A Burkholderia cepacia complex non-ribosomal peptide-synthesized toxin is hemolytic and required for full virulence.

2012·1.3 MB·English

by  Thomson, Euan L.S.

Virulence3:3,286–298;May/June2012;G2012LandesBioscience
A Burkholderia cepacia complex non-ribosomal
peptide-synthesized toxin is hemolytic and
required for full virulence
Euan L.S. Thomson and Jonathan J. Dennis*
DepartmentofBiologicalSciences;UniversityofAlberta;Edmonton,ABCanada
Keywords: Burkholderia, Burkholderia cepacia, hemolysin, bacterial pathogenesis, mutagenesis, non-ribosomal peptide synthesis,
.
bacterial toxin e
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MembersoftheBurkholderiacepaciacomplex(Bcc)haverecentlygainednotorietyassignificantbacterialpathogensdue ri
totheirextremelevelsofantibioticresistance,theirtransmissibilityinclinics,theirpersistenceinbacteriostaticsolutions, t
s
andtheirintracellularsurvivalcapabilities.Aspathogens,theBccareknowntoelaborateanumberofvirulencefactors i
d
including proteases, lipases and other exoproducts, as well as a number of secretion system associated effectors.

Throughrandomanddirectedmutagenesisstudies,wehaveidentifiedaBccgeneclustercapableofexpressingatoxin t
o
that is both hemolytic and required for full Bcc virulence. The Bcc toxin is synthesized via a non-ribosomal peptide
n
synthetase mechanism, and appears to be related to the previously identified antifungal compound burkholdine or
occidiofungin.Furthertestingshowsmutationstothisgeneclustercauseasignificantreductioninbothhemolysisand o
Galleria mellonella mortality. Mutation to a glycosyltransferase gene putatively responsible for a structural-functional D
toxin variant causes only partial reduction in hemolysis. Molecular screening identifies the Bcc species containing this

genecluster, ofwhich severalstrains produce hemolytic activity. .
e
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Introduction that only approximately 4% of Bcc isolates exhibit β-hemolytic
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activity when sheep erythrocytes were tested.23 However, later c
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Members of the Burkholderia cepacia complex (Bcc) are Gram- studies showed that up to 39% of clinical Bcc isolates possessed
o
negative bacilli ubiquitous in soil and water sources. These hemolyticactivitywhenusingsheeperythrocytes.24Onesourceof
i
bacteria embody a diverse array of metabolic capabilities, among this hemolytic activity may arise from a β-hemolysin,24 although B
them environmental contaminant degradation,1,2 fungal phyto- unlike Pseudomonas aeruginosa, Bcc phospholipase activity does
s
pathogensuppression3-7andnitrogenfixation.8Forthesereasons, notcorrelatewiththepresenceofthishemolyticactivity.In1994, e
the Bcc has been identified as a powerful class of bioremediation the isolation and purification of two related hemolysins with d
and plant growth promoting agents.9 However, the potential antifungal properties was published by Abe and Nakazawa.3 The n
usefulness of the Bcc has been compromised by their oppor- hemolytic activity of these “cepalysins” was inhibited by sterols, a
tunistic pathogenicity and concerns surrounding their extensive suggestingthattheyrequireaninteractionwithcholesterolinthe L
antibiotic and biocide resistance.10-12 For immune-compromised erythrocyte membrane to produce a biological effect. Hutchison
2
patients, such as those with the heritable genetic disease cystic etal.25showedthatalipopeptidetoxinproducedbyBurkholderia 1
fibrosis (CF), the Bcc represent a singular threat because of cenocepacia could cause hemolysis of erythrocytes, apoptosis of 0
their adept transmissibility in clinical settings,13,14 their extremely human neutrophils, and an increase in neutrophil degranulation. 2
high antibiotic resistance,15 their persistence in disinfectant Fehlner-Gardineretal.26identifiedcomponentsoftheBccgeneral ©
solutions16-18 and their ability to survive in vivo.19-22 Upon secretorypathwaythatwereinvolvedinthesecretionofhemolytic
gainingaccesstothelung,membersoftheBccproduceanumber and phospholipase C activities, but that were not necessary for
ofvirulencefactorstoxictothehostandinstigateimmunesystem Bcc intracellular survival within Acanthamoeba polyphaga. More
stimulation that ultimately severely reduces lung function. As recently, Bevivino et al.,27 using ram erythrocytes, found that
well, the Bcc occasionally create what has been termed “cepacia almostallBurkholderiaambifariaisolateswerehemolyticandthat
syndrome,” a disease characterized by an acute and severe the percentage of hemolytic environmental B. cenocepacia isolates
necrotizing pneumonia, sepsis and death.9 was markedly higher than the percentage of hemolytic clinical
Toxic factors produced by the Bcc include proteases, lipases isolates.Similarly,Carvalhoetal.28indicatedthatalmostallofthe
and type III secretion system effectors.9 Early reports suggested 59 Bcc clinical isolates from a reference CF center in Rio de
*Correspondenceto:JonathanJ.Dennis;Email:[email protected]
Submitted:09/02/11;Revised:01/11/12;Accepted:01/13/12
http://dx.doi.org/10.4161/viru.19355
286 Virulence Volume3Issue3
RESEARCHPAPER
Janeiro, Brazil produced several different exoproducts except for geneclustercontainingNRPS-encodinggenes,aswellasregulator
hemolysin (as tested against human erythrocytes). From these and transporter-encoding genes. Further insertion mutation
results, it is evident that some Bcc members produce hemolytic analysis identified genes essential to the hemolytic activity, and
toxins,althoughtheirimportancetowardpathogenicityisunclear. alloweddelineationoftheendsofthegenecluster.DNAsequence
Severalsecondarymetabolitecompoundswithbiologicaleffects analysis of the B. vietnamiensis DBO1 gene cluster demonstrated
arealsoproducedbytheBcc,eitherthroughpolyketidesynthetase 99.9%identitytoanearlyidenticalgeneclusterinthesequenced
(PKS) or non-ribosomal peptide synthetase (NRPS) enzyme genome of B. ambifaria strain AMMD. As shown in Figure1,
complexes. Known secondary metabolite toxins produced by the and with functional activity for each gene shown in Table 1, the
Bccincludethebroad-spectrumantifungalagent“pyrrolnitrin,”29 core Bcc NRPS gene cluster is approximately 50.5 kb in length
a group of antifungal and antitumor agents deemed “cepafun- andiscomprisedof13ORFsarrangedidenticallyinbothstrains.
gins,”30 hemolytic peptides named “cepalysins,”3 related anti- In B. ambifaria AMMD, the NRPS cluster of open reading
fungal compounds “cepacidine A1 and A2”7,31 and the recently frames (ORFs) is located on chromosome 3 and extends from .
e
described antifungal cyclic peptide “occidiofungin” or “burk- basepair 1,101,558 to 1,152,073 for a total of 50,515 base pairs.
t
holdine,”32-34 which may be similar to molecules previously BLASTN analysis39 identified only one other closely related gene u
isolated from Bcc strains as “xylocandins.”35,36 While these latter cluster in the GenBank database, a gene cluster of Bcc member b
compounds show a broad range of activity against fungi, in this B. contaminans strain MS14 encoding occidofungin biosynthesis ri
t
manuscript, we identify through random and directed muta- proteins.Notonlyarethese13genesinMS14similarlyarranged s
genesis a gene cluster in Burkholderia vietnamiensis strain (syntenic) to the NRPS genes in AMMD/DBO1, each ORF in i
d
DBO137 that is homologous to the gene cluster that synthesizes the AMMD/DBO1 gene cluster has as its most closely related

occidofungin/burkholdine, and demonstrate that this gene pro- database entry an ORF in the MS14 occidofungin biosynthetic ot
duct is also strikingly β-hemolytic. This study also examines the cluster. For example, the B. ambifaria AMMD 3176 amino acid n
prevalence of this gene cluster within the Bcc and the lethality of adenylation protein Bamb_4672 is 89% identical (2812/3176)
o
this compound in an invertebrate infection model. and93%similar(2944/3176)tothesameproteininBurkholderia
D
contaminansMS14,alongtheentiresequence(12/3176gaps).For
Results comparison, the next closest database relative is an uncharac- .
e
terized protein identified in Acidobacterium capsulatum ATCC
c
Identification of an NRPS cluster through plasposon muta- 51196, that is only 52% identical (1629/3138) and 68% similar
n
genesis.ThehemolyticpatternsoftheBccspeciesB.vietnamiensis (2119/3138)toBamb_4672across3138/3176aminoacids,with
e
DBO1 and B. ambifaria AMMD, which form β-hemolysis 72/3138 gaps. This analysis indicates that the gene clusters
i
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(yellow clearing) on sheep blood agar, were investigated by identified to be responsible for the hemolytic activity observed
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randomly mutagenizingDBO1with plasposonpTnMod-OTp'.38 in strain DBO1 (and by homology AMMD), are closely related
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Mutantswerescreenedonsheepbloodagar+100mg/mLTp,and to the occidofungin biosynthesis gene cluster identified in
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those deficient in hemolysis were carried forward for analysis, B. contaminans MS14, and to few other genes in the known
which revealed plasposon insertion sites in an uncharacterized bacterial genetic complement. s
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Figure1.TheNRPSclustergivingrisetotoxicactivityinB.vietnamiensisDBO1.TheclusterappearssimilartothatdescribedinB.contaminansMS14
byGuetal.32exceptthatanadditionaltwogenes,homologsof6477and6478inB.ambifariaAMMD,arepresentupstreamofthedescribedMS14cluster
thatarerequiredforvirulenceinDBO1.Filledverticalarrowsindicategenesdisruptedbyplasposonmutagenesis;unfilledverticalarrowsindicategenes
disruptedthroughtargetedmutagenesis.GenesarenamedaccordingtothesequencedB.ambifariaAMMDgenome:6465,encodinganFAD-linked
dioxygenasehomolog;6466,encodingaLuxRhomolog;6467,encodingahypotheticalproteinwithaDNAbindingmotif;6468,encodingaLuxR
homolog;6469,encodingacyclicpeptidetransporter;6470,encodingahypotheticalprotein;6471,encodingaglycosyltransferase;6472,encoding
anadenylationdomain-containingprotein(ADCP);6473,encodinganADCP;6474,encodinganADCP;6475,encodingab-lactamase;6476,encoding
anADCP-polyketidesynthetase(PKS)hybrid;6477,encodingaPKS;6478,encodingaPKS;6479,encodingataurinedioxygenase.Onlygenes6466
through6478appeartobeinvolvedintoxinproduction.
www.landesbioscience.com Virulence 287
Table1.Bacterialstrainsandplasmidsconstructedforthisstudy
Bacterialstrains Genotypeorrelevantphenotype Hemolytic Source
activity
B.vietnamiensis
DBO1 Parentstrain + 37
6465::TpR DBO1withtargetedoriTp’insertioningene6465,encodinganFAD-linkeddioxygenase + Thisstudy
6466::TpR DBO1withtargetedoriTp’insertioningene6466,encodingaputativeLuxRregulator 2 Thisstudy
6468::TpR DBO1withoriTp’plasposoninsertioningene6468,encodingaputativeLuxRregulator 2 Thisstudy
6469::TpR DBO1withoriTp’plasposoninsertioningene6469,encodingaputativepeptidetransporter 2 Thisstudy
6470::TpR DBO1withtargetedoriTp’insertioningene6470,encodingahypotheticalprotein + Thisstudy
6471::TpR DBO1withtargetedoriTp’insertioningene6471,encodingaputativeglycosyltransferase +/2 Thisstudy e.
withintheNRPSgenecluster t
u
6472::TpR DBO1withoriTp’plasposoninsertioningene6472,encodingaputativeadenylation 2 Thisstudy
b
domain-containingprotein
i
6473::TpR DBO1withoriTp’plasposoninsertioningene6473,encodingaputativeadenylation 2 Thisstudy r
t
domain-containingprotein s
6474::TpR DBO1withoriTp’plasposoninsertioningene6474,encodingaputativeadenylation 2 Thisstudy di
domain-containingprotein
t
6476::TpR DBO1withoriTp’plasposoninsertioningene6476,encodingaputativeadenylation 2 Thisstudy o
domain-containingproteinandpolyketidesynethetase n
6477::TpR DBO1withtargetedoriTp’insertioningene6477,encodingaputativepolyketidesynthetase 2 Thisstudy o
6478::TpR DBO1withoriTp’plasposoninsertioningene6478,encodingaputativepolyketidesynthetase 2 Thisstudy D
withintheNRPSgenecluster

6479::TpR DBO1withtargetedoriTp’insertioningene6479,encodingaputativetaurinemetabolismprotein + Thisstudy e.
DBO1/pSCRhaTc DBO1carryingpSCRhaTc + c
n
6466::TpR/pSCRhaTc DBO1mutant6466::TpRcarryingpSCRhaTc 2
e
6466::TpR/p6466 6466::TpRcarryingpSCRha-6466His + Thisstudy
i
c
E.coli
s
DH5a Cloninghoststrain NA Invitrogen o
i
Plasmids B
pJET1.2/blunt Cloningvector,AmpR NA Fermentas
s
pTnMod-OTp’ Plasposonusedforrandommutagenesis,TpR NA 38 e
pSCRha-6466His Rhamnose-inducibleplasmidpSCRhaB2modifiedwithTcresistancecassetteand NA Thisstudy d
6xhistidine-taggedgene6466forgeneticcomplementation n
pSCRhaTc pSCRha-6466Hiscarryingno6466Hisinsert NA 43 a
L
+,fullclearing;+/2,partialclearing;2,noclearing;NA,notapplicable.

2
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Uniquely structured toxin is a broad-specificity hemolysin. a panel of Gram-negative and Gram-positive bacterial species 0
Bcc strains DBO1 and AMMD were grown on blood agar with including various pseudomonads, Escherichia coli, Staphylococcus 2
hemocytes from various sources, including bovine, rabbit, sheep, and Bacillus species, or 25 different Bcc strains. In both condi- ©
horseandhuman,todeterminewhetherhemolysiswaslimitedto tions,theBcchemolyticactivitywasnotinhibitorytothegrowth
certain mammals. Both strains exhibited hemolytic activity on of any other bacteria tested, even though hemolytic activity was
all types of blood agar (data not shown). Clearing on sheep observable in the underlying blood agar.
blood agar produced large yellow zones of lysis, indicative of a To quantify the effect of different NRPS mutations on
β-hemolysis (Fig.2), while cleared zones on other blood types hemolyticactivity,wedevelopedahigh-throughputliquidhuman
appeared colorless. Mutants in DBO1 defective in producing blood hemolysis assay using hemoglobin release (absorbance at
zones of clearing on one type of blood agar were defective in wavelength 570 nm) as a marker. As shown in Figure3, we first
producing zones of clearing on the other types of blood agar. In tested this assay against wild-type strain DBO1, and two con-
order to determine whether the diffusible hemolytic activity structed mutants including 6466::TpR, a putative luxR homolog
observed was active against bacterial cells, Bcc strains producing mutant and 6477::TpR, a putative polyketide synthetase mutant,
hemolytic activity were streaked on sheep blood plates and cross- neither of which produce zones of clearing on sheep blood agar.
streaked(eitherconcurrentlyorafteroneday’sgrowth)witheither Although hemocytes were inoculated with similar numbers of
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Figure2.BcchemolyticactivityonTSA5%sheepbloodagar.B.vietnamiensisstrainDBO1andB.ambifariastrainAMMD(“V”streaked)producelarge o
transparentyellowhemolyticzonesofclearingonaninvertedsheepbloodplatefollowingovernightgrowthat30°C.B.cenocepaciastrainsC4455and D
C6433andB.multivoransstrainC3430producenosuchhemolyticzonesofclearingandarebarelyvisiblethroughtheopaquegrowthmedia.

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bacterial cells (Fig.3B), only wild-type strain DBO1 was able to activity, the absence of NRPS product glycosylation in DBO1
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release substantial amounts of hemoglobin (Fig.3A) or reduce reduces but does not eliminate the hemolytic activity of the
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the counted number of red blood cells (Fig.3C). NRPS-derived compound in B. vietnamiensis DBO1. Finally,
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As described previously,33,34 the NRPS product occidiofungin/ there is a complete abrogation of hemolysis in strains containing
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burkholdine is a cyclic lipopeptide comprising eight amino acids. mutations to genes 6466 (encoding a LuxR regulator), 6469
o
Atleasttwoformsofthepeptideexist.Oneformcontainsaxylose (encoding a cyclic peptide transporter), 6472 [encoding an
i
B
side chain (occidiofungin/burkholdine 1229), which is presum- adenylation domain-containing protein (ADCP)], 6473 (encod-
ably added to the peptide by the predicted glycosyltransferase ing an ADCP), 6474 (encoding an ADCP), 6476 [encoding an s
encoded within the NRPS gene cluster. The other form contains ADCP-polyketide synthetase (PKS)], 6477 (encoding a PKS), e
no xylose side chain (burkholdine 1097). While the xylose- and 6478 (encoding a PKS). To prove that the DBO1 genes d
containing peptide was shown to have antifungal activity, the identified by mutagenesis were indeed responsible for the defect n
peptide lacking xylose displayed little activity.32,33 To determine in hemolytic activity observed, genetic complementation was a
whether we would observe differences in hemolytic activity achieved for cluster gene 6466 (Fig.4). When gene 6466 is L

produced by the different B. vietnamiensis DBO1 NRPS cluster cloned behind a rhamnose inducible promoter in plasmid 2
mutants, including the glycosyltransferase mutant 6471::TpR, pSCRhaTc-6466His (forming plasmid p66), and introduced into 1
DBO1 mutants were incubated with human erythrocytes for a DBO1mutant6466::TpR,theadditionof0.2%rhamnoseresults 0
period of two days, and the absorbance at 570 nm of the in full restoration of hemolytic activity as measured by hemo- 2
supernatants was taken each day. As shown in Figure4, there globin release, whereas 6466::TpR/p66 without added rhamnose ©
are three potential hemolytic phenotypes demonstrated by the remains non-hemolytic.
strains.First,thereisafullyhemolyticphenotypecharacteristicof Galleria mellonella infection model. B. vietnamiensis DBO1
both wild-type DBO1 and strains containing mutations to genes strains (wild-type, 6466::TpR and 6466::TpR/p66) were grown
outside of the NRPS gene cluster (6465 and 6479). In addition, overnightandinoculatedat5(cid:1)106cfuintoG.mellonella(greater
genes 6469 and 6470, although by location included as a part of wax moth) larvae. Larval death counts were taken every 24 h.
the NRPS gene cluster, are not essential to the production of TheresultsindicatethattheDBO1NRPSproductistoxictothe
hemolytic activity, as their mutation does not significantly alter G. mellonella moth larvae, whereas a gene 6466 LuxR knockout
wild-type hemolytic activity. Second, there is an intermediate mutant lacking the ability to produce the hemolysin has greatly
hemolytic phenotype exhibited by mutant 6471::TpR, likely due reduced virulence. Genetic complementation of this gene knock-
to the lack of a xylose addition to the final product, as described out, as described above for hemoglobin release, showed similar
for occidofungin/burkholdine.30,33 Whereas xylose addition to restoration of the toxic effects toward G. mellonella upon the
occidiofungin/burkholdine appears to be essential for antifungal additionofrhamnose.Sincemothlarvaedonotcontainredblood
www.landesbioscience.com Virulence 289
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Figure3.(A)Absorbanceofbloodbrothsupernatantsacrossarangeofwavelengthsafter24hincubationwithwtDBO1andtwoNRPSclustermutants. B
Dottedblackline,wild-typeDBO1;solidgrayline,6466::oriTpR(luxRmutant);dottedgrayline,6477::oriTpR(polyketidesynthetasemutant).(B)Viableplate
countsafter24h.Black,wild-typeDBO1;white,6466::oriTpR;gray,6477::oriTpR.(C)Erythrocytecounts.Black,wild-typeDBO1;white,6466::oriTpR;light s
gray,6477::oriTpR;darkgray,blankcontrol(nobacteriaadded). e
d
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cells, the results illustrated in Figure5 suggest that the hemolytic Prevalence of the NRPS gene cluster in the Bcc species. In a
NRPSproductisnotsimplyahemolysinsensustricto,butrather order to determine the prevalence of occidiofungin/burkholdine- L

a cytotoxin, possibly active against several cell types including like compounds across members of the Bcc, we screened all 2
fungi (as demonstrated for B. contaminans MS14 occidofungin/ available Bcc isolates in our library using PCR primers designed 1
burkholdine), insects, amoebae and higher organisms. To further from homologous regions between known sequences of 0
thishypothesis,BccstrainsexhibitingNRPShemolyticactivity,as B. contaminans MS14 and B. ambifaria AMMD. The primers 2
well as isogenic mutants defective in hemolysin production, were were also designed to amplify the regions within these genes ©
tested in a Dictyostelium discoideum feeding infection model.40 showing no homology to other sequenced genes. Of 54 isolates
We were unable to detect differences in virulence toward grazing screened, 13 yielded PCR products for at least two of the primer
D. discoideum in Bcc strains either producing or not producing sets,andofthose13,tenyieldedproductsinallthreeprimersets
the NRPS toxin/hemolysin. We also observed that constructed (Table 2). Using the same assay as described above, hemolysis of
mutant 6471::TpR, which is unable to produce the NRPS toxin human erythrocytes by each of the 13 strains was investigated.
glycosyltransferase, and that produced intermediate levels of OfthetenBccstrainspositiveforallthreePCRproducts,sevenof
hemoglobinreleaseintheliquidhemolysisassay,didnotproduce them demonstrated high levels of hemolytic activity against
intermediatelevelsofdeathintheG.mellonellainfectionmodelas human erythrocytes. Of the three Bcc strains positive for only
compared with wild-type DBO1 and NRPS structural mutants two of the three PCR products, only one of these exhibited any
such as 6477::TpR (data not shown). Instead, 6471::TpR behaved hemolytic activity, and this was low level of activity (Fig.6). In
similarly to wild-type NRPS toxin-producing strains and addition, analysis of other Burkholderia genomes currently
produced wild-type levels of G. mellonella death. available in the GenBank database indicate that no additional
290 Virulence Volume3Issue3
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Figure4.LysisofhumanerythrocytesbyB.vietnamiensisDBO1requirestheintactNRPScluster,includingbiosyntheticgenes,Luxregulatorsand c
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transporter.Supernatantabsorbance(570nm)wastakenofeachstrainincubatedinTSB+5%humanerythrocytesaftershakingat30°Cfor48h.
Wt=wildtype;6466::TpR/p66–rhaand6466::TpR/p66+rha=complementedmutant6466::TpR/pSCRhaTc-6466Hisincubatedwithandwithout e
0.2%rhamnose,respectively.Errorbarsdenotestandarddeviationaroundthemeanfromthreeindependenttrialsperformedintriplicate.Means ci
labeledwithdifferentdigits(1–3)arestatisticallydifferent(p,0.05,n=3). s
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Bccgenomes(n=8)containNRPSgenesorgeneclusterssimilar mutant in DBO1 suggest that the NRPS-derived compounds
to those identified in AMMD, DBO1 or MS14 (Table 2). It from the three Bcc strains are similar. Prior structural analysis of s
is important to note that the apparent presence of these NRPS the MS14 NRPS compound suggests that the ring peptide can e
genes is required for hemolytic activity but does not guarantee be glycosylated by xylose, and that without this glycosylation, d
its production, possibly due to small, acquired mutations in the the antifungal properties of occidiofungin/burkholdine are lost. n
NRPS gene cluster in some Bcc strains. Overall, hemolytic or However, genetic inactivation of B. vietnamiensis DBO1 NRPS a
toxic activity produced by an NRPS-derived occidiofungin/ gene cluster homolog 6471, encoding the putative glycosyltrans- L

burkholdine-like compound appears to be limited to the Bcc ferase, doesnot eliminatehemolyticactivity completely, butdoes 2
species B. ambifaria, B. contaminans,32,33 Burkholderia pyrrocinia reduce hemolytic activity by approximately half, whereas in 1
and B. vietnamiensis. G. mellonella, the unglycosylated compound still exhibits wild- 0
type bacterial toxicity. This suggests that the unglycosylated 2
Discussion form of the occidofungin/burkholdine-like compound is fully ©
toxictowardssomeorganismssuchasininsects,butonlypartially
Bcc species B. contaminans MS14 produces cellular toxins that active against other cell types, including erythrocytes and fungal
are synthesized using a non-ribosomal peptide synthase pathway, cells. Because constructed mutant 6471::TpR does not produce
which is characteristic of complex secondary metabolite com- intermediate levels of killing in the G. mellonella infection model
pounds.Thesecompounds,knownasoccidiofunginsorburkhol- ascomparedwithwild-typeDBO1andNRPSstructuralmutants,
dines,havebeenpreviouslyshowntohaveantifungalactivity,32,33 thissuggeststhattheorganismalassaysusedtotesttoxicityareless
buthereinweshowthatthesecompounds,orcompoundsclosely sensitive than the hemoglobin release cellular lysis assay.
related to occidiofungins/burkholdines (based partially on bio- To examine whether other Bcc strains possess the NRPS gene
synthetic gene cluster similarities), also possess high levels of cluster, PCR primer sets localized to unique sequences were used
hemolytic activity. Besides individual ORF homology and to probe for the presence of two NRPS genes. This analysis
syntenic organization between the gene clusters in MS14 and identified13of55BccstrainspotentiallycarryingtheNRPSgene
AMMD/DBO1,thehemolysisresultsfromtheglycosyltransferase cluster. Further analysis with another unique PCR primer pair
www.landesbioscience.com Virulence 291
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Figure5.DBO1requiresaLuxRhomologregulatorforvirulenceagainstGalleriamellonellalarvae.Fivemicrolitersofbacterialsuspension,corresponding n
to5(cid:1)106colonyformingunits,wasinoculatedinto10larvaeinnindependenttrials.Theinfectedlarvaewereincubatedat30°Candviabilitycounts e
weretakenat24hintervals.[66TpR=strain6466::TpR;p66=6466::TpRcarryingcomplementationvectorpSCRha-6466His].Allresultsaremeansshown i
+/−SD.Meanslabeledwithdifferentdigits(1–3)arestatisticallydifferent(p,0.01). c
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reduced this number to ten strains that carried three NRPS The literature on NRPS and PKS systems indicates that
genes. Upon functional testing, only seven of these ten strains associated dedicated glycosyltransferases are not uncommon.
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exhibited hemolysis of human erythrocytes, suggesting that two Although the putative xylose moiety appears to enhance hemo- e
strains (B. pyrrocinia LMG 14294 and B. vietnamiensis LMG lytic activity of the Bcc NRPS-derived compound, the exact d
10929) possessed undefined mutations to the hemolytic gene mechanism through which this is achieved awaits further n
cluster. In addition, only two PCR products were amplified investigation. Walsh et al.41 describes a possible function of such a
from B. ambifaria CEP0996; however, this strain exhibited glycosyltransferases as having a role in imparting polarity or L
hemolytic activity in this assay. This suggests that either one set solubility to hydrophobic compounds, thus improving their
2
of primer pairs was degenerate from the CEP0996 genomic access into target cell surfaces. The results presented in this and 1
sequence to allow amplification of the NRPS ortholog from previous publications on occidiofungin/burkholdine32,33 suggest 0
this strain even though a functional protein was expressed, or that glycosylation is important for full toxicity of the compound. 2
that this strain produces a hemolytic toxin different from Several key questions remain with respect to the function of ©
occidiofungin/burkholdine. In terms of prevalence, it is interest- some ORFs found within the NRPS cluster. For example, gene
ing to note that this NRPS gene cluster has not been identified 6470 downstream of the glycosyltransferase 6471 encodes a
in the two most clinically important Bcc species, Burkholderia hypothetical protein that upon mutagenesis, appears to have no
multivorans and B. cenocepacia. Although these two species are effect on hemolytic or toxic activity. Gene 6467, overlapping
found both as environmental and clinical isolates, and clinical the two luxR homolog ORFs, is a small gene encoding a potent-
Bcc strains can exhibit relatively little difference from strains ial product of only 110 amino acids, with no homology to any
foundintheenvironment,27theidentificationofthisNRPSgene known protein. This may be a truncated ORF that was intro-
cluster in Bcc species better adapted to soil environments than duced along with the upstream luxR family transcriptional
inassociationwithhumanssuggeststhatthisgeneclusterevolved regulator gene 6468 during acquisition and assembly of the
to protect the Bcc from ecological niche predators such as fungi NRPS cluster. Finally, gene 6475, situated between two large
and amoeba rather than as a virulence factor to assist invasion of genes encoding proteins with amino acid adenylation domains,
human tissue. encodes a 538 amino acid protein that contains a β-lactamase
292 Virulence Volume3Issue3
Table2.Bacterialstrainsanalyzedinthisstudy
Species Strain PCRproducts Hemolysis Source/info
B.cepacia ATCC25416T ND NT Onionrot
B.cepacia LMG18821 ND None CFisolate
B.cepacia ATCC17759 ND None Soil
B.cepacia CEP521 ND None CFisolate
B.multivorans LMG13010T ND None CFisolate
B.multivorans ATCC17616 ND None Anthranilateenrichment
B.multivorans PC249–2 ND None ATCC17616mutant
B.multivorans C5393 ND None CFisolate
.
B.multivorans C3430 ND None CFisolate e
t
B.multivorans C5274 ND None CFisolate u
B.multivorans C5568 ND NT CFisolate b
i
B.multivorans CGD1 ND* NT CGDisolate r
t
B.multivorans CGD2 ND* NT CGDisolate s
i
B.cenocepacia J2315T ND(*) None CFisolate d
B.cenocepacia K56–2 ND None CFisolate t
o
B.cenocepacia PC184 ND NT CFisolate
n
B.cenocepacia 715j 6472 None CFisolate
o
B.cenocepacia K63–3 6472 NT CFisolate
D
B.cenocepacia C1257 ND None CFisolate

B.cenocepacia C4455 ND None CFisolate e.
B.cenocepacia C5424 ND NT CFisolate c
n
B.cenocepacia C6433 ND None CFisolate
e
B.cenocepacia BC7 ND None CFisolate
i
c
B.cenocepacia CEP511 ND None CFisolate
s
B.cenocepacia AU1045 ND* NT CFisolate o
B.cenocepacia HI2424 ND* NT Soilrhizosphere Bi
B.cenocepacia MC0–3 ND* NT Soilrhizosphere
s
B.cenocepacia D1 ND NT Environmentalisolate e
B.stabilis LMG14294T 6472,6474,6476 None CFisolate d
B.stabilis LMG18870 ND None CFisolate n
a
B.vietnamiensis LMG10929T 6472,6474,6476 None Soilrhizosphere
L
B.vietnamiensis DB01 6472,6474,6476 High Phthalateenrichment
2
B.vietnamiensis LMG18835 6472,6474,6476 High CFisolate
1
B.vietnamiensis G4 ND(*) NT Trichloroetheneenrichment 0
B.dolosa LMG18943T ND None CFisolate 2
B.dolosa L06 ND NT CFisolate ©
B.dolosa AU0645 ND NT CFisolate
B.dolosa STM1441/LMG21443 ND None Soilrhizosphere
B.dolosa CEP021 ND NT CFisolate
B.dolosa E12 ND NT CFisolate
B.ambifaria AMMDT 6472,6474,6476 High Soilrhizosphere
B.ambifaria CEP0996 6472,6474,6476 High CFisolate
B.ambifaria LMG17828 6472,6474,6476 None Soil
B.ambifaria M53 6472,6474,6476 High Soil
B.pyrrocinia LMG14191T 6472,6474 Low Soilrhizosphere
www.landesbioscience.com Virulence 293
Table2.Bacterialstrainsanalyzedinthisstudy(continued)
Species Strain PCRproducts Hemolysis Source/info
B.pyrrocinia LMG21822 6472,6474,6476 Moderate Soil
B.pyrrocinia LMG21823 6472,6474,6476 None Water
B.pyrrocinia LMG21824 6472,6474,6476 High CFisolate
B.anthina W92T ND NT Soilrhizosphere
B.anthina J2552 ND NT Soilrhizosphere
B.anthina C1765 ND NT CFisolate
B.anthina AU1293 6472 NT CFisolate
B.ubonensis Bu ND* NT Soil
.
B.contaminans MS14 6472,6474,6476* NT Soil e
B.lata 383 ND* NT Soil ut
Burkholderiasp JS150 ND NT p-dichlorobenzeneenrichment b
R.pickettii ATCC27511T ND NT Patientisolate ri
t
R.pickettii YH105 6472,6474 None p-nitrobenzoateenrichment s
i
Superscript “T” following the strain name denotes the Type strain of each species. ND, not detected. NT, not tested. Asterisks indicate the result was d
obtainedusinggenomicanalysis.CF,cysticfibrosis;CGD,chronicgranulomatousdisease.
t
o
n
domain. Rather than providing cellular resistance to antibiotics, Given the interest in developing the Bcc NRPS-derived com-

it is likely that this protein is somehow involved in modifying pound known as occidiofungin or burkholdine as an antifungal o
D
the cyclic peptide structure, as all homologs to this particular agent for agricultural (crop preservation and protection) or phar-
proteinareassociatedwithNRPSandPKSgeneclusters.Because maceutical use, it is important to note its toxic and hemolytic
.
we were unable to mutagenize gene 6475, a definitive function activities. Clearly, the development of biological-based fungicides e
for the associated protein remains to be assigned. All other should include extensive analysis of their toxic properties. A pre- c
n
genes in the strain DBO1 NRPS gene cluster are essential for vious study33 has noted that occidiofungin/burkholdine produces
e
hemolytic activity. aberrant cell membrane morphology in fungi similar to what has
i
c
s
o
i
B

s
e
d
n
a
L

2
1
0
2
©
Figure6.HumanerythrocytelysisgenerallyrequiresthepresenceoftheNRPSclusterinB.cepaciacomplexisolates.Forty-sevenBccstrains,oneuntyped
Burkholderiaspecies,andtwoR.picketiistrainsandwerescreenedusingPCRprimersforthreeofthenon-ribosomalpeptidebiosynthesisgeneswithin
thecluster.Ofthe50strains,13werepositiveforatleasttwoofthegenes,and10werepositiveforallthreegenestested.Whitecolumns=strains
positivefortwoofthegenes,blackcolumns=strainspositiveforallthreegenestested.A ofthesupernatantsweretakenafter48hincubationof
570
thestrainsinTSB+5%humanerythrocytesintriplicate.Errorbars=+/2SDoftriplicates.
294 Virulence Volume3Issue3
beendescribedfortheechinocandinclassofantifungalcompounds, Targeted mutagenesis. Once the NRPS cluster had been
whichdisruptβ-glucanpolymerization.A recentstudy42 describes identified through random plasposon mutagenesis, mutations in
occidiofungin’sinvitropotencyagainstCandidaspecies,aswellas the remaining genes within the cluster were created through a
itschemicalstabilityinthepresenceofhumanserum.Thisfinding targeted insertion mutagenesis strategy using homologous recom-
suggests that if occidiofungin/burkholdine displays pathogenic bination. Primers were designed to amplify open reading frames
traitsagainsthumans,thecompoundmayresistdegradationwithin (ORFs) and XbaI sites at either end. A full list of primers is
thebody. Furthermore,a toxicological evaluation of occidofungin shown in Table 3. PCR amplification was performed using
is described where B6C3F1 mice were given a single dose of TopTaq (Qiagen Inc.). In many cases, because of imperfect local
occidiofunginupto20mg/kgbodyweightoradailydosefor5d homologybetweentheAMMDandDBO1sequences,techniques
at2mg/kgbodyweight.Histologicalexaminationoftreatedmice were used to enhance primer binding in order to obtain PCR
did not identify organ toxicity; however, overall effects were a products. DNA bands were extracted from agarose gel using
reduction in body and organ weights, and anemia or neutropenia GeneClean (Fermentas), ligated to pJET1.2 (Fermentas) and .
e
were not tested. Our results suggest that the Bcc occidofungin/ clonedinE.coliDH5a(InvitrogenCorp.),andDNAsequencing
t
burkholdine-likecyclicpeptidetargetsanddisruptscomponentsof of the cloned fragments was performed to confirm the identity u
the membranes of eukaryotic cells (but not prokaryotic cells), of the product. To generate plasmids containing insertions, the b
especiallyerythrocytes,potentiallybindingtocholesteroloranother OriTpR region was extracted from pTnMod-OTp' by digestion ri
t
cell scaffolding carbohydrate component. Future experiments will with BglII and XbaI, ligated to the PCR products using T4 s
beaimedatfurtherinvestigationofBccNRPS-derivedhemolysins ligase (Promega Corp.) and the three-way ligation product was i
d
andtheirmechanismofactionagainsterythrocytes.

Table3.Oligonucleotidesusedinthisstudy t
o
Materials and Methods
Oligonucleotides Sequence n

Mutagenesis* o
Bacterial strains, plasmids, antibiotics and culture conditions.
m6465–1 GGTTCGACATTCTGACGTT D
Luria-Bertani (LB) broth was prepared in distilled water to half
m6465–2 CCTCTATGTGCCGAACAG
concentration for Bcc strains and full concentration for Escheri- .
chia coli strains. Sheep, horse, bovine and rabbit blood broth m6466–1 AGAGTCAGATGTTCGCGAAG e
c
(Dalynn Biologicals) and human blood broth (Canadian Blood m6466–2 GTCTCGACCGTGCTTTCC
n
Services) were prepared to 5% v/v blood in trypticase soy broth m6470–1 AAGCGGCGTTCGTCAAGTC e
(TSB).Agarplateswerepreparedwith1.5%w/vagar.Antibiotics m6470–2 AGGTGGCTGAGTTCGACATTG i
c
were included where necessary to the following concentrations:
ampicillin (amp), 100 mg/mL; trimethoprim (Tp), 100 mg/mL; m6471–1 AAGGTCTGCATCAATCTGG s
tetracycline(Tc),100mg/mL.Allantibioticswerepurchasedfrom m6471–2 AGGGAATAGGTCAGCGGC o
i
Difco through BD Canada. E. coli DH5a was used for plasmid m6477–1 GCCGTTCTGCAACTACATCC B
manipulation. E. coli strains were grown at 37°C, while DBO1 m6477–2 AGGCGGTCGGTCAGTTCG s
was grown at 30°C. All liquid cultures were shaken at 225 rpm. m6479–1 ATGGTACCGGCGTCCTTCGAATC e
High-copy number plasmid pJET (AmpR) was employed in m6479–2 ATAAGCTTGGCAGACGTCGGGTT d
cloning experiments. For random plasposon mutagenesis, n
Complementation*
pTnMod-OTp' (TpR) was used.38 To complement gene 6466, a
pSCRhaB2wasused.43Alistofbacterialstrainsandplasmidsused c6466–1 ATAATACATATGCATCATCACCATCACCACGTT- L
CGCGAAGCTTG
or constructed is shown in Table 1. 2
c6466–2 ATTCTAGACTACGCCGCCGACGCGCAC
Random plasposon mutagenesis. A random plasposon inser- 1
tion mutant library was created in B. vietnamiensis DBO1 using Sequencing 0
plasposonpTnMod-OTp'aspreviouslydescribed.38Cellscontain- pJET-F CGACTCACTATAGGGAGAGCGGC 2
ing integrated plasposons were selected on 1/2 LB + 100 mg/mL pJET-R AAGAACATCGATTTTCCATGGCAG ©
trimethoprim. Twenty thousand mutants were patched onto
pBBR-F AACAGCTATGACCATG
5% sheep blood TSA agar, incubated at 30°C and observed for
pBBR-R AATACGACTCACTATAG
hemolysis after 48 h. Mutants exhibiting loss of hemolysis were
Bccclusterscreening
carried forward and their plasposon insertion sites were isolated
as previously described.38 To ensure that the random plasposon s6472–1 TACATGCTCGACGACGCGCT
mutants obtained carried authentic mutations responsible for the s6472–2 ATGTTGTAGGTGGCCGACGGG
observed phenotype, plasposon site of insertion plasmid clones s6474–1 TCTCGACCAGCGGGCAATACC
were reintroduced into wild-type DBO1, and following recom- s6474–2 TCCTCGATCATGAAGCGCAG
bination and selection on 1/2 LB + 100 mg/mL trimethoprim,
s6476–1 AAGGTCACGTGGTTCGGCTCG
and PCR analysis to ensure proper integration, the reconstructed
s6476–2 ATTTCGCGGACCAGTTCGGC
mutants were again tested for hemolytic activity on 5% sheep
blood TSA agar. *Restrictionsitesshowninbold.6xhistidinetagunderlined.
www.landesbioscience.com Virulence 295