Phenotypic Variation and Modulation in Bordetella ...

1 , ,June1984, $ , * ,NationalInstituteofAllergyandInfectious Diseases,RockyMountainLaboratories,Hamil ton,Montana59840 Received23 September1983/Accepted15 March1984 MostoftheisolatesofBordetellabronchisept icaobtainedbythislaboratorypossessedacha racteristiccolonialmorphologywhengrownon Bordet-Gengouagar(BGA)at37 (Dom')withasmoothcolonialsurface(Scs+)an daclearzoneofhemolysis(Hly+).FromtheseDo m'Scs+Hly+BGAcolonytypesaroseflat(Dom-), smoothcolonialsurface(Scs+)andnonhemolyt ic(Hly-) 'Scs+Hly+andDom-Scs+Hly-BGAphenotypevari ants(BGA-PVs) ,andtheirwholecelllysateswerecomparedwit heachotherbysodiumdodecylsulfate-polyacr ylamidegelelectrophoresis(SDS-PAGE).Char acteristicSDS-PAGE profileswereobservedforeachoftheDom'Scs+ Hiy+andDom-Scs+Hly-BGA-PVswithregardto(i )surface-exposedproteins,basedonautoradi ographsof125I-Iodogen-labeledorganisms,( ii)polypeptidedifferences,basedongelssta inedwithCoomassiebrilliantblueR-250,and( iii) 'Scs+Hly+andDom-Scs+Hly-BGA-PVstransferr edandgrownonbrucellaagar,Trypticasesoyag ar, ,theDom'Scs'Hly+BGA-PVsfromsixofeightstr ainsshowedSDS-PAGE profilesidenticaltothoseofDom-Scs+ ,sinceDom'Scs+Hly+organismspassedbackont oBGAexpressedbothDom'Scs+Hly+colonialmor phologyandDom'Scs+Hly+ (9,10)whereinbloodagarwasusedfordiscrimi natingamongthephenotypevariants(PVs) (Table1).

2 ,morerarely,to"roughphases." (1)whereinbrucellaagar(BA) (Table2). ,regardlessoftheanimalspeciesfromwhichth eyhadbeenisolated(1).Inadditiontothestab lephenotypesjustdescribed,bothLacy(7)and Nakase(9,10) ,whenNakasegrewphaseIorganismsonnutrient agar(NA)oronbloodagarcontaining1%magnesi umsulfate, (BGA), ,medium-dependentphenotypicshiftwascalle dantigenicmodulationbyLacey(7). (11).Wewereimpressedwiththe* :DepartmentofMedicalMicrobiology,TheUniv ersityofAlberta,Edmonton,Alberta, , , (SDS-PAGE)witheitherCoomassiebrilliantbl uestain-ing,1251-Iodogenlabeling,orperio date-silverstainingforlipopolysaccharide (LPS), ,2320,Rab10,22067, (RockyMountainLaboratories).Strain10540w asobtainedfromtheNationalCollectionofTyp eCultures,London,England, (virulent)andstrain201(avirulent) ,UniversityofAlabama, ,UniversityofTennessee,Knoxville;thephas edesignationsforthesestrainsarethosedefi nedpreviouslybyBemisetal.(1):110H,501,an dRat1(allphaseI);87,17640 SAC,andBTS(allintermediatephase);11 ONH, ,andCo-lumbus(allroughphase).

3 AllstrainsandtheirPVsweregrownonBGAprepa redaspreviouslydescribed(11). (eitheraStereozoom4oraStereozoom7;Bausch &Lomb,Inc.,Rochester, )withbothreflectedlightasdescribed681 on November 28, 2018 by from 'Agglutinatedbyantiserumtoantigenb:NameC olonymorphologyCapsulestainFlagellaLH010 5 PhaseISmall,round,transparent,convex,cle arzone++-++++ or--ofhemolysisPhaseIIVariablecombinatio nofphaseIandphaseIII+or-++or-+or-+++Phas eIIIL arge,smooth,entire,lowconvex,-+--++++'or -d-cor++dnonhemolyticaAccordingtoNakase( 9,10).bAntigens:L,heat-labile(70 C,1h)capsularantigen;S,slightlyheat-stab le(100 C,1h)surfaceantigen;H,heat-labile(70 C,1h)flagellarantigen;01,05,heat-stable( 120 C,1h) (11)andalsowithsubstagelightingwithasilv eredmirrorasdescribedbySwanson(13).Singl ecoloniesofthedesiredcolonialmorphologyw erepassedat2-to3-dayintervals,andtheplat eswereincubatedat37 (DifcoLaboratories,Detroit,Mich.)perlite rtoeithercommercialbrucellabroth(AlbimiL aboratories,Flushing, )ortobrucellabrothmadeaccordingtoaformul adescribedprevi-ously(15).

4 Trypticasesoyagar(TSA)waspreparedbyaddin g15gofBactoagarto1literofTrypticasesoybr oth(BBLM icrobiologySystems,Cockeysville,Md.). ,theorganismswereharvestedanddilutedin50 mMTris-43mMsodiumglutamate-90mMsodiumchl oride(TGS; ). , (11). (13by100mm). (BeckmanInstruments,PaloAlto,Calif.) , (1mm),pulvinate,-+smooth,opaque,entire,h e-molyticonbloodagarIntermediateMedium(1 -2mm),convex,++or-smooth,opaque-transluc ent,entire,nonhemolyticRoughphaseLarge(2 mm)umbonate,rough,++or-translucent,undul ateedge,nonhemolyticaByBemisetal.(1). (basedontheLowryassaywithbovineserumalbu minasastandard[8]).Totalacrylamideconcen -trationintheslabgels(12by14cm) (wt/vol) ,N,N' (wt/vol),anda5%(wt/vol) ,gelswerefirstfixedovernightin5%aceticac id-25%isopropanol(vol/vol) (11).PutativeLPSbandswerevisualizedbythe silverstainingtechniqueofTsaiandFrasch(1 4).Fiftymicrolitersofwholecelllysate,pre paredasabove,wasincubatedwith10pgoffresh lypreparedprotein-aseK(BoehringerMannhei mCorp.,Indianapolis,Ind.)at56 Cfor3hwithvigorousvortexmixingat30-minin tervals(5,12).

5 Tenmicrolitersofthedigestedlysatewasthen appliedperlaneofa16%(wt/vol) (13by100mm). ,ulofTGS,andtheentirevolumewastransferre dtoadramvialpreviouslycoatedwith1,ugof1, 3,4,6-tetrachloro-3x,6a-diphenylglyco-lu ril(lodogen;PierceChemicalCo.,Rockford,I ll.)(11).TwentymicrocuriesofNa125I(highs pecificactivity;NewEnglandNuclearCorp.,B oston,Mass.)in10 RIofTGSwasthenadded, ,thenresuspendedin50 RIofSDS-solubilizingsolution, , (Kodak,Rochester, )at-70 Cfor11hinthepresenceofaDuPontCronexLight ningPlusintensifyingscreen( ,Wilmington,Del.). on November 28, 2018 by from ,theircolonialmorphologyonBGA, :domed,smoothcolonialsur-face,andhemolyt ic(Dom'Scs+Hly+)andflat,smoothcolonialsu rface,andnonhemolytic(Dom-Scs+Hly-).Othe rBGA-PVsofintermediatemorphologies, 'Scs+Hly-,Dom'Scs-Hly-,etc., , ,isogenicDom-Scs+Hly-couldbeeasilyisolat edfromtheDom'Scs+Hly+colonytypeofanystra insincethefrequencyofvariationfromDom'Sc s+Hly+toDom-Scs+Hly-rangedfrom10-2to10-3 forsixstrainstested(214,2320,10540,22067 ,Rab10,and899L).

6 Oncecloned, + ,labeledwith1251bylodogen, ,subtle,butneverthelesscharacteris-ABtic ,Coomassieblue-stainedpolypeptideprofile scouldbeassignedtoorganismsofeithertheDo m'Scs+Hly+orDom-Scs+Hly-colonytype(excep t899L;seebelow).ArrowheadsdenoteCoomassi eblue-stainedbandsseeninDom'Scs+Hly+orga nismsbutabsentorinlowerconcen-trationsin Dom-Scs+ ,whicharecharacteristicfororganismsofeac hcolonytype,anexceptionagainbeingstrain8 99L,whoseintermediatecolonialmorphologie sbothhaveCoomassie( )and1251profiles( )whichappeartobemixturesoftheDom'Scs+Hly +andDom-Scs+ ,subjectedtoSDS-PAGEin16%(wt/vol)totalac rylamidegels, ,therefore,representstheLPSprofiles(5,12 )fromthetwomajorphenotypesandshowsthatDo m'Scs+Hly+ ,slower-migratingbandaandalightlystained bandc,whereasDom-Scs+ ,whichmigratedfasterthanbandd,wasalsoobs ervedintheDom-Scs+Hly-PVsofstrainsRat1an d8442(notshown).Additionalbroadsilver-st ainedbands(brackets) (f-;tDom"S(--c;-eDfornrScs' Con(A)BGAand(B) ,1984 on November 28, 2018 by from ConBGAorBABGABAC olonyColonydiamSSPGConyColonySDS-PAGES ourceStrainphenotype(hemolysisSDSfPleCol onydiamPrfEphenotype'diam)profiletypephe notype'(MM)bprfltye(mm)bMunoz214 Dom'Scs+Hly+ ( )1 Dom'Scs+ + (0)2 Dom'Scs+ 'Scs+Hly+ ( )1 Dom'Scs+ + (0)2 Dom'Scs+ 'Scs+ ( )1 NDdNDNDDom-Scs+Hly+ ( )1 NDNDNDRab10 Dom'Scs+Hly+ ( )1 NDNDNDDom-Scs' (0)222067 Dom'Scs'Hly' ( )1 Dom-Scs' (0)2 NDNNCTC10540 Dom'Scs+Hly+ ( )1 Dom'Scs+ + (0)2 Dom'Scs+ 'Scs+Hly+ ( )1 Dom'Scs+ + (0)2 Dom'Scs+ + (0) 'Scs+Hly+ ( )1 Dom'Scs+ + (0) 'Scs+Hly+ ( )1 Dom'Scs+ + (0) 'Scs+Hly+ ( )1 Dom'Scs+ + (0)2 Dom'Scs+ 'Scs+Hly+ ( )1 Dom'Scs+ + (0)1 Dom'Scs+ 'Scs+ (0)1 Dom'Scs+ (0)2 Dom+Scs+ 'Scs+Hly+ ( )1 Dom'Scs+ + (0)2 Dom'Scs+ + (0) ' (0) + (0) 'Scs+ (0) + (0) ( , (16)frombothDom'Scs+Hly+andDom-Scs+Hly-B GA-PVs(Peppler,unpublisheddata).)))

7 ,1251-Iodogenlabeling, ,inadditiontocolonymorphology,thevarious BGA-PVsdescribedinTable3couldalsobechara cterizedbySDS-PAGE profilesintooneoftwoclasses:type1forprof ilescharacteristicoftheDom+Scs+Hly+BGA-P Vandtype2forprofilescharacteristicoftheD om-Scs+Hly-BGA-PV(Table3). ( 20%). ,forexam-ple,thetwomajorBGA-PVsfrommosts trainsbothshowedaDom'Scs+morphology( ).Thisoccurredwithallthreeintermediate-p haseprototypemorphotypesofBemisetal.(1), oneofthreeoftheirphaseIprototypes,fourof fivestrainsfromMunoz,NCTC strain10540,andaprototypevirulentstrain- phenotype,8442,fromCassell(Table3).TheDo m' (relativetotheirgrowthonBGA) ,TSA, , ,infact,revealedthatmostoftheDom'Scs+Hly + on November 28, 2018 by from ,1984~~~~~~~~~ ~~~~a.~~~~~CSiistzin,rr-);set(1c-tPf~A__ __423222? (a)Dom'Scs'Hly'and(b)Dom-Scs'Hly-PVsfrom strains214,2320,22067,Rab10,and10540,and isogenic(c)Dom'Scs'Hly'and(d)Dom-Scs'Hly 'PVsofstrain899L.(A) 'Scs'Hly'PVsandabsent(orinreducedamounts )intheDom-Scs'Hly-PVs.(B) 'Scs'Hly'PVs,whereaslower-molecular-weig htbandstendtobelabeledintheDom-Scs'Hly-P Vs, 'Scs'Hly'typeprofileandtype2fortheDom-Sc s'Hly-typeprofile.

8 (C)SamesamplepreparationsasinA, , 'Scs'Hly' ' ,broadbandsofsilver-stainedmaterialarese enineachPVpreparation(brackets). ,however, ,asinglepassageontoBGAoftheoriginallyDom 'Scs'Hly' 'Hly-BGA-PVwaslittleaffectedbygrowthmedi umasjudgedbythesameSDS-PAGE criteria( ). 'Scs'Hly'PVofstrain214onBA(lane2),TSA(la ne3),andNA(lane4)affectsitsSDS-PAGEC oomassieblue( )andLPS( )profileswhencomparedtoitsprofilesafterg rowthonBGA(lane1).Incontrast,theSDS-PAGE C oomassieandLPSprofilesfromtheDom-Scs' 'Scs'Hly'BGA-PVdescribedhereisanalo-gous tothephaseIofNakase(9,10).LikewisetheDom -Scs' (9),itsadherencetocells(17),anditsposses sionofadenylatecyclase(2).PhaseIII,incon trast, [_lodogen-labeledwhole-cellprofilesshown herearereminis-centofthoseobtainedwithth edomed,hemolyticandflat, (11) ,thenthemethodswe' , ,themajorityofourclearlydistinguishableB GA-PVshadsimilarcolonialmorphologiesonBA ,and,asjudgedbyCoomassiebluestaining,125 1labeling,andperiodate-silverstaining,th eDom'Scs'Hly' (10)hasalsoshown,byserologicalmethods, (includingheat-stable0antigens) , ,1984 on November 28, 2018 by from 'HlyNABANABA1\iNI1I\ ;BGAEBABCANABRA% ,"a, *.]

9 1,_:' :g30,-,-s'Dom-Scs-HIyDomScsHlyNABANABAto v\1N\1 NBR1,-,,. '251-Iodogen-labeledwholecelllysatesfrom theDom'Scs+Hly+andDom-Scs+ ,organismsweretrans-ferred(arrows) ,theorganismswereharvestedoffeachmediuml abeledwith1251,andsubjectedtoSDS-PAGE.(A )Coomassiebrilliantbluestained.(B) 'Scs+Hly+BGA-PVonNAandBAareindistinguish ablefromtheDom-Scs+ ,however,theDom'Scs+Hly+ , ,bytheSDS-PAGE techniquesdescribedhere, (1).AsTable3summarizes, ,manyofwhichmodulatedtotype2 SDS-PAGE profileswhentransferredontoBA,despitethe irbeingDom'Scs+Hly' 'Scs+Hly+BGA-PVsasavirulentBAmorphotypes inthesystemofBemisetal.(1).Theuseofmedia orgrowthconditionsinwhichmodulationoccur shasitsobviousdrawbacks, ,theSDS-PAGE profilesofwholecelllysatescanrevealpolyp eptides,surface-exposedproteins, ,furtherinvestigationmayrevealthattheCoo massieblue-stainedbandsuniquetotheDom,Sc s+Hly+ ,pili, 'Scs+Hly+andDom-Scs+Hly-PVs( ) ( ) , , (9).Andfinally,thedifferencesseenintheLP SprofilesoftheDom'Scs+Hly+andDom-Scs+Hly -PVs( )mayhelpdefinethephysicalandchemicalbasi sfortheantigenicheterogeneityoftheheat-s table(120 C,1h) (9).

10 Inadditiontoantigenicdiffer-ences, , (6).Fortheabovereasons, ,butalsoyieldsmoreinformationthantheSDS- PAGE profilesofeithercell-associatedproteins( 3)orsupernatantsofcellsonicates(4) , (3,4),piliation(1),flagellation(1,9,10), and0antigenexpression(10). *M:iw,;,w:j-lssamweso on November 28, 2018 by from +Hly+DomScs+Hly12341234_I--4__---_--V[ 'Scs+Hly+andDom-Scs+ (lane1),BA(lane2),TSA(lane3),orNA(lane4) andthensubjectedtoSDS-PAGE.(A)Coomassieb rilliantbluestained.(B) 'Scs+Hly+ ,TSA,orNA,however, +Hly-BGA-PVof214givesitscharacteristicb- dpatternonallmedia, ,dotheyreallyrepresentnaturaldiversityor aretheylaboratoryartifactscausedbyindisc riminantpassage? ,GailCassellandDavidBemisforsupplyingstr ainsandfortheirhelpfuldiscussions, , , , ,M., , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,reagentsandstains, , , ,Jr., (ed.),Manualofclinicalmicrobiology, ,Washington, ,O., ,Y., ,1984 on November 28, 2018 by from]