Virulence3:6,504–509;October1,2012;G2012LandesBioscience luxS Disrupting the quorum sensing gene does Bacillus anthracis not significantly affect virulence in mice or guinea pigs Joel Bozue,* Bradford S. Powell,† Christopher K. Cote, Krishna L. Moody,4 H. Carl Gelhaus,1 Nicholas J. Vietri and David A. Rozak TheUnitedStatesArmyofMedicalResearchInstituteofInfectiousDiseases;FortDetrick,MDUSA Currentaffiliations:†CernomicsSolutions;Frederick,MDUSA;‡DepartmentofMicrobiology;BostonUniversitySchoolofMedicine;Boston,MAUSA; 1BattelleBiomedicalResearchCenter;BattelleMemorialInstitute;Columbus,OHUSA Keywords: Bacillus anthracis, LuxS, anthrax, animal models of infection Manybacterialspeciesusesecretedquorum-sensingautoinducermoleculestoregulatecelldensity-andgrowthphase- dependent gene expression, including virulence factor production, as sufficient environmental autoinducer concentrations are achieved. Bacillus anthracis,the causativeagentof anthrax, containsa functional autoinducer (AI-2) system,whichappearstoregulatevirulencegeneexpression.TodetermineiftheAI-2systemisnecessaryfordisease,we constructedaLuxSAI-2synthase-deficientmutantinthevirulentAmesstrainofB.anthracis.Wefoundthatgrowthofthe LuxS-deficient mutant was inhibited and sporulation was delayed when compared with the parental strain. However, sporesof theAmes luxSmutantremained fully virulent inboth mice andguineapigs. Introduction A functional AI-2 molecule was identified in the Sterne strain of B. anthracis.8 Researchers found subsequently that AI-2 QS Bacillus anthracis is a rod-shaped, Gram-positive bacteria and the inhibitors limit growth and toxin gene expression in the etiological agent of anthrax.1 In response to nutrient depletion, bacterium.9 Recently, a luxS-deficient mutant of the B. anthracis B.anthracisformshighlyresistantsporesandcanremaindormant Sterne strain was shown to exhibit similar phenotypic defects.13 and viable in the soil for decades.2 When these spores are These findings suggest that opportunities might exist to treat deposited in the lungs, gastrointestinal tract, or skin lesions of a B. anthracis infections using QS inhibitors. Therefore, we susceptible animal, they germinate3 to form toxin-producing, challenged mice and guinea pigs with a luxS-deficient mutant of vegetativebacilli,whichcanrapidlyproliferateandoverwhelmthe thefullyvirulentAmesstraintodeterminewhetherdisruptingthe host.4 Improved and novel anthrax treatments are needed to B. anthracis AI-2 QS pathway limited the severity of B. anthracis address the risks posed by possible antibiotic- and/or vaccine- infections in small animal models. As reported here, our study resistant B. anthracis strains.5 failed to reveal statistically-significant differences in survival rates Many Gram-negative and -positive bacteria use methods of between animals challenged with B. anthracis Ames wild-type or intracellularcommunications,referredtoasquorumsensing(QS), luxS mutants. to coordinate gene expression in response to local cell density. One QS pathway that is common to both Gram-negative and Results -positive bacteria is the autoinducer-2 (AI-2) pathway. This system has been well described in Vibrio harveyi where the LuxS Disrupting the luxS gene eliminates AI-2 production in synthase produces the AI-2 signal molecule from S-ribosylhomo- B. anthracis Ames. The luxS gene (BA5047) encodes an AI-2 cysteine,abyproductofS-adenoxylmethioninemetabolism.6The synthase,whichproducestheAI-2QSreportermolecule.Because membrane-permeable AI-2 signal molecule binds to LuxP in the luxS gene is monocistronic and downstream open reading neighboring cells to initiate a phosphate transfer cascade, which frames (BA5045 and BA5046) are encoded in the opposite leadstothedeactivationofthenegativeresponseregulatorLuxO.7 direction,14,15 disruption of the luxS gene would not have a AI-2signalmoleculeshavebeenshowntoregulategenesencoding downstream polar effect. Therefore, using a technique previously a variety of functions, including toxin expression and other applied by ourselves and others to study B. anthracis pathogen- bacterial virulence determinants.8-12 esis,16-20wedisruptedtheB.anthracisAmesluxSgenebyinserting *Correspondenceto:JoelBozue;Email:[email protected] Submitted:07/03/12;Revised:08/14/12;Accepted:08/22/12 http://dx.doi.org/10.4161/viru.21934 504 Virulence Volume3Issue6 BRIEFREPORT the erythromycin-resistant pEO3 plasmid into the middle of the while the luxS mutant is not significantly defective in spore codingregionbyhomologousrecombination.Wethenconfirmed production, it is defective in the progression through at least the proper formation of the merodiploid mutant by PCR-amplifying earlystages oftheprocess. Asaresult,luxS cells progress through a gene fragment that spanned the junction of the pEO3 plasmid sporulation less efficiently or less rapidly than wild type. and the luxS open reading frame. Similarly assaying multiple Finally, to assess the stability of luxS cointegrate mutants, 200 colony isolates from media- and animal passaged-mutants coloniesfromthesporulationassaywerepickedandpatchedonto confirmed stable retention of the insert, even in the absence of Luria-Bertani (LB) agar plates with and without erythromycin. the selective antibiotic (data not shown). Noerythromycin-sensitivecoloniesweredetected,indicatingthat We created the B. anthracis Ames luxS mutant to eliminate the cointegrate mutant was genetically stable during the in vitro endogenous production of the AI-2 reporter molecule and cultureconditions.WefurthervalidatedmutantstabilitybyPCR evaluatetheeffectofAI-2-deficientbacteriaonmouseandguinea analysis of DNA derived from several of the antibiotic-resistant pig survival. Therefore, to show that we successfully eliminated colonies.PCRanalysisindicatedthattheluxSgeneineachofthe AI-2 signal molecule production in the luxS mutant, we used sampled colonies were disrupted by a pEO3 integrant (data not filtered conditioned medium from wild-type and mutant shown). B. anthracis cultures to induce bioluminescence in the V. harveyi LuxS-deficient mutants remain as virulent as wild-type BB170 reporter strain.21 Filtered medium collected from bacteria in mouse and guinea pig models. Because QS has been stationary phase cultures of both the wild-type and luxS bacteria linkedtovirulencegeneexpression inmanypathogens,including were added to the reporter strain and luminescence measured. toxin gene expression in B. anthracis,9,13 we evaluated the Ames The conditioned medium from the wild-type strain was able to luxSmutantinmultiplesmallanimalmodelsofanthraxinfection. induce bioluminescence, thus demonstrating the production of a First, we compared survival of BALB/c mice challenged with functional AI-2 molecule in the Ames strain. However, when either wild-type or luxS spores via intranasal (~2.65 (cid:1) 106 spores conditioned medium from the Ames luxS mutant was added to in 50 ml of water) or intraperitoneal (~2,700 spores in 100 ml of the reporter strain, the bioluminescence detected was no greater water) delivery. For these challenge models, previous studies than what was observed with buffer alone (Fig.1). These data calculatedtheAmesstrainLD formicetobe3.7(cid:1)104and500 50 confirm that the AI-2 signal molecule is not produced by the spores, respectively.23,24 As shown in Figure4, there were no Ames luxS mutant. statistically significant differences in survival or time to death The luxS mutant is deficient in growth and delayed in between mice challenged with wild-type or luxS mutant bacteria, sporulation. A previous report indicated that a luxS mutation in regardless of challenge route. theattenuatedSternestrainofB.anthracisledtodecreasedgrowth In addition to the mouse models of infection, guinea pigs in liquid culture.13 Given this relationship between LuxS activity (n = 10 for luxS and n = 5 for Ames wild-type) were challenged and bacterial growth, we compared the growth kinetics of the intramuscularly (~350 spores in 200 ml of water). A previous Ames wild-type and luxS mutant strains in brain heart infusion studycalculatedtheAmesstrainLD foraguineapigtobe~100 50 (BHI)broth.ConsistentwiththefindingsforB.anthracisSterne, spores.25 Our study used a relatively low challenge dose so as to theAmesluxSmutantgrewslowerthanwild-typeAmesinliquid avoidoverwhelming theanimalsandtoensure thatalowlevel of culture (Fig.2). attenuation in the virulence of spores could be observed. WealsofoundthattheluxSmutantwasimpairedforgrowthin However,allanimalssimilarlysuccumbedtoinfectionwitheither Difco Sporulation Medium (DSM) when compared with the strain by day 3, and no differences in survival or time to death parental strain (data not shown) and measured about a log were observed (data not shown). reductioninthetotalnumberofcolony formingunits(CFU)/ml Finally, to exclude the possibility that reversion of the luxS at the 24 h time point (Fig.3). While the drop in measured mutanttothewild-typeformcouldbeoccurringduringinfection, CFU/ml is likely due in part to slower growth, it may also be guinea pig spleens were removed and bacteria recovered. A attributabletothemoreextensiveaggregationofvegetativebacilli representative sample of bacteria (100 CFU from each mutant- inluxS cultures than inwild-typecultures, which exhibited more challenged animal) was screened on LB agar plates with and free-floating spores under a light microscope (data not shown). without erythromycin. All plated CFU remained antibiotic AlthoughluxSmutantwasimpairedforgrowthinDSM,thetime resistant, indicating that the guinea pigs had succumbed to at which the culture left exponential growth and began to infection by luxS mutants and not wild-type revertants. sporulate was similar to that of the wild-type bacteria. To confirm that the luxS mutant was producing fewer spores Discussion thanwild-typebacteria,weanalyzedsporulationbymeasuringthe number of heat-resistant spores at 24 and 48 h after the onset of Many bacterial species possess density- and growth phase- sporulation. Heat-resistance appears at an intermediate point in dependent genes, which respond to extracellular signaling sporulation and is a reliable measure of progression through all molecules that accumulate in the environment. These QS signals butthefinalstagesofsporeformation.22At24h,only15%ofthe regulatevariousbacterialfunctionsandhavebeenshowntoaffect viableluxSmutantcellswereheat-resistantcomparedwith92%of the expression of numerous virulence factors.8-12 B. anthracis wild-typebacteria.However,by48h,100%oftheluxScellswere containsafunctionalAI-2systemthatregulatesthegeneencoding resistant to high temperature (Fig.3). These data suggest that the S-layer protein as well as other virulence genes, such as pagA, www.landesbioscience.com Virulence 505 Figure1.ConditionedmediumfromtheB.anthracisluxSmutantdoesnotinduceaV.harveyiQSresponse.AI-2activityisshownasluminosityexpressed fromV.harveyistrainBB170duringgrowthinresponsetotheadditionofautoinducerbufferorfiltered,conditionedmediumfromV.harveyi(BB170),the wild-typeB.anthracisstrain(Ameswt),ortheB.anthracisluxSmutant(AmesluxS).(A)Luminosityvaluesat3hofgrowth.Theerrorbarsrepresentthe standarddeviationfromthereadingsofsixreplicates.Thedifferencebetweenthewild-typeAmesandluxSmutantwasstatisticallysignificant(p=3.15 (cid:1)10−6).(B)Luminosityvaluesatvariousgrowthtimesoftheassay.Thesedataarerepresentativeoftheresultsoftwoindependentexperiments. pagR, lef and cya.8,9,13 These findings suggest that the AI-2 QS observed for completion of sporulation with the luxS mutant system might provide an effective therapeutic target against when compared with the parental strain (Fig.3). To date, a role B. anthracis infection. To further evaluate this possibility, we fortheLuxSAI-2synthaseproteininB.anthracissporulationhas constructed a LuxS-deficient mutant of the fully virulent not been demonstrated. Furthermore, a recent microarray study, B. anthracis Ames strain to determine if the LuxS AI-2 synthase which compared the parental and luxS mutant Sterne strains,13 is necessary for virulence in mouse and guinea pig models of did not reveal expression differences of known sporulation genes. infection. However,aluxSmutantintherelatedBacillussubtilisnattostrain The B. anthracis Ames luxS mutant exhibited an in vitro exhibited delayed formation of fruiting bodies during spore growth defect (Fig.2) similar to that previously observed in a development.26 B. subtilis sporulation is regulated by both the luxS-deficient Sterne strain.8 Additionally, a temporary delay was ComXpeptideautoinducer27andsmallRNAs(sRNA).28Because 506 Virulence Volume3Issue6 The main goal of this study was to determine if the AI-2 QS pathway is significant for B. anthracis virulence. Therefore, we created and then evaluated virulence of an Ames luxS mutant in severalsmallanimalmodelsforB.anthracisinfection.BALB/cmice were tested by both intranasal and intraperitoneal routes of infection.Inaddition,guineapigswerechallengedintramuscularly. DespitepreviousstudieslinkingAI-2signalmoleculestotoxinand virulence factor regulation and the fact that our B. anthracis luxS mutant was deficient in growth, we saw no statistically significant differencesineitherthetimetodeathorsurvivalratesforanimals challenged with mutant and wild-type isolates (Fig.4). These resultsdemonstratethattheintactAI-2QSpathwaydoesnotplaya significantroleinB.anthracisinfectionintheseanimalsandsuggest that the AI-2 synthase enzyme would not provide an effective therapeutic target. Although LuxS activity is known to affect virulenceinotherbacterialspecies,30-32ourstudyusingtheserodent Figure2.TheB.anthracisluxSmutantexhibitedimpairedgrowthinBHI modelsofanthraxinfectiondemonstratesthatB.anthracismaybe broth.ComparisonismadeofgrowthbetweentheAmeswild-type amongthose bacteria forwhich theAI-2 synthase doesnotplay a (circles)andtheluxS(squares)bacteriainBHIbroth.Thesedataare significantroleininfection.33-36 representativeoftheresultsofatleastthreeindependentexperiments. Methods AI-2 and sRNA are synergistic in regulating Vibrio cholera29 and possibly B. anthracis13 toxin expression, perhaps such combined Bacterial growth and sporulation. Escherichia coli were cultured regulation may explain the delayed sporulation results observed in LB medium supplemented with 50 mg/ml of ampicillin. The with the luxS mutant of B. anthracis. AmesstrainofB.anthracis37wasculturedineitherLBmediumor Figure3.LuxS-deficientbacteriashowsignsofdelayedsporulation.Ameswild-typeandluxSbacteriawereinoculatedatequalconcentrations(byOD ) 600 inDSM.CFUcountswereobtainedat24hand48hbyplatingculturesamplesbeforeandafterheatingfor30minat65°C.Thepercentagesofheat- resistantsporesateachtimepointareindicatedabovethecolonycountsandarebasedonthefractionofheat-resistantcellsinthesampledmedia. Thesedataarerepresentativeofthreeindependentexperiments. www.landesbioscience.com Virulence 507 merodiploid mutant as previously described.16 PCR analysis was used to confirm stabile disruption of the luxS gene in both culture- and animal-passaged bacteria. AI-2production.Apreviouslypublishedbioluminescenceassay was used, with minor modifications, to detect endogenously producedAI-2signalmoleculesinwild-typeandluxSB.anthracis cultures.21Briefly,anovernightcultureofV.harveyistrainBB170 (ATCC) was grown in LB at 30°C, diluted 1:5,000 into autoinducer buffer, and 990 ml aliquots were distributed into optical-grade micro-titer plates preloaded with 10 ml of condi- tioned medium. The filtered, conditioned medium was prepared from liquid bacterial cultures of B. anthracis Ames wild-type or luxS strains grown to stationary phase in BHI at 37°C. Similarly prepared filtered medium from V. harveyi was used as a control. The assay plate was incubated at 30°C with orbital shaking, and luminescencewasmeasuredat490nmeveryhourusingaVictor2 Multilabel Counter (Perkin-Elmer). Each test subject was averaged from six replicates across a single microtiter plate. Animal challenges. To assess potential changes in virulence associated with disrupting the B. anthracis Ames AI-2 QS pathway, spores from both wild-type and mutant strains were used in mouse intraperitoneal and intranasal models,39 as well as the guinea pig intramuscular model.25 Research was conducted under an IACUC-approved protocol in compliance with the Animal Welfare Act, PHS Policy and other federal statutes and regulationsrelatingtoanimalsandexperimentsinvolvinganimals. Thefacilitywherethisresearchwasconductedisaccreditedbythe Association for Assessment and Accreditation of Laboratory Animal Care, International and adheres to principles stated in Figure4.LossofLuxSAI-2synthaseactivitydoesnotaffectB.anthracis the Guide for theCare and Use ofLaboratory Animals, National virulenceinmice.Therewasnostatisticaldifferencebetweenthe Research Council, 2011. survivalcurvesforBALB/cmicechallengedwithB.anthracisAmesluxS Statistics. Survival rates were compared between groups by spores(squares;n=10)orwild-typespores(circles;n=10)regardlessof Fisher exact tests with permutation adjustment for multiple whetherthesporeswereadministeredintranasally(A)(~2.65(cid:1)106 comparisons using SAS Version 8.2 (SAS Institute Inc., SAS spores;p=0.72)orintraperitonally(B)(~2,700spores;p=0.49). OnlineDoc, Version 8). For comparing data from biolumin- escence experiments and time to death studies of mouse BHI broth. For construction and selection of the luxS mutant, challenges, statistical significance (p , 0.05) was determined by medium was supplemented with 5 mg/ml of erythromycin, the two-tailed Student’s t-test. otherwisethemutantwasgrownunderthesameconditionsasthe wild-typestrain.Toinducesporulation,B.anthraciswasgrownin Disclosure of Potential Conflicts ofInterest DSM38 and spores were purified as previously described.16 No potential conflicts of interest were disclosed. Because B. anthracis spores are resistant to heat, spore formation was measured by counting CFUs before and after incubating the Acknowledgments bacterial cultures at 65°C for 30 min. We thank Gabriel Rother for his invaluable technical assistance, Mutant construction. The luxS mutant was constructed by Diane Fisher for completing the statistical analysis and Adam PCR-amplifying an internal fragment of the B. anthracis Ames Driks and Brad Stiles for their helpful comments and review of luxS gene (nucleotides 120–224, where ATG = 1–3) using this manuscript. The research described herein was sponsored by two internal primers (5'-TTGCCAACCGAATAAAC-3' and the Defense Threat Reduction Agency JSTO-CBD project 5'-TCAAAATGTGGATAACGAT-3') and cloning the PCR 1.1A0021_07_RD_B. 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CoteCK,ReaKM,NorrisSL,vanRooijenN,Welkos growthandvirulencefactorexpression.Virulence2010; Bacteriol 2006; 188:4442-52; PMID:16740951; SL.Theuseofamodelofinvivomacrophagedepletionto 1:72-83;PMID:21178420;http://dx.doi.org/10.4161/ http://dx.doi.org/10.1128/JB.00165-06 study the role of macrophages during infection with viru.1.2.10752 27. WatersCM,BasslerBL.Quorumsensing:cell-to-cell Bacillusanthracisspores.MicrobPathog2004;37:169-75; 14. Jones MB, Blaser MJ. Detection of a luxS-signaling communicationinbacteria.AnnuRevCell DevBiol PMID:15458777; http://dx.doi.org/10.1016/j.micpath. moleculeinBacillusanthracis.InfectImmun2003;71: 2005;21:319-46;PMID:16212498;http://dx.doi.org/ 2004.06.013 3914-9; PMID:12819077; http://dx.doi.org/10.1128/ 10.1146/annurev.cellbio.21.012704.131001 IAI.71.7.3914-3919.2003 www.landesbioscience.com Virulence 509