Variability of the response of human vaginal Lactobacillus crispatus to 17β-estradiol 2021 Maximilien Clabaut.pdf

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OPENVariabilityoftheresponse
ofhumanvaginalLactobacillus
crispatusto17β‑estradiol
MaximilienClabaut1,,AmineBenMlouka2,AmandineSuet3,AliTahrioui1,
JulienVerdon4,MagalieBarreau1,OlivierMaillot1,AgatheLeTirant5,MadinaKarsybayeva6,
CoralieKremser7,GérardRedziniak8,CécileDuclairoir‑Poc1,ChantalPichon3,JulieHardouin2,
PascalCosette2,SylvieChevalier1&*
Wepreviouslyshowedthatthephysiologicalconcentrationof17β‑estradiolinthevaginal
environmentissufficienttoaffectthemembranedynamicsandadhesionphenotypeofthe
,,
weinvestigatedtheeffectof17β‑,

thesamemedium,,incontrast
toCIP104459,‑estradiolpromotedthis
‑surfacepolarityandLewisacid/base
,weobservednoeffectonthemembranedynamics,contrarytoCIP104459.
TheseresultscanbeexplainedbydifferencesinthepropertiesandorganizationoftheSlayerbetween
,asforCIP104459,17β‑estradiolincreasedbiosurfactantproductionofL.
‑estradiolagonistswouldbe
valuabletoolstofavorastablere‑.
Thevaginalmicrobiotaofhealthynon-pregnantreproductiveagedwomenischaracterizedbyalimitednumber
ofbacterialspecies,mostofwhichbelongtotheLactobacillusgenus,,,
,,,
heterogeneous,,thereappearstobecompetitionbetweenL.
crispatusvariants,resultinginamutualexclusionprocessandconsequentlytohighlyhomogeneousandalmost
,,areknown
,asrecentlydemon-
strated,suchantimicrobialactivityisalsostraindependent5,possiblyexplainingthevariabilityoftheprotection
.
Theseobservationsareessential,asvariousstrategieshavebeendevelopedtomaintainorre-implantL.
crispatusinthevaginalmicrobiota,particularlyinpost-menopausalwomen,butareoftennotsuccessfulover

,sub-micromolarconcentra-
tionsof17β-estradiolaffectbacterialmembranehomeostasisandpromotebiosurfactantproduction,leading

bysignaltransductionandaputative17β-estradiolsensorprotein,themembranelipidrafts-associatedSPFH
domain-containingprotein,showinghomologywiththeeukaryoticestrogen-relatedreceptorgamma(ERR3)
andtheestradiolbindingproteinprohibitin-2(PHB2),.
However,ourknowledgeofthedirectimpactofestradiolonLactobacillusphysiologyisstillverylimited,and

1LaboratoryofMicrobiologySignalsandMicroenvironment(LMSMEA4312),RouenNormandieUniversité,
55rueSaint-Germain,27000Evreux,«Polymères,Biopolymères,Surfaces»(UMR6270
CNRS),ProteomicPlatformPISSAROUniversityofRouenNormandy,Mont-Saint-Aignan,
BiophysiqueMoléculaire,UPR4301FrenchNationalCentreforScientificResearch,Orléans,
EBI,UMRCNRS7267,UniversitédePoitiers,Poitiers,,Porcheville,
Laboratory,Paris,,Longjumeau,,Antony,France.*email:
marc.******@univ-
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ofhighimportance,,whichcould
,theresponsetohostfactorscan
differbetweenstrainsandevenribotypes,asdemonstratedforskinassociatedbacteria10.
Here,wetookadvantageoftheidentificationandavailabilityofthedraftgenomesequenceofanewvaginal
,designatedasV411,,particularly
-estradiol
wassubsequentlystudiedusingproteomic,physiological,physicochemical,morphological,andcellbinding
approaches.
Results

-
sibleintheRefSeqNCBIReferenceSequenceDatabase(accessedonNovember3rd,2020).Thepercentageof
,forwhichthe
responseto17β-estradiolwaspreviouslystudied8,%( 1).Agenome-to-genomedis-
tanceanalysisindicatedaninsilicoDNA-DNAhybridizationvalue(isDDH)%.Bothresultssuggesta
,thetwostrainsarerelatedtodifferent
phylogeneticclusters(Fig. 1).,

,whichcontain2178and2034openreadingframes(ORFs),respec-
tively(Fig. 2).Thecoregenomesharedbythetwostrainsislimitedto1638ORFs(%of
theaveragecompletegenomes),
(540and386,respectively).Noticeably,full-lengthcopiesofgenesequencessimilartotheglgXgene,encoding
aglycogendebranchingenzymedesignatedasatype1pullulanase,werepresentinboththegenomesequences
-basedgenomeannotationandmanualcuration,thegenome
sequencesofbothstrainsalsoappearedtocontaingenesencodingotherputativeglycosyltransferases,including
csbB,epsH,andglyD,.

bynanoLC–MS/,420±6

proteome(374)wererelatedto14differentfunctions,includingDNAsynthesis,transcriptionandregulation
(25),translation,ribosomalstructureandbiogenesis(73),posttranslationalmodification,proteinturnover,and
chaperones(32),energyproductionandconversion(21),carbohydratetransportandmetabolism(56),amino-
acidtransportandmetabolism(46),nucleotidetransportandmetabolism(15),inorganiciontransportand
metabolism(3),fatty-acidandphospholipidmetabolism(11),secretionandexport(1),membranefunction(1),
Slayer,cellwall,andenvelopebiogenesis(16),andadaptationandprotection(32).Wealsodetectedhypotheti-
calproteins(42)( 2).
similar(409±4).However,only246(outof420,.,%)
,whereas332proteinsofCIP104459(%)weredetectedintheV4

the332proteinsoftheV4proteomecommontoV4andCIP104459,226showeda>
(%)wereoverexpressedinV4( 33).Remarkably,onlyoneproteininvolvedinproteinsecre-
tion,thedipeptide-bindingproteinDppE,wasdifferentiallyexpressedinV4(×).Twomembraneproteins
werealsooverexpressed,particularlytheoligopeptide-bindingproteinOppA(×).Envelope-associated
proteins,suchasFtsZ,alsoappearedtobeupregulatedinV4.
QuantificationofproteinsexpressedbyLcrispatusV4intheabsenceorpresenceof17β-estradiolshowed
>twofold,consideredtobethelimitof
significance,–8 Mof17β-estradiolandonlyoneprotein,elongation
factorG(MHJPMHAL_00289),showedanincrease>twofoldafterexposureto10–10 Mof17β-estradiol(+).
17β‑
that10–6to10–10 Mof17β-( 1A).
.
-estradiol(Fig. 3A).We
quantifiedtheaggregationphenotypeusingthesedimentationtechnique14(Fig. 3B),whichshowedaninverted
dose-relatedeffectfor17β--estradiolat10–6 Mhadnodetectableeffect,whereaswe
observedanon-significantincreaseoftheaggregationphenotypewithbacteriaexposedto10–8 M17β-estradiol.
17β-estradiolat10–10 ,reaching+±6%of
thecontrol(p<).
analysis(Fig. 3C).Themeansizeofdetectedstructureswasplottedonthehorizontalaxis(forwardscattering
fractionofthelight,FSC)andthesurfaceheterogeneity(granularity)ontheverticalaxis(sidescatterlight,
SSC)–6 M17β-estradiolresultedinasignificantincreaseinmeanparticlessize
(+±%ofthecontrol;p<).Thesignalwasdividedbetweentwopeaks,thesmalleronelikelycor-
,thesurfaceheterogeneityofdetectedparticleswashigherthanthat
ofthecontrol(p<).Weobservedthesametendencyforbacteriaexposedto10–8 M17β-estradiolandthe
FSCsignalwasdividedbetweentwopeaks,asbefore,whereasthesurfaceheterogeneityrevertedtoaunique
group,–10 M17β-estradiolatshowed
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Figure (ANI)illustratingthephylogenetic
relationshipsbasedonthepercentageidentity[high(red)tolow(blue/grey)]sharedbetweenthewhole
sequenceofLactobacilluscrispatusV4(blueboxesandarrows)
genomesequenceswereaccessibleintheNCBIRefSeqdatabase(accessedon3rdNovember,2020),including
thatofLactobacilluscrispatusCIP104459fromClabautet (yellowboxesandarrows).Thephylogenictree
wasrootedusingthegenomesequenceofanunrelatedbacterialspecies,presentlyLeuconostocmesenteroides
subspeciesmesenteroides,ATCC8293.
(aggregates)wassignificant(p<),as
observedwith10–6 M17β-estradiol.
17β‑estradiolaffectsthesurfacepolarityandLewisacid/
-surfaceintheaggregationphenotype16,westudiedthesurface
(MATS)-
ityforapolarsolvents,particularlychloroform(±%)anddecane(±%).Conversely,itsaffinityfor
ethylacetate,amorepolarsolvent,waslimited(±%)(Fig. 4A).Exposureto17β-estradiolinducedagen-

andchloroform,,
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