INNATE!IMMUNE!EVASION!OF!MICROBES!

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1 INNATEIMMUNEEVASIONOFMICROBES HANNEAMDAHL DepartmentofBacteriologyandImmunology HaartmanInstitute UniversityofHelsinki,Finland and ResearchProgramsUnit,Immunobiology FacultyofMedicine UniversityofHelsinki,Finland ACADEMICDISSERTATION Tobepubliclydiscussed,withthepermissionoftheMedicalFaculty, Universityof Helsinki, in the Auditorium 1 of the Haartman Institute, on 28 th May, 2014, at 12 o clock noon

2 SUPERVISORS DocentT.SakariJokiranta,SeniorLecturer,MD,PhD DepartmentofBacteriologyandImmunology, HaartmanInstitute UniversityofHelsinki,Finland and DocentTaruMeri,PhD DepartmentofBacteriologyandImmunology HaartmanInstitute UniversityofHelsinki,Finland and DepartmentofBiosciences UniversityofHelsinki,Finland REVIEWERS ProfessorBenitaWesterlundNWikström,PhD DivisionofGeneralMicrobiology DepartmentofBiosciences UniversityofHelsinki,Finland and ProfessorIlkkaJulkunen,MD,PhD DepartmentofVirology InstituteofBiomedicine, UniversityofTurku,Finland and DepartmentofVirology TheNationalInstituteforHealthandWelfare Helsinki,Finland OPPONENT ProfessorKristianRiesbeck,MD,PhD MedicalMicrobiology DepartmentofLaboratoryMedicineMalmö UniversityofLund,Sweden 2014HanneAmdahl CoverfigurebyLinus ISBN978N952N10N9933N5(Paperback) ISBN978N952N10N9934N2(PDF) PrintedatOasisMediaOy,Finland,2014

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5 CONTENTS TABLEOFCONTENTS Publications...7 Abbreviations...8 Abstract...9 1Introduction Reviewofliterature Theimmunesystem Innateimmunity Adaptiveimmunity Thecomplementsystem Recognition Activation Regulation Phagocytosisofmicrobesbyneutrophils Transmigration Chemotaxisandprimingofneutrophils Opsonizationandphagocytosis Eliminationofmicrobes Microbialevasionofcomplementandphagocytes Microbialcomplementevasion Microbialevasionofphagocytosis Examplesofinnateimmuneevasion Stapylococcusaureus immuneevasionstrategiesofagramnpositive bacterium BordetellapertussisNimmuneevasionstrategiesofaGramNnegativebacterium Aimsofthestudy Materialsandmethods Materials Complementproteins Bacterialproteins Otherproteinsandsera Antibodies Microbes Methods GenerationofC3b Radiolabelingofproteins Direct 125 INproteinbindingassays(I,II) Radioligandassays(II,III,IV) CofactorassaysforC3binactivation(I,II,III,IV)...59

6 4.2.6DecayNaccelerationactivityofFH(III) Bactericidalassays(studiesI,III) Serumandproteinabsorptionassays(I) Flowcytometricanalysis(I,III,IV) Isolationofneutrophilsandredbloodcells(IV) C3bbindingtoneutrophilsanderythrocytes Neutrophilbindingandphagocytosisassays(IV) Structuralmodeling,curvefitting,andstatisticalanalysis Results ComplementevasionofB.pertussisbyFHacquisition(I) MicrobialcomplementevasionbybindingFH19N20(II) Effectoftripartitecomplexformationoncomplementevasion(III) InhibitionofCR1Nmediatedrecognitionbystaphylococcalproteins(IV) Discussion InnateimmuneevasionNdifferencesbetweenGramNpositiveandGramNnegative bacteria SurfacestructuresofGramNpositiveandGramNnegativebacteria Importanceofcapsuleinimmuneevasion ComplementevasionatC3stage Controlbyhost'ssolubleregulators MicrobialproteinsactonC Whyoverlappingevasionmechanisms?S.aureusasanexample Examplesofevasionstrategiesofmicrobesindifferentbodyparts Vaccinedesignandtherapeutics Conclusions...81 Acknowledgements...82 References

7 PUBLICATIONS, Thethesisisbasedonthefollowingoriginalarticlesandmanuscript: I. II. III. IV. Amdahl,H.,Jarva,H.,Haanperä,M.,Mertsola,J.,He,Q.,Jokiranta,T.S.,Meri,S InteractionsbetweenBordetellapertussisandthecomplementinhibitor factorh.molecularimmunology48:697n705. Meri,T.,Amdahl,H.*,Lehtinen,M.J.*,Hyvärinen,S.,McDowell,J.V.,Bhattacharjee, A.,Meri,S.,Marconi,R.,Goldman,A.,Jokiranta,T.S.2013.Microbesbind complementinhibitorfactorhviaacommonsite.plospathogens9(4): e Amdahl,H.Jongerius,I.,Meri,T.,Pasanen,T.,Hyvärinen,S.,Haapasalo,K.,van Strijp,J.A.,Rooijakkers,S.H.,Jokiranta,T.S.2013.StaphylococcalEcbproteinand hostcomplementregulatorfactorhenhancefunctionsofeachotherinbacterial immuneevasion.journalofimmunology,191(4):1775n84 Amdahl,H,Tan,L.,Haapasalo,K.,Meri,T.,vanStrijp,J.A.,Rooijakkers,S.H., Jokiranta,T.S.2014.StaphylococcalproteinEcbimpairscomplementreceptorN1 mediatedrecognitionandphagocytosisofopsonizedbacteria.submitted. *Equalcontributions 7

8 ABBREVIATIONS,, AP Alternativepathway BSA Bovineserumalbumin C4BP C4bbindingprotein CD Clusterofdifferentiation CP Classicalpathway CR Complementreceptor CRP CNreactiveprotein DAF Decayacceleratingfactor Ecb Extracellularcomplementbindingprotein Efb Extracellularfibrinogenbindingprotein Fab FragmentantigenNbinding FB FactorB Fc Fragmentcrystallizibleregion FD FactorD FcγR Fcgammareceptor FH FactorH FHA Filamentoushemagglutinin FHLN1 FactorHNlikeprotein1 FHR FactorHNrelatedprotein fmlp Formylatedpeptides(NNformylNMetNLeuNPhe) FPR Formylpeptidereceptor GPCR GNproteincoupledreceptor HIS Heatinactivatedserum HSA Humanserumalbumin Ig Immunoglobulin IL Interleukin LPS Lipopolysaccharide LP Lectinpathway MBL Mannosebindinglectin MCP Membranecofactorprotein NADPH Nicotinamideadeninedinucleotidephosphate NETs Neutrophilextracellulartraps NHS Normalhumanserum PAMP PathogenNassociatedmolecularpattern PBS Phosphatebufferedsaline PRR Patternrecognitionreceptor Ptx Pertussistoxin ROS Reactiveoxygenspecies SCIN Staphylococcalcomplementinhibitor SCR Shortconsensusrepeat SpA StaphylococcalproteinA SSL StaphylococcalsuperantigenNlike TLR TollNlikereceptor TNFN Tumornecrosisfactoralpha TP Terminalpathway VBS Veronalbufferedsaline 8

9 Abstract ABSTRACT, Innateimmunityisthefirstlineofdefenseagainstmicrobes encounteringthehuman body. A central player in initiating and augmenting the innate immunity is the complementsystem.complementisaproteolyticandselfnamplifyingcascadeconsisting of soluble plasma proteins and membrane bound regulatory proteins. This system is abletorecognizeanddiscriminatebetweenhostandnonnhoststructuresviaactivation of three pathways: classical, lectin, and alternative pathway. All the pathways lead to formationofc3activatingenzymesresultingincovalentc3bdepositionontotargetcell surfaces. Deposition of C3b results in amplification of the activation, release of chemotacticmolecules,attractionofphagocytes,andinitiationoftheterminalpathway resultinginformationoflyticmembraneattackcomplexes. Toavoiddestructionofhostcellscomplementhastobestrictlyregulated.FactorH(FH) isthemajorregulatorofthealternativepathway.itconsistsoftwentydomainswhere thedomains1n4bindtoc3bandmediateinhibitionofthecomplementactivationwhile domains 19N20 are essential for the discrimination between host from nonnhost surfaces.fhdownregulatescomplementactivationbythreemeans:actingasacofactor in proteolytic inactivation of C3b, preventing formation of the C3 convertase, and acceleratingdecayofthec3convertase. Several pathogens utilize host FH to protect themselves against complement attack. Binding of FH provides the microbe with a powerful tool to evade, not only C3bN mediatedopsonophagocytosis,butalsoinflammation,chemotaxis,andlyticmembrane attack complexes. Several microbes have previously been shown to acquire surfacen boundfhviaitsdomains5n7and19n20.whilethenumberofpathogensreportedto bindfhisincreasing,itisknownthatsomepathogenssuchasstaphylococcusaureusdo notbindfhbutinsteadbindc3b. In this thesis there are three specific aims.first,to determinewhether the important human pathogen, Bordetella pertussis, evades complement attack by the alternative pathway.second,todiscoverbywhichmechanismsmicrobesbindfhviathecnterminal domainsfh19n20.third,tostudyhows.aureuspreventstheattackbythealternative pathwayofcomplementalthoughitdoesnotbindfh. Tostudythefirstaim,variousimmunologicalassayswereusedtoanalyzebindingofFH bydifferentbordetellastrains.theresultsshowedthatbothb.pertussisandtheclosely related B. parapertussis bound FH. The bound FH retained its regulatory activity for 9

10 Abstract factorimediatedcleavageofc3b,suggestingthatthebindingwasimportantforserum survival. Tomeetthesecondaim,i.e.toexplainwhytheFHdomains19N20isacommontarget for most FHNbinding microbes, we used several FH19N20 point mutant proteins to analyzethebindingsitesonpathogensrepresentinggramnpositiveandgramnnegative bacteria,ayeast,andthreepurifiedmicrobialproteins.theresultsrevealedaconserved binding site on the domain 20 for all the studied microbes. An explanation for the common binding site was provided by the fact that by binding to the domain 20, the microbial proteins enhanced the interaction between C3b and FH. This resulted in a moreefficientinactivationofc3bandsubsequently enhanced evasion of complement mediateddamage. The third aim of this thesis was to determine how pathogenic S. aureus evades the alternativepathwayactivationindependentlyofdirectfhnbinding.forthiswestudied smallsecretedc3bnbindingmoleculesofs.aureus,e.g.extracellularcomplementbinding protein(ecb)thatbindstothesameregionasfhonc3b.weshowedthatfhdeposition on the surface of S.aureus could be induced by the formation of tripartite complexes betweenecb,c3b,andfh.inthecomplextwooftheregulatoryfunctionsoffhwere maintained and,mostinterestingly,evenenhanced.thesignificanceofthetripartite complex formation in complement evasion was verified using a serum sensitive Haemophilusinfluenzae strain.ecbaddedtotheserumbeforeh.influenzae cells were included protected the bacteria from complementnmediated lysis, and the effect was increased by addition of the FH19N20. These results suggest that upon formation of tripartitecomplexess.aureusmayusefhforitsownprotectiontoeliminatec3b. Studies with S. aureus revealed that the formation of the tripartite complex (Ecb:C3b:FH) did not enhance inactivation of the major opsonin C3b. This was surprising, since C3b is the ligand for complement receptorn1 (CR1) expressed on phagocytes.therefore,westudiedtheroleofthesecretedproteinsininhibitingbinding ofc3btoneutrophils.theresultsrevealedthatecbblockstheinteractionbetweenc3b andcr1,whichimpairedthephagocytosisofc3bnopsonizeds.aureusandpresenceof FH enhanced this. S.aureus is able to utilize host FH in three ways to promote the tripartite complex formation so that the C3 convertase cannot be formed, to prevent CR1Nbinding leading to impaired opsonophagocytosis, and to prevent degradation of C3btoiC3btherebypreventingrecognitionofS.aureusbycomplementreceptors3and 4onphagocytes. 10

11 Abstract In conclusion, these studies have revealed an immune evasion mechanism for B. pertussisandidentifiedinmoleculardetailasiteonthedomain20offhimportantfor various microbes in complement escape. These studies also explained how S. aureus usessolublec3bnbindingmoleculestorecruithostfhforimmuneevasion,despitenot binding directly to this regulator. Taken together, these results revealed detailed knowledge on structural and functional basis that is relevant for vaccine and antimicrobialdevelopment. 11

12 1Introduction 1,INTRODUCTION, Theenvironmentisdominatedbybacteriaandeverydayourbodyhastodefenditself againstmicrobes.ourimmunesystem,however,isveryeffectiveandonlyaminorityof thesemicrobesisabletoenterandsurvivewithinthebody.theinnateimmunesystem representsthefirstlinedefenseagainstmicrobesanditcanquicklyprevent,control,or eliminate the incoming pathogen. It can also stimulate the second line of defense, the adaptive immune system, which reacts moreslowly but is highly specific and has the abilitytorememberthesamemicrobeifencounteredinthebodyagain. Skinandmucosalsurfacesprovideeffectiveprotectionagainstpathogenicmicrobesby inhibitingtheentryofmicrobestotissuesandblood.ifapathogensucceedsinbreaking through the host s physical barriers, molecules of the innate immunity are activated, such as antimicrobial agents and the complement system. The complement system targetsforeignsurfacesandmediatesdirectlysisanduptakebyphagocyticcells. The history of complement (originally called alexin by Buchner) began in the late 1880 s when several researchers observed bactericidal activity in normal serum (Lachmann 2006). A Belgian microbiologist, Jules Bordet, performed the experiment thatfinallyprovidedanexplanationforthisphenomenon.hedemonstratedthataheatn labile blood component (complement) together with a heatnstable component (antibody)hadthecapacitytolysecertainbacteria. For more than fifty years it was believed that complement required antibodies to be activated until Louis Pillemer demonstrated that complement can be activated independentlyfromantibodies(pillemeretal.1954).heoriginallynamedthepathway properdinpathway,nowknownasthealternativepathway(ap).theaptogetherwith the lectin pathway (LP) appeartohavebeenfunctionallongtimebeforetheadaptive immunity(farries&atkinson1991;pintoetal.2007).duringthelastdecadesseveral importantdiscoverieswithinthecomplementfieldhasbroadenedtheresearchanditis no longer considered to be a nonnspecific immune response, but capable of discriminatingbetweenselfandnonself.theimportanceofcomplementinimmunityis emphasizedbydeficienciesormutationsofcomplementcomponents. 12

13 2,REVIEW,OF,LITERATURE,, 2Reviewofliterature 2.1,THE,IMMUNE,SYSTEM, A wellnorganized network of cells and molecules comprises the immune system that respondstomicrobesorotherforeignsubstancesenteringthehumanbody.itspurpose is to recognize, immobilize and eliminate invading microbes and develop an immunologicalmemoryagainstpreviouspathogens.thetwobranchesoftheimmune system,theinnateandadaptiveimmunity,includeahumoralandacellnmediatedpart (Fig.1).Althoughtheyaredifferentintheirmechanismofaction,synergybetweenthe partsisessentialforafullyeffectiveimmuneresponse. Figure,1.,Schematicoverviewoftheimmunesystem., 2.1.1INNATEIMMUNITY Innateimmunityistheevolutionarilyoldestbranchofhostprotectionagainstinvading pathogens. Tears and saliva contain lysozyme, defensins, and cathelicidins with antimicrobial activity that protect and hamper the growth of pathogens (Hancock & Diamond 2000; Ganz 2003). In addition, our microbiota present on the skin and on mucousmembraneshelptoprotectagainstpathogensbyoccupyingsitesthatmightbe colonized by pathogens. If microbes manage to cross those barriers they encounter a 13

14 2Reviewofliterature powerfulandcomplexdefensesystemconsistingofproteinsofthecomplementsystem (section2.2)andphagocyticcells,includingneutrophils(section2.3). Thekeytoproperfunctionoftheimmunesystemisitsabilitytodiscriminateselffrom nonself.microbescanberecognizedbypatternnrecognitionreceptors(prrs)presenton host cell surfaces, endosomal vesicles, and even in the cytoplasm. The PRRs have signaling or phagocytic functions. Examples of phagocytic PRRs are the CNtype lectin receptors,scavengerreceptors(peiseretal.2002),andcomplementreceptors(section 2.3.3).TheGNproteincoupledreceptors(GPCRs)aresignalreceptorsandtheyinclude the NNformyl peptide receptors (FPRs) (Rabiet et al. 2007) (section 2.3.2). The evolutionary conserved TollNlike receptors (TLRs) recognize commonly expressed molecules on microbes but not the host s own cells, and they are found both on cell surfacesandonendosomalvesicles(section2.3.2).thenodnlikereceptors(nlrs)are locatedinthecytoplasmwheretheydetectintracellularmicrobes(franchietal.2009). Therearethreeclassesofphagocyticcells:monocytesandmacrophages,dendriticcells, and granulocytes which include neutrophils, basophils, and eosinophils. Monocytes differentiateintomacrophagesuponenteringvarioustissueswheretheycandivideand are important in fast recognition and phagocytosis as well as in a later phase of the infection.thegranulocytesarecirculatinginthebloodstreamandmigrateintotissues inresponsetoaninfection.ofthese,neutrophilsmediatethefastestresponseandcan migrate in large numbers within a few hours to the microbial entry site (Kim et al. 2008). Macrophages and granulocytes are central in innate immunity since they can recognize and eliminate an invader without the help from the adaptive immunity. In contrast, the functions of the dendritic cells are primarily to process and present antigens to T cells, thereby linking innate and adaptive immunity (Nussenzweig et al. 1980;Steinman2012). Natural killer (NK) cells are found in blood and various tissues where they sense infected or abnormal cells without specific sensitization and eliminate them via apoptosis. Moreover, natural killer cells secrete cytokines that activate macrophages (Vivieretal.2008). Phagocytes are attracted to the site of infection by molecules of microbial or host derived origin such as complement fragments, chemokines and cytokines. Also, mast cells and e.g. histamine are involved in this process. These chemoattractants activate phagocytes and assist their migration through the blood vessel wall (Campbell et al. 1998). 14

15 2Reviewofliterature Cytokines are proteins secreted by phagocytes and are essential in regulating and mediating cellular effector functions. Tumor necrosis factor alpha (TNFNα) activates endothelialcellstoexpressselectinsforrecruitmentofphagocytestotheinfectionsite. Host defense against certain pathogens such as mycobacteria and some viruses is significantly impaired if TNFNα or its receptor are lacking (Havell 1989; Mohan et al. 2004). Mononuclear phagocytes are stimulated by TNFNα to secrete interleukin (IL)N1 thathassimilarfunctionsastnfnα.inlocalinfections,lowconcentrationsoftnfnαare generated,however,inseveregramnnegativesepsis(septicshock)tnfnαtogetherwith othercytokines,areproducedinlargeamountsinresponsetolipopolysaccharide(lps). This cytokine storm affects the complement and coagulation cascades, leading to endothelialdamageandmultiplenorgansystemfailure. Chemokines are small polypeptides that stimulate and regulate the migration of phagocytes and other leukocytes from blood vessel to inflamed tissues. Chemokine receptorsaregpcrsfoundmainlyontcells ADAPTIVEIMMUNITY The evolutionally more recent adaptive immune system provides a more versatile althoughaslowerprotectionagainstmicrobialinfections.thecellsininnateimmunity are, however, needed for initiation of the adaptive immune response. Antigen presenting cells, such as dendritic cells, take up pathogens and migrate to the local lymphnodeswheretheypresenttheprocessedantigentorestingtcells.thisresultsin activationandproliferationintothelpercells(cd4+)andcytotoxictcells(cd8+).at thesiteofinfection,thelpercellsassistinactivationofmacrophagesformoreefficient phagocytosis whereas cytotoxic T cells directly kill infected cells or cells that lack protectivereceptors.aportionofthehelpertcellsassistsantigenactivatedbcellsto differentiateintoantibodynproducingplasmacells.althoughtheresponsetotheantigen isslow,alonglastingmemorytothespecificantigensisdeveloped. Antibodies are soluble glycoproteins produced by B cells in response to antigens. Recognitionandbindingofantibodiestoantigenscanleadtoneutralizationofavirus, toxin, or labeling (opsonization) of the target for attack by other components of the immunesystem. Antibodies are comprised of two identical heavy chains and two identical light chains andbothheavyandlightchainshaveaconstantandvariableregion(fig.2).basedon theconstantdomaintheyaredividedintofivetypes:immunoglobulin(ig)a,igd,ige, 15

16 2Reviewofliterature IgG,andIgM.IgGandIgMhaveimportantfunctionsincomplementactivationandIgG has also a direct role in opsonization. IgA is produced mainly by mucosal lymphoid tissue,andsubsequentlyformsanimportantpartofdefenseagainstmicrobesentering the respiratory or gastrointestinal tract. IgD has a function in defense against respiratorypathogensbydirectlybindingtopathogensortheirvirulencefactors.italso inducessignalsthatactivatetheproductionofantimicrobialmoleculesbyseveralcells (Chenetal.2009).IgEhasafunctionindefenseagainsthelminths. The heavy and light chains form two variable antigen binding fragments (Fab). The heavychainshavealsoaconstantcrystallizabledomain(fc)thatisconnectedtofabs viaaflexiblehingeregion.eachfabfragmentrecognizesandbindsitstargetwithhigh affinity,whereasthefcregioninteractswithfcγnreceptors(fcγrs)onleukocytes. Figure, 2., Structure of an IgG antibody. The YNshaped antibody consists of two heavy and two light chains linked together by disulphide bonds (sns) as indicated. The antigen binds to the variable region of the Fab domain while Fc region interacts with the receptor on the cell and mediates complement activation. HumanIgGscomprisefourstructurallydifferentsubclasses,IgG1N4,thatdifferintheir heavychain(γnchain).theglycosylationoftheimmunoglobulinshelpstostabilizethefc region(mimuraetal.2001)andglycansonthefcregionofiggareevenrequiredfor optimalinteractiontoitsspecificfcγrandsubsequentlytheeffectorfunctions(jefferis & Lund 2002). Immunoglobulins can also bind directly to glycans on pathogens and mannosereceptorsonphagocytes(malhotraetal.1995;dongetal.1999).thecapacity to activate complement is highest for IgG3 followed by IgG1 and IgG2, whereas IgG4 completelylacksthisability. 16

17 2Reviewofliterature 2.2,THE,COMPLEMENT,SYSTEM, An organized network of more than thirty soluble or membranenassociated proteins comprisesthepowerfulcomplementsystem.basedonitsrecognitioncapacityandits effector mechanisms, leading to either activation or downregulation of the cascade, affect both innate and adaptive immunity. By marking pathogens with opsonizing moleculesandgeneratingchemotacticmolecules,complementattractsphagocytesand enhances phagocytosis of the invader. Receptors on erythrocytes bind C3bNopsonized immunecomplexes,debrisfromthebodyfortransportationandclearanceintheliver or spleen. Furthermore, the cleavage products of the complement cascade are recognized by B cells leading to downstream activation, antibody production and amplification of the immune response against those structures that have activated complement (Fearon & Locksley 1996; Carroll 2004; Kemper & Atkinson 2007; Pekkarinenetal.2013) RECOGNITION Effectorfunctionsofcomplementdependontargetversushostdiscriminationandeach ofthethreecomplementpathwaysusesitsownuniquerecognitionmechanism.based on the recognition, complement proteins act together and make sure that the complementcascadeisregulatedcorrectlybyeitherpromotingorhaltingthecascade. Threestrategiesareusedbytheimmunesystemtodetectunwantedcellsorparticles forelimination:microbialnonself,missingself,andalteredself(medzhitov&janeway 2002).Recognitionoftargetsbythecomplementsystemcanalsobedividedintothese threecategories MICROBIALNONSELF The host PRRs can detect unique conserved patterns on microbes referred to as pathogennassociatedmolecularpatterns(pamps).thesepatternsarenotfoundonhost cellsbutonmicrobesandareusuallyvitalmicrobialproductslikelps,peptidoglycan,or flagella (Janeway 1989). Direct recognition of microbial nonself by complement is mediated via molecules such as C1q, mannosenbinding lectin (MBL), and ficolins. C1q recognizes antigennbound antibodies, MBL recognizes mannose and NN acetylglucosamine, whereas ficolins bind NNacetylglucosamine residues on microbial surfaces(matsushita&fujita1992;matsushita2010). 17

18 2Reviewofliterature MISSINGSELF Host cells possess membranenbound complement proteins that downregulate the complementactivityonownsurfaces.therefore,theabsenceoftheseregulatorsmakes thesurfacemuchmorepronetocomplementactivation.eveninthiscasethefluidphase APregulatorfactorH(FH)canprotectselfstructures.Discriminationbetweenselfand nonselftargetsismediatedbybindingoffhtosialicacidsandglycosaminoglycanson selfcells(kazatchkineetal.1979;meri&pangburn1990;pangburn2002;kajanderet al.2011).ifthecellismissingmoleculesthatmarkthemasselftheywillbedestroyed bytheimmunesystem RECOGNITIONOFALTEREDSELF Removal of apoptotic and necrotic cells is crucial for the cell homeostasis and complement is suggested to have a role in the late apoptotic phase. The classical pathway component C1q, as well as CNreactive protein (CRP) (Jiang et al. 1991) and pentraxin3(ptx3)(bottazzietal.1997;debanetal.2010)havebeenshowntobind apoptotic and necrotic cells leading to opsonization and phagocytosis of the target (Elward et al. 2005; Trouw et al. 2007). The recruitment and function of soluble regulators such as C4bNbinding protein (C4BP) and FH, are most probable to inhibit excessivecomplementactivationandavoidinflammation(elwardetal.2005;trouwet al.2005;trouwetal.2007) ACTIVATION After recognition, the activation of complement occurs via the classical, lectin, or alternativepathway.allpathwaysconvergeintothec3moleculeandformationofthe C5convertasethatinitiatestheterminalpathway CLASSICALPATHWAY(CP) TheCPisactivatedwhentheC1qmoleculeoftheC1complexbindstoitsligand(Table 1). The major ligands are the FcNregions of IgG or IgM antibodies that have formed complexes with an antigen (Bindon et al. 1988; Collins et al. 2002a). At least two moleculesofiggboundincloseproximityonthetargetarerequiredforinteractionwith C1q (HughesNJones et al. 1984). However, C1q can also bind several targets in the absenceofantibodies,likeptx3(bottazzietal.1997;debanetal.2010)crp(jiangetal. 1991),lipidAofLPS(Morrison&Kline1977)andviralenvelopeglycoproteins(Cooper etal.1976).c1qisacomplexproteinconsistingofmonomerswithcollagennlikestalks 18

19 2Reviewofliterature and globular heads arranged in trimers. Six trimers form the C1q molecule which appearslikeabunchoftulipsinelectronmicroscopy(calcott&mullerneberhard1972; Gadjeva et al. 2008). There is no enzymatic activity in C1q. C1q binding to its ligand resultsinconformationalchangesandactivationoftheproteasesc1randc1s(naff& Ratnoff1968;Doddsetal.1978;BudayovaNSpanoetal.2002).TheC1smoleculeinthe activated C1 complex cleaves C4 and C2 in a Ca 2+ Ndependent manner (Lepow 1963; Ziccardi&Cooper1977;Ballyetal.2009). TheC4moleculeconsistsofthreechains(α,β,γ)thatareboundtogetherbydisulfide bridgesandnonncovalentinteractions.uponactivation,c1scleavesc4toyieldc4band C4a(Schreiber&MüllerNEberhard1974)andathioestergroupisexposedonC4b.This highlyreactivegroupcanattachcovalentlytoanearbysurfacewhilethec4afragment actsasaweakchemoattractant(gorskietal.1979;isenman&kells1982;doddsetal. 1996). In the presence of Mg 2+, the membrane bound C4b binds the single chain C2 moleculeallowingitscleavagebyc1sresultinginformationofthec3convertasec4b2a (MüllerNEberhardetal.1967;Nagasawa&Stroud1977).Itisnotexactlyclearwhatrole the smaller dissociated C2b fragment has. One study suggested that it enhances the vascularpermeabilityleadingtoedema(strangetal.1988),however,thefindingsare notconfirmed.nevertheless,domainswithinc2bareimportantforbindingofintactc2 toc4b(nagasawa&stroud1977). Table,1.Componentsoftheclassicalpathway Component Molecularmass(kDa) Function C1 780 C1q 460 BindstoFcofIgG,IgM C1r 80 CleavesC1s C1s 80 CleavesC4andC2 C4 205(α,β,γchains) Bindstosurfaces C4a 9 Chemoattractant C4b 198 Covalentlyboundtothesurface C2 102 BindsC4b C2a 73 CleavesC3 C2b 34 Unknown 19

20 2Reviewofliterature LECTINPATHWAY(LP) ThelectinpathwayisactivatedwhencarbohydratebasedligandssuchasmannoseorNN acetylglucosamine on microbes are recognized by MBL or ficolins (1N, 2N, or 3Nficolin) (Table2).Interactionoftheserecognitionmoleculeswiththetargetleadstoactivation ofthemblnassociatedserineproteases(maspn1,maspn2,andmaspn3),whichactivate downstreamcomplementmolecules(matsushita&fujita1992;satoetal.1994;thielet al. 1997). MASPN2 cleaves both C4 and C2 forming the C3 convertase, C4b2a, while MASPN1canactivateMASPN2andissuggestedtohaveamajorroleintheactivationof thelectinpathway(degnetal.2012;hejaetal.2012;megyerietal.2013).bothmaspn1 andmaspn2haveautoactivatingpropertiesastheyarefoundtoassociateinthesame complex indicating that they act in a similar fashion as C1r and C1s do (Degn et al. 2012).ThephysiologicalroleofMASPN3isnotyetclearbut,however,murineMASPN3 and MASPN1 have shown to cleave the pronfactor D, indicating involvement in the AP activation(takahashietal.2010;sekineetal.2013). Table,2.Componentsofthelectinpathway Component Molecularmass(kDa) Function MBL 3N6x32kDa BindsmannoseorNNacetylglucosamine Ficolins1,2,and3 12N18x34N40kDa Bindacetylatedmolecules MASPN1 100 CleavesC2/activatesMASPN2 MASPN2 76 CleavesC4andC2 MASPN3 94 CleavesproNfactorD? ALTERNATIVEPATHWAY(AP) AllthreecomplementactivationpathwaysconvergeatthestageofC3,thekeymolecule of complement activation. It has the highest concentration of the complement componentsinplasma(0.7n1.5mg/ml).the185kdamoleculeiscomposedofα(115 kda) and β (75 kda) chains which are held together by one disulphide bond and noncovalentforces(tacketal.1979a;janssenetal.2005)(table3). ActivationofAPinafluidphaseisspontaneoussinceC3ishydrolyzedatalowrateto formc3(h 2O)(Pangburnetal.1981)(Fig.3).ThemetastableC3(H 2O)ishighlyreactive and within milliseconds it can bind factor B (FB) in the presence of Mg 2+ making it susceptible to cleavage by factor D (FD) and generating the initial fluid phase convertase,c3(h 2O)Bb. 20

21 2Reviewofliterature The initial fluid phase convertase, C3(H 2O)Bb, can cleave C3 into C3b and the chemoattractantc3a(hugli1975).theformedc3bcanattachtobasicallyanysurface viaitsexposedthioesterdomainwithinaveryshorttime(fearon&austen1975a;law & Levine 1977; Pangburn & MüllerNEberhard 1980; Sim & Sim 1981; Fishelson et al. 1984). Table,3.Componentsofthealternativepathway Component Molecularmass(kDa) Function C3 185 Bindscovalentlytosurfaces C3a 9 Chemoattractant C3b 175 BindsFB,FH,CR1 FB 93 BindsC3 Ba 30 Bb 60 CleavesC3andC5 FD 26 CleavesproNfactorD? Properdin 2N5x53 StabilizesC3/C5convertase A conformational change occurs when FB binds to C3(H 2O) or C3b and exposes a bindingsitefortheserineproteasefd(lesavre&müllerneberhard1978;janssenetal. 2009; Hourcade & Mitchell 2011). This results in a release of the Ba fragment while leavingtheactivesubunitbbboundtothec3convertase,c3bbb(medicusetal.1976b; Lesavre et al. 1979). Amplification of the AP occurs when C3 convertase cleaves and activates other C3 molecules to C3b resulting in opsonization of the target (MüllerN Eberhard & Götze 1972). The C3 convertase has a halfnlife of only ninety seconds (Medicus et al. 1976a; Pangburn & MüllerNEberhard 1986) but can be stabilized by associating with properdin (Fearon & Austen 1975b). Even though C3b surface depositionisaresultofactivationofallpathways,mostofthedepositedc3bmolecules aregeneratedbytheapc3convertase(harboe&mollnes2008). 21

22 2Reviewofliterature Figure, 3., ActivationoftheAP.C3isspontaneouslyhydrolyzedatalowrateinplasmaforming C3(H 2O)whichbindsfactorB(FB)andbecomescleavedbyfactorD(FD).Theformedfluidphase convertase, C3(H 2O)Bb, cleaves C3 into C3b and C3a. On an activator surface, FB binds to the depositedc3bmoleculemakingitsusceptibletocleavagebyfdresultinginthereleaseoftheba fragmentandformationofthec3convertase,c3bbb.anewc3bmoleculebindstothesurface depositedc3bandformsthec5convertase,c3bbbc3b.properdin(p)increasesthehalfnlivesof the C3 and C5 convertases when bound to the complexes. The C3 convertase cleaves other C3 moleculesasindicatedbythedottedarrow(amplification) TERMINALPATHWAY(TP) The binding of an additional C3b on or near the C3 convertase generates the C5 convertase, C4b2aC3b (CP/LP) or C3bBbC3b (AP) (Daha et al. 1976; Medicus et al. 1976a;Rawal&Pangburn2001;Pangburn&Rawal2002)andinitiatestheTP(Fig.4) andformationofthemembraneattackcomplex(mac)(müllerneberhard1986).thec5 moleculebindstosurfaceboundc3binthec5convertaseandiscleavedbyc2aorbb intoc5bandthechemoattractantc5a(medicusetal.1976b).c5aisrapidlymetabolized byplasmaandcellsurfacepeptidasestoformc5andesarg(bokisch&müllerneberhard 1970). C5a, and to a lesser extent C5aNdesArg, are important chemotactic peptides in recruitment and activation of phagocytes to the site of infection or inflammation by bindingtothec5areceptor(c5ar)onphagocytes.c5bremainsattachedtothec3bin the complex when the single chain molecule C6 binds to C5b (Tack et al. 1979b). A 22

23 2Reviewofliterature bindingsiteisexposedforthenextmolecule,c7thatresultsinaconformationalchange withinthec5bn7complexwhichisreleasedtothefluidphase(podacketal.1978a).the formed complex is very unstable and is inactivated by hydrolysis or other plasma proteins,unlessitbindstoamembranewithinshorttime(podacketal.1978b).when boundtoamembrane,itattractsthethreenchainedc8molecule,whichusesitsβnchain tobindtoc7resultinginthec5bn8complex(tamuraetal.1972).thelastcomponentin the pathway, the singlenchained C9, binds to the C8αNchain and undergoes a conformationalchangeexposingabindingsiteforadditionalc9molecules.numerous C9moleculesformaringNlikestructureinthemembrane,apore,knownastheMAC.The functionalconsequenceofthemacformationisosmoticlysisofthecell. Figure, 4., Activation of the terminal pathway. The C5 convertase cleaves bound C5 to C5aandC5b.C5binacomplexwithC6andC7bindstothemembraneandattractsC8 andc9resultingintheformationofthemembraneattackcomplex(mac),followedby lysisofthecell REGULATION Since complement is continuously activated at a slow rate and is amplified in plasma, uncontrolledactivationwouldleadtolocaltissuedamageandarapidconsumptionof thecomplementcomponents.therefore,thecascadeisstrictlyregulatedatalmostevery step by several regulators in the fluid phase and on membranes. The importance of regulators is evident in individuals who are deficient in, or have altered regulators leadingtovariousseverediseases.microbescanalsousethehostregulatorstoprotect themselves against complement attack (section 2.4). Most of the regulators are members of the regulators of complement activation (RCA) cluster located in chromosome1q32.regulatorsshareasimilarstructurecalledshortconsensusrepeat (SCR), complement control protein (CCP) domains, or Sushi repeats (Medof et al. 1987b). A single SCR is a globular domain containing 60N70 amino acids with a 23

24 2Reviewofliterature hydrophobic core wrapped in βnsheet held together by two intradomain disulphide bonds (Klickstein et al. 1987). The RCA proteins contain multiple copies of these domains, from four (as for decay accelerating factor, DAF; and membrane cofactor protein,mcp)tothirty(complementreceptor1,cr1).domainsareorganizedinsucha waythatproteinshaveatypicalelongatedstructure SOLUBLEREGULATORS C1Cinhibitor,(C1INH)isasinglechainhighlyglycosylatedserineprotease(Johnsonet al.1971;gregoreketal.1991)belongingtotheserineproteaseinhibitor(serpin)family (Table4).InadditionofbeingabletoinhibittheCP/LP,itisalsocapableofinhibiting the fibrinolytic and contact/kinin system of coagulation such as Factor XIa and XIIa, kallikreinandplasmin(ratnoff&naff1969;forbesetal.1970;giglietal.1980).c1inh hastwoimportantrolesinregulationofc1.intheabsenceofantibody,thec1complex willautoactivateatlowlevelsandc1inhisneededtopreventthisbystabilizationofthe complex and therefore preventing the consumption of C1, C2 and C4 (Ziccardi 1982; Hosoi et al. 1987; Bianchino et al. 1988). The second role of C1INH is to directly inactivate C1 by binding to the activated C1r and C1s which results in dissociation of thosefromc1qyieldingonecomplexofonec1r,onec1sandtwomoleculesofc1inh (Ziccardi&Cooper1977;Perkinsetal.1990).AdeficiencyofC1INHresultsinadisease hereditary angioedema (HAE) which is characterized by episodic edema of the extremities,face,larynxorgastrointestinaltract. Table,4.Solublecomplementregulators. Regulator kda Serumconc. (μg/ml) Function Reference C1INH N250 InactivatesC1r,C1s (Gregoreketal.1991) C4BP 540N CA 1,DAA 2 (Fujita et al. 1978; Fujita & Nussenzweig1979;Dahlbäck& Hildebrand1983) FH N269 CA,DAA (Pangburn et al. 1977; Ross et al.1983) FHLN N50 CA,DAA (Kühnetal.1995) FactorI CleavesC3b/C4b (Harrison&Lachmann1980) Properdin StabilizesC3bBb (Fearon&Austen1975b) Vitronectin N540 InhibitsMAC 3 (Podacketal.1984) Clusterin N420 InhibitsMAC (Murphyetal.1989) 1cofactoractivity; 2 decayacceleratingactivity; 3 membraneattackcomplex 24

25 2Reviewofliterature C4bCbinding, protein, (C4BP) regulates both CP and LP. By interfering with the C4b moleculewithinthec4b2aenzymeoncellsurfaces,c4bpacceleratesitsnaturaldecay andpreventsanassociationoftheconvertasesubunits(giglietal.1979).furthermore, itactsasacofactorforfactoriinproteolyticcleavageofc4btothefragmentsc4cand C4d in both fluid phase and on membranes (Fujita & Nussenzweig 1979; Fujita & Tamura 1983). At high concentrations it can also accelerate the decay of the AP C3 convertase(blometal.2003)butthephysiologicalroleofthisisnotverifiedinvivo. C4BPisalargeglycoproteinproducedmainlyintheliver.Ithasseveralisoforms,and themajorformconsistsofsevenαnchains(each70kda)andasingleβnchain(45kda) (Dahlbäcketal.1983).αandβNchainsarelinkedbydisulphidebridgesandorganizedin aspidernlikestructurewherethechainsarelinkedtoacentral core inthecarboxyln terminalends(dahlbäcketal.1983;chungetal.1985;hillarp&dahlbäck1990).each αnchainconsistsofeightscrdomainswheretheaminonterminaldomains1and2are required for binding to C4b and the cofactor activity (Chung et al. 1985; Blom et al. 2000).TheβNchainhasthreeSCRdomainsanditbindswithproteinS,mostlikelyvia thefirsttwodomains(hillarp&dahlbäck1990;vandepoeletal.1999).proteinsisa vitamin KNdependent protein involved in the coagulation cascade. Although the C4BPN protein S complex has been shown to have some anticoagulant activity its role is not fullyunderstood. Factor, H,(FH),originallynamedβ1HNglobulin,isthemajorfluidphaseregulatorofAP (Nilsson & MüllerNEberhard 1965). FH controls the AP at the C3 level by three mechanisms as illustrated in figure 5. It regulates the activation of C3 by acting as a cofactorforfactoriincleavageofc3bresultinginaninactiveform,ic3b(pangburnet al.1977).invivoitisobservedthatfhcanassisttheserineproteasefactoriincleaving C3b at two sites while under nonnphysiological conditions invitro further cleavage of C3bcanbeobserved(Rossetal.1983).Inaddition,FHcompeteswithFBforbindingto C3b and accelerates the natural decay of the AP C3 convertase, C3bBb (Weiler et al. 1976; Whaley & Ruddy 1976), by dissociating the BbNfragment from the complex (Pangburn&MüllerNEberhard1978). 25

26 2Reviewofliterature, Figure,5.,RegulationoftheAPbyfactorH.1)Cofactoractivity:FHassociateswithC3b andpromotesproteolyticcleavageofc3bbyfactori(fi)resultinginaninactiveformof C3b(iC3b).2)FHcompeteswithFBinbindingtoC3b.3)Decayacceleratingactivity:FH associateswithc3bbbandenhancesthedissociationofthebbfromthecomplex. FHisanelongatedmoleculeconsistingoftwentySCRs(Sim&DiScipio1982;Ripocheet al.1988)(fig.6).theregulatorysiteoffhtoc3bthatcontainsthecofactoractivityand decay accelerating activities is located within the domains 1N4 (FH1N4) (Gordon et al. 1995;Kühnetal.1995;Kühn&Zipfel1996;Wuetal.2009).TheotherimportantC3b bindingsiteiswithinthecnterminaldomains19n20(fh19n20),whichisalsothesurface recognition site and has high affinity for anionic molecules like sialic acids and glycosaminoglycans abundantly found on host cells (Jokiranta et al. 2000; Pangburn 2002; Ferreira et al. 2006). FH is able to discriminate between host and nonnhost structuresbybindingviathedomain20tosialicacidsorglycosaminoglycansonhost cellsandsimultaneouslyviathedomain19onc3bleavingtheregulatorydomainsfree (Kajander et al. 2011). A third binding site for C3b has been located within FH8N18 (Sharma & Pangburn 1996; Jokiranta et al. 2000; Jokiranta 2006). Interactions with heparin are mapped to the domains 7 and 20 (Pangburn et al. 1991; Blackmore et al. 1996; Blackmore et al. 1998) and a possibly weaker interaction within the domain 9 (Ormsbyetal.2006).Moreover,FH16N20hasbeenshowntointeractwithsialicacidson the surface of Neisseria (Ram et al. 1998; Lewis et al. 2012) and it has also been proposed,basedonbindingstudiesoftrypticcleavagefragmentsoffhtopolyanions, thatthedomain13offhbindspolyanions/sialicacids(pangburnetal.1991). 26

27 2Reviewofliterature Figure, 6., Presentation of the elongated FHNmolecule. The main C3bNbinding sites are located in the NNterminal domains 1N4 and domains 19N20 (shaded domains) and heparin binding sites are on the domains 7, 9, and 20 as indicated. The domains for surfacerecognitionarelocatedinthecnterminus(fh19n20). MutationsinFHmoleculeareassociatedwithrenaldiseasessuchasatypicalhemolytic syndrome(ahus)anddensedepositdisease(ddd)(warwickeretal.1998;richardset al.2001).themajorityofmutationsfoundinahusareclusteredwithinfh19n20,the binding site for the C3d part of C3b, sialic acids, and glycoasaminoglycans. The cell damage that is characteristic in ahus occurs when mutated FH fails to recognize C3b depositedonglomerularbasementmembrane(kavanagh&goodship2010).dddisa disease associated with an FH deficiency or dysfunction of FH. Characteristic for pathogenesisofdddistheinabilityoffhtoprotectglomerularbasementmembranes. ThisleadstooveractivationoftheAPanddepositionofC3bandTPcomponentsinthe glomeruli. In 2005, five groups reported a polymorphism in the domain 7 of FH that is strongly associated with a condition called agenrelated macular degeneration (AMD), an eye disease causing visual loss in the elderly (Edwards et al. 2005; Hageman et al. 2005; Hainesetal.2005;Kleinetal.2005).Intheriskalleleatyrosineatposition402(402Y) in the domain 7 is mutated to a histidine (402H). This site is in the proximity to the bindingsiteofheparin,crp,andthemproteinofstreptococcuspyogenes(giannakiset al. 2003). Indeed, the Y402H polymorphism was shown to affect binding of FH to heparin and surface glycosaminoglycans (Clark et al. 2006; Prosser et al. 2007). The FH(402H) allotype showed an impaired binding to CRP partly explaining the inflammationinmaculaofpatientswithamd(laineetal.2007). FHClike,protein,1,(FHLC1)isanalternativelysplicedproteinfromthesamegeneasFH. ThisproteinconsistsofsevendomainsidenticaltotheNNterminusofFHfollowedbyan unique tail of four amino acids (Ripoche et al. 1988). As expected on the basis of its similaritytothennterminusoffh,ithasonebindingsiteforc3bandcofactoraswellas 27

28 2Reviewofliterature decay accelerating activities within FHL1N4, but lacks the target recognition capacity (Kühnetal.1995;Kühn&Zipfel1996). FHCrelated,proteins,(FHRs)areencodedbydifferentgenesnexttothegenecodingfor FH(andFHLN1)andsharestructuralsimilaritywithFH(DiazNGuillenetal.1999).There arefivefhrproteinscirculatinginplasmaandbasedontheirconserveddomainsand abilitytoformdimers,fhrscanbedividedintotwogroups(skerkaetal.2013).groupi consists of FHR1, FHR2, and FHR5, which is characterized by a highly conserved NN terminus. These proteins are present in plasma exclusively as dimers (Goicoechea de Jorgeetal.2013;Tortajadaetal.2013).GroupIIincludesFHR3andFHR4whichlack the NNterminal dimerization motif in the NNterminus domains. All five FHR proteins show high homology to the CNterminal domain of FH and can probably discriminate between self and nonself surfaces similarly to FH (Hellwage et al. 1999; McRae et al. 2005; Heinen et al. 2009; Eberhardt et al. 2013; Goicoechea de Jorge et al. 2013; Tortajadaetal.2013).FHR1hasbeensuggestedtobindC5andinhibitthefunctionof C5 convertase and the formation of MAC (Heinen et al. 2009). FHR2 was previously describedasac3bandheparinbindingproteinandrecentlywassuggestedtoinhibitc3 convertasemediatedcleavageofc3(eberhardtetal.2013).bothfhr3andfhr5have been found to have cofactor activity for FI cleavage of C3b but this has not been confirmed. FHR1 has two different forms, FHR1α (37 kda) and FHR1β (42 kda), dependent on glycosylation(timmannetal.1991).thefhr4geneencodestwoproteins,fhr4a(86 kda)andfhr4b(42kda)(jozsietal.2005).fhr4bindstoc3bandcofactoractivityis reportedforbothfhr4aandfhr4b(hellwageetal.1999;hebeckeretal.2010). ProperdinstabilizesbothAPC3andC5convertasesbydecreasingtheirnaturaldecay. Thus it is the only positive regulator in the complement activation system (Fearon & Austen1975b;Medicusetal.1976a;Alcorloetal.2013).Moreover,thereareindications that properdin acts as a pattern recognition molecule binding to negatively charged moleculesonapoptoticandnecroticcellsandonsurfacesofmicrobessuchasneisseria gonorrhoeaeandchlamydophilapneumoniae(formerlychlamydiapneumoniae)(spitzer etal.2007;cortesetal.2011).boundtothesurface,properdincontrolsc3bdeposition andassemblyofc3bbbtherebyamplifyingcomplementactivation(spitzeretal.2007). IndividualsdeficientinproperdinaremoresusceptibletorecurrentNeisseriainfections (Densen1989). 28

29 2Reviewofliterature Vitronectin,originallycalledSNprotein,isinvolvedinregulationoftheTP.Itbindsthe fluidphasec5bn7complexpreventingitfrombindingtoanearbysurface(podacketal. 1978a; Podack et al. 1984). In addition to its role in complement regulation, it has a crucialroleincelladhesion,migration,andangiogenesis(preissner&seiffert1998).in plasma,vitronectinexistseitherasasinglechainmolecule(84kda)orasatwonchain moleculewiththechainslinkedtogetherbydisulfidebonds(69and15kda). Clusterin, (SPC40,, 40,, Apolipoprotein, J).Inasimilarfashionasvitronectin,clusterin interacts with the C5bN7 complex and inhibits its attachment to cell membranes (Murphyetal.1989).Moreover,clusterinbindstheterminalcomponentsC7,C8,andC9. AlthoughitsroleintheregulationofMACisunclear,theinteractionwithC9suggests inhibitionofformationofthetransmembranepore(tschoppetal.1993).clusterinhasa functioninclearingofcelldebrisandlipidtransportation(jenne&tschopp1992). Factor,I,(C3bCinactivator)isaserineproteasethatdegradesbothC3bandC4binthe presence of a cofactor. Cleavage of C3b occurs at three specific sites resulting in the fragments C3a, C3b, C3c, and C3dg (Harrison & Lachmann 1980; Medof et al. 1982a). ThetwofirstcleavagesareassistedbyFH,CR1,orMCP,andresultsininactivationof C3btoiC3bandreleaseofthesmallnonfunctionalC3ffragment(Pangburnetal.1977; Fearon1979;Seyaetal.1986).FurthercleavageofiC3bbyfactorI(CR1asacofactor) results in the release of C3cNmolecule in the fluid phase leaving the C3dgNfragment attachedtothecellsurface.underphysiologicalconditionsthethirdcleavageonlytakes placeinthepresenceofcr1asacofactor(ross&lambris1982).c3bandic3battached on membranes are major opsonic fragments that can be recognized by receptors on phagocyticcells.therefore,itiscrucialthatic3biscleavedintothenonnopsonicc3dg onhostcells(vanlookerencampagneetal.2007). DegradationofC4bissupportedbyC4BP,MCP,orCR1(Iida&Nussenzweig1981;Seya et al. 1986). Factor I cleaves the αnchain of C4b at two sites releasing a large C4c fragmentandleavingthesmallerc4dattachedonthemembrane. The plasma concentration of factor I is on average 35 μg/ml but it increases during inflammation since it is an acute phase protein. It consists of a heavy (50 kda) and a lightchain(38kda)heldtogetherwithdisulphidebonds.thec3bnbindingsiteislocated in the heavy chain while the protease activity is in the light chain (Goldberger et al. 1987; DiScipio 1992). Deficiency of factor I can lead to recurrent infections of encapsulated bacteria such as Neisseria meningitidis, Haemophilus influenzae, and 29

30 2Reviewofliterature Streptococcus pneumoniae (Vyse et al. 1994; Nita et al. 2011; AlbaNDominguez et al. 2012) MEMBRANEBOUNDREGULATORS Decay, accelerating, factor, (DAF,, CD55) accelerates the decay of AP and CP C3 convertases.itbindstomembraneboundc3borc4bandinactivatestheconvertaseby dissociating the BbN or C2aN fragments from the convertases (NicholsonNWeller et al. 1981).TwoisoformsofDAFareknown,amembraneNboundandasolubleform(Medof etal.1982b;medofetal.1987a).themembraneboundproteinisattachedtothecell membrane via a glycosylphosphatidylinositol (GPI) anchor, followed by a serine/threonine/proline(stp)nrichregionandfourscrdomains(medof,etal.,1987). ThefunctionalsiteswithinDAFarelocatedwithindomains2N4forC3bBbanddomains 2N3 for C4b2a, respectively (Brodbeck et al. 1996). Recently, two novel membrane boundisoformsofdafwereidentified(vaineretal.2013). Membrane,cofactor,protein,(MCP,,CD46)isexpressedubiquitouslyonthesurfaceof nucleatedcellsandfunctionsasacofactorinthefactorinmediatedcleavagesofc4band thefirstcleavageofc3b(seyaetal.1986;seyaetal.1988).ithasstructuralsimilarity withdafwithfourscrdomainsandahighlyglycosylatedstpnregion,butitlacksthe GPINanchor. Instead, it consists of a transmembrane region followed by a cytoplasmic domaincontainingasignalingmotif.thestpnandcytoplasmicregionsarealternatively splicedresultinginseveralisoformsthatcanbeexpressedonthesamecell(postetal. 1991). Crucial domains for the regulatory activity are located to SCR2N4 and the domains3and4areimportantforthecofactoractivity(adamsetal.1991). CD59,(Protectin)isabundantlyexpressedonallcellstypes(Merietal.1990b).Itbinds tothemembraneboundc5bn8andpreventsincorporationofc9tothecomplexthereby blocking the formation of MAC (Meri et al. 1990a). Similarly to DAF, CD59 is a GPIN anchoredprotein(daviesetal.1989).asomaticmutationinhematopoieticstemcells canaffectsynthesisofthegpinanchorinthebloodcellsderivedfromstemcellsresulting inerythrocytesdeficientincd59anddaf.thisleadstocomplementnmediatedlysisof thissubsetofbloodcellscausingintravascularanemiaandthromboembolicphenomena characteristic of paroxysmal nocturnal hemoglobinuria (PNH) (NicholsonNWeller et al. 1983). Complement, receptor, 1, (CR1,, CD35) is expressed on all human blood cells (except some T cells), follicular dendritic cells, and Kupffer cells (Fearon 1980; Tedder et al. 30

31 2Reviewofliterature 1983; Wilson et al. 1983; Fischer et al. 1986). It is involved in several important functionssuchascomplementregulation,phagocytosis(describedin2.3),andimmune complexclearance(fearon1979).inadditiontoitscrucialroleasacofactorforfactori it has a decay accelerating activity on the AP C3 convertase and the C3 and C5 convertases of CP (Fearon 1979; Iida & Nussenzweig 1981). A soluble form, scr1, is circulating in plasma at low concentration (30 ng/ml) (Yoon & Fearon 1985), and is most probably released from leukocytes by enzymatic cleavage (Hamer et al. 1998). ThereisalsoevidencethatCR1isareceptorforC1q(Klicksteinetal.1997)andMBL (Ghiran et al. 2000; Jacquet et al. 2013). The ectodomain of CR1 is organized into repeatingunitsofsevenscrs,calledlonghomologousrepeats,andtwoadditionalscrs atthecnterminalend.thetwomostcommonformsarecomposedof30scrsor37scrs (Dykmanetal.1984). Complement, receptor, of, the, immunoglobulin, family, (CRIg) is an important complement receptor on Kupffer cells, the liver resident macrophages, where it mediatesphagocytosisofc3bnoric3bboundparticles(helmyetal.2006).moreover, CRIg regulates the AP by preventing the interaction of C3 and C5 with the AP convertases(wiesmannetal.2006). Table,,5.Membraneboundcomplementregulators, kda, Function, Reference, CR1 190N220 CA 1,DAA 2 (Fearon1979) DAF 70 DAA (NicholsonNWelleretal.1981) MCP 45N70 CA (Seyaetal.1986) CD59 20 InhibitsMAC 3 (Merietal.1990b) CRIg 44 InhibitsAPconvertases (Wiesmannetal.2006) 1cofactoractivity; 2 decayacceleratingactivity; 3 membraneattackcomplex CUB,and,Sushi,multiple,domains,1,(CSMD1)isatransmembraneproteinthatinhibits CPandLPbypromotingcleavageoftheactivatedC3bandC4bbyfactorIandinhibition of MAC formation at the C7 level (EscuderoNEsparza et al. 2013). Containing 29 SCR domains,theproteinactsasacomplementinhibitorhighlyexpressedincentralnervous systemandepithelialtissuesinaratmodel(krausetal.2006). 31

32 2Reviewofliterature 2.3,PHAGOCYTOSIS,OF,MICROBES,BY,NEUTROPHILS, Phagocytosis is a complex process where specialized cell types recognize and engulf foreign particles, such as microbes. Cells involved in microbial phagocytosis are macrophages,monocytes,dendriticcells,andneutrophils.neutrophilscompriseabout 60% of the total leukocyte population and are crucial in defense against pathogens (Amulicetal.2012).Furthermore,uponinfectionthenumberofneutrophilsincreases dramatically(kimetal.2008).neutrophilshaveacharacteristiclobulatednucleusand forthatreasontheyarealsocalledpolymorphonuclearleukocytes(pmns).importantly, neutrophils are equipped with unique storage structures known as granules that contain antimicrobial compounds and are essential in elimination of the engulfed microbes.sincemostofthepathogensareencounteredatmucosalsurfacesorintissues, neutrophilshavetomigrateoutofthebloodvesselsthroughtheepitheliumandmove towardstheinfectionsitebeforetheycanphagocytoseandeliminatetheinvader TRANSMIGRATION At the sites of infection the bacterianderived components such as LPS and formylated peptides (fmlp; NNformylNMetNLeuNPhe) and hostnproduced factors such as cytokines (e.g.tumornecrosisfactors,andinterleukins)stimulateendothelialcellsinthevicinity. The activated endothelial cells produce molecules that slow down the neutrophils on endothelialcellsneartheinfectionsite,causing rolling oftheneutrophilsonthevessel wall(mooreetal.1995).next,inaprocessknownas firmadhesion theneutrophils interact with the endothelial cells via binding of neutrophil β2nintegrins, including complementreceptor3(cr3),toendothelialintercellularadhesionmolecule(icam)n1 (Diamondetal.1990).Neutrophilrollingresultsinclusteringofβ2Nintegrinreceptors ontheneutrophils,whichallowstransmigrationofthecellsfromthebloodvesseland across the endothelial junctions and even transcellularly through the endothelial cells (Campbelletal.1998;Phillipsonetal.2006).Althoughinsometissuesotherreceptors andmechanismsarelikelytobeinvolved,thedescriptiongivenaboveisanoverviewof thegeneralconcepthowneutrophilsarerecruitedtotheinfectionsite CHEMOTAXISANDPRIMINGOFNEUTROPHILS Afterthephagocyteshavecrossedtheendothelialbarrier,theyencounteranexcessive amount of chemotactic factors that direct them to the site of infection where inflammatorystimulantsprimeandactivatethecells.recognitionofpampsbythecells (e.g.neutrophils,macrophagesanddendriticcells)withintheinfectionsiteinducethe productionofinflammatorycytokinesandinterferons. 32

33 2Reviewofliterature The chemotactic factors are released by host cells or fragments generated after complementactivation.thesmallcomplementpeptidesc5a,c3a,andtolesserextent C5aNdesArgareproducedafterproteolyticcleavageofC3andC5.Thefragmentsbindto membraneboundreceptorsofthegpcrsuperfamily(joost&methner2002).although thereceptorssharehighhomologytheyhavedifferentligandspecificityandfunctions (Leeetal.2001).C5areceptor(C5a 1R)hashighaffinityforC5aandmoreweaklyforits truncatedform,c5andesarg,whilec5a 2Nreceptor(C5a 2R,C5areceptorNlike2)bindsC5a andc5andesargequally(cain&monk2002;okinagaetal.2003).c3anreceptor(c3ar) recognizesc3a(wilkenetal.1999),whichisamuchweakerchemotacticstimulusthan C5a(Fernandezetal.1978). Other important GPCRs in neutrophil chemotaxis are FPRs that recognize the NN formylatedproteinsandpeptidessecretedbybacteria(schiffmannetal.1975;gaoetal. 1999). Theprimarycellularsensorsofpathogens are the TollNlike receptors(tlrs) of which tendifferenttlrsareidentifiedinhumans(medzhitov2007).tlr1,2,4,5,and6are expressedonthecellsurfaceandareessentialinrecognizingmolecularpatternsfrom bacteria, fungi and protozoa. TLR3, 7, 8, and 9 are expressed within endocytic compartments and recognize nucleic acids derived from bacteria or viruses. TRL10 is alsoinvolvedinviralrecognitionbymacrophages(leeetal.2014).essentialbacterial TLR ligands are LPS in the outer membrane of GramNnegative bacteria, which is recognizedbytlr4(poltoraketal.1998).tlr2,insteadsensespeptidoglycanofgramn positive bacteria (Schwandner et al. 1999). Furthermore, the three TLRs 1, 2, and 6 recognizelipoproteins(brightbilletal.1999;takeuchietal.2002),flagellinistheligand for TLR5 and cytidinenphophateguanosine (CpGN)Nrich bacterial DNA is recognized by TLR9 as well as TLR5, respectively (Hayashi et al. 2001). TLRs are transmembrane glycoproteins and most of them form homodimers upon ligand binding. For example, formation of the TLR2NTLR6 heterodimer recognizes diacylated lipopeptides from GramNpositive bacteria and mycoplasma (Takeuchi et al. 2001; Kang et al. 2009). DimerizationbringsthecytoplasmicdomainsoftheTLRstogetherleadingtoactivation and recruitment of signaling components. Roughly, the TLR signaling is distinguished intothemyd88nandthetrifn(tirndomainncontainingadapterninducinginterferonnβ) dependent pathways. Both signaling pathways eventually result in activation of cells depending one or more transcription factors leading to generation of inflammatory cytokinesandchemokines. 33

34 2Reviewofliterature 2.3.3OPSONIZATIONANDPHAGOCYTOSIS Opsonizationwithcomplementcomponents,immunoglobulins,orotherinnateimmune factors is crucial for neutrophil phagocytosis. Pathogens that are covered with complementcleavageproductsarerecognizedbycomplementreceptors1,2,3,and4 (CR1, CR2, CR3, CR4), or CRIg. Two receptors, CR1 and CRIg, have complement regulatoryfunctionsaswell(section2.2.3). CR1hasthehighestaffinityforC3bfollowedbyC4bandiC3b(Rossetal.1983;Medof& Nussenzweig 1984). The number of CR1 molecules expressed on erythrocytes is low (average500percell),however,sincethesecellsareabundantinbloodthenumberin bloodexceedsthatonleukocytes.immunecomplexes(ic)aretransportedintotheliver and spleen for phagocytosis where adenosine triphosphate (ATP) released from erythrocytes is suggested to have a stimulatory effect on phagocytosis of immune complexes(melhornetal.2013).releaseofthecomplexoccurswhenc3bisdegraded toic3bwhichhasloweraffinityforcr1(medicusetal.1983)andhigherforcr3and CR4allowingerythrocytestoreturntothecirculation(Nelson1953;Medofetal.1982b; Schifferli et al. 1986) leaving immune complexes bound to the spleen or liver cells. Resting neutrophils have approximately 5000 CR1 molecules per cell, and CR1 expression is rapidly upnregulated by C5aNdesArg, fmlp, and increased temperatures (Fearon & Collins 1983; Berger et al. 1984). Opsonization facilitates phagocytosis of pathogensbystimulatingthecr3andfcnreceptors(ehlenberger&nussenzweig1977). Moreover,neutrophilsreleaselargeamountsofsCR1whenstimulatedwithfMLPand TNFNα (Danielsson et al. 1994) suggesting a physiological role for unbound CR1. The highestlevelofcr1isexpressedonbcells(20,000n40,000percell)(fearon&collins 1983).ThehighnumbermaybeneededforthefactorItocleaveC3btogenerateC3dg andbindingtothenearbycr2onbcells. CR2, (CD21) is composed of 15 or 16 SCRs, a transmembrane domain, and a short cytoplasmic tail. It has a key function in B cell activation and in bridging innate and adaptiveimmunesystems.cr2hasahighaffinityforc3dgandic3bfragmentswhereas aloweraffinityforc3bandc3(h 2O)(Rossetal.1983;vandenElsen&Isenman2011). In B cell activation the antigen is recognized by B cell receptor (BCR) while the C3dg attached to the antigen is recognized by CR2. Crosslinking of BCR and CR2 leads to couplingoftheconreceptorscd19andcd81.uponcrosslinking,thecytoplasmictailof CD19isphosphorylatedandadownstreamsignalingcascadeisactivatedenhancingthe Bcellactivation(Fearon&Carter1995).Inamousemodel,C3dgwasshowntoactasan adjuvant, increasing the immunogenicity of an analysed antigen 1,000 to 10,000 fold, 34

35 2Reviewofliterature depending on the number of C3dg fragments bound to the antigen (Dempsey et al. 1996). CR3, (CD11b/CD18,, MacC1) is a β2nintegrin receptor expressed on neutrophils, monocytes, macrophages, dendritic cells, and natural killer cells (Underhill & Ozinsky 2002). It is a heterodimer consisting of a unique αnsubunit (CD11b) and a nonn covalentlyassociatedβsubunit(cd18)(corbietal.1988;larson&springer1990).the ligands for CR3 are ic3b and C3b and this interaction has an important function in phagocytosis. CR4, (CD11c/CD18) is a β2nintegrin, similar to CR3, and it differs from that receptor onlyinitsαnchain,cd11c.itismainlyexpressedontissuenderivedmacrophageswhere ithasthesameligandsandfunctionsascr3(myonesetal.1988). Immunoglobulinscontributetoeliminationofapathogeninmanyways.Viabindingto microbial epitopes they prevent spreading by immobilization (agglutination), block microbial attachment to host cells (neutralization), and mediate opsonization directly and by activating the CP. IgM and IgG1, IgG3 classes and subclasses, respectively, are effectiveinactivation of thecp and in neutralizationof bacterial toxins and virulence factors(walport2001).themajoropsoninsigg3andigg1arerecognizedbyfcγrson neutrophils.thesereceptorsaredividedintotwoclasses,theactivatingandinhibitory FcγRs(Daeron1997),andexpressionoftheIgGsubclassesdeterminesthethresholdfor phagocytosisoftheiggnopsonizedparticles.furthermore,onthebasisofstructuraland biochemical differences the activating receptors are divided into three, FcγRI, FcγRII, andfcγriii.thesubclassfcγriiaisthemajorreceptorforphagocytosisonneutrophils (van de Winkel & Capel 1993). Upon crossnlinking by binding to a ligand the CRs and FcγRs induce uptake of the pathogen and the plasma membrane of the neutrophil extendsarounditstargetandcreatesavacuoleknownasaphagosome(nordenfelt& Tapper2011).NeutrophilsareextremelyefficientandcanphagocyteanIgGNopsonized targetwithin20seconds(segaletal.1980) ELIMINATIONOFMICROBES Neutrophils are equipped with antimicrobial molecules that efficiently kill pathogens. Whenapathogenisphagocytosed,theassemblyofanicotinamideadeninedinucleotide phosphate(nadph)noxidaseisinducedandhighamountsofrosaregeneratedleading tooxidativeburst(borregaard&cowland1997;babior1999;leto&geiszt2006).in addition,avarietyoftoxicantimicrobialcompoundsarestoredwithingranulesthatare 35

36 2Reviewofliterature utilizedwhenmicrobesarephagocytosed.inacalciumdependentmannerthegranules are fused with the phagosome where the content is released (Jaconi et al. 1990; Nordenfelt et al. 2009) and the microbe is exposed to a high concentration of antimicrobialpeptides.thephinsidethephagosomesofneutrophilsisclosetoneutral (Jankowskietal.2002),whereasinmacrophagesanacidicpHismaintained(Clausetal. 1998). Neutrophilsusealsoastrategytotrapthemicrobesbyexpellingtheirnuclear,granular andcytosoliccontentformingastickymesh,calledneutrophilextracellulartraps(nets) (Brinkmannetal.2004).MostlikelyNETsareimportantinthebloodstreamwherethe antimicrobial protein concentration is too low for efficient elimination of microbes. However, when associated with NETs the antimicrobial proteins could reach their criticalconcentrationforefficientmicrobialkilling. 2.4,MICROBIAL,EVASION,OF,COMPLEMENT,AND,PHAGOCYTES,, An invading microbe is quickly recognized as a target for elimination by the immune system in a healthy person. However, microbes have evolved various mechanisms to escape from targeted killing. Since the complement and phagocytes are the major components of the first line of defense, survival of the microbes in the human body dependsonefficientevasionstrategies MICROBIALCOMPLEMENTEVASION Thetightlyregulatedcomplementcascadeinvolvesseveralenzymes,proteincomplexes andreceptorsthatprovidemanysitesthatcouldbeinterferedbymicrobialmolecules. Avarietyofmicrobesproduceacapsuleconsistingoflooselyattachedpolysaccharides tomasktheirsurfaceantigens,therebypreventingapactivationintheabsenceofantin capsularantibodies.examplesofwellnknowncapsulatedpathogenicbacteriaaresome types or strains of the GramNnegative bacteria Escherichiacoli (Bortolussi et al. 1983; Corbett&Roberts2009),Haemophilusinfluenzae,andNeisseriameningitidisaswellas the GramNpositive bacteria Staphylococcus aureus and Streptococcus pneumoniae (Watsonetal.1995). C3isakeyplayerinthecomplementcascadeandthereforeanidealtargetforinhibition bymicrobes.severalproteinsfroms.aureusareshowntodirectlyinteractwiththec3n or C5N molecules through various mechanisms and are discussed in the section

37 2Reviewofliterature Other microbes known to bind C3 include S. pneumoniae via the cholinenbinding pneumococcal surface protein A (PspA) (Cheng et al. 2000), Moraxellacatarrhalis via ubiquitous surface protein A (UspA) (Hallström et al. 2011), Candidaalbicans via phn regulatedantigen1(pra1)(luoetal.2010),yersiniaenterocoliticaviayersiniaadhesion Aprotein(YadA)(Schindleretal.2012).Interestingly,herpessimplexvirus1(HSVN1) and the flagellate Trypanosoma cruzi trypomastigotes express proteins with decay acceleratingactivityonc3bbb(friesetal.1986). Severalmicrobesacquireproteasesorproteaseprecursorsfromplasma.Plasminogenis circulating in the blood as an inactive precursor of the serine protease plasmin that dissolvesfibrinclots.inaddition,plasminbindsanddegradesc3(bartheletal.2012). Anincreasingnumberofmicrobeshavebeenshowntorecruitplasminogenorplasmin onto their surfaces by various receptors. Streptococcus pyogenes has an enzyme that binds and cleaves plasminogen to active plasmin which subsequently cleaves IgG and C3b, leading to impaired recognition of the targets by the complement and FcγR on phagocytes(lähteenmäkietal.2001).secretionofproteinsthatinactivatecomplement proteinsisacommonmechanismamongmicrobes.recently,theproteasenaipfromn. meningitidiswasshowntodegradec3intoalongerformofc3bthatresultedinreduced C3bNdeposition on the bacterial surface (Del Tordello et al. 2014). Other bacterial proteasesthattargetthec3moleculeincludes.pyogenesspeb(teraoetal.2008),anda fewpseudomonasaeruginosaproteases(hong&ghebrehiwet1992;schmidtchenetal. 2003).TheE.coliproteaseEspPcleavesC3andC5(Orthetal.2010),whiletheyeast secreted aspartyl proteases (Saps) of C. albicans and the surface protease PgtE of SalmonellaentericadegradesC3b,C4b,andC5(Ramuetal.2007;Groppetal.2009). Host cells are protected by the presence of complement regulators on their surfaces, either the membranenbound regulators or soluble regulators that have been acquired ontothehostcellsurface.mostpathogensdonotexpressregulatorsbuthaveevolved means to protect themselves from complement attack by acquisition of complement regulators onto their surfaces. Tables 6 and 7 give a summary of microbes that have beenreportedtobindhostcomplementregulators. 37

38 Table,6.,MicrobialacquisitionofFH 2Reviewofliterature Microbe, Ligand, Reference, Actinobacillusactinomycetemcomitans Omp100 (Asakawaetal.2003) Aspergillusfumigatus? (Behnsenetal.2008) Borreliaburgdorferis.l. BbCRASP 1 (Kraiczyetal.2001;Hartmannetal.2006) Borreliaburgdorferis.l. OspE (Hellwage,etal.,2001) Borreliahermsii FhbA 1 (Hovis,etal.,2006) Borreliarecurrentis HcpA 2 (Grosskinsky,etal.,2009) Candidaalbicans Gpm1p 1 (Poltermann,etal.,2007) Candidaalbicans Pra1 1 (Luo,etal.,2009) Fusobacteriumnecrophorum? (Friberg,etal.,2008) Haemophilusinfluenzae,Hib? 1,2 (Hallström,etal.,2008) Haemophilusinfluenzae,NTHi P5 (Rosadini,etal.,2014) Loaloa? (Haapasalo,etal.,2008) Neisseriagonorrhoeae Por1A 1,2 (Ram,etal.,1998) Neisseriagonorrhoeae Por1B 1,2 (Ngampasutadol,etal.,2008) Neisseriameningitidis Fhbp (Schneider,etal.,2009) Neisseriameningitidis NspA (Lewis,etal.,2010) Neisseriameningitidis PorB2 (Lewis,etal.,2013) Onchocercavolvulus? (Meri,etal.,2002) Pseudomonasaeruginosa Tuf 2 (Kunert,etal.,2007) Rickettsiaconorii OmpB (Riley,etal.,2012) Salmonellaenterica Rck (Ho,etal.,2010) Streptococcusagalactiae βprotein (Areschougetal.2002) Streptococcusagalactiae SHT (Maruvadaetal.2009) Streptococcuspneumoniae Hic (Jarva,etal.,2004) Streptococcuspneumoniae PspC (Hammerschmidt,etal.,2007) Streptococcuspyogenes Fba 1 (Pandiripally,etal.,2002) Streptococcuspyogenes Mprotein 1 Horstmannetal.,1988 Streptococcuspyogenes Scl 2 (Reuter,etal.,2010) Streptococcussuis Fhb (Pian,etal.,2012) Treponemadenticola? (McDowell,etal.,2009) Yersiniaenterocolitica Ail (BiedzkaNSarek,etal.,2008) Yersiniaenterocolitica YadA (China,etal.,1993) 1BindsalsoFHLN1, 2 BindsalsoFHR1 Abbreviations:Ail,attachmentNinvasionlocusprotein;BbCRASP,Borreliaburgdorfericomplement regulatornacquiringsurfaceproteins;fba,fibronectinnbindingprotein;fhba,factorhnbindingprotein;fhbp, factorhbindingprotein;gpm,phosphoglyceratemutase;hcp,humancomplementandplasminogen bindingprotein;hic,factorhnbindinginhibitorofcomplement;nsp,neisserialsurfaceprotein;omp,outer membraneprotein;osp,outersurfaceprotein;por,porin;pra,phnregulatedantigen;psp,pneumococcal surfaceprotein;rck,resistacetocomplementkilling;scl,streptococcalcollagennlikeprotein;sht, streptococcalhistidinetrial;tuf,elongationfactortu;yad,yersiniaadhesin *nonntypeable BindingoftheCPandLPregulator,C4BP,isalsoextensivelystudiedandthebindingis mediatedviadomainswithintheαnchain(blometal.2009).manypathogensbindc4bp withthesamemoleculetheyuseforbindingtofh.examplesofthosearepor1aofn. gonorrhoeae (Ram et al. 2001), Rck of S.enterica (Ho et al. 2011), YadA and Ail of Y. enterocolitica(kirjavainenetal.2008),andpra1ofc.albicans(luoetal.2011).c4bpis furthermore bound by B. pertussis FHA (Berggård et al. 1997), M. catarrhalis UspA (Nordström et al. 2004), N. meningitidis PorA (Jarva et al. 2005), E. coli K1 OmpA 38

39 2Reviewofliterature (Prasadaraoetal.2002),BorreliarecurrentisCihC(Grosskinskyetal.2010),aswellas severals.pyogenesmnproteins(thernetal.1995). The long and still growing list of pathogens binding FH and C4BP emphasizes the importance of the mechanism in microbial immune evasion. Other soluble regulators, suchasc1inh,vitronectin,clusterin,andfactoriarealsodescribedbeingusedbysome microbes(table2).membranenboundregulatorsareusedbyonlyalimitednumberof pathogens but some use them for entry into the host cells. CR1s on erythrocytes are utilized by Plasmodium falciparum (Spadafora et al. 2010; Tham et al. 2010) and Leishmania major (Da Silva et al. 1989). Coxackie A 21 virus (Shafren et al. 1997), Echovirus7(Wardetal.1994),andDrfimbriatedE.coli(Nowickietal.1993)useDAF whereasmcpareusedbymeaslesvirusforinvasion(nanicheetal.1993;manchesteret al.2000). 39

40 2Reviewofliterature Table,7.MicrobialacquisitionofcomplementregulatorsothersthanFH Protein, Microbe, Ligand, Reference, C1INH Bordetellapertussis Vag8 (Marr,etal.,2007,Marr,etal.,2011) Borreliarecurrentis CihC (Grosskinsky,etal.,2010) Escherichiacoli StcE (Lathemetal.2004) C4BP Bordetellapertussis FHA (Berggård,etal.,1997) Borreliaburgdorferis.l.? (Pietikäinenetal.2010) Borreliarecurrentis CihC (Grosskinskyetal.2010) Candidaalbicans Pra1 (Luo,etal.,2011) Escherichiacoli OmpA (Prasadarao,etal.,2002) Moraxellacatarrhalis UspA (Nordström,etal.,2004) Neisseriagonorrhoeae Por1A (Ram,etal.,2001) Neisseriameningitidis PorA (Jarva,etal.,2005) Porphyromonasgingivalis? (Potempa,etal.,2008) Salmonellaenterica Rck (Ho,etal.,2011) Staphylococcusaureus (Hair,etal.,2012) Streptococcuspneumoniae Enolase (Agarwal,etal.,2012) Streptococcuspyogenes Mprot., proteinh (Thern,etal.,1995) (Ermertetal.2013) Yersiniaenterocolitica YadA,Ail (Kirjavainenetal.2008) Yersiniapseudotuberculosis Ail (Ho,etal.,2012) Clusterin Staphylococcusaureus? (Partridgeetal.1996)(Chhatwalet al.1987) Staphylococcusepidermis? (Li&Ljungh2001) Streptococcuspyogenes SIC (Chhatwaletal.1987;Åkessonetal. 1996) Escherichia? (Chhatwal,etal.,1987) FactorI Staphylococcusaureus Prevotellaintermedia ClfA? (Hairetal.2008) (Malmetal.2012) CR1 Plasmodiumfalciparum PfRh4 (Spadaforaetal.2010;Thametal. 2010) CD59 Escherichiacoli? (Rautemaaetal.1998) Helicobacterpylori? (Rautemaaetal.2001) MCP Streptococcuspyogenes Mprotein (Okadaetal.1995) Vitronectin Haemophilusinfluenzae,Hib Hsf (Hallströmetal.2006) Moraxellacatarrhalis UspA2 (Singhetal.2010) Neisseriameningitidis Msf (Griffithsetal.2011) Rickettsiaconorii Adr1 (Rileyetal.2013) Streptococcuspneumoniae PspC (Vossetal.2013) Abbreviations:Adr,adhesionofRickettsia;Ail,attachmentNinvasionlocusprotein;Cih,complement inhibition;clf;clumpingfactor;crasp,complementregulatornacquiringprotein;fha,filamentous hemagglutinin;hsf,haemophilussurfacefibrils;msf,meningococcalsurfacefibrils;omp,outermembrane protein;pfrh,reticulocytenbindingnlikehomologprotein;por,porin;pra,phnregulatedantigen;psp, pneumococcalsurfaceprotein;rck,resistancetocomplementkilling;sic,streptococcalinhibitorof complement;stc,secretedproteaseofc1esteraseinhibitorfromehec;usp,ubiquitoussurfaceprotein; Yad,Yersiniaadhesin 40

41 2Reviewofliterature 2.4.2MICROBIALEVASIONOFPHAGOCYTOSIS Foramicrobe,preventingdetectionbyphagocytesiscriticalforavoidingphagocytosis and subsequent destruction. As described in the section 2.3, phagocytes recognize microbesmainlyviaprrs,crsandfcγrs.forthesereceptorstoidentifythetargetthe phagocyteneedstofindthemicrobeandthisismediatedbychemotaxis.microbescan inhibit any of these phenomena and, in addition, microbes may directly inhibit phagocytesbycausingdamagetothesehostcells EVASIONOFRECEPTORNMEDIATEDRECOGNITION Inadditiontoblockingdepositionofopsoniccomplementcomponentsontheirsurface, microbes have been reported to use some other strategies to inhibit complementn mediatedphagocytosis.secretedmicrobialproteinscandirectlybindtocr3andblock the chemotactic and phagocytic functions of neutrophils. The βnglucan surface on the yeastc.albicansismainlyrecognizedbycr3onneutrophils(vanbruggenetal.2009). However, the secreted protein Pra1 interacts with CR3 and inhibits the uptake and subsequent phagocytosis of the pathogen (Soloviev et al. 2007; Jawhara et al. 2012). Interestingly,sinceCR3recognizesFH,someFHNbindingmicrobeshavebeensuggested to utilize the FHNCR3 interaction for their own internalization into CR3 expressing epithelial cells, such as N.gonorrhoeae and S.pneumonia (Agarwal et al. 2010). Some microbialleukotoxinsbindtoβ2nintegrins(cr3orleukocytefunctionantigenn1;lfan1) onneutrophilsandmanipulatethecellulardownstream.theseincludethertx(repeat intoxin)familycytotoxinsofgramnnegativegenera,suchasthebordetella,escherichia, Moraxella,andVibrio(Welch2001). In the presence of antibodies, most pathogenic strains of S. pyogenes, produce the proteasesides,speb,andendosthattargetandcleavethesurfaceboundigg,thereby hinderingneutrophilrecognition(collin&olsen2001).secretoryiga(siga)interacts with FcγRI expressed on phagocytes and strains of the mucosal bacteria Neisseria, S. pneumoniae, and H. influenzae express IgA proteases (Plaut et al. 1975; Male 1979; Murphyetal.2011).Recently,EsiB(E.coliSIgAbindingprotein)fromextraintestinalE. coli was found to bind SIgA leading to impaired FcγRI crossnlinking and further downstreamsignaling.asaresult,neutrophilchemotaxiswasinhibitedaswellasthe activationofthenadphnoxidase(pastorelloetal.2013). Upon release of bacterial and hostnderived chemotactic fragments, phagocytes move towardstheinfectionsite.however,manypathogenshaveevolvedwaystoimpairthis 41

42 2Reviewofliterature recruitment.theendopeptidasescpaofs.pyogenescleavesc5aandadehydrogenase bindsandinactivatesc5a(teraoetal.2006) OTHEREVASIONMECHANISMS Toxins that form pores on neutrophils and subsequently lyse the cells are important playersininnateimmuneevasion,suchasstreptolysinsfoundons.pyogenes(miyoshin Akiyama et al. 2005). GramNnegative bacteria have evolved type III secretion systems (T3SS)thatallowdirectinjectionofbacterialproteinsintothecytosoloftheneutrophil to disrupt the neutrophil signaling network and the production of ROS. Examples includetheyersiniaouterproteins(yops)(ruckdescheletal.1996;diepoldetal.2010), and exotoxins of Pseudomonas and Salmonella (Geddes et al. 2007). Several other microbeshavemechanismswhichdownregulatetheproductionofros,likec.albicans (Wellington et al. 2009). Moreover, individuals with defects in the assembly of the NADPHNoxidase are susceptible to recurrent infections of S. aureus, C. albicans and Aspergillusspp.(Segal1996). After phagocytic uptake the microbe faces an array of bactericidal mechanisms which several pathogens manipulate for their own advantage. Francisellatularensis does not triggeranoxidativeburst,likelybydisturbingintracellularsignaling(sandströmetal. 1988).HelicobacterpyloriblocksNADPHNoxidasebybindingtothephagosomethereby redirectingthegranulestotheplasmamembraneleadingtoextracellularrosrelease (Allen et al. 2005). Intracellular fate of bacteria within neutrophils is not as well characterized as compared to macrophages, however, S.pyogenes and N.gonohorreae resistkillingbydelayingthefusionofgranulestothephagosomewhichmaycontribute tothepersistenceinthehost(staalietal.2006;johnson&criss2013). Escape from the NET formation is described for a few pathogens. The polysaccharide capsule, together with a modulation of its lipoteichoic acids, protects S. pneumoniae against elimination by NETs (Wartha et al. 2007). H.influenzae entrapped in biofilms, which consists of both host derived DNA from NETs and bacterianderived DNA, is resistant to both NET killing and elimination by phagocytes recruited to the site of infection (Thornton et al. 2013). Another mechanism utilized by S. pyogenes and S. pneumoniaeistheproductionofdnases(sumbyetal.2005;buchananetal.2006). 42

43 2Reviewofliterature 2.5,EXAMPLES,OF,INNATE,IMMUNE,EVASION, 2.5.1STAPYLOCOCCUSAUREUS IMMUNEEVASIONSTRATEGIESOFA GRAMNPOSITIVEBACTERIUM EversinceOgstonidentifiedS.aureusasoneofthefirstbacterialpathogensin1880this bacterium has remained a major human pathogen (Smith 1982), causing a variety of diseases such as superficial and deep skin infections and more severe conditions includingabscesses,sepsis,andendocarditis. S.aureus naturally colonizes the mucosal membranes of the nose and the skin of 20N 30%ofthehumanpopulation(Lowy1998).Skinandsofttissueinfections(SSTI)may occurifthereisawoundoranotherlesionintheskinallowingthebacteriatoenter.a previous infection does not generate protective immune responses and recurrence of SSTI is not uncommon. Usually the infections are mild but in a number of cases the infection leads to lifenthreatening disease (Lowy 1998). In Europe S.aureus has been isolated from 10% of the hospitalnacquired bloodstream infections (Fluit et al. 2001). Moreseldom,S.aureusisthecausativeagentforrespiratory,bone,andjointinfections. Some staphylococcal diseases result from effects of a specific toxin, such as staphylococcal food poisoning, staphylococcal scalded skin syndrome, and toxic shock syndrome.forexample,toxicshocksyndromeisinitiatedwiththelocalizedgrowthof toxinnproducingstrainsfollowingsystemiceffectsoftheabsorbedandbloodcirculation distributedsuperantigentoxin;thefatalityrateishighunlessappropriateantibioticsare rapidlyadministered(devriesetal.2011) CELLWALL The ability of S.aureus to cause infection is due to its various cell surface proteins as well the secreted ones. Moreover, several strains are covered with a polysaccharide capsule and an additional slimenlayer consisting of monosaccharides, proteins and peptides.thecellwallisbuiltupoftwomajorparts,apeptidoglycanlayerandteichoic acid. The peptidoglycan layer consists of glycans with two alternating sugars, NN acetylmuramic acid (MurNAc) and NNacetylglucosamine (GlcNAc), which form a disaccharide unit. Peptide subunits of LN and DN amino acids are linked to the NN acetylmuramic acid and the peptide subunits are highly crossnlinked by interpeptide bridges that make the cell wall rigid (Amako et al. 1982). Host lysozyme naturally breaks the bonds between NNacetylmuramic acid and NNacetylglucosamine aiming at killing the bacteria, but a staphylococcal enzyme which causes ONacetylation of the 43

44 2Reviewofliterature peptidoglycan makes S. aureus resistant to lysis by lysozyme (Bera et al. 2005). The other major component of the cell wall, the teichoic acids, are phosphatencontaining polymers covalently bound to the peptidoglycan layer (wall teichoic acids) or to membranenbound lipids (lipoteichoic acids). Modified phosphatidylglycerol with DN lysinehasbeenshowntoprotects.aureusagainstantimicrobialpeptidesproducedby thehost(pescheletal.1999;collinsetal.2002b).bymodulatingthecompositionand chargeofthelipoteichoicandthecellswallteichoicacidsandphospholipidsinthecell membrane,s.aureusprotectsitselfagainstdefensinsandcathelicidinsgeneratedbythe host(peschel2002) ESCAPEFROMOPSONIZATION Themajorityofthestaphylococcalsurfaceproteinsserveasadhesinsandinvasinsbut someareclearlyapartoftheinnateimmuneevasionmachinery(fig.7,modifiedfrom Spaanetal,2013).StaphylococcalproteinA(SpA)preventsCPactivationviabindingto the C1q molecule (Forsgren & Sjöquist 1966; Sjöquist & Stalenheim 1969). More importantly,spaalsointeractswiththefcregionofiggtherebyinhibitingrecognition by FcγR on neutrophils (Palmqvist et al. 2005). By binding to Fab regions on IgM it crossnlinks the B cell receptor and modulates downstream signals and inhibits the adaptive immune responses against staphylococcal antigens (Falugi et al. 2013). Recently,SpAwasshowntobereleasedfromthebacterialwallsuggestingthatithasa broaderroleandfunctionssimilartosecretedpeptides(beckeretal.2014). S.aureusclumpingfactorA(ClfA)interactsnotonlywithfibrinogenandfibrinbutalso factorileadingtoenhanceddegradationofc3b(cunnionetal.2004;hairetal.2008). StaphylococcalbinderofIgG(Sbi)hasalsoamembraneboundandasecretedformand bothformshavebeensuggestedtocontributetoimmuneevasion(smithetal.2011). The NNterminal part of the molecule binds the IgG Fc region similar to SpA and apolipoprotein H (Zhang et al. 1998; Atkins et al. 2008), and the CNterminus interacts withthec3molecule(hauptetal.2008). InadditiontoSbi,S.aureushastwoothergroupsorfamiliesofsecretedproteinsthat interact with C3, the SCINN (staphylococcal complement inhibitor) and the EfbN (extracellularfibrinogenbinding protein)proteinfamilies.thescinnfamilyconsistsof SCINNA, NB, and NC all of which share 46N48% sequence identity with each other (Rooijakkersetal.2005b;Jongeriusetal.2007).AfourthSCINprotein,SCINND(ORFND), hasalsobeenidentifiedbutitdoesnotinteractwithanyofthec3fragmentsandaclear functionforitiscurrentlynotknown(rooijakkersetal.2007).scinproteinstargetc3 44

45 2Reviewofliterature convertasesofbothapandcp/lpandstabilizetheconvertasesonbacterialsurfaceand inafluidphase,therebyslowingdowngenerationofnewconvertases(rooijakkersetal. 2005b;Jongeriusetal.2007;Rooijakkersetal.2009).Itwasshownthatdimerizationof SCINNAinacomplex,(C3bBb/SCINNA) 2,wasimportantforitsfullfunctions(Jongeriuset al.2010b).amutantscinnawasabletoblocktheconvertaseactivityequallytothewild typebuthadnoeffectontherecognitionofc3bbycr1andcrig,whilethewildtype disruptedthisrecognition. Originally,Efbwasidentifiedasa16.8kDafibrinogenbindingprotein(Boden&Flock 1994),andlateritwasfoundthatitsCNterminalfragment(EfbNC)interactswithC3,C3b, andc3d(leeetal.2004;hammeletal.2007b;jongeriusetal.2007).anearbygenewas independently found by two groups to encode a 9.8 kda protein with 44% sequence homology to EfbNC (Hammel et al. 2007b; Jongerius et al. 2007). Due to its binding activitiesforc3,c3b,andc3d,itwasnamedextracellularcomplementbindingprotein (Ecb),butitisalsoknownasEfbhomologousprotein(Ehp).Bothproteinswereshown tocontributetopathogenesisofs.aureusinfectionsinvivoinaratwoundmodelorin variousmousemodels(mamoetal.1994;palmaetal.1996;jongeriusetal.2012).the EcbandEfbproteinsblockC3bNcontainingconvertasesleadingtodecreasedproduction of C5a (Jongerius, etal., 2007). A molecular explanation for this event was suggested when Ecb was shown to enhance the interaction between FB and C3b, suggesting stabilizationofthec3bnfbcomplex,sincecleavageoffbtoitsbbandbafragmentswas impaired (Jongerius, et al., 2010). However, contrasting results were reported in anotherstudywithefbnc,whichinhibitedinitialfbbindingtoimmobilizedc3b(chenet al. 2010). The binding between C3d and EfbNC/Ecb also disrupts the C3dNCR2 interaction(ricklinetal.2008),whichhaspotentiallyfunctionalconsequencesforbcell activationandadaptiveimmuneresponses. Also, Sbi and Efb are shown to recruit plasminogen for degradation of C3 and C3b S. aureus has several molecules that can proteolytically cleave complement components. Thesecretedstaphylokinase(SAK)targetsplasminogentothebacterialsurface,which is subsequently cleaved into plasmin (Lähteenmäki et al. 2001). Plasmin cleaves the opsoninsigg,c3b,andic3bleadingtoimpairedneutrophilphagocytosis(rooijakkerset al.2005a).also,sbiandefbareshowntorecruitplasminogenfordegradationofc3and C3b (Koch et al. 2012). The metalloprotease aureolysin cleaves C3 and subsequently blocksallcomplementpathways(laarmanetal.2011).s.aureushasalsobeenshownto acquiretheregulatorfhviasdre(sharpetal.2012)andviaasbinc3bcomplex(haupt etal.2008)resultingindecreasedic3bconversion. 45

46 2Reviewofliterature S. aureus has several staphylococcal superantigennlike proteins (SSL). One of them SSLN10,interactswithIgG(Itohetal.2010)whileSSLN7bindshumanIgA(Langleyetal. 2005) leading to impaired interaction of IgG and IgA with FcγRI on neutrophils. Moreover, SSLN7 binds to and prevents the cleavage of C5 (Bestebroer et al. 2010; Laursenetal.2010).Alltheabovementionedmechanismsleadtoimpairedneutrophil uptake. Figure,7.,S.aureus,escape,from,complement,and,opsonization.Proteinsindarkgrey indicatehost scomplementproteins.membranenboundorsecretedvirulenceproteins markedinredarebindingtoandblockingthefunctionsofcomplementcomponentsor immunoglobulins. The proteins in blue are proteases that degrade complement components.abbreviations:clf,clumpingfactor;ecb,extracellularcomplementbinding protein; Efb, extracellular fibrinogen binding protein; SAK, staphylokinase; Sbi, staphylococcal IgGNbinding protein; SpA, staphylococcal protein A; SSL, staphylococcal superantigennlikeprotein.(picturemodifiedfrom(spaanetal.2013). 46

47 2Reviewofliterature ESCAPEFROMNEUTROPHILSANDNETS The S.aureus SSLN5, SSLN11, and extracellular adherence protein (Eap) interfere with ligandsrequiredforneutrophilstorollandadhereontoendothelialcells(chavakisetal. 2002;Bestebroeretal.2010).SSLN5isalsoaninhibitorofchemokinereceptorsaswell asc5ar(bestebroeretal.2009).staphylococcalchemotaxisinhibitoryprotein(chips) blocksthec5araswellasfprsonneutrophilsandseveralother(fpr)nlikeinhibitory proteins are shown to bind to FPR. This eventually results in impaired phagocytic responses(dehaasetal.2004;pratetal.2006;kretschmeretal.2010).secretionofthe protease Staphopain A inhibits the CXC chemokine receptorn2 (CXCR2) dependent chemotaxis and activation (Laarman et al. 2012). S. aureus also has mechanisms that directly eliminate neutrophils. Secreted cytolytic toxins, such as the twoncomponent leukocidins, attack the cell membrane resulting in leakage and lysis of the cells. For example,thepantonnvalentineleukocidinispresentinmoststrainscausingcommunityn acquired methicillinnresistant S. aureus (MRSA) infections (Naimi et al. 2003). A schematicoverviewofmoleculesinvolvedinescapefromneutrophilsisshowninfig.8 (figuremodifiedfromspaanetal.,2013).inaddition,thereisevidencethats.aureuscan surviveinsidehostcells(thwaites&gant2011),aswellaslysingtheneutrophilsafter phagocytosis(kobayashietal.2010). S. aureus escapes from NETs by secreting the two staphylococcal nucleases staphylococcalnuclease(nuc)(berendsetal.2010)andadenosinesynthasea(adsa) (Thammavongsa et al. 2009; Thammavongsa et al. 2011) that degrade the DNA. The golden pigment staphyloxanthin is an antioxidant that protects against ROS (Liu et al. 2005).OthermoleculescontributingtoS.aureusresistanceagainstROSarecatalasethat converts hydrogen peroxide to oxygen and water (Mandell 1975), and SOK (surface factorpromotingresistancetooxidativekilling)(liuetal.2005). The large number of molecules that act and inhibit distinct stages of the innate immunityaswellasfactorsthatformabridgebetweeninnateandadaptiveimmunity has turned S. aureus into a master of immune evasion. On top of all the virulence mechanisms,s.aureusiseffectiveinacquiringantibioticresistanceandtheemergence of resistant strains has become a global problem (DeLeo et al. 2010) which offer challengesforthefuture. 47

48 2Reviewofliterature Figure,8.S.aureus,escape,from,neutrophils.MoleculesintheredovalsareS.aureus factorsthatcanbindandblockthefunctionsofthereceptors.theproteinintheblue oval is a protease that cleaves the NNterminal of CXCR2. Abbreviations: C5aR, C5a receptor; CHIPS, chemotaxis inhibitory protein of staphylococcus; CXCR2, chemokine receptor;gpcr,gnproteincoupledreceptor;fpr,formylproteinreceptor;flipr,fprn like inhibitory proteins; ScpA, staphopain A; SSL, staphylococcal superantigennlike (FiguremodifiedfromSpaanetal.,2013) BORDETELLAPERTUSSIS IMMUNEEVASIONSTRATEGIESOFAGRAMN NEGATIVEBACTERIUM Bordetellapertussisisthecausativeagentofwhoopingcough(pertussis, hundredday cough )andwasfirstisolatedin1906bybordetandgengou(mattoo&cherry2005). The highly contagious bacteria colonize the ciliated epithelial cells of the upper respiratory tract in humans. Symptoms include paroxysmal coughing with whooping, often followed by postncoughing vomiting. Coughing can persist for weeks or even months,butinindividualswithpartialimmunitythesymptomsarelikelytobemilder (Cherryetal.2004). The earliest vaccines that consisted of killed whole B. pertussis cells brought the epidemicundercontrolbuttheywereassociatedwithsevereadverseevents(codyetal. 1981). Today these vaccines are mostly replaced by the second generation vaccines 48

49 2Reviewofliterature containing one to five purified bacterial components being filamentous hemagglutinin (FHA),pertactin,twotypesoffimbriae,anddetoxifiedpertussistoxin(Ptx)(seesection ).Protectionaftervaccinationvariesdependingonthequalityofthevaccineand immunizationscheduleinuseandimmunity,butmaylastfrom4n12years(lugaueret al. 2002; Wendelboe et al. 2005). The vaccine coverage in the population is high, in Finlandashighas98%(Mertsola2001),whichkeepsthediseaseundercontrol.Inspite ofthisthecirculationofstrainsinthecommunityhasnotdecreased LIPOPOLYSACCHARIDE The complex LPS (endotoxin) of GramNnegative bacteria consists of lipids covalently linkedtopolysaccharidescomprisingtheinnerandoutercoreregions.thecoreregions may be linked to an ONside chain (ONantigen) which is composed repetitive glycomers (Caroff&Karibian2003).B.pertussislackstheONchain(Peppler1984;Burnsetal.2003) andissuggestedtohaveanadvantageincomplementevasion(pishkoetal.2003).lipid A is a part of the outer leaflet of the outer membrane and anchors the core oligosaccharidetothecellwall.thelpsofb.pertussisappearsastwodistinctbands,a and B, in a silver stained SDSNgel (Peppler 1984). Band B of the core oligosaccharide contains the sugars heptose and glucose, and glucuronic acid, glucosamine, and galactosaminuronic acid (GalNAcA) (Caroff et al. 1990). The slower migrating band A consists of band B connected to a trisaccharide of NNacetylNNNmethylfucosamine (FucNAcMe), 2,3NdeoxyNdiNNNacetylmannosaminuronic acid (2,3NdiNAcManA), and NN acetylglucosamine (GlcNAc) (Caroff et al. 1990). In general, the lipid A on LPS is recognizedandopsonizedbysurfactantproteinasecretedbyalveolarcellsinthelungs. TheB.pertussisLPS,however,hasastructurethatprotectsagainstsurfactantproteinA, possiblybysterichindrance(schaefferetal.2004).thisleadstodestabilizationofthe bacterial outer membrane, aggregation of bacteria thereby hindering adhesion to alveolarepithelia,andpromotionofphagocytosis(vaniwaardenetal.1994) VIRULENCEFACTORS ExpressionofmostvirulencefactorsofB.pertussisareregulatedbythetwoNcomponent control system, BvgAS. BvgAS is a transmembrane sensor protein that upon environmental stimulation controls the three phenotypic phases. The Bvg + phase expresses molecules needed during colonization and invasion while during the Bvg N phase the phenotype is avirulent. It is unclear whether the avirulent phenotype is functional in B. pertussis. A third intermediate phase, Bvg i, is suggested to have an essential function in the transmission and early respiratory tract colonization 49

50 2Reviewofliterature (Stockbauer et al. 2001). Important B. pertussis virulence factors are schematically describedbelowandpresentedinfigure9. Colonization.Thefirststepforamucosalpathogenistoadheretotheepithelialcellsand B.pertussisexpressesvariousproteinsthatfacilitatethecolonizationoftherespiratory tract. The major adhesins are FHA, fimbriae (Relman et al. 1989; van den Berg et al. 1999;Rodriguezetal.2006),andpertactin(Leiningeretal.1991).FHAisalarge(232 kda)proteinpresentassurfacenattachedandsecretedforms(renauldnmongenieetal. 1996).ItsCNterminaldomainisrequiredforcolonizationoftherespiratorytractinarat model (Julio et al. 2009). The long, thin structures of fimbriae extend from the cell membraneandcontainheparinnbindingregions,whichmostlikelyareinvolvedinthe adherence(geuijenetal.1998).thefimbriaeconsistofoneofthemajorsubunits,fim2 or Fim3, and an additional FimD subunit (Willems et al. 1993; Geuijen et al. 1997). Pertactinisanautotransporterproteinwhichischaracterizedbyanabilitytodirectits ownsecretionacrosstheoutermembrane(leiningeretal.1991).itcontainsonergd (ArgNGlyNAsp) domain that most likely is involved in adhesion. Another virulence molecule in the autotransporter family is the tracheal colonization factor (Tcf). Most likely it has an important function in colonization of B. pertussis, although the mechanismisstillunclear(finnetal.1991;finn&stevens1995). Formation of biofilms is a rather complex and not extensively characterized phenomenon in B. pertussis. It is, however, important for mucosal pathogens by facilitating adherence and protection against antimicrobial agents and host immune defenses(conoveretal.2010;serraetal.2011). Escapefromopsonizationandphagocytosis.TheproteinsVag8(Finn&Amsbaugh1998) andfhainteractwiththecpofcomplementbybindingc1inh(marretal.2007;marr et al. 2011) and C4BP (Berggård et al. 1997; Berggard et al. 2001), respectively. Moreover, the Bordetella resistance to killing protein (BrkA) mediates complement resistancebyinhibitingdepositionofc3b,c4b,andformationofthemac(fernandez& Weiss 1994; Barnes & Weiss 2001). The pertactin protein, which has 29% homology withbrka,didnotcontributetoserumresistance(fernandez&weiss1998).however, colonization with the related animal pathogen B. bronchiseptica in a mouse model showedthatastrainlackingpertactininhibitedclearancebyneutrophils(nicholsonet al.2009;inatsukaetal.2010). Toxins are important in B.pertussis pathogenesis and most of them have functions in inhibitionofneutrophilrecruitment.ptxisproducedexclusivelybyb.pertussisamong 50

51 2Reviewofliterature the genus Bordetellae. It is composed of five subunits, S1NS5 (Tamura et al. 1982) of which the S1 subunit (A subunit) maintains the enzymatic activity. The A subunit is bound to a ringnlike structure formed by the S2NS5 subunits (B oligomer) (Carbonetti 2010).PtxbindstohostcellsviaitsBoligomerandentersthecytosolbyendocytosis where it targets the GPCR resulting in modulation of the signaling pathway and intoxication of the cell (Katada et al. 1983). By binding to epithelial cells in the respiratory tract Ptx inhibits chemokine release from the cells leading to impaired chemotaxis and neutrophil recruitment (Thomazzi et al. 1995; Kirimanjeswara et al. 2005;Andreasen&Carbonetti2009).Furthermore,invivostudieshaveshownthatPtx can suppress the antibody responses to several B. pertussis antigens (Mielcarek et al. 1998;Carbonettietal.2004). Trachealcytotoxin(TCT)isapartofthecellwallofGramNnegativebacteriaandwhile other bacteria, such as E.coli, recycle the disaccharidentetrapeptide by transporting it back to its cell cytoplasm (Jacobs et al. 1995), B. pertussis releases it into the environment(rosenthaletal.1987).tctdamagestheairwayepithelialcells(wilsonet al. 1991). In addition, it inhibits neutrophil migration toward formylated peptides (Cundell et al. 1994). Adenylate cyclase toxin (ACT) is secreted, however, most of it interactswithfhaandremainsassociatedwiththebacterialsurface(grayetal.2004). Itquicklyformsinactiveaggregatesinsolutionandaclosecontactwiththetargetcellis needed for its activity. ACT inhibits neutrophil phagocytosis by increasing cyclic adenosine monophosphate (camp) cellular levels leading to actin cytoskeletal rearrangements and subsequently inhibition of chemotaxis and formation of ROS (Friedmanetal.1987;Kamanovaetal.2008). Invitro studies have demonstrated that both IgG and IgA are important in neutrophil phagocytosis of B. pertussis (Hellwig et al. 2001a; Rodriguez et al. 2006) and in vivo studies showed that the neutrophil FcγR is important in the clearance of the bacteria fromthelungs(hellwigetal.2001b;andreasen&carbonetti2008).intheabsenceof antibodies the bacteria can be bound via a FHANCR3 interaction and subsequently phagocytosed by neutrophils, macrophages and monocytes. Interestingly, this process does not seem to promote an oxidative burst (Berton et al. 1992) and suggest that B. pertussisexploitscr3foruptakeandintracellularsurvival.furthermore,bothptxand FHAenhancetheexpressionofCR3byneutrophils(MobberleyNSchuman&Weiss2005) providingfurtherevidencethatuptakeviacr3isadvantageousforb.pertussis. 51

52 2Reviewofliterature OTHERBORDETELLASPECIES InadditiontoB.pertussis,thegenusBordetellaincludeseightotherspecies,whichmost likelyoriginatefromacommonenvironmentalb.petriinancestor.theb.pertussisandb. parapertussisspecieshaveevolvedashumanspecificpathogensfromb.bronchiseptican likeanchestorsandtogetherthesethreespeciesareknownastheclassicalbordetella.a small portion of pertussis is caused by B. parapertussis, and sporadically also by B. bronchiseptica and B. holmesii, the two latter ones mostly isolated from immunocompromised individuals (Woolfrey & Moody 1991; Yih et al. 1999). Other membersofthebordetellareportedtobeisolatedfromimmunocompromisedpatients are B. hinzii (Vandamme et al. 1995), B. avium (Harrington et al. 2009), B. trematum (Vandammeetal.1996),andB.ansorpii(Fryetal.2007). 52

53 2Reviewofliterature, Figure, 9., Presentation of important B. pertussis virulence factors. The adhesins, fimbriae, pertactin,fha,vag8,brka,andtcfareshowninblue;hostmoleculesingreen;thetoxinsact andptxinredandtctinbrown.fhaandvag8bindtheclassicalpathwayregulatorsc4bpand C1INH,respectively,andBrkAinhibitscomplementopsonizationbyanunknownmechanism.Ptx stimulatestheexpressionofchemokinesfromepithelialcellsresultingininhibitedrecruitment ofphagocytes,includingneutrophilsandalveolarmacrophages.ptxdirectlyintoxicatealveolar macrophages,andinthebloodstreamitsuppressesantibodyresponsestob.pertussisantigens. ACT together with FHA induces apoptosis of macrophages in a CR3N dependent manner. They block CR3 on dendritic cells and ACT bound to phagocytes modulate the signaling leading to inhibited chemotaxis and superoxide production. TCT is released from the peptidoglycan in periplasmicspaceandreleasedintotheextracellularenvironmentbyanunknownmechanism.it attacksanddamagesciliatedcells.abbreviations:ab,antibody;act,adenylatecyclasetoxin;brk, Bordetella resistance to complement; C4BP, C4b binding protein; C1INH, C1 inhibitor; FHA, filamentoushemagglutinin;im,innermembrane;om,outermembrane;ptx,pertussistoxin;tcf, trachealcolonizationfactor;tct,trachealcytotoxin. 53

54 3Aimsofthestudy 3,AIMS,OF,THE,STUDY, I. TostudyhowtheimportantpathogenBordetellapertussisevadestheattackby complementalternativepathway. II. To analyze the molecular mechanisms of how and why various microbes bind complementalternativepathwayinhibitorfactorhviathecnterminus. III. To determine how Staphylococcus aureus prevents the attack by alternative complementpathwayattack. 54

55 4Materialsandmethods 4,,MATERIALS,AND,METHODS, 4.1,MATERIALS, 4.1.1COMPLEMENTPROTEINS FH and C3 were isolated from plasma of healthy laboratory workers according to Koistinen et al. (Koistinen et al. 1989) with minor modifications described elsewhere (Haapasalo,etal.,2008)orobtainedfromCalbiochem(LaJolla,CA).C3bwasprepared fromc3asdescribedinthesection4.2.1.thec3dproteinwasexpressedine.coli(nagar etal.1998)andwasakindgiftfromprofessordavide.isenman(universityoftoronto, Canada).FactorsBandDwerepurchasedfromCompTech(Tyler,TX)andFactorIfrom Calbiochem/MerckMillipore (Darmstadt, Germany). Soluble CR1 was obtained from CellDexTherapeutics(Needham,MA). ThegenesencodingrecombinantproteinsFH1N5,1N6,1N7,8N11,11N15,and15N20were cloned and the recombinant proteins produced in the baculovirus expression system described earlier (Kühn & Zipfel 1995). The genes encoding recombinant fragments FH5N7andFH5N7variant(FH5N7 var) withan Y402Hsubstitution, were cloned and the gene products produced as described by Haapasalo, etal. (2008). Cloning of the gene and purification of FH19N20 was described previously (Jokiranta, et al., 2006). GenerationoftheFH19N20substitutions D1119G/Q1139A,D1119G,Q1139A,L1189R, E1198A,R1215Q,andW1157A,R1182A,W1183L,T1184R,K1186A,K1188A,R1203A, R1206A,andR1210Aweredescribedearlier(Lehtinenetal.2009).Theconstructswith several point mutations in the domain 19 (FH19 del N20, with substitutions Q1137A, Q1139A, and Y1142A), or the domain 20 (FH19N20 del, with substitutions T1184G, K1202A,R1203A,andY1205A)havebeendescribed(Kajander,etal.,2011).FH1N4was expressedinpichiapastorisbymscs.hyvärinenasdescribed(blancetal.2012) BACTERIALPROTEINS The genes encoding outer surface proteins OspE and OspA were cloned from B. burgdorferi sensu stricto strain N40 and the recombinant protein was purified (Hellwageetal.2001).ThegeneencodingFhbAwasclonedfromB.hermsiistrainMAN (Hovisetal.2006)andTufgenewasclonedfromaP.aeruginosabloodisolatestrain, and the recombinant proteins were purified as previously described (Kunert et al. 2007).ThegenesencodingS.aureusproteinsEcb,Ecb N63E/R75E/N82E,andEfbwerecloned, expressedandtherecombinantproteinspurifiedasdescribedearlier(jongerius etal. 2007;Hammeletal.2007;Jongeriusetal.2010). 55

56 4Materialsandmethods 4.1.3OTHERPROTEINSANDSERA Bovine and human serum albumin (BSA and HSA, respectively), gelatin and heparin werepurchasedfromsigmaaldrich(st.louis,mo).normalhumanserum(nhs)was obtainedfromatleastfivehealthypersonsbelongingtothelaboratorypersonneland the NHS was stored at N70 C until used. To inactivate complement, NHS was heatn inactivated at 56 C for 30 min (HIS). NHS deficient in FHRN1 was obtained from an individualknowntobefhrn1deficient.forserumsurvivalassaysinthestudyi,nhs with IgG titer <1500 was used. The classical and lectin pathways were blocked with ethyleneglycoltetranaceticacid(egta)andmgcl 2addedtoNHStofinalconcentrations of 10 mm and 5 mm, respectively (MgEGTANNHS). C3 depleted serum was purchased fromquidel(sandiego,ca) ANTIBODIES AntibodiesusedinthethesisarelistedinTable8. Table,8.Primaryandsecondaryantibodiesusedinthestudies Antibody Description Supplier Study Primary,antibodies, AntiNFH GoatpAb 1 Calbiochem,CA I AntiNFH GoatpAb Quidel,SanDiego,CA III AntiNC3c RabbitpAb DAKO,Denmark IV AntiNC3d RabbitpAb DAKO III AntiNFB RabbitpAb HoechstNBehring,Marburg, III Germany Secondary,antibodies, AntiNgoat PeroxidaseNconjugated JacksonImmunoresearch, I WestGrove,PA AntiNrabbit PeroxidaseNconjugated JacksonImmunoresearch III AntiNgoat FITC 2 Nconjugated JacksonImmunoresearch I AntiNrabbit AlexaFluor 488N Invitrogen,Eugene,OR IV labeled AntiNhumanC3 FITCNconjugated ProtosImmunoresearch, Burlingame,CA IV 1polyclonalantibody; 2 fluoresceinisothiocyanate 56

57 4Materialsandmethods 4.1.5MICROBES Allmicrobialstrainsusedinthisthesis,includingtheirgrowthconditionsarelistedin Table9. Table,9.Microbialstrainsandgrowthconditions Bacteria Strainandrelevantfeature Referenceorsource Study,,,, B.pertussis* TohamaI,referencestrain (Kasugaetal.1954) I B.pertussis* B32,mutationinPtxP (Black&Falkow1987) I B.pertussis* B.p.175,Clinicalisolate, TheNationalPublicHealth I Fim3,Prn2,PTXS1A Institute,Turku,Finland B.pertussis* B.p406,Clinicalisolate, TheNationalPublicHealth I Fim2,Prn2,PTXS1A Institute,Turku,Finland B.parapertussis ATCC15251 (Eldering&Kendrick1952) I B.holmesii ATCC51541,human (Weyantetal.1995) I isolate B.avium ATCC35086,frompoultry (Hinzetal.1978) I S.aureus Wood46 (Jongeriusetal.2007) III S.aureus Newman (Jongeriusetal.2012) IV S.aureus Clinicalisolatefromblood HUSLAB,Helsinki,Finland II,IV H.influenzae Clinicalisolate HUSLAB,Helsinki,Finland III H.influenzae Clinicalisolatefromblood HUSLAB,Helsinki,Finland II S.pneumoniae Bloodisolate HUSLAB,Helsinki,Finland II S.pyogenes emm8,nonnbinderoffh Haapasalo,etal.,2008 II S.pyogenes st369,bindsfh Haapasalo,etal.,2008 I P.aeruginosa # Bloodisolate HUSLAB,Helsinki,Finland II C.albicans Bloodisolate HUSLAB,Helsinki,Finland II * Growth conditions: 36 C; on charcoal agar without blood and containing cephalexin (0.04 mg/ml) or StainerNScholtebroth. Growthconditions:37 C;otherwisesameasB.pertussis Growthconditions:35 C(B.holmesii)or37 C(B.avium),5%CO2,oncharcoalplateswithoutantibioticsor brainbheartinfusionbroth. Growthconditions:37 C,5%CO2;onbloodbagar,chocolatebagarplates,orToddbHewittbroth Growthconditions:37 C,5%CO2;onchocolatebagarplatesorbrainbheartinfusionbroth #Growthconditions:37 C,5%CO2;oncledplates Growthconditions:33 C;onSaboraudagar 57

58 4Materialsandmethods 4.2,METHODS, 4.2.1GENERATIONOFC3b The C3b was generated from C3 by trypsin cleavage. Trypsin (final volume 1% w/w; Sigma Aldrich) was added to C3 in phosphate buffered saline (PBS: 137 mm NaCl, 10 mmna 2HPO 4,2.7mMKCl;pH7.4)mixed,andincubatedinawaterbathat37 Cfor2 min. The reaction was stopped by adding soy bean trypsin inhibitor (SBTI; Sigma Aldrich) at a final concentration of 2% (w/w). The generation of C3b and C3a was indicatedbyashiftinthesizeoftheαnchaintoα NchainvisualizedafterSDSNPAGEand subsequentcoomassiestaining.thesampleswerecentrifugedandproteinsseparated bygelfiltration(hiload16/60sephadex200,gehealthcare) RADIOLABELINGOFPROTEINS Proteinswerelabeledwith 125 I(PerkinElmer)usingtheIodoGenmethodaccordingto themanufacturer sinstructions(thermoscientificpierce,rockford,il).briefly,iodogen (1,3,4,6NtetrachloroN3a,6aNdiphenylNglycoluril, approx. 1 mg/ml) (Thermo Scientific or Sigma)wasmixedwithchloroform(25ml)andallocated(50µl)intoglasstubesand airndried. The protein (20N50 µg in 100 µl of PBS) was mixed with 4 µl of 125 I in the coatediodogentubeandafterincubationfor10minthemixturewastransferredintoa glass tube. The nonnbound 125 I was separated from bound by running the mixture throughapdn10column(amershambiosciences) DIRECT 125 INPROTEINBINDINGASSAYS(I,II) AfterharvestingthebacteriaoryeasttheywerewashedthreetimeswithVBS(142mM NaCl; 5 mm diethyl barbiturate, ph 7.3) or PBS. The indicated number of cells were incubatedwithradiolabeledprotein(25,000 40,000cpm/reaction)inVBS, or50%pbs containing0.1%gelatin(gvbsandgpbs,respectively),or0.33%vbs.inhibitorsofthe interactionwereanalyzedbyaddingunlabeledproteintothereactionmixture.after20n 30minofincubationat37 Cwithagitation(1200rpm),themixtureswerecentrifuged through20%sucroseingvbsorgpbstoseparatecellnassociatedandfreeradioactive proteins. Radioactivity in the pellet and supernatants were measured with a gammacounter(wallac,turku,finland). 58

59 4Materialsandmethods 4.2.4RADIOLIGANDASSAYS(II,III,IV) For the radioligand assays BreakApart microtiter plates (NALGE NUNC, Roskilde, Denmark)werecoatedwithbacteria(1x10 6 CFU/wellinPBSat37 Cfor12hours)or variousproteins(2n25μg/mlinpbsat4 Cfor12N18hours).Afterwashingthreetimes withpbs,thewellswereblockedwith0.5%bsainpbs,or0.5%bsain50%pbs,for60 min at 22 C. In a separate nonadherent microtiter plate (Greiner Bio One, Frickenhausen, Germany), variable concentrations of nonlabeled proteins were mixed withtheradiolabeledproteinsin50%pbsorpbscontaining0.1%bsa.mixtureswere addedtothewellsandincubatedat37 Cfor60min.ThewellswerewashedwithPBSor 50% PBS, dried, and bound radioactivity from wells was measured with a gammacounter. When 125 INFB binding to C3b was analyzed, coating and washing buffer was VBS and blockingbufferwasvbscontaining0.5%bsa.theunlabeledandlabeledproteinswere incubatedin½vbsnnicl 2(0.75mMNiCl 2,2.5mMbarbituralacid,71mMNaCl) COFACTORASSAYSFORC3bINACTIVATION(I,II,III,IV) CleavageofC3bbyFIwasanalyzedbyincubatingFH,FH1N4,orsCR1(atconcentrations indicated in the studies II, III, and IV) with 125 INC3b (100,000 cpm/reaction) in the absenceorpresenceofbacterialproteinsat37 Cfor5N60min.Amixturecontaining 125 IN C3b and FI served as a negative control and 125 INC3b, FI and FH served as a positive control.thesamplesweretreatedwithβnmercaptoethanol(sigmanaldrich),heated(3 minat93 C)andrunonSDSNPAGEgels,fixedwith5%aceticacid,dried,andcleavage fragmentsdetectedbyautoradiography.inthestudiesiiandiv,theintensityofthec3b α NchainwasdeterminedwithaGelEvalNprogramme(FrogDanceSoftware,Dundee,UK). For detection of the cofactor activity on a bacterial surface, approximately 2x10 9 bacteria/ml were exposed to 2% HIS in GVB at 37 C for 30 min with agitation. After washing,radiolabeledc3b(50,000cpm/reaction)andfactori(100ng/reaction)were addedandincubated(37 C,30min,1200rpm).Thesupernatantswereseparatedfrom thepelletsandtreatedasdescribedaboveanddetectedbyautoradiography. Analysisofthecofactoractivityonacellsurface(studyIII)wasperformedasdescribed previously(jokirantaetal.1996).normalrabbiterythrocyteswerecoatedwithc3bby usingfreshlypurifiedc3,fb,andfd.afterwashes,cellswereincubatedwithfactori (62.5nM),FH(1.3μM),andEcb(11 110nM)for10minat37 Cwithagitationfollowed bytreatmentwith2nmercaptoethanol.cellswerecentrifuged(4min,500 g),andthe 59

60 4Materialsandmethods supernatantswererunonsdsnpage,transferredtoanitrocellulosemembranefollowed by blocking with 3% nonfat milk in PBS for 60 min at 22 C. The membranes were incubated with a mixture of rabbit antinhuman C3d and C3c Abs (dilution 1:2000 for both) for 17 h at 4 C, washed with PBS, and incubated further with a peroxidasen conjugatedgoatantinrabbitab(1:5000)for60minat22 C.Afterwashing,theproteins weredetectedbyenhancedchemiluminescence(ecl) DECAYNACCELERATIONACTIVITYOFFH(III) Analysis of the decay accelerating activity of FH was based on a previously described protocol(krychngoldbergetal.1999).microtiterplateswerecoatedwith5µg/mlc3b inpbs(4 Cfor18h).AfterwashingswithVBS,wellswereblockedwithVBS/0.5%BSA (22 Cfor60min).TheconvertaseswereformedbyincubatingthecoatedwellswithFB (4ng)andFD(0.4ng)inVBSNNiCl 2containing0.05%Tween(37 Cfor60min).After washing twice with VBSNNiCl 2, FH (0N2 nm) with either Ecb (0N2.2 nm) or buffer was added and incubated for 20 min at 22 C. The wells were washed twice and C3bBb convertasesweredetectedwithapabrabbitantinfbantibody(1:5000)followedbyan antinrabbit antibody (1:5000). Bound conjugate was detected by adding on phenylenediaminedihydrochloride(dako)andmeasuringabsorbanceat492nm BACTERICIDALASSAYS(STUDIESI,III) Freshly harvested Bordetella grown to midnlog phase were washed three times with GVBSandincubatedwith10%NHS,MgEGTANNHS,orHISat37 Cwithgentleagitation. Samples were taken after 0, 15, 30 and 60 min of incubation and the complement activationwasstoppedonicebeforedilutingandplatingthesamplesoncharcoalagar. Bacterial survival was calculated as the number of viable colonies at different times, relativetobaselinecolonycountsatthestart. TheeffectofEcbinthepresenceorabsenceofFHandFH19N20onsurvivalofbacteria wasassayedbyusingaserumsensitivestrainofh.influenzae.nhsorhis(12%inpbs) wasmixedwithecborbsatogetherwithfhorfh19n20.afterincubationfor5minat 37 Cwithcontinuousagitation,bacteria(31µlof1x10 7 CFU/ml)inPBSwereaddedand incubatedfor10min.complementactivationwasstoppedbyplacingthesamplesonice for2min,dilutedwithpbsfollowedbyplatingontochocolateagarplates. 60

61 4Materialsandmethods 4.2.8SERUMANDPROTEINABSORPTIONASSAYS(I) Bacteria were incubated with 10% HIS, HIS deficient in FHRN1, or recombinant FH fragments for 30 min at 37 C. After washing five times with PBSNTween, the bound proteinswereelutedwithanacidicbuffer(0.1mglycinenhcl,ph2.7),thesupernatants were collected and ph adjusted (ph 7) with 1M TrisNHCl (ph 9.5). Aliquots of the fractionsweresubjectedtosdsnpageandtransferredontonitrocellulosemembranes. Bound proteins were analyzed with a pab antinfh antibody and an HRPNconjugated secondaryantibodyanddetectedbyecl FLOWCYTOMETRICANALYSES(I,III,IV) Washed bacteria were exposed to NHS, HIS, or C3Ndepleted serum (10N30%) in the presenceorabsenceofproteinsasindicatedinthestudiesandincubatedat37 Cfor20 or30minwithvigorousagitation.afterwashingwithpbscontaining1%bsaor0.05% TweenN20,thecellswereincubatedwithantiNFHpAb(1:50or1:300dilution)at37 C for30minwithagitation,or4 Cfor60min.Afterwashes,bacteriawereincubatedwith a FITCNconjugated antingoat antibody (Bordetella; 1:200, 37 C for 30 min) or Alexa fluor488nlabeledantingoatantibody(s.aureus;1:100,4 Cfor60min)inPBS.Samples were washed and bound antibody was detected by flow cytometry (FACScan, Becton Dickinson,orCyAN TM ADP,BeckmanCoulter) ISOLATIONOFNEUTROPHILSANDREDBLOODCELLS(IV) Isolation of neutrophils used in binding and phagocytosis assays was performed as previouslydescribed(troelstraetal.1997)withminormodifications.bloodwasdrawn from healthy laboratory individuals into tubes containing hirudin (Roche Diagnostic, Mannheim,Germany)anddiluted1:1withPBS.Cellswereseparatedbycentrifugation throughagradient(histopaque 1.119and1.077,SigmaAldrich)at320xgfor20min at22 C.NeutrophilswerecollectedandwashedoncewithRPMIMedia1640(GIBCO ) containing0.05%hsa(rpminhsa).theremainingbloodcellswerelysedwithicencold waterandisotonicconditionswerereestablishedwithpbsbeforetheneutrophilswere washedanddilutedtoaconcentrationof1x10 5 N1x10 6 cells/ml. Erythrocytes were isolated from whole blood anticoagulated with EDTA. After centrifugation(500x g) at 4 Cfor15min, the plasma, buffy coat, andtheuppermost layer of erythrocytes were removed and the cells were washed three times with PBS containing0.5%bsabeforeusedintheassays. 61

62 4Materialsandmethods C3BBINDINGTONEUTROPHILSANDERYTHROCYTES All assays were analyzed by flow cytometry and incubations and centrifugations involvingneutrophilsorerythrocyteswereperformedat4 C. NeutrophilswereincubatedwithS.aureussupernatantorpurifiedbacterialproteinsfor 30min,washedwithRPMINHSAandcollected(1200rpm,5min).Afterincubationwith C3b(15μg/ml,60min),cellswerewashedandincubatedwithFITCNlabeledantiNhuman C3(10μg/ml,60min)beforeanalysis. Alternatively,C3b(100μg/ml)waspreincubatedwithstapylococcalproteins(0.1or1 μm)at22 Cfor15min beforeneutrophilswereadded.after60minincubation,cells were washed with RPMINHSA and incubated with rabbit antinc3c (1:50, 20 min) and AlexaFluor 488Nlabeledantibody(1:100,20or30min). A similar assay using erythrocytes was performed where the preincubation step was excluded,i.eproteinsanderythrocyteswereaddedatthesametime NEUTROPHILBINDINGANDPHAGOCYTOSISASSAYS(IV) S.aureusbacteriawereincubatedwithfluoresceincoupledtoNNhydroxysuccinimideNester (NHSNfluorescein,ThermoScientific)intheneutrophilbindingassays,orpHrhodo TM Green STP ester (Molecular probes, Eugene, OR) in phagocytosis assays according to the manufacturer sprotocol.thelabeledbacteriawereopsonizedinnhs(20%)for15min before bacterial proteins (0.09 or 0.9 μm) and neutrophils were added (bacteria:neutrophilratiowasapproximately20:1)inrpminhsafor60minat37 C.The reactionswerestoppedbyaddingicecoldrpminhsaandneutrophilswerecollectedby centrifugation (400 x g, 10 min). After washing they were fixed with 1% paraformaldehydeandsubjectedtoflowcytometry. ToanalyzetheeffectofEcbandEfbonbindingofneutrophilstoS.aureusinwholeblood, the bacteria were incubated (60 min at 37 C) with 450 µl of hirudinn or EDTAN anticoagulatedbloodinthepresenceof1.6μmofthebacterialproteins.thereactionwas stoppedbycentrifugationasdescribedaboveandredbloodcellswerelysedwithicencold waterandisotonicconditionsrestoredwithpbs.cellswerewashed,fixedandanalyzedby flowcytometry. 62

63 4Materialsandmethods STRUCTURALMODELING,CURVEFITTING,ANDSTATISTICAL ANALYSIS Structural modeling of the OspE:FH19N20:C3b complex in study II was based on the publishedstructuresofc3b(wuetal.2009),c3dandfh19n20(jokiranta,etal.,2006; Kajander, et al., 2011) and was performed by MSc Arnab Bhattarcharjee under the supervisionbysakarijokiranta(bhattacharjeeetal.2013).inthestudyiii,modelingof the Ecb/EfbNC:C3d:FH19N20 was based on C3d in complex with Ecb (Hammel et al. 2007b),EfbNCincomplexwithC3d(Hammeletal.2007a)andFH19N20incomplexwith C3d (Kajander, et al., 2011). The structural illustrations were done using PyMOL software( Curve fitting was performed using nonnlinear regression models implemented in GraphPadPrism.StatisticalanalyzesweredoneusingMicrosoftExcel andgraphpad Prism.ComparisonofmeanvalueswasdoneusinganunpairedtwoNtailedtNtestand resultswerepresentedwithmeanusingstandarddeviationtoindicatetheerror. 63

64 5Results 5,,RESULTS, 5.1,COMPLEMENT,EVASION,OF,B.PERTUSSIS,BY,FH,ACQUISITION(I) SeveralGramNnegativerespiratorytractpathogensbindsolublecomplementregulators to escape the complement attack (Kunert et al. 2007; Hallström et al. 2008). The causative agent of whooping cough, B. pertussis, is also known to utilize the CP regulators C4BP (Berggård et al. 1997) and C1INH (Marr et al. 2011) for immune evasion,butnodataonacquisitionofhostapinhibitorshasbeenreported.however,it hasbeenpreviouslyshownthatb.pertussisisparticularlyresistanttoapattackinvivo (Pishkoetal.2003). We analyzed CP and AP sensitivity of seven Bordetella strains and found that all the BordetellastrainscapableofinfectinghumanswereresistanttokillingbytheAPinvitro (Fig.1,studyI).Whenbothofthepathwayswereactive,twoB.pertussisclinicalisolates (strains175and406)werefullyresistanttokilling.analysisoffhbindinginadirect bindingassaydemonstratedthatallbordetellastrainstested,exceptanavianpathogen B.avium,boundFH(Fig.2A,studyI).Thebindingrangedbetween13%and32%ofthe totalproteinoffered.asapositivecontrolweusedastrainofs.pyogenesthatisknown to be a strong FH binder (Haapasalo et al. 2008). Binding of FH to Bordetella was confirmedinananotherassaywherebacteriawereexposedtoheatinactivatedserum andbindingoffhwasanalyzedbyflowcytometry(fig.3,studyi). Two strong FHNbinding strains, the clinical isolates of B.pertussis (strain 175) and B. parapertussis(strain15311),wereselectedforfurtherstudies.bothstrainsboundfhln 1andFHR1fromNHS(Fig.4,studyI).FurtheranalysisusingrecombinantFHfragments demonstratedthatthemainbindingsitewithinfhislocatedinthecnterminalpartof FH(FH19N20).Asecond,muchweakerbindingsitewithinFH5N7wasdemonstratedby protein absorption experiments and direct binding assays (Figs. 5 and 6, study I). Finally,weshowedthatFHdepositedontothesurfaceofboththestrainsanalysedand bacteriandepositedfhwasfunctionallyactiveandcleavedthebacterianbounddeposited C3b into its inactive fragment ic3b (Fig. 7). From this study we concluded that the respiratorypathogensb.pertussisandb.parapertussisutilizefhforprotectionfromapn mediatedcomplementattack. 64

65 5Results 5.2,MICROBIAL,COMPLEMENT,EVASION,BY,BINDING,FH19C20,(II), ThemajorityofthepathogenswhichutilizeFHasanevasionmechanismbindFHeither viadomains5n7,19n20,orboth,asdemonstratedalsoforbordetellainthestudyi.inthe studyiiweaimedtofindaplausibleexplanationforwhyvariousmicrobesbindfhvia itscnterminusbyinvestigatingthebindinginteractionatamolecularlevel. We selected five microbes that have previously been shown to bind FH via FH19N20, including the GramNnegative bacteria P.aeruginosa (Kunert et al. 2007), H.influenzae (Hallström et al. 2008), and B. pertussis (study I), the GramNpositive S. pneumoniae (Hammerschmidt et al. 2007), as well as the yeast C. albicans (Meri et al. 2002). All microbes were isolates from blood cultures, except for B. pertussis that was isolated fromthenasopharynx.allthestrainsemployedboundfh19n20andfourteendifferent FH19N20 fragments containing surfacenexposed point mutations were tested for their capacitytoinhibitbindingoffh19n20tothemicrobes(fig.1,studyii).inaddition,we analyzed three nonhomologous bacterial proteins: the surface proteins OspE from B. burgdorferi,fhbafromb.hermsii,andtuffromp.aeruginosafortheirbindingtofh19n 20 and capacity to inhibit the interaction with FH19N20. We showed that only the fragments with a mutation within domain 20 decreased the binding in a statistically significant manner (Fig. 2ANC, study II). Furthermore, it was revealed that three key residuesoffh(r1182a,r1203a,andr1206)wereinvolvedinthebindingoffh19n20 toallthemicrobesandmicrobialproteinsanalyzed. Sinceonebindingsiteforheparinandglycosaminoglycansislocatedinthedomain20 (Blackmore et al. 1998), we hypothesized that microbes might mimic the host cell surfacesbybindingfhviatheheparinsiteondomain20whileleavingdomain19free forc3binteraction.wetestedtheeffectofheparinontheinteractionbetweenfh19n20 andthemicrobialproteinsanddemonstratedthatthemicrobialbindingsiteoverlapped onlypartiallywiththeheparinbindingsite(fig.4,studyii).previously,itwasshown thatfhcanbindthec3dpartofc3beitherviadomain19ordomain20(kajanderetal. 2011). We next asked whether the FH19N20:microbe interaction was affected by C3d andshowedthatc3ddidnotinhibitbindingoffh19n20tothemicrobialproteins(fig.4, study II). This important finding suggested that microbes can bind FH in a similar manner as host cells, i.e. FH19N20 can bind to the microbial surface and C3d simultaneously. This phenomenon is explained structurally by the studies of Bhattacharjeeetal.(Bhattacharjeeetal.2013). 65

66 5Results ResultsoftheinhibitionassaysindicatedthatC3ddoesnotblockbutenhancesbinding offh19n20toallthetestedmicrobialproteins.byusingospe,whichisunabletobindto C3b(Hellwageetal.2001),itwasnextdemonstratedthatthedomain19isessentialfor the C3b:FH19N20 interaction as well as for the formation of the tripartite complex C3b:FH19N20:OspE (Fig. 5, study II). The functional consequence of formation of the complexwasanalyzedincofactorassayswithfhusingthennterminalpartoffh(fh1n 4)asanegativecontrol(Fig.6,studyII).WefoundanenhancedcofactoractivityofFH whenthiswasboundtothemicrobialprotein.itwasfinallyconcludedthataconserved microbialbindingsitewithindomain20onfhwascrucialforbindingtofhandthat microbial proteins enhance the FH:C3b interaction leading to a more efficient downn regulationofthecomplementattack. 5.3,EFFECT,OF,TRIPARTITE,COMPLEX,FORMATION,ON,COMPLEMENT, EVASION,(III), S. aureus has several secreted C3 binding proteins. The two staphylococcal secreted proteins, extracellular complement binding protein (Ecb) and extracellular fibrinogen binding protein (Efb), bind to the same region on C3b as FH. Therefore, we asked whetherthebacterialproteinsaffecttheinteractionbetweenc3bandfh. AshasbeendemonstratedinthestudiesIandII,depositionofFHontoamicrobeisan importantimmuneevasionmechanismandthefhcnterminusplaysacrucialroleinthis phenomenon. In the study III we found that the deposition of FH from NHS onto the surface of S.aureus was clearly dependent on the presence of both C3 and functional Ecb,sinceintheabsenceofeitheroftheseproteinsFHdepositionwasnotdetected(Fig. 1,studyIII).Inthebindingassaysusingpurifiedproteins,wefoundthatEcbenhanced the binding of FH to C3b and C3d and, moreover, this enhanced interaction was mediatedbyfh19n20(fig.2,studyiii).fromtheseexperimentsweconcludedthatecb formedtripartitecomplexeswithc3bandfh19n20. OnthebasisoftheresultsobtainedwithRIA,theC3dregionofC3bandtheCNterminal end of FH play essential roles in the enhancement of Ecb to C3b. Therefore, we determinedwhichsiteoffh19n20isinvolvedintheenhancementusingthesamesetsof mutant FH19N20 fragments as in the study II. In contrast to the study II where we observed that domain 20 mediated the formation of the tripartite complex, here we demonstratedthatasiteondomain19wascriticalformediatingthetripartitecomplex formation (Fig. 3, study III). Moreover, in a structural model based on previous 66

67 5Results structuresofc3d:ecb(hammeletal.2007b)andfh19n20:c3d(kajanderetal.2011)we foundnodirectcontactbetweenecbandfh19n20,althoughtheywerelocatedcloseto eachother(fig.4,studyiii). InthestudiesIandII,weshowedexamplesofbacteriaorbacterialproteinsthatacquire FH in a functional manner such that the regulatory activity in cleaving C3b to ic3b is retained. Surprisingly, the presence of Ecb only in very high concentrations led to enhancedcofactoractivityinthefluidphaseandonthesurfaceofrabbiterythrocytes (Fig.5ANC,studyIII).ThedecayacceleratingactivityofFHwasweaklyinhibitedbyEcb andinhibitionoffbbindingtoc3bbyfhwassomewhatenhancedinthepresenceof Ecb(Fig.5DNF,studyIII). Next,weanalyzed whether the enhanced functions of FH by Ecb had an effect on the survivalofbacteria.whenserumsensitiveh.influenzae bacteriawereincubatedwith NHSinthepresenceofEcbthesurvivalofbacteriaincreasedsignificantly(Fig.6A,study III).ThesurvivalwasfurtherenhancedwhenFHorFH19N20wasadded(Fig.6B,study III).Incontrast,thebacteriawerekilledinthepresenceofNHSonlyorNHStogether withfhorfh19n20butintheabsenceofecb.weconcludedthatbycausingformation of the tripartite complexes, Ecb:C3b:FH, S. aureus enhances the FHNmediated defense againstcomplementattack. 5.4,INHIBITION,OF,CR1CMEDIATED,RECOGNITION, BY,STAPHYLOCOCCAL, PROTEINS,(IV), In study III we showed that Ecb enhanced the deposition of FH onto the surface of S. aureusbut,surprisingly,thisdidnotresultinincreasedcleavageofc3bintoic3b.since ic3bisthemainopsoninrecognizedbycr3onphagocytes,itisclearlyanadvantagefor S.aureustoavoidiC3bdepositiononitssurface.Nevertheless,alsoC3bisrecognizedby neutrophils via CR1. This led us to speculate that S. aureus produces one or several secretedmoleculesthatarecapableofinhibitingrecognitionofc3bbycr1. WeusedstaphylococcalculturesupernatantininhibitionassaystostudybindingofC3b toneutrophilsandidentifiedefbasamediatoroftheinhibition(fig.1,studyiv).since thecnterminusofefbishighlyhomologouswithecbandbothbindc3b(hammeletal. 2007b; Hammel et al. 2007a; Jongerius et al. 2007), we included Ecb in our binding assays and showed that both the staphylococcal proteins inhibit C3b binding to neutrophils (Fig. 2B). Only Ecb inhibited attachment of neutrophils to S. aureus after 67

68 preopsonizings.aureuswithc3bgeneratedduringincubationinnhs(fig.6a,studyiv). Whenwerecreatedtheinvivo situationandincubateds.aureus in whole blood, both EcbandEfbsignificantlyinhibitedbindingofS.aureustoneutrophils(Fig.6B,studyIV). ResultsfromthedirectbindinganalyseswithpurifiedproteinsclearlyshowedthatEcb andefbblockedtheinteractionofscr1withc3b(fig.3a,studyiv).inthepresenceof FH, the binding was further enhanced (Fig. 3CND, study IV). In the study III we had discoveredthatecbdoesnotenhancethecofactoractivityoffhandinthestudyivwe foundthatecbalsoblockedthecofactoractivityofscr1forc3b(fig.4,studyiv). ErythrocytesareimportantforclearanceofbacteriafrombloodandtheyexpressCR1, butnottheothercomplementreceptorsontheirsurface.weshowedthatecbandefb efficientlyinhibitbindingofc3btoerythrocytes(fig.5,studyiv).toanalyzetheeffect ofecbandefbonphagocytosisofs.aureusbyneutrophils,welabeledthebacteriawith a dye that increases in fluorescence in acidic conditions, i.e. upon phagocytosis of the bacteria.inthepresenceofecb,significantlyfewerbacteriawerephagocytosedwhilein thepresenceofefbnodifferencewasseen(fig.6c,studyiv).inconclusion,itisclear thatatleastecbimpairsrecognitionofc3bnopsonizeds.aureusbycr1onneutrophils, whiletheroleforefbininhibitionofphagocytosisremainsunknown., 68

69 6Discussion 6,,DISCUSSION, 6.1,INNATE,IMMUNE,EVASION,C,DIFFERENCES,BETWEEN,GRAMCPOSITIVE, AND,GRAMCNEGATIVE,BACTERIA, 6.1.1SURFACESTRUCTURESOFGRAMNPOSITIVEANDGRAMNNEGATIVE BACTERIA TheGramNpositiveandGramNnegativeclassesofbacteriaareprimarilydistinguishedon thebasisofthestructureoftheircellwall.thecellmembraneofgramnpositivebacteria issurroundedbyathickwalltowhichthemajorityofcellsurfaceproteinsareanchored. Incomparison,GramNnegativebacteriaarecomposedoftwocellmembranesseparated by a periplasmic space and the outer cell membrane is utilized as a platform for anchoringtheouterproteins(figure10). Figure,10.SchematicstructuresofthecomponentsofthecellwallsofGramNpositiveandGramN negativebacteria.,, 69

70 6Discussion It is generally accepted that GramNpositive bacteria are protected from complementn mediatedlysisbythethickpeptidoglycanlayerthatphysicallypreventtheformationof MAConthebacterialcellmembrane(Joineretal.1984).InGramNnegativebacteria,long ONantigens(smoothstrains)ontheLPScausesterichindrance,whileroughstrainswith short or even without ONantigens are susceptible to MACNmediated lysis (Clay et al. 2008; Goebel et al. 2008). In spite of that, the study I demonstrated that strains of B. pertussis, naturally lacking ONantigen (Burns et al. 2003), were resistant to killing by human serum. In particular, the bacteria were protected against the AP attack, which suggeststhattheypossessfactors,eitheronthesurfaceorsecreted,thatinhibittheap. ThisisinlinewithfindingsfromPishko,etal.,(2003)whoshowedthatB.pertussiswas abletosurviveinvivobyacquiringafactorfromserum IMPORTANCEOFCAPSULEINIMMUNEEVASION The polysaccharide capsule outside the outer cell membrane is clearly important for virulence of several GramNnegative and GramNpositive bacteria, as capsule deficient strainsofs.aureus,s.pneumonia,andh.influenzaeareshowntobelesswellprotected againstcomplementmediatedattack(thakkeretal.1998;hallströmetal.2006;hyams etal.2010).uponcleavageofthec3nmoleculeareactivethioesterinc3bisexposedand reacts rapidly with hydroxyl and amine groups on the target surface (Pangburn & MüllerNEberhard 1980). Although C3b molecules are deposited on the surface of bacteria,thecapsuleissuggestedtophysicallyinhibitthemactotargetthemembrane (Joineretal.1984). When strains of B.pertussis are grown at 36 C, as in this study, thestrains are inthe virulentphase(bvg+).duringthevirulentphasethecapsuleofb.pertussisisverythin andiscalledamicrocapsule(hotetal.2003;neoetal.2010).themicrocapsuledidnot contributetoprotectionagainstcomplementattackorphagocytosisanditsfunctionin pathogenesis remains elusive (Neo et al. 2010). A capsule is, however, important for optimalorientationofthemembraneproteinssothattheycanactasimmuneevasion molecules. Althoughacapsuleseemstoefficientlyhinder the deposition ofmac onthebacterial membrane, several encapsulated bacteria bind the terminal pathway regulator vitronectin, for instance H.influenzae (Hallströmetal.2006) and some GramNpositive bacteria(chhatwaletal.1987).inaddition,certainstrainsofs.aureusands.pyogenes secretemoleculesthatinterferewithcomponentsoftheterminalpathway.forexample, S.aureusbindsC5viaitsSSL7andconsequentlyblocksMACassembly(Laursenetal. 70

71 6Discussion 2010), and S.pyogenes streptococcalinhibitorofcomplement (SIC)proteinblocksthe membraneninsertion site on C5bN7 inhibiting MAC formation (Åkesson et al. 1996). Recently, it was shown that C5bN9 complexes are deposited on the surface of several GramNpositive bacteria, including S. aureus and S. pyogenes (Berends et al. 2013). AP activation results in the generation of C5bN9 and eventually impaired survival of the bacteriainblood(haapasaloetal.2012).thesefindingsareinteresting,becausethey emphasize the significance of C5bN9 complexes on GramNpositive bacteria and their possibleroleindirectlysisbymeansoftheterminalpathway. 6.2,,COMPLEMENT,EVASION,AT,C3,STAGE, All microbial evasion mechanisms studied in this thesis target the C3 molecule. ImpairingC3stageanddownstreamfunctionsisclearlyanadvantageforthemicrobes sinceallthreecomplementpathwaysconvergeonthisstage CONTROLBYHOST'SSOLUBLEREGULATORS C3b can bind to any cellular surface, including the host s own cells, therefore the regulationofcomplementisstrongestalsoonhumancellsatthec3level.humancells areprotectedbythepresenceofmembranen boundregulatorssuchascr1,daf,and MCFaswellasbyseveralsolubleregulators.ThemainAPregulator,FH,regulatesC3b depositiononhostcellsurfacesbybindingc3bviathedomain19andanionicmolecules onselfsurfacesviathedomain20,leavingtheregulatorydomainfreetoinactivatec3b (Kajanderetal.2011). NumerousmicrobesacquireFHontotheirsurfacesleadingtogenerationoftheinactive C3bfragment,iC3b,resultingindecreasedMACformation.Strikingly,themajorityofthe microbes exploit two regions for FHNbinding: domains 5N7 and domains 19N20 as presentedinfigure11. 71

72 6Discussion Figure,11.,MicrobialbindingsiteswithinFH.Theidentifiedbindingsitesareindicatedwithlines. From light to dark grey: GramNpositive bacteria, GramNnegative bacteria, GramNnegative spirochetes,andyeasts.themostimportantreferencesareindicated.thefigureismodifiedfrom thestudyii. DependingonthemicrobeoneofthesitesmaybemoreimportantforFHNbinding,as demonstratedforb.pertussisandb.parapertussisinthestudyiwherefh19n20seemed to be essential for the binding. Despite being an important pathogen, complement evasion by B.pertussis has not been extensively studied. On the other hand, it can be questioned whether B.pertussis needs to be serum resistant since it lives on mucosal surfacesandnotinblood.bordetellastrainssuchasb.holmesii(tartofetal.2014),b. hinzii (Cookson et al. 1994), B. bronchiseptica (Katzenstein et al. 1984), and B. parapertussis (Wallihan et al. 2013), have been reported to cause bacteremia in immunocompromised patients, but it has not been described for B. pertussis. Complementcomponentsarepresentonmucosalsurfacesintheairwaysthatareinthe range 10N20% of the concentration of that in the blood (Boackle 1991; Persson et al. 1998).Therefore,respiratorypathogenssuchasB.pertussisneedtoprotectthemselves fromcomplement. Previous studies have demonstrated that B. pertussis escapes the CP (Berggård et al. 1997;Barnes&Weiss2001;Marretal.2011)bybindingC4bpandC1INH.AB.pertussis strainlackingfha,whichistheligandforc4bp,showednodifferenceinserumsurvival ascomparedtothewildtypestrain(fernandez&weiss1998).however,fhamayact in synergy with other virulence factors as described for FHA and Ptx (Relman et al. 1989)andFHAandACT(Grayetal.2004),whichmayexplaintheresults.ThestudyI providesimportantinformationonhowb.pertussisandb.parapertussisevadetheapof 72

73 6Discussion complement.futurestudiesneedtoelucidatetheligandsforfhandtheirsignificancein thosepathogens. TheCNterminaldomainsFH19N20arecentralinmicrobialevasionbutithasremained elusivehowandwhythesedomainsareinvolvedinthisprocess.domain20isutilized by host cells for discrimination between host and nonnhost surfaces (Lehtinen et al. 2009; Ferreira et al. 2010; Kajander et al. 2011). Therefore the hypothesis was that microbescoulduseasimilarmechanismorevensoncalledmolecularmimicry.indeed, thestudyiirevealedaconservedbindingsiteondomain20usedbyseveraldifferent microbes,butthebindingsitewasnotidenticalwiththe one that is used by the host cells.thisfindingwasimportant,sinceanoverlappingbindingsiterevealspossibilities fordesigningmoleculesthatblockthemicrobesfrombindingfhwithoutblockingthe importantdiscriminationonowncells. Notably, the three structurally distinct proteins analyzed, OspE, FhbA, and Tuf, efficiently enhanced the cofactor activity of FH by formation of a tripartite complex consistingofc3b,fh,andmicrobialprotein.althoughafunctionalcofactoractivityhas been reported for most microbes that acquire FH, this was the first time enhanced regulationwasdescribed. Avarietyofcomplementevasionfactorshavebeendescribedfor S.aureus, but direct FHNbinding has not been demonstrated. Although one study has reported FHNbinding, normal human serum instead of purified protein was used in almost all the assays in that study (Sharp et al. 2012). Therefore, it was likely that other components from serumwereinvolved.thestudyiiigivesanexplanationtohows.aureuscanutilizefh for its own protection. The secreted staphylococcal protein Ecb binds C3b using the same area as FH (Hammel et al. 2007b; Hammel et al. 2007a; Jongerius et al. 2007; Kajanderetal.2011)andourstudiesrevealedthatdepositionofFHontothesurfaceof S. aureus was dependent on both C3 and Ecb. The formation of a tripartite complex between C3b, Ecb, and FH occurred, similar to what was described in the study II. In contrasttothatstudywheredomain19boundtoc3b,herethedomain20offhwas responsible for C3bNbinding. Surprisingly, increased concentration of the S. aureus proteinecbdidnotleadtoenhancedcofactoractivityasdemonstratedfortheproteins inthestudyii.thedecayacceleratingactivityoffh,however,wasslightlyenhancedin thepresenceofecb.thisindicatesthatuponformationoftheecb:c3b:fhcomplex,the regulatory part of FH (FH1N4) remains free to decay the same or even a nearby convertase.thefbinteractionwithc3bwasmostlikelyinhibitedviaaconformational 73

74 6Discussion change within C3b upon EcbNbinding, as previously suggested (Hammel et al. 2007b; Chen et al. 2010). The formation of the C3b:Ecb complex did, however, enhance FH bindingtothecomplex,whichinturnresultedintheinhibitionoffbnc3binteraction. These results demonstrated that two out of the three functions of FH, illustrated in figure 5, were affected by the formation of a C3b:Ecb:FH complex. Increased concentration of FH significantly increased the survival of a serum sensitive strain, comparedtoonlyecb,andpinpointedthecontributionoftheenhancedfunctionsoffh indownregulationofc3b MICROBIALPROTEINSACTONC3 TheevasionmechanismdescribedinthestudyIIIrevealedthatincreasedconcentration ofbacterialproteinecbdidnotleadtoenhancedformationofic3b,butonthecontrary, blockedthegenerationofic3b.thiswasapuzzlingresult,sincec3bisthemajorligand for CR1 expressed on most blood cells and C3b deposition eventually leads to phagocytosis. Either the C3b on bacteria is recognized by CR1 on erythrocytes and transported to the liver and spleen for elimination, or CR1 on neutrophils facilitates phagocytosis by stimulation of CR3 and CR4 (Berger et al. 1984). Moreover, the ic3b fragmenthasa100nfoldloweraffinityforcr1ascomparedtothatofc3b(kallietal. 1991).Takentogether,forS.aureusitseemstobebeneficialtoinactivateC3bintoiC3b. Therefore,thehypothesisinthestudyIVwasthatS.aureushasoneormoresecreted moleculesthatinhibitc3bncr1interaction.indeed,boththestaphylococcalproteinsecb and the fibrinogennbinding Efb inhibited the direct C3bNCR1 protein interaction. However, only Ecb impaired the phagocytosis of S.aureus by neutrophils after serum exposure.whenexposedtowholebloodalsoefbboundtoneutrophils(fig.6b,study IV), which indicated that a factor from blood contributes to this interaction. It was recently demonstrated that the Efb:C3b complex attracts fibrinogen to the microbial surfaceandhindersrecognitionbyphagocyticreceptors(koetal.2013),whichmayat least partially explain the phenomenon observed in the study IV. It is not known whether other bacteria exploit the same strategy, but another staphylococcal protein (SCIN) prevents CR1 recognition of the C3b within the convertase (Jongerius et al. 2010a). SincetheinteractionsiteofCR1toC3bisnotidentifiedindetailitisnotclearbywhich mechanismsecb,efb,andscinblocktheinteractionon amolecularlevel.mostlikely theyhavedifferentinhibitionstrategies.uponbindingtothec3dpart(teddomain)of C3b, EfbNC induces conformational changes within C3b (Hammel et al. 2007a) which 74

75 6Discussion mayresultintheimpairedrecognitionbycr1.scinbindstothennterminaltailofc3b α (Rooijakkersetal.2009)locatedintheoppositeendthantheTEDdomain.Therefore, itislikelythatscininhibitsthec3bncr1interactionbysterichindrance,whichisthe suggestedmechanismoftheinhibitionofc3bncriginteractionbyscin(wiesmannetal. 2006;Jongeriusetal.2010b). Since CR3 on neutrophils is known to be important for elimination of GramNpositive bacteria(fällmanetal.1993;jongstranbilenetal.2003;nilssonetal.2005),itismost likely important for S. aureus and other GramNpositive bacteria to avoid ic3b depositions,asthefindingsofthestudiesiiiandivsuggest. OnhostsurfacesC3bdepositionsarerapidlydegradedtoC3dgbyCR1expressedonthe cellsurfaceoronadjacenterythrocytes.thegeneratedc3dgcanbindtocr2onbcells and in a conreceptor complex (CR2NCD19NCD81) lowers the threshold for activation leading to stimulation of antibody production, which is an important bridge between innate and adaptive immunity (Dempsey et al. 1996). The study IV revealed that Ecb blockedthecofactoractivityofcr1incleavageofc3binasimilarmannerasfhwas showntodointhestudyiii.thisisanimportantfindingsinceitindicatesthats.aureus, by preventing the formation of C3dg, targets both the innate and adaptive immunity. PreviousreportsshowthatS.aureushasdevelopedseveralmechanismstodisablethe C3dNCR2interaction,sinceEfb,Ecb,andSbialloccupyareasofC3dthatisimportantfor CR2binding(Burmanetal.2008;Ricklinetal.2008;vandenElsen&Isenman2011).So fartherearenotmanyexamplesofmicrobesusingthisstrategy.theuspa1anduspa2 proteinsofm.catarrhalisinteractwithc3d(hallströmetal.2011),butitisnotknown whetherthebindinghampersthecr2nc3dinteraction.neitheroftheuspaproteinsdid induceaconformationalchangeinc3,suggestingthattheyuseadifferentstrategythan thestaphylococcalproteinsefbandecb.moreover,theuspasdidinhibitgenerationof C3a that eventually lead to impaired phagocytosis and inflammatory response, demonstrating that these proteins are important virulence factors of M.catarrhalis. A recentstudyshowedthatthec3dpartofic3bplaysamajorroleinrecognitionbycr3 (Bajicetal.2013),suggestinganadditionalexplanationwhymicrobialproteinsbenefit frombindingc3d. In conclusion, by targeting the C3 molecule, microbes inhibit opsonization, MACN mediatedlysis,phagocytosis,generationofthechemotacticfragmentsc3aandc5a,and tothebridgetoadaptiveimmunity. 75

76 6Discussion 6.3,,WHY,OVERLAPPING,EVASION,MECHANISMS?,S.AUREUS,AS,AN, EXAMPLE, A principal goal of immune evasion by pathogenic microbes is to avoid phagocytosis. Thisgoalcanbeachievedbyproducingathickcapsulethatrequiresexpressionofmany proteinsinvolvingseveralgenes.anotherstrategyusedbys.aureus,istoexpresssmall molecules that inhibit many steps in innate immunity, including the complement cascade. The majority of these molecules, such as Ecb and Efb studied in the present work, are expressed by most clinical isolates (Jongerius et al. 2010a). On top of that, manyofthemoleculeshavemultiplefunctionsasillustratedbytheefbprotein.itsnn terminal binds fibrinogen and inhibits platelet functions (Palma et al. 2001), whereas the CNterminal end binds C3b and interacts with complement leading to impaired phagocytosis(jongeriusetal.2010a;koetal.2013)studiesiiiandiv).anotherexample is Sbi whereits CNterminusofsecretedSbibindsC3(Burmanetal.2008; Smithetal. 2011)andtheNNterminusbindsIgGwhenantibodyisattachedtothecellwall(Smithet al.2011).moreover,bothefbandsbirecruitplasminanduseittocleavec3(kochetal. 2012).Thesmall9kDaEcbmoleculeenhancestwofunctionsofFHindownregulationof C3b(studyIII)andblocksinteractionwithCR1(studyIII)andCR2(Ricklinetal.2008). Sinceallthestaphylococcalmoleculesthattargetthecomplementcascadeareableto inhibit,butnotcompletelyblockthedifferentstages,theyworkinconcertandprovidea powerfulattackagainsttheimmunesystemleadingtobacterialsurvivalinthehuman host. 6.4,,EXAMPLES,OF,EVASION,STRATEGIES,OF,MICROBES,IN,DIFFERENT, BODY,PARTS, Host pathogeninteractionsareconstantlyrespondingtochangesinimmunestatusand niche availability. Thus different microbial evasion mechanisms are required for survivalindistinctbodyparts. Mostofthebacteriaanalyzedinthisthesiswereisolatedfromblood,exceptB.pertussis. Infectionofblood, however, isfrequentlyprecededbycolonizationofothertissuesof the body and the respiratory tract is one of the most important routes of entry for pathogens.althoughtheairwaysarecontinuouslyexposedtovarioustypesofmicrobes, themucosalimmunityiseliminatingthemajority oftheintruders.theepithelialcells are covered with a glycoproteinsncontaining mucus layer that traps the microbes and preventsthemfromattachingtothehostcellsurface.withthehelpofciliatedcellsmost 76

77 6Discussion ofthemicrobesareefficientlymovedofoutofthearea.inaddition,secretorycellsare generatingawiderangeofantimicrobialagentssuchassiga,andserumcomponents, includingcomplement(perssonetal.1998;greiffetal.2003). Oftenmixedbacteriacauseinfectionsintherespiratorytractandbacteriathatarenot abletoescapethecomplementsystemcanbenefitfromconcolonization,asillustratedby unencapsulatedstrainsofh.influenzae. SomestrainsofM.catarrhalis releasevesicles containing UspA that degrade C3 (Nordström et al. 2004), offering C3Ndepletion and protectionfromcomplementattackforalsoh.influenzaeinthenearbyarea(tanetal. 2007).Interestingly,thestudyIIIshowedthataserumsensitivestrainofH.influenzae was protected against complementnmediated killing in the presence of the secreted staphylococcalc3nbindingecbprotein.thismechanism,however,ismostlikelydueto EcbboundtosurfaceNdepositedC3bleadingtoinhibitionofMACNmediatedlysis. S. aureus seldom causes infection in the airways but more commonly participates in formationofabscessesorevenbacteremiaandsepsis.afterenteringthebloodstream, some bacteria, such as S. aureus, can form abscesses by dissemination into tissues (Lowy, et al., 1998). The infection leads to inflammatory responses that attract phagocytestothesiteandformspus,whichisacollectionofdeadandlivephagocytes, dead tissue and bacteria. Studies suggest that S.aureus protects itself by organizing a pseudocapsule composedoffibrindeposits(chengetal.2010),whichincludeseveral virulence proteins. SpA and coagulases interfere with factors from the coagulation cascade,suchasfibrinogenandvonwillebrandfactor(friedrichetal.2003;bjerketorp etal.2004).inamousemodel,ecbandefbcontributedtoabscessformationintheheart andkidneys(jongeriusetal.2012).thiswasmostlikelycausedbymodifyingimmune responses, since S. aureus strains lacking Ecb and Efb caused smaller abscesses and enhancedneutrophilinfiltration.thisisinlinewiththestudyivthatshowedthatecb and Efb prevented interaction with neutrophils. Recently, it was demonstrated that S. aureus degrades DNA of the neutrophil extracellular traps resulting in apoptosis of macrophages and increases the possibility of bacterial survival inside the abscess (Thammavongsaetal.2013). Occasionally, some strains of S. pneumoniae and H. influenzae may cause invasive diseasessuchassepsisandmeningitiseitherbya commensalstrainoranothermore virulent strain. The yeast C. albicans is a commensal on the skin, oral cavity, gastrointestinal and urogenital tracts of healthy individuals. Immunocompromised patientstypicallyhavelowneutrophilsorthelpercelllevels,butnormalcomplement 77

78 6Discussion concentrations.inthoseindividualsc.albicanscanpenetrateintodeepertissues,which leadstolocalorsystemicinfections.thespirochetebacteriaborreliaentersthehuman bodyviaatickbiteandmaycausedifferentdiseases,suchascardiac,neurological,or arthriticdisorders(burgdorferetal.1982;nadelman&wormser1998).somespecies canevenpersistforlongperiodsoftimes inmammals,indicatingthattheyefficiently evadethehost simmunesystem. Takentogether,microbeshaveevolvedvirulencefactorsneededforthesurvivalinthe human body. Strains that are usually not pathogenic for humans can cause severe diseaseiftheyaccessareasofthebodythatarelesswellprotected. 6.5,VACCINE,DESIGN, HostNpathogenresearchhasoneobviousmajorgoalNtodevelopprotectivemeansfor hostsagainstpathogens.microbialvirulencefactorsorproteinsthatareinvolvedinthe escapefromtheimmunesystem,couldbegoodvaccinecandidates.therefore,microbial proteinsmediatingcomplementevasionareattractivevaccinecandidates,suchasfhbp of N. meningitidis, which is the antigen in an approved vaccine that targets group B meningococci(madicoetal.2006;granoff2013). B. pertussis should theoretically be possible to eradicate, as the situation was for smallpoxin1978,sincehumansaretheonlyknownreservoir.however,thebacteria arepersistentandhaveahighrateoftransmission,asforeachinfectionwithb.pertussis another fifteen transmission events or secondary infections may occur (Bjornstad & Harvill 2005). We now know that new isolates of B. pertussis that express proteins differentfromthoseincludedinthevaccinehaveevolved(octaviaetal.2011;advaniet al.2013).oneexampleisamorevirulentb.pertussislineageassociatedwithenhanced pertussistoxinproduction(mooietal.2009).recently,anewvaccineadministeredby thenasalrouteshowedpromisingresultsinaphasenistudy(thorstenssonetal.2014). Thatvaccineincludeslivebacteriawithgeneticallyeliminateddermonecrotictoxinand tracheal cytotoxin, as well as genetically detoxified Ptx. In that manner the vaccine mimicthenaturalinfectionwithoutcausingthedisease(mielcareketal.2006). TheFHligandsofthemicrobesusedinthestudyIIarestructurallydifferentandsome have been extensively studied. PspC has shown potential as a protective antigen in a mouse model against sepsis, pneumonia, and colonization of lungs (Rosenow et al. 1997).TheTufNproteinisinvolvedinpeptideelongationduringproteinsynthesisandis 78

79 6Discussion abundantlypresentinthecytosolofmicrobes.afractionoftheproteinisidentifiedon thecellsurfaceofseveralmicrobesandisthereforeincludedinthegroupofsoncalled moonlighting proteins (Jeffery 1999; Kunert et al. 2007). Although surfacenattached TufonP.aeruginosabindsFH,itscontributioninserumsurvivalisnotknown.Infuture experimentsamutantstrainlackingtufshouldbeanalyzedforitssensitivitytohuman serumbeforeitcanbeconsideredasavaccinecandidate. Pra1islocatedonthesurfaceofyeastcellsandhyphaeofC.albicans.Inaddition,itis foundintheculturesupernatant.secretedpra1isdemonstratedtobindc3andinhibit thecleavageofc3intoc3bandc3a(luoetal.2010),andfurthermore,itblockscr3on neutrophils (Soloviev et al. 2007). The surface attached Pra1 is shown to bind the solublecomplementregulatorsfhfhln1,andc4bp(luoetal.2009;luoetal.2011). Together the secreted and surfacenbound Pra1 inhibits C3b deposition, impairs the recruitmentofphagocytesandblocksphagocytosisbyneutrophils,aswellasitinhibits theinflammatoryresponse(solovievetal.2007;luoetal.2009;luoetal.2010;luoet al. 2011). Therefore, neutralization of this multifunctional protein would most likely inhibitthesurvivalofc.albicans.vaccinesagainstc.albicansandp.aeruginosawould be useful as tailored treatments for immunocompromised individuals and patients sufferingfromcysticfibrosis,respectively. OspEisproposedtobeagoodvaccinecandidatesinceitisessentialforinfection,itis expressedbythemajorityofclinicalisolatesanditishighlyconserved(hellwageetal. 2001;Bhattacharjeeetal.2013). Study II demonstrated that different microbes bound FH in a similar manner and the bindingsitewasmappedonamolecularlevel.sincemembraneproteinsoftenarerather largeproteins,thisinformationisessentialinfutureworktodesignsmallmoleculesthat could block the interaction site thereby neutralizing several microbes. A challenge is that many of the virulence factors are humannspecific, which complicates the analysis sinceusingavalidanimalmodelisnotalwaysavailable. Often FH binding correlates with bacterial survival in serum, as demonstrated for S. pyogenesandn.meningitidis(madicoetal.2006;haapasaloetal.2008).somestrainsof S.pyogenesstronglybindFH5N7.SinceFH5N7lacksaregulatorysite,thefragmenthas beenproposedasatherapeuticmolecule.byaddingfh5n7,fhbindingtothemicrobial proteinisblocked(haapasaloetal.2012)leadingtoimpairedphagocytosis.exploitinga humanproteinminimizesthechancesforanantigenicresponse.thisapproachwould not be possible for FH19N20 since in the studies II and III it was found that FH19N20 79

80 6Discussion enhances the binding of microbial protein to C3b leading to an effect in favor of the microbe. 80

81 7""Conclusions" 7""CONCLUSIONS" The" innate" immunity," including" the" complement" system," is" efficient" in" the" first" line" protection"against"microbial"infections."complement"can"directly"destroy"the"microbes" by"mac>mediated"lysis"and"recruit"and"activate"phagocytes,"such"as"neutrophils,"to"the" site"of"infection."in"order"to"survive"within"humans,"microbes"have"evolved"various"and" distinct" strategies" to" evade" the" complement" attack." For" example," several" microbes" acquire" soluble" complement" regulators." The" present" studies" demonstrated" the" importance"of"binding"of"the"c>terminal"domain"of"the"main"ap"regulator"fh"to"bacteria." By"binding"to"FH19>20,"the"respiratory"tract"pathogen"B.#pertussis"can"escape"the"AP"of" complement" and" increase" the" survival" in" human" serum." It" was" shown" at" a" molecular" level"that"a"common"site"on"domain"20"of"fh"was"essential"for"gram>negative,"including" B.#pertussis,"and"Gram>positive"bacteria"as"well"as"the"yeast"C.#albicans"in"order"to"escape" from" complement" attack." Binding" of" microbial" proteins" to" FH" demonstrated" an" enhanced" C3b:FH" interaction" leading" to" increased" downregulation" of" C3b" that" eventually"results"in"increased"survival"of"the"microbes.""" The"important"pathogen"S.#aureus"does"not"directly"bind"FH"and"these"studies"gave"an" explanation" on" how" it" uses" soluble" C3b>binding" molecules" to" recruit" host" FH." Importantly," the" functional" consequences" of" the" C3b:FH:Ecb" complex" were" demonstrated"both"on"a"protein"level"and"for"bacterial"survival"in"serum."furthermore," the" studies" demonstrated" how" S.# aureus" avoids" recognition" by" CR1" and" subsequently" phagocytosis"by"neutrophils."" A"better"understanding"of"the"host>pathogen"interaction"is"essential"for"the"development" of"new"vaccines,"novel"antimicrobial"compounds"and"new"mechanisms"to"fight"against" microbial" infections." The" results" in" this" thesis" revealed" detailed" knowledge" on" both" functional" and" structural" levels" of" host>pathogen" interactions" and" provide" important" contribution"to"this"research"field." " " 81"

82 Acknowledgements. ACKNOWLEDGEMENTS. The. work. presented. was. carried. out. at. the. Department. of. Bacteriology. and. Immunology,.Haartman.Institute,.University.of.Helsinki..I.want.to.thank.Seppo.Meri,.for. giving. me. the. opportunity. to. work. at. the. department,. for. introducing. me. to. the. complement.field.and.for.sharing.his.knowledge.. I.want.to.give.my.warmest.thanks.to.my.supervisors,.Sakari.Jokiranta.and.Taru.Meri.. Sakari.has.guided.me.through.this.work.by.sharing.his.vast.knowledge.and.creativeness.. He. has. always. been. supportive. and. optimistic. and. has. always. had. time,. even. in. the. busiest.periods..it.has.been.very.rewarding.to.have.taru.as.a.second.supervisor..she.has. guided.me.in.the.lab,.encouraged.me,.and.we.have.had.lots.of.discussions..also.nong scientific.ones..thank.you.for.making.such.a.good.working.atmosphere.and.for.all.the. good.laughters.i.acknowledge.both.my.supervisors.for.being.very.patient.. I.am.grateful.to.my.reviewers.Benita.WesterlundGWikström.and.Ilkka.Julkunen.for.their. good.comments.and.their.quick.responses..i.want.to.thank.kristian.riesbeck.for.his.kind. promise.to.act.as.an.opponent... I.appreciate.the.stay.at.Medical.Center.Utrecht,.The.Netherlands..I.warmly.thank.Jos.van. Strijp,. Suzan. Rooijakkers,. Ilse. Jongerius,. and. Lydia. Tan. for. sharing. their. time,. knowledge,. material,. and. for. the. fruitful. scientific. collaboration. during. these. years.. I. thank. all. the. members. of. the. lab. in. Utrecht. for. good. moments. and. especially. Ilaria. Pastorello.from.Siena,.who.visited.at.the.same.time.as.me... Thank. you. all. the. present. and. past. colleagues. at. the. Department. of. Bacteriology. and. Immunology. for. creating. such. a. good. atmosphere. I. specifically. want. to. thank. the. members.of.the.jokirantaglab:.karita.haapasalogtuomainen.for.the.advices,.discussions. and.for.her.excellent.skills.in.blood.drawing,.satu.hyvärinen.for.giving.practical.advices. with. assays. and. for. sharing. her. proteins. (and. food. recipes).. Markus. Lehtinen. for. introducing. the. RIA,. Arnab. Bhattacharjee. for. solving. my. computer. problems,. Aino. Koskinen.and.Hannah.Söderholm.for.sharing.their.medical.knowledge..I.also.appreciate. the.friendship.and.discussions.with.hannah.in. svorsk..i.acknowledge.heidi.sillanpää. for.practical.advices.in.the.last.phase.of.the.project..i.also.want.to.thank.tanja.pasanen,. Tiira.Johansson,.Maarit.Myöhänen,.Sonja.Rajakaari,.and.Valentina.la.Verde.. I.am.grateful.to.Hanna.Jarva.who.helped.me.in.the.beginning.of.my.studies,.Derek.Ho. who.kindly.edited.the.language.in.this.thesis..nathalie.friberg.for.always.being.positive,. Nina. Vivi.Palkola.for.taking.care.of.me.when.I.arrived.at.the.Haartman.Institute,.and. Laura.Bouchard.for.the.good.laughters.and.good.coffee..I.also.thank.the.other.members. of.the.meriggroup:.anna.jalava,.annagleena.saariaho,.annika.kalanti,.antti.lavikainen,. Ayman. Khattab,. Eija. Nissilä,. Hanna. Dyggve,. Hanna. Järvinen,. Inkeri. Lokki,. Johanna. Pietikäinen,.Jorma.Tissari,.Kirsi.Vaali,.Laura.Degerstedt,.Marcel.Messing,.Marta.Barroso,. Martin. Reichard,. Matti. Laine,. Rauna. Riva,. Sami. Junnikkala,. Tobias. Freitag,. and. Ville. Holmberg... Thanks.go.to.Mikael.Skurnik.for.always.being.helpful.and.for.arranging.the.Pathogenesis. Day.every.year.and.to.his.group:.Anu,.Elif,.Juho,.Julia,.Katarzyna,.Laura,.Mabruka,.and. Martha..I.thank.the.Petteri.Arstila.labGmembers:.Anni,.Eliisa,.Helga,.Heli,.Iivo,.Laura.R.,. 82.

83 Acknowledgements Nelli, Pirkka, Tamàs, Tuisku. I thank Laura S. from the Tuuminen reseach group, Sari fromthekanteleresearchgroup,anneandjudithfromwillemdevos sresearchgroup, and Amarjit, Dawit, and Hanne from Päivi Saavalainen research group. I acknowledge PenttiKuuselaforsharinghisknowledgeandprovidingstaphylococcalstrains. IamgratefulforthehelpfromthetechniciansMarjattaAhonen,PirkkoKokkonen,Kirsti Widing,MarjoRissanen,andEilaKetolainen.MarkoHietavuoandIlkkaVanhatalohave maintained the technical equipment. I thank the office ladies Carina Wasström, Kirsi Udueze,andTaijaPietiläfortheirkindhelpinpracticalmatters. ManythankstoKirstiIivonenandthekindpersonnelatthe elatusaineosasto andat thehuslab. I acknowledge my collaborators Quishui He, Jussi Mertsola, Marjo Haanperä, John V. McDowell,RichardT.Marconi,andAdrianGoldmanforsharingtheirtime,knowledge, andmaterial. IwanttothankKåreBerghandthepersonnelatDepartmentofMicrobiology,NTNU,for introducing me to the microbial world. Special thanks to my friends Bente, Bodil and Janniche. ManythanksgotomyparentsIngrunnandTerjefortheirgeneroussupportandcare,as wellasmymotherninnlaw,tuula.ialsowanttothankmysportysisterskirstiandeva andtheirfamiliesforalwaysfeelingwelcomewhenvisitingnorway. MywarmestthanksgotomyhusbandMarcoforhissupport,patience,andlove,andour boys Marcus and Linus (who made the yellow staphylococci and eating cells on the cover).iloveyou. This study was financially supported by the Sigrid Jusélius Foundation, the FinnishN Norwegian Medical Foundation, the Finnish Cultural Foundation, and the Academy of Finland. HanneAmdahl Helsinki,May

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