Table Of ContentMethods in
Molecular Biology 1921
Carmen Buchrieser
Hubert Hilbi Editors
Legionella
Methods and Protocols
Second Edition
M M B
ETHODS IN OLECULAR IO LO GY
SeriesEditor
JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
Hatfield, Hertfordshire AL109AB,UK
Forfurther volumes:
http://www.springer.com/series/7651
Legionella
Methods and Protocols
Second Edition
Edited by
Carmen Buchrieser
Institut Pasteur, Biologie des Bactéries Intracellulaires, Paris, France
CNRS UMR 3525, Paris, France
Hubert Hilbi
Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland
Editors
CarmenBuchrieser HubertHilbi
InstitutPasteur,Biologiedes InstituteofMedicalMicrobiology
Bacte´riesIntracellulaires UniversityofZu¨rich
Paris,France Zu¨rich,Switzerland
CNRSUMR3525
Paris,France
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-4939-9047-4 ISBN978-1-4939-9048-1 (eBook)
https://doi.org/10.1007/978-1-4939-9048-1
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Coverillustration:Humanprimarymacrophages(hMDMs)infectedwithLegionellapneumophila(green)stainedwith
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Preface
Summary
Legionella species are the causative agents of a life-threatening form of pneumonia called
Legionnaires’ disease, which was identified more than 40 years ago. The opportunistic
pathogens can occur in epidemics of several hundreds of cases and preferentially infect
elderly and immune-compromised people. Therefore, Legionnaires’ disease will very likely
gainincreasedsocietalimportanceintheyearstocome.Intensemolecularresearchoverthe
last two decades on the most prevalent clinical species, Legionella pneumophila, provided
insights into the ecology, physiology, genetics, virulence, evolution, and epidemiology of
this facultative intracellular bacterium. Further in-depth understanding of Legionnaires’
diseaseandpossibleinterventionswiththepathogenesisofLegionellaspp.requiressophis-
ticated approachesand methods.Thesecondeditionofthismethodbookassemblesstate-
of-the-artprotocolsfromexpertsintherespectivefieldstostudyL.pneumophilaandother
clinically relevant Legionella species. Specifically, the volume includes chapters on the
ecology, physiology, genetics, virulence mechanisms, host responses, as well as metage-
nomics, proteomics, and host microbiomes. The protocols will hopefully contribute to an
efficientandfruitfulanalysisofanemergingandpotentiallydeadlybacterialpathogen.
Introduction
Legionellapneumophilaisanenvironmentalbacteriumparasitizingaquaticprotozoasuchas
Acanthamoeba castellanii or Hartmannella vermiformis [1–3]. When reaching the human
lungthroughcontaminatedaerosols,thesebacteriacanalsobehumanpathogens,causinga
severepneumoniacalledLegionnaires’disease,anatypicalpneumoniawithahighmortality
rate of up to 15%. Persons at risk are elderly and immune-compromised individuals, two
demographic groups that are steadily increasing in size [1]. Recently, it was observed that
theincidenceofLegionnaires’diseaseisrisingandthemortalityrateisstillhigh,particularly
for immune-compromised patients. Thus, Legionnaires’ disease remains an important
publichealththreat[4].
Legionellaspp.areevolutionaryoldbacteria,but“young”fromahumanpointofview,
astheywererecognizedonlyin1977duringalargeoutbreakofpneumoniaataconvention
oftheAmericanLegioninahotelinPhiladelphia[5].Alreadyshortlyafteritsidentification,
L. pneumophila was characterized as a ubiquitous water-borne bacterium that parasitizes
free-living environmental protozoa, wherein it is able to grow intracellularly [6, 7]. The
similarity between aquatic protozoa and human macrophages allows the bacteria to also
infectandreplicateinmammaliancells,inparticularalveolarlungmacrophages[8–10].The
tight coevolution of Legionella with protozoa is also seen in its genome sequence, as a
considerable number of virulence factor and genes encoding secreted effectors have been
acquired by horizontal gene transfer from protozoan hosts [11–16]. Transmission of
Legionella to humans occurs mainly through contaminated aerosols produced by many
artificial water systems created by men, such as air conditioning systems, cooling towers,
thermal bath, showers, etc. [4]. Today, over 60 Legionella species have been described.
v
vi Preface
However, L. pneumophila and Legionella longbeachae are responsible for nearly all clinical
cases in Europe and the USA. Whereas L. pneumophila is responsible for over 90% of
Legionnaires’ disease in the northern hemisphere, L. longbeachae is predominant in
AustraliaandNewZealand,whereitcausesover30%ofhumancases[1].
Duetoitspublichealthimportancebutalsoitsdualhostsystem,humanmacrophages
and aquatic protozoa, L. pneumophila – the by far best studied Legionella species – is a
uniquemodeltostudyevolutionandhost-pathogeninteractions.Accordingly,researchon
L.pneumophilaover thelasttwodecadesfocusedontheevolution,ecology,epidemiology,
and pathogenesis of this opportunistic pathogen. This second edition of the Methods in
Molecular Biology book on Legionella covers updated and new protocols and methods to
study the ecology and physiology of Legionella spp., genetics, cellular microbiology, bio-
chemical assays to study Legionella effectors and enzymes, immunity and host response
againstLegionella,aswellasmetagenomicsandproteomeanalysisandhostmicrobiomesof
Legionella.Manyoftheseprotocolsarealsowidelyapplicableforthestudyofotherbacteria.
Ecology and Physiology of Legionella
In the environment Legionella spp. grow inside several different species of protozoa and
ciliates,fromwhichtheysequesternutrientstoproliferate.Thisinteractionleadstomultiple
physiological changes in the host and the bacteria. Among these, complex metabolic
adaptations that are necessary for the proliferation of the pathogen in the host cells are
induced. Indeed, when Legionella is phagocytosed by protozoa or human macrophages, a
hostcelldefenseprogramisinitiated,includingantimicrobialmetabolicreactionsaimingat
thedegradationofthepathogen,whilethebacteriatrytosequesternutrientsfromthehost
cell and to counteract the antimicrobial reactions [17, 18]. Thus,the analysis of metabolic
changes induced during host-pathogen interactions is an important, emerging field of
investigation. Isotopologue profiling using 13C-labeled compounds is a powerful method
recently applied to analyze the metabolic pathways and fluxes employed by extra- and
intracellular L. pneumophila [18–22]. The approach allows studying catabolic pathways in
detail due to the incorporation of label into amino acids or other metabolites that can be
traced.Indeed,isotopologueprofilingshowedthatthemajorregulatoryproteinCsrAhasa
determiningroleinsubstrateusageandcarbonpartitioningduringtheL.pneumophilalife
cycleandregulatesaswitchfromaminoacidusageinreplicativephasetoglycerolipidusage
duringtransmissivegrowth[22].
A keynutrientfor most bacteriain particular forpathogensisiron [23].However,the
mechanisms by which microbes gain access to iron despite host iron restriction when
residing in a vacuole are poorly understood. L. pneumophila has evolved different systems
toacquireiron,suchastheFeoBsystemfor theuptakeof ferrousironandsiderophoresto
acquireferriciron[24,25].Thesiderophorelegiobactinwasshowntobeimportantinlung
infection [26], and bacterial pyomelanin [27] and a pyoverdine-like siderophore [28] play
roles in iron acquisition, too. Interestingly, L. pneumophila also uses its Icm/Dot type IV
secretion system (T4SS; see below) to secrete effectors such as IroT/MavN to facilitate
intravacuolarironacquisitionwithinA.castellaniiandhumanmacrophages[29,30].
If iron or other nutrients become limited, or if Legionella encounters non-optimal
temperatures or is exposed to disinfectants, the bacteria may enter a so-called viable but
non-culturable (VBNC) state [31]. These cells are metabolically active and have an intact
cell wall, but cannot be grown on classical culture media. However, the addition of
Preface vii
A.castellaniitothedormantbacteriamayresultintheresuscitationofL.pneumophila,and
as deduced from intracellular survival in human monocytes and intraperitoneally infected
guineapigs,virulenceisnotreducedinthereactivatedcells[32].Thus,thequantificationof
VBNC L.pneumophila isof utmost importance for the surveillance ofcontaminated water
systems. Accordingly, several methods and kits have been developed in the last years to
achievecorrectsurveillanceofartificialwatersystems.
As Legionella spp. are mainly environmental bacteria, which have learned to infect
human macrophages from the interaction with environmental protozoa and ciliates, the
analyses of these host cells should shed light on the virulence strategies used to infect
macrophages. Researchers use mainly three amoeba models to study Legionella-protozoa
interactions, which are A. castellanii, H. vermiformis, and the genetically amenable model
amoeba Dictyostelium discoideum, although over 14 species of protozoa and ciliates have
been shown to support the growth of Legionella [33]. This is partly due to the fact that
working with other environmental protozoa is not easy. Yet, new methods have been
developed to facilitate these important studies, which may teach us also more on how
Legionella causes disease in humans. These include among others the analyses of grazing
ofdifferentenvironmentalamoebaonLegionellatoisolateenvironmentalprotozoa[34]or
the analyses of biofilm architecture [35–38], as this is a main mode of life of Legionella, as
wellasthequantificationofgrazingofamoebawithinthesebiofilms.
Combinedthesedifferentmethodsandanalysesshouldallowtogetabetterpictureof
the life and survival of Legionella in the environment. This is a prerequisite to develop
countermeasurestoavoidcontaminationofartificialwatersystemsanddiseaseinhumans.
Legionella Genetics
Tounderstandbacterialphysiologyandhowabacteriuminteractswithitshosttoreplicate
intracellularly and to cause disease in the case of human infection, one needs to attribute
functions to specific genes. The classical way is to disrupt/delete a gene, search for a
phenotype, and complement it to confirm its function. Classical genetics approaches can
be used to study Legionella, which are even supported by the fact that this bacterium is
naturallycompetent[39,40],andmethodshavebeendevelopedtoexploitcompetencefor
genedisruption[41].
Inthelastyears,severalnewmethodsforscar-freegenomeeditingaswellasunbiased,
high-throughputmethodshavebeendevelopedtohelpidentifynewfunctionsencodedby
L. pneumophila. Powerful high-throughput methods are the use of transposon libraries
followed by sequencing (Tnseq). However, there are important bottlenecks, if one wants
tousethesemethodstodefinefactorsimportantduringinfection.Asthousandsofmutants
needtobescreenedtoobtainstatisticallysignificantresults,thenumberofbacteriaentering
a cell in infection assays biases these analyses [42]. In order to overcome this, Shames and
colleagues developed a targeted Tnseq method, named INseq that contained the
300 known secreted effectors of L. pneumophila as a subset of transposon mutants. This
allowed,indeed,identifyingneweffector functionsnotknownbefore[43].
Unfortunately,mostifnotallgeneticmethodstomanipulateLegionellaweresetupto
manipulate L. pneumophila, the by far most studied Legionella species. However, other
species, in particular L. longbeachae, also cause considerable disease burden. Thus, under-
standingthepathogenesismechanismsofL.longbeachaeandothernon-pneumophilaLegio-
nellaspeciesisimportantforclinicalintervention.Whenprotocolsforgeneticmanipulation
viii Preface
developedforL.pneumophilawereemployedforL.longbeachae,itbecameapparentthatthis
bacterium could not be manipulated in the same way. Genome sequence analyses allowed
identifyingthereason:incontrasttoL.pneumophila,L.longbeachaeproducesacapsulethat
seemstohindertheentryofDNAbytransformationandevenbyelectroporation[44].This
knowledge paved the way for developing new methods for genetic manipulation of
L.longbeachae,whichweresuccessfulasmutantstrainslackingtheIcm/DotT4SScompo-
nentdotBor theeffectorsidCwererecentlyconstructed[45–47].
Cellular Microbiology of Legionella
Employing an apparently conserved mechanism, the facultative intracellular pathogen
L. pneumophila replicates in free-living protozoa, as well as in mammalian macrophages
[1,48].Hence,amoebaearenotonlynaturalhostcellsforLegionellaspp.butalsoversatile
modelstostudypathogen-hostcellinteractions[49].Inparticular,Acanthamoebaspp.and
the genetically tractable social amoeba D. discoideum (albeit likely not a natural host) have
beeninstrumental to gaininsightsinto cellular andmolecular aspectsof Legionella-phago-
cyteinteractions[50].L.pneumophilaemploystheIcm/DotT4SStoestablishinhostcells
an elaborate replication compartment, the Legionella-containing vacuole (LCV) [51–55].
LCVscircumvent thefusion with bactericidal lysosomesand instead extensively communi-
catewiththeendocytic,secretory,andretrogradetraffickingpathways[52,56,57].Inthe
course of LCV maturation, the nascent compartment is engulfed by the endoplasmic
reticulum(ER);yet,thebenefitsofthisprocessfor thepathogenarestillunclear.
Over the last 15 years, a number of host factor have been identified, which play
important roles for L. pneumophila-host cell interactions. These include small GTPases of
theArf[58,59],Rab[52,60],Ran[61]andRap[62]families,largedynamin-likeGTPases
such as ER-resident atlastin3 (Atl3/Sey1) [63] and mitochondrial Dnm1l [64], as well as
componentsoftheretrogradetraffickingpathway,includingtheretromercomplex[65–68]
and the phosphoinositide (PI) phosphatase OCRL [69, 70]. PI lipids represent another
crucial class of regulators of L. pneumophila-phagocyte interactions. The nascent LCV
undergoes a PI conversion from PtdIns(3)P to PtdIns(4)P and thereby is rerouted from
theendosomaltothesecretorytraffickingpathway[63,71–74].
To study Legionella-host interactions on a cellular and molecular level, a range of
powerful techniques have been established and employed. For the investigation of host
cell factors involved in LCV formation, gene silencing by RNA interference has been
implemented for Drosophila melanogaster phagocytic cells [75–77], as well as for mamma-
lian cells [60, 61, 63, 65]. L. pneumophila strains producing fluorescent proteins such as
GFP,DsRed,mCherry,mPlum,ormCeruleanarenotimpairedforvirulenceand,thus,find
multipleapplicationsinmicroscopyandflowcytometry[63,66,70,74,78,79].
Confocal laser scanning microscopy has proven very useful for the spatiotemporal
analysis of LCV formation using either fixed samples and antibodies [58, 69, 71] or live
cells [74, 80]. Live-cell imaging has the advantage of an improved preservation of cellular
structuresandarchitectureandhasbeenparticularlysuccessfulusingsinglyorduallylabeled
D. discoideum [63, 66, 74]. The applications of flow cytometry – traditionally used to
quantify uptake, intracellular replication, and cytotoxicity of L. pneumophila [71, 81] –
haverecentlybeenenhancedbyimagingflowcytometry,allowingaquantitative,non-biased
high-throughput analysis of host and pathogen factors involved in LCV formation [63,
66, 70]. Electron microscopy still provides maximum resolution for investigations of
Preface ix
morphology and ultrastructure of Legionella and Legionella-infected eukaryotic cells [63,
82–84].
Another powerful method to identify host factors implicated in LCV formation is the
isolationofintactpathogenvacuolesfollowedby(comparative)proteomics[85].Originally
establishedforD.discoideum[77,86,87],thisapproachwasmorerecentlyalsosuccessfully
implemented for macrophages [60, 62, 88, 89]. The proteomics approach allowed a
hypothesis-driven validation and characterization of small and large GTPases and other
factorsinvolvedinLCVmaturation[61,63,89].
Biochemical Assays to Study Legionella Effectors and Enzymes
TheLegionellaIcm/Dot(intracellularreplication/defectiveinorganelletrafficking)T4SSis
composedof27subunits,andrepresentsanessentialvirulencefactorthattranslocatesmore
than 300 different “effector” proteins into host cells [90]. Using transmission electron
microscopy, the biochemically isolated “core complex,” composed of the five proteins
DotC, DotD, DotF, DotG, and DotH, was found to adopt a ring-shaped architecture,
whereDotGformedacentralchannelspanningtheinnerandoutermembranes[91].Using
electroncryotomographyahigh-resolutioninsitustructureofthecomplexrevealedawell-
orderedcentralchannelthatopensupintoalarge(~32nmwide)secretionchamber witha
13-fold symmetry [92]. Molecular structures of mutant T4SSs allowed the placement of
some of the subunits in the complex. Moreover, the Icm/Dot T4SS was found to be
localized at the bacterial poles, which represents a critical requirement for the virulence
functionoftranslocatedeffectors[93].
SincethediscoveryofthefirstIcm/Dotsubstratein2002[58],substantialprogresshas
beenmade to functionally characterizethe effectorsusing structural, biochemical, genetic,
and cellular approaches [53, 94, 95]. The about 60 effectors studied to date adopt a wide
rangeof(novel)biologicalactivities,includingtheactivation/inactivationofsmallGTPases
(guanine nucleotide exchange factor, GTPase activation protein, (de)AMPylase, (de)phos-
phocholinase) [52, 59], subversion of the ubiquitination machinery (E3 ubiquitin ligase,
ADP ribosyltransferase/nucleotidase) [96–101], exploitation/modification of PI lipids
(interactors, kinases, phosphatases) [71, 73, 76, 102–104], hydrolysis of phospholipids
[105], the modulation of trafficking complexes such as the retromer [65–68, 106], the
inhibition of the ribosome [106–108], or the activation of the inflammasome [110–112].
Effector targetshavebeenidentifiedbytheabovementionedLCVpurification/proteomics
approach [61], by pull-down experiments with PI-coupled beads [45, 76], or by tandem
affinity purification/mass spectrometry using A549 cells stably producing the Escherichia
coli biotin ligase BirA [113]. In the latter approach, the cells are infected with
L. pneumophila producing the effector of choice fused to a BirA-specific biotinylation site
and a polyhistidine tag, followed by tandem Ni2+ nitrilotriacetic acid/streptavidin affinity
chromatography.
Immunity and Host Response Against Legionella
Legionella spp. are facultative intracellular and opportunistic pathogenic bacteria, which
colonize a variety of niches, including protozoa and nematodes that habitually feed on
bacteria [1, 49]. Hence, protozoan organisms represent valid and versatile models to
x Preface
investigate Legionella virulence traits as well as cell autonomous defense mechanisms [50,
110, 114]. In addition to Acanthamoeba spp., the social soil amoeba D. discoideum has
emergedasaversatilemodeltoanalyzeLCVformationandL.pneumophilavirulence(albeit
likely not a natural host of the pathogen) [50, 114]. The haploid amoeba D. discoideum is
easilygrownaxenically,andaseriesofsophisticatedgenetic,biochemical,andcellbiological
tools are available. Many defined deletion mutant strains have been produced, and the
production of single or tandem fluorescent fusion proteins allows investigating in real-
time the dynamics of L. pneumophila uptake, LCV formation, and intracellular replication
[74,80].
Inadditiontoprotozoa,nematodessuchasCaenorhabditiselegans[115–117],orlarvae
ofthewaxmothGalleriamellonella[118,119],havebeenestablishedasinfectionmodels
to investigate the molecular mechanisms of Legionella virulence. In these models
L. pneumophila virulence was dependent on the Icm/Dot T4SS and distinct effector
proteins.
MammalianmacrophagesplayacentralroleininitiatingthehostresponsetoLegionella
infection. These innate immune cells produce and activate caspase-1, which by processing
the cytokines pro-IL-1β and pro-IL-18 triggers the fulminant inflammation characteristic
for Legionnaires’ disease [112]. The activation of caspase-1 is catalyzed by the “inflamma-
some” complex, which contains the adapter proteins Asc or the Nod-like pattern recogni-
tionreceptor(NLR)proteinIpaf(Nlrc4)[120,121].TheIpafinflammasomeisassembled
upon direct binding of bacterial flagellin to Naip5 (Birc1e) [122], an NLR protein that
restrictsL.pneumophilareplicationinmousemacrophages[123–125].
Most inbred mouse strains restrict L. pneumophila infection and disease progression.
However, the A/J mouse strain is susceptible and presents with acute pneumonia that
resembles the human disease [126]. The A/J mouse strain is permissive for
L. pneumophila infection, since it harbors a Naip5/Birc1e allele that does not recognize
flagellin[127].Finally,humanlungtissueexplantshaverecentlybeenestablishedtoinvesti-
gate the virulence and pathogenesis of L. pneumophila [128]. In summary, the plethora of
cellularandmetazoanmodelsavailabletostudyvariousaspectsofLegionnaires’pneumonia
provide a solid basis for continued research aiming at a molecular understanding of the
diseaseetiology.
Metagenomics, Proteomics, and Host Microbiomes of Legionella
Therapiddevelopmentofsequencingmethodsandthepossibilitytoanalyzefromoneupto
thousandsofgenomesallowedtounderstandthegeneticbasisofvirulenceofLegionella,to
learn about the evolution of the species and the genus and to analyze the emergence of
particularly predominant clones [11, 44, 129–132]. Furthermore, proteomics approaches
are employed to study extracellular Legionella spp. [133] or L. pneumophila in protozoan
andmammalianhostcells[60,62,77,85].
LikemostorganismsLegionellaspp.arelivingincommunitiesintheenvironment,and
also during disease. Accordingly, the development of microbiome studies of the niches
Legionella inhabits during disease is now under way, as well as microbiome and metage-
nomicsanalysesofenvironmentalwatersystemstolearnaboutthecommunitycomposition
that may further or hinder the development of Legionella. Little is known about the lung
microbiomeanditsroleinhealthanddisease.Thelungisnotsterileaspreviouslythought
[134, 135], and thus, it is possible that susceptibility to respiratory pathogens such as
