MASTERARBEIT / MASTER’S THESISothes.univie.ac.at/41599/1/2016-03-20_1108033.pdf · 2016-04-08 ·...

MASTERARBEIT / MASTER’S THESIS

Titel der Masterarbeit / Title of the Master‘s Thesis

Description of two novel and as-yet uncultured endosymbionts of Acanthamoeba spp.

verfasst von / submitted by

Stefanie Michels BSc

angestrebter akademischer Grad / in partial fulfilment of the requirements for the degree of

Master of Science (MSc) Wien, 2016 / Vienna 2016

Studienkennzahl lt. Studienblatt / degree programme code as it appears on the student record sheet:

A 066 830

Studienrichtung lt. Studienblatt / degree programme as it appears on the student record sheet:

Masterstudium Molekulare Mikrobiologie, mikrobielle Ökologie und Immunbiologie

Betreut von / Supervisor: Univ.-Prof. Dr. Matthias Horn

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Tableofcontent

1. Introduction.........................................................................................................................................7

1.1 Endosymbiosisandotherdefinitions.............................................................................................7

1.2 Diversityofbacterialendosymbiontsinfree-livingamoebae........................................................8

1.3 Generalphysiologyofbacterialendosymbiosis...........................................................................11

1.3.1 Theintracellularlife-style......................................................................................................11

1.3.2 Chlamydialmetabolismandphysiologicactivity..................................................................12

1.4 Aimofthestudy...........................................................................................................................15

2. MaterialandMethods........................................................................................................................17

2.1 Technicalsupplyandcomputersoftware....................................................................................17

2.2 Consumables................................................................................................................................18

2.3 Molecularbiologykits..................................................................................................................18

2.4 Probesandprimers......................................................................................................................19

2.5 Growthmedia,buffersandotherchemicalsolutions..................................................................20

2.6 Methods.......................................................................................................................................24

2.6.1 Isolationofamoebaeoffree-livingamoebaeusingnon-nutrientagarplatesseededwithfoodbacteria.....................................................................................................................................24

2.6.2 Axenizationoffree-livingamoebae......................................................................................25

2.6.3 Fluorescencein-situhybridization(FISH)..............................................................................25

2.6.4 16SrRNAgenesequencingofaxenizedamoebacultures....................................................26

2.6.5 CultivationofidentifiedAcanthamoebaspp.HSC................................................................31

2.6.6 CuringofAcanthamoebaspp.HSC.......................................................................................31

2.6.7 Definingthehostrangeofendosymbionts...........................................................................32

2.6.8 IsolationofgenomicDNA......................................................................................................32

2.6.9 HeLacellinfectionassay.......................................................................................................33

2.6.10 Growthcomparison............................................................................................................34

2.6.11 Infectioncycles...................................................................................................................36

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2.6.12 ExtracellularinfectivityAssay.............................................................................................37

2.6.13 GlucoseAssay.....................................................................................................................38

3. Results................................................................................................................................................41

3.1 Isolationoffree-livingamoebaeusingnon-nutrientagarplatesseededwithGram-negativefoodbacteriaandaxeniccultivation..............................................................................................................41

3.2 IdentificationofnovelendosymbiontsofAcanthamoebaspp....................................................42

3.2.1 Fluorescencein-situhybridizationresults.............................................................................42

3.2.2 16S/18SrRNAgenesequencingresultsofamoebaculturesharboringendosymbionts......43

3.3 Phylogeneticanalysis...................................................................................................................45

3.4 HostrangeoftheendosymbiontsHSC3andHSC8......................................................................47

3.4.1 CuringofthenaturalAcanthamoebasp.HSChost...............................................................47

3.4.2 InfectionofdifferentAcanthamoebaspp.,humanandinsectcelllines...............................48

3.5 CharacterizationofEndosymbiontofAcanthamoebasp.HSC3..................................................49

3.5.1 TheendosymbiontHSC3hasabiphasicinfectioncycle........................................................49

3.5.2 UninfectedAcanthamoebasp.HSCshowfastergrowthincomparisontoacanthamoebaeharbouringtheendosymbiontHSC3.................................................................................................51

3.5.3 InfluenceoftheendosymbiontHSC3onacanthamoebaviability........................................53

3.5.4 Influenceofnutrientavailabilityoninfectivity.....................................................................55

3.6 EndosymbiontofAcanthamoebasp.HSC8..................................................................................58

3.6.1 TheinfectioncycleofendosymbiontHSC8...........................................................................58

3.6.2 UninfectedAcanthamoebasp.HSCshowfastergrowthincomparisontoacanthamoebaeharbouringtheendosymbiontHSC8.................................................................................................61

3.6.3 InfluenceoftheendosymbiontHSC8onacanthamoebaviability........................................63

3.6.4 Maintenanceofinfectivityduringhost-freeincubation.......................................................65

4. Discussion...........................................................................................................................................69

4.1 Detectionofendosymbiontsoffree-livingamoebae..................................................................69

4.1.1 Isolationandaxenizationoffree-livingamoebae.................................................................69

4.1.2 Detectionofendosymbiontswithinfree-livingamoebaeusingFISH...................................69

4.2 IdentificationandphylogeneticanalysisofendosymbiontsofAcanthamoebasp......................70

4.3 CuringofnaturalAcanthamoebaspp.HSChost..........................................................................72

4.4 HostrangeofendosymbiontsofAcanthamoebaspp.HSC3andHSC8......................................73

4.4.1 HSC3andHSC8areabletoinfectavarietyofAcanthamoebaspp......................................73

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4.4.2 HSC3andHSC8areincapabletoinfectmammalianandinsectcells....................................74

4.5 CharacterizationoftheendosymbiontofAcanthamoebasp.HSC3............................................76

4.5.1 ThedevelopmentalcycleofthechlamydiaHSC3.................................................................76

4.5.2 Influenceoftheendosymbiontongrowthandfitnessofamoebaehosts...........................78

4.5.3 Host-freesurvivalcapabilityandmaintenanceofinfectivityinrelationtonutrientavailability..........................................................................................................................................80

4.6 CharacterizationoftheendosymbiontofAcanthamoebasp.HSC8............................................82

4.6.1 TheinfectioncycleoftheendosymbiontHSC8....................................................................82

4.6.2 Influenceoftheendosymbiontongrowthandfitnessofamoebaehosts...........................84

4.6.3 Host-freesurvivalcapacityandmaintenanceofinfectivity..................................................86

5. Abstract..............................................................................................................................................89

6. Zusammenfassung..............................................................................................................................91

Appendix....................................................................................................................................................93

16S/18SrRNAgenesequenceoftheendosymbiontsandAcanthamoebahosts.............................93

UnculturedBetaproteobacteriathatarebestblasthitsofendosymbiontsHSC8............................95

Glossary.....................................................................................................................................................98

References.................................................................................................................................................99

Acknowledgements.................................................................................................................................111

CurriculumVitae(German)......................................................................................................................113

CurriculumVitae(English).......................................................................................................................117

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1. Introduction

1.1 Endosymbiosisandotherdefinitions

Symbiosisisubiquitousinnatureandisbecomingincreasinglyrecognizedasasignificantfieldofstudy,because of the fundamental and unifying role across ecological interactions, physiological anddevelopmentalprocesses,aswellasevolutionarydiversification,eventheimpactsonhumans.Thetermsymbiosis originates from the ancient Greek and in the literal sense it describes a “way of life incompany”.Itisdefinedasanintimateandlong-termassociationbetweendifferentdissimilarorganisms,whethertheoutcomeoftheinteractionsisbeneficial,neutralorharmfulforoneofthepartiesinvolved.Insuchasymbiosis,thehostorganismisdefinedastheproviderofresources,whilethesymbiontsaretheconsumersofthoseresourcesandmayormaynotoffersomethinginreturn(Ferrièreetal.,2007;Leung&Poulin,2008).AccordingtoCampbell’sbiologicaltextbook(Campbell&Reece,1984),symbioticassociations are divided in three distinctive categories: mutualism, commensalism and parasitism.Mutualism is characterized as an interspecies relationship, where host and symbiont directly benefitfrom each other. In this case both partners have elevated fitness. If the symbiontmakes use of thehost’sserviceswithoutaffectingitinaneitherharmfulnorbeneficialway,itisconsideredcommensal.Asymbiontqualifiesasparasiticifitutilizesthehostasaresourcereducingitsfitness.Anewerperceptionofsymbiosispointsoutacontinuousgradationand fine linesbetweenmutualism,commensalismandparasitism.Ithasfrequentlybeenarguedhowthiscontinuumcanbehighlyvariableandcircumstantial,andhoweasilysuchsymbioticassociationscanswitchfromonetoanotherinresponsetotheslightestenvironmentalchange(Leung&Poulin,2008).

Incontrasttoectosymbiosis,wherepartnersliveincloseproximitytoeachother,butstillmaintaintheircellular integrity, endosymbiosis involves the internalization of bacterial organisms by a variety ofeukaryotes.Oftensymbiontsdirectlybenefit fromnurturing,protectionfromhostileenvironmentsforstablereplicationandtransportandthehostinreturnreceivesaccesstouniquemetabolites,assistancein defense against natural enemies or sometimes enhanced reproduction (Hétérier et al., 2008).Interspeciesinteractionsofbothecto-andendosymbiosisinvolveabroadvariationofobligateandnon–obligateorfacultativeassociations.Facultative intracellularsymbiontsor intermsofmicrobialecologysecondary symbionts are still capable of growing and replicating outside a host cell; a characteristicfeaturewellknownforLegionellapneumophila,thecausativeagentofLegionnaire’sDisease(Horwitz&Silverstein, 1980) or Listeria monocytogenes. However, such a facultative association still increasesfitness and survival of Legionella pneumophila. In contrast to facultative endosymbiosis, obligateassociationsbetweentwoormorespecies,suchasCoxiellaburnetiiaredisadvantageousinmanyways.They are unable to survive and replicate alone outside of the host and in tight and long-termrelationshipstheylosegeneticmaterial(Nikohetal,2011;Ochman&Moran,2001).

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1.2 Diversityofbacterialendosymbiontsinfree-livingamoebae

Free-living amoebae, such as Acanthamoeba, Hartmanella and Naegleria are ubiquitous unicellulareukaryotes (protozoans), livingmostly in the soil,water and the air aswell as several anthropogenicecosystems.Protozoansplayakeyroleinmaintainingthevarietyandamountofmicrobialspeciesandthediversityofmicrobialcommunities (Brown&Barker,1999;Rodríguez-Zaragoza,1994). Insidesuchbiofilmstheyhavekeyfunctionsintheregulationofthemicrobialbiomassandmaintenanceofahighbacterialmineralizationrateoforganicmatter throughpredation (Molmeret,Horn,Wagner,&Santic,2005). It is well known that free-living amoebae are predators of prokaryotic and eukaryoticmicroorganisms,performed throughphagocytosis andpinocytosis (Greub&Raoult, 2004;Weekersetal.,1993).Acanthamoebapreferentiallygrazeongram–negativebacteria;afactwidelyusedinisolationassays(Khan,2006).Someareopportunisticpathogensofanimalsandhumans,suchasAcanthamoebaspp., a free–living and amphizoic protozoa causing amoebic keratitis (Province, 2012) andGranulomatous Amebic Encephalitis (Khan, 2006). However, most importantly amoebae representfrequenthostsfordiversebacterialendosymbionts.

Basically,onecandistinguishbetweentwodifferentdevelopmentalstages.Trophozoitesresemblethemetabolically active and vegetative form (Bowers&Korn, 1968). Cellmigration ismainly achievedbypseudopodia,temporaryprojectionsofthecell.Byconstantlychangingthecellshapethroughreversibleextensionandcontractionoftheircytoskeleton,amoebaemoveforward,inducedbyanutrientgradientlike free–living bacteria. These pseudopods facilitate attachment to a variety of surfaces and areresponsibleforthetypicalamoebalcellmorphologyandirregularshape.Cystsaremetabolicallyinactiveand provide increased protection from hazardous environmental conditions like temperature and pHfluctuations aswell asUV irradiation anddesiccation (Bowers&Korn, 1969). Cysts ofAcanthamoebaexhibit double walls unlike Hartmannella cysts, which are characterized by a round, smooth–walledmorphology(Khan,2006).Additionally,cystsarecapableoffacilitatingtherecurrenceof infectionandtherebyenablingamoebaetowithstandperiodsofunfavorableenvironmentalconditions(Khan,2006).The developmental cycle is closed upon the emergence of trophozoites from cysts, if favorableconditionshavereoccurred.

Establishing an intracellular interspecies relationship is often initialized through bacteria taken up byphagocytosis but somehow evading host cell defense mechanisms and becoming resistant tointernalization and digestion by the host’s phago-lysosomes. This has first been shown in 1956(Drozanski,1956).Resistancetodigestioncanbemanagedthroughinhibitionoftheformationofphago-lysosomes, modulation of endosomes through host manipulation (Isberg & Heidtman, 2009), simpleescape from phagosomes or capability to grow at acidic pH inside the phago-lysosome (Khan, 2006)usingdefensemechanismssuchastoxins,toxicpigmentsoroutermembranestructures(Weekersetal.,1993).

Oncetheadaptiontoanintracellularlifestyleiscomplete,bacteriamaybegintoexploitthehostcellformassivereplicationandspreadutilizingmetabolicpathwaysandsubstrates. Inductionofhostcell lysisthrough host cell apoptosis is frequent among obligate intracellular pathogens (Ojcius et al., 1998).

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Indeed, the constant pressure of adapting to hostile intracellular environment of protozoa,maywellprimepathogenicbacteriaforvirulence,extensionoftheirhostrange(Horn&Wagner,2004;Molmeretetal.,2005)andguaranteethetransportintohumans(Barker&Brown,1993).Avarietyofreportshaveconfirmed, that Acanthamoeba and Hartmannella provide a general resort for intracellular bacteriaresistanttofree–livingamoebae(Greub&Raoult,2004a),whichareestablishedpathogensofhumans.These includethefacultativepathogenicmycobacteria likeMycobacteriumavium, thecausativeagentofrespiratorydisease(Krishna-Prasad&Gupta,1978;Tayloretal.,2003)orLegionellapneumophilathecausative agent of Legionnaire’s Disease, (Holden et al. , 1984; Kwaik, 1996; Rowbotham, 1980)orChlamydophila pneumonia. Examples of emerging pathogens are Simkania negevensis andParachlamydia acanthamoebae. However, various bacteria will in return confer access to its ownmetabolic capabilities, thereby the utilization of primary or secondary metabolites that had beeninaccessiblebeforeor reprogrammingofhostgeneexpressionpatterns.Eventually,host cellsmaybeabletoconquerandsubdueuniqueecologicalniches(Oldroyd,2009;Wernegreenetal.,2004).

Mostofthesebacteriahavedevelopedintimateandlong–termassociationswiththeirhostsandincludemembersofthephylaAlphaproteobacteria(Birtlesetal.,2000;Fritsche,1999;Hornetal.,1999;Xuanetal.,2007),Betaproteobacteria(Heinzetal.,2007a;Hornetal.,2002),Bacteroidetes(Horn,Harzenetter,Linner,Michel,etal.,2001),Chlamydiae(Collingro,2005a;Collingro,2005b;Fritscheetal.,2000;Heinzetal.,2007a;Hornetal.,2000)andveryrecentlyalsoGammaproteobacteria(unpublished).Diversityofobligateintracellularsymbiontsofamoebae,theirhostrangeandglobaldistributionintheenvironmenthavebeenhighlyunderestimated(Horn&Wagner,2004;Schmitz-Esseretal.,2008).Inthelastdecade,an increased diversity has been recognized within the phylum of environmental chlamydiae inparticular. Molecular evidence indicates that the actual diversity is even larger. According to formerstudies(Fritsche,1993)about5%ofallAcanthamoebaisolatescarrychlamydia-likeendosymbionts.Forexample, sequences related to chlamydiaewere recovered from various aquatic, terrestrial and evenputativeenvironments,suchasactivatedsludge,marinesediment,hydrothermalvents,permafrostsoil,lavacaveorhotspringmicrobialmats(Northupetal.,2011;Siegletal.,2012).ThesampleanalyzedinthisstudywasagreencoloredmicrobialbiofilmofalittoralcavewallfromtheHawaiianIslands.

Such an intimate symbiotic interaction is the key feature that totally undermines traditional culture–dependentidentificationassays.Deeperinsightsintothiscomplexitycouldonlybegainedthroughthedevelopment of culture–independent techniques, for example the 16S rRNA full–cycle approach incombinationwithvariousphylogenetictools.

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Figure 1.1 16S rRNA based phylogenetic tree ofAcanthamoeba endosymbionts. (A) Proteobacterialsymbionts,(B)theBacteroidetessymbiontsand(C)thechlamydialsymbionts(Schmitz-Esseretal.,2008)

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1.3 Generalphysiologyofbacterialendosymbiosis

1.3.1 Theintracellularlife-style

Some microorganisms have evolved to resist the internalization by the protists or become able tosurvive, grow, and exit free-living amoebae after engulfment. Reports suggest that the fate ofinternalizedbacteriafallsintothreemaingroups;thosewhichmultiplyandcauselysisofamoebalhostsuch as Legionella and Listeria; those which multiply without causing cell lysis, thus consideredendosymbionts with maintenance of a stable host-parasite ratio; and those which survive withoutmultiplication(Barker&Brown,1993;Fritscheetal.,1993;Hall&Voelz,1985;Preeretal.,1974).Weareparticularlyinterestedintheobligateintracellularbacteriathatagainshowavarietyofdifferentlife-styles.Eithertheylivedirectlyinthecytoplasmorenclosedinhost-derivedvacuolessuggestingfurtherdifferencesinthemechanismsofhost-cellinteractions.

The discovery that Legionella pneumopbila infects and multiplies within some species of free-livingamoebae (Rowbotham, 1980) has been the first that confirmed the ability of bacteria to exploit anormallyhostileintracellularenvironmenttoensuresurvival.Indeed,survivalandintracellulargrowthofbacterial species in protozoamaywell prime pathogenic bacteria for virulence (Swanson&Hammer,2000). Following phagocytosis by acanthamoebae, legionellaemultiply within the cytoplasm, evadingthehostlysosomalattackbyblockingthephagosome-lysosomefusionandmodulatinghostprocessestouseitforreplication.Thefinaleffectofinfectionislysisofthecellandliberationofmanymotilebacteriaintotheenvironment(Barker&Brown,1993;Isberg&Heidtman,2009).GiventhefactthatRickettsia-likeendosymbionts apparently have a narrowhost range and thatRickettsia spp.may be consideredcommensalendosymbiontsof ticks, thisdeepbranchingmaycorrespondto the timeofdivergenceofthe protozoan and arthropods or to the time of their acquisition by ancestral ticks. The humanpathogenicity of this Rickettsia-like lineage remains to be defined, as do its host range, prevalence,distribution, and interactions with free-living amoebae. Ralstonia pickettii of the Burkholderiaceaefamily and Procabacter acanthamoeba of the Procabacteriaceae are as yet the only two species ofBetaproteobacteria shown to naturally infect free-living amoebae. R. pickettii may act as anopportunistic pathogen. The pathogenic role of Procabacter acanthamoeba is largely unknown, butgiven itsobligate intracellular lifestyle, infectionmay remainundiagnosed if axenic culturesaloneareused.

During the past decade, the Chlamydia-like organisms have been defined as obligate intracellularbacteria that naturally infect a large variety of organisms, such as insects, mammalians, fish andunicellular eukaryotic species. Additionally, several have been considered as emerging pathogens,responsibleforrespiratorytractinfections(GGreub,2009;Haideretal.,2008;Lamoth&Greub,2010),abortionandprematurebirthinhumansaswellasanimals(Baudetal.,2008;Lamoth&Greub,2010;Lamoth et al., 2011; Ruhl et al., 2009). The Parachlamydiaceae family in particular, comprising theobligate intracellular bacterial species Parachlamydia acanthamoebae and Protochlamydiaamoebophila, is associatedwith free-living amoebae. The infectious bacterial form is endocytosed by

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eukaryoticcellsandresideswithinacytoplasmic inclusion,where it transforms intoavegetative formandreplicatesbybinary fission (Mouldert,1991),describedmore indetail in thenextparagraph.Theendosymbiont of free-living amoebae Protochlamydia amoebophila modify the inclusion membranethrough insertion of unique proteins,which are involved in interactionwith andmanipulation of thehostcell(Heinzetal.,2010).Parachlamydiaacanthamoebaehaveevenbeenshowntobeabletoresistthemicrobial effectors of humanmacrophages, lung fibroblasts aswell as pneumocytes and the firstmember of the order Chlamydiales to be shown to traffic through the endocytic pathway withinmonocytederivedmacrophages(Greub,2009;Greubetal.,2005).Apparentlythebacteriareplicateinvacuolesbymodifyingthevacuolebiogenesis,preventingthefusionofthePCVwiththehydrolases-richlysosomalcompartmentandpartiallyinhibitingtheacquisitionofthev-ATPase,amultisubunitcomplexthat is involved in the acidification of the endocytic pathway. The strategy of P. acanthamoebae tosubvert innate immune cells is very similar to that used by Salmonella (Meresse et al., 1999; Steele-mortimeretal.,1999).IncontrasttheChlamydiaceaeappeartocompletelybypasstheearlyendocyticpathwayandreplicatewithinaninclusionthatistraffickedtotheperi-Golgiregionwhereitfuseswithexocyticvesicles(Greubetal.,2005;Hackstadtetal.,1995;Scidmore-Carlson&Hackstadt,2000).

1.3.2 Chlamydialmetabolismandphysiologicactivity

TheChlamydiaeceaeareagroupofobligateintracellularbacteriaincludingsomeofthemostimportanthuman and animal pathogens such asChlamydia trachomatis aswell as endosymbionts of protozoa.Intracellularbacteriaoftenhavedeepimpactontheirhosts,whicheithermighthavelyticeffectsoronwhichthehostsdependforsurvivalandreproduction.Asaconsequenceofthisspecializedlife-styleintheinteriorofeukaryoticcells,thesebacteriahavehighlyreducedmetaboliccapabilitiesandrelyonalargevarietyofhost-derivedmetabolites(Omslandetal.,2014;Zientzetal.,2004).

All members of the Chlamydiales share similar features in their biphasic developmental cycle, whichconsistsofthealternationbetweentwomainmorphologicalforms.Aninfectionisthusinitiatedbytheattachment of the extracellular elementary body (EB) known as the infectious form followed by theinternalization into a membrane-bound compartment termed inclusion. EBs differentiate into theintracellular replicative form called reticulate body (RB) which then start dividing by binary fission.Finally, the largely accumulated RBs differentiate back to infectious EBs and are released into theenvironment by extrusion or host cell lysis. One of the essential characteristics of the small, round,electron dense form of Chlamydia spp. EBs is their high resistance to harsh extracellular conditionsincluding osmotic and physical stress due to their rigid and highly cross-linked outer membrane(Abdelrahman&Belland,2005;Hatch,1996;Hybiske&Stephens,2007;Mouldert,1991;Omslandetal.,2014;Sixtetal.,2013).TheultrastructureofEBsischaracterizedbyhighlycondensedchromatin,whichmayleadtoinactivationoftranscriptionandareducedRNAtoDNAratio.Thus,EBshavebeenthoughttobetotallymetabolicallyinertparticlesforalongtime(Pedersen,1996).

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Figure 1.2 The biphasic chlamydial developmental cycle. The host cell cytoplasmic membrane isindicated as a red line and illustrates the interactions with Chlamydial EBs. The formation of theinclusionmembrane,aswellas themajorevents in thedevelopmentalcyclearedescribed in thetext(Abdelrahman&Belland,2005).

In amore recent study, a profound characterizationof themetabolic activities of EBsof the amoebaendosymbiontProtochlamydiaamoebophilawasperformed(Sixtetal.,2013).Especially, theeffectofnutrientavailabilityordeprivationonchlamydialinfectivitywascloselyinvestigated.Thestudyrevealedthat extracellular Protochlamydia amoebophila EBs maintain their respiratory activity as well as theuptakeandutilizationofD-glucose.ByreplacingthesubstratewiththenonmetabolizablestereoisomereL-glucose, a rapid decline in the amount of infectious particleswasmeasured. Themajor route ofD-glucose catabolism was identified as the pentose phosphate pathway, but to some extend host-independentactivityofthetricarboxylicacid(TCA)cyclewasalsoobserved.Genomesequenceanalysisalso revealed thatProtochlamydia amoebophilaandmany other environmental chlamydiae encode aglucokinase, which enables them to phosphorylate and activate D-glucose. Furthermore D-glucoseavailabilitywasevendemonstratedtobeessentialforthesurvivalandmaintenanceofProtochlamydiaamoebophilainfectivity.

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Figure 1.3 The comparative metabolic repertoire of environmental chlamydiae and theChlamydiaceae.(Omslandetal.,2014)

In contrast to this, genomic studies reveal that theChlamydiaceae lack a glucokinase geneaswell assubstrate-specific componentsof thephosphotransferasesystem.Thus, thesebacteriadependon theimport of the phosphorylated D-glucose-6-phosphate from the host by a sugar-phosphate/inorganicphosphateantiporterUhpC(Iliffe-Lee&McClarty,1999) insteadofphosphorylatingD-glucoseontheirown. Comparative genome analyses, including sequences of four environmental chlamydiae,Protochlamydia amoebophila, Parachlamydia acanthamoebae, Simkania nevegensis and WaddliachondrophilaindicateasignificantdivergenceandlittlesyntenytoeachotheraswellastogenomesoftheChlamydiaceae(Bertellietal.,2010;Collingroetal.,2011;Hornetal.,2004;Omslandetal.,2014).Anumber of the genes that were only found in environmental chlamydiae comprised such encodingproteinsinvolvedinmetabolicprocesses.ThissuggeststhattheseenvironmentalchlamydiaestillretainahighermetabolicpotentialthantheChlamydiaceae,butthattherearesignificantspecies-andstrain-specificdifferencesamongtheenvironmentalchlamydiaconcerningtheirmetabolicactivity.Wearefar

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fromunderstandingtheinteractionofchlamydiaewiththeirintracellularandextracellularenvironment,nutrientrequirementandthephysiologicalchangesduringthecourseofthedevelopmentalcycle.

1.4 Aimofthestudy

Until now, the diversity of amoeba endosymbionts has been underestimated and the bacteria haverarely been identified or poorly described. (Horn, 2008) Therefore, the aim of this project was theisolation of amoebae from a biofilm sample, their axenization, as well as the identification andcharacterizationoftheirendosymbionts.InthisworkacombinationofFluorescentin-situHybridizationassaysandmolecularmethodswasapplied.

First,amoebaehadtobeisolatedfromtheenvironmentalsample.Aswaspreviouslyshown,amoebaeareabletomovebymeansofcytoplasmic flow,thus isolationofenvironmentalamoebae isbasedonmigrationonagarsurfacescoveredwithaliveE.coli layer.(Neff,1958)Theamoebaespreadoutfromthe inoculation site and can be transferred to new agar plates. In order to obtain axenic amoebaecultures, amoebae are eventually grown in E. coli-free growthmedia (TSY, PYG), supplementedwithampicillin.

As illustrated in fig. 3 an axenic amoebae culture can be used for the molecular approach offluorescence in-situ hybridization to detect intracellular bacterial endosymbionts. Identification andphylogeneticassociationwaspossiblethrough16SrRNAgenesequencing.

Here,thedetectionoftwonovelspeciesoftheChlamydialesandtheBetaproteobacteria,encouragesafurther in-depth characterization of the endosymbionts. These enclose the description of infectioncycles in hosts, analysis of host range, their impact on amoebae growth and fitness and finally theirability of extracellular survival and maintenance of infectivity. To lead on the study, whole genomesequencingof thestrainsHSC3andHSC8willprovideanefficientmeansofacquiringdatarelevanttothemoredetailedmolecularcharacterizationoftheendosymbionts.

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Figure 1.4 Flow chart illustrating the steps of amoebae isolation and axenization, followed by theidentificationandcharacterizationofamoebaeendosymbionts.

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2. MaterialandMethods

2.1 Technicalsupplyandcomputersoftware

Mostof theworkdescribed in this thesiswasdoneat theDivisionofMicrobialEcology (UniversityofVienna),withoneexception.TheTecanInfiniteM200microplatereaderneededforthequantificationoftotalamountofDAPIstainedDNAaswellaspropidiumiodidestainedcellswaslocatedattheDivisionofTerrestrialEcosystemResearch(UniversityofVienna).

Table2.1Technicalequipmentwithindicationofthecorrespondingsoftware.

Machine Software ManufacturerPowerPacBasic(electrophoresispowersupply)SmartSpecTM3000(spectrometer)Sub-CellGT(agarosegelelectrophoresissystem)Sub-cellGTgeltrayUST-C30M-8R(UVtransilluminator)Centrifuge5804REppendorfResearch®pipettesMiniSpin®plusIncubationwaterbath1004Mikro20(centrifuge)LaminAirModel1.2(laminarflowhood)CLSM(confocallaserscanningmicroscope)Neubauerhaemocytometer(countingchamber)HybridizationovenMilli-QWaterPurificationSystemC-5050Zoom(digitalcamera,transilluminator)BL3100(balance)BL6100(balance)VortexGenie2Infinite®M200(microplatereader)Haake®DC10-P5/U(heatingcirculator)NanoDropND-1000(UV-visiblespectrometer)GalaxyMiniC12XX(microcentrifuge)InoLab®pHLevel1(pHmeter)AxioImagerM1(epifluorescencemicroscope)Axioplan2imaging(epifluorescencemicroscope)AxioCamHRc(digitalcamera,epifluorescencemicroscope)

ArgusX1(4.1)ND-1000(3.2)AxioVision(4.7)i-controlTM(1.6)

BioRadBioRadBioRadBioRadBiostepEppendorfEppendorfEppendorfGFLHettichJOUANNordicA/SLeicaMarienfeldMemmertMilliporeOlympusSartoriusSartoriusScientificIndustriesTecanThermoScientificThermoScientificVWRWTWZeissZeissZeiss

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2.2 Consumables

Consumablesusedforthecultivationandinfectionassayswereonlyopenedandusedinalaminarflowhoodtoavoidcontamination.Theoutersurfaceswereadditionallycleanedwith70%(v/v)ethanolpriortomoving them into the laminar flow hood. PCR consumables were only opened at designated PCRhoods and radiated with UV light for 15 min before use. The work benches designated for DNAisolations, includingconsumablessuchaspipettesandtipboxeswerecleanedwithDNAAWAYbeforestartingtowork.

Table2.2Consumables.

Consumable Manufacturer0.6mlreactiontubes0.2mlPCRtubesPremiumTips(withfilters)Omnifix®single-usesyringes,30and50mlMicroscopeslidesPipettetips1.5mlreactiontubes2mlreactiontubes15mlreactiontubes50mlreactiontubesMicroplates,96-well,flatbottom,chimney,blackMicroscopeslides,polyethylenecoated,blackmaskwith10wellsGlasscoverslips,24x50mmNalgenesyringefilters(0.2µm)Standardcellcultureflasks,NuclonTMΔsurface,vent/closecap,25cm2TripleFlaskTMcellcultureflasks,NuclonTMΔsurface,vent/closecap,500cm2Minisart®NMLsyringefilters,1.2and5.0µmGlassbottlesPetridishes,90mmCellcultureflasks,standardcap,182cm2Serologicalpipettes,2,10and25ml

BiozymBiozymBiozymBraunCarlRothCarlRothGreinerBio-OneGreinerBio-OneGreinerBio-OneGreinerBio-OneGreinerBio-OneMarienfeldMarienfeldNalgeneNuncNuncSartoriusSchottSterilinVWRVWR

2.3 Molecularbiologykits

Table2.3Molecularbiologykits.

Kit ManufacturerTOPO®TACloningKitDNeasy®Blood&TissueKitQIAquick®GelExtractionKitQIAquick®PCRPurificationKit

InvitrogenQIAGENQIAGENQIAGEN

19

2.4 Probesandprimers

Oligonucleotide probes were generally single-labelled with 5 (6)-Carboxyfluorescein-N-hydroxysuccinimideester(FLUOS),Cy3orCy5andsometimesdouble-labeledandarelistedinTable2.4.AllprimersandprobeswereproducedbyThermoFisherScientific.

Table2.4OligonucleotideprobesforrRNAtargetedfluorescenceinsituhybridization.

Probe Targetorganism 5'-3'sequence %FA AuthorAcRic90 Rickettsia-like TGC CAC TAG CAG AAC TCC 20 Fritscheetal.AcRic1196 Rickettsia-like CCT ATT GCG TCC AAT TGT 10 Fritscheetal.Alf968 α-Proteobakteria GGT AAG GTT CTG CGC GTT 20 Neefetal.Aph1180 Amoebophilus

asiaticusCTG ACC TCA TCC CCT CCT 20 Hornetal.

CC23a Caedibacter-related

TTC CAC TTT CCT CTC TCG 20 Springeretal.

Chls-0523 Chlamydiales CCT CCG TAT TAC CGC AGC 25 Poppertetal.CFB286 Bacteroidetes TCC TCT CAG AAC CCC TAC 20 Welleretal.Gam42a γ-Proteobacteria GCC TTC CCA CAT CGT TT 35 Manzetal.Bet42a β-Proteobacteria GCC TTC CCA CTT CGT TT 35 Manzetal.EUB338-IEUB338-IIEUB338-III

MostbacteriaPlanctomycetalesVerrucomicrobiales

GCT GCC TCC CGT AGG AGT GCA GCC ACC CGT AGG TGTGCT GCC ACC CGT AGG TGT

0-600-600-60

Amannetal.Daimsetal.Daimsetal.

HSC8 Betaproteobacteriasp.endosymbiontHSC8

20 Unpublished

Proca438 Procabactersp. CGA TTT CCT CCC RGA CAA 20 Hornetal.Ric1395 Rickettsia-like GGC TTG ACG GGC AGT GTG 20

Table2.5Primersusedinstandard16Sand18SPCR.

Primer Targetorganism

Targetmolecule

5'-3'sequence Author

616V Bacteria 16SrRNA AGA GTT TGA TYM TGG CTC AG Kimetal.1492R Bacteria 16SrRNA RGY TAC CTT GTT ACG ACT T McAllisteretal.PanF Chlamydiales 16SrRNA CGT GGA TGA GGC ATG CRA GTC G Corsaroetal.PanR Chlamydiales 16SrRNA GTC ATC RGC CYY ACC TTV SRC RYY TCT Corsaroetal.18SF Eukaryota 18SrRNA GTA GTC ATA TGC TTG TCT C Whiteetal.18SR Eukaryota 18SrRNA CGR ARA CCT TGT TAC GAC Whiteetal.

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2.5 Growthmedia,buffersandotherchemicalsolutions

Thelistedgrowthmedia,buffersandsolutionswereautoclavedat121°Cand1013x105Pafor20minandwerestoredatroomtemperature, ifnotstatedotherwise.Solidificationoftheculturemediawasassured by adding 15 g/l agar prior to autoclaving, supplemented with antibiotics (if required) andpouredinto90mmpetridishes(Sterilin).

Table2.6GrowthmediaforAcanthamoebacultivationandE.colicultivation.

Ingredients Amountfor1LTSYBroth(TrypticaseSoybrothwithYeastextract)TrypticaseSoyBroth(DIFCO,Detroit,USA)YeastextractH2Odest. pH7.3PYG(Peptone-Yeast-Glucose-Medium)Pepton Glukose Hefeextrakt Natriumcitrat MgSO4*7H2O Na2HPO4*7H2O KH2PO4 Fe(NH4)2(SO4)2*6H2O H2Odest. pH6.5 Autoclavedat110°C NNA(NonNutrientAgar)Page’sAmoebicSaline(10x) H2Odest. Agar LB-medium(Luria-Bertani-medium)Trypton NaCl Yeastextract H2Odest. pH7.0-7.5DMEM(Dulbecco'sModifiedEagleMedium)

30g10g

add1000ml

20g18g2g1g

980mg355mg340mg20mg

add1000ml

100ml900ml

15g

10.0g5.0g5.0g

add1000ml

21

Table2.7Buffers.Ingredients Amountfor50ml Amountfor1LPage’sAmoebicSaline(10xPAS)NaCl MgSO4*7H2O CaCl2*2H2O Na2HPO4 KH2PO4 H2Odest. pH6.9PhosphateBufferedSaline(10xPBS)NaClKClNa2HPO4 KH2PO4H2Odest. pH7.4Dulbecco’sPhosphateBufferedSaline(10xDPBS)pH7.2-7.6Tris/HClbufferTrisH2OreinstpH8.010xTBE(Tris/Borate/EDTA)bufferTrisBoricacidEDTAdisodiumsaltdehydrateH2OreinstpH8.350xTAE(Tris/Acetate/EDTA)bufferTrisAceticacidEDTAdisodiumsaltdehydrateH2OreinstpH8.3-8.71xTEBufferAWithoutEDTA35mMTris-HCl250mMSucrose

22g4,28g

1.2g0.04g0.04g1.42g1.36g1000ml80g2.0g14.4g2.4g1000ml121.1gAdd1000ml107.8g55.0g7.4gAdd1000ml242g57.1ml18.6gAdd1000ml4,4g85,6g

22

25mMKCl10mMMgCl2H2OdestpH7.5BufferAWith250mMEDTA35mMTris-HCl250mMSucrose 250mMEDTA25mMKCl 10mMMgCl2H2OdestpH7,5 Hybridizationbuffer5MNaCl 1MTris/HClpH8.0 Formamid H2OreinstWashingbuffer1MTris/HClpH8,0 5MNaCl 0.5MEDTA H2Oreinst

0,09g0,1gadd50ml0,44g8,56g9,3g0,18g0,2gadd100ml

1,8g2gadd1000ml4,4g85,6g93,1g1,8g2gadd1000ml180µl20µlxµl*add1000µl1mlyµl*500µl(above20%formamidinhybridizationbuffer)add50ml

Table2.8Antibiotics.Allantibioticslistedwerefiltersterilized(0.2µmfilter)andstoredat-20°C.Antibiotic Mechanism AntibioticClass Stock

concentrationDissolvent

Ampicillin Inhibitionoftranspeptidaseneededforcellwallsynthesis

Aminopenicillin 100mg/ml 50%(v/v)96%EtOH

Kanamycin Interactionwithbacterial30SRSUandinhibitionoftranslocation

Kanamycin 100mg/ml H2Oreinst

Rifampicin InhibitionofbacterialDNA-dependentRNA-polymerase(RNAsynthesis)

Rifamycin 10mg/ml 50%(v/v)DMSO

Tetracyclin Bindingofbacterial30SRSUandblockingofchargedaminoacyl-tRNA

Tetracyclin 10mg/ml H2Oreinst

Doxycyclin Bindingofbacterial30SRSUandAminoacyl-tRNA

Tetracyclin 10mg/ml H2Oreinst

23

Fortheproperpreparationofthesesolutions,onlydistilled,UV-lighttreated,filtered,deionizedwaterfromtheMILLI-QWaterPurificationSystemwasused.OnlyPCR reagentswerepreparedwithdoubledistilledwater.PHwasadjustedusingNaOHplateletsorHClsolution.Table2.9Chemicalsolutions.Ingredients Amountfor1LSodiumchloridesolutionNaClH2OreinstParaformaldehyde(PFA)solutionFormaldehyde(37%solution)H2OreinstFiltersterilized,notautoclavedEDTA(Ethylenediaminetetraaceticacid)solutionEDTAdisodiumsaltdihydrateH2OreinstpH8.0

292.2gAdd1000ml

Amountfor50ml

5.4gAdd50ml

Amountfor250ml46.5g

Add250ml

Table2.10Hybridizationbuffer(46°C).Amountsareindicatedinµl.FA% 0% 5% 10% 20% 25% 30% 35%NaCl 180 180 180 180 180 180 180Tris 20 20 20 20 20 20 20MQ 800 750 700 600 550 500 450FA 0 50 100 200 250 300 350SDS 1 1 1 1 1 1 1Table2.11Washingbuffer(48°C).Amountsareindicatedinµl.FA% 0% 5% 10% 20% 25% 30% 35%NaCl 9ml 6.3ml 4.5ml 2150 1490 1020 700Tris 1ml 1ml 1ml 1ml 1ml 1ml 1mlEDTA 0 0 0 500 500 500 500MQ Ad50ml Ad50ml Ad50ml Ad50ml Ad50ml Ad50ml Ad50ml

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2.6 Methods

2.6.1 Isolationofamoebaeoffree-livingamoebaeusingnon-nutrientagarplatesseededwithfoodbacteria

In nature, prokaryotic and eukaryotic microorganisms are an important food source for free-livingamoebae (Khan,2006).Therefore, in thisstudyamoebaewere isolatedby luringthemaway fromtheinoculationsiteastheagarplateiscoveredwithabacteriallayer,preciselyEscherichiacoli(NEFF,1958).

Accordingtoamodified“walkout”methoddescribedbyLagkouvardosandcolleagues(Lagkouvardosetal.,2014), twoNon-nutrientagarplates (NNA)werecoveredwith100µl liveTolC-E.colimutantsandenvironmentalsamplewasaddedtothecenter.OnePetridishwasincubatedat27°C,theotheroneatRT,andtheamoebaewere lettomigrateawayfromthe inoculum.Thismethodisusedextensively intheisolationofamoebaefrombothenvironmentalandclinicalsamplesworldwide.

Figure2.1Biofilmplated inthemiddleofaPetridishwithNNAcoveredwithTolC-E.coli. (A)SampleincubatedatRT,(B)sampleincubatedat27°C.

Eachplatewasobserveddailyforthepresenceofthetypicalamoebaetrophozoitesbylightmicroscopy,and then6and9 locations respectivelywerechosenandexcisedasagar cubeswitha spatula.Thesewere transferred upside down onto a new E. coli NNA plate and incubated at the respectivetemperatures until the amoebae reached the edge of the plate. In order to keep the plates free ofcontaminants, agar cubes with amoebaewere transferred another two times, until 4 pieces of eachisolate were dedicated to be grown in a 12-well plate with 2.5 ml liquid media (TSY and PYG)supplemented with 2.5 µl TolC- E. coli and 0.25 µl ampicillin (100 mg/ml stock solution to a finalconcentrationof10µg/ml).

(A) (B)

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2.6.2 Axenizationoffree-livingamoebae

Anaxenicculture istypicallygrownintheabsenceofexternal livefoodbacteria. Inordertoeliminatethenecessityofamoebaeforthefoodsourceovertimeasingle-geneknockoutE.colimutantwasused,whose gene coding for the outer membrane transporter TolC is replaced by a kanamycin resistantcodinggene.Thus,theTolC-E.coliarehypersensitivetoampicillinandgentamycinandcanselectivelybeeliminatedby sublethalamountsof theseantibiotics (Babaetal.,2006; Lagkouvardosetal.,2014;Tamaeetal.,2008).

Assoonasanincreaseinthenumbersofamoebaecouldbeobservedbylightmicroscopy,1mlamoebaculturewas transferred toa6-wellplate, filledwith9mlofgrowthmedium, supplementedwith1µlampicillin.NoTolC-E.coliwereaddedinordertogetridofthenecessityofamoebaetofeedonthem.Furtherenhancedamoebalgrowth indicatedthepassagetoaxenizationandtheculture is transferredinto 25 cm2 polystyrene culture flasks, containing 8-10ml TSY or PYGmedium. Thesewere emptiedeveryweek,refilledwithfreshgrowthmediumandsupplementedwith1µlampicillin.Theculturecouldbeconsideredaxenic,whenamoebaecontinuedgrowingwithoutanyfurtheradditionofantibioticsandnoextracellulargrowthofbacteriacouldbeobserved.

Figure2.2Workingschemeforisolationandaxenizationofamoebae.Thetransferstepsofagarcubescontainingamoebaeareindicated.

2.6.3 Fluorescencein-situhybridization(FISH)In order to detect and identify endosymbiotic bacteria of free-living amoebae, a hybridization usingspecificoligonucleotideprobescanbeapplied.Thebestwaytogettheprobestoenterthecellsistofixtheamoebaewith4%formaldehyde,actingasdenaturingagentformembraneproteins.

The stringencyof thehybridizationofoligonucleotideprobes toany target16S rRNAdependson theconcentrationofformamidaddedtotheHybridizationbufferandtheNaClconcentrationinthewashing

26

buffer. These conditions differ with every single probe, thus have to be adapted most efficiently.Formamidhasastrongerdipolemomentthanwaterandthereforedestabilizeshydrogenbonds,whileNaClstabilizesnucleicacidhybrids.

Anoligonucleotideprobecontainsasequencecomplementarytothetargetsequenceononehand,onthe other hand a fluorescent dye (typically Cy-3 with the red emission, or FLUOS in green) whichvisualizestheorganismofinterestunderafluorescencemicroscope.

FixationofamoebaecellsonFISHslide

Fortheinsituhybridizationpreferentiallyaxenizedamoebaecultureswereused.Thecultureflaskswerevigorously shaken, so that theamoebaeattached to thesurfacewere loosened,1-2mlof theculturewere pipetted into a 2 ml Eppendorf tube, centrifuged for 5 min at 5000 rpm. After removing thesupernatant, the pellet was resuspended in a small amount of 1 x PAS (50-120 µl). 20 µl of the cellsuspensionwas lefttoattachonaTeflon-slidewellduring1-2hoursatRT,untilthesupernatantdropwasalmostdry.Afteradding20µlof4%formaldehyde,thesamplewasincubatedfor10min,beforecarefullyremovingthesupernatantdropbypipetting.Towashthewellsandgetridofanyremaining4%formaldehyde20µlMilliQwereaddedandremovedagainimmediately.Theslidewasthenair-dried.

Fluorescencein-situhybridization

For the in situ hybridization 10 µl hybridization buffer and 1 µl fluorescently labeled oligonucleotideprobewasdroppedontoeachwellandcarefullymixed.Theslidewasputintoa50mlscrew-toptube,togetherwith awetted tissue paper. The tubewas placed horizontally into a hybridization oven andincubatedfor1.5hat46°.Afterthattheslidewaswashedduring10minin50mlwashingbufferthathas been preheated to 48 °C in a water bath, dipped for 2 s into ice-cold ddH2O and dried withcompressedair. Prior to analysis under anepifluorescencemicroscope (ZeissAxioplan2) the samplesweremountedwithCitifluorandcoveredwithaglasscoverslip.

All sampleswere labeledwith the probe EUB338-mix that targetsmost bacteria, labeled in green byFLUOSandanotherspecificprobetargetingsingleclassesofbacteriainredbyCy3.Theamoebahostswere marked by a probe EUK516 targeting eukaryotic cells in general and a probe specific forAcanthamoebasp.bothlabelledinbluebyCy5.

2.6.4 16SrRNAgenesequencingofaxenizedamoebaculturesThe 16S rRNA is part of the 30S subunit of the bacterial ribosome and memorizes evolutionaryinformationaboutaprokaryoticorganism.Theribosomeitselfistheproteinmachineryofacellandhasoneofthemostessentialfunctionsinanorganism.Thefactthatthesefunctionsareresponsiblefortheviabilityofthebacteriaappliesastrongselectivepressureontheorganismsandeliminatesthemutatedones immediately. The rRNA genes are functionally homologous between all known bacterial speciesand contain several highly conserved regions, which can be targeted by “universal primers” like theforwardprimer616Vandthereverseprimer1492Rusedinthisstudy.Primersspecificforchlamydiae,

27

such as the forward primer PanF and the reverse primer PanR target the conserved regions ofchlamydial16SrRNAgenes.

Along with the conserved regions there are hyper variable sequences that differ from one genus orspeciestoanotherduetomutations.Thereforetheribosomal16SrRNAgenesactasamolecularclockandallowaclassificationofunidentifiedbacteriaandaphylogeneticanalysis.

Figure2.3BacterialrRNAoperonwithindicationofprimerbindingregionsusedforthe16SrRNAgeneamplification.

DNAextraction

BacterialDNAwasextractedaccordingtotheBloodandTissueKit(Qiagen).1mlofanaxenicamoebaculture inTSY/PYGmediawascentrifugedfor10minat7’500rpmandthepelletwasresuspended in180 μl Buffer ATL, 20 μl proteinase Kwas added and thoroughly vortexed, before the sampleswereincubatedforanother2hat55°C.Afterthelysiswascomplete,thesampleswerevortexedfor15s,200μlBufferALwereaddedandmixedimmediatelyandthoroughlybyvortexing.Afteranotherincubationat70°Cfor10min,200μlethanolabsolutewereaddedandvortexed.ThemixturewasthenpipettedontoaDNeasyspincolumnplacedina2mlcollectiontube.Afterafirstcentrifugationstepat8’000rpmfor1min,theflowthroughwasdiscardedandinordertogetridoftheremainingethanolthesampleswerewashedtwice:500μlBufferAW1wasadded, followedbyasecondcentrifugationstepat8’000rpmfor1min,thenanother500μlBufferAW2waspipettedontothecolumnandcentrifugedfor3minatfullspeed,todrytheDNeasymembrane.TheextractedDNAcouldafterwardsbeelutedintoanew2mltubebyadditionof100μlBufferAE,incubationduring1minatRTandthencentrifugationduring1minat8’000rpm.Thiswasrepeatedtwice,sothatintotalanamountof200μlofdissolvedDNAcouldbe used for further analysis. The concentration of extracted DNA was measured by a NanoDropSpectrophotometerusingBufferAEasablank.

28

RibosomalRNA-geneamplificationbyconventionalPCRandclean-upofthePCRproductConventionalPCRwasdonetoamplifybacterial16SrRNAgenesorspecificchlamydial16SrRNAgenesononehandandamoebal18SrRNAgenesontheotherhand.TheMasterMixcontainedthefollowingreagentsrespectivelyandwasrunwithPCRprogramsthatperform35cycles.

Bacterial16SPCRDNAMgCl2PCRBufferNucleotideMixTaqpolymeraseForwardPrimer616VReversePrimer1492RMilliQ

1μl4μl5μl5μl0.2μl1μl1μl32.8μl50μl

Chlamydial16SPCRPanFPanR


Amoebal18SPCR18SF18SR


°C Sec

Denaturation 95 5min

Denaturation 95 30sec

Annealing 55 40sec 35x

Elongation 72 1min

FinalElongation 72 10min

°C Sec




Elongation 72 1.5min


29

°C Sec




Elongation 72 3min


ForthecontrolofthePCRproductformation,agelelectrophoresisona1.5%agarosegelwasmade.A1:10 KBL (5 μl) was used for PCR product verification and 8 μl PCR productwas added to a drop ofloadingdyeandfilledintothegelpockets.

Tocleanupthe16SPCRproducts,theQiaQuickPCRPurificationKitwasapplied.Acolumnwasfilledwithamixtureofthe40μlPCRsamplecomplementedwith200μlofPB(5xthevolume).Aftera1min13’000rpmcentrifugationstep,thesamplewaswashedwith750μlPEandcentrifugedagainfor1minat13’000rpmintoanew2mlEppendorfTube.TheDNAwaselutedthenbyadditionof35μlElutionbuffer, 1min incubation timeatRT, before a spindownat 13’000 rpm for 1min. TheobtainedDNAconcentrationcouldbemeasuredbyaNanoDropSpectrophotometerusingElutionbufferasablank.

Asecondmethodwasusedtocleanupthe18SPCRproducts,theQiaQuickGelExtractionKit.42μlPCRsamplewere loadedontoanother1.5%agarosegel complementedwith7μl loadingdye.ThebandswereshortlycheckedundertheUVlight.Asmorethanonebandcouldbedetectedthefragmentwiththeappropriate lengthwasexcised fromthegelusingaclean, sharpscalpel, thenput intoEppendorftubesthatwerepreviouslyweighted.3volumesofBufferQCwasaddedto1volumegel(100mg~100ml) and incubated at 50 °C for 10min until the gel slice had completely dissolved. 1 gel volume ofisopropanolwasaddedtothesampleandmixed.TobindtheDNAthesamplewasappliedtoaQiaquickspin columnand centrifuged for 1minat 13’000 rpm. The flow-throughwasdiscardedand500μl ofBufferQCwasadded.Aftercentrifugationfor1minat13’000rpmanddiscardingoftheflow-through750 μl Buffer PE was added and centrifuged for 1 min at 13’000 rpm. Again the flow-through wasdiscarded and another centrifugation step followed for 1min at 13’000 rpm. To elute DNA 50 μl ofMilliQwasaddedtothecenterofthecolumnandcentrifugedfor1minat13’000rpmintoanew1.5mlEppendorftube.Thislaststepwasrepeatedusing30μlMilliQ.

Cloningoftheamplified16SrRNAgenesequence

Asthesamplesanalyzedinthisstudywerenopurecultures,butpossiblyamorecomplexcompositionof diverse bacterial endosymbionts, as is very common among environmental samples, a mereamplification and sequencing of 16S rRNA genes would not be efficient. The diverse PCR productsamplifiedby theuniversalprimershad tobe integrated into single cloningvectors.Each recombinantDNAmoleculewas transferred intoa livinghostorganism (typicallyE. coli),and then replicatedalongwiththehostDNA,before16SrRNAgenesequencing.

30

TheTaqDNApolymeraseused for thePCRaddsanoverlappingadenine (dATP) to the3‘endofeveryamplificateduringthelastelongationstep.Becauseofthelacking3’-5’proofreadingactivityoftheTaqDNA polymerase themistake is not being adjusted. Fortunately, this fact can be used for TA cloningwhereavectorinitslinearizedformisused,carryingacomplementary3’thymineoverhang(ddTTP)oneachbluntend.Hybridizationiscatalyzedbyatopoisomerase.Noteverycloningvectorincorporatesthe16SrRNAinsertofinterest,thusselectionprocessesarebeingneeded.Anewermethod,alsoappliedinthis study thevectorpCR4contains twoantibiotic resistancegenes (kanamycinundampicillin)andasuicidegene,whichkillsitshostcell.Thisgeneisthoughttoserveasinsertionsite,whichmeansthatiftheplasmidtakesupaninsertthesuicidegeneisdestroyedandkeepsthehostorganismalive.

ThecloningofthePCRproductswasdoneaccordingtotheTOPOTACloningKit.4μlofeachamplificatewasmixedwith1μlsaltsolutionand1μlsuicidevector(pCR4),beforeshortcentrifugationand15minincubationatRT.

Afterasuccessful ligation, thetransformationstepwasprepared.ChemicallycompetentE.coliTOP10werethawedonice,SOC-mediumwaspreheatedto37°CandLBagarplatesweresupplementedwithafinalampicillinconcentrationof100μg/ml.3μloftheligationproductwasaddedtothetubewiththechemicallycompetentE.coliTOP10cellsandstirredcarefullyinordernottodamagethecells.After30min incubation on ice, the cells were heat-shocked at 42 °C in a water bath, followed by 2 minincubationonice.250μlSOC-mediumwasaddedtothereactionandthetubeswerefixedonashakerfor 1 h at 37 °C and 200 rpm. The reactionmixwas then plated on the previously prepared LB agarplates,100μland150μleach,whichwerethenstoredover-nightat37°C.BecauseofthesuicidegeneofthevectorpCR4,onlyE.coliTOP10thathavetakenupthevectorwithaninsertcanbuildcoloniesontheLB-ampicillinagarplates.

E.coli transformants:A96-wellplatewasfilledwith150μlLBmediumandampicillin,and inoculatedwithoneclone.OnonehandaforwardprimerT3,ontheotherhandareverseprimerT7wasappliedforeachcloneinordertoobtainthewholesequenceofabout1000bp.Theplatewasincubatedfor4hwithgentleshakingat37°Candlabeledwiththerespectivebarcode,priortosendingittothecompanyMicrosynthforsequencing.

PurifiedPCRProducts:IncaseswhereaconventionalPCRreactionwassufficient,usingthechlamydia-specific forward primer PanF and the reverse primer PanRor the universal forward primer 616V andreverseprimer1492R,agelwasruntoensure,thattheinsertofinterestwaspresent,beforepurifyingthePCRproductusingtheQIAQuickPCRPurificationKit,asdescribedonpage12and13.Microsynthrequires22.5ngper100bp in15µl totalvolume,whichwasaliquoted intoSarstedtscrewcaptubeslabeledwiththerespectivebarcodeandsendforsequencing.

31

2.6.5 CultivationofidentifiedAcanthamoebaspp.HSC

Continuous axenic cultures of Acanthamoeba spp. HSC were transferred from 25 cm2 polystyrenecultureflaskscontaining8-10mlTSYto500cm2polystyrenecultureflasksandfedwith150mlofthesamemedium.Thisisaboveallnecessarytoattainahigherbiomassthatwillbeneededfortheeventualisolationofextracellularendosymbionts.Foragoodmaintenanceoftheamoebaculturesmediumwasexchangedevery2weeksand in general kept at the room-temperatureor if needed forexperimentscultivatedat20°Cor27°Crespectively.Regularobservationsofmorphologicalcharacteristicsby lightmicroscopy guaranteed the usage of a well-grown culture indicated by densely packed amoebaeattachedtothesurfaceofthecultureflask.

2.6.6 CuringofAcanthamoebaspp.HSC

The identified Acanthamoeba sp. cultures infected with the endosymbionts HSC3 and HSC8 wereharvested in 12-well plates filled with 2.5 ml TSY. The cultures were treated with differentconcentrationsof5classesofantibiotics, inordertoeliminatethebacteriawhilekeepingtheamoebaculturealive.Allantibioticsusedhavespecifiedmechanismsofaction,mentionedintable2.8.Severalantibiotictreatmentswereappliedaccordingtothefollowingtable2.12.

Table2.12TypeofantibioticsusedforthecuringoftheAcanthamoebaspp.Hostswithconcentrationsindicated.

Isolate Antibiotic FinalconcentrationinmediaHSC3 Doxycycline 50,70,100ng/ml Tetracycline 50,70,100ng/mlHSC8 Rifampicin 100ng/ml Kanamycin 50,70,100ng/ml Ampicillin 50,70,100ng/ml

The plates were kept at 20 °C and the same concentration of antibiotics was added every 3 days.Generalconditionoftheamoebaewascheckedregularlyby lightmicroscopyand infectionrateswereobservedbyrepeatedscreeningusingspecificprobesforFISHuntilinatleastonewellonlyuninfectedAcanthamoeba spp. were detected. After 8 weeks of several dilution steps an Acanthamoeba spp.culturefreeofendosymbiontswasgrown.

32

2.6.7 Definingthehostrangeofendosymbionts

TheendosymbiontsHSC3andHSC8wereaddedatanMOIof500tofourdifferentAcanthamoebasp.strainsaswellastoanS2insectcell linelistedintable2.13.TheinfectionwasthenincubatedforoneweekanddetectedbyFISHandconfocallaserscanningmicroscopyasdescribedin2.6.2.

Table2.13ListofhoststhatweresubjecttoinfectionwithendosymbiontsHSC3andHSC8.

Host MOI TemperatureAcanthamoebasp.C1 500 20°CAcanthamoebasp.5a2 500 20°CAcanthamoebapolyphaga 500 20°CAcanthamoebacastellaniiNEFF 500 20°CS2cells 500 27°C

2.6.8 IsolationofgenomicDNA

Whole-genome sequencing is a useful tool for understanding the evolution, diversity, physiology andbiology of symbiosis of the endosymbionts described in this study. Many different methods areavailable for isolating the total genomic DNA of bacteria. Choosing the appropriate method needsconsiderationoftheorganismthattheDNAcomesfrom,aswellastheyield,purityandintegrityoftheDNAthatisneededintheendforsubsequentsteps.

In these cases, the bacteria are obligate intracellular endosymbionts of Acanthamoeba spp., whichmeanstheculturesfrequentlycontainlowlevelsofendosymbiontDNA,yetlargeamountsofamoebalDNA.Thustheprocedureacquiresalotoftechnicaladaptationsandprecautions. First of all several 500 cm2polystyrene culture flasks ofwell-grown infectedAcanthamoeba spp.HSCweregatheredinto50mlGreinertubesandcentrifugedat6’600rpmfor5minat4°C.Thepelletswereresuspended incold10mlBufferAcomplementedwithEDTAandhomogenized15timeswithatightDounceHomogenizer.Thelysatewasthenspinneddownat300xgfor2minat4°C.Thesupernatantwasstoredat4°C,whereasthepelletwasresuspendedoncemorein10mlBufferAcomplementedbyEDTAtoincreasetheyieldandhomogenized15timeswiththetightdouncer.Thelysatewascentrifugedat300xgfor2minat4°C.Allsupernatantswerethenpooled,filteredwith5µmandcentrifugedagainat11’000 rpm for5minat4 °C.Thepelletwaswashedwith5ml coldBufferA, centrifugedagainat11’000rpmfor5minat4 °Candresuspended in1mlBufferA.Thesuspensionwas thentransferredinto a new 2 ml Eppendorf tube, where 10 µl DNase 1 (Thermo Scientific) were added. After anincubationof1hat4ºCoronicethedigestionwasstoppedbyadding1/10Vol.0.5MEDTA(100µl)andcentrifuged at 11’000 rpm for 5 min at 4 °C. The pellet was washed once with 1 ml cold Buffer AcomplementedwithEDTA,againcentrifugedat11’000rpmfor5minat4°Candresuspendedin250µl

33

TEbuffer.Thesuspensionwasthenmixedwith675µlofDNAextractionbufferand20µlProteinaseK,before incubating it for 30min at 37°Cby invertingonce after 15min. Then, 75µl of 20%SDSwereaddedandincubatedina65°Cwaterbathfor1hwithgentle inversionsevery15-20min.AftermixingwithanequalvolumeofroomtemperaturePhenol/Chloroform/Isoamylalcohol(25:24:1,v/v;pH>7.8),theEppendorftubewascarefullyinvertedafewtimesthencentrifugedat14’000rpmfor5minatRT.Theaqueousphasewasrecoveredandtransferred intoanewEppendorftube,avoidinganymaterialsfrom the interphase or phenol. This was mixed with an equal volume of room temperatureChloroform/Isoamylalcohol (24:1, v/v) and again carefully inverted a few times. The followingcentrifugationstepat14’000rpmfor5minatRTproducedanotheraqueousphasethatwastransferredoncemoreintonewEppendorftube.A1/10thvolumeof3MSodiumacetateor2MSodiumchlorideand1µlofglycogenwasaddedtothesample.Thenucleicacidswerethenprecipitatedbyadding0.7volumeofisopropanolatroomtemperatureforatleast1huntiltheywerepelletedbycentrifugationat16’000xgfor30minat4°C.Thepelletwaswashedwith500µlice-cold70%EtOHfor5minandspinneddownat14’000 rpm for 10 min at RT. After that, the pellet was air-dried for 15 min, resuspended in 30 µlTris/HClcomplementedwithRNase[1µlRNase(Qiagen,100mg/ml)+1mlTris/HCl]andincubatedfor20minat37°C.

2.6.9 HeLacellinfectionassayTheChlamydiaecomprise thewell-known familyChlamydiaceae that includes a numberof importantpathogens causing human diseases, such as Chlamydia trachomatis and Chlamydia pneumonia.Furthermore,thereisevidenceforapotentialpathogenicityofsomeenvironmentalchlamydiae,suchasSimkania negevensisandWaddlia chondrophilawhich have been shown to infect both amoebae andmammalian cells and associatedwith respiratory andmiscarriage in humans. Even somemembers ofthefamilyofParachlamydiaceaeseemtoohavealimitedcapacitytogrowinanumberofnonprotozoanhost cells (Omsland, 2014). Within the Betaproteobacteria, some important agents of a number ofhuman diseases include the pathogenic Neisseria gonorrhoeae (causing gonorrhea), Neisseriameningitides (the cause ofmeningococcalmeningitis), and the genus Burkholderiawhose pathogenicmembers include Burkholderia mallei (responsible for glanders, mostly in horses), Burkholderiapseudomalleia(causativeagentofmelioidosis)andBurkholderiacepacia(causingpulmonaryinfectionsin people with cystic fibrosis). Ralstonia pickettii is one of the only two known species ofBetaproteobacteria shown to naturally infect free-living amoebae, but also acts as an opportunisticpathogen. The pathogenic role of the second obligate endosymbiont of amoebae Procabacteracanthamoebaislargelyunknown(GilbertGreub&Raoult,2004b).BecauseofthecloserelationoftheendosymbiontsofAcanthamoebaspp.HSC3andHSC8accordingtothephylogenetictreesshownin4.2,tothelistedpathogensitseemedimportanttotesttheirabilitytonotonlyinfectamoebalstrains,butalsohumancelllines,suchasHeLa229.TheHeLacellisacelltypeinanimmortalcellline,derivedfromcervicalcancercellsandmostcommonlyusedinscientificresearch.

34

ThefirststepconsistedinharvestingandseedingofHeLa229cells.TheDMEMHeLacellmediumwascarefully removed fromtheculture flasks, then3mlDPBSwasadded towashcellsbutwas removedinstantly. 0.5 ml 1 x TE was added and the cells were incubated for 10 min at 37 °C and 5% CO2.Afterwards, 5mlDMEMwere added, the suspensionwas transferred to50mlGreiner andmixedbypipetting. A small aliquot was used for cell counting with a Neubauer haemocytometer, then aconcentration of 7 x 104 cells per well were transferred onto glass coverslips in a 24-well plate andincubatedforseveralhoursat37°Cuntilthecellswerefullyattached.ThenthemediumwasremovedandfreshDMEMwasadded.

FortheinfectionofHeLa229cells,extracellularHSC3andHSC8respectivelywereharvestedfromwellgrowncultures, filteredwith1.2µmor5µm,resuspended in5-10mlDMEMthenaddedtotheHeLacells.The24-wellplatewasspinneddownfor15minat37°Cfollowedbyanincubationof24hat37°C,5%CO2.

AfixationoftheinfectedHeLa229cellsfollowed.After24hincubationat37°C,5%CO2themediumwasremovedand1ml4%PFAwasaddedtofixthecellsduring1houratRT.Then4%PFAwasremovedagainandthewellswerewashedwith1mlDPBS.Theglasscoverslipsweretransferredtoanew24-wellplatecontaining1mlDPBS/wellandcouldbestoredat4°C.

The DPBS was removed and an EtOH series was applied, starting with a 0.5 ml 50% EtOH solutionincubatedfor3minatRTandremoved.Then0.5ml80%EtOHwasadded incubatedfor3minatRT,removedandfinally0.5ml96%EtOHwasadded,incubatedfor3minatRT,removedagainbeforethecoverslips were let to dry out completely. For fluorescent in-situ hybridization 200 µl hybridizationbufferand20µlprobewereaddedtoeachwellandtheplatewassealedwithPCRfilm,greentapeandaluminumfoil.Hybridizationtakesplaceinanovenfor3hat46°C.1.5mlpreheatedwashingbuffer(48°C)was added to eachwell of a new 24-well plate, the coverslipswere transferred to thewells andincubatedfor10minat48°C.Thenthecoverslipsweretippedinice-coldMilliQwateranddriedundercompressedair,beforetheywereembeddedupsidedownonglassslidesinmowiol.Theslideswerelettodryovernight.

2.6.10 Growthcomparison

Agrowthcomparisonbetweenendosymbiont-freeAcanthamoebaspp.HSCandinfectedAcanthamoebaspp. HSC was used to study the influence of the endosymbionts on amoebal growth. In parallel alive/deadstainingwithpropidiumiodidewasappliedtoanalyzeapossiblehostcelllysiscausedbytheendosymbionts.

Propidium iodide (PI) binds to DNA by intercalating between the bases with little or no sequencepreferenceand ismembrane impermeant,whichmeans it isgenerallyexcludedfromviablecells.PI iscommonlyusedforidentifyingdeadcells inapopulation, inthiscaseasaparameterforhostcell lysis

35

(Riccardi&Nicoletti,2006).Duetoitssufficientlylargeshift,itallowssimultaneousdetectionofnuclearDNA,forexamplebyDAPIstainingtargetingthetotalamountofDNA.

First of all, endosymbiont-free Acanthamoeba spp. HSC as well as infected Acanthamoeba spp. HSCwere collected for a growth comparison, starting with a centrifugation step at 6’000 rpm for 6min,takenup in10mlTSYandanamountof105cellsperwellweredistributedintoa12-wellplate(threewellspertimepoint).

Figure2.4AnexperimentalsetupforthecomparisonofHSC8orHSC3infectedAcanthamoebaspp.andtheiruninfected originalAcanthamoeba spp.hosts bymaking growth curves andmeasuring the lyticactivityoftheendosymbionts.

After 2 h, 24 h, 48 h and 72 h incubation at 20 °C in 2 ml TSY, amoebae were harvested in 2 mlEppendorftubes.AsmallamountwasusedforquantificationwithaNeubauerhaemocytometer,whilethe remaining cells were stained with propidium iodide (PI) for live/dead differentiation. First, thesampleswerespundownusinganEppendorf5804Rmicrocentrifugeat6’000rpmfor6minatRT.Thesupernatantwasdecantedandthepelletresuspendedin100µl1xPASwith1µMPIsolutionpriortoincubation for 20 min in the dark at RT. For additional quantification of the total DNA, 10 µl of a1:10’000dilutionofDAPIsolutionwasaddedtogetherwiththePIsolution.Afteranothercentrifugationstepat6’000rpmfor6minatRTthepelletwasresuspendedin150µl1xPASandtransferredtoablack96-well plate readerdish. ThePI andDAPI fluorescence intensitywasmeasuredwith a Tecan InfiniteM200microplatereaderaccordingtothefollowingprotocol.

36

Table2.14ProtocolforthemeasuringofPIandDAPIfluorescenceintensity.

Propidiumiodide DAPIShaking 5s 5sFluorescencetopreading withoutlid withoutlidGain 100 125Reads 4readsperwell 4readsperwellExcitationwavelength Aexc=535nm Aexc=350nmEmissionwavelength Aem=617nm

Aem=470nm

2.6.11 Infectioncycles

To analyze the endosymbiont’s infection cycle, emptyAcanthamoeba castellanii NEFF as well as theoriginalAcanthamoebaspp.HSChostwerecollectedbycentrifugationat6’000rpmfor6min,takenupin10mlTSYandanamountof105cells/wellwerefilledintoa12-wellplate(threewellspertimepoint).ExtracellularHSC8werefilteredfromthesupernatantofanAcanthamoebaspp.culturewith2x5µmsyringe filters, and then centrifuged at 7’500 rpm for 6 min. The pellet was resuspended in a smallvolumeoffreshculturemediumTSYofwhich10µlweredissolvedinPASandstainedwith500µlDAPI(dilution1:5’000)forquantificationanddeterminationofMOI(averagecellcountx15’126xDilution=cells/ml).ThesameprocedurewasdonewithextracellularHSC3,excepttheywere isolatedwith1x5µmand1x1.2µmsyringefilters,centrifugationstepsweredoneat12’800xgfor10min.ExtracellularHSC8orHSC3 respectivelywereadded toendosymbionts-freeAcanthamoebaspp.atamultiplicityofinfection of 50, incubated for 15min at RT followedby centrifugation at 130 x g during 15min. Theinfectionwasthenincubatedat21°Cfor2h,washedwithPASandfreshculturemediumwasadded.Cellswere fixedontomicroscopeslidesafter2h,8h,24h,48h,72hand96h incubationusing4%formaldehyde, followed by awashing stepwithMilliQ. Cellswere visualized by FISH using either theHSC8-specific probe or the Chlamydia-specific probe. Amoebae were counted by distinguishing fullyinfectedcells(>30bacteria),intermediateinfectedcells(6-30bacteria),lowinfectedcells(1-6bacteria)anduninfectedcells.

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Figure2.5AnalysisoftheinfectioncyclesoftheendosymbiontsofAcanthamoebaspp.HSC8andHSC3inuninfectedAcanthamoebaspp.hosts.

2.6.12 ExtracellularinfectivityAssay

The endosymbiont ofAcanthamoeba sp.HSC8 from simple observation seems to be a novel obligateintracellularbacteriumoffree-livingamoebae.InordertodemonstrateasomehowlimitedextracellularsurvivalandmaintenanceofinfectivityintherichmediaTSY,thefollowingassaywasplanned.

ExtracellularHSC8wereharvestedfromahostcellcultureandfilteredtwicewith5µmsyringefilters,thencollectbycentrifugationat7’500rpmfor6min.ThepelletwasresuspendedinasmallvolumeoffreshTSYofwhich10µlofthesuspensionwasdilutedin5mlPBSforquantificationwithDAPI(500µlofa 1:5000 dilution in PBS). The bacterial suspension was then transferred into 12-well plates forextracellular incubation at 20 °C, at a finalmultiplicity of infection of 50. In order to investigate thesurvivalandmaintenanceofinfectivity,emptyAcanthamoebasp.HSCwereaddedinanamountof105

cells per well after 2 h, 24 h, 48 h, 72 h and 96 h. The amoebae were fixed then after 48 h ontomicroscopeslidesusing4%formaldehyde,followedbyawashingstepwithMilliQ.CellswerevisualizedbyFISHusingaHSC8-specificprobe.Amoebaewerecountedbydistinguishingfullyinfectedcells(>30

38

bacteria), intermediate infected cells (6-30 bacteria), low infected cells (1-6 bacteria) and uninfectedcells.

Figure2.6Experimentalset-ups fortheanalysisofhost-freesurvivalandmaintenanceof infectivityoftheendosymbiontofAcanthamoebasp.HSC8,bycalculatingtheinfectivityratesofdifferentincubationtimes.

2.6.13 GlucoseAssay

The endosymbiont of Acanthamoeba sp. HSC3 as is shown later in the phylogenetic tree under 4.2clusters together with Protochlamydia acanthamoebaeUWE25within the family Parachlamydiaceae.Protochlamydia acanthamoebaeUWE25 is one of the better studied environmental chlamydiae andformerfindingsindicatethatProtochlamydiaacanthamoebaeUWE25EBsdependontheavailabilityofD-glucose in a host-free environment that is converted to D-glucose-6-phosphate by a self-encodedglucokinase (Sixt et al., 2013). To investigate whether the metabolic activities of the closely relatedendosymbiontofAcanthamoebasp.HSC3EBsactinasimilarmanneranassayforsubstrateavailabilitywasapplied.

39

TheendosymbiontHSC3washarvested fromhost cell cultureand filteredwith1.2µmsyringe filters,thencollectedbycentrifugationat12’800xg for10min.Thepelletwaswashedwith20mlPBSandresuspendedinasmallvolumeofPBS.10µlofthesuspensionwasdilutedin5mlPBSforquantificationwithDAPI(500µlofa1:5000dilutioninPBS).Thebacterialsuspensionwasthentransferredinto1.5mlEppendorf tubes for parallel host-free incubation in triplicates of differentmedia. To investigate theimportanceofnutrientavailabilityforHSC3’shost-freesurvival,thesemediaincludedthenutrient-richDGM-D containing D-glucose as a supplement, a modified medium DGM-L, in which D-glucose isreplacedbyL-glucoseaswellasanutrient-freesaltsolution0.6%NaCl(0.1Mor100mM)andabufferPBSofsamepHandosmolarity.Allofthemwereincubatedat27°Cand90rpmfor2h,48h,94h,and168hrespectively,thenaddedto105amoebaein24-wellplateatMOI=5containing1mlTSYmedium.

Theplatewasincubatedfor15minat27°Cfollowedbyspinningdownat130xgfor15minat23°C.Fixationhappenedafter48hofincubationat27°Cusing4%formaldehyde,followedbyawashingstepwith MilliQ. Cells were visualized by FISH using the Chlamydiae-specific probe Chls-0523. Infectedamoebae containing at least six intracellular bacteria were counted (~600 cells per sample) andinfectivity was expressed relative to the infectivity of bacteria incubated for 2 h in DGM-D.

40

Figure2.7Experimentalset-upsfortheanalysisofhost-freesurvivalandthemaintenanceofinfectivityoftheendosymbiontofAcanthamoebasp.HSC3inthepresenceorabsenceofdifferentnutrients.

41

3. Results

3.1 Isolationoffree-livingamoebaeusingnon-nutrientagarplatesseededwithGram-negativefoodbacteriaandaxeniccultivation

ThetwoenvironmentalsamplesanalyzedinthisstudywereagreencoloredbiofilmofaHawaiianseacavewallandasamplefromaHawaiiancoralsandbeach. Intotal15amoebaecultureswere isolatedfromthebiofilmof the littoralcave. Inorder to includepossiblegrowthpreferencesofamoebae intothestudy,wechosetwodistinct incubationtemperatures.SixamoebaecultureswereincubatedatRTandnineculturesweregrownat27°C.ThreeoftheRTcultureswereaxenizedsuccessfullytheother3were no well-grown cultures. Two of the three axenized cultures harbored endosymbionts.Simultaneously, sevenout of nine amoebae cultures grown at 27 °Cwere axenized and twoof themcarriedintracellularbacteria.Noamoebaewereisolatedfromthesampleofcoralsand.

Table3.1The isolationofamoebaefromtwoenvironmentalsamples,axenizationandthefollowingdetectionofendosymbionts.Intotal15amoebaecultureswereisolatedfromthebiofilmofthelittoralcaveofwhichfourwereshowntocarryintracellularbacteria.

EnvironmentalSample

IncubationT(°C) Amoebaecultures Axenization Infection

I–Littoralcave RT 3unknown1uninfectedamoebaeculture2cultureswithendosymbionts

-++

--+

27°C 2unknown5uninfectedamoebaecultures2cultureswithendosymbionts

-++

--+

II–Coralsandbeach RT Noculture - - 27°C Noculture - -

42

3.2 IdentificationofnovelendosymbiontsofAcanthamoebaspp.

3.2.1 Fluorescencein-situhybridizationresults

Forafirstinsightintothepresenceofendosymbiontsallamoebaeculturesweresubjecttoascreeningby Fluorescence in-situ hybridization. This method gives then additional information about themorphology,sizeandahintofintracellularlocationofbacteriaaswellastheamoebahosts.

A B

C D

E F

43

Figure 3.1 (A-B) Isolates HSC3 hybridized with Chlamydiales specific probes in Cy3, Chls-0523. (C-D)IsolatesHSC6,hybridizedwithprobesthatarespecific forRickettsiales-relatedmicroorganisms inCy3,AcRic90 and Rick1395. (E-F) Isolates HSC8 and HSC9 hybridizedwith EUB338-mix in FLUOS, targetingmostbacteria.

InFigure3.1,thedifferentendosymbiontsareshowninsidetheirnaturalAcanthamoebasp.HSChost.ConfocallaserscanningmicroscopicanalysisrevealedthatAcanthamoebasp.HSCtrophozoitesarefullyinfectedwithcoccoid,chlamydia-likeendosymbiontsthatresideinsingle-cellinclusionvacuoles(figure3.1AandB).Twomorphological formscouldbedifferentiated;anextracellularstagewhich is slightlysmaller than the intracellular one of about 0.5-0.7 µm. Figure 3.1 C and D show the identity andintracellularlocationofasecondisolateusingFISHincombinationwithlaserscanningmicroscopy.Theendosymbiontsweremoreorlessrod-shaped,cytoplasmicandvariedinsize.Athirdamoebaeculturelabelledwith FISH and subsequently analyzedwith confocal laser scanningmicroscopywas shown toharborrod-shapedbacteriaspreadevenlythroughoutthecytoplasmofitsAcanthamoebasp.HSChost.Thetrophozoiteswerefullyinfectedandadditionallyplentyofextracellularbacteriacouldbedetectedinthemedium.(figure3.1EandF)

3.2.2 16S/18SrRNAgenesequencingresultsofamoebaculturesharboringendosymbionts

There were several amoebae isolated from the Non-nutrient agar plates, harboring differentintracellularbacteria.Firstofall,theendosymbiontofAcanthamoebasp.HSC3wasidentifiedasacloserelativeoftheendosymbiontofAcanthamoebasp.UWE1withasequencesimilarityof99%andbelongsto the order Chlamydiales. It was isolated from theAcanthamoeba sp. UWE1 from a soil sample inwesternWashingtonState.Detailsare indicatedinTable3.2.Thebestblasthitaccordingto16SrRNAgenesequencingoftheendosymbiontofAcanthamoebasp.HSC6wasshowntobetheendosymbiontofAcanthamoebasp.UWE8aswellastheAcanthamoebasp.UWC36withamaximumidentityof99%.Both isolates were recovered from the Acanthamoeba sp. UWC8 and Acanthamoeba sp. UWC36respectively found in infectedhumancorneal tissuesandassigned to theorderRickettsiales. The tworod-shapedendosymbiontsofAcanthamoebasp.HSC8andHSC9werefoundtobeidenticalwith100%sequence similarity and as-yet unknown and uncultured bacteria. According to the 16S rRNA genesequencingbothclusterinthesuborderBetaproteobacteriaandthebestblasthitwasgivenwith94%sequencesimilarityasunculturedproteobacteriumclonessf-42andsf-25.Table3.2givesthedetailedinformation of all best blast hits for the endosymbionts ofAcanthamoeba sp. HSC3,HSC6,HSC8 andHSC9,suchasthemaximumidentity,accessionnumber,habitatandreference.

C

E

GIK

DK

44

Table3.2Isolated,identifiedendosymbiontsby16Sor18SrRNAgenesequencing.

Isolate Bestblasthitbasedon16SrRNAgenesequencing

Max.Identity

Accessionnumber

Habitat Reference

HSC3 EndosymbiontofAcanthamoebasp.UWE1

99% AF083614 Acanthamoebasp.UWE1soil,westernWashingtonState

Fritscheetal.(2000)

HSC6 EndosymbiontofAcanthamoebasp.UWC8EndosymbiontofAcanthamoebasp.UWC36

99%99%

AF069963AF069962

Acanthamoebasp.UWC8humancornealtissueAcanthamoebasp.UWC36humancornealtissue

Fritscheetal.(1999)Fritscheetal.(1999)

HSC8 Unculturedbetaproteobacteriumclonesf-42Unculturedbetaproteobacteriumclonesf-25Unculturedbetaproteobacteriumclone26-2_44Unculturedbetaproteobacteriumclone14-1_27

94%94%92%92%

JQ278953JQ278943FJ517704FJ517688

groundwatergroundwaterepitheliumofhydraepitheliumofhydra

UnpublishedUnpublishedFrauneetal.(2009)Frauneetal.(2009)

HSC9* Unculturedbetaproteobacteriumclonesf-42Unculturedbetaproteobacteriumclonesf-25Unculturedbetaproteobacteriumclone26-2_44Unculturedbetaproteobacteriumclone14-1_27

94%94%92%92%

JQ278953JQ278943FJ517704FJ517688

groundwatergroundwaterepitheliumofhydraepitheliumofhydra

UnpublishedUnpublishedFrauneetal.(2009)Frauneetal.(2009)

*Sequencedwithonlyforwardprimer

ThenativeamoebaehostsoftheendosymbiontsHSC3,HSC6,HSC8andHSC9werefoundtobeidenticalwithasequencesimilarityof100%.Basedon18SrRNAgenesequencingthebestblasthitwasgivenwith100%maximumidentityasAcanthamoebasp.KA/MSG18andKA/MSG10.

Table3.3Isolated,identifiedhostsbasedon18SrRNAgenesequencing.

Isolate Bestblasthitbasedon18SrRNAgenesequencing

Max.Identity

Accessionnumber

Habitat Reference

HSC Acanthamoebasp.KA/MSG18Acanthamoebasp.KA/MSG10

100%100%

AY173009AY173005

MarineSedimentMarineSediment

UnpublishedUnpublished

45

3.3 Phylogeneticanalysis

Comparative sequence analysis using maximum-likelihood and neighbor joining revealed thephylogenetic relationships of the endosymbiont isolates to other bacteria. The endosymbiont ofAcanthamoeba sp. HSC3 showed 99 % sequence identity with each other and were classified asmembers of the Chlamydiales order. Blast search revealed a 99 % sequence similarity with theendosymbiontofAcanthamoebasp.UWE1isolatedfromasoilsamplefromwesternWashingtonState(Fritscheetal.,2000).PhylogeneticanalysisconfirmedthatthestrainsclusteredwithothermembersoftheChlamydialesandmoreoverformedalineagewiththeendosymbiontofAcanthamoebasp.UWE1,Parachlamydiaceae bacterium CRIB38 andmore distantly Ca.Metachlamydia lacustris,NeochlamydiahartmannellaeandNeochlamydiasp.CRIB37.

Figure 3.2 16S rRNA based phylogenetic tree of the endosymbiont of Acanthamoeba sp. HSC3. Amaximum-likelihood and neighbor-joining dendrogram shows phylogenetic relationships of thesequencedendosymbiontofAcanthamoebasp.HSC3toothermembersoftheChlamydiales.

46

TheendosymbiontofAcanthamoebasp.HSC6appearstoberelatedtomembersofthealphasubclassof Proteobacteria, among which the closest neighbors are the endosymbiont of Acanthamoeba sp.UWC8andtheendosymbiontofAcanthamoebasp.UWC36withasequenceidentityof99%,aswellasRickettsiaaustralis,RickettsiasibiricaandRickettsiatyphi(Fritscheetal.,1999).Furtheranalysisrevealsthat the endosymbiont ofAcanthamoeba sp. HSC6 form an independent, distinct lineage within theRickettsiales,togetherwiththeUWC8andUWC36endosymbionts.

ComparativesequenceanalysisrevealsthatthetwoendosymbiontofAcanthamoebasp.HSC8andHSC9were almost identical with a sequence identity of 99% and assembled with members of theBetaproteobacteria.When taking a closer look the isolatedoesnotonly showclose relationshipwithmembers of Procabacteriaceae, Nitrosomonadaceae and Neisseriaceae but formed a distinct lineagewith a number of uncultured Betaproteobacteria among which the closest relative has a sequencesimilarityof94%.Thereforethis isolatemight representanovelspeciesandmost likelyevenanovelfamilywithintheBetaproteobacteria.

Figure3.3 ‘EndosymbiontofAcanthamoeba sp.HSC8’.16S rRNAbasedphylogenetic relationshipsofthe‘endosymbiontofAcanthamoebasp.HSC8’toothermembersoftheBetaproteobacteria.

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3.4 HostrangeoftheendosymbiontsHSC3andHSC8

3.4.1 CuringofthenaturalAcanthamoebasp.HSChost

TheoriginalAcanthamoebasp.HSChostwassuccessfullycuredfromitsendosymbiontsHSC3andHSC8respectively. Cultures were cured from HSC8 with Rifampicin and Kanamycin, whereas HSC3 waseliminatedbyDoxycyclinwithoutharmingtheamoebahosts.

Table3.4.ListofAntibiotics thatsuccessfully freedacanthamoebacultures fromendosymbiontsHSC3andHSC8respectively.

Targetorganism

Antibiotic Mechanism Finalconcentration

Successfulcuring

Observations

HSC8 Ampicillin Inhibitionoftranspeptidaseneededforcellwallsynthesis

50,70,100ng/ml No Nocuring

Kanamycin Interactionwithbacterial30SRSUandinhibitionoftranslocation

50,70,100ng/ml Yes Amoebaeinverygoodcondition

Rifampicin InhibitionofbacterialDNA-dependentRNA-polymerase(RNAsynthesis)

100ng/ml Yes Amoebaeingoodconditionbutseveralreinfectionsobserved

HSC3 Tetracyclin Bindingofbacterial30SRSUandblockingofchargedaminoacyl-tRNA

50,70,100ng/ml No Nocuring

Doxycyclin Bindingofbacterial30SRSUandAminoacyl-tRNA

50,70,100ng/ml Yes Amoebaeingoodcondition

The uninfected culture is represented in the image made with confocal laser scanning microscope(figure 3.4). The blue Cy5 signal was the only one detected, showing the typical acanthamoebatrophozoitemorphology. The absences of FLUOS or Cy3 signals indicate that all endosymbiontswereeliminatedfromtheamoebaeculture.

48

Figure 3.4 Cured natural Acanthamoeba sp. HSC host of endosymbiont HSC8. The bacteria wereeliminated by the antibiotic Rifampicin and the amoebaewere visualizedwith FISH using the probesHSC8-Cy3,EUB338-FluosandAcanth412a-Cy5.

3.4.2 InfectionofdifferentAcanthamoebaspp.,humanandinsectcelllines

In order to evaluate the potential of the endosymbiont of Acanthamoeba sp. HSC8 and theendosymbiontofAcanthamoebasp.HSC3toinfectcellsotherthantheoriginalAcanthamoebaspp.HSChost, purified bacteria were added to various Acanthamoeba strains and cell lines. All fourAcanthamoebaspp.listedintable3.5weresusceptibletobothsymbionts,andreachedaninfectionrateof100%aftersevendays.Incontrast,thehumanHeLa229celllineandtheDrosophilaSchneider2(S2)cellscouldnotbepermanentlyinfectedbyeitherofthem.

Table3.5InfectionofvariousAcanthamoebaspp.,humanandinsectcelllines.

Host EndosymbiontofAcanthamoebasp.HSC3

EndosymbiontofAcanthamoebasp.HSC8

Acanthamoebasp.C1 + +Acanthamoebasp.5a2 + +Acanthamoebapolyphaga + +AcanthamoebacastellaniiNEFF + +HeLa229cells - -S2cells - -

49

3.5 CharacterizationofEndosymbiontofAcanthamoebasp.HSC3

3.5.1 TheendosymbiontHSC3hasabiphasicinfectioncycle

Fluorescenceinsituhybridizationandphasecontrastmicroscopygaveinsightsintotheinfectioncycleofthe chlamydia endosymbiont HSC3 was in its natural Acanthamoeba sp. HSC host as well as inAcanthamoeba castellanii NEFF. Cell morphologies, infection levels and metabolic activity of theendosymbiont HSC3 were illuminated by these synchronized infection assays. In a second part, theinfectionratesofHSC3initsoriginalhostwascomparedtotheinfectionlevelsofHSC3inthelaboratorystrainA.castellaniiinasecondexperimentalsetup.Atdifferenttimepointsbeforeandafterinfection,the overall cell condition was determined visually. Most cells were attached to the surface and introphozoitecellstage,indicatingwellgrownamoebacultures.

Aswe distinguished low infected, intermediate infected and fully infectedAcanthamoeba spp., thesenumbers were expressed in relation to each other as well as to the number of uninfectedAcanthamoeba spp. The infection ratewas calculated for both amoebae cultures and at several timepoints after infection by counting all infected amoebae and dividing it by the total number of cellsincluding the uninfected amoebae. The average number of counted uninfected, low infected,intermediate infected and fully infected amoebae as well as the infection rate throughout one fullinfectioncycleisillustratedinfigure.TheinfectionrateofHSC3inAcanthamoebacastellaniiNEFFwasthencomparedtotheinfectionlevelsintheoriginalAcanthamoebasp.HSChost.

TheinfectionofHSC3inAcanthamoebaesp.HSCstartedwithapercentageof42.7%ofwhichmostlyallamoebaewereonlylowinfectedat2hpi.Only4.3%oftheamoebaecontainedmorethan5particles.AtthisearlytimepointtheparticleswerehardlydetectablebyFISH,buttheDAPIsignalsshowedsmallcoccoidbacteriaveryclosetothehostsoutermembrane.After24hmostFISHsignalscorrelatedwiththeDAPIsignalsandthemorphologyoftheparticlesswitchedtoslightlybiggercoccoid-shapedcells.Atlater timepointsafter48hpia largernumberofamoebaewere fully infectedwithchlamydiae.Upto40.4%of thehostsharboredover30particlesseeminglydistributed throughout thecell in singlecellinclusions.Additionallytheproportionofinfectedamoebaeslowlyincreasedupto63.5%.Theinfectioncycle seemed to be complete after 96 hpi, indicated by 100% full infection and a larger number ofparticlesintheamoebaesurroundingmedium.

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Figure3.5ThecourseofinfectionoftheendosymbiontHSC3indifferentAcanthameobaspp.strains.PercentageofamoebaecontainingHSC3over96hourspostinfection.Green:Noinfectionmeaningthatno bacteria had infected a host cell. Orange: Low infection, 1-5 symbionts have entered a cell. Red:Intermediate infection, 5-30 cells per amoebaewereobserved. Black: Full infection for > 30 cells perhost. (A) The infection cycle of HSC3 in the original hostAcanthamoeba sp. HSC. The number of ofinfected cells increased over time and 96 hours post infection (hpi) all acanthamoebae were fullyinfectedwiththeendosymbiontHSC3.(B)TheinfectioncycleofHSC3inAcanthamoebacastellaniiNEFF.TheinfectionrateinA,castellaniiNEFFstartedlow,only10%ofacanthamoebaewereinfectedafter3to8hourspostinfection,butincreasedovertimeuntilthecycleendsbetween72hpiand96hpiwitha100%infectionrate.(C)TheinfectionratesofendosymbiontHSC3inA.castellaniiNEFFcomparedtoitsoriginalhost,Acanthamoebasp.HSC.throughoutoneroundoftheinfectioncycle.White:HSCforthe

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infectioninAcanthamoebasp.HSC;black:NEFFfortheinfectioninA.castellaniiNEFF.At2-3hpiHSC3waspresentinin40%ofallAcanthamoebasp.HSCwhereasonly10%ofAcanthamoebacastellaniiNEFFwereinfectedwhenthesamemultiplicityofinfectionwasapplied.After24hpitheinfectionlevelswerehigherA.castellaniiNEFFcomparedtoAcanthamoebasp.HSC.Only60%ofAcanthamoebasp.HSCwasfully populatedat 48hpi in comparison to the95% infection rate inA. castellaniiNEFF.Neverthelessbothstrainsreachaninfectionrateof100%after96hpi.

In contrast to the infectionofHSC3 in thenaturalAcanthamoeba sp.HSC, the startingpercentageofinfectedAcanthamoebacastellaniiNEFFat3hpiseemedmuchlowerwith7.3%.Mostofallamoebaecontainedsingleparticlesorrarelymorethanfivechlamydiae.Attheearlytimepointsafter2-8hpi,itseemed even harder to detect the infectious particles by FISH, than it was in the study ofAcanthamoebae sp. HSC. After 24 h most FISH signals correlated with the DAPI signals and showedslightlybiggercoccoidcellmorphology.Nowtheproportionofinfectedamoebaerapidlyincreasedupto78.5%ofwhich50.7%of theamoebaecontainedup to30particles.Thepercentagewas still slowlyincreasing after 48 hpi with now 94.7 % infected amoebae, most harboring over 30 particles (69.7%).The chlamydia seem tobewidelydistributed throughout theamoebahostandorganized in singlecellinclusions.The100%fullinfectionwasreachedbetween72and96hpi,indicatingcompletionoftheinfectioncycleinA.castellaniiNEFF.Thebacteriawerereleasedintothesurroundingmedium.

The evaluation of the endosymbiont ofAcanthamoeba sp. HSC3 infection cycle inAcanthamoeba sp.HSCaswellasinAcanthamoebacastellaniiNEFFusingFISHandDAPIstaining,suggeststhatthebacteriaaretakenupafter2-3hourspostinfection.AsisknownforEBs,theirdetectionbyFISHisweak,duetolowmetabolicactivityand therefore less rRNAtargetmolecules.As theamountofEBs thatenter thehostsatthefirsttimepointsislow,especiallyintheinfectioncycleinA.castellaniiNEFF,thisindicatesthatmostbacterialparticleswerestillEBs.After24hmostchlamydiaeseemedtohaveswitchedfromEBs to the more active RBs, as the FISH signals mainly correlated with the DAPI signals. A longerreplicativephasefollowedthatlasteduntil72hpiwithanincreaseinnumbersfromfourto30bacteriaperamoeba.Acanthamoebaspp.werefullyinfectedwithHSC3after96hpi,whichwerethenreleasedintotheenvironmentfornewinfections.

3.5.2 UninfectedAcanthamoebasp.HSCshowfastergrowthincomparisontoacanthamoebaeharbouringtheendosymbiontHSC3

Theinfluenceofanendosymbiontonamoebalgrowthwasanalyzedbyphasecontrastmicroscopyandbycalculatingthetotalcellnumberineachwell.Theoverallcellconditionwasdeterminedvisuallyaftereachtimepoint, inorder tobesureofwellgrowncultureswheremostamoebaeareattachedto thesurface and in trophozoite cell stage. To avoid bias in cell countingwe detached all amoebae,mixedthemcarefullyandonlyusedasmallamountofthesubstrateforcounting.Asiscommonlyrecognized

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amoebae have amobile lifestyle, resulting in an unequal distribution throughout thewells. A higherdensitycanoftenbeobservedinthecentercomparedtotheareasneartheborder.

The average number of counted uninfected and infected amoebae are represented in figure andcomparedtoeachother.TheuninfectedacanthamoebaegrewmuchfasterthantheonesinfectedwiththechlamydiaendosymbiontHSC3.Asignificantdifferenceincellnumberscouldfirstbeobservedat48hpswithuninfectedamoebaedisplayinghighernumbers.Acanthamoebasp.HSCculturesinfectedwithHSC3startedgrowingfrom54’167cellsupto81’300cellsinaverage,whichindicatesthattheykeepthegrowth rate relatively constant over time. In comparison, the uninfected Acanthamoeba sp. HSCculturesgrowexponentiallyandreachtheten-foldamountofcellsthreedaysafterseeding.

Tofurtherdefinethedifferenceinacanthamoebaereplicationrate,theduplicationtimeforuninfectedaswellasfor infectedamoebaewerecalculatedandrepresentedintable.Uninfectedacanthamoebaedoubled their numbers afteronly 9hourspost seeding,which is very fast compared to the amoebaeculturesharbouringHSC3.Inaverage,theirduplicationtimewaslaterthan72hours,notrepresentedinthis study. Table3.7 shows theaverageduplication timeofuninfectedand infectedacanthamoebae.Moreover,emptyAcanthamoebasp.HSCofthefirstrundisplayedthehighestnumberofcellsintotal.

Figure3.6Growthofuninfectedandinfectedacanthamoebae.Thecurvesillustrateaveragevaluesfortwoseparateruns.White:firstandsecondrunofamoebacultures infectedwithHSC3.Black:firstrunand second runofemptycontrols. Thegrowthofuninfectedamoebaewas faster than thegrowthofamoebaeinfectedwithHSC3.

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Table3.6TheequationandR2valueofthetrendlineofeachcultureinfigure.

Equation R²value

UninfectedHSC_Run1 y=61994e0.033x R²=0.9946

UninfectedHSC_Run2 y=53287e0.0294x R²=0.948

InfectedHSC_Run1 y=64534e0.0032x R²=0.7217

InfectedHSC_Run2 y=46026e0.009x R²=0.7171

Table 3.7 Duplication time of uninfected and infected amoebae in the first and second runrespectively.Theduplication timeofuninfectedacanthamoebae rangedaround20hpost seeding. Incontrast, acanthamoebae that harbor the endosymbiont HSC3 need at least 61.4 h post seeding toduplicatetheirnumber.

Run_1 Run_2

UninfectedHSC 20.9 20.3

InfectedHSC 234.5 61.4

3.5.3 InfluenceoftheendosymbiontHSC3onacanthamoebaviability

ThepresenceofhostcelldeathwithinHSC3infectedAcanthamoebasp.HSCcultureswascomparedtouninfectedAcanthamoebasp.HSCusingpropidiumiodideforvisualizationofdeadcells.NosignificantPI fluorescence intensitypeakhappensthroughoutthecourseoftheexperiment,which indicatesthatnocelllysiswascausedbytheendosymbiontofAcanthamoebasp.HSC3(Figure).Additionally,amoebalgrowthincontinuousculturesdidnotseemtobeinfluencedbythepresenceofHSC3attemperaturesbelow27°C.Theamoebalfitnessisconstantovertimeinbothuninfectedandinfectedacanthamoebaecultures. Nevertheless, a slow increase in PI values was observed in both cultures until 48 hps. Thedifferencesbetweeninfectedamobaeanduninfectedcontrolswereonlysignificantlydivergentatthatvery time point. The trends were rather contradictory after 48 hps, decreasing in the first run andfurtherincreasinginthererunobservedinbothcultures.Forbetterunderstanding,aDAPIstainingwasapplied in parallel, which visualizes the total amount of DNA in the sample. The DAPI fluorescenceintensitymeasuredcouldthenbeputinrelationtothePIfluorescenceintensity,inordertorepresenttherelativemortalityintheculture.ThetotalamountofDNAisconstantovertimeinculturesinfectedwith HSC3, whereas an increase of DAPI fluorescence intensity is observed in uninfected amoebaecultures. Both cultures show a drop after 24 h. nevertheless, the values are very low, indicating aninsignificantdifferencebetweenallcultures,independentoninfectionwithendosymbiontHSC3.

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Figure 3.7Acanthamoeba sp. HSC fitness depending on the presence or absence of the chlamydialendosymbiontHSC3.Thedatarepresentaveragevaluesfortwodifferentruns.White:firstandsecondrun of amoeba cultures infected with HSC3. Black: first run and second run of empty controls (A)Propidium iodide fluorescence intensity measured for infected amoebae cultures and uninfectedcontrols.Theamoebalviabilityisconstantoverthemeasuredtimeinallcultures,independentonHSC3

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infection.(B)DAPIfluorescenceintensityinuninfectedandinfectedamoebacultures.ThetotalamountofDNAisincreasingovertimeinallamoebaculturestested,withhighervaluesforuninfectedculturescompared to cultures containing the endosymbiont HSC3. (C) The mortality rate in uninfected andinfected amoebae cultures. The relativemortality of a culture results from the relation between thepropidiumiodidefluorescenceintensityandtheDAPIfluorescenceintensitydifferentculturesovertime.We observe increasedmortality rates in infected amoebae cultures, but an overall constant relativemortalityovertimeinallcultures.

The relativemortalitywas calculated for eachuninfectedand infectedamoebae culture and for eachincubationperiodasthetotalnumberofdeadcellsdividedbytheinitialamountofDNA.Nosignificantincrease inrelativemortalitywasobservedovertime inallcultures.Althoughthevalueswereslightlyhigherintheinfectedcultures,therelativemortalityratesbasedonthesemeasurementsofPIandDAPIfluorescenceintensitiesmaybebiasedandthedifferencecannotbedescribedassignificant.

3.5.4 Influenceofnutrientavailabilityoninfectivity

The chlamydial endosymbiontofAcanthamoeba sp.HSC3were incubated in theabsenceofhosts forseveral days in different nutrient-rich and nutrient-free media (2.6.11). After infection of emptyAcanthamoeba sp. HSC with host-free HSC3 at defined time points, the infectivity, relative to thatobserved for 2 h incubation in DGM-D, was calculated. The average values for relative infectivity ofeverygrowthconditionareillustratedinfigureandcomparedtoeachother.

Ingeneral,theamountofinfectiouschlamydiaappearedtodeclineovertimeinallfourmediathatweretested.Infact,undertheappliedincubationandinfectionconditionstheinfectionratewasreducedtoanaverageof30%afterahost-freeperiodof7days.Theinitialinfectivity,observedafter2hincubationrangedaroundthe100%infection,exceptforthechlamydiaeincubatedinsaltsolutiononlyinfected80%oftheamoebae.Thenarapidincreasefollowedafter2daysandthetrendoftheinfectivitycurvewassimilartoallothertestedmedia,butthevaluesstayedhigher.Thesefindingssuggestthatdespitethelackofessentialsubstrateschlamydialsurvivalandinfectivitywasmaintainedoveralongperiodoftime.

To further determine the differences between the courses of infectivity in the fourmedia, the exacttime point where the relative infectivity of the chlamydiae declined to 50 %, 25 % and 0 %, wascalculated using the equations from table. The relative infectivity of chlamydiae incubated in DGM-Lbegan to decline between 48 h and 96 h, reached an infectivity of 50 % after 142 h. Although nomeasurementsweredoneafter168h,theequationgaveanapproximatetimepointof180.5h,wherenoinfectivitywouldbeobserved.TheinfectivityofchlamydiaeincubatedinDGM-Dseemedtoslightlyincreaseatthestart,butthensankdownto50%after155.4huntilafter186hnoamoebaewouldbeleftinfected.Thenutrient-freebufferPBSshowedaratherconstantinfectivityduringthefirstfourdays.But then decreased and 50 % infected amoebae remained after 157 h and no infectivity would be

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detectedafter196h.Thehighestvalueswereobservedforchlamydiae incubated innutrient-freesaltsolution. Starting with lower values compared to all other chlamydiae, a rapid and striking increasereached a relative infectivity of 140 %, which declined only after 96 h. Seven days of host-freeincubationdidnotsinklowerthantheinitialinfectivity.Accordingtotheequationofthetrendlinetherelative infectivitywouldbezeroafterahost-free incubationof212.5h,whichmeansthechlamydiaeapproximatelysurvivefor9daysinnutrient-freesaltsolution.

Table3.8TheequationandR2valueofthetrendlineofeachgrowthmediainfigure.

Equation R²value

DGM-D y=-7E-05x2+0.0077x+0.9934 R²=0.9981

DGM-L y=-5E-05x2+0.0031x+1.0703 R²=0.9971

PBS y=-5E-05x2+0.0049x+0.9638 R²=0.9724

NaCl y=-9E-05x2+0.0154x+0.7919 R²=0.9674

Table 3.9 Hours of host-free incubation needed until a fraction of 50 %. 25 % and 0 % infectivityremained in the culture. The relative infectivity of chlamydiae incubated in DGM-L was the first todecline, followedbychlamydiae inDGM-DandPBS.Thehighestvalueswereobservedforchlamydiaeincubatedinnutrient-freesaltsolution.

Relativeinfectivity

DGM-D DGM-L PBS NaCl

50% 155.4h 142h 157h 188.3h

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0% 186h 180.5h 196h 212.5h

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Figure3.8Influenceofsubstrateavailabilityonmaintenanceofinfectivityafterhost-freeincubation.Relative infectivityofHSC3previously incubatedover aperiodof 7days in the absenceof host cells.Green:HSC3 incubated in the innutrient-richmediumDGM-D,containingD-glucoseasasupplement.Red:chlamydiaeincubatedinamodifiedmedium,containingL-glucose.Black:chlamydiaeincubatedina nutrient-free PBS buffer Blue: HSC3 incubated in nutrient-free salt solution. The figure indicatesaverage relative infectivity values from three biological replicates. The endosymbionts incubated inDGM-D and in salt solution experienced an initial increase of infectivity, whereas the chlamydiae inDGM-LandPBSkeepaconstantinfectivityratefor4days.After96htheinfectivitydeclinedinalltestedmedia. The infectivity of the endosymbionts incubated in DGM-L was the first to drop. The onesincubatedinNaClwerestillhighlyinfectiveafter7daysofhost-freeincubation.

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3.6 EndosymbiontofAcanthamoebasp.HSC8

3.6.1 TheinfectioncycleofendosymbiontHSC8

The infectioncycleof theendosymbiontHSC8 in itsnaturalAcanthamoeba sp.HSChostaswell as inAcanthamoebacastellaniiNEFFwasanalyzedby fluorescence in situhybridizationandphase contrastmicroscopy.Thesesynchronizedinfectionassaysgiveinsightintocellmorphologies,infectionlevelsandmetabolicactivityoftheendosymbiontHSC8.FurthermoretheinfectionrateofendosymbiontHSC8initsoriginalhostwascomparedtotheinfectionlevelsofHSC8inthelaboratorystrainA.castellaniianddifferenceswerepointedout.

Wedistinguished low infected, intermediate infectedand fully infectedAcanthamoebaspp.over timeand expressed the numbers relative to each other as well as to the number of uninfectedAcanthamoeba spp.Atdifferent timepointsbefore andafter infection, theoverall cell conditionwasdetermined visually.Most cellswere attached to the surface and in trophozoite cell stage, indicatingwellgrownamoebacultures.Theinfectionratewascalculatedfortwoamoebaeculturesandatseveraltime points after infection by summing up the low infected, intermediate infected and fully infectedAcanthamoeba spp. divided by the total number of counted cells. The infection rate of HSC8 inAcanthamoebacastellaniiNEFFwasthencomparedtotheinfectionlevelsintheoriginalAcanthamoebasp.HSChost.

TheinfectionofHSC8inAcanthamoebaesp.HSCstartedwithaveryhighpercentageof82.3%at2hpiofwhichmostamoebaewereinfectedwithonetofiveparticles.Only16.7%oftheamoebaecontainedmorethanfiveintracellularbacteria.FISHandDAPIsignalsshowedsmallrod-shapedbacteriainsidethecytoplasm.After 24 hpimost amoebaeharboredbetween five and 30 endosymbiontswith a relativepercentageof 55%and somewerealready fully infected (13.8%). It took themonlyuntil 48hpi forreplicationinsidethecells,soafter48hpiwith96.9%almosteverycellwasfullyinfectedwithHSC8.Thebacteriawereveryevenlyspreadthroughout thecytosolThe infectioncycleseemedtobecompletedbetween48and72hpi,indicatedby100%infectionandalargenumberofextracellularparticlesinthesurroundingmedium.Formoredetail,theinfectionrateofHSC8inAcanthamoebacastellaniiNEFFwasthen compared to the infection levels in the original Acanthamoeba sp. HSC host. The startingpercentageofinfectedAcanthamoebacastellaniiNEFFwassignificantlylowerthaninAcanthamoebasp.HSCwith44.6%.Between3hpiand48hpi,thenumbersofinfectedcellsincreasesto64.1%then78.4%andreachesalmostafullinfectionafteronly48hpiwith99.7%.Attheearlytimepoints26.5%werealreadyintermediatelyinfectedwhereasonly17.2%oftheamoebaeharboredlessthanfivecells.Thenumberoffully infectedcells increasedupto70.6%after48hpiandat72hpiallcellswereinfected,the majority of them containing over 30 endosymbionts. This indicates that the infection cyclecompletesat72hpi.

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Figure 3.9 The course of infection of HSC8 within different Acanthamoeba spp. strains. (A) Theinfection cycle of HSC8 in the original Acanthamoeba sp. HSC host. The number of of infected cellsincreasedovertimeandafter48hourspostinfection(hpi)theendosymbiontHSC8reachedaninfectionrateof100%. (B)The infection cycleofendosymbiontHSC8 inA. castellaniiNEFF.The infection rate

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increasedover timeuntil48hourspost infection (hpi)all acanthamoebaewere fully infectedand thecycle ended. (C) The infection rate of endosymbiont HSC8 in A. castellanii NEFF compared to theinfectionrateofHSC8initsoriginalAcanthamoebasp.HSChostthroughoutoneroundoftheinfectioncycle.The infection levels inbothamoebaculturesweresimilar fortheperiodsbetween8hpiand72hpi. At 2-3 hpi HSC8 was present in in 80% of all Acanthamoeba sp. HSC whereas only 40% ofAcanthamoeba castellanii NEFF were infected when the same multiplicity of infection was applied.Neverthelessbothstrainsreachedaninfectionrateof100%after48hpi.

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Figure3.10VisualizationoftheinfectioncycleoftheendosymbiontHSC8inAcanthamoebasp.HSC.Thecourseof infectionwasanalyzedoveraperiodof96handvisualizedwithFISHusing theprobesEUK516 (light blue), a combinationof EUBmix andHSC (orange) and theDNAdyeDAPI (blue). Singlebacteriastartenteringthecellafter2-3hourspostinfection,thenreplicatebetween8hpiand24hpi.After48hpi,amoebaewerefullyinfectedandbacteriawerefinallyreleasedbetween48and72hpi.

In summary, theanalysisof the infectioncycleof theendosymbiontofAcanthamoebasp.HSC8 in itsnaturalAcanthamoebasp.HSChostaswellasinAcanthamoebacastellaniiNEFFusingFISHrevealedaquite similar process. Bacteria enter the cell after 2-3 hours post infection, followed by a replicativephase after 8 hpi. Numbers of bacteria per amoeba increase from four up to 30 observed at 24 hpi.Acanthamoebaspp.were fully infectedwithHSC8after48-72hpi,whichwere then released into theenvironment for new infection. Differences were only observed as early as 2-3 hpi. Starting withinfection rates of 40 % and 80 % after 2-3 hpi, a fully completed cycle was observed after 48 hpiinfectioninbothcultures.

3.6.2 UninfectedAcanthamoebasp.HSCshowfastergrowthincomparisontoacanthamoebaeharbouringtheendosymbiontHSC8

The influence of an endosymbiont HSC8 on amoebal growth was determined by phase contrastmicroscopyandthetotalcellnumberwascalculatedovertime.Atdifferenttimepointsafterseeding,theoverallcellconditionwasdeterminedvisually.Indeedafter2hours,mostcellswereattachedtothesurfaceandintrophozoitecellstage,whichsuggestsgoodgrowthconditions.Astheamoebaearenotequally distributed throughout the wells, but higher density can often be observed in the centercomparedtotheareaneartheborder,wedetachtheamoebaanduseasmallamountofthesubstratetoavoidbiasincellcounting.

The average number of counted uninfected and infected amoebae are represented in figure andcompared to each other. The growth rate of uninfected acanthamoebae was much faster than thereplication time of infected acanthamoebae. A significant difference in cell numbers could first beobservedat48hpswithuninfectedamoebaedisplayinghighernumbers.Theresults indicate that theAcanthamoeba sp. HSC cultures infectedwithHSC8 keep a low, even relatively constant growth rateresultinginanaverageof195’600cellsafter72hincubation.Thetotalcellnumberdoubledaround72hpostseeding,whereastheuninfectedculturegrowsmuchfaster.Thenumbersweredoubledaround24hpostseedingandreachasixfoldmaximumofabout602’000cellsinaverageafterthreedays(Figure3.11).Indeed,theuninfectedamoebacultureofthefirstrunreachedthehighestcellnumbers.

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Table3.10TheequationandR2valueofthetrendlineofeachcultureinfigure.

Equation R² value

Uninfected HSC_Run1 y=88716e0.028x 0.9726

Uninfected HSC_Run2 y=76488e0.0286x 0.9603

Infected HSC_Run1 y=98470e0.0108x 0.9346

Infected HSC_Run2 y=145196e0.0047x 0.6423

Tofurtherdefinethedifferenceinacanthamoebaereplicationrate,theduplicationtimeforuninfectedaswellasforinfectedamoebaewerecalculatedwiththeequationsofthetrendlinesfromtable3.10.Table3.11indicatesthecalculatedduplicationtime.

Figure 3.11 Amoeba growth in the absence and presence of endosymbiont HSC8. Total cell countswerecalculatedforanincubationperiodof72hours.Everyinfectiontypeisillustratedasaveragevaluesfor twodifferentruns.White: firstandsecondrunofamoebacultures infectedwithHSC8.Black: firstrunandsecondrunofemptycontrols.ThegrowthofuninfectedamoebaewasfasterthanthegrowthofamoebaeinfectedwithHSC8.

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Table 3.11 Duplication time of uninfected and infected amoebae in the first and second runrespectively.Uninfectedacanthamoebaereachdoubleofthestartingnumberbetween20.7hand31.1h post seeding, which means it takes them about one day. Acanthamoebae that harbor theendosymbiontHSC8need67.2hormoretoduplicatetheirnumber,inaverageatleastthreedays.

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Infected HSC 67.2 128.1

3.6.3 InfluenceoftheendosymbiontHSC8onacanthamoebaviability

HostcelllysisofinfectedAcanthamoebasp.HSCcultureswasanalyzedandquantifiedusingalive/deadstaining and was then compared to uninfected Acanthamoeba sp. HSC as a negative control. Thepropidiumiodidevisualizesdeadcellswithleakingcellmembranes.NosignificantPIpeakindicatinghostcell death could be observed throughout the course of the experiment (Figure 3.12A). Additionally,amoebal fitness in continuous cultures did not seem to be influenced by the presence of HSC8 attemperaturesbelow27°Candstaysconstantovertimeinbothuninfectedandinfectedacanthamoebaecultures.

The PI fluorescence intensity is slightly increasing over time in both uninfected and infectedacanthamoebae.AmoebaeharboringtheendosymbiontHSC8haveconsistentlyhigherPI fluorescenceintensity compared to the uninfected control. The differences in amoebal fitness observed betweeninfectedamoebaeanduninfectedcontrolsduringthe72hofincubationweresignificantatallthetimepoints,butingeneralsolowthattheeffectofHSC8onhostcellgrowthisnegligible.Moreover,atthelatertimepointof72hpsthePIvaluesoftheinfectedcultureduringthefirstrundecreased,incontrasttoallotheramoebacultures. Inconclusion,sincethevaluesarevery lowtheresults indicatethat thepresenceofHSC8didnotseemtohaveaninfluenceonamoebagrowthincontinuousculturesandthatingeneralnocelllysistakesplaceintheoriginalAcanthamoebasp.HSChostat27°C.

Forbettercomparison,asimultaneousDAPIstainingdetectedthetotalamountofDNAinthesample.The DAPI fluorescence intensities are illustrated in figure 3.12B. The total amount of DNA increasedconstantly over time in all cultures, independent on infection with the endosymbiont HSC8, butexperienced a drop at the later time points in the infected cultures. DAPI fluorescence intensitywasthendepictedinrelationtothePIfluorescenceintensity.TheresultingrelativemortalitywascalculatedforeachcultureandforeachtimepointasthetotalnumberofdeadcellsdividedbytheinitialamountofDNA.Although the valueswere slightly higher in the infected cultures, the relativemortality rateswere constant over time. We observe elevated mortality rates for amoeba cultures containingendosymbionts,butanoverallconstantrelativemortalityovertimeinallcultures.

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Figure3.12Acanthamoebasp.HSC fitnessdependenton thepresenceorabsenceofendosymbiontHSC8.Thedatarepresentaveragevaluesfortwodifferentruns.White:firstandsecondrunofamoebacultures infected with HSC3. Black: first run and second run of empty controls (A) PI fluorescenceintensityovernincubationperiodof72h.Theamoebalviabilityisconstantinemptyamoebae.Hostcelldeathiselevatedinculturescontainingendosymbiontsandincreasingovertime.(B)DAPIfluorescencesignaling represents the total amount of DNA in uninfected and infected amoeba cultures. The DAPIfluorescenceintensitiesincreasedinallculturesfortwodays,followedbyadropintheinfectedculturesandacontinuous increase inuninfectedcultures.Animportantdifferencecanbeobservedafter72h.(C)Therelativemortalityrateofuninfectedand infectedacanthamoebaefordifferentculturesoveraperiodof72h.Weobserveelevatedmortalityratesininfectedamoebacultures,butanoverallconstantrelativemortalityovertimeinallcultures.

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3.6.4 Maintenanceofinfectivityduringhost-freeincubation

The first step in analyzing the infectivity of the HSC8 consisted in the comparison of extracellular tointracellular HSC8 both recovered from a well-grown amoeba culture. An infectivity assay wasperformedbyincubatingextracellularandintracellularsymbiontshost-freeduringsixdays,whileaddinguninfectedAcanthamoebacastellaniiNEFFeverythirdday.Nosignificantdifferenceininfectivitycouldbeobservedinbothstages.The infectivitycurvesweresimilar,startingwithan infectivityrateof20%followed by a rapid decrease after 3 days. No infected amoebae were detected after 6 days ofextracellularincubation.

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Figure3.13ThemaintenanceofinfectivityoftheextracellularcellstagecomparedtotheintracellularcellstageofHSC8afterhost-freeincubationinTSY(MOI50).(A)PercentageofhostcellsinfectedwithextracellularHSC8previouslyincubatedoveraperiodof6daysinhost-freemedium.(B)Percentageofhost cells infectedwithHSC8previously isolated from the cytoplasmof amoebae (intracellular stage)followedbyan incubationperiodof6days inhost-freemedium.Lightblue:No infection,meaningnobacteriahad infectedahostcellafter24hourspost infection(hpi).Darkblue:Lowinfection,meaningthat1-5symbiontshaveenteredacellafter24hpi.Green:Intermediateinfection,when5-30bacteriaperamoebaewereobservedafter24hpi.Orange:Fullinfection,for>30cellsperhostafter24hpi.(C)Infection rate of both stages over the course of 6 days.White: extracellular HSC8 isolated from themedium.Black:intracellularHSC8isolatedfromthecytosolofhostcellsbylysis.Anobviousdecreaseofinfectedamoebaehostswasdetectedinbothextracellularandintracellularbacteria,resultinginalossofinfectivityaftersixdaysofhost-freeincubation.Nodifferencewasobservedbetweenbothstages.

Noticing that in this study, only 20 % of amoebae were infected right from the beginning a secondinfectivity assaywas applied. To further analyze the infectious behaviour of extracellularHSC8 in theoriginalAcanthamoebasp.HSChost,amoebaewerefixedafter48hourspost infection.Adecreaseofinfected cells is observed over time,with an especially dramatic drop of fully infected amoebae. Thenumberoffullyinfectedcellsdroppeddownto10%afteronedayofhost-freeincubation.After5dayshost-freeincubation,allinfectivityofHSC8waslost.

Inaddition,wecalculatedtheperiodoftimenecessaryfortheinfectivityofHSC8tosinkdownto½and¼ in relation to the infection ratemeasured after 2 h of host-free incubation.Weused the equationshowninfigure3.14B.Halfofthe infectivitywas lostasearlyas14.6handafteronedayofhost-freeincubationonly25%oftheendosymbiontswereobservedwithininfectedamoebae.

Table3.12Half-livesoftheendosymbiont`sabilitytoinfecttheacanthamoebaehosts.Theamountoftimerequired for the infectivity to fall to½and¼ itsvalueasmeasuredat thebeginningof the timeperiodwascalculatedwiththeequationshowninfigure,After14.6htheinfectivitydecreaseddownto50%andafteronedayonly25%oftheamoebaehavebeeninfected.

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Figure 3.14 Maintenance of infectivity of HSC8 after host-free incubation in TSY (MOI 50). (A-B)Infectivityrates indicatingtheabilityoftheobligateendosymbiontHSC8tomaintain infectivityafteraperiodof5dayshost-freeincubation.(A)PercentageofamoebaeinfectedwithHSC8,startingwith95%after 2 h host-free survival, followed by a quick decline over the following 4 days. Light blue: Noinfection,meaningnobacteriahadinfectedahostcellafter48hourspostinfection(hpi).Darkblue:Lowinfection,meaning that1-5symbiontshaveenteredacellafter48hpi.Green: Intermediate infection,when5-30bacteriaperamoebaewereobservedafter48hpi.Yellow:Full infection, for>30cellsperhostafter48hpi.Errorbarsindicatestandarddeviation(B)Theexponentialdecreaseininfectivityovertime, after 4 days only few survivors of extracellular HSC8 was observed. The equation underlinesexponentialdecline.

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Figure3.15Visualizationofmaintenanceofinfectivityafterhost-freeincubationinTSY.Thehost-freeincubabtion and successive infectionwasmonitored over a period of 5 dayswith FISH using EUK516(light blue), a combination of EUBmix and HSC8 (yellow) and by DAPI stain (blue). (A)Nearly everyamoebaintermediatetofullyinfectedwithHSC8after2hourshost-freeincubation(B)Lessthanhalfofthe total number of amoebae on the pannel contain endosymbionts, most are intermediate to fullyinfected(C)Onlyfewinfectedcellsareobservedontheseconddayofhost-freeincubation.(D-E)Oneorfew surviving and infective particles left among empty amoebae hosts (F) No bacterial infectionobservedwithinamoebae,onlyendosymbiont-freetrophozoitesdistributedthroughouttheplate.

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4. Discussion

4.1 Detectionofendosymbiontsoffree-livingamoebae

4.1.1 Isolationandaxenizationoffree-livingamoebae

The isolationof free-livingamoebae frombothenvironmental andclinical samplesusingnon-nutrientagar plates seeded with Gram-negative bacteria (E. coli) is used extensively and worldwide(Lagkouvardosetal.,2014).Themethodwassuccessfullyappliedinthisstudy.Weisolated15amoebaecultures in total, of which 10 were successfully grown in TSY or PYG media in the absence ofsupplementaryfoodorganisms.Withinseveraldays,amoebaeswitchfromgrazingtopinocytosisofTSYorPYGmediumasfoodsource.Assoonasmultiplicationofamoebaecanbeobservedintheabsenceofexternal live food microorganisms, the culture is typically referred to as axenic. The screening byfluorescence in situ hybridization resulted in the detection of three distinct strains of intracellularbacteriaamongwhichonestrainmaymostlikelyrepresentanovelspeciesandcanthusberecognizedas a valuable tool. However, the approach has its limitations simply due to the restricted ability ofcultivation. The media used may not be suitable for many amoebae that remained unrecognized,together with all possible endosymbionts. Furthermore not only the cultivation is critical, but someamoebae might not be able to adapt to axenic culture conditions and will be dependent on foodbacteria, mostly Escherichia coli, that may mask the detection of endosymbionts. If a prospectiveinvestigationwouldconcentrateonthediversityofamoebalendosymbiontsinadefinedenvironmentalsample, this method will have to be complemented by other tools, media and supplements. Onepossibility might be to use alternative food sources, another one would be an alternative isolationprocedure. Cocultivation of environmental samples containing symbiont-free axenic amoebae hasalreadybeensuccessfullyusedinthepast,buthasitsownlimitations(Collingroetal.,2005b).

4.1.2 Detectionofendosymbiontswithinfree-livingamoebaeusingFISH

Fluorescence insituhybridizationusingrRNA-targetedoligonucleotideprobeshasbeenavaluabletoolfor investigations of uncultured bacteria in complex microbial communities. For the detection ofendosymbiontswithin environmental amoebae, all 15 isolated cultureswere treatedwith a universalprobe(EUBmix) inFLUOStogetherwithaspecificprobe.Thespecificprobemostly labeled inCy3wasfirst chosen among a list of common bacterial orders, families and species, known to be frequentendosymbiontsofotheramoebae.AmongtheseprobeswereoligonucleotidestargetingAlpha-,Beta-,andGammaproteobacteria aswell asChlamydialesandBacteroidetes ingeneral,more specificallyweusedprobeslabelingRickettsia-likeandCaedibacter-relatedbacteria,aswellasAmoebophilusasiaticus

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andProcabactersp.TheisolateHSC3gaveastrongsignalwhenlabeledwiththeprobespecificfortheorderChlamydiales.

We detected another endosymbiont using a unpublished probe targeting Rickettsia-like bacteria(Rick1395).Totakeadeeper look into theAlphaproteobacteria,weappliedtwootheroligonucleotideAcRic90andAcRic1196originallydesignedbyFritsche(Fritscheetal.,1999)totargetthecloselyrelatedendosymbiontsUWC36andUWC8. Interestingly, our isolateHSC6gavea fluorescent signal onlywithRick1395 and AcRic90, and not AcRic1196, which suggests a mismatch in the target region of HSC6comparedtotheendosymbiontsUWC36andUWC8.

A successful detection of the endosymbiont HSC8 and HSC9was achieved by the probe that usuallytargets Gammaproteobacteria, Gam42a. The fact that according to the phylogenetic classification(under 3.3) the strain HSC8 was assigned to the order Betaproteobacteria is contradictory to thesefindings.Gam42a(5'-GCCTTCCCACATCGTTT-3')targetsthe23SrRNAofGammaproteobacteriaanddiffersfromtheprobetargeting23SrRNAofBetaproteobacteriaBet42a(5'-GCCTTCCCACTTCGTTT-3'), only by one single base. The simple inability to detect HSC8with theBetaproteobacteria-specificprobeBet42ahasalsobeenreportedforthedistantlyrelated“CandidatusProcabacteracanthamoeba”.The 23S rRNA gene sequence analysis of these Betaproteobacteria revealed the presence of onepolymorphismatthetargetsiteoftheoligonucleotideprobeBet42athatisotherwiseconservedinallasyetsequencedBetaproteobacteria.ThepresenceofthissingleT-Umismatchinthecenterofthetargetsequencedestabilizestheprobe-rRNAinteraction.Consequently,theapplicationoftheoligonucleotideBet42resultsinanearlyundetectablyweakfluorescentsignal(Hornetal.,2002).Tofurtherinvestigateifasimilarpolymorphismisdisplayedinthe23SrRNAgeneoftheendosymbiontofAcanthamoebasp.HSC8,resulting inatargetsitewithstrongeraffinityfortheoligonucleotideprobeGam42a,a23SPCRcan be done followed by a target sequence analysis. In themeantime, a specific probe targeting theendosymbiontHSC8wasdesigned.

4.2 IdentificationandphylogeneticanalysisofendosymbiontsofAcanthamoebasp.

ThesampleanalyzedinthisstudywasagreencoloredmicrobialbiofilmofalittoralcavewallfromtheHawaiianIslands.Suchaseacaveistypicallyformedalongafaultordikebythewaveactionofthesea.Hawaii’s warm tropical climate, ocean shore and volcano landscape make it an interesting naturalscenerywithgreatbiologicaldiversity.Formerstudiescompareddifferentcavetypesbyanalyzingtheirmicroflora, and found a high overlap. Especially Actinobacteria, Proteobacteria, Acidobacteria,Verrucomicrobia, Planctomycetes, Nitrospirae and Bacteroidetes have been recovered from all cavetypes.Theonlyphylafoundinlavatubesbutnotyetdocumentedaspartofothercavemicroflora,wereChlamydiaeandKtedonobacteria(Northupetal.,2011).

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Inthisstudy,theisolatedbacteriawereidentifiedasobligateintracellularendosymbionts,whichmeansthattheyareinneedofanamoebalhosttoreplicate.Thishasbeenreportedformanyotherbacterialendosymbionts isolated from free-living protozoa, such as Parachlamydia sp., Protochlamydiaamoebophila,“CandidatusParacaedibacteracanthamoebae”,“CandidatusAmoebophilusasiaticus”and“CandidatusProcabacteracanthamoebae”(Beieretal.,2002;Collingroetal.,2005;Everettetal.,1999;Horn et al., 2002; Horn et al., 2001; Schmitz-Esser et al., 2008). The effects that these intracellularorganismsmayhaveontheirhostsarevaried.Previousstudiestellusthatnotonlytheendosymbiontdepends on the host`s intracellular environment, but there exist some free-living protozoa whoseendosymbiontsare justasnecessary for their survival (TRFritscheetal., 1993).Wesuggest that thestudyofsuchrelationshipsincombinationwithpolymerasechainreactiontargetingthe16SrRNAgeneand sequencing of the 16S rRNA gene are to be important sources of phylogenetic and evolutionaryanalysis.Unfortunately the furthercharacterization is still very limiteddue to the inability tocultivatemostbacterialendosymbiontsfreeoftheirprotozoalhostandthisfactnothingbutemphasizesthehighdegreeofadaptationtoanintracellularlife-style.

Phylogenetic analysiswas done usingMEGA andARB software and indicates the relationships of thenew isolates with their closest relatives and the assignment to orders and families. Comparativesequenceanalysis revealedthat the isolateHSC3showedhighest16SrRNAsequencesimilarity (97-99%) to previously described members of the Parachlamydiaceae, including the endosymbiont ofAcanthamoeba sp. UWE1 (sequence similarity of 99 %) and Parachlamydiaceae bacterium CRIB38(sequence similarity of 97 %) recovered from a water treatment plant. Thus the isolate HSC3undoubtedly clusters together with the two strains, forming a distinct lineage within the familyParachlamydiaceae. Furtherobligateendosymbiontsoffree-livingamoebaewerereportedwithinthisfamily.WhileProtochlamydiaamoebophilaUWE25andParachlamydiaspp.countasthebetterstudiedintracellular chlamydiae, the more recently identified bacteria such as “CandidatusMesochlamydiaelodaea”,“CandidatusMetachlamydialacustris”andNeochlamydiahartmanellaeseemtobethecloserrelativestothisisolateHSC3accordingtoFigure8(Corsaroetal.,2010;Corsaroetal.,2013;Hornetal.,2000).

We applied the techniques to characterize another isolate HSC6 and it was revealed that theseendosymbiontsofAcanthamoebasp.wereclusteredunequivocallywithintheRickettsiales.Infactmostmembersofthisorderareassociatedwitharthropods;howeverthefindingisconsistentwithpreviousidentifications of endosymbionts of Acanthamoeba sp. that demonstrate their reliance uponintracellulargrowth.ThetwoclosestrelativeswerefoundtobethepreviouslydescribedendosymbiontofAcanthamoebasp.UWC8andtheendosymbiontofAcanthamoebasp.UWC36(sequencesimilarity99%).Morespecifically,accordingtoformerstudiestheendosymbiontofAcanthamoebasp.UWC8andUWC36showsequence identitiesof99.6%and thus the threeorganismsmay formawell-separatedlineagewithintheRickettsiales.TheoligonucleotideprobesAcRic90(5’-TGCCACTAGCAGAACTCC-3’)and AcRic1196 (5’- CCT ATT GCG TCC AAT TGT -3’) were designed complementary to shared targetregionson16S rRNAofboth strainsUWC8andUWC36 (Fritscheetal., 1999).Although in this study,onlyAcRic90was able toproperlydetectHSC616S rRNA.Because this organismhaspreviouslybeendescribedbyFritscheetal.,nofurtherinvestigationshavebeendoneinthisstudy.

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Itiscommonlyrecognizedthatthemajorityofthedescribedbacteriawithanintracellularlife-stylearemembers of the Alpha- or Gammaproteobacteria (Schmitz-Esser et al., 2008). Currently, only a fewendosymbiotic organisms have been identified as Betaproteobacteria, including the obligateintracellular “CandidatusProcabacter acanthamoebae”, recovered fromAcanthamoeba spp. (Heinz etal.,2007a;Hornetal.,2002).So rather surprisingly,we found that twoof thestrainsHSC8andHSC9couldbeassignedtotheBetaproteobacteria,withasequenceidentityof100%tooneanother,butlessthan94%sequencesimilaritytothebestblasthitsintheGenbank.Phylogeneticanalysisrevealedthatthe isolated endosymbionts of Acanthamoeba sp. HSC8 and HSC9 might represent a distinct novelspecieswithintheBetaproteobacteriaandverylikelyanewlineage.Mostphylogenetictreesshowthesequence within a clade of uncultured Betaproteobacteria and more distantly related to a fewpreviously described members of theNeisseriaceae family. The uncultured Betaproteobacteria wereisolated from a number of different locations, such as the epithelium of basalmetazoan hydra, ironoxidizing biofilms or lake and seawater samples, listed in Table S1, whereas the isolate HSC8 wasrecovered from a littoral cave in Hawaii. As protozoa hosts are ubiquitous in aquatic and terrestrialhabitats such as freshwater, soil and air, these findings are not especially surprising. However thepresenceofsimilarbacterialsequencesinsamplesfromaquaticenvironmentsworldwidemightindicateapreferencefortheseconditions.

4.3 CuringofnaturalAcanthamoebaspp.HSChost

Kanamycin is an aminoglycoside antibiotic, used for treatmentof infections causedbyGram-negativebacteria.Kanamycin interactswith thebacterial30Ssubunitof ribosomes, inducesmistranslationandinhibitstranslocationduringproteinsynthesis(Misumietal.,1978;Misumi&Tanaka,1980).Aftertheregular treatment of the amoebae culturewith Kanamycin, the endosymbiont HSC8was successfullyeliminatedandasaresultweobtainedawell-grownendosymbiont-freeamebaeculture.

Rifampicin is abactericidaldrugof theRifamycingroup, and is typicallyused to treatMycobacteriuminfections, but also Neisseria, Listeria or Legionella infections. Rifampicin inhibits bacterial DNA-dependent RNA-polymerase by binding to the active center, thus inhibits RNA synthesis by physicallyblocking the formation of the phosphodiester bond in the RNA backbone. This “steric occlusion”mechanismpreventsanextensionofRNAproducts longer than2-3nucleotides (E.A.Campbelletal.,2001).Rifampicinresistancedevelopsquicklyduringtreatmentduetomutationsthatalterresiduesofthe Rifampicin binding site on RNA polymerase. The affinity for Rifampicin decreases, and as aconsequence monotherapy bears great risks. The amoebae culture was successfully cured withRifampicin,butthefinalculturefreeoftheendosymbiontHSC8wasachievedonlyaftertwoattemptsduetoreinfection.AlternativelyRifampicinshouldbeused incombinationwithotherantibiotics,suchaskanamycin,DoxycyclinorErythromycin,whichwould inaddition increase theselectivepressureonbacteriaandacceleratetheprocess.

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AmpicillinispartoftheaminopenicillinfamilyandisactiveagainstanumberofGram-positiveandsomeGram-negativebacteria.Ampicillinactsintheinhibitionofanenzymecalledtranspeptidaseneededforcell wall synthesis. Asmost intracellular bacteria are Gram-negative, Ampicillinmight not be able topenetratetheoutermembraneoftheendosymbiontHSC8.ThecuringoftheamoebaecultureinfectedwithHSC8wasnotsuccessfulusingAmpicillinastheonlyantibiotic.Nevertheless,itmaybeinterestingtoapplyitincombinationwithotherdrugs,suchasRifampicin.

Tetracyclinisabroad-spectrumantibioticandthenamereferstothefour-ringsystemofthecompound.Althoughitcanbeusedagainstavarietyofbacterialinfections,wepointoutthatitremainsespeciallyuseful in the treatment of infections by certain obligate intracellular pathogens such as Chlamydia,MycoplasmaandRickettsia.Tetracyclininhibitsproteinsynthesisbybindingthebacterial30Sribosomalsubunit and by blocking the A site for attachment of the charged aminoacyl-tRNA. Bacteria acquireresistance to Tetracyclin from horizontal gene transfer of a gene that encodes an efflux pump thatactively eliminate tetracycline from the cell cytoplasm (Chopra & Roberts, 2001), or a ribosomalprotectionproteinthatdislodgestetracyclinefromtheribosome.Tetracyclinalsobindseukaryotic40Sribosomalsubunit,butaseukaryoticcellsdonotactivelypumptheantibioticintotheircytoplasmevenagainstaconcentrationgradientasbacteriado,theyremainlessvulnerable(Connelletal.,2003).AstheamoebaeculturetreatedwithTetracyclinwasnotsuccessfullycured,wesuggestthateitherthebacteriamighthavedevelopedresistanceveryquicklyortheconcentrationofantibioticswastoolow.Doxycyclinis part of the Tetracycline antibiotic class and has a similar mechanism of action as the otherTetracyclinesdo.Ithasbeenusedsuccessfullytotreatsexuallytransmitted,respiratoryandophthalmicinfections including the generaChlamydia, Streptococcus, UreaplasmaandMycoplasma. Interestinglydoxycycline is used as antiprotozoal drug in the prophylaxis againstmalaria, but notmuch is knownabout the effects on amoebae. In this study the amoebae treatedwith Doxycyclin did not show anyimpairmentduetotheantibioticsandthechlamydialendosymbiontHSC3wassuccessfullyeliminatedfromtheculture.

OtherantibioticssuchasErythromycin,Gentamycin,PhosphomycinorOfloxacinwerenottested,butincombinationwithRifampicinworthatrialifanyfurtherstudiesinthisareawereplanned.Ingeneral,thecuringwassuccessful,buttostudythedrugresistancesofthesetwointracellularbacteriaindetail,newcombinationswouldbeneeded.

4.4 HostrangeofendosymbiontsofAcanthamoebaspp.HSC3andHSC8

4.4.1 HSC3andHSC8areabletoinfectavarietyofAcanthamoebaspp.

Byextendingthehostrange,endosymbiontsmightbeabletofurtherassuretheirsurvivalintheharshenvironments,theircontinuousdistributionaswellaslimitpossiblebottlenecksduetotransmission.Itis also commonly recognized that various amoebae species in distinct geographic regions can harborclosely related strains of endosymbionts (Molmeret et al., 2005; Schmitz-Esser et al., 2008). Such a

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globaldistributionindicatesthatfree-livingamoebaesuchasAcanthamoebaspp.functionasauniqueecologicalnicheforintracellularbacteria,morethanthesurroundinghabitatdoes.Withinthenutrient-rich intracellular of amoebal trophozoites these bacteria are protected against unaccustomedenvironmentalconditions.Consideringtheextendofhowthesebacteriaevolvedtheabilitytoadapttothe intracellular life-stylewithinearlyeukaryoticcells itseemspossiblethatmanyvirulencestrategiesweredevelopedduring these interactions, longbeforeplantoranimal cellsexisted.Examplesare theATP/ADP translocase importing ATP from the host in exchange for ADP, the nucleoside triphosphatetransporters, taking up nucleotides or the type III secretion system, injecting effector proteins intoeukaryotic cells used by several bacterial pathogens. Nowadays pathogenic chlamydiae use thesestrategies for the infection of humans (Horn&Wagner, 2004). HSC3 aswell asHSC8were shown topermanently infectvariousAcanthamoebaspp. strains,whicharemembersof thesame familyas thenativehostsofHSC3andHSC8,Acanthamoebasp.HSC.However, theyremainedunableto infectanyeukaryotesofhigherdevelopmentalorder,neithermammaliannorinsectcells.OtheramoebaesuchasHartmanella,Naegleria orDictosteliumwere not tested, but should be studied in the near future. Inparticular, it is known that theclosely relatedAcanthamoebasp.SymbiontUWE25 isable tomultiplynot only within various Acanthamoebae hosts but also in the distantly related amoebaDictosteliumdiscoideum(Fritscheetal.,1998;Skriwanetal.,2002).

4.4.2 HSC3andHSC8areincapabletoinfectmammalianandinsectcells

The study of intracellular bacteria or environmental chlamydiae in particular, represents a uniqueopportunitytofurtherdefinecharacteristicfeaturesofhostadaptionaswellastoidentifyfactorsthatcontributetopathogenicity.HSC3andHSC8botharedistantlyrelatedtoestablishedpathogens,suchasC.trachomatis,NesseiriagonorrhoeandBurkholderiacepacia.Therealreadyisevidenceforapotentialpathogenicity of environmental chlamydiae, such as Simkania negevensis andWaddlia chondrophila,which are able to infect both amoebae and mammalian cells (Corsaro & Greub, 2006; Horn, 2008).However, members of the family Parachlamydiaceae, including Parachlamydia acanthamoebae andProtochlamydia amoebophila appear to have limited ability to thrive in nonprotozoan host cells(Collingro et al., 2003;Maurin et al., 2002;Omsland et al., 2014).No permanent infection of neitherHSC3(Parachlamydiaceae)norHSC8inHeLa229andDrosophilamelanogastermacrophage-likecelllineS2couldbeobserved.IncontrasttoAcanthamoebaspp.andotheramoebae,whereuptakeofbacteriais achieved through the classical phagocytosis, HeLa 229 cells are non-phagocytic cells. Receptor-mediated endocytosis is a mechanism common among eukaryotic cells for the uptake ofmacromolecules or small particles such as viruses. This pathway involves plasmamembrane domainsthat lateronare transformed intoclathrin-coatedvesicles (Clerc&Sansonetti,1987).Several invasivebacteriasuchasShigellaflexneriinduceamechanismsimilartophagocytosis,involvingcondensationsoffilamentousactinbeneaththeplasmamembraneofHeLacellsaswellasmyosinaccumulationsattheentry site. Shigellae, yersiniae, salmonellae, rickettsiae and chlamydiae are all capable of entering

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nonphagocytic cells (Moulder, 1985). S2 cells derived from a macrophage-like cell line of a lateembryonic stage ofDrosophilamelanogaster andmight as well take upmacromolecules by clathrin-dependentendocytosis(Schneider,1972).

Receptor-mediated endocytosis has also been suggested for the internalization of chlamydiae. Theobligate intracellularbacteriumChlamydiatrachomatis induces itsownentry intohostcells,aprocessmediated by a tyrosine-phosphorylated protein at the site of attachment of surface-associatedchlamydiae. This chlamydial protein, termed Tarp (Translocated actin-recruiting phosphoprotein) israpidly translocated across the membrane into the host cell by type III secretion and recruits actin(Clifton et al., 2004). Furthermore, the analysis of total chlamydial genomes has given us valuableinformationaboutproteingroups, includingtwoimportantones,atpresent,uniquetoallChlamydiae.Inclusionmembrane (Inc) proteins and the polymorphicmembrane proteins (Pmp proteins) are bothinvolved in adhesion and formation of inclusionmembrane (Rockey et al., 2000). However, genomicsequencing of theHSC3 related strainProtochlamydia amoebophilaUWE25 indicated that the outer-membrane of this organism lacks a number of these proteins thought characteristic for allChlamydiaceae. Among these proteins the Pmp proteins, which might be involved in adhesion tomammaliancellsmightbea reason for the low infectivityofmammaliancells (Collingroetal., 2004).LittleinformationisavailableontheinternalizationmechanismsofBetaproteobacteria.Asweobservedno actual infection of eitherHeLa229 cells or insect S2 cells by the endosymbiont strainHSC8 in thisstudy, we suggest a similar lack of outer membrane proteins. The analysis of the whole genomesequenceofthestrainsHSC3andHSC8wouldadddepthtoourunderstandingofthisinabilitytoinfectmammalian cells andof course further informationof chlamydia-specificprocessesnot coveredhere.Professionalphagocytes, suchasmacrophages, i.e. themousemacrophagesTHP-1werenot tested inthisstudy,butwouldgenerallybeagoodattemptforfurtherstudies.

Anotheraspectofnon-phagocyticcellsistheircriticalroleintheintrinsicimmunedefensemechanisminresponse to microbial infection: triggering apoptotic, necrotic or pyroptotic host cell death. On onehand, apoptosis kills pathogens at a very early stage; on the other hand it induces dendritic cells toeliminate infected apoptotic bodies and inducing further protective immune responses. Somepathogenic bacteria have highly evolved strategies to manipulate cell death pathways in order toenhance their own survival and replication. Examples are various gastrointestinal pathogens such asSalmonella Typhimurium, Yersinia, Shigella flexneri, Helicobacter pylori and enteropathogenic E. coli(EPEC)aswellasChlamydiatrachomatisandLegionellapneumophila(Ashidaetal.,2011).IfHSC3andHSC8do not possess the ability to inhibit host cell death, itmight also be a reasonwhy they do notsucceedininfectingnon-phagocyticcells.

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4.5 CharacterizationoftheendosymbiontofAcanthamoebasp.HSC3

4.5.1 ThedevelopmentalcycleofthechlamydiaHSC3

Fluorescence in situ hybridization with rRNA-targeted oligonucleotide probes is commonly used forinvestigationsofunculturedcomplexmicrobialcommunities.InthisstudyithasbeenasuccessfultooltotrackthedevelopmentalcycleoftheendosymbiontHSC3inboththenativeAcanthamoebasp.HSCandthewellcharacterizedA.castellaniiNEFF.

Knownchlamydialsymbiontsdisplayanumberofremarkablecharacteristicssharedbyallmembersofthe Chlamydiales, especially concerning the unique biphasic developmental cycle. It consists of thetransition between two main stages, the infectious extracellular elementary bodies (EBs) and theintracellularreplicativereticulatebodies(RBs).TheendosymbiontofAcanthamoebasp.HSC3makesnoexception, as the combination of fluorescence in situ hybridization and the DNA stain DAPI clearlydifferentiatesbetweentheEBsexhibitingalowerribosomalcontent,andthemetabolicallyactiveRBs.ItiswellestablishedthatFISHdoesnotresultinstrongfluorescentsignalsearlierthan12hpi(Poppertetal., 2002). Theonly knownexception so far isSimkanianegevensiswhosedevelopmental stagesbothhavebeendescribedasinfectioussuggestingahigherextracellularstabilityofreplicativeforms(Kahaneet al., 2002). However, further insight into the infection cycle of this novel member of the familyParachlamydiaceaecouldrevealyetunknowncharacteristicfeatures.

TheuptakeofEBsintotheAcanthamoebaspp.hostoccurredafter2-3hoursportinfection.AsbacterialFISH signals were very rare and at this time point we suggest that the EBs might still have a lowmetabolical activitybut that thenumberof ribosomes is already increasing. Presumably, thebacteriabegin the transition from infectious stage to replicativecell formuntil the firstappearanceofmatureRBsat24hourspost infection.Themoreextensivemultiplicationcontinuesuntilup to72hourspostinfection,whenat lastallAcanthamoebaspp.hostswerefully infectedwithHSC3after96hourspostinfection. At times, it seems that the intracellular life form of HSC3 has a slightly larger and moreelongatedshapebuttorevealthedetailedmorphologicaldifferencestheultrastructurecanbeanalyzedbytransmissionelectronmicroscopy.Thiscouldaswellbeauseful tool to lookforpossible infectiouscrescent bodies, as have been observed by Greub and coworkers in Parachlamydia acanthamoebae(Greub&Raoult,2002a).Accordingtothestudy,crescentbodiesaredefinedasathirddevelopmentalstage found extracellularly as well as inside the vacuoles of the host. To further investigate thereplicativecourseofHSC3anadditionalquantitativereal-timePCRcanbedone.

It is commonly recognized, that all Chlamydiae replicate within a host plasma membrane derivedvesicular compartment, termed inclusion that is formed during the entry process (Heinz, 2010). Wedistinguish single cell inclusions frommultiple cell inclusions, but unfortunately little is known aboutneithertheintracellulartraffickingofinclusionsnorhowitisinvolvedintheresistanceagainstthehostdefensemechanisms.HSC3seemstoformsinglecellinclusions,whichwouldsimilartoProtochlamydiaamoebophila,which form small inclusions containingoneor fewparticles (Collingro et al., 2005), but

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differfromothermembersoftheParachlamydiaceae.Parachlamydiaacanthamoebacontainshugefullypacked replicative vacuoles (G. Greub & Raoult, 2002; Gilbert Greub et al., 2005). Still it cannot beexcluded that theobserved features correspond tomultiple cell inclusionsdue to thedenselypackedamoebahosts. It remainsuncertainas longas it isnot further investigatedby repeating the infectioncyclesusingalowermultiplicityofinfection.Transmissionelectronmicroscopyhasalwaysbeenausefultoolfortheevaluationofsuchmodalitiesandhasbeenusedfrequentlytoelucidatethelifecycleofavarietyofobligateintracellularbacteria(Greub&Raoult,2002b;Kahaneetal.,2002)

Weconclude, that theendosymbiontofAcanthamoeba spp.HSC3completed itsdevelopmental cycleafter96hourspostinfection,whichisconsistentwiththeinformationwehaveaboutrelatedorganisms.Protochlamydia amoebophila releases its progeny after 96 hours post infection. In contrast otherChlamydia-like bacteria can show huge differences concerning the time of cycle completion.Parachlamydia acanthamoebae and Waddlia chondrophila both exhibit release of first infectiousparticlesafter24hpiand36hpifollowedbylysisofthemacrophages(Goyetal.,2008;GilbertGreubetal., 2003). Undoubtedly, the experimental setup is crucial when comparing two or more organisms,includingthetemperature,mediacomposition,multiplicityofinfectionatthebeginningoftheinfectionassayandmostimportantlythehost.

0hpi - Attachmentofelementary bodies

2-3hpi – Uptake and formationof vacuole

48-72hpi - Replication

24hpi – Differentiationintoreticulate bodies

96hpi – Transitioninto elementarybodies and release

Elementary body

Reticulate body

Figure 4.1 The infection cycle of chlamydial endosymbiont HSC3. Note the two distinct cell typesindicatingthetypicalbiphasicdevelopmentalcycleoftheChlamydialesandthesinglecellinclusions.

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4.5.2 Influenceoftheendosymbiontongrowthandfitnessofamoebaehosts

Enumeration of amoebae and additional PI staining followed by PI fluorescence measurement giveinsightsintoamoebalgrowthandfitnessoverthecourseofanexperiment.Weanalyzedtheinfluenceof an endosymbiont on amoebal growth by phase contrast microscopy and calculating the total cellnumberforallculturesduringreplication.Firstofall,weobservedthattheuninfectedacanthamoebaegrew much faster than the ones infected with the chlamydia endosymbiont HSC3. These findingssuggestthatHSC3definitelyslowsdowntheexponentialgrowthobservedintheemptycontrols.

A first difference in cell numbers could be observed as early as 24 hps with uninfected amoebaedisplayinghighernumbers,butaremarkabledifferenceoccurredat48hps.Itseemsasifthe24hpsarecrucial within the course of amoebal replication. This is consistent with the fact that in generalAcanthamoebadisplayreplicationratesbetween8–24hoursdependingonthespecies/genotypeunderoptimal growth conditions. (Khan, 2006) However, it is delicate to claim that hosting bacterialendosymbionts is no optimal growth condition, as it is the symbiosis occurs naturally, and thecontinuous amoeba cultures infected with HSC3 kept at 20 °C are stable. We therefore propose aparasiticlifestyleofHSC3withinAcanthamoebasp.HSC,exploitingthehostcellforreplicationutilizingmetabolic pathways and substrates, and thereby slowing down the amoebal growth. Reports haveshown that endosymbionts can affect host cell growth of diverse eukaryotic hosts in many ways(Collingroetal.,2004).

Another explanation for the striking difference between infected and uninfected amoebae culturesmight involve a bias resulting from density, followed by enhanced nutrient utilization that ultimatelyleads toa fasterdepletionof substrates.Our results indicate that suchanutrientdepletionmightbedelayedintheabsenceofendosymbiontHSC3.Additionaldensity-dependentquorumsensingsystemspresentinmanyGram-negativebacteriacomprisecaninfluenceamoebalgrowth.Quorumsensingisasystem of cell–cell communication mediated by diffusible effector N-acyl homoserine lactone (AHL)molecules,alsoknownasautoinducers.Onceacertainthresholdofautoinducersignalingmolecules isachieved, the regulationof virulence genes amongothers occurs.High cell density has typically beensynonymouswithcessationofexponentialgrowth(Brown&Barker,1999),whichleadsustothesecondpossibleexplanationfortheconstantgrowthrateof infectedAcanthamoebasp.HSCcomparedtotheuninfectedones.We speculate that thepresenceof bacterial endosymbionts in the amoebae cultureleadstoa fasterdepletionofnutriments in themediaandas intracellulargrowthresults inearlyhighcell density this could give rise to an early expression of such density-dependent phenomena.Consequentlyanearlierentry intostationaryphasewillbeachieved incontrast toendosymbiont-freeamoebae cultures. However, there is little evidence for such phenomena, either in chlamydiae noramoebaeletaloneinasymbiosisbetweenthetwoorganismsandwillhavetobefurtheranalyzed.Forexample,suchanapproachcouldincorporatelowerstartingnumberstopreventanybiasresultingfromdensity.Consideringthataperiodof3daysisnotalongperiodoftimewecouldspeculatethatindeedtheendosymbiontshaveaninhibitingeffectonamoebagrowth,butthattheculturemightrecoverafterafewmoredays.Thiswouldindicatethatinfectedamoebaeculturesjustshowadelayedstartingphase.

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Further experimentsmay prove such a phenomenon ifmeasurementswere done over a longer timeperiod.

BasedontheresultswefoundregardingtheinhibitingeffectofHSC3onitshost,itseemedinterestingtoinvestigatethedegreeofdamagecausedbytheendosymbiont.Inductionofhostcelllysisisfrequentamongobligateintracellularbacteria;thereforeweusedapropidiumiodidestainingforvisualizationofdead cells. Thepresenceofhost cell deathwithinHSC3 infectedAcanthamoeba sp.HSC cultureswascomparedtouninfectedAcanthamoebasp.HSC.Asiscommonlyknown,thecellularexitofChlamydiaecanbemediatedbytwodistinctmechanisms.Thefirstpathwayiscalcium-dependentpermeabilizationwithin the cell, starting with rupture of the inclusion membrane followed by other intracellularcompartments, resulting in the ultimate lysis of the plasma membrane. The second mechanism isreferredtoasextrusionpathway,apackagedreleaseprocessthatleavesthehostcellintact.(Hybiske&Stephens, 2007) In this study, no significant PI fluorescence intensity peak happens throughout thecourse of the experiment, indicating that no cell lysis was caused by the endosymbiont ofAcanthamoeba sp. HSC3 (Figure). Nevertheless, we did not observe packaged release, which isconsistentwith the findings thatHSC3 formssinglecell inclusions.Theclosely relatedProtochlamydiaamoebophila UWE25 resides within small inclusions containing only one or few particles, dispersedthrough the cytoplasm. Both differ from other members of the Parachlamydiaceae forming largeinclusionswithhighernumberofcellsinsidetheamoebaehosts(Fritscheetal.,2000;Hornetal.,2000;Ludwigetal.,1997).Wecanonlyspeculatethateverychlamydialparticlemightbepackagedwithinasingle cell extrusionandexits theamoebaehostby leaving it intact. Furtherexperiments,wouldgiveinsight into the HSC3 specific mechanism of host cell exit. We could investigate whether extrudedchlamydialinclusionswerereleasedoutoftheamoebaeentirelyorwhethertheyremainedsurroundedby plasma membrane. In former studies, the cytoplasm and plasma membrane of HeLa cells werelabelledwithcytosolic-redfluorescentproteinandpalmitoyl-GFPrespectively.ThesecellswereinfectedwithChlamydiaandobservedwith livefluorescencevideomicroscopyafter72h.(Hybiske&Stephens,2007)

Additionally,amoebalgrowthincontinuousculturesdidnotseemtobeinfluencedbythepresenceofHSC3at temperaturesbelow27 °C.Theamoebal fitness is constantover time inbothuninfectedandinfected acanthamoebae cultures. Nevertheless, we have no information about howHSC3 can affectamoebal fitness at higher temperatures or different growth conditions (e.g.media). Chlamydiamightgrow a lytic effect on its host cells at temperatures around 30 °C. Such investigations would giveadditional insight into adaption of endosymbiont HSC3 to temperatures higher than in its naturalhabitat.

The slow increase inPI values inboth culturesuntil 48hps, thegreaterdivergencebetween infectedamobaeanduninfectedcontrolsat48hpsandthecontradictorytrendsafter48hpscanbeexplainedbytheextremelylowPIvaluesingeneral.Regardingthethresholdof300,differencesinthevaluesrangingbetween 3 and 19 are quite negligible. The small fluctuations are likely underestimated, due to thegreater chance of losing parts of the pellet duringwashing steps andmeasuring errors of the TecanInfiniteM200microplatereader.

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ADAPI stainingwas applied in parallel to visualize the total amount of DNA in the sample, includingamoebal as well as bacterial DNA. Results are relatively consistent with the findings of cell counts.IndeedanincreaseofDAPIfluorescenceintensityisobservedinuninfectedamoebaecultures,whereasthe totalamountofDNA is constantover time incultures infectedwithHSC3. It seems logic that theDAPIvaluesarenotas strikinglydivergentas thecell countscanbeexplainedby thesimple fact thatinfectedculturescontainmoreDNAduetotheendosymbionts,butstilllessamoebalDNA.Interestinglybothcultures showadropafter24h,most likelydue toacontinuousmeasurementerrorat the firsttimepoint.Nevertheless,thevaluesareverylow,indicatingthatthevariationsmightaswellberandomandthereforeinsignificantbetweenallcultures,independentoninfectionwithendosymbiontHSC3.At48 hps a significant divergence between uninfected and infected cultures occurred, which is keptconstantuntil72hps.ThefirsttwodaysseemtobearemarkabletimepointasthefindingscollidewiththedivergenceinPIfluorescenceintensities.

DAPI fluorescence intensity put in relation to the PI fluorescence intensity represents the relativemortalityinanamoebaculture.Therelativemortalitywasconstantovertimeinallcultures.Thevalueswereslightlyhigherintheinfectedcultures.WemightassumethattheendosymbiontHSC3hasaveryslighteffectontheamoebalgrowthandfitness.However,astherelativemortalityratesarebasedonmeasurementsofPIandDAPIfluorescence intensities,theresultsoftheuninfectedamoebaeculturesmaybebiasedandunderestimated.Inthatcasethedifferencewouldbemoreorlessnegligible.Furtherinvestigationsshouldbedonewithanimprovedexperimentalset-uptoavoidmeasurementerrors.

In conclusion, considering the more or less continuously increasing amoebal numbers and nosignificantly elevated PI fluorescence intensities, we assume an overall non-detrimental effect onamoebalfitness.Bacteria-inducedhostcelllysismostlikelyplayednomajorroleinthetransmissionofinfectiveparticles.Wesuggestanon-lyticmodeofexitofHSC3fromthehost,ashasbeenreportedtooccur inProtochlamydiaamoebophilaUWE25 (Schulz, 2011) in contrast to the lyticoneproposed forParachlamydia sp. (GilbertGreub&Raoult,2002c)and forC. trachomatis (Hybiske & Stephens, 2007).However in contrast to other free-living protozoa who rely on the endosymbionts for survival,Acanthamoeba sp. HSC grow perfectly well and show improved replication rates in absence ofendosymbiontsHSC3.

4.5.3 Host-freesurvivalcapabilityandmaintenanceofinfectivityinrelationtonutrientavailability

Livinginsideeukaryotichostcells,chlamydiaeinhabitanecologicalnicheinwhichenergyrichmetabolicintermediates are readily available. Consequently, many metabolic reactions are dispensable for theChlamydiaceaeandthereforetheseorganismsoftenlackkeyenzymesofseveralbiosyntheticpathwaysand are auxotrophic formost amino acids, nucleotides and cofactors (Iliffe-Lee&McClarty, 1999). Asimilar dependency is also reflected in the genome of the environmentalChlamydia. The chlamydialendosymbiontofanAcanthamoebasp.,forwhichacompletegenomesequenceisavailable,andaclose

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relativeofHSC3,hasbeentermedProtochlamydiaamoebophilaUWE25(Collingroetal.,2005;Fritscheetal.,2000).Glycolysisaswellastheoxidativepentosephosphatepathwayneedmetabolicallyactive,phosphorylated hexoses as a starting point. In contrast to the Chlamydiaceae, ProtochlamydiaamoebophilaUWE25isnotonlyabletoimporthost-derivedphosphorylatedglucoseusingaglucose-6-phosphate transporter uhpC; (Schwöppe et al., 2002), but encodes a glucokinase (glk) and is thusadditionallyabletogeneratephosphorylatedhexosesindependentlyfromitsamoebahostcell.

Our findings of this study suggest sustained metabolic activity in HSC3 EBs as their infectivity washypotheticallymaintainedfor8-9days indifferentnutrient-richandnutrient-freegrowthmedia intheabsenceofhostcells.Asourtestedtimeperioddidnotinvolvemeasurementsbeyond7days,wecanonlyguessasurvivalof9days, furtherexperimentswith later timepointsneedtobeassessed inthisregard. After 7 days of host-free incubation the infectivity rate was reduced to 30 % in average.Nevertheless, they support previous studies about the respiratory activity in ProtochlamydiaamoebophilaEBs. Interestingly,theEBssurvived inhost-freeenvironmentsapparently independentofthe nutrient availability tested in this study, with particular reference to presence or absence of D-glucose.

Indeed,notallhost-freemetabolicactivitiesdetectedinProtochlamydiaamoebophilaEBsareexpectedtooccurinexactlythesamemannerinotherchlamydialspeciesduetothedifferencesintheirgenomerepertoire. Thus, the obviously indifference in infectivity for the tested growth conditions, we couldsuggestthatHSC3mightalsolackessentialproteinsforphosphorylationofglucoseandthereforerelyonimport of D-glucose-6-phosphate from their host cell similar to the Chlamydiaceae. Hopefully, theanalysis of theHSC3genome sequencewill give adeeper insight into themetabolic pathwaysof thisnew member of the environmental chlamydiae, especially to enlighten if, for example, it generallyencodesthecompletepathwayforD-glucosecatabolism,liketheProtochlamydiaamoebophilagenomedoes or if their metabolism is totally independent from the uptake and/or use of D-glucose in theenvironment.Forexample,host-freeHSC3mayutilizeotherinternalorexternalcarboncompoundsorsubstrates that enter downstream of the central carbon metabolism. A similar co-occurrence ofalternativecarbonsubstratescompensatingD-glucoseshortagehasbeensuggested inProtochlamydiaamoebophila(Sixtetal.,2013).AsHSC3EBssurvivedespitethelackofallegedlyessentialsubstratesinbuffer and salt solution, we could propose a complete independency from the environmentalconditions, explainedbyglycogen storagesor similar storage compounds.Evenmore likelyHSC3maymetabolize products from imported amino acids (in case of the nutrient richmedia), protein or lipiddegradation.

Nevertheless, most of these speculations do not explain the constantly higher infection rates of EBsgrown in nutrient free PBS and salt solution. Even after 7 days of host-free incubation in NaCl theinfectivityratedidnotsinkbelow80%,whichcorrespondstotheinitialinfectionrate.Comparedtotheothermedia thiswas the lowestof the initial infectivity rates.Allothers ranged round100% relativeinfectivity.Interestingly,weobservedarapidincreaseimmediatelyafterwards,reachingapeakof140%after2days.ThereisapossibilitythattheadaptionofEBstothesaltsolutionwasdelayed.AstheNaClsolutionaloneshouldnotvaryinosmolarityorpH,wespeculatethattheEBsarenotinhibiteddirectly,but maybe self-induced stress factors or the absence of signaling that trigger survival and infective

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activitydelayedtheprocess.ThemechanismsbywhichHSC3cansurvivethehost-freenutrientdeprivedconditions and the source of energy or substrates that are crucial for themaintenance of infectivity,remainstobeelucidated.

Altogether, the HSC3 EBs seem to be very effective in the adaptation for survival in host-freeenvironments.AsEBsareindeedtheinfectivestagesuchabilitiescanbecrucialintheirbiologicalroleasdispersalstagedependingonthedensityofprotozoahostsintheirnaturalhabitat.Despitetheevidenceofquickadaptiontoadversehost-freeenvironmentsprovidedinthisstudy,theexactrequirementsforhost-freesurvivalandametabolicpotential thathasbeensuggested forProtochlamydiaamoebophilaEBs(Sixtetal.,2013)remaintobeenlightened.Furtherinvestigationsusingdifferentgrowthconditions,changing the nutrient availability, testing other incubation media supplemented with a diversity ofsubstratesneedtobeassessed.

4.6 CharacterizationoftheendosymbiontofAcanthamoebasp.HSC8

4.6.1 TheinfectioncycleoftheendosymbiontHSC8

In thisstudy,Fluorescence insituhybridizationwithrRNA-targetedoligonucleotideprobeshasbeenasuccessful tool to track the developmental cycle of the endosymbiont HSC3 in both the nativeAcanthamoebasp.HSCandthewellcharacterizedA.castellaniiNEFF(4.5.1). Inthesamemanner,weanalyzedthecharacteristicfeaturesofthelifecycleoftheendosymbiontHSC8inbothAcanthamoebasp.HSCandA.castellaniiNEFF.AsweexploredthehostrangeofHSC8,wenoticedthattheywerewellable to infect a variety ofAcanthamoeba spp., but small differences in the developmental cycles areprobable(4.4.1).

TheuptakeofHSC8intotheAcanthamoebaspp.hostoccurredasearlyas2-3hourspostinfection.Mostcells containing one or few particles and begin theirmassive replication between 8 and 24 hpi. It iscommonlyrecognized,thatthemodeofentryofendosymbiontsintoamoebahostsisbyphagocytosis(Khan,2006).However,wehavenoinformationthemechanismofleavingtheendocyticphathwayandintracellular trafficking for thisnovelorganism.Moreextensivemultiplicationcontinuesuntilup to48hpi,when at last allAcanthamoeba spp. hostswere densely packedwithHSC8.Chlamydiae replicatewithin a host plasma membrane derived vesicular compartment, termed inclusion which is formedduring the entry process (Heinz et al., 2010). Other obligate intracellular bacteria, members of theAlphaproteobacteria formvacuoles.Procabacter sp., a closer relativeofHSC8,wereobservedequallydistributedinthecytoplasm(Hornetal.,2002)orenclosedbyahost-derivedmembraneinamultiple-partner association (Heinz et al., 2007b). As was already proposed for the chlamydial endosymbiontHSC3,transmissionelectronmicroscopyisasuitabletoolfortheevaluationof intracellular localizationandtraffickingincombinationwiththevisualizationofvacuoles.FISHrevealedacontinuousrod-shapedformofthebacteriawithin,butintheprocessofinfectionthedetailedmorphologieswerelostbecause

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of excessive density. To further analyze the ultrastructure, we could use transmission electronmicroscopy. In thepast, ithasbeenused frequently toelucidate the lifecycleofavarietyofobligateintracellular bacteria (Gilbert Greub & Raoult, 2002a; Kahane, 2002). The replicative course of otherobligateintracellularbacteriahasbeenelucidatedusinganadditionalquantitativereal-timePCR,whichwouldgiveinsightintofurthercharacteristicsoftheHSC8lifecycle.Intheend,bacteriaweredetectedinmassive amounts in the extracellular environment after 72 hpi, indicating that the developmentalcycle iscomplete.Dependingonhowthebacteriaareenclosedorfreelytrafficking insidetheamoebahostcell,themodeofexitremainstobeinvestigated.

0hpi – Attachmentofextracellular bacteria

2-3hpi – Uptake of bacteria

8-24hpi – Replicationinthe cytosolof the amoeba host

48hpi – Full infection

72hpi – Releaseof bacteriainto the environment

Figure 4.2 The infection cycle of the bacterial endosymbiont HSC8. Rod-shaped betaproteobacteriaspreadevenlythroughouttheamoebahostcell,noapparentsinglecellinclusionsorclustering.

Inconclusion,theendosymbiontofAcanthamoebaspp.HSC8completed itsdevelopmentalcycleafter72hourspostinfection,whichisalittleearlierthanobservedwiththeendosymbiontofAcanthamoebasp.HSC3.Thechlamydia-relatedorganismendeditslifecycleafter96hourspostinfection.OthermorecloselyrelatedBetaproteobacteria,suchasCandidatusProcabacteracanthamoebaeareas-yettheonlyknown obligate intracellular symbiont of amoebae in this phylum. Ralstonia picketti is frequentlyisolated from patient and environmental samples, but has as well been detected inside free-living

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Acanthamoeba sp., where they form large parasitophorous vacuoles and leave the amoeba hosts byrupture or lysis. They have been shown capable of infecting a variety of Acanthamoebae strains.Burkholderia pseudomalleiandBurkholderia thailandensisare related pathogens of theBetaproteobacteriainvadingavarietyofcelltypes,replicateinthecytoplasm,anduseacrypticflagellarsystemforintracellularmovementandcell-cellspread(Frenchetal.,2011;Inglisetal.,2000;Michel&Hauröder,1997;Ralstonetal.,1973).

ThedirectcomparisonoftheinfectionrateofendosymbiontHSC8initsoriginalAcanthamoebasp.HSChosttoA.castellaniiNEFFpointedoutdifferencesbetweenthetwoamoebaestrainsatthebeginningofthedevelopmentalcycle.Indeed,especiallyaround2-3hpi,whenHSC8startedtoentertheiramoebalhosts, a discrimination between symbionts attached to the outer part of the cell membrane andsymbionts that just penetrated the cellmembrane to the intracellular. These visual assessmentsmayhaveledtoerroneousresultsduringthefirsttimepointandthenumberofinfectedA.castellaniiNEFFmay be underestimated. In previous studies FISH slides were therefore manually screened throughdifferentfocalplanesoftheamoebae.FISHsignalsthataredistributedthroughoutthecellinmorethanonefocalplane,thesymbiontismostlikelyintracellular,whereasifFISHsignalsarefoundinonesinglefocal plane, the symbionts might still be attached to the outside of the amoebae cell membrane(DiplomarbeitHarreither,2013).AsthecourseofinfectionA.castellaniiNEFFisveryconsistentwiththeoneinAcanthamoebasp.HSClaterinthedevelopmentalcycle,thevariationatthetimeofinvasionmayindicatea socalleddelayed infection.A.castellaniiNEFF isnot theoriginalhostof theendosymbiontHSC8andmayneedtimetoadapttotheabsenceofanativehost.

4.6.2 Influenceoftheendosymbiontongrowthandfitnessofamoebaehosts

Obligate intracellular bacteria can have a variety of effects on the growth and fitness of eukaryotichosts. As we already observed during the investigations of the chlamydial endosymbiont HSC3, thebacteriasloweddowntheexponentialgrowthof infectedamoebae.Similar resultswere foundduringthecountingandcalculatingofuninfectedandHSC8infectedamoebaecellnumbers.ThegrowthrateofuninfectedacanthamoebaewasmuchfasterthantheacanthamoebaeinfectedwiththeendosymbiontHSC8. The endosymbiont somehow reduces amoebaehost cell replication, indicatedby the relativelyconstant, non-exponential growth rate observed for uninfectedAcanthamoeba sp. HSC.We supposethisisduetoHSC8utilizingnutrientsfromthehostasenergysource.

A significant difference in cell numbers could first be observed at 48 hps,when uninfected amoebaedoubled their numbers compared to infected ones. In 4.5.1, we spoke of density-dependent cell-cellcommunicationsystemsthatcaninfluenceamoebalgrowthbydiffusibleAHLmolecules.Onceacertainthresholdofautoinducersignalingmoleculesisachieved,theregulationofvariousgenesoccurs,suchasvirulence genes. High cell density has typically been correlatedwith cessation of exponential growth(Brown&Barker,1999).Additionally,thepresenceofbacterialendosymbiontsintheamoebaecultureleadstoafasterdepletionofsubstratesandasintracellulargrowthresultsinearlyhighcelldensitythis

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could explain the constant growth rate of infected amoebae. In our case, regarding the startingnumbers,suchterminationofexponentialgrowthinthepresenceofHSC8mayindeedhavehappenedasearlyas24hps.

The membrane impermeable DNA dye propidium iodide was used to verify any lytic effect of theendosymbiontHSC8onAcanthamoebasp.HSC.Indeed,PIfluorescenceintensitywasslightlyincreasingovertimeinbothuninfectedandinfectedacanthamoebae.AmoebaeharboringtheendosymbiontHSC8have consistently elevated PI fluorescence intensity compared to the uninfected control. Despite thefewdifferencesinamoebalfitnessbetweeninfectedamoebaeanduninfectedcontrols,ingeneralsuchasmallproportionwasstainedwithpropidiumiodide,thatwesupposethehostcellmembranesstayedintact in both cases. Consequently no host cell lysis was observed and the influence of HSC8 onamoebae fitnesswas negligible at temperatures below 27 °C. The threshold of 300was not reachedthroughout the course of the experiment and even the slightly elevated values of infected cellsincreasingbetween23and64arenegligible.Moreover,atthelatertimepointof72hpsthePIvaluesofthe infected culture during the first run decreased, in contrast to all other amoeba cultures. Thevariationsaremostlikelyduetotechnicalissues,suchaslossofpartsofthepelletduringwashingstepsor measuring errors of the Tecan Infinite M200 microplate reader. However, the PI fluorescenceintensitieswere higher for HSC8 infected than HSC3 infected amoebae.We could assume that HSC8mighthaveamoreprominenteffectonhostcell fitnessatthesetemperaturesthanHSC3has.Ontheother hand, the chlamydial endosymbiont HSC3 was incubated with a lower starting density. As wealready explained, cell density can have an inhibiting effect on amoeba growth due to enhancedsubstrate turnoverwhich ultimately leads to a faster depletion of nutriments in themedia. Host celldeathcomesalongwithdensity-dependentsubstratedepletionandmayconsequentlybeincreasedinsuchaway.Additionally,asindicatedinourresultsnutrientdepletionmightaswelloccurratherearlyinthepresenceofintracellularbacteria,duetoparasiticbehaviorandexploitationofresources.Amoebalexponentialgrowthisinhibitedaccompaniedbyhostcelldeathduetostarvation.Thiswouldnotimplyany direct symbiont-induced host cell lysis, but neither canwe deny such events. Further analysis isnecessary to make conclusions about the proportion of cells that die of density-dependent nutrientdepletionandtheproportionthatislysedduetoevasionofintracellularparasites.

For more precise comparison between two intracellular organisms concerning the influence on hostamoeba replication and lytic behavior, further experiments must involve lower as well as the samestartingdensities.Moreover,recentstudiesreportedthattemperaturedoesplayanessentialroleinthedevelopmentofhost–symbiontinteractions(Fels&Kaltz,2006;LaScolaetal.,2002,2004;Ohnoetal.,2008), themodeoftransmissionandevenmodulationofvirulencefactorsofparasites(Restif&Kaltz,2006). Parachlamydia acanthamoeba UV7 grows lytic effects on its Acanthamoeba spp. host attemperaturesabove30°C,while lowertemperatureskeep itendosymbiotic forthehostcell (Greubetal., 2003). Varying the incubation temperatures for both organismswould give insight into their lyticbehavior,thustheirinfluenceonhostcellfitnessinanygivenenvironment.

The second DNA dye DAPI is a marker for the total amount of DNA present in the culture. DAPIfluorescenceintensityincreasedconstantlyovertimeinallcultures,independentoninfectionwiththeendosymbiontHSC8,until48hps.A firstdiversificationwasobservedat72hps,where thevalues for

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infected amoebae experienced a drop or cessation of increase, whereas uninfected cultures showcontinuouslyrisingDAPIsignals.Ingeneral,theseresultsareconsistentwiththefindingsofamoebacellgrowthinpresenceorabsenceofintracellularbacteria.However,thedifferencebetweeninfectedanduninfectedcultureswassignificantlymoreoutstandingbyusingtheenumerationtechniquethanDAPIfluorescencemeasurements.DAPI is indeedauniversalDNAstainingdyethatbindsnotonlyamoebalbutalsobacterialDNA.Consequently,thevaluesforinfectedculturesmaybeoverestimated.

TherelativemortalityratewascalculatedbyputtingthetwoDNAbindingdiesinarelationandwasthusused to reveal differences between the cultures. The slightly elevated values in the infected culturesunderlined our idea of a growth inhibiting, parasitic life style ofHSC8withinAcanthamoeba sp.HSC,exploiting thehost cell for replication,utilizingmetabolicpathways aswell as substrates and therebyslowingdowntheamoebalfitnessallthewhilekeepingthemalive.

4.6.3 Host-freesurvivalcapacityandmaintenanceofinfectivity

Host-free survival capacityandmaintenanceof infectivityofHSC8 innutrient-rich incubationmediumwas our first attempt to provide a link to their biological role as extracellular survival forms.Simultaneously,host-freesurvivalislikelyacriticalfactorforcell-cellspreading.Theextracellularphaseofthedevelopmentalcycledemandsquickadaptiontoahost-freeenvironmenttoensuresurvivalanddispersal.Indeed,along-termextracellularmetabolicactivityhasbeenassessedforchlamydiae(Haideret al., 2010; Matsuo et al., 2010), such as Protochlamydia amoebophila EBs (Sixt et al., 2013) andParachlamydiaacanthamoebae surviving for severalweeks in liquidmedium (Fukumotoet al., 2010).Morerecentstudiesreportedthatchlamydia-likeorganismsremaininfectiveforweeksinanumberofdifferent conditions, even in sterile tapwater.Parachlamydia acanthamoebae lose infectivity after 2weeks,butafewsurvivorsremaindisplayedevenafter10weeksofhost-freeincubationinPYG(Coulonetal.,2012).ExtracellularsurvivalofendosymbiontHSC3formorethanaweekhasbeenobserved inthisstudy(4.5.1).Consequently,wesupposethatChlamydialesmightbeenvironmentallypersistent.Incontrast, our results indicate that the infectivity rate of endosymbiont HSC8 encounters a quickdecrease over time, with no survivors displayed after 5 days of host-free incubation in TSY. Theinfectivityratehassunkendownto25%afteronly24hours.ExtracellulardispersalformsofHSC8donot seem to be as environmentally persistent as Chlamydiales are, and might not display suchspecializedfeaturesforfacingadverseextracellularconditions.Consideringthedifferentgrowthmediaused, the dramatic decline of infected amoebae after 24 hmay aswell be due to the differences inmediacomposition.DGMisachemicallydefinednutrient-richincubationmedium,supplementedwithvarious amino acids among other components, whereas TSY is a standard growth medium used foramoebaculturing.Furthermore,asthequickdrophappenedduringthefirst24h,adilutioneffectmightplay an additional role. Generally, Acanthamoebae display replication rates between 8–24 hoursdepending on the species/genotype under optimal growth conditions (Khan, 2006). UninfectedamoebaemayatonetimereplicatefasterthaninfectedamoebaeandthisdilutioneffectmayleadtoabiasoftheactualinfectivityofHSC8.

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Studies also revealed that Chlamydia-like organisms present better survival capacities than thepathogenC.trachomatis.Infact,pathogensinfectingmammalianandhumancelltissuedonotneedtoremaininhost-freeconditionsforlongertimeperiods,becausethecelldensityismuchhigherthantheamoebaldensityinenvironmentalsources.WhenwerefertothenativeenvironmentofAcanthamoebaHSCanditsendosymbiontHSC8inthelittoralcave,wecouldspeculatethatthedensityofamoebaeishighenoughforHSC8tosurviveafterevadingoneamoebahostuntilencounteringanotherone.

Anumberofstudiesnowrevealedaextracellularmetabolicactivityinchlamydiae(Haideretal.,2010;Matsuoetal.,2010).ProtochlamydiaamoebophilaEBsforexample,areabletoutilizeD-glucoseevenintheabsenceofamoebae(Sixtetal.,2013).Thisismostlikelyacriticalfactorforlong-termsurvivalunderhost-freeenvironmentalconditions.ExtracellularHSC8hasnotyetbeentestedformetabolicactivity.Inourpreliminary infectivityassayon thedifferencebetweenextracellularand intracellularbacteria,weobserved none. Both stages were similarly infective, no infective and replicative forms could bedistinguishedasistypicalforchlamydiae.Theinfectivitycurvesweresimilar,startingwithaninfectivityrateofnomorethan20%.The lowvaluesaremost likelyduetotechnicalproblemsand issues intheexperimentalset-upasitwasaverypreliminaryexperiment.Nevertheless,thisunderlinesthefactthatextracellular HSC8 might not be as perfectly adapted to host-free environment and need a healthyenvironment with relatively high amoebal density to assure survival and dispersal. Indeed the exactrequirementsforhost-freesurvival,ametabolicpotentialashasbeenassessedforchlamydialEBsortheprocess of adaption to the extracellular conditions remain to be enlightened. We need to pursuepossiblechangestriggeredduringextracellularstages,onagenomic,transcriptomicandproteomicleveltogetdeeperinsightintotheissue.

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5. Abstract

Endosymbiosis involves the internalizationofbacterialorganismsbyavarietyofeukaryotes,a fieldofstudygainingmoreandmore recognition.Mostof thesebacteriahavedeveloped intimateand long–term associations with their amoeba hosts and include members of the phyla Alphaproteobacteria,Betaproteobacteria, Bacteroidetes, Chlamydiae very recently alsoGammaproteobacteria. Diversity ofobligateintracellularsymbiontsofamoebae,theirhostrangeandglobaldistributionintheenvironmenthavebeenhighlyunderestimatedandthebacteriahaverarelybeenidentifiedorpoorlydescribed.

Inthisstudy,thesampleanalyzedwasagreencoloredmicrobialbiofilmofalittoralcavewallfromtheHawaiianIslands.Free-livingamoebaewereisolatedfromthebiofilmusinga“walkout”method,thenaxenizedtoeliminatetheirnecessityoffoodbacteria.AFluorescenceinsituhybridizationand16SrRNAfull–cycle approach allowed identification of two novel endosymbiotic bacteria. According to variousphylogenetictrees,HSC3clusterswithintheParachlamydiaceaefamilyandHSC8isverylikelyamemberof a novel familywithin theBetaproteobacteria. Such an intimate and complex symbiotic interactionbetweenhostamoebaandendosymbioticbacteriaisthekeyfeaturethatencouragesafurtherin-depthcharacterizationofthenovelendosymbionts.Theseenclosefirstofalltheanalysisofhostrange,asbyextending the host range, endosymbionts might be able to further assure their survival in harshenvironmentsandglobaldistribution.

BothHSC3andHSC8wereabletopermanentlyinfectvariousAcanthamoebaspp.strains,butremainedincapable of invading non-phagocytic eukaryotic cells neither human HeLa229 nor insect S2 cells. Incontrast to their pathogenic relatives, these endosymbionts might not be able to trigger receptor-mediated endocytosis or lack important membrane proteins unique to Chlamydiales. By a furtherdescriptionofinfectioncyclesinhostamoebae,theirimpactonamoebaegrowthandfitnessandfinallytheirabilityofextracellularsurvivalandmaintenanceofinfectivity,wecharacterizedHSC3andHSC8asendosymbiotic bacteria, rather than parasites. Indeed, they seemed to slow down the amoebalreplication rate, but overall no direct detrimental effect on amoebal fitness was observed. Bacteria-inducedhostcelllysismostlikelyplayednomajorroleinthetransmissionofinfectiveparticles.Forbothendosymbionts,wesuggestanon-lyticmodeofexitfromthehost.Ourfindingsofhost-freeincubationof chlamydialHSC3 showed sustained infectivity overmore than 7 days in different nutrient-rich andnutrient-freegrowthmediaintheabsenceofhostcells.AsHSC3EBssurvivedespitethelackofallegedlyessential substrates, we suppose theymight be environmentally persistent. Indeed host-free survivalcapacityandmaintenanceof infectivityofHSC3andHSC8very likelyprovidea link to theirbiologicalroleasextracellularsurvivalanddispersalformsaswellastoquickadaptiontoahost-freeenvironment.IncontrasttoHSC3,fewsurvivorsofBetaproteobacteriaHSC8wereobservedafter4daysofhost-freeincubation,suggestingalesspersistentextracellularform.

Toleadonthestudy,wholegenomesequencingofthestrainsHSC3andHSC8willprovideanefficientmeansofacquiringdatarelevanttothemoredetailedmolecularcharacterizationoftheendosymbionts.

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6. Zusammenfassung

Eine Endosymbiose beschreibt die völlige Internalisierung eines Bakteriums durch eine ReiheunterschiedlicherEukaryotenundgewinntalsneueresStudienfeldmehrundmehranBedeutung.DiemeistendieserBakterienhabeneineengeundandauerndeVerbindungzuihrenWirtszellenentwickeltund schließen viele Vertreter der Phyla Alphaproteobakterien, Betaproteobakterien, Bakteroidetes,Chlamydien und seit kurzem auch Gammaproteobakterien ein. Die Diversität der obligatenintrazellulären Symbionten von Amöben, ihre Bandbreite an Wirten und globale Verbreitung in derUmwelt wurde bisher stark unterschätzt und die Bakterien sind selten identifiziert oder mangelhaftbeschriebenworden.

IndieserStudiegalteseinengrünfarbenen,mikrobiellenBiofilmzuanalysieren,dervonderWandeinerMeereshöhle der hawaiianischen Inseln stammte. Wir isolierten freilebende Amöben mithilfe einer„Auswanderungs“-Methode um nach erfolgreicher Axenisierung mögliche intrazelluläre Bakterien zudetektieren. Fluoreszenz in situ Hybridisierung und 16S rRNA Gensequenzierung waren Mittel zurIdentifizierungvonzweineuartigenEndosymbionten.DiversephylogenetischeBäumezeigten,dasssichHSC3innerhalbderParachlamydiaceaeFamilieeinreihtundHSC8sehrwahrscheinlicheineneueFamilieinnerhalbderBetaproteobakterienbildet.Solch intimeundkomplexe InteraktionenzwischenAmöbenund intrazellulären Bakterien machen eine tiefgehendere Charakterisierung dieser neuartigenEndosymbiontensehr interessant.DeswegensolltezuallerersteineAnalysederAusbreitungskapazitätdurchgeführt werden, da diese ausschlaggebend für das Überleben unter rauen UmweltbedingungenunddieglobaleVerbreitungderBakterienist.

SowohlHSC3alsauchHSC8gelangeinepermanenteundvolle InfektiondiverserAcanthamoebaspp.,konnten aber keine nicht-phagozytischen Zellen, wie menschliche HeLa229 Zellen oder S2Insektenzellen infizieren. Im Gegensatz zu ihren nächsten pathogenen Verwandten, scheinen dieseEndosymbionten keine rezeptorabhängige Endozytose hervorzurufen oder es fehlen ihnen dienotwendigenMembranproteine,wiesiezumBeispielbeidenVertreternderChlamydialesvorkommen.DieBeschreibungderLebenszykleninnerhalbderWirtszellen,derEinflussderBakterienaufÜberlebenundWachstum der Amöben, sowie die Fähigkeit ohneWirtszelle infektiös zu bleiben, belegten, dassbeide Organismen HSC3 und HSC8 als Endosymbionten statt Parasiten eingestuft werden können.Obwohl wir beobachtet haben, dass die Bakterien die Vermehrung der Wirtszellen verlangsamen,konnte kein direkt zerstörerischer Effekt (Lyse) bei der Übertragung infektiöser Partikel festgestelltwerden. Höchstwahrscheinlich liegt bei beiden Endosymbionten ein nicht-lytischerAusscheidungsmechanismus vor.WeitereBefunde zeigten, dass die ChlamydienHSC3 ihre Infektivitätüber7Tage inverschiedenstennährstoffreichenund–armenWachstumsmedienbeibehalten,obwohlkeineWirtszellenvorhandenwaren.DassHSC3Ebsebensolangeüberleben,wenngarkeineNährstoffezugegen sind, kann ein Hinweis auf ihre extreme Persistenz sein. Sowohl das reine Überleben inAbwesenheiteinerWirtszelle,alsauchdieFähigkeitübereinenlängerenZeitraumtrotzdeminfektiöszubleiben,sindwahrscheinlichwichtigeKriterienfürihrebiologischeRolleals extrazelluläreÜberlebens-und Verbreitungsformen. So ist es ihnen möglich, sich sehr schnell und effektiv an ungewohnte

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Lebensräumeanzupassen. ImGegensatz zudenChlamydien, scheinendie BetaproteobakterienHSC8weit weniger persistent zu sein. Für eine weiterführende und detailliertere, molekulareCharakterisierung,wäredieSequenzierungundAnalysedesgesamtenGenomsderbeidenOrganismenHSC3undHSC8sehreffizient.

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Appendix

16S/18SrRNAgenesequenceoftheendosymbiontsandAcanthamoebahosts

HSC3CGGTTGGAAACGATCGCTAATACCAAATATGGTGCAAGAAGTATCTTCTTGTTATTAAAGTGGGGGATCGCAAGACCTCGCGGTTAAAGAGGGGCCCATGAGATATCAGCTAGTTGGTGAGGTAAGAGCTCACCAAGGCTAAGACGTMTAGGCGGATTGAGAGATTGACCGCYAACACTGGGACTGCAGCAACTGCCCTAGACTCCTACGGGAGGCTGCAGTCGAGAATCATTCGCAATGGGCGAAAGCCTGACGATGCGACGCTGTGTGAGTGATGAAGGCCTTCGGGTCGTAAAGCTCTTTCGCCTGGGAAAAAGAGAGGTAAGCTAATATCTTACCGATTTGAGAGTATCAGGTAAAGAAGCACCGGCTAACTCCGTGCCAGCAGCTGCGGTAATACGGAGGGTGCAAGCATTAATCGGATTTATTGGGCGTAAAGGGCGCGTAGGCGGAAAAATAAGTCGGATGTGAAATCCCGGGGCTCAACCCCGGAACAGCATTTGAAACTATTTTCCTTGAGGGTAGGCGGAGAAAACGGAATTCCACAAGTAGCGGTGAAATGCGTAGATATGTGGAAGAACACCCGTGGCGAAGGCGGTTTTCTAGCTTACTCCTGACGCTGAGGCGCGAAAGCAAGGGGATCAAACAGGATTAGATACCCTGGTAGTCCTTGCCGTAAACTATGTATACTTGGTGTAACTGGACTCAACCCTAGTTGTGCCGTAGCTAACGCGATAAGTATACCGCCTGAGGAGTACGCTCGCAAGGGTGAAACTCAAAAGAATTGACGGGGACCCGCACAAGCAGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCCAGGTTTGACATGCAAAGGACAATACTAGAGATAGTATCTCCCTTCGGGGCCTTTGCACAGGTGCTGCATGGCTGTCGTCAGCTCGTGCCGTGAGGTGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCATTAGTTGCCAACATTTAAGGTGGGAACTCTAATGAGACTGCCTGGGTTAACCAGGAGGAAGGTGAGGATGACGTCAAGTCCGCATGGCCCTTATGTCTGGGGCTACACACGTGCTACAATGGTCGGTACAGAAGGCAGCGAAGCCGCGAGGTGAAGCAAATCCCGAAAACCGATCTCAGTTCAGATTGTAGTCTGCAACTCGACTACAAGAAGACGGAATTGCTAGTAATGGCGAGTCAGCAACATCGCCGTGAATACGTTCCCGGGTCTTGTACA

HSC6TTATGGCTCAGAGTGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGAGTAAGAGCCGTAGCAATACGGAGTTCTGCTAGTGGCAGACGGGTGAGTAATACATGGGAATCTACCTTAAAGTCTGGGATAACTGTTGGAAACGACAGCTAATACCGGATATTGCCGAGAGGTGAAAGATTTATTGCTTTAAGATGAGCCCATGCAAGATTAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCTACGATCTTTAGCTGGTTTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCGACGCCGCGTGAGTGATGAAGGCCTTCGGGTTGTAAAGCTCTTTTAGTAGGGAAGATAATGACGGTACCCACAGAAAAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCAAGCGTTACTCGGAATTACTGGGCGTAAAGCGTGCGTAGGCGGCTTGGTAAGTTGGAAGTGAAAGCCTAGGGCTCAACCTTAGAATTGCTTTCAAAACTGCCTGGCTAGAGTACTAGAGAGGATAGCGGAATTCCTAGTGTAGAGGTGAAATTCGTAGATATTAGGAGGAACACCGGAAGCGAAAGCGGCTATCTGGCTAGACACTGACGCTGTTGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCAGTAAACGAAGAGTGCTAGATATTGGAATTTAATTTTCAGTGTCAAAGCTAACGCGTTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACTCGCACAAGCGGTGGAACATGTGGTTTAATTCGATGCTACGCGAAAAACCTTACCAGGCCTTGACATGTTGGTCATATCATGAAGAGATTCATGAGTCAGCTCGGCTGGACCATCACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTTAGTTACCAACAGGTTATGCTGGGCACTCTAAGGAAACTGCCGGTGATAAGCCGGAGG

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AAGGTGGGGATGACGTCAAGTCAGCATGGCCCTTACGGCCTGGGCTACACACGTGTTACAATGGTGGTGACAATTGGACGCAATAGGGCGACCTGGAGCAAATCCCTAAAAGCCACCTCAGTTCGGATTGTACCCTGCAACTCGGGTACATGAAGTCGGAATCGCTAGTAATCGCAGATCAGCATGCTGTGGTGAATACGTTCTCGGGTCTTGTACACACCGCCC

HSC8TTCGCCCTTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCAGCATGCTGATCTGCGATTACTAGCGATTCCGACTTCATGCACTCGAGTTGCAGAGTGCAATCCGGACTACGATAGGCTTTCTCAGATTAGCTCCCCCTCGCGGGTTGGCAACCGTTTGTACCTACCATTGTATGACGTGTGAAGCCCTGCTCATAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCCGGCTTAGCACCGGCAGTCCCACTAGAGTTCCAAACTTAATTAGGGCAACTAGTAGTAAGGGTTGCGCTCGTTGCGTCACTTAAGACAACATTTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTCCAGGTTCCCGAAGGCACAATCACATCTCTGCGATCTTCCTGGCATGTCAAGAGCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCACATCATCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGTCTACTTCTCGCGTTAGCTTCGCTACTAAGATTACTCCCAACAGCAAGTAGACATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTACCCACGCTTTCGTGCCTGAGCGTCAGTTCTATCCCAGGGGGCTGCCTTCGCCATCGGTATTCCTCCAAATCTCTACGCATTTCACTGCTACACTTGGAATTCTACCCCCCTCTGACAAACTCTAGACATACAGTCTTAAATGCCGTTCCCAGGTTAAGCCCGGGGATTTCACATCTAACTTATATATCCGCCTGCGCACGCTTTACGCCCAGTAATTCCGATTAACGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCTTCTGGTACTCTCAGTCGCTCATGCTGTTAACACCAGCGGTTTGCTCCCAGATAAAAGAACTTTACAACCCGAAGGCCTTCTTCATTCACGCGGCGTTGCTGGATCAGGGTTCCCCCCATTGTCCAAAATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCTGATCTTCCTCTCAGAACAGCTACCGATCATCGCCTTGGTAAGCCGTTACCTCACCAACTAGCTAATCGGCCATCGGCCGCTCTTGTAACGCCAGGCCCGAAGGTCCCCAGCTTTCCTCCTCAGAGATTATGCGGTATTAGCCAATCTTTCGATTGGTTATCCCCCTCTACAAGGCACGTTCCGATGGTTTACTCACCCGTTCGCCACTTGCCATCAACCCGAAGGTCATGCTGCCGTTCGACTTGCATGTGTAAAGCACGCCGCCAGCGTTCAATCTGAGCCATGA

HSCATACGGCGAGACTGCGGATGGCTCATTAAATCAGTTATAGTTTATTTGATGGTCTCTTTTGTCTTTTTTTTACCTACTTGGATAACCGTGGTAATTCTAGAGCTAATACATGCGCAAGGTCCCGAGCGCGGGGGACGGGGCTTCACGGCTCTGTTCTCGCATGCGCAGAGGGATGTATTTATTAGGTTAAAAACCAGCGTAGCCAGCAATGGCTACTCAATCTCCTGGTGATTCATAGTAACTCTTTCGGATCGCATTCATGTCCTCCTTGTGGGGACGGCGACGATTCATTCAAATTTCTGCCCTATCAACTTTCGATGGTAGGATAGAGGCCTACCATGGTCGTAACGGGTAACGGAGAATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAATGGCTACCACTTCTAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCCGACACGGGGAGGTAGTGACAATAAATAACAATACAGGCGCTCGATAAGAGTCTTGTAATTGGAATGAGTACAATTTAAACCCCTTAACGAGTAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGGATCTAGGGACGCGCATTTCAAGCGCCCGTGCCATCGGGTCAAACCGGTGGCTGCGTT

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UnculturedBetaproteobacteriathatarebestblasthitsofendosymbiontsHSC8

TableS1.ListofunculturedBetaproteobacteriathatwereisolatedfromvariouslocations,complementedwiththeaccessionnumbersofthesequences.

Acc.Nr Name Source Title Author

FJ517739 UnculturedbetaproteobacteriumcloneAEP-eGFP-peri_16

embryonicstagesofthebasalmetazoanHydra(spikeandcuticle)epithelium

Inanearlybranchingmetazoan,bacterialcolonizationoftheembryoiscontrolledbymaternalantimicrobialpeptides

Frauneetal.

FJ517688 Unculturedbetaproteobacteriumclone14-1_27

H.magnipapillata,4daysaftertemperaturetreatment,epithelium

DisturbingepithelialhomeostasisinthemetazoanHydraleadstodrasticchangesinassociatedmicrobiota

Frauneetal.

EU937888 Unculturedbacteriumclone3BR-5CC

riparianironoxidizingbiofilm

BiogeochemistryofIronOxidationinacircumneutralFreshwaterHabitat

O.W.Duckworthetal.

AB240515 UnculturedbacteriumcloneSRRB42

PCR-derivedsequencefromroot-base(80to160mm)ofPhragmitesatSoseiRiverinSappro,Japan

AnalysisofmicrobialcommunitystructureinrhizospherebiofilmofPhragmitesatSoseiRiverinSapporo,Japan

Y.Nakamuraetal.

JN609350 Unculturedbacteriumclone15-4-27

activatedsludgefromlab-simulatedsequencingbatchreactors

ComparativeimpactsofnanoscaleAg,ZnOandTiO2onwastewatertreatmentprocess

J.Chenetal.

AJ867904 unculturedbetaproteobacteriumcloneA11-B10

lakewater EnvironmentalFluctuationsSignificantlyInfluencetheMicrobialCommunityCompositionPresentinNivalLakes

M.Yuhana

AJ289984 UnculturedbacteriumFukuN108

threedifferentlakes(LakeGossenköllesee,LakeFuchskuhle,andLakeBaikal)

Comparative16SrRNAanalysisoflakebacterioplanktonrevealsgloballydistributedphylogeneticclusters

F.O.Glöckneretal.

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includinganabundantgroupofActinobacteria

AB478649 Unculturedbetaproteobacteriumclone004-Cadma

submergedbiofilmmatsofamontanewetland

SSUrRNAsequencesofbacteriafromsubmergedbiofilmmatsofamontanewetlandintheAlpsclosetoCadagnodiFuori

T.D.HorathandR.Bachofen

FJ382075 Unculturedbacteriumclone11W_04c09

winterhospitalshowerwaterfromaBMTunit

Potentiallypathogenicbacteriainshowerwaterandairofastemcelltransplantunit

S.D.Perkinsetal.

FJ825812 UnculturedmarinebacteriumcloneBM1-1-44

filteredsurfaceseawaterintheperiodofdiatombloom

SuccessionofbacterialcommunityduringspringdiatombloomintheYellowSea

L.Min

JF178540 Unculturedbacteriumclonencd2077d12c1

skin,poplitealfossa Temporalshiftsintheskinmicrobiomeassociatedwithdiseaseflaresandtreatmentinchildrenwithatopicdermatitis

H.H.Kong

AB199579 Unculturedbacteriumclone:RVW-12

PCR-derivedsequencefromriverwaterinJapan

Selectivephylogeneticanalysistargetedat16SrRNAgenesofthermophilesandhyperthermophilesindeep-subsurfacegeothermalenvironments

H.Kimura

JF737894 UnculturedbacteriumcloneRT2-ant07-a11-S

RioTinto ComparativemicrobialecologystudyofthesedimentsandthewatercolumnoftheRíoTinto,anextremeacidicenvironment

A.Garcia-Moyano

HM445124 UnculturedbacteriumcloneGBL17O32

yellowmicrobialmatfromlavatubewalls,Portugal

InvestigationofNovelMicrobialDiversityinAzoreanandHawaiianLavaTubes

J.J.Hathaway

AY963481 UnculturedbacteriumcloneBS43

soil 16SrRNAgeneanalysesofbacterialcommunitystructuresinthesoilsof

O.C.Chan

97

evergreenbroad-leavedforestsinsouth-westChina

FJ946566 UnculturedbetaproteobacteriumcloneMWR-B12v

arcticmeltwater Microbialsequencesretrievedfromenvironmentalsamplesfromseasonalarcticsnowandmeltwater

C.Larose

EU104028 UnculturedbacteriumcloneM0111_24

activatedsludge EvidencefortheComamonadaceaeasdeterminantsofactivatedsludgesettlingperformance

C.E.Brown

FJ236051 UnculturedbetaproteobacteriumcloneJoinville2

drinkingwater Assessmentofphylogeneticdiversityofbacterialmicrofloraindrinkingwaterusingserialanalysisofribosomalsequencetags

J.B.Poitelon

EU156142 UnculturedbetaproteobacteriumclonepCOF_65.7_B2

CoffeePotsHotSpring

MolecularcharacterizationofthediversityanddistributionofathermalspringmicrobialcommunitybyusingrRNAandmetabolicgenes.

J.R.Hall

HM481366HM481365HM481313HM481335

UnculturedbacteriumcloneFL185UnculturedbacteriumcloneFL176UnculturedbacteriumcloneFL29UnculturedbacteriumcloneFL89

TCE-contaminatedfieldsite

PhylogeneticmicroarrayanalysisofamicrobialcommunityperformingreductivedechlorinationataTCE-contaminatedsite

P.K.Lee

DQ129259 UnculturedbacteriumcloneAKIW598

urbanaerosol Urbanaerosolsharbordiverseanddynamicbacterialpopulations

E.L.Brodie

AJ583179 unculturedbeta-proteobacteriumcloneS15D-MN120

groundwaterfromamonitoringdeep-wellataradioactivewastedisposalsite

Molecularanalysisofbacterialcommunitiesingroundwatersofthedeep-wellinjectionsiteTomsk-7,Siberia,Russia

M.Nedelkova

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Glossary

%(v/v)%(w/v)%(w/w)16SrRNA18SrRNA23SrRNAbpDAPIDNasedNTPEDTAemexcFAFISHFLUOShpihpskbLSMMOImRNAntNTCo/nPCRPFAPIqPCRRNaserpmrRNARTSDSTEMUV

volume/volumepercentageweight/volume(or,moreaccurately,mass/volume)percentageweight/weight(or,moreaccurately,mass/mass)percentageprokaryoticrRNAfromthesmallribosomalsubuniteukaryoticrRNAfromthesmallribosomalsubunitprokaryoticrRNAfromthelargeribosomalsubunitbasepairs(lengthunitfordoublestrandednucleotidechains)4,6diamidino2phenylindoleʹDeoxyribonucleaseDeoxyribonucleotideethylenediaminetetraaceticacidemissionwavelengthexcitationwavelengthformamidefluorescenceinsituhybridization5(6)CarboxyfluoresceinNhydroxysuccinimideesterhourspostinfectionhourspostseedingkilobases(1kb=1000bp)laserscanningmicroscopemultiplicityofinfectionmessengerRNAnucleotides(lengthunitforsinglestrandednucleotidechains)notemplatecontrol(e.g.inPCRorqPCR)overnightpolymerasechainreactionparaformaldehydepropidiumiodidequantitativerealtimePCRribonucleaserevolutionsperminuteribosomalRNAroomtemperaturesodiumdodecylsulfateT3SStypethreesecretionsystemT6SStypesixsecretionsystemtransmissionelectronmicroscopyultraviolet

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Acknowledgements

Hereby,IwouldliketoshowmygratitudetowardsProf.MatthiasHornfortakingmeupintohisworkgroupandallowingmetoparticipateintheseveryexcitingprojects.Thankyouforshowinginterestinmywork and for supportingmeduringmy presentations. I also thank Prof. Dr.MichaelWagner, theheadofDOMEforcreating thisexceptionaland inspiringdepartment in the fieldofmicrobialecologyandforconvincingmewithhislecturesthatthisiswhatIwanttodo.

Moreover,IwouldliketothankAlexanderSiegl,forhisguidancethroughoutmymasterproject,forhispatience and kindness, for the valuable discussions before and after every successful or failedexperiment. You always inspired me with your scientific knowledge and experience. Thank you forteachingmehowtoworkoutexperimentalset-ups,preparescientifictalksandcopewithunexpectedresults.

Of course, Iwould like to express great appreciation to Allen for his unwavering patience and highlyvaluablepresenceinthelab.Thankyousomuchforguidingme,alwayslisteningtomeandforhelpingmesolveeverylittle(orbigger;))problemthatcameupfromtimetotime.Ihopeyou’lltakegoodcareofmy“chewbactersp.”

Furthermore,Ineedtothankmyparentsfortheirunconditionallovingandfinancialsupportthroughoutmyyearsofstudying,mymotherforcalmingmynervesoneveryfortnightofexams,myfatherfortryingtounderstand90pagesofscientificwriting,mybrotherandsisterforalwayskeepingmeworried;)justlikefamilyhastodo!

I’dalso like to thankallmy friends,mostof themspreadover theworld,butstillbeing there forme.Veryspecialthanksgotothoseclosetomeduringmylabwork,forbeingpatient,whenIhadtospendweekendsoreveningsinthelabwhentheywerepromisedtothem.ThankstoallofyouTina,Jil,Kevin,Pit,ChristineandMichel foryourmoralsupport, forevery“downfall”we livedthrough,yoursenseofhumorandmostofallfortakingmymindoffthebiologicalworldfromtimetotime.

Bigthanksalsotothesymbiosisgroup,especiallytoLisaandKarinforguidanceinthelabandforsharingmyenthusiasmwhenIhadchlamydia;).EvenmoreimportantlyIwouldliketothanktheothermasterstudentsStephan,Flo&Flo,Martin,Michi,Kathi,Esther,JasminandphDstudentsBelaandClausfortheenjoyablecompanyduringlabwork,themoralsupportandthegoodtimeswehadtogetherduringmystay.Coffeebreaks,sessionsoftabletennisandbeeronthepatioalwaysmadetheDOME-lifeenjoyableanditstillputsasmileonmyfacewhenIrememberthenightwhenIwasnotsittingonthefloor.Noregrets!

ThanksalsototherestoftheDOMEcrewforthegreatanduniqueworkingatmosphere.

113

CurriculumVitae(German)

ANGABENZURPERSONName:

StefanieMichels

Adresse:

4,rueMichelEngelsL-1465Luxemburg

Telefon:

+352671038497+4369910176867

E-Mail:

[email protected]

AngestrebterGrad:MasterderNaturwissenschaftengeborenam6.März1987inLuxemburgLuxemburgischeStaatsbürgerschaft

AUSBILDUNG

April2013–now

MasterarbeitanderDivisionofmicrobialecology,UniversitätWienThema:‚Descriptionoftwonovelandas-yetunculturedendosymbiontsofAcanthamoebaspp.‘,unterUniv.Prof.Dr.MatthiasHorn

Oktober2011–Juni2014

UniversitätWienMasterstudiumderMolekulerenMikrobiologieundImmunologie

Oktober2006–Dezember2010

UniversitätZürichBachelorstudiumderBiologieSpezialisierungim5.und6.SemesterindenGebieten:-Mikrobiologie-MolekulareBiologieBachelorofScienceinBiologieam7.März2011

1999–2006AthénéedeLuxembourg,LuxemburgSektionC(SchwerpunktinNaturwissenschaften)Maturaam4.Juli2006

114

BERUFSERFAHRUNG

Januar2015

GreenpeaceCEE,WienDirektDialogFundraiserin,PräsentationderKampagnenundProjekteandieÖffentlichkeit,MenscheninspirierenundüberzeugendieOrganisationzuunterstützen,BetreuungderFörderer,AusfüllenvonBankformularen.

August2011–September2011

DupontdeNemours,LuxemburgPraktikantin,technischesProjekt,"ImpactofnaturalandacceleratedageingonProshield®,Tyvek®andTychem®productsproperties",AusführungundAuswertungphysikalischerLabortestsvonFliessprodukten.

März2011–Juni2011

DepartementfürLebensmittelmikrobiologie,ETHZ,ZürichPraktikantin:eigenesForschungsprojekt,Isolierung,KultivierungundSequenzierungvonkomplexerMikrofloraaufOberflächevonHartkäse,mikrobiologischeundmolekularbiologischeMethoden

Januar2011–Februar2011

WildliferescuecenterParadero,CostaRicaFreiwilligendienst:BetreuungundmedizinischeVersorgungvondiversenWildtieren

Juli2010–August2010

DupontdeNemours,LuxemburgPraktikantin,AuswertungdiverserLaborberichteüberSchutzvorbiologischenGefahrendurchSpezialkleidung,Internetrecherche,Normen

AUßERBERUFLICHETÄTIGKEITEN2015 AktivistinbeiGreenpeaceCEEinWien,NVDA-TrainingimFebruar2015.

2011

MitgliedimAnimationsteambeiderviertägigenChallenge2011inAnzere,einjährlichstattfindenderWettbewerbzwischendenbeidenrenommiertestenSchweizerHochschulen,derETHZürichundderEPFLausanneaufundabseitsderSkipiste.VorbereitungdesAbendprogrammsundAnimationwährenddessen.

2009–2010

MitgliedimOrganisationsteamfürdieviertägigeREEL,réunioneuropéennedesétudant(e)sluxembourgeois(es),inZürichimOktober2010.OrganisationderEventswährenddervierTage,Besichtigungen,VeranstaltungenzurDiskussionrundumdasStudium,RestaurantsundAbendprogramm.

115

2006–2007 VorstandsmitgliedimVereinderLuxemburgerStudenteninZürich(LSZ)OrganisationdiverserEventsundAusflüge,ZüricherBal.

2004–2007

WöchentlicheLeitungeinerPfadfindergruppe,BetreuungvonKindernimAlterzwischen5und8Jahren,OrganisationvonVersammlungen,AusflügenundPfadfinderlagern.TeilnahmeaninternationalenPfadfinderlagerninMoldavien,Polen,Österreich,SpanienundItalien.FormationmitDiplomalsACC(AssistantChefdeColonie)undCC(ChefdeColonie)

SPRACHLICHEUNDSONSTIGEKENNTNISSELuxemburgisch Muttersprache

Deutsch Muttersprache

Englisch Fließend

Französisch Fließend

Spanisch Grundkenntnisse

PC

MSOffice(fortgeschritten),RStudio(Grundkenntnisse)

TechnikenMikrobiologischesArbeiten;SterilesArbeiten;DNA-Isolierung&SequenzierungausUmweltproben;KultivierungvonAmöbenundBakterien;PCR;KlonierenvonPlasmiden;

Arbeitserfahrungmit Escherichiacoli,Protochlamydiaacanthamoeba,Acanthamoebacastellanii

AuslandsaufenthalteVolontariatCostaRica,Wildliferescuecenter,2011,sechsWochenStudienreiseFrankreich,StationbiologiqueRoscoff,2010,zweiWochen

HOBBYSUNDINTERESSENHobbys Individualreisen,Pfadfinder,Sport,Lesen,Musik

SportlicheAktivitäten Schwimmen,Indoor-undBeachvolleyball,InlineSkaten,wandern,joggen,Yoga,Snowboarden,Wellen-,Wind-undKitesurfen,Segeln

Wissenschaftliches Umwelt,Naturschutz,Ernährungswissenschaften,Medizin,Zoologie

117

CurriculumVitae(English)

PERSONALDATAName:

StefanieMichels

Address:

4,rueMichelEngelsL-1465Luxemburg

Phonenumber:

+352671038497+4369910176867

E-Mail:

[email protected]

In partial fulfillment of the requirements for the degree of MasterofScience(MSc)bornon6thMarch1987inLuxembourgLuxembourgishcitizenship

EDUCATION

April2013–now

MasterthesisattheDivisionofmicrobialecology,UniversityofViennaTopic:‚DescriptionoftwonovelendosymbiontsofAcanthamoebaspp.‘,underUniv.Prof.Dr.MatthiasHorn

October2011–June2014

UniversityofViennaMasterstudyinMolecularMicrobiologyandImmunology

October2006–December2010

UniversityofZurichBachelorstudyinbiologyAreasofspecialization:-Microbiology-MolecularbiologyBachelorofScienceinBiologyon7thMarch2011

1999–2006AthénéedeLuxembourg,LuxembourgSectionC(Scientific)Graduationon4thJuly2006

118

EMPLOYMENTHISTORY

January2015

GreenpeaceCEE,ViennaDirectdialoguer,face-to-facefundraising,presenting information about the campaigns to the public, inspiring people to want to be part of the organization, supporter care, completing bank forms.

August2011–September2011

DupontdeNemours,LuxembourgIntern,technicalproject,studyingtheimpactofnaturalandacceleratedageingonproductsproperties,Executionandanalysisofphysicallaboratorytests,internetresearch.

March2011–June2011

Departementoffoodmicrobiology,ETHZ,ZurichIntern,researchproject,isolation,cultivationandsequencingofacomplexmicrofloraonthesurfaceofhardcheese,microbiologicalandmolecularbiologicalmethods

January2011–February2011

WildliferescuecenterParadero,CostaRicaVolunteer:Sittingandmedicalcareofvariouswildanimals

July2010–August2010

DupontdeNemours,LuxembourgIntern,analysisofvarioustestreportsaboutbiologicalhazardsandprotectivewear,internetresearch.

EXTRA-PROFESSIONALACTIVIT IES

20152011

GreenpeaceCEEactivist,NVDAtraining,February2015MemberoftheanimationteamattheChallenge2011inAnzere,ayearlycontestbetweenthetworenomatedSwissuniversities,theETHZurichandtheEPFLausanneonandofftheskislope.Preparationandanimationoftheevening’sentertainmentduringfourdays.

2009–2010

MemberoftheorganisationcommiteefortheREEL2010,réunioneuropéennedesétudiant(e)sluxembourgeois(es),inZurichinOctober2010.Organisationofeventsduringfourdays,visitations,discussionsaroundandaboutthestudies,restaurantsandevening’sentertainment.

2006–2007MemberoftheorganisationcommiteeoftheLuxemburgishStudentsinZurich(LSZ)Organisationofvariouseventsandexcursions,„ZüricherBal“

119

2004–2007

Weeklyleadershipofascoutsgroup,careofchildrenagedbetween5and8years,organisationofgatherings,excursionsandscoutscamps.ParticipationatinternationalscoutscampsinMoldova,Poland,Austria,SpainandItaly.FormationwithdiplomaasACC(AssistantChefdeColonie)andCC(ChefdeColonie)

SPOKENLANGUAGESANDOTHERSKILLSLuxembourgish Motherlanguage

German Motherlanguage

English Fluent

French Fluent

Spanish Basicknowledge

PC

MSOffice(advanced),RStudio(Basicknowledge)

Workingtechniques

Microbiologicalwork;sterilework;DNAisolation&sequencingfromenvironmentalsamples;cultivationofamoebaeandbacteria;cellculturing;PCR;cloningofplasmids;Fluorescentin-situhybridization

Workingexperience Escherichiacoli,Protochlamydiaacanthamoeba,Acanthamoebacastellanii

Staysabroad VolunteeringinCostaRica,Wildliferescuecenter,2011,sixweeksStudytriptoFrance,stationbiologiqueRoscoff,2010,twoweeks

HOBBIES AND INTERESTSHobbies Travelling,scouting,sports,reading,music

SportsSwimming,indoorandbeachvolleyball,inlineskating,running,hiking,Yoga,snowboarding,surfing,windsurfingandkiteboarding,sailing

Scientificinterests Environment,nature,nutritionalsciences,zoology,medicine

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