Type III Secretion Effectors with Arginine N-Glycosyltransferase Activity
Abstract
Type III secretion systems are used by many Gram-negative bacterial pathogens to inject proteins, known as effectors, into the cytosol of host cells. These virulence factors interfere with a diverse array of host signal transduction pathways and cellular processes. Many effectors have catalytic activities to promote post-translational modifications of host proteins. This review focuses on a family of effectors with glycosyltransferase activity that catalyze addition of N-acetyl-d-glucosamine to specific arginine residues in target proteins, leading to reduced NF-κB pathway activation and impaired host cell death. This family includes NleB from Citrobacter rodentium, NleB1 and NleB2 from enteropathogenic and enterohemorrhagic Escherichia coli, and SseK1, SseK2, and SseK3 from Salmonella enterica. First, we place these effectors in the general framework of the glycosyltransferase superfamily and in the particular context of the role of glycosylation in bacterial pathogenesis. Then, we provide detailed information about currently known members of this family, their role in virulence, and their targets
Full text
Mic oo ganisms2020,8,357;doi:10.3390/mic oo ganisms8030357www.mdpi.com/jou nal/mic oo ganisms
Re iew
TypeIIISec e ionE ec o swi hA ginine
N‐Glycosyl ans e aseAc i i y
JuanLuisA aujo‐Ga ido,JoaquínBe nal‐Baya dandF anciscoRamos‐Mo ales*
Depa amen odeGené ica,Facul addeBiología,Uni e sidaddeSe illa,41012Se illa,Spain;
[email p o ec ed](J.L.A.‐G.)[email p o ec ed](J.B.‐B.)
*Co espondence:[email p o ec ed]
Recei ed:12Feb ua y2020;Accep ed:29Feb ua y2020;Published:2Ma ch2020
Abs ac :TypeIIIsec e ionsys emsa eusedbymanyG am‐nega i ebac e ialpa hogens oinjec
p o eins,knownase ec o s,in o hecy osolo hos cells.These i ulence ac o sin e e ewi ha
di e sea ayo hos signal ansduc ionpa hwaysandcellula p ocesses.Manye ec o sha e
ca aly icac i i ies op omo epos ‐ ansla ionalmodi ica ionso hos p o eins.This e iew ocuses
ona amilyo e ec o swi hglycosyl ans e aseac i i y ha ca alyzeaddi iono N‐ace yl‐
D
‐
glucosamine ospeci ica ginine esiduesin a ge p o eins,leading o educedNF‐κBpa hway
ac i a ionandimpai edhos celldea h.This amilyincludesNleB omCi obac e oden ium,NleB1
andNleB2 omen e opa hogenicanden e ohemo hagicEsche ichiacoli,andSseK1,SseK2,and
SseK3 omSalmonellaen e ica.Fi s ,weplace hesee ec o sin hegene al amewo ko he
glycosyl ans e asesupe amilyandin hepa icula con ex o he oleo glycosyla ioninbac e ial
pa hogenesis.Then,wep o idede ailedin o ma ionabou cu en lyknownmembe so his
amily, hei olein i ulence,and hei a ge s.
Keywo ds:glycosyl ans e ases; ypeIIIsec e ion;e ec o s;NleB;SseK;Ci obac e ;Esche ichia;
Salmonella;dea hdomains
1.In oduc ion
TypeIIIsec e ionsys ems(T3SSs)a e undamen al ools o hein e ac ionbe weenmany
pa hogenicandsymbio icG am‐nega i ebac e iaand hei hos s.T3SSs,alsoknownasinjec isomes,
span hebac e ialinne andou e memb anesand o minjec ionnanomachines ha deli e p o eins,
knownase ec o s,in o a ge euka yo iccells omodula ea a ie yo cellula unc ions[1,2].
E ec o sha edi e seac i i iesand a ge s,and ypicallyha eanN‐ e minalsec e iondomainand
oneo mo eC‐ e minal unc ionaldomains[3,4].E ec o scanbeclassi iedin ou non‐mu ually
exclusi eca ego iesacco ding o hei mechanismso ac i i y[5]:(i)e ec o s ha media e hei
ac i i ies h oughphysicalin e ac ionswi hhos a ge s;(ii)e ec o s ha use unc ionalo s uc u al
mimic y oal e hos cellp ocesses;(iii)e ec o s ha p omo epos ‐ ansla ionalmodi ica ionso
hos p o eins;and(i )e ec o swi hp o easeac i i y.Exampleso ca aly icac i i ieso e ec o sa e
ubiqui ina ion,deubiqui ina ion,phospho yla ion,dephospho yla ion,ace yla ion,deamida ion,
ADP ibosyla ion,cys eineme hyla ion,andglycosyla ion.This e iew ocusesonaspeci ic amily
o T3SSe ec o swi hglycosyl ans e aseac i i y ha a e oundinSalmonellaen e icaandin
a aching/e acing(A/E)pa hogens.
Salmonellaea e acul a i ein acellula G am‐nega i ebac e ialpa hogensbelonging o he
amilyEn e obac e iaceae.ThegenusSalmonellaincludes hespeciesS.bongo iandS.en e ica, he
la e di idedin osixsubspeciesandmo e han2500se o a s[6].S.en e icapossesses wo i ulence‐
ela edT3SS[7–9],T3SS1andT3SS2, ha a eencodedbySalmonellapa hogenici yisland1(SPI1)and
Salmonellapa hogenici yisland2(SPI2)(Figu e1), espec i ely,whe easS.bongo ilacksSPI2[10].
Mic oo ganisms2020,8,3572o 23
TheT3SS1 ansloca ese ec o sac oss hehos cellplasmamemb ane ha p omo eSalmonella
in asionin oepi helialcellsin hein es ineands imula elocalizedin lamma ion[11].TheT3SS2is
exp essedin acellula lyin esponse oacidicpHandnu ien limi a ion oundin helumeno he
Salmonella‐con aining acuole(SCV),amodi iedphagolysosomewhichis he ypicalin acellula
nicheo hispa hogen[12].T3SS2e ec o sa e ansloca ed h ough heSCVmemb ane,a e
impo an inin es inalanddissemina edin ec ions,anda e equi ed o g ow hwi hindi e en hos
cell ypes[13].Toge he ,T3SS1andT3SS2sec e emo e han40e ec o s,someencodedbySPI1o
SPI2,bu mos encodedou side heseislands,ino he ho izon allyacqui ed egionso hegenome
[14–17].
En e ohemo hagicEsche ichiacoli(EHEC)anden e opa hogenicE.coli(EPEC)a eex acellula
humanpa hogens ha a ach o hein es inalepi heliumande aceb ushbo de mic o illi o ming
A/Elesions[18].EHECcauseshemo hagiccoli isandhemoly icu emicsynd ome,whe easEPEC
isacauseo in an iledia hea.Ci obac e oden iumisano he A/Epa hogenwhichis hecausa i e
agen o mu ine ansmissiblecolonichype plasia[19].LesionsinducedbyC. oden iuminmicea e
simila o hosecausedbyEHECo EPECinhumansand hesepa hogenssha eanumbe o
i ulencede e minan sincluding helocuso en e ocy ee acemen (LEE)[20,21](Figu e1).TheLEE
isa34kbpa hogenici yislandwi h41genes ha encodeall hes uc u alcomponen so aT3SS,
se eno i se ec o sand hei chape ones,p o einsin ol edinadhe ence,and egula o yp o eins
[22].Manye ec o sa eencodedou side heLEE,and he o alnumbe o e ec o sisa leas 29inC.
oden ium,22inEPEC,and39inEHEC[22].Asecondgeneclus e ,po en iallyencodingaT3SSand
knownasETT2, o E.coliT3SS2,isp esen inmanyE.colis ains[23,24].ETT2 esembles he
SalmonellaT3SS1bu inmos casesi hasbeensubjec ed omu a ionala i ionand he eisno
e idence o hesec e iono e ec o sby hissys em[25].Howe e , his egionisin ac insome
s ainso E.coliandEsche ichiaalbe ii,andappea s obeimpo an o bac e ialpa hogenesis[25–
27].
Figu e1.Gene ics uc u eo Salmonellapa hogenici yisland1(SPI1)andSalmonellapa hogenici y
island2(SPI2)o S.en e icase o a Typhimu iums ain14028,and helocuso en e ocy ee acemen
(LEE)islando E.coliO127:H6s ainE2348/69.Genesa ecolo edacco ding o hei unc ional
ca ego ies[22,28].
Mic oo ganisms2020,8,3573o 23
2.Glycosyl ans e ases
Glycosyl ans e ases(GTs)ca alyze he o ma iono glycosidebondsusinganac i a eddono
suga subs a e ha con ainsanucleosidephospha eo alipidphospha elea ingg oup[29].
Enzymes ha usesuga mono‐o diphosphonucleo idesasdono sa eknownasLeloi GTs[30].
Non‐Leloi GTsusenon‐nucleo idedono s,suchaspolyp enolpy ophospha es,polyp enol
phospha es,suga ‐1‐phospha es,o suga ‐1‐py ophospha es.Suga scanbe ans e ed oa a ie y
o biomoleculesincludingglycans,lipids,p o eins,pep ides,andsmallmolecules.The esul ing
glycosidiclinkagecanbeanO‐,S‐,N‐o C‐co alen bondconnec ingamonosaccha ide oano he
esidue.
2.1.Classi ica ion
T adi ionally,GTswe eclassi iedon hebasiso hei dono ,accep o ,andp oduc speci ici y,
acco ding o he ecommenda ionso heIn e na ionalUniono Biochemis yandMolecula Biology
(IUBMB),unde heEnzymeCommissionnumbe EC.2.4.Gi en helimi a ionso hissys em,anew
scheme ha in ol es heclassi ica iono GTsin o amilieson hebasiso aminoacidsequence
simila i iesisnowaccep ed[31,32].Today, heca bohyd a eac i eenzymes(CAZy)da abase
(h p://www.cazy.o g)includeso e 620,000GTmodulesclassi iedinmo e han100 amiliesand
mo e han13,000nonclassi iedmodules.
Dependingon hei mechanismo ans e ,GTscanbeclassi iedasin e ingo e aining,
esul inginin e sion(α‐>β)o e en ion(α‐>α)o hes e eochemis yo hedono ’sanome ic
bonddu ing he ans e , espec i ely[33].Theo e icalandexpe imen als udiessuppo asingle‐
displacemen SN2(subs i u ion,nucleophilic,bimolecula )‐likemechanism o in e ingGTs[29].
Thismechanismin ol es he o ma iono anoxoca beniumion ansi ions a e(Figu e2).Incon as ,
he eac ionmechanismo e ainingglycosyla ioniscon o e sial[34].Adouble‐displacemen
mechanism,inwhich hesuga is i s ans e ed o heenzymeand hen ans e ed o heaccep o ,
wassugges edbyanalogy o e ainingglycosidases[35].Someexpe imen alsuppo wasp o ided
o hismechanism[36,37],pa icula ly o heGT6 amilymembe s ha ha eaca boxyla ep ope ly
o ien ed oac asaca aly icnucleophile.Bu hisisno hecase o mos e ainingGTs,leading o he
p oposalo anal e na i ein e nal e u nSNi(subs i u ion,nucleophilic,in e nal)‐likemechanism,
also e med on ‐ acemechanism,inwhich henucleophilichyd oxyg oupo heaccep o a acks
heanome icca bona om om hesameside omwhich helea ingg oupdepa s[38,39].An
o hogonalmechanismhasalsobeenp oposed ha di e s om heSNimechanismin he eac ion
p o ileand he imeo bond o ma ionandb eakage[40](Figu e2).
Figu e2.Glycosyl ans e asemechanisms.AnSN2(subs i u ion,nucleophilic,bimolecula )p ocessis
accep ed o in e ingglycosyl ans e ases(GTs).Se e almechanismsha ebeenp oposed o
e ainingenzymes.Theo hogonalmechanismisdepic edhe e.‡:T ansi ions a e.ImagebyD .
Mic oo ganisms2020,8,3574o 23
B ockSchumann(WikimediaCommons,CCBY‐SA3.0,h ps://c ea i ecommons.o g/licenses/by‐
sa/3.0/deed.en).
2.2.S uc u alS udies
GTs uc u es(Figu e3) e ealed ha hemajo i yo heseenzymes allin o wogene al olds,
calledGT‐AandGT‐B,eacho whichincludesmembe s ha a e e ainingandin e ingGTs.This
led oaclassi ica iono GTsin ou clans:clanI o in e ingenzymeswi h heGT‐A old,clanII o
in e ingenzymeswi h heGT‐B old,clanIII o e ainingenzymeswi h heGT‐A old,andclanIV
o e ainingenzymeswi h heGT‐B old[32].Bo h old ypescon ainaRossmann old[41],a
nucleo ide‐bindingdomain esponsible o dono nucleo ide ecogni ion.Aseconddomainisusually
esponsible o accep o ecogni ionandismo e a iable.Enzymeswi h heGT‐B ype oldcon ain
woRossmann‐like oldssepa a edbyadeepwidec e ice[42].Howe e ,no allGTs i in o hese
clans.A hi d old ype,calledGT‐C old,wasp edic edusingcompu a ionalme hods[43–45],and
hes uc u eo se e alGTswi h his old,mos o hembelonging o amilyGT66,ha ebeen
desc ibed[46].TheseGTsusuallycon ainanN‐ e minal ansmemb anedomainandaC‐ e minal
globula domainwi hGTac i i y.Inaddi ion, hes uc u eo heenzymeDUF1792 e ealedano el
olddesigna edasGT‐D ype[47]andde ined amilyGT101.Membe so heGT51 amilyalsoha e
adis inc s uc u ede inedaslysozyme‐ ype[48,49],and he ecen lypublisheds uc u eo TagA,a
membe o heGT26 amily,sugges s hede ini iono ano el old e medGT‐E[50].Acommon
ea u e oundinmanymembe so heGT‐AandGT‐Csupe amiliesisaDxDmo i in ol edin
binding odi alen me alionandca alysis.Howe e , heoccu enceo hismo i isno enough o
p edic apo en ialGT unc ionsincemanyGTsdono possessi and heDxDmo i is oundinmo e
hanahal o alldesc ibedp o eins[32].Finally,gi en hemodula na u eo ca bohyd a e‐ac i e
enzymes,anumbe o GTscon ainnonca aly icdomainspo en iallyin ol edinsubs a e
ecogni iono p o ein–p o einin e ac ion,andsomepolypep idescon ain woGTmodules
belonging o hesameo di e en amilies.
Figu e3.Glycosyl ans e ase olds.(a)Diag amo heGT‐A oldp o einSpsA omBacillussub ilis,
belonging o amilyGT2(PDBID:1QG8)[51];(b)diag amo heGT‐B old‐ ypep o einG B om
Amycolap osiso ien alis,belonging o amilyGT1(PDBID:1IIR)[52];(c)diag amo heGT‐C old
p o einPglB omCampylobac e la i,belonging o amilyGT66(PDBID:3RCE)[46].D awingsc ea ed
wi hNGL[53].
Table1showsaclassi ica iono GT amiliesbasedon eac ionmechanismsand old ypes.
Mic oo ganisms2020,8,3575o 23
Table1.Classi ica iono glycosyl ans e ase amilies.
Reac ionMechanism
In e ingRe ainingUnknown
Fold ype
GT‐A21
,
7,12,13,14,16,21,29,31,42,43,
82,84,109
6,8,15,24,27,
34,44,55,62,
64,78,81,88
GT‐B1,9,10,18,19,23,28,30,33,38,41,
52,63,65,68,70,80,90,104
3,4,5,20,35,
52,72,99,107
GT‐C22,39,50,53,57,58,59,66,83,85,87
O he s26(GT‐E),51(lysozyme‐ ype), 101(GT‐D)
Unknown
11,17,25,37,40,47,48,49,54,56,
61,67,73,74,75,76,92,94,97,98,
100,102,103,105,106,108
32,45,60,69,
71,77,79,89,
93,95,96
91,110
1Numbe sco espond o heGT amilynumbe sassignedin heca bohyd a eac i eenzymes
(CAZy)da abase.
3.Glycosyla ioninBac e ialPa hogenesis
Asmen ionedin hein oduc ion,e ec o s ompa hogenicbac e iao enexhibi speci ic
enzyma icac i i yagains hos cell a ge saspa o i sin ec ions a egy.P o einglycosyla ion, he
mos commonpos ‐ ansla ionalmodi ica ion,hasbeen adi ionally hough obe es ic ed o
euka yo ico ganisms.Howe e ,in helas yea s,manyexampleso bac e ial i ulence ac o sand
oxinsexhibi ingGTac i i yha ebeencha ac e ized[54].Bac e ialglycosyla ionisb oadlylinked
obac e ialpa hogenesis[55].I ul ills womain unc ionsdu ingin ec ion: i s ly,p o ein
glycosyla ionenablesbac e ialadhesion ohos cellsand,secondly,leads omodi ica iono speci ic
hos a ge swi h heconsequen manipula iono hos cellula p ocessesby hepa hogen o hei own
bene i .
Bac e ialGTspe o m wo ypeso p o einmodi ica ions:N‐linkedglycosyla iono O‐linked
glycosyla ion[29].Whe easN‐GTsconjuga eglycans osidechainamideni ogeno anaspa agine
inanAsn‐X‐Se /Th consensussequence,O‐glycosyla ionhappenson hehyd oxylg oupo ase ine
o h eonine esidue.Bo hN‐linkedandO‐linkedglycosyla ionpa hwaysmaymodi ymul iple
p o eins.In e es ingly, he ea eexampleso pa hogenicbac e ia,likeC. oden ium,pe o mingN‐
andO‐glycosyla ion ha a ebo hc i ically equi ed o coloniza iono hegas oin es inal ac in
in ec edmice[54].
In hissec ionwep o ide ep esen a i eexampleso wellcha ac e izedbac e ialGTs,a ending
o hei speci icbiological oledu ingin ec ionand he ypeo modi ica ion heype o m.
3.1.Glycosyla iono Adhesins
Oneessen ials ep oini ia ein ec ionisbac e ialadhesion o hehos epi helium.This i s s ep
dependssome imesonglycosyla iono bac e ialadhesins.Someexamplesa edesc ibedbelow.
Awell‐knownexampleis hehigh‐molecula ‐weigh (HMW)sys emo Haemophilusin luenza.
HMW1/HMW2a e woadhesins ha a eglycosyla eda hecy oplasmbyGTsHMW1Cand
HMW2C, espec i ely[56,57].Then, heya e anspo ed o hebac e ialsu aceandmedia e
adhe ence o espi a o yepi helialcells[57].Glucoseandgalac osea eadded ospeci ic
aspa agine esiduesin heeuka yo ic‐likeAsn‐X‐Se /Th consensus[58].
Pgl( omP o einglycosyla ion)sys eminCampylobac e jejuniisano he exampleo a
glycosyla edadhesin ha hasbeenconside edap o o ype o he ele anceo bac e ial
Mic oo ganisms2020,8,3576o 23
glycosyla ioninbac e ial–hos in e ac ion[55].I isanN‐linkedp o einglycosyla ionsys em
composedo se e alp o eins,including a iousGTs ha modi yC.jejunip o einsleading o
bac e ial–hos celladhesionandin asiono hos epi helium[59].Dis up iono hePglpa hway
esul sin educ iono humanepi helialcellsadhesionanddec eases heabili y ocolonize
mouseandchickenin i o[59–61].Modi ica ionca alyzedby hePglsys emconsis so he
addi iono ahep asaccha ide oaspa agine esidueswi hin heeuka yo ic‐likeAsn‐X‐Se /Th
glycosyla ionconsensus.Theoligosaccha ideisassembledon hecy oplasmicsideo heinne
memb ane.Then,i is ansloca edbyanABC‐ anspo e (PglK)[62].Oncein hepe iplasmic
space,PglB,anoligo‐saccha yl ans e ase, ans e s hehep asaccha ide oaspa agine esidues
o a ge ligands[63,64].
Inaddi ion oN‐glycosyla ion‐ ela edmechanisms oimp o ebac e ialadhesion,o he sys ems
basedonO‐glycosyla ionha ebeencha ac e ized.Oneexampleis heeme gingbac e ial
au o anspo e hep osyl ans e ase(BAHT) amily oundinse e alG am‐nega i ebac e ia
[65].Au o anspo e swhichbelong o heT5SS amilyha eamodula composi ionwi hanN‐
e minalpassenge domainandaC‐ e minalβ‐ba eldomain ha d i es hepassenge ac oss
heou e memb ane.Thepassenge domainmaybehep osyla edbyaBAHTmembe on
nume ousse ine esidues.Thismodi ica ionoccu sbe o e ansloca iono hep o einac oss he
inne memb aneandis equi ed o bac e ialadhesion o hehos .Twowell‐cha ac e ized
BAHTmembe a eAAH(au o anspo e adhesinhep osyl ans e ase), omdi uselyadhe ing
E.coliisola e2787,andTibC omen e o oxigenicE.colis ainH10407[65,66].Inaddi ion o
pa hogenicE.coli,BAHTmembe sha ebeeniden i iedino he bac e ia,includingCi obac e ,
Shigella,Salmonella,andBu kholde ia[65].Fu he s udiesa enecessa y o ullycha ac e ize hese
in e es ingbac e ialenzymes.
Pos ‐ ansla ionalmodi ica iono bac e ialp o eins oenhancebac e ialadhesion o hehos is
no es ic ed oG am‐nega i ebac e ia.Awell‐knownexampleo modi iedbac e ialp o eins
inG am‐posi i ebac e iaa e heO‐glycosyla edse ine‐ ich epea p o eins(SRRPs)[67].These
la geglycop o eins,uniqueinG am‐posi i ebac e ia,a ec ucial o bio ilm o ma ionand,
mo eimpo an ly,hos celladhesion[68].Thebes ‐cha ac e izedSRRPisFap1 om
S ep ococcuspa asanguinis.Glycosyla iono Fap1 elieson heac i i yo a leas sixdi e en
GTs,al hough hespeci ic unc ionandglycan ans e ed emainsunclea [69].This amilyo
glycop o einsishighlyconse edamongs ep ococcalands aphylococcalspecies[67], hus,
cha ac e iza iono SRRPsGTsandunde s andingo hei biological olea ekey oiden i ynew
he apeu icd ug a ge sino de o ackle ele an G am‐posi i epa hogens.
3.2.Glycosyla iono Flagellins
Glycosyla iono lagellinisanimpo an componen o nume ous lagella sys emsinA chaea
andBac e ia,playinganimpo an oleinassemblyand i ulence[70],since hiso ganelle,in
addi ion ocon e ingmo ili y,canplaya oleine e ys epo hein ec ioncycle[71].FlagellaO‐
glycosyla ionhasbeendemons a edinmanypola lagellins omG am‐nega i e[72]andG am‐
posi i ebac e ia[73–80],andalsoinalimi ednumbe o la e al lagellins[72,81,82].Bac e iacan
glycosyla e lagellina a a yingnumbe o accep o si es, omasinglesi einBu kholde iaspp.o
Lis e iamonocy ogenes omul iplesi esinC.jejunio Selenomonasspu igena[80,83–85].Mo eo e ,
whe eassomebac e iauseasinglemonosaccha ide,o he sshows ain‐ o‐s ainglycan
he e ogene i y[72].Selec edexamplesa edesc ibedbelow.
InAe omonasca iaeSch3N, hepola lagellins,FlaAandFlaB,a eglycosyla edwi h
pseudoaminicacidglycansand hisglycosyla ionis equi ed o lagella assembly.Genes
in ol edinglycosyla iona emappedin heO‐an igenbiosyn he icclus e [86,87].InAe omonas
hyd ophilaAH‐3,la e al lagellinismodi iedwi hade i a i eo pseudaminicacid,whe eas
pola lagellaa eglycosyla edwi hahe e ogeneousglycan[88].
C.jejuniFlaAisglycosyla eda up o19si es ha con ibu e o he lagellumse ospeci ici y.FlaB
isalsoglycosyla ed,bu henumbe o si es emains obees ablished.Thep edominan O‐
glycansa ede i a i eso pseudoaminicacidandlegionaminicacid.ThegenesencodingGTs
Mic oo ganisms2020,8,3577o 23
in ol edin lagellinglycosyla iona eloca edadjacen o he lagellins uc u algenes, laAand
laB[85,89,90].
InHelicobac e pylo i,bo h heFlaAandFlaBs uc u alp o einsa eglycosyla edwi h
pseudomainicacida se enand ensi es, espec i ely.Biosyn hesiso pseudaminicacidhas
beenex ensi elys udiedinH.pylo iandC.jejuniandin ol essixconsecu i eenzyma ics eps
be o ebeing ans e edon o lagellins iaO‐linkedglycosyla ion[91,92].
TheGT ans e ingpseudaminicacidon o lagellinhasbeens udiedinMagne ospi illum
magne icumAMB‐1[93].ThisGT,knownasMa ,displayssigni ican sequenceiden i ywi h
p o eins omo he G am‐nega i ebac e ia,includingMa 1 omA.ca iaeandPseE omC.
jejuni, ha ha ebeenshown o ans e pseudaminicacidon o lagellinsin heseo ganisms
[94,95].Thes uc u eo M.magne icumMa displaysamodi iedGT‐A opologyas oundin
CAZy amiliesGT29andGT42.In ac , hecloses s uc u alhomologueisC.jejuni
sialyl ans e aseCs ‐II(PDB1RO7),belonging o amilyGT42[93].
Glycosyl a iono Clos idiumdi icile lagellinisessen ial o mo ili y.Two ypeso modi ica ion
ha ebeende inedas ypeAandB.TypeAmodi ica ionin ol esanO‐linkedN‐
ace ylglucosamine(GlcNAc)linked oame hyla ed h eonine iaaphosphodies e bond.Th ee
GTsa ein ol edin ypeBmodi ica ion:GT1andGT2a e esponsible o hesequen ialaddi ion
o aGlcNAcand wo hamnoses, espec i ely,andGT3isassocia edwi h heinco po a iono
asul ona edpep idyl‐amidosuga moie y[74].
3.3.Modi ica iono Hos CellTa ge sbyBac e ialGTs
Inaddi ion obac e ialGTs ha glycosyla e hei ownp o eins,we indano he in e es ing
g oupo bac e ialp o eins ha modi yhos cellp o eins,manipula ing hecellphysiology o
coun e ac hehos de ense esponse.Thela geclos idial oxinsTcdA/TcdB, heLegionellacy o oxic
glycosyl ans e ase amily(Lg ),andN‐GTs om heNleB/SseK amilya eincludedin hisg oup
[54].
La geclos idialglycosyla ing oxins:pa hogenesiso Clos idiumdependson heac i i yo wo
simila oxins,TcdAandTcdB, ha exhibi GTac i i yin hei N‐ e minaldomains[96,97].Bo h
oxinspe o mO‐glycosyla iono smallGTPaseso heRho amily,inhibi ing he egula o y
unc ionso hesep o eins[98].Thismodi ica ionoccu son hesidechainhyd oxylg oupo a
h eonine esidue,whichisspeci ic o each a ge edRhoGTPase[99].Ano he exampleo his
amilyo oxins ecen lyiden i iedisPaTox omen omopa hogenicPho o habdusasymbio ica
[100].
LegionellapneumophilaLg amily:L.pneumophilais hecausa i eagen o legionellosis,ase e e
lungdisease.This acul a i ein acellula pa hogenusesaT4SS oinjec mo e han300e ec o s
in o hehos cell[101].Among hem,L.pneumophilasec e esse e alGTs(Lg 1‐3) ha pe o m
speci icglycosyla iono ase ine esidue(Se 53)in heeuka yo icelonga ion ac o 1A,ala ge
GTPaseandacomponen o heelonga ioncomplexinp o einsyn hesis.Thismodi ica ion
esul sininhibi iono p o einsyn hesisandsubsequen lycauses hedea ho in ec edcells
[102,103].In e es ingly,Lg 1sha ehighs uc u alhomologywi h heclos idial oxinTcdB.
Bo hGTsa emembe so heclanIIIo e ainingenzymeswi h heGT‐A old[103–105].
NleB/SseK amily:we e e o his amilyo T3SSe ec o sin henex sec ion.
4.Glycosyl ans e aseso heNleBFamily
4.1.Membe so heFamily
T3SSe ec o s ha ca alyze he ans e o GlcNAc oa ginineso hos p o einsha ebeen
s udiedinC. oden ium,EHEC,EPEC,andS.en e ica(Table2).
Mic oo ganisms2020,8,3578o 23
Table2.NleB/SseKglycosyl ans e ases.
O ganism.
Re e ence
S ainE ec o NameP o einSize
(aa)GenomicCon ex Re e ences
C. oden iumICC168NleB329GenomicislandGI4[106,107]
En e ohemo hagic
E.coli(EHEC)
O157:H7
EDL933
NleB1329O‐island122/SpLE3
[106,108]
NleB2326O‐island36/PhageCP‐
933K/Sp3
En e opa hogenicE.
coli(EPEC)O127:H6E2348/69
NleB1329In eg a i eelemen IE6
[109,110]
NleB2326PhagePP4
S.en e icase o a
Typhimu ium14028
SseK1336
[111–113]
SseK2348
SseK3335PhageST64B
NleBwasiden i iedinC. oden ium oge he wi ho he po en ial ypeIIIe ec o sencoded
ou side heLEE.Theywe edesigna edNleA,NleB,NleC,NleD,NleE,NleF,andNleG o non‐LEE‐
encodede ec o s[106].Twohomologso NleBa eencodedin hegenomeso EHECandEPEC:
NleB1andNleB2[106,108,109].NleB1is89%iden ical oNleB omC. oden ium,and he eisa
di e enceinse enaminoacidsbe ween heEHECandEPEC e sions.NleB2isiden icalinbo hE.
colipa ho ypesbu isonly60%iden ical oC. oden iumNleB,al houghano hologo nleB2isp esen
in hegenomeo C. oden iumasapseudogene[107].In e es ingly,nleB2isanon‐neu ale ol ing
gene,p obablydue oposi i eselec ion,sugges ing ha hisgeneiss illin hep ocesso op imizing
i s unc ion[114].S.en e icas ainsencode h eehomologso NleB:SseK1,SseK2,andSseK3
[111,112].Al hough hegenesencoding hesee ec o sa eno loca edin hepa hogenici yislands
whe e hecogna eT3SSa eencoded,allo hemha ee idenceo ho izon al ans e andsomea e
associa edwi ho he pa hogenici yislandso p ophages(Table2).In e es ingly,whe eassseK1and
sseK2a ep esen inmos a ailableSalmonellagenomesequences, hephage‐encodedSseK3e ec o
hasalimi eddis ibu ion[111].ABLASTsea choncomple egenomesa ailablea heNa ional
Cen e o Bio echnologyIn o ma ion(NCBI) e eals ha p o einsiden icalo e ysimila oNleB1
o NleB2a eencodedindi e en s ainso heeme gingA/Epa hogenEsche ichiaalbe ii[115],and
signi ican lysimila p o einsa e oundinEsche ichiama mo ae[116]andYe siniahibe nica[117].
Pe cen ageso iden i ybe ween ep esen a i eNleB/SseKp o einsa eshowninTable3,anda
phylogene icanalysisisshowninFigu e4.
Table3.Pe cen iden i yda a o NleB/SseKp o einsp o idedbyClus alW.
NleBC.
oden ium
NleB1
EPEC
NleB1
EHEC
NleBE.
albe ii
NleBE.
ma mo aeNleB2SseK1SseK2SseK3
NleBC. oden ium
NleB1EPEC88.75
NleB1EHEC89.0697.87
NleB1E.albe ii88.4597.5799.09
NleBE.ma mo ae71.4271.7371.7371.43
NleB2EPEC/EHEC59.8261.0461.3560.4360.43
SseK1S.en e ica57.4558.0559.5758.9758.9753.68
SseK2S.en e ica52.5852.2853.1952.5851.0646.9355.36
Mic oo ganisms2020,8,3579o 23
SseK3S.en e ica51.9853.1953.5052.8954.1047.5554.9375.22
NleBY.hibe nica32.5232.8334.0434.0434.0433.1335.1233.1433.43
Figu e4.Phylogene icanalysiso NleB/SseKp o eins.Alignmen andphylogene ic econs uc ions
we epe o medusing he unc ion“build”o ETE3 3.1.1[118]asimplemen edon heGenomeNe
(h ps://www.genome.jp/ ools/e e/).The eewascons uc edusingFas T ee 2.1.8wi hde aul
pa ame e s[119].Valuesa nodesa eSH‐likelocalsuppo .
4.2.Exp ession,T ansloca ion,andSubcellula Localiza ion
E ec o so T3SSsa eexpec ed obeexp essedincoo dina ionwi h hesyn hesiso hei
cogna esec e ionappa a us.Thisis hecase o e ec o so heLEE,SPI1o SPI2sys ems ha a e
encodedin heislands,bu heissueisno soob ious o e ec o sencodedou side heislands,
including hemembe so heNleB/SseK amily.
InA/Eo ganisms,Le is hemain ansc ip ional egula o o LEE.I isencodedin heisland
andi sle eliscon olledbyo he ansc ip ional egula o s ha espond odi e en en i onmen al
s imuli,includinghos ho mones,mic obio a‐libe a edsuga sandme aboli es,molecula oxygena
hegu epi helium,andcon ac o bac e iawi hhos epi helialcells[120].Exp essionisunde ec able
whenbac e iaa eg ownin ichlabo a o ymedia,bu apidexp essionandsec e iono p o eins o
hesupe na an isobse edupon ans e oMEM‐HEPESmedium,whichisassocia edwi h he
he e ogeneousexp essiono heEspADB ansloconappa a us[121].Se e alnlegenes,including
nleB,a e ansc ibedinEHECunde hesesec e ion‐pe missi econdi ions,al houghinduc iono
nleBexp essionwassligh ( wo old)andwasno coo dina edwi h hesyn hesiso EspA ilamen s
a hesingle‐cellle el[122].Sec e iono NleB(NleB1inEPEC)by heLEE‐encodedT3SShasbeen
demons a edinC. oden iumandEPEC[106,110].T ans ec ionwi hGFP‐ usionso ansloca iono
TEM‐1β‐lac amase usions[123] omEPECin oHeLacells e ealsadi usecy osoliclocaliza iono
NleB1[110,124].The egula o y egiono EPECnleB2con ainsamo i conse edamongasubse o
genesencodinge ec o p o einsinA/Epa hogens ha wasnamedNRIR(nle egula o yin e ed
Mic oo ganisms2020,8,35716o 23
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