BURPS1106A A0094Km

BURPS1106A 1131

1131IF AAGGAGTTGGGTACGTCG Internal PCR1131R CCCTTGTGCCATTGATAG Internal PCRpUT-R TTTGAGTGACACAGGAACAC Mutant confirmationC1131F GTGGTTCAGCCAGGCACG Complementation of BURPS1106A 1131KmC1131R GTATGTGTCGTCCGCATTTG Complementation of BURPS1106A 1131Km

BURPS1710A 1419

1419IF GCGATAGCGATTGGAAAACT Internal PCR1419IR GAATCCAGACCCATTCCGT Internal PCRKmF ATGAGCCATATTCAACGGGA Mutant confirmationC1419F TGCACAAGCTGTTCAAATG Complementation of BURPS1710A 1419KmC1419R TTAAGGCTTGGGTGCAAG Complementation of BURPS1710A 1419Km

BPSL3147

3147IF GACCAGTACGCGCTCAAG Internal PCR3147IR GAACGAATACTTGTCGATCG Internal PCRM3147F GATGTACACGTTCAACGACAAG Mutant confirmationM3147R CTCTTCCGGCATCTCGTA Mutant confirmationC3147F TTCAGGGTTACGAAGCGAAG Complementation of BPSL3147KmC3147R TCAGTGCAGCCGGATGCTCG Complementation of BPSL3147Km

for 24 h and then allowed to equilibrate for 24 h at roomtemperature before coculturing with the host worms Thirtyage-matched hermaphrodites were individually transferredto freshly lawned plates by using the flattened tip of a wormpick (platinum wire) The plates were incubated at room

temperature and virulence was tracked by counting thenumber of live and dead worms every 24 h for 3 days Threeindependent experiments were carried out for each strainand each test was performed in triplicate (with a total of 270worms) A worm was considered dead on failure to respond


2033IFand

pUT-R

2033IRand

KmF

M2033Fand

M2033R

M2033F M2033R

2033IF pUT-R KmF 2033IR

(bp)

6000

2000

1000

500

L M W M W M W

1056bp

Wild type

Homologousregion

599bp 1700bp

6256bp

sim6256bp

sim1700bp

sim1056bp

sim599bpMutant

Figure 1 Representative of mutant construct Verification of the construction of BPSL2033Km mutant by PCR using specific alignmentprimers M and W represent mutant and wild-type strains respectively while L indicates 1 kb DNA ladder from Fermentas (USA)

to gentle touch by the worm pick E coli OP50-1 was usedas a negative control The resulting data was analyzed withGraphPad Prism 5 software and plotted using the Kaplan-Meier survival plot

3 Results

31 Insertion Mutant Construction and Growth Positivechromosomal integration mutants were successfully con-structed for the 7 candidate genes on the first or secondattempt and a representative of construct is shown inFigure 1 All 7 mutant strains demonstrated similar growthrates to the parental strain in liquid media over 8 h

32 Bacterial Replication and Survival Assay Overall wild-type bacteriawere able to survive and replicate inmacrophagecells over the course of the experiments In contrast the5 mutant strains BPSL2033Km BP1026B I2780KmBURPS1106A A0094Km BURPS1710A 1419Km andBPSL3147Km showed reduced intracellular survival insideRAW2647 cells at 4 and 8 h post infection but onlyBPSL2033Km reached statistical significance (119875 = 0049)at 8 h post infection Another 2 mutant strains BP1026BI2784Km and BURPS1106A 1131Km demonstrated nodifference in survival in RAW2647 cells at 4 and 8 h postinfection

Most of these plasmid-complemented strains partiallyrestored intracellular survival and replication exceptfor BP1026B I2780Km BURPS1106A A0094Km andBURPS1106A 1131Km The results suggest that 3 genesBPSL2033Km (119875 = 0049) BURPS1710A 1419Km (119875 =

0165) and BPSL3147Km (119875 = 0076) individually hadlittle effect on the intracellular survival of B pseudomallei inphagocytic cells (Figures 2(a) to 2(c))

33 Caenorhabditis elegans Killing Assay The 6 mutants(BPSL2033Km BP1026B I2784Km BP1026B I2780KmBURPS1106A 1131Km BURPS1710A 1419Km andBPSL3147Km) exhibited low levels of attenuated virulencein C elegans where survival rates of worms were only 2-foldhigher than that of the wild type (data not shown) Amongthem the most attenuation of virulence was observed inBURPS1710A 1419Km as 51 worms were able to survivefollowed by BPSL2033Km (47) compared to the wild-type parental strain (23) after 3 days (Figures 3(a) and 3(b))The complemented strains of both mutants BURPS1710A1419Km and BPSL2033Km showed at least partialrestoration of virulence thus suggesting a minor role in thenematode infection model

4 Discussion

There was no difference in the growth rates of all 7 mutantscompared to that of the wild type when assayed in richmedia(data not shown) ruling out the possibility that these genesare not affecting the growth but involved in other aspectssuch as pathogenicity The infection assay on phagocyticcells showed that without the presence of gene BPSL2033(putative transport-related membrane protein) the mutantsdemonstrated reduced ability to replicate and survive over8 h This result was supported by a plasmid-encoded com-plemented strain which demonstrated partial restoration of


160

140

120

100

80

60

40

20

0

Bact

eria

l sur

viva

l (

)

2 4 8

Time after infection (h)

100100

100

134

67

96

138

30

76

lowast

Bp-CMS Complemented strain

BPSL2033Km

(a) BPSL2033Km and complemented strain

160

140

120

100

80

60

40

20

0

Bact

eria

l sur

viva

l (

)

2 4 8


100 100100


134

87

83

138

44

84

BURPS1710A 1419Km

(b) BURPS1710A 1419Km and complemented strain

160

140

120

100

80

60

40

20

0

Bact

eria

l sur

viva

l (

)

2 4 8


100 100100

134


BPSL3147Km

82

111

138

46

82

(c) BPSL3147Km and complemented strain

Figure 2 Bacterial survival and replication within RAW2647 macrophage-like cells infected at MOI of 100 using wild-type strain andthe insertion mutants as well as their complemented strains Data represents means and standard errors of 3 separate experiments eachexperiment was carried out in 3 technical replicates for each time point Asterisk indicates significant differences (P lt 005) relative to thewild-type strain Bp-CMS at each time point

100

80

60

40

20

0

Surv

ival

()

0 24 48 72

Duration of coculture (h)

BPSL2033KmComplemented strain

Wild-type Bp-CMSE coli OP50-1

(a) BPSL2033Km and its complemented strain

100

80

60

40

20

0

Surv

ival

()

0 24 48 72


Complemented strainWild-type Bp-CMSE coli OP50-1

BURPS1710A 1419Km

(b) BURPS1710A 1419Km and its complemented strain

Figure 3 Kaplan-Meier survival curves for C elegans infected with different strains of B pseudomallei Values are the pooled data fromtriplicate of 3 separate experiments (119873 = 270) (E coli OP50-1 was the negative control)


intracellular survival when compared to wild-type Bp-CMSSimilar outcomes were seen with BURPS1710A 1419 (putativelipoprotein) and BPSL3147 (lipoprotein)

At 8 h post infection loss of gene BPSL2033 showed a5-fold reduction in survival time in RAW2647 cells whileboth BURPS1710A 1419 and BPSL3147 resulted in a 3-foldreduction in survival indicating that these genes may beinvolved in intracellular survival There was a weak but stillsignificant difference (119875 = 0049) exhibited by BPSL2033Kmcompared to Bp-CMS Thus BPSL2033 may act in concertwith other genes and play an essential role in virulenceSubtractive hybridization as reported in our earlier studydemonstrated that Bp-CMS contained 6 DNA sequencesthat were not found in the attenuated strain indicating thatmaximal virulence probably requires multiple genes actingtogether in concert [13] This hypothesis is further supportedby the present study in which B pseudomallei virulence inthe phagocytic cell line model was not critically dependenton any single putative gene tested

Several studies have suggested that a double mutant andnot a single mutant of B pseudomallei contributed signif-icantly to the growth inside murine macrophage [19 20]Future experiments involving the use of double mutants (ieBPSL2033 and BURPS1710A 1419) may prove this possibilityIn the context of BPSL3147 there may have been otherunidentified gene(s) acting together for full virulence inB pseudomallei infections It is unclear why these genesBPSL2033BURPS1710A 1419 andBPSL3147 with their corre-sponding complemented plasmids only restored intracellularsurvival and replication to approximately 50 of the wild-type level One possible explanation for this may be due tothe gene being present on multiple copies of the plasmid

C elegans has been used as a simple surrogate host formodeling bacterial diseases [21] It has been shown thaton a low nutrient nematode growth medium (NGM) Bpseudomallei killed C elegans strain N2 within 3 days andthis type of killing is referred to as ldquoslow killingrdquo [22ndash24]In our study wild-type strain Bp-CMS killed 74 of thenematode population at 72 h time point in NGM Our resultsare in agreement with published reports that differencesin killing efficiency of C elegans occur among wild-typestrains of B pseudomallei [23 25 26] For instance thepercentage of killing of worms by various B pseudomalleistrains that is ATCC23343 EY4 number 40 andKHWwasapproximately 50 60 75 and 90 respectively at 72 h timepoints [23] Virulence of B pseudomallei for the nematode islikely to be variable due to the different genetic determinantsin the strains

In the present study all the constructed mutants wereless effective in killing C elegans under slow-killing condi-tions that is twice as many worms survived when fed onmutant strains compared to worms fed onwild-type Bp-CMSafter 72 h of coculture However there was no significantdifference of C elegans killing between the mutants andwild type Two complemented strains of BPSL2033Km andBURPS1710A 1419Km achieved at least partial restoration ofvirulence at 72 h coculture thus suggesting that both genesare most probably involved in bacterial virulence

The low level attenuation of virulence in the mutantscompared to the wild type may possibly be due to thefollowing

(i) A single gene was insufficient to mediate full killingin the animal model At least 2 genes are probablyrequired to act together for achieving virulence inB pseudomallei A double mutant ΔrelAΔspoT wasreported to exhibit significant and severe attenuationin larva of the wax moth Galleria mellonella andC57BL6 inmicemodels which was not seen with thesingle mutant [20]

(ii) C elegans possesses mechanisms to avoid or moveaway from pathogenic bacteria like B pseudomalleiIt has a simple nervous system that consists of 302neurons that facilitate the identification of moleculesneurons and circuits involved in their behavior [27]It uses chemotaxis to find food on the plate andis able to discriminate food both physically basedon size and chemically based on taste and olfaction[28] Worms can modify their olfactory preferencevia the neurotransmitter serotonin to avoid odoursfrom pathogens and this learning occurs with expo-sure as short as 4 h [29] In our study C eleganswas cultivated on E coli OP50-1 that best supportsgrowth so the worms had already experienced goodfood which might have increased their exploratorybahaviour when switching to very bad food suchas B pseudomallei especially in leaving bahavioursNonetheless it is impossible to raise worms on Bpseudomallei alone because of the virulence of theorganism

BPSL2033 is a 428-amino-acid protein with a molecularmass of 46 kDa as a transport-related membrane proteinBURPS1710A 1419 is a 74-amino-acid protein with a calcu-latedmolecular mass of 8 kDa which is a putative lipoproteinFurther bioinformatics analysis suggests that amino acids 23-324 of BPSL2033 encode a domain belonging to major facil-itator superfamily (MFS) MFS transporters are ubiquitousand found in all classes of organisms and in several pathogenssuch as Francisella tularensis [30] and Legionella pneumophila[31] Chatfiled and colleagues [32] have shown that MFSprotein plays a role in virulence by promoting bacterial iron-siderophore import

The Phyre 2 model [33] predicts that BPSL2033 formsa major facilitator superfamily fold and shares very lowsequence identity (16) to glycerol-3-phosphate transporterprotein from E coli with known three-dimensional structure(PDB template 1pw4A) The results imply that BPSL2033might exhibit new structural andor functional charac-teristics and further X-ray crystallography analysis mayprovide valuable information At present we postulatethat BPSL2033 transports nutrients (probably glycerol-3-phosphate) that are essential for the replication of B pseudo-mallei Database searches with theNCBI blastp tool identifiedBURPS1710A 1419 as a putative lipoprotein within genuslevel is diverged from 37 to 82 with no conserved domainidentified as well as a lack of three-dimensional structural


information possibly suggesting that BURPS1710A 1419 geneproduct has new or different functional characteristics

In conclusion our results suggest that BPSL2033 andBURPS1710A 1419 individually are likely to contribute to aminor role in virulence and provide a basis for furthercharacterization of their role in pathogenesisWe hypothesizethat the combination effect of both genes can provide a clearvirulence role in B pseudomallei

Conflict of Interests

The authors declare that there is no conflict of interests regar-ding the publication of this paper

Acknowledgments

The authors would like to thank all members from Lab R739especially Dr Tzu-Lung Lin Dr Pei-Fang Hsieh Dr Chun-Ru Hsu and Dr Meng-Chuan Wu (Department of Micro-biology National Taiwan University College of MedicineTaipei) for their guidance in the construction of the mutantsand Prof Dr Sheila Nathan from National University ofMalaysia for the E coli OP50-1 This work was supportedby University of Malaya Research Grant (RG409-12HTM)FRGS FP037-2013A and High Impact Research MoE GrantUMC6251HIRMoEE000044-20001

References

[1] W JWiersinga B J Currie and S J Peacock ldquoMelioidosisrdquoTheNew England Journal of Medicine vol 367 no 11 pp 1035ndash10442012

[2] AC Cheng andB J Currie ldquoMelioidosis epidemiology patho-physiology and managementrdquo Clinical Microbiology Reviewsvol 18 no 2 pp 383ndash416 2005

[3] M T Holden R W Titball S J Peacock et al ldquoGenomicplasticity of the causative agent of melioidosis Burkholderiapseudomalleirdquo Proceedings of the National Academy of Sciencesof the United States of America vol 101 pp 14240ndash14245 2004

[4] A Tuanyok B R Leadem R K Auerbach et al ldquoGenomicislands from five strains of Burkholderia pseudomalleirdquo BMCGenomics vol 9 article 566 2008

[5] S L Reckseidler D DeShazer P A Sokol and D EWoods ldquoDetection of bacterial virulence genes by subtractivehybridization Identification of capsular polysaccharide of Bur-kholderia pseudomallei as a major virulence determinantrdquoInfection and Immunity vol 69 no 1 pp 34ndash44 2001

[6] Y Yu H S Kim H C Hui et al ldquoGenomic patterns ofpathogen evolution revealed by comparison of Burkholderiapseudomallei the causative agent of melioidosis to avirulentBurkholderia thailandensisrdquo BMC Microbiology vol 6 article46 2006

[7] J Cuccui A Easton K K Chu et al ldquoDevelopment of signa-ture-taggedmutagenesis in Burkholderia pseudomallei to iden-tify genes important in survival and pathogenesisrdquo Infection andImmunity vol 75 no 3 pp 1186ndash1195 2007

[8] D A Rholl L A Trunck and H P Schweizer ldquoIn vivo Himar1transposon mutagenesis of Burkholderia pseudomalleirdquo Appliedand Environmental Microbiology vol 74 no 24 pp 7529ndash75352008

[9] G Shalom J G Shaw and M S Thomas ldquoIn vivo expressiontechnology identifies a type VI secretion system locus inBurkholderia pseudomallei that is induced upon invasion ofmacrophagesrdquoMicrobiology vol 153 no 8 pp 2689ndash2699 2007

[10] S Chieng L Carreto and S Nathan ldquoBurkholderia pseudoma-llei transcriptional adaptation inmacrophagesrdquoBMCGenomicsvol 13 article 328 2012

[11] S H Yoon C-G Hur H-Y Kang Y H Kim T K Oh and J FKim ldquoA computational approach for identifying pathogenicityislands in prokaryotic genomesrdquo BMC Bioinformatics vol 6article 184 2005

[12] L Zheng Y Li J Ding et al ldquoA comparison of computationalmethods for identifying virulence factorsrdquo PLoS ONE vol 7 no8 Article ID e42517 2012

[13] S D Puthucheary S M Puah H C Chai K L Thong andK H Chua ldquoMolecular investigation of virulence determinantsbetween a virulent clinical strain and an attenuated strain ofBurkholderia pseudomalleirdquo Journal of Molecular Microbiologyand Biotechnology vol 22 no 3 pp 198ndash204 2012

[14] T Suzuki T Murai I Fukuda T Tobe M Yoshikawa and CSasakawa ldquoIdentification and characterization of a chromoso-mal virulence gene vacJ required for intercellular spreading ofShigella flexnerirdquo Molecular Microbiology vol 11 no 1 pp 31ndash41 1994

[15] Y P Chuang C T Fang S Y Lai S C Chaing and J T WangldquoGenetic determinants of capsular serotype K1 of Klebsiellapneumoniae causing primary pyogenic liver abscessrdquo Journal ofInfectious Diseases vol 193 no 5 pp 645ndash654 2006

[16] T-L Lin C-Z Lee P-FHsieh S-F Tsai and J-TWang ldquoChar-acterization of integrative and conjugative element ICEKp1-associated genomic heterogeneity in a Klebsiella pneumoniaestrain isolated from a primary liver abscessrdquo Journal of Bacte-riology vol 190 no 2 pp 515ndash526 2008

[17] M W Tan S Mahajan-Miklos and F M Ausubel ldquoKillingof Caenorhabditis elegans by Pseudomonas aeruginosa used tomodel mammalian bacterial pathogenesisrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 2 pp 715ndash720 1999

[18] T Stiernagle Maintenance of C elegans Oxford UniversityPress New York NY USA 1999

[19] R Balder S Lipski J J Lazarus et al ldquoIdentification ofBurkholderiamallei andBurkholderia pseudomallei adhesins forhuman respiratory epithelial cellsrdquo BMC Microbiology vol 10article 250 2010

[20] C MMuller L Conejero N Spink M EWand G J Bancroftand R W Titball ldquoRole of RelA and SpoT in Burkholderiapseudomallei virulence and immunityrdquo Infection and Immunityvol 80 no 9 pp 3247ndash3255 2012

[21] M J Gravato-Nobre and J Hodgkin ldquoCaenorhabditis elegans asa model for innate immunity to pathogensrdquo Cellular Microbiol-ogy vol 7 no 6 pp 741ndash751 2005

[22] K L Chua Y Y Chan and Y H Gan ldquoFlagella are viru-lence determinants of Burkholderia pseudomalleirdquo Infection andImmunity vol 71 no 4 pp 1622ndash1629 2003

[23] Y-H Gan K L Chua H H Chua et al ldquoCharacterization ofBurkholderia pseudomallei infection and identification of novelvirulence factors using a Caenorhabditis elegans host systemrdquoMolecular Microbiology vol 44 no 5 pp 1185ndash1197 2002

[24] Y Song C Xie Y M Ong Y H Gan and K L Chua ldquoTheBpsIR quorum-sensing system of Burkholderia pseudomalleirdquoJournal of Bacteriology vol 187 no 2 pp 785ndash790 2005


[25] S H Lee S K Ooi N M Mahadi M W Tan and S NathanldquoComplete killing of Caenorhabditis elegans by Burkholderiapseudomallei is dependent on prolonged direct association withthe viable pathogenrdquo PLoS ONE vol 6 no 3 Article ID e167072011

[26] A LOrsquoQuinn EMWiegand and J A Jeddeloh ldquoBurkholderiapseudomallei kills the nematodeCaenorhabditis elegansusing anendotoxin-mediated paralysisrdquoCellularMicrobiology vol 3 no6 pp 381ndash393 2001

[27] J G White E Southgate J N Thomson and S Brenner ldquoThestructure of the nervous system of the nematodeCaenorhabditiselegansrdquo Philosophical Transactions of the Royal Society BBiological Sciences vol 314 no 1165 pp 1ndash340 1986

[28] Y Kiyama K Miyahara and Y Ohshima ldquoActive uptake ofartificial particles in the nematode Caenorhabditis elegansrdquoTheJournal of Experimental Biology vol 215 no 7 pp 1178ndash11832012

[29] Y Zhang H Lu and C I Bargmann ldquoPathogenic bacteriainduce aversive olfactory learning in Caenorhabditis elegansrdquoNature vol 438 no 7065 pp 179ndash184 2005

[30] M E Marohn A E Santiago K A Shirey M Lipsky S NVogel and E M Barry ldquoMembers of the Francisella tularensisphagosomal transporter subfamily of major facilitator super-family transporters are critical for pathogenesisrdquo Infection andImmunity vol 80 no 7 pp 2390ndash2401 2012

[31] J Sauer M A Bachman and M S Swanson ldquoThe phagosomaltransporter A couples threonine acquisition to differentiationand replication of Legionella pneumophila in macrophagesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 28 pp 9924ndash9929 2005

[32] C H Chatfield B J Mulhern D M Burnside and N P Cian-ciotto ldquoLegionella pneumophila LbtU acts as a novel TonB-independent receptor for the legiobactin siderophorerdquo Journalof Bacteriology vol 193 no 7 pp 1563ndash1575 2011

[33] L A Kelley andM J E Sternberg ldquoProtein structure predictionon the Web a case study using the Phyre serverrdquo Natureprotocols vol 4 no 3 pp 363ndash371 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


subtractive hybridization successfully demonstrated 6 sub-tracted DNA fragments that were unique to the virulentstrain of B pseudomallei whereas these DNA fragments werenot seen in its ldquoattenuatedrdquo strain [13] Sequencing of thesubtracted DNA fragments revealed 6 unique genes withunknown functions as follows BPSL2033 BP1026B I2784BP1026B I2780 BURPS1106A A0094 BURPS1106A 1131 andBURPS1710A 1419

Besides the 6 putative ldquovirulencerdquo determinantsBPSL3147 is another potential virulence determinant iden-tified by Cuccui et al [7] This gene encodes a putativelipoprotein and is reported to be a putative lipoproteincontaining 3916 amino acid that is identical to a VacJlipoprotein in Shigella flexneri The Tn10 mutant of S flexneriYSH6000T VacJ lipoprotein was unable to spread from cellto cell suggesting VacJ is important for intercellular spreadof the organism [14]

The aim of this study was to extensively characterizethe 6 putative virulence determinants which were absent inthe ldquoattenuatedrdquo strain (av) that is believed to have reducedvirulence that occurred after several subcultures and long-term storage in the laboratory together with an additionalcandidate BPSL3147 using the same methodology that isgene knockout approach using in vitro and in vivo assays

2 Material and Methods

21 Bacterial Strains Media and Culture Conditions Bacter-ial strains and plasmids used are listed in Table 1 Theclinical B pseudomallei strain was isolated from the bloodof a patient CMS at the University Hospital Universityof Malaya Kuala Lumpur who died from melioidosis asdescribed in previous report [11] and this strain was usedthroughout the study (henceforth referred to as Bp-CMS)The Escherichia coli strains DH10B CC118120582pir S17-1120582pir vec-tor pUT-Km and chloramphenicol acetyltransferase (CAT)cassette were obtained from Prof Dr Wang Jin-Town(National Taiwan University Taiwan) All strains were grownin Luria-Bertani (LB)mediumat 37∘Candwhen appropriateantibiotics were used at the following final concentrationsampicillin 100 120583gmL kanamycin 50 120583gmL chlorampheni-col 100 120583gmL and streptomycin 100 120583gmL The mouseleukaemic monocyte macrophage cell line RAW2647 wasobtained from American Type Culture Collection (ATCCUSA) It was cultured and maintained in flasks (CorningUSA) with DMEM (Gifco USA) supplemented with 10(vv) fetal bovine serum (Gifco USA) 4mM L-glutamineand an antibioticmixture containing 100UmLpenicillin and01mgmL streptomycin at 37∘C in 5 CO

2

22 Construction of Mutants Genes BPSL2033 BP1026BI2784 BP1026B I2780 BURPS1106A A0094 BURPS1106A1131 BURPS1710A 1419 and BPSL3147 were disrupted byhomologous 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 conju-gation and recombinant events) was amplified by polymerasechain reaction (PCR) using primer pairs as listed in Table 2

The amplicon was subcloned into pGEM-T easy vectordigested with EcoRI and subcloned into the suicide vectorpUT-Km [15] Selection of transformants by plating onto LBagar containing kanamycin and rapid screening for desiredinserts by colony PCR was performed using primers KmFand KmR The construct was then transformed into E coliS17-1120582pir and introduced into the wild-type B pseudomallei(Bp-CMS) by conjugation An insertion mutant was selectedusing LB agar supplementedwith kanamycin and site-specificchromosomal integration was verified by PCR using vector-and corresponding gene-specific primer pairs

23 Construction of Complemented Plasmids Intact genesBURPS1710A 1419 BPSL2033 BP1026B I2784 BP1026BI2780 BURPS1106A A0094 BURPS1106A 1131 andBPSL3147and their promoters were amplified by PCR and cloned into apGEM-T-CAT plasmid These complemented plasmids werethen reintroduced into the corresponding insertion mutantsby transformation The resulting complemented strains wereselected using LB agar containing chloramphenicol

24 Bacterial Growth Curve The growth of B pseudomalleistrains in LB broth was monitored over 8 h by taking 1mLof culture broth every hour to perform OD600 readings Thewild-type Bp-CMS was used as a positive control for growthin LB

25 Bacterial Replication Assays Intracellular bacterial sur-vival and replication was assayed in the mouse macrophage-like cell line RAW 2647 Cells were seeded at a density of1 times 10

5 cellswell into 24-well culture plates (Corning USA)and incubated overnight at 37∘C in 5 CO

2 An overnight

culture of B pseudomallei strain was diluted 1 100 and grownat 37∘C for 3 h with shaking to reach mid-log phase Cellmonolayers were then washed twice with PBS and incubatedin fresh DMEM without antibiotic for 1 h prior to infectionwith bacteria The bacterial suspension was added at a MOIof 100 1 and the coculture was immediately centrifuged atroom temperature at 170timesg for 5min to bring the bacteriain direct contact with the host cells After 1 h the cells werewashed twice with PBS and incubated in fresh DMEM con-taining 300 120583gmL of tetracycline to suppress the growth ofresidual extracellular bacteria Tetracycline was used insteadof gentamicin as Bp-CMS is resistant to gentamicin At 24 and 8 h after infection infected monolayers were washedtwice with PBS and lysed with 01 (vv) Triton X-100 for15min Serial dilutions of the released bacteria (expressedas colony forming units CFU) were plated on tryptic soyagar (TSA) plates to enumerate bacterial loads by directcolony counts The number of internalized bacteria obtainedat 2 h after infection represented the initial entry of bacteriawhereas at 4 and 8 h after infection represented intracellularbacterial replication Bacterial survival was normalized tocounts obtained at 2 h after infection and the relative survivalrate presented as a percentage

26 Virulence Testing with C elegans The wild-type C ele-gansN2 was obtained from Carolina Biological Supply Com-pany (USA) The nematode was propagated on nematode




Strains

B pseudomallei


Current study



CmR
















E coli


Prof DrJin-TownWang





Plasmid

pUT-Km









BPSL2033


BP1026B I2784


BP1026B I2780


BURPS1106A A0094


BURPS1106A 1131


BURPS1710A 1419


BPSL3147





2033IFand

pUT-R

2033IRand

KmF

M2033Fand

M2033R

M2033F M2033R


(bp)

6000

2000

1000

500

L M W M W M W

1056bp

Wild type

Homologousregion

599bp 1700bp

6256bp

sim6256bp

sim1700bp

sim1056bp

sim599bpMutant



3 Results






4 Discussion



160

140

120

100

80

60

40

20

0

Bact

eria

l sur

viva

l (

)

2 4 8


100100

100

134

67

96

138

30

76

lowast


BPSL2033Km


160

140

120

100

80

60

40

20

0

Bact

eria

l sur

viva

l (

)

2 4 8


100 100100


134

87

83

138

44

84

BURPS1710A 1419Km


160

140

120

100

80

60

40

20

0

Bact

eria

l sur

viva

l (

)

2 4 8


100 100100

134


BPSL3147Km

82

111

138

46

82



100

80

60

40

20

0

Surv

ival

()

0 24 48 72





100

80

60

40

20

0

Surv

ival

()

0 24 48 72



BURPS1710A 1419Km



















Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Strains

B pseudomallei


Current study



CmR
















E coli


Prof DrJin-TownWang





Plasmid

pUT-Km









BPSL2033


BP1026B I2784


BP1026B I2780


BURPS1106A A0094


BURPS1106A 1131


BURPS1710A 1419


BPSL3147





2033IFand

pUT-R

2033IRand

KmF

M2033Fand

M2033R

M2033F M2033R


(bp)

6000

2000

1000

500

L M W M W M W

1056bp

Wild type

Homologousregion

599bp 1700bp

6256bp

sim6256bp

sim1700bp

sim1056bp

sim599bpMutant



3 Results






4 Discussion



160

140

120

100

80

60

40

20

0

Bact

eria

l sur

viva

l (

)

2 4 8


100100

100

134

67

96

138

30

76

lowast


BPSL2033Km


160

140

120

100

80

60

40

20

0

Bact

eria

l sur

viva

l (

)

2 4 8


100 100100


134

87

83

138

44

84

BURPS1710A 1419Km


160

140

120

100

80

60

40

20

0

Bact

eria

l sur

viva

l (

)

2 4 8


100 100100

134


BPSL3147Km

82

111

138

46

82



100

80

60

40

20

0

Surv

ival

()

0 24 48 72





100

80

60

40

20

0

Surv

ival

()

0 24 48 72



BURPS1710A 1419Km



















Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




BPSL2033


BP1026B I2784


BP1026B I2780


BURPS1106A A0094


BURPS1106A 1131


BURPS1710A 1419


BPSL3147





2033IFand

pUT-R

2033IRand

KmF

M2033Fand

M2033R

M2033F M2033R


(bp)

6000

2000

1000

500

L M W M W M W

1056bp

Wild type

Homologousregion

599bp 1700bp

6256bp

sim6256bp

sim1700bp

sim1056bp

sim599bpMutant



3 Results






4 Discussion



160

140

120

100

80

60

40

20

0

Bact

eria

l sur

viva

l (

)

2 4 8


100100

100

134

67

96

138

30

76

lowast


BPSL2033Km


160

140

120

100

80

60

40

20

0

Bact

eria

l sur

viva

l (

)

2 4 8


100 100100


134

87

83

138

44

84

BURPS1710A 1419Km


160

140

120

100

80

60

40

20

0

Bact

eria

l sur

viva

l (

)

2 4 8


100 100100

134


BPSL3147Km

82

111

138

46

82



100

80

60

40

20

0

Surv

ival

()

0 24 48 72





100

80

60

40

20

0

Surv

ival

()

0 24 48 72



BURPS1710A 1419Km



















Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


2033IFand

pUT-R

2033IRand

KmF

M2033Fand

M2033R

M2033F M2033R


(bp)

6000

2000

1000

500

L M W M W M W

1056bp

Wild type

Homologousregion

599bp 1700bp

6256bp

sim6256bp

sim1700bp

sim1056bp

sim599bpMutant



3 Results






4 Discussion



160

140

120

100

80

60

40

20

0

Bact

eria

l sur

viva

l (

)

2 4 8


100100

100

134

67

96

138

30

76

lowast


BPSL2033Km


160

140

120

100

80

60

40

20

0

Bact

eria

l sur

viva

l (

)

2 4 8


100 100100


134

87

83

138

44

84

BURPS1710A 1419Km


160

140

120

100

80

60

40

20

0

Bact

eria

l sur

viva

l (

)

2 4 8


100 100100

134


BPSL3147Km

82

111

138

46

82



100

80

60

40

20

0

Surv

ival

()

0 24 48 72





100

80

60

40

20

0

Surv

ival

()

0 24 48 72



BURPS1710A 1419Km



















Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


160

140

120

100

80

60

40

20

0

Bact

eria

l sur

viva

l (

)

2 4 8


100100

100

134

67

96

138

30

76

lowast


BPSL2033Km


160

140

120

100

80

60

40

20

0

Bact

eria

l sur

viva

l (

)

2 4 8


100 100100


134

87

83

138

44

84

BURPS1710A 1419Km


160

140

120

100

80

60

40

20

0

Bact

eria

l sur

viva

l (

)

2 4 8


100 100100

134


BPSL3147Km

82

111

138

46

82



100

80

60

40

20

0

Surv

ival

()

0 24 48 72





100

80

60

40

20

0

Surv

ival

()

0 24 48 72



BURPS1710A 1419Km



















Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Molecular Characterization of Putative Virulence …downloads.hindawi.com/journals/tswj/2014/590803.pdf · 2019-07-31 · Research Article Molecular Characterization