Novel gain of function approaches for vaccine candidate identification in Burkholderia pseudomallei.

0.68 MB·English

by  Dowling, Andrea J.

MINIREVIEWARTICLE
published:09January2013
CELLULAR AND INFECTION MICROBIOLOGY doi:10.3389/fcimb.2012.00139
Novel gain of function approaches for vaccine candidate
identification in Burkholderia pseudomallei
AndreaJ.Dowling*
Biosciences,CollegeofLifeandEnvironmentalSciences,UniversityofExeter,Penryn,UK
Editedby: The Gram-negative bacterium Burkholderia pseudomallei is a serious environmental
MarkEstes,UniversityofGeorgia, pathogen and the causative agent of the often fatal melioidosis. Disease occurs
USA following exposure to contaminated water or soil, usually through cuts in the skin
Reviewedby: or via inhalation. However, the underlying mechanisms of pathogenicity remain poorly
Xiao-LianZhang,WuhanMedical
understood.B.pseudomallei isendemictoSouthEastAsiaandNorthernAustraliawhere
School,China
YongqunHe,UniversityofMichigan infectionsareassociatedwithantibioticresistanceandhighmortalityrates.Categorization
SchoolofMedicine,USA of the pathogen as a potential biowarfare agent has also made research into vaccine
*Correspondence: developmentahighpriority.Recentgenome-scalescreeninghasproducedalargenumber
AndreaJ.Dowling,Biosciences, of putative gene candidates from B. pseudomallei with the potential for development
CollegeofLifeandEnvironmental
into vaccines. This mini-review will discuss the advantages and limitations of this novel
Sciences,UniversityofExeterin
Cornwall,TremoughCampus, approach, how these new techniques can complement existing strategies, and outline
PenrynTR109EZ,UK. aimsforfutureresearch.
e-mail:[email protected]
Keywords:Burkholderia,vaccine,melioidosis,novelscreening,virulencefactors
INTRODUCTION of B. pseudomallei makesuccessful antibiotic treatment difficult
Burkholderiapseudomalleiisaseriousenvironmentalpathogenof therefore vaccine development is an important strategy against
manandthecausativeagentoftheoften fataldiseasemelioido- melioidosis.
sis.Infectionoccursfollowingexposuretocontaminatedwateror
soil, usually through cuts in the skin or via inhalation, but the CHALLENGINGMACROPHAGESTOIDENTIFYANTI-IMMUNE
underlying mechanisms of pathogenicity of B. pseudomallei to CELLFACTORS
humansremainpoorly understood(Adler etal.,2009). B.pseu- Evidencesuggeststhatmacrophagesareanessentialearlydefence
domallei is endemic to South East Asia and Northern Australia against Burkholderia infection (Breitbach et al., 2006). These
where infections are associated with both antibiotic resistance innate immune cells are one of the first responding phagocytes
andhighpatientmortality (∼50%). Thehighrates of infection to arrive at the site of infection where they phagocytose invad-
and subsequent mortality make B. pseudomallei a high prior- ing bacteria and (along with other cells) release chemokines
ityforresearchandvaccinedevelopment,asnoeffectivevaccine andcytokines to govern immune responses andpromote adap-
currentlyexists.Acombinationofthelackofvaccine,highinfec- tive immunity. To establish intracellular infection Burkholderia
tionrateviaaerosolinhalation,andlimitedutilityofantibiotics must evade macrophage killing and disrupt the normal cell
has resulted in classification of this bacterium as a Category B signaling and regulation processes. In order to detect poten-
biowarfarepathogen(Rotzetal.,2002). tial vaccine candidates a simple “gain of function” screen was
DuringtheestablishmentofinfectionB.pseudomalleiadheres designed aimed at identifying genes encoding anti-macrophage
to, survives and replicates within both host epithelial cells and virulence factors across the B. pseudomallei genome (Figure1).
macrophages. The bacterium is therefore capable of interfering This screen identifies recombinantly expressed genes that equip
withthehostcellularmechanismsthatwouldotherwisedestroy naïve Escherichia coli with anti-macrophage abilities in three
it. Known bacterial factors affecting this interaction with host different ways: (1) the production of cytotoxic moieties e.g.,
cellsincludethebacterialcapsule,andeffectors delivered bythe proteins, secondary metabolites (toxins, effectors), (2) the abil-
typeIIIandtypeVIsecretionsystems(T3SSandT6SS)(Stevens ity to evade phagocytosis or destruction within the phagolyso-
et al., 2003; Shalom et al., 2007; Galyov et al., 2010; Burtnick some(intracellularsurvival),and(3)overwhelminge.g.,nutrient
etal.,2011). Onceinsidethemacrophagethepathogeninduces depletionandstarvation(biofilmformation,hydrogenperoxide
cell fusion leading to the formation of “Multi-Nucleated Giant scavenging)(Dowlingetal.,2010).
Cells” or MNGCs, a process key to both intracellular replica-
tion and bacterial persistence (Kespichayawattana et al., 2000). POTENTIALVACCINECANDIDATESIDENTIFIED
Followingtheinvasionofhostcellspathogenreplicationreaches A large proportion of melioidosis vaccines tested to date have
a critical point at which the bacteria induce cell death and are focused on the use of live-attenuated strains of B. pseudoma-
released to establish secondary infections (Adler et al., 2009). llei. However, a major issue with live-attenuated vaccines is the
Both the intrinsic drug resistance and the intracellular lifestyle risk of reversion to virulence and although they are currently
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Dowling NovelvaccinecandidateidentificationinBurkholderia
FIGURE1|Brief overview of the anti-macrophage screen workflow. by 40% or more are classed as positive and the end sequences of
Recombinant genomic libraries of the sequenced clinical isolate these positive clones are aligned onto the genome sequence (3). Each
B. pseudomallei K96243 were produced in E. coli (1), end-sequenced anti-macrophage locus, or region of interest (ROI), is then described
and subsequently screened for cytotoxicity against murine as the minimum region of genetic overlap covered by two or more
macrophages (2). Library clones shown to reduce macrophage viability positive clones (4).
the most effective vaccines studied in mice it is unlikely that exposed to the immune system upon B. pseudomallei infection
theywouldbeusedinhumans(Sarkar-TysonandTitball,2010). (GarmoryandTitball,2004;LazarAdleretal.,2011).ABCtrans-
Less research has been focused on the development of subunit portershaveaknownroleinbacterialsurvivalandpathogenicity,
vaccines (mainly as a putative antigen needs to first be iden- 22ofthe105predictedK96243ABCtransportersweredetected
tified) and those investigated are far less effective (Patel et al., bythescreen(Harlandetal.,2007b;Dowlingetal.,2010).Ofpar-
2011). This screen represents a novel method that can be used ticularinterestamongthedetectedlociarethelipopolysaccharide
to directly identifypotential recombinantsubunitvaccines.The (LPS) biosynthesis operon and wzt ABC transporter (Table1).
design of the screen means that any active anti-macrophage ThisparticularfindingsuggeststhatanactiveLPScanbesuccess-
productsidentifiedaresuccessfullyexpressedwithinrecombinant fullyconstructedinarecombinantsystem.GiventhatLPSelicits
E. coli. Recombinantsubunitvaccines areanattractive proposi- strongimmuneresponsesthiscouldmakeitanattractivevaccine
tion as they eliminate the safety issues associated with subunit candidate. Further, this screen detected 5 out of the 11 docu-
purificationfromlargecultures ofthepathogenitself(Liljeqvist mentedautotransportersinK96243andinterestinglydetectsboth
andStahl,1999). BPSS0796 (BoaA) and BPSL1705 (BoaB), these trimeric auto-
Theoriginalscreenrevealedover100anti-macrophagerelated transportedadhesinsareimplicatedinadhesiontoepithelialcells
loci on both chromosomes 1 and 2 of B. pseudomallei strain and are shown to have immunogenic properties (Tiyawisutsri
K96243. Several functional classes were repeatedly identified; etal.,2007;Balderetal.,2010).
putative secondary metabolite synthesis gene clusters (NRPSs Recent advances have been made regarding Type VI secre-
and PKSs), signal response and regulation systems (methyl- tion machinery proteins; in particular the Hcp family, as sub-
accepting chemotaxis proteins, two-component sensor systems, unit vaccine candidates (Burtnick et al., 2011). Three of the
AraCandLysRfamilyregulators),phage-relatedelements,resis- six documented hcp encoded proteins; Hcp5 (BPSS099), Hcp4
tance (drugs, metals, reactive oxygen species), adhesion and (BPSS0171), and Hcp6 (BPSL3015), were within ROI detected
biofilm formation. The largest number of coding sequences bythisscreen.However, research sofardemonstrates thatpuri-
(CDSs) detected however related to toxin/enzyme production fied Hcp2 gives the highest-level protection (80%) to BALB/c
(hemolysins,proteases, phospholipases)or transport andsecre- mice inoculated with a lethal dose of B. pseudomallei. Burtnick
tion (ABC transporters, autotransporters, efflux transporters, et al. demonstrated T6SS 1 as a major virulence determinant
and components of Type III and Type VI secretion systems). for B. pseudomallei with deletion of hcp1 resulting in higher
Examples of putative vaccine candidates from different func- LD andgrowthdefectscomparedtowild-typeB.pseudomallei.
50s
tional classes discussed in the following text are summarized in DeletionofvgrG1fromthissystemalsodisplayedthesameresults
Table1. demonstrating its importance in secretion of Hcp1 (Burtnick
et al., 2011). VgrG proteins play an important role in host cell
TRANSPORTERSANDSECRETIONSYSTEMS cytotoxicityaswellasformingthesecretionmachineryitselfand
ABCtransporter andautotransporter-related proteinsaredraw- are present on the surface of the pathogen, several VgrG CDSs
ingconsiderableinterestaspotentialvaccinecandidatesgiventhe wereidentifiedbythescreen(Pukatzkietal.,2009;Suarezetal.,
surface association of their component proteins which may be 2010).
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Dowling NovelvaccinecandidateidentificationinBurkholderia
Table1|Examplesofputativevaccinecandidatesidentified.
Functionalclass Examplevaccinecandidate CDS References
ABCtransporter LPSbiosynthesisoperonpluswztABCtransporter BPSL2672- GarmoryandTitball,2004;Harlandetal.,2007b
BPSL2688
Autotransporter BoaA BPSS0796 Tiyawisutsrietal.,2007;Balderetal.,2010;
BoaB BPSL1705 LazarAdleretal.,2011
TypeVIsecretion Hcpproteins(inc.Hcp4,Hcp5,Hcp6)andVgrG numerous Pukatzkietal.,2009;Suarezetal.,2010;
proteins Burtnicketal.,2011
Toxin BurkholderiaLethalFactor1 BPSL1549 Cruz-Migonietal.,2011
Enzyme PhospholipaseD BPSS1381 McKeanetal.,2007;Driskelletal.,2009
Non-hemolyticphospholipasePLC-3 BPSS0067 Tuanyoketal.,2006
Adhesion Filamentoushemagglutinin-like BPSS1721- Satoetal.,1981;Kimuraetal.,1990;Dowling
BPSS1735 etal.,2010
Secondarymetabolism SylA-like(BylA) BPSS1263- Grolletal.,2008;Dowlingetal.,2010
BPSS1269
Hypotheticalprotein Putativetoxins BPSL0590 Dowlingetal.,2010
BPSL0591
TOXINS,ENZYMES,ANDHYPOTHETICALPROTEINS and has been named “Burkholderia Lethal Factor 1” or BLF1
Oneof the anti-macrophageregions identified containsa puta- (BPSL1549) (Cruz-Migoni et al., 2011). BLF1 has the ability to
tive Phospholipase D-like protein (BPSS1381) (Dowling et al., inhibithelicaseactivityandthusproteinsynthesis.BLF1waspre-
2010). Mutationsof PhospholipaseDproteins inthepathogens viously described as a hypothetical protein given that similarity
Corynebacterium pseudotuberculosis andRickettsia prowazekii,in toCNF1isbasedonstructural ratherthansequencehomology.
whichtheproteinisamajorvirulencefactor,haveshownpoten- BLF1 mutants were attenuated in their ability to kill mice and
tial as live-attenuated vaccines (McKean et al., 2007; Driskell purifiedtoxinwaslethaluponinjectionindicatingtheimportant
etal.,2009). Furthercharacterizationiswarrantedtodetermine nature of this toxin in B. pseudomallei virulence (Cruz-Migoni
if this phospholipase D-like protein may be of use as a poten- et al., 2011). The BPSL1549 CDS that encodes BLF1 was iden-
tialvaccinecandidateforB.pseudomallei.Anotherlociofinterest tifiedwithinapositivelocus pulledoutbytheanti-macrophage
encodestheputativetoxinPLC-3(non-hemolyticphospholipase) screen.Ofnote,thetoxinisflankedbytwoABCtransportersys-
which has been shown to be strongly upregulated in a hamster tems (BPSL1545-BPSL1546 andBPSL1548) alsocontainedwith
modelofmelioidosisandamutantdisplaysanLD significantly the anti-macrophage region of interest that may be involved in
50
higher than wild-type B. pseudomallei parent strain (Tuanyok secretion of BLF1. The utility of BLF1 as a toxoid vaccine has
et al., 2006). These data highlight PLC-3 as a potential vaccine yet to be explored. Thereare a further 28 anti-macrophageloci
candidatewarrantingfurtherinvestigation. outofthe113describedinthescreenwhichcontainatleastone
Approximately30%ofB.pseudomalleigenesencodehypothet- hypotheticalprotein.Theseareyettobeinvestigatedviadetailed
icalproteinswhosebiologicalfunctionsareyettobeestablished. protein and structural prediction algorithms to identify active
These hypothetical proteins are a potential source of novel vir- sitesorhomologies.However,asexemplifiedbyBLF1,suchhypo-
ulence factors, and indeed potential vaccine candidates. The theticalproteinswarrantfurtherresearchasasourceofpotential,
anti-macrophage screen promotes the identification of hypo- currentlyhidden,virulencefactors,andhopefullyfuturevaccine
thetical proteins with anti-macrophage functions. One positive candidates.
region described by the screen contained two large adjacent
CDSs, BPSL0590 and BPSL0591, encoding hypothetical pro- ADHESINS
teins. The position-specific-iterative BLAST (psi-BLAST) algo- Adhesinsmakeattractive vaccinecandidates,astheyaresurface
rithm predicted homology within BPSL0590 and BPSL0591 to expressed and likely therefore to be presented to the immune
knowntoxinsfromotherpathogens.Bothcontaindomainswith systemuponinfection.Adhesinsareoftenkeyvirulencedetermi-
homology to the Toxin complexes (Tc’s) from Photorhabdus, nants allowing the initial attachment of the invading pathogen
further the predicted N-terminal of BPSL0590 has homology to host cells. The effectiveness of the Filamentous hemagglu-
to the Salmonella enterica virulence associated protein or SpvB. tinin (FHA) of Bordetella pertussis as an antigen has long been
MacrophagestreatedwithlysatefromrecombinantE.colilibrary utilized in vaccines against Pertussis (Sato et al., 1981). The
clones expressing BPSL0590 and BPSL0591 show altered actin region BPSS1721-BPSS1735 identified as an anti-macrophage
cytoskeletal morphology, multi-nucleation, and apoptosis illus- locus encodes proteins with predicted homology to B. pertus-
tratingthatthesemaybenoveltoxins(Dowlingetal.,2010). sisFHA.Macrophagestreated withlibraryclonesexpressingthe
Recently a novel toxin hasbeen described in B. pseudomallei region showed dramatic actin alterations and apoptotic nuclei.
K96243 with similarity to the potent CNF1 toxin from E. coli Of note, B. pertussis FHA is successful at preventing adherence
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Dowling NovelvaccinecandidateidentificationinBurkholderia
of the pathogen to the respiratory epithelium when introduced library strain E. coli may not result in production of cytotoxic
as a protective antigen via intranasal immunization (Kimura moietiesorcytotoxicityasaresultofenhancedbacterialgrowth
et al., 1990). The role of this hemagglutinin-like protein in andoverwhelming.
B. pseudomallei infection has not so far been characterized.
Determination of whether this FHA homolog may prove useful ADVANTAGESANDLIMITATIONS
inprotectionagainstmelioidosis,inparticulardiseasecontracted There are several advantages of using gain of function screens
via inhalation, could help in the formulation of a successful for discoveringvirulencefactors andvaccine candidates. Firstly,
vaccine. theyovercome functional redundancy to unmask virulencefac-
tors which may be compensated in mutants. B. pseudomallei
SECONDARYMETABOLITES has a large genome and the ability to survive in a wide range
Manyoftheanti-macrophageregionsidentifiedcontainedlarge of environmental niches. This suggests that functional redun-
gene clusters associated with the non-ribosomal peptide syn- dancy is likely among virulence factors with various genes or
thetase(NRPS)orpolyketidesynthase(PKS)systemsusedinthe geneticregionsabletocompensatemutationtoproducesuccess-
productionofpeptidesandsmallmolecules.Theroleofsuchsec- fulinfection.Secondly,thescreenscannotonlybeusedtodirectly
ondarymetabolites invirulenceislargelyundescribed,however, isolate candidates, which may be useful in the development of
they may be used to promote intracellular survival or produc- recombinant subunit vaccines, but also subunit vaccines, DNA
tion oftoxic products andcouldprovidepartoftheunderlying vaccines,andlive-attenuatedvaccines.Thescreenworksbyiden-
metabolismsupportinglatentdisease.Onesuchanti-macrophage tifyingfactorsthatareeffectiveagainstimmunecellsviasuccessful
region described by the screen encodes an NRPS gene cluster recombinant expression in E. coli. Production of vaccines via
(BylA)withhomologytotheSylA(SyringolinA)producinggenes recombinantexpressionissafer,andmoreefficient,thanpurify-
fromPseudomonassyringae(Dowlingetal.,2010).SylAisapro- ingfromthepathogenitself.Further,thetechniquenarrowsdown
teasomeinhibitorandpotentcytotoxin(Grolletal.,2008).NRPS theminimumgeneticareaneededtoproducearesponse.Sofar
and PKS gene clusters and their secondary metabolite products purifiedproteinshavehadlimitedsuccessasvaccines,mainlyas
areanunexploredclassofvaccinecandidates.Purifiedsecondary thisapproachrequiresactualidentificationofvirulencefactorsor
metabolitesmaybedirectlyusefulasantigens,andthereisalsothe antigenicdeterminantsascandidatesfirst.Thisproblemistackled
potentialformutatingNRPS/PKSgeneforuseaslive-attenuated by the screening process, which is also able to identify hypo-
vaccines. thetical proteins with a role in macrophage toxicity. Identifying
virulencefactorsand/orantigenshaspreviouslyproveddifficult
PREVIOUSLYEVALUATEDVACCINECANDIDATESIDENTIFIEDBYTHE forintracellularbacteria(Titball,2008;Pateletal.,2011).Weare
SCREEN currentlydevelopingafurther novelgenomicscreen tolookfor
The subunit vaccine candidate PotF and the asd (aspartate-β- virulencefactorsinvolvedinintracellularsurvivalandpersistence.
semialdehyde dehydrogenase) gene whose mutation has been Thirdly,suchscreeningisrapidandcost-effective,withoutneed-
investigatedforuseasalive-attenuatedvaccinewerere-identified ingClassIIIsafetyfacilitiesandtrainedstaff.Suchrequirements
(Harlandetal.,2007a;Norrisetal.,2011).Asdaminoacidprod- can restrict the progress of vaccine candidate identification and
ucts have a role in cell wall biosynthesis and are likely to be testing.
necessary for growth and replication (Norris et al., 2011). The OnelimitationofthescreenisthesizerangeofDNAfragments
asd gene may have equipped recombinant library E. coli with clonedintotheBACandfosmidlibrariesused(10kband40kb
an increased growth ability enablingthem to cause cytotoxicity respectively) therefore anyfactors abovethis sizewill bemissed
byoverwhelmingthemacrophages.PotFisanABCtransporter- (forexamplecompletetypeIIIsecretionsystems).Forsuccessful
associated proteininvolvedinputrescine transportdocumented screening high library quality, in particular good genomic cov-
asnecessaryforvirulenceinotherbacterialpathogens,putatively erage, is necessary to allow repeated identification of cytotoxic
involvedinhostcellattachmentandvirulenceasinahomologous clones containing the same genetic region in order to generate
ABC transporter in the plant pathogen Agrobacterium tumefa- confidenceintheROIs identified. TheE. coli used inscreens to
ciens and for invasion and intracellular survival in Salmonella datearenotmodifiedwithforexample,regulatorysystems,func-
enterica(Matthysseetal.,1996;Jelsbaketal.,2012).Outermem- tional secretion systems, or promoters of secondary metabolite
braneproteinA(OmpA)membersOmp3andOmp7(Haraetal., production. Although this may limit what is detected, it is also
2009) and also Omp85 (Su et al., 2010) have been researched servesasanadvantage,asanycytotoxicactivitydetectedissimply
assubunitvaccinecandidates,however, protection affordedwas aresultofsuccessfulproductionbytherecombinantlibraryE.coli
no greater than with other strategies. Several Omps were iden- expressing a B. pseudomallei DNA fragment. This immediately
tified by this screen, but not those so far tested as candidates. allowsustonarrowdownwhatcanbeproducedinarecombinant
Interestingly, the majority of previously studied vaccine candi- system and importantly identify the minimum genetic region
dateswerenotre-identified.Themainreasonforthisisthatthe necessarytoproducebioactivityinsucharecombinantsystem,as
screenlooksspecificallyfortoxicitytowardmacrophages.Existing wouldberequiredforcultureandproductionofarecombinant
subunit candidates were selected for potential immunogenicity subunit vaccine. Successful secretion/release from recombinant
and are not necessarily cytotoxic. Most of the genes selected E.coliandeffectoruptakebymacrophagesmayalsolimitthefac-
for mutationinthedevelopmentof live-attenuated vaccines are tors detected. Some factors may achieve access to macrophages
involved in biosynthetic processes and expression of these in asthecellstakeinsolutesnon-specificallyfromtheextracellular
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Dowling NovelvaccinecandidateidentificationinBurkholderia
media (inoculated with clone lysate preparation) via pinocyto- persistencewithincellsinthehostcouldhelptomakethistypeof
sis/macropinocytosis. Further, some viable E. coli in the lysate vaccinemoreviableforuse(Pateletal.,2011).Thesegainoffunc-
preparationappearsnecessaryfortoxicityinsomecases. tionscreenscanbeusedto“mixandmatch”recombinantvaccine
candidates to investigate combinations for possible multivalent
PATHWAYSFORFUTURERESEARCH subunit vaccines. A combination vaccine composed of polysac-
The number of candidates identified by the gain of function charideandproteinpresentsthemostpromisingformulationfor
screenislarge(>100).Combinedapproachescorrelatingdatasets amelioidosisvaccinetodate(Pateletal.,2011).
obtainedfromtechniquessuchasinvivoexpressionwiththistype
ofrecombinantscreenwillfurthernarrowdownkeyvaccinecan- CONCLUDINGREMARKS
didates for follow-up. Use of advanced cytotoxicity screens and BydirectlyaskingmacrophageswhichgenomicregionsofB.pseu-
high-contentphenotypescreeningwillallowdetailedfunctional domallei express anti-macrophage gene products in a recombi-
characterization of candidates. Further investigation of positive nantlibrarysystemwecanbegintoisolatevirulencefactorsand
regions identified will involve transposon mutagenesis to iden- potentialvaccinecandidates.Wecanexploreandcharacterizethe
tifyminimumregionofactivity/genesinvolvedandsub-cloning effects of single virulence factors provided that activity can be
ofthesegenestoconfirmactivity.Anotherusefulnextstepwould reproducedinrecombinantE.coli.This“gainoffunction”tech-
betomeasuretheextentoftheimmuneresponseofmacrophages niquecanbestrengthenedbycombiningwithothermethods,for
challengedwiththeanti-macrophagefactorsidentifiedfromthe example, in vivo expression data, providing a powerful tool for
originalscreenby,forexample,assayingnitricoxideproduction. discovery,andprioritizationofkeyvaccinecandidatesforfuture
Screeningforadherenceandintracellularpersistencewillidentify research.
factorsinvolvedintheseaspectsofB.pseudomalleiinfection.This
couldprovevaluablefordevelopmentoflive-attenuatedvaccines ACKNOWLEDGMENTS
forwhichtheissuesofsafetyandpotentiallatencyofthemutant Iwouldliketothankallofthoseinvolvedinthedevelopmentof
B.pseudomalleibacteriaareofmajorconcern.Introducingaddi- the screening process and Prof. R. ffrench-Constant for critical
tional mutations which will limit or prevent adherence to or readingofthemanuscript.
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