Pathogenesis of Human Diffusely Adhering Escherichia coli Expressing Afa/Dr Adhesins

47 Pages·2014·4.76 MB·English

Pathogenesis of Human Diffusely Adhering Escherichia coli Expressing
Afa/Dr Adhesins (Afa/Dr DAEC): Current Insights and Future
Challenges
AlainL.Servin
CNRSUMR8076BioCIS,FacultyofPharmacy,Châtenay-Malabry,France,andUniversityParis-Sud,FacultyofPharmacy,Châtenay-Malabry,France
SUMMARY..................................................................................................................................................824
INTRODUCTION............................................................................................................................................824
EPIDEMIOLOGY ............................................................................................................................................825
Detection.................................................................................................................................................825 D
UrinaryTractInfections...................................................................................................................................825 o
Diarrhea..................................................................................................................................................826 w
n
IntestinalAsymptomaticPortage.........................................................................................................................826
VIRULENCEFACTORS ......................................................................................................................................826 lo
a
Afa/DrAdhesins..........................................................................................................................................826 d
Afaadhesins...........................................................................................................................................827 e
d
Dradhesins............................................................................................................................................828

F1845adhesin.........................................................................................................................................829 fr
o
Flagella...................................................................................................................................................829 m
SecretedAutotransporterToxin..........................................................................................................................829

Hemolysin................................................................................................................................................830 h
t
OtherFactors.............................................................................................................................................830 tp
MECHANISMSOFPATHOGENICITY .......................................................................................................................831 :
/
HostCellReceptorsforAfa/DrAdhesins.................................................................................................................831 /c
hDAF...................................................................................................................................................831 m
(i)Structureandfunctions..........................................................................................................................831 r
.
(ii)ReceptorforAfa/Dradhesins....................................................................................................................831 a
s
(iii)Receptorformicrobialpathogensandviruses..................................................................................................833 m
hCEACAMs.............................................................................................................................................833 .
o
(i)hCEACAM1structureandfunctions..............................................................................................................834
r
(ii)hCEAstructureandfunctions....................................................................................................................834 g
/
(iii)hCEACAM6structureandfunctions.............................................................................................................834 o
(iv)ReceptorsforAfa/Dradhesins...................................................................................................................834 n
(v)Receptorsformicrobialpathogens..............................................................................................................835 J
BasementmembranetypeIVcollagen................................................................................................................835 a
(cid:2)1Integrin.............................................................................................................................................835 nu
ReceptorClusteringandCellSignaling...................................................................................................................835 a
Mobilizationofadhesinreceptorsandconstituentsofcellmembrane-associatedlipidrafts.........................................................835 ry
hDAF-dependentsignaling............................................................................................................................838 2
hCEACAM-dependentsignaling.......................................................................................................................838 ,

UrinaryTractInfectionsandPregnancyComplications..................................................................................................838 2
0
Internalization.........................................................................................................................................839 1
Intracellularlifestyle....................................................................................................................................840 9
Celldetachment.......................................................................................................................................842 b
Inflammatoryresponses...............................................................................................................................842 y

AnimalmodelsofUTIs.................................................................................................................................842 g
u
PregnancyComplications................................................................................................................................843 e
IntestinalTractInfection..................................................................................................................................843 s
Structuralandfunctionalinjuriesattheintestinalepithelialbarrier...................................................................................843 t
(i)Structurallesionsatthebrushborder............................................................................................................844
(ii)Functionallesionsatthebrushborder...........................................................................................................845
(iii)Structuralandfunctionallesionsatthejunctionaldomain......................................................................................846
(continued)
[email protected].
ThisarticleisdedicatedtothememoryofArletteDarfeuille-Michaud(UMR1071
“Microbes,Intestine,InflammationandHostSusceptibility,”Inserm,Inra,and
Universitéd’Auvergne,Clermont-Ferrand,France).
Copyright©2014,AmericanSocietyforMicrobiology.AllRightsReserved.
doi:10.1128/CMR.00036-14
October2014 Volume27 Number4 ClinicalMicrobiologyReviews p.823–869 cmr.asm.org 823
Servin
Inflammatoryresponses...............................................................................................................................846
Angiogenesis..........................................................................................................................................848
EMTevents............................................................................................................................................848
pks-relatedcellinjuries.................................................................................................................................849
Hostcelldefenseresponses...........................................................................................................................850
CLINICALCONSIDERATIONS ..............................................................................................................................851
ReservoirandTransmission...............................................................................................................................851
Treatments...............................................................................................................................................851
AntibioticResistance.....................................................................................................................................851
VaccinesandPilicides....................................................................................................................................851
CONCLUDINGREMARKSANDFUTUREDIRECTIONS......................................................................................................852
ACKNOWLEDGMENTS......................................................................................................................................853
REFERENCES................................................................................................................................................854
AUTHORBIO................................................................................................................................................869
D
o
w
SUMMARY adheringE.coli(DAEC),werefirstdefinedbyJamesP.Nataroand
n
The pathogenicity and clinical pertinence of diffusely adhering JamesB.Kaper(3).Recently(4,5),aseventhgroupofentericE. lo
EscherichiacoliexpressingtheAfa/Dradhesins(Afa/DrDAEC)in colistrainshasbeendefined,theCrohn’sdisease-associatedad- ad
urinarytractinfections(UTIs)andpregnancycomplicationsare herent-invasiveE.colipathotype(AIEC)(6),whichhaveparticu- e
d
wellestablished.Incontrast,theimplicationofintestinalAfa/Dr larmechanismsofpathogenesis(7).Itisnoticeablethat,distinct
f
DAECindiarrheaisstillunderdebate.Thesestrainsareagede- fromenterovirulentE.coliinexpressingparticularvirulencede- ro
pendently involved in diarrhea in children, are apparently not terminantsanddevelopingpathogenesisinextraintestinaltissues, m

involvedindiarrheainadults,andcanalsobeasymptomaticin- ExPECstrainsincludeuropathogenicE.coli(UPEC)(8),sepsis- h
t
testinalmicrobiotastrainsinchildrenandadult.Thiscomprehen- associatedE.coli(SEPEC)(9),andneonatalmeningitis-associated tp
:
sive review analyzes the epidemiology and diagnosis and high- E.coli(NEMEC)(10). /
/
c
lightsrecentprogresswhichhasimprovedtheunderstandingof ThediffuselyadherentE.coli(DAEC)classofpathogenicE.coli m
Afa/DrDAECpathogenesis.Here,IsummarizetherolesofAfa/Dr (1,3)waspreviouslysubdividedintotwosubclasses:DAECex- r
.
a
DAECvirulencefactors,includingAfa/Dradhesins,flagella,Sat pressingAfa/Dradhesins(Afa/DrDAEC)andDAECnotexpress-
s
toxin, and pks island products, in the development of specific ing Afa/Dr adhesins (11). The subclass of DAEC that does not m
mechanisms of pathogenicity. In intestinal epithelial polarized express Afa/Dr adhesins has recently evolved. Indeed, the main .o
r
cells,theAfa/Dradhesinstriggercellmembranereceptorcluster- memberofthissubclass,i.e.,thediarrhea-associatedDAECex- g
/
ingandactivationofthelinkedcellsignalingpathways,promote pressingtheaidAgene,encodinganadhesininvolvedindiffuse o
structuralandfunctionalcelllesionsandinjuriesinintestinalbar- adherence(AIDA-I)(12–15),belongstothenewlydefinedsecond n

rier,induceproinflammatoryresponses,createangiogenesis,in- classofEPECdesignated“atypicalEPEC”(aEPEC)sinceitiseae Ja
stigateepithelial-mesenchymaltransition-likeevents,andleadto positive.TheEPECclassofenterovirulentE.colihasbeenrecently n
u
pks-dependent DNA damage. UTI-associated Afa/Dr DAEC subdividedintotwosubclasses:typicalEPEC(tEPEC)andatypi- a
strains, following adhesin-membrane receptor cell interactions calEPEC(aEPEC)(4).TheaEPECsubclass(16)compriseseae- ry

and activation of associated lipid raft-dependent cell signaling positivestrainsthatexpressawiderangeofgenes,suchasaida-1, 2
,
pathways,internalizeinamicrotubule-dependentmannerwithin fimA,ecpA,csgA,elfA,hcpA,andlda,whichcodeforknownadhe- 2
urinarytractepithelialcells,developaparticularintracellularlife- sivefactorstriggeringlocalizedadherence-like(LAL),DA,orag- 01
style,andtriggeratoxin-dependentcelldetachment.Inresponse gregative(AA)patternsofadhesion,andthatdonotexpress-bun- 9

toAfa/DrDAECinfection,thehostepithelialcellsgenerateanti- dle forming pili (BFP), a type IV pilus encoded by the EPEC b
y
bacterialdefenseresponses.Finally,Idiscussahypotheticalroleof adherencefactor(EAF)plasmid(pEAF),whichallowsintercon- g
intestinalAfa/DrDAECstrainsthatcanactas“silentpathogens” nection between bacteria within the dense microcolonies that u
e
withthecapacitytoemergeas“pathobionts”forthedevelopment formthelocalizedadhesion(LA)patternoftEPEC. s
t
ofinflammatoryboweldiseaseandintestinalcarcinogenesis. Afa/DrDAECstrainsareassociatedwithurinarytractinfec-
tions(UTIs),pregnancycomplications,anddiarrheainchildren
INTRODUCTION
ofages18monthsto5years,buttheycanalsobeasymptomatic
HumanEscherichiacolistrainsareclassifiedascommensalmi- intestinalmicrobiotastrainsinchildrenandadults(11,17).Five
crobiota E. coli, enterovirulent E. coli, and extraintestinal phylogeneticgroups,includingthemainphylogeneticgroupsA,
pathogenicE.coli(ExPEC)onthebasisoftheirgeneticfeatures B1,B2,andD,havebeenidentifiedinGram-negativespeciesus-
andclinicaloutcomes(1).Theirserotypesarebasedonvirulence ingmultilocusenzymeelectrophoresisandsequencetypingmeth-
factorspresentinsmallorlargevirulence-associatedplasmidsor ods. Afa/Dr DAEC strains belong to the phylogenetic B2 group
chromosomalpathogenicityislands(PAIs)(2)andthemolecular (18, 19). In commensal E. coli from humans (in Europe, the
andcellularmechanismsbywhichtheintestinaldiseaseisthought UnitedStates,Australia,andJapan),B2groupE.colistrainsare
to be provoked. For the pathogenic enteric E. coli strains, six predominant(20),anditisnoteworthythattheseE.colistrains
pathotypes,i.e.,enterotoxigenicE.coli(ETEC),enteropathogenic displayedahighcapacitytocolonizeepithelia(21–23).Thename
E.coli(EPEC),enterohemorrhagicE.coli(EHEC),enteroaggre- “Afa/DrDAEC”wasproposedin2005todefineafamilyofhuman
gativeE.coli(EAEC),enteroinvasiveE.coli(EIEC),anddiffusely UTI-ordiarrhea-associatedclinicalE.coliisolatesharboringad-
824 cmr.asm.org ClinicalMicrobiologyReviews
Afa/DrDAECPathogenesis
hesinsencodedbytheafa(24–28),dra(29,30),anddaa(31,32) (64);andprimersafa-fandafa-r,whichflankeda672-bpDNA
operons, having a similar genetic organization and displaying a segmentinternaltotheafaCgeneoftheafa-3,afa-7,andafa-8
similar receptor specificity for human decay-accelerating factor operons. Yamamoto et al. (65) established a multiplex PCR to
(hDAF)andmembersofthefamilyofhumancarcinoembryonic detect UPEC-associated genes, including afa genes. Multiplex
antigencelladhesionmolecules(hCEACAMs)(11).Itisimpor- PCRstodetectUPEC-associatedgeneshavebeendescribed,in-
tanttonotethatthename“Drfamily”hasbeenusedbyBogdan cludingafa1(65)orgenesexpressedbydiarrheagenicE.coli,in-
Nowickiandcoworkersasdictatedbythereceptorspecificityof cludingdaaD(66–68).
Afa,Dr,andF1845adhesinsfortheDrbloodgroupantigen(33, The specificity of the daaC probe for the characterization of
34). In this review, I summarize recent advances in our under- diarrheagenicAfa/DrDAEChasrecentlybeenquestioned.Smith
standingofAfa/DrDAECpathogenesisintheurinaryandintes- etal.(69)foundthat80ofthe86EAECstrainspositiveforthe
tinaltractsbyanalyzinghowtheAfa/DrDAECvirulencefactors AEAC probe (1-kb EcoRI-PstI fragment from pCVD432) (70)
contributetocausediseaseinhumans. also hybridized with the probe derived from the daaC gene.
Gomesetal.(71)reportedthat5ofthe197daaC-positiveE.coli
isolateshybridizedwiththeAAECprobe(eaeA).Recently,Snel-
EPIDEMIOLOGY
D
lingetal.(72)haverevealedthatthedaaCprobecross-reactswith
o
Detection strainsbelongingtoEAEC.Thisisdueto84%identitybetween w
InordertodetectE.colibearingAfa/Dradhesins,phenotypeand thedaaClocusandtheEAECfimbriaIIclustergeneaafCatthe n
lo
genotypemethodshavebeendeveloped.Scaletskyetal.(35)and nucleotidelevel(73).Moreover,Montieroetal.(74),inastudy a
Nataroetal.(36),investigatingtheadhesionofdiarrheagenicE. investigating the presence of dispersin in pathogenic and non- d
e
coli onto cultured, nonintestinal, undifferentiated epithelial pathogenic intestinal E. coli isolates, observed the presence of d

Hep-2andHeLacells,werethefirsttoobservethreespecificpat- agg3C-positiveE.colistrainsdespitetheabsenceofexpressionof fr
o
ternsofadhesion:diffuseadherence(DA),resultinginadherent thepilin-encodinggeneagg3Aandhavesuggestedthattheagg3C m
bacteriabeingrandomlydistributedonallthewholecellsurface; primersmayhavecross-reactedwithanAfa/Drusher-encoding
h
localizedadherence(LA),whereadherentbacteriaformorganized gene(s). Indeed, the biogenesis of the well-characterized EAEC t
t
p
microcoloniesrandomlydistributedonthecellsurface;andag- adhesinsAAF-I,-II,and-IIIandHda(75–78)involvedaperiplas- :
/
gregativeadherence(AA),inwhichadherentbacteriaformtypical micchaperone,anoutermembraneusherprotein,amajoradhe- /c
m
“stacked-brick” microcolonies randomly distributed on the cell sin subunit, and a capping subunit (79–81). Afa/Dr DAEC and
r
surface.However,thiscelladhesionassayisnotsuitableforthe EAECstrainsarecauseofdiarrhealillnessinyoungchildren.The .a
detection of enteric Afa/Dr DAEC, since several aEPEC strains cross-reactionmakesitdifficulttoestablishaclearidentification s
m
alsodevelopedaDApatternofadhesion(16).Moreover,DAad- ofAfa/DrDAECinrelationtodiarrhea,notablyinregionsofthe
.
o
hesionontoHep-2orHeLacellshasbeenalsoobservedforUPEC worldinwhichEAECandAfa/DrDAECstrainsareknowntobe r
g
strainsexpressingAfa-I(37),Afa-III(28),andDr(38). responsibleforacutediarrheainchildren.Itisobviousthatnew /

Goluszkoetal.(39)haveproposedaHeLacellreceptorassay PCRprobesthataremorespecificfordiarrhea-associatedAfa/Dr o
n
designated the diffuse clustering assay (DCA), which associates DAECarecalledforfutureepidemiologicalstudies.Blanc-Potard
J
the cell diffuse adhesion of Afa/Dr DAEC with the property of etal.(60)haveidentifiedM030,S109,andS111sequencesinthe a
n
Afa/Dr adhesins to promote hDAF receptor clustering around diarrhea-associated,wild-typestrainC1845.Thesesequencesare u
a
adheringbacteria(40–42).However,theDCAdidnotdetectall highly widespread (77 to 80%) among Afa/Dr strains, but have
r
y
theE.colistrainsbearingAfa/Dradhesins,consideringthatAfaE- lowprevalence(12to23%)innon-Afa/Drstrains.Additionally,
2
VIIandAfaE-VIIIadhesinsdidnotrecognizehDAF(26).Thisisa analysis shows that only the M030, S109, S111, and S164 se- ,

2
particulardrawbackforthedetectionofhumanAfaE-VIIIadhe- quencesarepresentindiarrhea-associatedAfa/DrDAECstrains
0
sin-positive ExPEC strains (43–45). Moreover, the DCA could andabsentfromnon-Afa/DrECORstrainsanddiarrhea-associ- 1
9
giveoverestimatedresultssinceseveralaEPECstrainshavebeen ated clinical isolates (60). Moreover, M030 positivity has been
b
foundtobedaaCpositive(13,46–53). foundinhumanentericDAECisolatesbelongingtophylogroups y

To detect daaC, daaE, afaB, and afaC sequences, probes and A,B2,andD(61).Incontrast,M030positivityhasbeenfoundto g
u
PCRprimershavebeendeveloped(32,45,54,55).DNAprobes beabsentinETEC,EAEC,EPEC,andEHECisolates(61).Epide- e
s
included the following: drb (56), a 260-bp PstI fragment of the miological studies associating probes designed from these se- t
pIL14 plasmid (afa-1 operon) coding for the AfaE-I adhesin of quences and associating probes specific for EAEC remain to be
uropathogenic Afa/Dr DAEC KS52 strain (25); daaC, a 300-bp conductedinareassuchasLatinAmerica,whereAfa/DrDAEC
PstIfragmentoftheplasmidpSSS1daaoperon(57);a390-bpI andEAEChavebeenfoundtobeprevalentinchildrenwithacute
fragmentofthepSLM852daaoperon(58);aDNAfragmentho- andpersistentdiarrheaillness(seebelow).
mologous to daaE (59); and a probe designed from the M030
sequencefoundtobespecificallypresentinwild-type,diarrhea- UrinaryTractInfections
associatedAfa/DrDAECstrainC1845(60,61). TheroleofUPECexpressingAfa/DradhesinsinrecurrentUTIs
PCR approaches have been developed, including primers de- has been clearly established (82, 83). afaBC/daaC positivity has
signedtoamplifya750-bpfragmentoftheafaBgene(62)andthe beenfoundinUPECstrainsbelongingtotheB2phylogroups(61).
390-bp PstI fragment of the pSLM852 daa operon (63). Others Epidemiological studies show that E. coli isolates expressing
PCRassayshavebeendevelopedtodetectalltheAfa/Dradhesins, Afa/Dradhesinsareinvolvedincystitisinchildren(25to50%)
including the afa1 and afa2 primers designed on the partial se- andpyelonephritisinpregnantwomen(30%)(34,53,55–57,84–
quenceoftheafa-1geneoperonoverlappingtheafaBandafaC 93). In addition, these pathogenic E. coli strains cause UTIs in
genes(54),primersforafaE-I,afaE-II,afaE-III/draE,andafaEV pregnantwomen(30,90,91,94–97).InpatientswithafirstUTI,
October2014 Volume27 Number4 cmr.asm.org 825
Servin
thepresenceofE.coliisolatesexpressingAfa/Dradhesinsleadsto ingtheUnitedKingdom(134)andFrance(135,136).InAfa/Dr
an elevated occurrence of a second UTI (56, 57, 64, 98, 99). In DAEC strains isolated from stools of children, the sat gene has
patientswithpyelonephritis,therewasavariabledistributionof beenfoundinalmosthalfofthediarrhea-associatedAfa/DrDAEC
afaEsubtypesinafa-positivestrains(100).Zhangetal.(64)have strainsandwasnotpresentinallthenon-diarrhea-associatedAfa/
found that UTI-associated and fecal E. coli isolates were afaE1 DrDAECstrains(137).WhyandhowAfa/DrDAECisolatesare
positive(18%),afaE2positive(1.3%),afaE3positive(1.3%),draE potential pathogens in children with an age-dependent occur-
positive(12%),daaEpositive(1.3%),anddraE-afaE3hybridpos- renceremaintobedetermined.Apossibleexplanation,butone
itive(12%).SomehumanpyelonephritisE.coliisolateshavebeen thatdoesnotexcludeotherpossiblecauses,isthatinchildrenin
foundtobepositiveforafa1-afa2andafa-f-afa-rPCRprobesand thisagerange,theintestinalepithelialbarrierisnotstructurally
insomecasestoexpresstheafaE1(5%),afaE8(39%),andafaEX and functionally mature, and therefore the strong host defense
(20%)operons(45).AUTI-associatedE.colistraingenerallyex- responsesagainstinfectionbyAfa/DrDAEC,whicharedescribed
pressesamultiplicityofadhesivefactors.Foxmanetal.(92),ana- below,arenotyetfunctional.
lyzing E. coli strains isolated from women with first-time UTIs, The observation of daaC positivity of some aEPEC strains is
observedthatdrb-probepositiveE.coliisolatesdisplayedpositiv- indicativethatAfa/DradhesinsareexpressedbyenterovirulentE.
D
itywiththetype1pilusprobe(80to100%positivity)andtheP colistrainsotherthanAfa/DrDAEC(13,46–53).Moreover,E.coli
o
fimbriaprobe(50%positivity)andnopositivityfortheSfimbria isolatesdisplayingdaaCandafaBCpositivitieshavebeenfound w
probe.Szemiakoetal.(101)haveobservedthatcombinationsof amongAIEC,inflammatoryboweldisease(IBD)-associated,and n
lo
genesencodingtwoadherencefactors(PandDrfimbriaeorSand intestinalcancer-associated(6,138–140)strains. a
Dr fimbriae) in UTI-associated E. coli isolates result in an in- d
e
creased risk of translocation to the vascular system, leading to IntestinalAsymptomaticPortage d

bacteremia.Moreover,daaC-positive,UTI-associatedclinicaliso- Mostoftheepidemiologicalstudiesconductedinvariousareasof fr
o
lateshavebeenfoundtoexpressaerobactin(89),hemolysin(56, theworldthatwereintendedtoidentifyAfa/DrDAECascauseof m
86,89,90,92,102,103),andcytotoxicnecrotizingfactor(CNF) diarrheainchildrenover5yearsofageandinadultswereincon-
h
(19,56,89,92).Thesamenumbersofstrainsexpressingthedrb clusive,asthesamenumbersofdaaC-positivestrainswerefound tt
p
probehavebeenfoundinisolatesfromtheurinarytractorrectum incasesandcontrols(13,48,50,71,99,107–115,141).Thisob- :
/
ofwomenwithUTIs(104). servationhighlightstheexistenceofasymptomaticcarriersofin- /c
m
Afa/DradhesinsarefrequentlyfoundinE.coliassociatedwith testinalAfa/DrDAECstrainsandsuggeststhatthesepathogenicE.
r
pyelonephritisinpregnantwomenwithgestationalcomplications colistrainscanbetoleratedorcontrolledifthematureintestinal .a
(30,62,90,96,97).Inaddition,Afa/DrDAECstrainsareassoci- epithelialbarrierisinahealthycondition. s
m
atedwithpretermlabor/birth(82,95,105).Sledzinskaetal.(97)
.
o
havereportedthepresenceofanE.colistrainharboringthecom- VIRULENCEFACTORS rg
binationofPandDrfimbriaeinacaseoffatalsepsisinapregnant /

womanwhodevelopedpyelonephritis. Afa/DrAdhesins o
n
TheprocessesbywhichepitheliaareinfectedbypathogenicE.coli
J
Diarrhea startbytheattachmentofbacteriatospecifichostcells.Todothis, a
n
ItiswellestablishedthatpathogenicETEC,tEPEC,aEPEC,and pathogenicbacteriaexpressawidevarietyofsurface-exposedad- u
a
AIECcolonizethesmallintestine,EAECcolonizessmallintestine hesinsresponsibleforspecificbindingtostructuralorfunctional
r
y
and/or colon, EHEC colonizes the distal ileum and colon, and cellmembrane-associatedmolecules(142).Theattachmentsonto
2
EIECcolonizesthecolon(4,5).Incontrast,theintestinalsite(s) thetargethostcellsallowentericandurinarytractbacterialpatho- ,

2
colonizedbydiarrhea-associatedAfa/DrDAECcurrentlyremains genstoresistclearancebyperistalsisandmicturition,respectively.
0
tobedetermined.ThereisanabsenceofaroleofAfa/DrDAECin The bacterial adhesion to target host cells can be more than a 1
9
diarrheainadults.Indeed,whenthewild-typestrainC1845was simpleattachmentduetopathogen-specificrecognitionofhost
b
inoculated in adult volunteers, none of the patients developed cellmembrane-associatedmolecules,sinceseveralofthesemole- y

diarrhea, despite the strain being detected in duodenal cultures culesfunctionedintrinsicallyassignalingmoleculesorafterrec- g
u
and stools (106). Moreover, examination of a large number of ognition/activationrecruitedcytosolicsignalingmolecules(143). e
s
humandiarrhea-associatedDAECstrainshasshownthatonlytwo Attachment by fimbrial or afimbrial structures allows bacterial t
carried the daaE gene, suggesting that the F1845 fimbria is rare pathogenstointeractwiththehostcellmembranetoensurethe
amongdiarrheagenicDAECstrains(59).Inaddition,epidemio- optimaldeliveryoftheircytotonicorcytotoxictoxinsinthevicin-
logicalstudiesinvariousareasoftheworldareinconclusivewith ity of their membrane-associated receptors, triggering signaling
regardtoaroleofdaaC-positiveE.colistrainsindiarrheainchil- eventsthataffecttransport/secretionfunctionsorthecellstruc-
drenoradults(13,48,50,99,107–115).However,therelationship turalorganization.Forotherpathogenicbacteria,adhesivefactors
betweenAfa/DrDAECanddiarrheainchildrenasafunctionof allowtheintimateassociationofbacteriawiththecellmembrane
agehasbeenmoreconvincinglydemonstratedinagecross-sec- that is necessary for the initiation and completion of signaling-
tionalstudiesshowinganincreasedincidenceinchildren(cid:3)1to5 controlled structural lesions, which in turn dramatically impair
yearsofage.ThesestudieswereconductedintheUnitedStates hostcellfunctions.Forinvasivebacterialpathogens,attachment
(116),Mexico(117),anddifferentSouthAmericancountries,in- initiatesanorderlyseriesofsignaling-controlledeventsthatlead
cluding Chile (118), Brazil (58, 71, 119–121), Colombia (122), tohostcellmembranerearrangementsthatarenecessaryforthe
Peru(123–127),andArgentina(128),aswellasinThailand(50), achievementofbacterialcellentryfollowedbythedevelopmentof
Bangladesh(129),Japan(119),NewCaledonia(45,63,130),var- sophisticatedbacterialintracellularlifestyles.
iousplacesinAfrica(131–133),andEuropeancountries,includ- Twomajorclassesofadhesinsarepresentonthebacterialsur-
826 cmr.asm.org ClinicalMicrobiologyReviews
Afa/DrDAECPathogenesis
TABLE1CharacteristicsofAfa/DradhesinsandAfa/Dr-relatedadhesins
Receptors
Adhesin Type Host TypeIVcollagen hDAF hCEACAMs
AfaE-I Afimbrial Human Negative Positive Positive
AfaE-II Afimbrial Human Unknown Positive Unknown
AfaE-III Afimbrial Human Negative Positive Positive
AfaE-V Afimbrial Human Unknown Positive Positive
AfaE-VII Afimbrial Bovine Unknown Negative Unknown
AfaE-VIII Afimbrial Human/animal Unknown Negative Negative
Dr Fimbrial Human Positive Positive Positive
Dr-II Afimbrial Human Negative Positive Negative
F1845 Fimbrial Human Negative Positive Positive
Distantmembersa
NFA-I Afimbrial Human Unknown Positive Unknown D
o
AAF-I Fimbrial Human Unknown Unknown Unknown w
AAF-II Fimbrial Human Unknown Unknown Unknown n
AAF-III Fimbrial Human Unknown Unknown Unknown lo
HdaA Fimbrial Human Unknown Unknown Unknown a
d
aLikeAfa/Dradhesins,AAF-I,-II,and-IIIandHdaApromoteanMRHAphenotypeinhumanerythrocytes(78). e
d

f
r
o
m
faceofGram-negativepathogens:thefimbrialadhesins,consist- havebeenusedtodefinethefunctionaldomainsoftheDraEand
h
ingoflinearhomopolymersorheteropolymers,andtheafimbrial AfaE-III-Dscadhesins,whicharerequiredforbindingtohostre- t
t
p
adhesins,formedofsingleproteinsorhomotrimers(142).Forthe ceptorssuchashDAF(157–160),hCEACAMs(160),andcollagen :
/
completionoffimbrialandafimbrialadhesinsinGram-negative type IV (161, 162), and to explain the differential sensitivity to /c
m
pathogens, different secretion systems have been identified, in- chloramphenicol(161,162).
r
cludingSec-independentandSec-dependentpathways(144).The Afaadhesins.AgnèsLabigneandChantalLeBouguénechave .a
major families of adhesive proteins (145) include the classical extensively described the pathogenicity mechanisms of E. coli s
m
chaperone/usher pathway-dependent fimbrial adhesins (146, strains bearing the afimbrial adhesins (Afa) encoded by the afa
.
o
147), the alternate chaperone/usher pathway-dependent E. coli operons (Table 1). The first Afa adhesin was isolated from the r
g
surface pili (148), the extracellular nucleation precipitation-de- wild-typeprototypeUPECstrainKS52byLabigneetal.(25,37). /

pendentcurlyorthinaggregativefimbrialadhesins(149),thetype The6.7-kbchromosomalDNAfragmentessentialforamannose- o
n
Isecretionsystem-dependentafimbrialadhesins(150),thetype resistanthemagglutination(MRHA)phenotypeinhumaneryth-
J
IIIsecretionsystem-dependentintegraloutermembraneproteins rocytes and for adhesion onto uroepithelial cells contains five a
n
(151),thepolymerization-assembledtypeIVpili(152),andthe genes:afaA,afaE,afaD,afaB,andafaC(24).AdhesinsAfaE-IIand u
a
type V secretion system-dependent nonfimbrial trimeric auto- AfaE-IIIwerethenisolatedfromtwootherUPECstrains,A22and
r
y
transportedadhesins(153). A30,byLabigneetal.(84).LeBouguénecetal.(27)isolatedfrom
2
TheAfa/Drfamilyofadhesinscontainsfimbrial(29,32,75,76, theUPECstrainA30a9-kbplasmidregioncontainingtheafa-3 ,

2
154, 155) or afimbrial (25–27, 30, 37, 44, 64, 84, 156) adhesins genecluster.Theafa-3geneclustercontainssixgenesdesignated
0
(Table1).Theseadhesinsareencodedbygenespresentinoperons afaAtoafaF(163).TheAfaE-IIIandDraEadhesinsubunitsdis- 1
9
containingfivemajorgenes,includinghighlyconservedgenesA played98%identity(of160aminoacids,157aresimilar)(27,30),
b
toD,encodingaccessoryproteins,andmoredivergentgenesE, andtheafa-3geneclusteranddaaoperonarecloselysimilar(31, y

encoding the adhesin subunits (Fig. 1A). Assembly via the FGS 32, 164, 165). The atomic resolution structure for the AfaE-III g
u
(with a short F1-G1 loop) and FGL (with a long F1-G1 loop) subunithasbeendetermined(158,159,166,167).Nuclearmag- e
s
classesofperiplasmicchaperoneshasbeendescribed,andtheFGL netic resolution and biophysical studies have revealed that the t
chaperone/usherproteinsecretionsystemassemblesAfa/Drad- structural organization of Afa-III adhesin develops by assembly
hesins (146, 147 ) (Fig. 1B). The structural organization of long ontothebacterialmembraneusherofAfaE-IIIadhesinssubunits
andshortAfa/Dradhesinsdevelopsbytheassemblyofthebacte- (158,159)cappedbytheAfaD-IIIsubunit(166–168).TheAfaD-
rialmembraneusher,successiveEadhesinsubunits,andoneD IIIsubunitalsohastheabilitytoseparatefromtheDrfimbriae
subunit capping the structure. It is interesting to note that the (42).Adiffuseandnotwell-orderedcellsurfacelocalizationofthe
chaperoneusherfunctionsinUPECtoformadhesivestructures AfaD-IIIsubunithasbeenobservedbyimmunoelectronmicros-
suchasthePpili,resultingintheorderlyassemblyofPapA,PapK, copy(42,168,169).Usingchimerasconstructedfromtheafa-3
PapE,PapF,andadhesivePapGsubunits,andtype1pili,formed anddaaoperons,astudyhasrevealedthattheafimbrialorfim-
byassemblyofFimA,FimF,FimG,andadhesiveFimHsubunits brialmorphologiesoftheadhesinswereinfluencedbytheorderin
(146,147).ThePapGsubunit,localizingatthetipofthefimbria, the genes coding for the afimbrial or fimbrial adhesin subunits
triggersrecognitionofhostcellmembrane-associatedgloboseries (28).TheAfaE-IIIadhesinsubunitisinvolvedinrecognizinghost
glycolipids,andtheFimHsubunittriggersthemannose-depen- cellreceptors(158,159)suchastheDraEandDaaEadhesinsub-
dent recognition. Crystallographic and nuclear magnetic reso- units(170).Asshownintheanalysisofepidemiologicalstudies
nance(NMR)studiescoupledornotcoupledwithmutagenesis below, Afa-possessing E. coli strains have been found to be ex-
October2014 Volume27 Number4 cmr.asm.org 827
Servin
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
c
m
r
.
a
s
m
.
o
r
g
/

o
n

J
a
n
FIG1GeneticorganizationofAfa/Droperons(A)andassemblyofDradhesinviathechaperone-usherpathway(B). u
a
r
y

2
pressedbyUPEC,diarrhea-associatedE.coli,andE.coliisolated opsMRHA,doesnotexerthemolyticactivity,anddoesnotpro- ,

2
fromthefecesofasymptomaticpatients. ducecolicin(155)(Table1).StrainIH11128exhibitsmannose-
0
Theafa-7andafa-8operonsencodetheAfaE-VIIandAfaE- resistant Dr fimbriae (29). Five Dr operon-associated proteins 1
9
VIII adhesins (26, 44) (Table 1). Like the Afa-III adhesin, the withmolecularmassesof15.5,5,18,32,and90kDaarenecessary
b
Afa-VIIandAfa-VIIIadhesinscanaggregatetoformamorphous forthedevelopmentofthecompleteMRHA(55). y

masses(26,42).AmongthefoursRNAgenes(171)presentinthe TheroleoftheFGLchaperone/usherbiogenesispathwayofDr g
u
PAI strain and expressed by afa-8-positive E. coli (44), the fimbriae has been investigated in detail (174–178). To allow e
AL862
s
AfaRsmallRNA,thetranscriptionofwhichistemperaturecon- properfoldingoftheDraEadhesinsubunitstooccur,theDraC t
trolled,regulatestheexpressionoftheAfaD-VIIIsubunit(172).It ushercreatesanassemblyandsecretionplatform,andpremature
isnoticeablethatdespitethepresenceoftheAfa-VIIIadhesinin DraEsubunit-subunitassociationispreventedbytheDraBchap-
human intestinal E. coli isolates (43), these E. coli isolates have erone. When the DraC is lacking, there are no protein subunit
neverbeenfoundtoberesponsiblefordiarrheainhumans(45). secretion and fimbria assembly, since the protein complexes
Incontrast,incalves,pigs,andpoultrywithdiarrheatherewasthe amass in the periplasm. Mutagenesis of the DraC N terminus
presence of E. coli isolates expressing sequences of the afa-8 shows that DraC-F4A, DraC-C64, DraC-C100A, and DraC-
operon(173). W142Aplayapivotalroleinthebioassemblyoffimbriae.Inthe
Dr adhesins. Bogdan Nowicki and coworkers have magnifi- caseoftheEsubunit,twoconservedcysteineresiduesforminga
centlydemonstratedtheroleofE.coliexpressingDradhesinsin disulfidebondareimportantforstabilizingelementsoftheim-
pyelonephritis,recurrentbladdercystitis,andpregnancycompli- munoglobulinfoldoftheDrfimbriae.WithregardtotheDraD
cationsandhavealsodissectedtheirmolecularandcellularmech- subunit(179),ithasbeenreportedthatwhentheDraEsubunit
anismsofinfection.Thehumanpyelonephritis-associatedwild- assembles,theDraDsubunitlocalizesatthetipofthefiber(158,
type prototype UPEC strain E. coli IH11128 (O75) has a K5 159,166,167,180).Jedrzejczaketal.(181)haveshownthatthe
capsule,lackstheflagellarantigen,expressesatype1pilus,devel- DraDsubunitlocalizesatthetip,becauseitlacksadonorstrand
828 cmr.asm.org ClinicalMicrobiologyReviews
Afa/DrDAECPathogenesis
and as a consequence functions only as an acceptor. However, regulatedbybothphasevariationandenvironmentalregulatory
expressionoftheDraDsubunithasbeenfoundtobeindependent mechanisms.White-Ziegleretal.(193)havereportedthatinre-
oftheDraCusher,andDraDappearsnottobenecessaryforthe sponsetomultipleenvironmentalsignals,thehistone-likeH-NS
polymerization of DraE subunits (182). Recently, Zalewska- actsasanoverallregulatorbycontrollingtranscriptionofthedaa
Piateketal.(183)showedthattheDraDsubunitcanbeproduced operon.
byachaperone/usher-independent,typeIIsecretion-dependent
processthatallowsthetranslocationoftheDraDsubunitontothe Flagella
cellsurfaceofDr-positiveE.coli. Thebiogenesisofflagellainvolvesthecoordinatedstructuralas-
ThemajorstructuralsubunitDraEisinvolvedinhostcellre- semblyofflagellarproteins(195).Avarietyofflagellarstructural
ceptorrecognition(170),asaretheAfaE-III(158,159)andDaaE proteins and capping proteins compose the flagellar propeller
adhesinsubunits(170).Calculationoftheelectrostaticpotentials (195), and cytoplasmic membrane proteins compose the force-
oftheDraEstructureshowsanelectronegativeareaaroundthe generatingunitoftheflagellarmotor(196).Inanaqueousenvi-
cluster of amino acids involved in binding onto hDAF (Asp61, ronment,manybacterialspeciesmovebyrotatingtheirflagella,
Asp63,andAsp75)(157).InthegenesencodingDrfimbriae,sin- allowingindividualbacteriatoswiminthreedimensions(197).
D
gle-nucleotidepolymorphismsconferringanadaptiveadvantage Moreover,flagellarswarmingcoordinatesthemovementofbac-
o
havebeenidentified(184).DrfimbriaeareuniqueamongAfa/Dr teriaacrossthehostcellsurface(198).FlagellaexpressedbyUPEC w
adhesins in expressing chloramphenicol sensitivity for binding contributetocolonizationoftheepithelium,disseminationtothe n
lo
onto host cell receptors, whereas binding of Afa-I to -III and kidney by ascending progression from the bladder, and biofilm a
F1845isnotaffected(27,185,186).Korotkovaetal.(187)have formation(199).IthasbeenobservedthatUPECstrainsexpress- d
e
interestinglyshownthatgenesencodingDrfimbriaeformeight ing type 1 pili or P fimbriae are less flagellated and display re- d

structuralgroupsdisplayingahighlevelofaminoacidsequence pressed motility, suggesting that when fimbrial expression is fr
o
diversityamongthem.Itisnoticeablethatafunctionalanalysis switchedoff,UPECstrainsaremotile(200,201).Afa/DrDAEC m
has revealed the presence of distinctly different binding pheno- strainsexpressordonotexpressflagella.Theprototypepyelone-
h
typescontrollingaffinitytohDAF,capabilitytobindcollagentype phritis-associated, wild-type Afa/Dr DAEC strain IH11128, ex- t
t
p
IV and hCEACAMs, and sensitivity of adhesiveness capacity to pressingatype1pilus,doesnotpossessflagellarantigens(155).In :
/
chloramphenicol.SincetheAfaD/DraD/DaaDsubunitslocalizeat contrast, the UPEC wild-type strain A30, which does express /c
m
thetipoftheAfa/Drfimbriae,theirpossibleinvolvementinadhe- AfaE-III adhesin, is positive for flagellar antigen (unpublished
r
sionontoepithelialcellsand,inaddition,thatoftheAfaE/DraE/ data),andtheanimalwild-typeAfa-VIII-positivestrainAL511is .a
DaaEsubunitshasbeenenvisaged.Conflictingresultshavebeen H8positive(202).Itisworthmentioningthatthattheprototype s
m
obtained. Recombinant DraE(cid:4)/DraD(cid:5) or AfaE(cid:4)/D(cid:5)-III E. coli diarrheagenicwild-typeAfa/DrDAECstrainC1845(32)doesnot
.
o
failedtoadheretodifferentiatedprimarybladdercells(188)and express flagellar antigens (unpublished data). According to r
CPiHatOek-heDtaAl.F(-1(cid:6)950(cid:2))1hcaevlelsr(e1p8o9r)t,erdetshpaetctiinveHlye.LIancceollns,trDarsat,EZ(cid:4)a/lDewrasDka(cid:5)- pAoriskitaiwvea,edtiaalr.r(h2e0a3-)a,sosonclyiasteevdenE.ocfotlhiei1so9laatfeasE1th-,eayfaeEx2am,oirnaefadEaXr-e og/
n
E. coli displays a low level of adhesion, (cid:7)3-fold lower that of motile.Incontrast,Merazetal.(107),whoexamined18DAEC
J
DraE(cid:5)/DraD(cid:5)E.coli.Incontrasttothechloramphenicol-sensi- isolates,foundthatallninediarrhea-associated,afaE1-orafaEX- a
n
tive adhesion of DraE, the DraD-induced binding is chloram- positive E. coli isolates are motile. These findings indicate that u
a
phenicolinsensitive(190). UPEC and diarrhea-associated Afa/Dr DAEC display heteroge-
r
y
TheDr-IIadhesinhasbeenisolatedfromthehumanpyelone- neousflagellumexpression.
2
phritis-associated strain EC7372 (Table 1). Compared to the ,

membersoftheAfa/Dradhesinfamily,theDr-IIadhesindisplays SecretedAutotransporterToxin 2
0
poorsequenceidentity(17to20%)(30).Dr-IIhas96%identity Secretedautotransportertoxin(Sat)belongstothetypeVsecre- 1
9
withthenonfimbrialadhesinI(NFA-I)expressedbyUTI-associ- tionpathway-dependentsubfamilyofserineproteaseautotrans-
b
atedE.coli(191).Interestingly,NFAsandAfa/Dradhesinshavea portersofEnterobacteriaceae(SPATE)toxins(81,153,204).Asthe y

verysimilargeneticorganization,andthenfageneclusterencodes resultofdifferencesinthetoxinsstructuresandactivities,there g
u
NfaAsubunitsassembledviathechaperone-usherpathway(191). aretwoclassesofSPATEtoxins.ClassIincludesplasmid-encoded e
s
F1845adhesin.SteveL.Moseleyandcoworkersdiscoveredthe toxin (Pet) of EAEC, extracellular serine protease, plasmid en- t
diarrhea-associatedE.coliexpressingF1845adhesinandbeauti- coded(EspP)ofEHEC,EspCofEPEC,SigAofShigellaflexneri
fully described the structural aspects of the interaction between andEAEC,SatofintestinalE.coliandExPEC,andthehypothet-
Afa/Dr adhesins and their epithelial cell hDAF and hCEACAM icalEspC-likeSPATEtoxinswithEcPCN033-C1sp(NCBIacces-
receptors. The human wild-type prototype diarrheagenic strain sionnumberEGP21815.1)ofExPEC,EcNA114-C1sp(NCBIac-
C1845expressesafimbrialadhesin,designatedF1845(Table1). cession number AEG39156.1) of UPEC, and EcM605-C1sp
TheorderandregulationofthegenesnecessaryforF1845adhesin (NCBIaccessionnumberZP_08351236.1)ofAIEC(204).ClassII
assembly have been identified (31, 32, 164, 165, 192–194). The includesproteininvolvedinintestinalcolonization(Pic)ofShi-
F1845andDradhesinsdisplay57%identity(91aminoacidsof gella,EAEC,andUPEC,SepAofShigella,EatAofETEC,vacuo-
160 are identical) (30). Five polypeptides (10, 95, 27, 15.5, and latingautotransportertoxin(Vat)-liketoxinsofUPEC,SEPEC,
14.3 kDa) are encoded by daaA, daaB, daaC, daaD, and daaE and NEMEC, EcRN587-C2sp (NCBI accession number
genes, respectively. The major structural subunit, DaaE, is in- EFZ76879.1)ofEAECandEPEC,andEpeAofShigatoxin-pro-
volvedinhostcellreceptorrecognitionliketheAfaE-III(158,159) ducingE.coli(204).ClassISPATEtoxinsaregenerallycytotoxic,
andDraEadhesinsubunits(170).Bilgeetal.(31)havedemon- whereasclassIIdisplaydiverseactivities,includingthecleavageof
stratedthatthefimbrialgeneexpressioninthe daaoperonwas mucus,whichprovidesacompetitiveadvantageforhostepithe-
October2014 Volume27 Number4 cmr.asm.org 829
Servin
liumcolonization(81,204).Thesatgenehasbeencharacterizedin hlyA,hlyD,hp1-hp4,papG,orpapF sequences(60).Aremnantof
theUPECprototypestrainCFT073(205),whereitresideswithin thepapoperonwhichhastheF10papAallelebutlacksmostofthe
PAI-II (206–209).ThesatgeneisprevalentinUPECstrains, centralregionofthepapoperonhasbeendetected.Itisnotewor-
CFT073
including those bearing Afa/Dr adhesins (8, 56, 137, 206, 208, thythatregionsofthePAI completegenomesequence(207,
CFT073
210–214),residentintestinalmicrobiotaE.colistains,andpatho- 209,224)havebeenfoundinE.colistrainsoftheB2phylogenic
genicstrainsofE.coli,includingEAEC(137,215–218),andShi- group (208) and are prevalently expressed in ExPEC strains of
gellaisolates(219,220).Thesatgenehasbeenfoundpresentin groupB2involvedinUTIs(8).Moreover,partsofPAI have
CFT073
daaC-positiveE.colistrainsisolatedfromstoolsofchildrenwith recentlybeenfoundinintestinalcommensalE.colistrains,partic-
diarrhea in Brazil and France (137, 218, 221). In Afa/Dr DAEC ularlythoseofphylogenicgroupB2(225–227),andinanAIEC
isolates,thesatgenehasbeenfoundtobeexpressedequivalently strain(228).
bydiarrheicandasymptomaticadults(222).Interestingly,thesat The PAI expressed by afa-8-positive E. coli strains (44)
AL862
geneisprevalentlyexpressedinAfa/DrDAECisolatedfromchil- includesthedeoKgene,whichconfersmetabolicadaptabilityand
dreninacontextofdiarrhea(222). increasesthecompetitiveadvantagewithregardtohostinfectivity
(229). The locus designated vpe (virulence-associated phospho-
Hemolysin D
transferase)containsthevpeA,vpeB,andvpeCgenes,whichen- o
ThepyelonephritogenicstrainEC7372,whichexpressesDr-IIad- code,respectively,theEIIA,EIIB,andEIICconstituentsofapu- w
hesin(30),istheonlyAfa/DrDAECstrainthatproducesafunc- n
tativecarbohydrate-specificpermeaseoftheSgaTBAfamily(230). lo
tional hemolysin. Indeed, unlike other Afa/Dr DAEC strains, This locus is present in the pyelonephritis-associated strain a
strain EC7372 promotes a strong cellular lysis in epithelial cells d
AL511,whichexpressestheafa-8operon(43),whichconfersan e
precededbyapoptosis(102).Onthebasisofresultsreportedby d
abilitytoadaptforkidneyandintestinalcolonization(231).The

Blanc-Potard et al. (60), the hemolysin-positive strain EC7372 presenceofthevpelocusinotherUTI-anddiarrhea-associated fro
carriesboththehlyandpapoperonsandseemstohaveacquireda Afa/DrDAECstrainshasnotbeendocumented. m
largerpartofthePAIs (207–209)thanAfa/DrDAEC.The
CFT073 Thecapacitytoformfilamentousformsresultsfromaplasticity h
recombinantE.colistrainEC901,whichcarriesplasmidpBJN406 t
capacitydevelopedbyabacterialpathogeninordertoescapehost tp
andcontainsthedraAto-EgenesinvolvedinexpressionofDr defenseswheninanintracellularlocationortoassembletoforma :/
fimbriae(223),hasbeenobservedtodisplayacurioushemolytic /c
biofilm-like structure that leads to resistance to anti-infective m
activity.InsertionmutationsindraDanddraE,butnotindraA,
treatments,suchasantibiotics(199).Someexcellentexperiments r
draB, and draC, abolish hemolytic activity, indicating that this havedemonstratedthattype1pilus-positiveUPECstrains,after .a
activityissupportedbytheextracellulardomainofDrfimbriae. s
internalizationintosuperficialepithelialcellsknownas“umbrella m
This observation is intriguing, since strain IH11128, gestational
cells”liningtheluminalsurfaceofthebladder,formbiofilm-like .o
pyelonephritis Dr-positive E. coli isolates (94, 155), and clinical r
bacterialassemblagesdesignated“intracellularbacterialcommu- g
Dr-positiveE.coliisolates(60)alllackeitherhemolyticactivityor /
hly gene expression. In contrast, the wild-type O75X strain nities”(IBCs)thatfunctionastransientprotectivestructuresfor o
UPECintracellulargrowth(199,232).UPECcellsinIBCsconsti- n
IH11032 does display hemolytic activity (155). Moreover, four
tute reservoirs of UPEC, which, after switching to filamentous J
afaE1-positive and one afaEX-positive diarrheagenic E. coli iso- a
forms,becomedetachedfromthebacterialcommunityandmay n
lateshavebeenfoundtotriggerhemolysis,while14otherafaE1- u
beflushedoutofthehostcells.Zalewska-Piateketal.(233)were a
positiveandoneafaEX-positiveisolatesdonot(203).Collectively,
r
thefirsttoobservethatthatDr-positiveE.coliformedbiofilms. y
thesefindingsshowthatAfa/DrDAECstrainsareheterogeneous
intermsof(cid:6)-hemolysinexpression,suggestingavariabledistri- ThisphenomenonmeansthatDr-positiveE.colistrainsformlive 2,
filamentousbacteria,dependingontheirnutritionalenvironment 2
butionofthepartofPAI containingthehlygeneamongthe
Afa/DrDAECstrains. CFT073 (190,233).IthasbeenobservedthatadheringDr-positiveE.coli 01
formsfilamentousformsatthecellsurfaceofCHO-hDAF-(cid:6)5(cid:2)1 9

OtherFactors (189) or CHO-hDAF (234) cells. Filamentous bacteria residing by
Blanc-Potardetal.(60)identifiedseveralshortsequences(73to withinthephagosomeescapedphagosomalkillingasthebacteria g
495bp)thatareprevalentinAfa/Dradhesin-positiveE.coliclin- manipulatedthephagosomecompartmentbyblockingtheacqui- ue
icalisolatesincomparisonwithE.coliclinicalisolatesnotexpress- sitionofhydrolyticcomponents(235,236).Eventhoughthein- st
ingAfa/Dradhesins(GenBankaccessionnumbersAZ935556to tracellular vacuole-containing Dr-positive E. coli in HeLa cells
AZ935604).Severalsequencesarehomologoustovirulencegenes lackthecharacteristicsofadegradativecompartment(189),no
expressedinotherpathotypesofE.coli,includinggenesfortwo filamentousformsofAfa-orDr-positiveE.coliresidingintracel-
siderophores (irp2 and iuc), a catechol siderophore receptor lularlyhaveeverbeenobserved.ThisaspectofAfa/Drpathogen-
(iroN),andtwotransportsystems(shuandmodD)(60).Interest- esisremainstobeexploredintheappropriatemodelofbladder
ingly, several C1845-specific sequences display no likeness with epithelialcells.BacterialbiofilmformedbyUPECafteraggrega-
knownsequences(60).Importantly,thediarrhea-associatedwild- tionofthree-dimensionalstructuredcellsconnectedbyself-pro-
typeC1845straindoesnotexpressthegenesencodingETECand ducedexopolysaccharidematrixplaysamajorroleinpersistent
EAECvirulencefactorsandisdevoidofgenesencodingEPECand and chronic UTIs (199). Exopolysaccharide production, which
EHECvirulencefactors,includingthegenesofthelocusofentero- playsapivotalroleinbiofilmcompletion,hasbeenfoundinUTI-
cyte effacement (LEE) island involved in the type III secretion associated E. coli strains expressing Dr (190, 233) or Afa-VIII
system(T3SS)orT3SS-associatedeffectorproteinsandnothy- (231) adhesins. Interestingly, exopolysaccharide production is
bridizedwitheaeprobes(60).Thewild-typeC1845andIH11128 controlledbythevpeBCgene(231),whichispresentinthevpe
strainsexpressedapartofPAI (207–209)notincludingthe locusofafa-8-positiveE.coli(43).DraE(cid:5)/DraD(cid:5)E.colistrains
CFT073
830 cmr.asm.org ClinicalMicrobiologyReviews
Afa/DrDAECPathogenesis
formdensebiofilms,andDraD,whetherassociatedwithfimbriae theintestinalmucosaviaadhesins(18,75–78),(ii)theformation
ornot,playsaroleinbiofilmformation(190,233). oftypical“stacked-brick”microcoloniesaseachbacteriuminter-
Alargevarietyofbacteriahavebeenfoundtoproducetoxins, actswithothers,(iii)productionofenterotoxinsandcytotoxins,
named cyclomodulins, that dramatically interfere with the cell and(iv)thedevelopmentofaseveremucosalinflammation.Boi-
cycle (237). Cyclomodulins produced by pathogenic E. coli in- senetal.(78),analyzingthissuperfamilyofadhesins,havepro-
cludedcolibactin,cycle-inhibitingfactor(Cif),cytotoxicnecrotiz- posedapertinentphylogramcomposedofthreedistinctclusters.
ingfactor(CNF),andcytolethaldistendingtoxin(CDT)(238). ThefirstclustercomprisesAfa-I,Afa-II,Afa-III,Afa-V,Dr,Dr-II,
Currently, the two known genotoxins are colibactin and CDT and F1845, the second comprises AAF-I, AAF-II, and AAF-III,
(238).Theclusterofgenesknownasthe“pksisland”(239)en- andthethirdcomprisesAfa-VII,Afa-VIII,andHda.Itisworth
codes a multienzymatic machinery for synthesizing the hybrid, mentioningthatcluster3(78)alsoincludesthenonfimbrialM-
nonribosomal,peptide-polyketidegenotoxincolibactin(240).It agglutininencodedbythebmageneclusterofUPEC(250).
hasbeensuggestedthatthepksislandmayaffectthehostimmune hDAF.Nowickietal.(33)werethefirsttoreportthathuman
responseandcouldbeinvolvedinchronicinflammation,inthe decay-acceleratingfactor(hDAF)(CD55)expressingtheantigens
accumulation of genomic instability, and in tumor progression oftheCromerbloodgroupsystem(251)actsasanepithelialcell
D
(241).Whetherthepksislandcontainsothergenesencodingad- receptorforE.coliexpressingAfa/Dradhesins(Fig.2)(Table1).
o
ditionalbacterialfactorsandwhetherthepks-relatedcolibactinis (i)Structureandfunctions.DAFisacomplement-regulating w
aprototypeofafamilyofmoleculesornotremaintobeinvesti- proteinwithanM of55,000to70,000(251).Thephysiological n
r lo
gated.ThepksgenomicislandispresentintheprototypeAfa/Dr functionofDAFistocontroltheamplificationofthecomplement a
DAEC wild-type IH11128 and C1845 strains (J. P. Nougayrede cascade by a direct interaction with membrane-bound C3b or d
e
andE.Oswald,unpublishedresult)andincolonicafa-I-positiveE C4b,whichinturnimpedestheulterioruptakeofC2andfactorB. d

colistrainsisolatedfrompatientswithIBDandcolorectalcancer Membrane-boundDAFisformedbyamembraneglycosylphos- fr
o
(140). The pks island has been also found in ExPEC strains of phatidylinositol (GPI) anchor followed by a serine/threonine/ m
phylogeneticgroupB2(242),infecalE.colistrainsisolatedfrom proline(STP)-richregionandbyfourcomplementcontrolpro-
h
healthypatientsbutnotinpathogenicEPECandEHECisolates tein repeat (CCP) domains, previously named short consensus t
t
p
(243),ingroupB2E.colistrainsthatarelong-termcolonizersof repeats(SCRs)(Fig.2).Modelingoftheextracellulardomainof :
/
theintestine(22),inE.coliisolatedfromthemucosaofpatients DAFrevealsthatCCPsareorganizedinahelicalmanner.While /c
m
with IBD (244), in mucosa-associated or internalized E. coli of CCP-1 had no effect on hDAF regulatory activity, deletion of
r
tumorsandmucosaofcolorectalcancerpatients(244–246),and CCP-2,CCP-3,orCCP-4entirelyabolishedtheregulatoryactiv- .a
inurosepsisE.colistrains(247).Itwasnotedthattheintestinal ity.InteractionofDAFwiththeconvertasesismediatedpredom- s
m
microbiota E. coli strain Nissle 1917 expresses the pks genomic inantly by two patches approximately 13 Å apart, one centered
.
o
islandanddisplayssimilaritieswiththeprototypeAfa/DrDAEC aroundArg69andArg96onCCP-2andtheotheraroundPhe148 r
g
wild-type C1845 and IH11128 strains, since it harbors parts of andLeu171onCCP-3(252).Phe123andPhe148,localizingatthe /

PAI thatlacktheexpressionof(cid:6)-hemolysinandPfimbriae interfacebetweenCCP-2andCCP-3,andalsoPhe154,whichis o
CFT073 n
butincludesironuptakesystems(225,227).ThisprobioticE.coli presentintheCCP-3cavity,arepivotalfortheregulatoryactivity
J
strainwithdiverseactivities(248)isintriguingsinceitspromotion (253). The GPI anchor increases the lateral mobility of DAF a
n
ofguthomeostasisactivityinresponsetomucosalinjurycannot withinthecellmembraneinrelationtoitslocalizationintomem- u
a
bedissociatedfromthepresenceofthepksisland(249).Whether brane-associatedlipidrafts,andtheO-glycosylatedSTPservesasa
r
y
thepresenceofthepksislandinintestinalE.coliandExPECstrains spacerfortheprojectionofthehDAFfunctionaldomainsatthe
2
isdeleteriousforthehostorwithoutpathologicalconsequences cellmembrane(253). ,

2
remainstobeinvestigated. (ii)ReceptorforAfa/Dradhesins.hDAFisoneofthereceptors
0
recognizedbyAfa/Dradhesinsinepithelialcells(Fig.2)(Table1). 1
9
MECHANISMSOFPATHOGENICITY ItisnoteworthythatAfa/DradhesinsbindspecificallytohDAF b
butnottorodentorpigDAF(254).Drfimbriabindingdevelops y
HostCellReceptorsforAfa/DrAdhesins inthedigestive,urinary,genital,andrespiratoryepitheliaandin g
u
Onthebasisofthedifferentialrecognitionofhumanepithelialcell skin(255),consistentwiththehDAFexpression(251).Onlyuro- e
s
membrane-associated receptors by Afa/Dr adhesins (Table 1), pathogenic and diarrhea-associated E. coli strains bearing the t
Afa/Dr DAEC strains have been subdivided into two subclasses F1845, AfaE-I, AfaE-III, AfaE-V, Dr, and Dr-II adhesins recog-
(11).ThefirstsubclassincludesE.colistrainsharboringtheAfa-I nizedhDAFasareceptor(62,160,256).Incontrast,theAfa-VIII
(25,37),Afa-II(27),Afa-III(27),Afa-V(64),Dr(29,155),Dr-II adhesin expressed by human ExPEC does not recognize hDAF
(30),andF1845(32)adhesinsrecognizinghDAF,whichalsomay (26,43,44).Ithasbeenestablishedfromfunctionalstudiesand
or may not recognize members of the hCEACAM family. The atomic resolution models that Afa/Dr adhesins recognize the
secondsubclassincludesstrainsthatexpressAfa-VII(26,44)and CCP-2andCCP-3onhDAF(41,158,159,257–259)(Fig.2).In
Afa-VIII(26,44)adhesinsthatdonotrecognizehDAF.Inaddi- contrast, gestational pyelonephritis-associated E. coli expressing
tion, the NFA-I adhesin of UPEC (191) belongs to the Afa/Dr dra-relatedXadhesinsrecognizedtheCCP-3andCCP-4domains
familyofadhesins(Table1).Moreover,despiteasimilargenetic ofhDAF(62).IntheCCP-3,asinglepointsubstitution(Ser155-
organization with the gene clusters triggering the biogenesis of AlaandSer165-Leu,mimickingtheDra-to-Drballelicpolymor-
Afa/Dr adhesins, the EAEC adhesins AAF-I (77), AAF-II (76), phisms)resultsinacompletelossofDrfimbriabindingtohDAF
AAF-III(75),andHda(78)aredistantpathogenicfactorsofthe (257, 260). The amino acids (148 to 171), in particular Ser155,
Afa/Drfamilyofadhesins(Table1).Thefourmajorcharacteris- presentatthesurfaceofCCP-3controlledtheDradhesinbinding
ticsofEAECpathogenesis(79–81)areasfollows:(i)adherenceto (260).AsurfaceplasmonresonancestudyofAfa-IIIadhesinbind-
October2014 Volume27 Number4 cmr.asm.org 831
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o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
c
m
FIG 2Membrane-associated proteins expressed by human epithelial cells that function as receptors for Afa/Dr adhesins. Center, representations of the
r
structuresofhDAFandhCEACAMs.Left,surfacerepresentationofhDAF.Right,homologymodelofhumanN-CEA.(RepresentationsofhDAFandN-hCEA .a
reprintedfromreference158withpermissionofElsevierandfromreference160withpermissionofthepublisher,respectively.) s
m
.
o
r
g
ing onto CCP domains of hDAF has revealed that a construct changesatpositions10,63,65,75,77,79,and131ofthemature /

formedofCCP-1and-2didnotshowanymeasurablebindingto DraEsequence(261). o
n
theAfaE-IIIadhesinsubunit,whileconstructsformedofCCP-2, BindingoftheDraEadhesinsubunitontohDAFissensitiveto
J
-3,and-4,CCP-2and-3,orCCP-3and-4allowedAfaE-IIIbind- chloramphenicol, which also inhibits the hDAF-dependent a
n
ingwithaffinitiescomparabletothatfortheentirehDAF,con- MRHAofhumanerythrocytes(chloramphenicol-sensitivehem- u
a
firming the previously observed importance of the combined agglutination[CSHA])(33,256).InHeLacells,thepresenceof
r
CCP-2 and CCP-3 domains for the recognition of hDAF by chloramphenicol diminished the adhesion of DraE(cid:5)/DraD(cid:5) E. y
Afa/Dradhesins(158,159).TheDradhesin-bindingandcomple- coli by (cid:7)3-fold and totally abolished the adhesion of DraE(cid:5)/ 2,

ment-regulatingepitopesofhDAFhavebeenfoundtobedistin- DraD(cid:4) E. coli but did not change the adhesiveness capacity of 2
0
guishableandareapproximately20Åapart(260).However,An- DraE(cid:4)/DraD(cid:5)E.coli(190).AccordingtoSwansonetal.(262),the 1
9
dersonetal.(158)observedthatthebindingofAfaE-IIItohDAF domainsinvolvedintheCSHAarepresentwithintheN-terminal
b
antagonizedthehDAFregulatoryactivity. domainoftheDraEsubunit.AccordingtoPettigrewetal.(161, y

TheAfaE-I,AfaE-III,AfaE-V,DraE,andDaaEsubunitsfunc- 162), a hydrophobic pocket including Gly113, Gly42, Pro40, g
u
tionasreceptorligandsforhDAF(27,160,170)(Fig.3AtoD).In Pro43,Ile111,Tyr115,andIle114playsapivotalroleinthechlor- e
s
DraE/AfaE-IIIsubunits,thehDAF-bindingsiteformsalargecon- amphenicol-bindingsiteintheDraEsubunit.Theinhibitionof t
vex surface involving seven (cid:2) strands (158, 159). The residues thebindingoftheDraEsubunitontohDAFbychloramphenicol
Asp61, Ile73, and Asn77 have been found to be important for hasreceivedastructuralexplanation,sincebycoveringthefunc-
binding to hDAF (158, 159). Mutagenesis and crystallographic tionalportionoftheadhesinsubunit,chloramphenicoldisrupts
studiesofDaaEhavebeenconductedinordertodefinethede- therecognitionofhDAF(161,162).Incontrasttothecaseforthe
tailedmolecularinteractionsbetweenAfa/DradhesinsandhDAF Dr adhesin, chloramphenicol does not affect the hDAF-depen-
(157).FivedaaEmutants(T8N,A60V,D61A,D63V,andT133S) dentMRHAexertedbytheAfaE-I,AfaE-III,andF1845adhesins
showeda30to50%reducedabilitytobindontoCHOcellstrans- (27,256).Thisisaresultofadifferenceinexpressionofamino
fectedforhDAFexpression(157).MappingthesitesofDaaEre- acidsbetweentheadhesinsubunits(161,162).Moreover,ithas
vealsthatpositionsAsp61andAsp63arenecessaryforbindingto beenestablishedthatbindingofchloramphenicolontotheDraE
hDAF,andcalculationoftheelectrostaticpotentialsoftheDaaE subunit develops via the interaction of its chlorine “tail” rather
structurehasrevealedanelectronegativeregionaroundthecluster than its benzene ring (161, 162). Analyzing structural chloram-
ofaminoacidsinvolvedinhDAFrecognition(Asp61,Asp63,and phenicolmodifications,Pettigrewetal.(162)havedemonstrated
Glu126)(157).Moreover,theabilityoftheDraEadhesintobind thatacylatingthe3-hydroxylgrouphasnoeffectonthebinding
hDAF has been found influenced by individual amino acid ontohDAF.
832 cmr.asm.org ClinicalMicrobiologyReviews