molecular mechanisms of blister formation in bullous impetigo and staphylococcal scalded skin...

8/12/2019 Molecular Mechanisms of Blister Formation in Bullous Impetigo and Staphylococcal Scalded Skin Syndrome

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Molecular mechanisms of blisterformation in bullous impetigo and

staphylococcal scalded skin syndromeYasushi Hanakawa 1, Norman M. Schechter 1, Chenyan Lin 1, Luis Garza 1, Hong Li 1,Takayuki Yamaguchi 2, Yasuyuki u!a"a 2, #o$i Nishi%u$i&, Motoyuki Sugai 2, Masayuki'magai & an! (ohn ). Stan*ey 1

1+e artment o% +ermato*ogy, -ni ersity o% /ennsy* ania Schoo* o% Me!icine,/hi*a!e* hia, /ennsy* ania, -S'2 +e artment o% 0acterio*ogy, Hiroshima -ni ersity Gra!uate Schoo* o% 0iome!ica*Sciences, Hiroshima, (a an& +e artment o% +ermato*ogy, #eio -ni ersity Schoo* o% Me!icine, Tokyo, (a an

'!!ress corres on!ence to (ohn ). Stan*ey, +e artment o% +ermato*ogy, -ni ersity o%/ennsy* ania, Schoo* o% Me!icine, 211 C*inica* )esearch 0ui*!ing, 13 Curie 0ou*e ar!,/hi*a!e* hia, /ennsy* ania 1415 , -S'. /hone 62137 8489&2 5: a; 62137 3mai*.me!.u enn.e!u.

/u"*ishe! (u*y 1, 2552)ecei e! %or u"*ication ' ri* 22, 2552, an! acce te! in re ise! %orm May 24, 2552.

0u**ous im etigo !ue to Staphylococcus aureus is one o% the most common "acteria*in%ections o% man, an! its genera*ize! %orm, sta hy*ococca* sca*!e! skin syn!rome

6SSSS7, is a %re?uent mani%estation o% sta hy*ococca* e i!emics in neonata* nurseries.0oth !iseases are me!iate! "y e;%o*iati e to;ins 6=Ts7, which show e;?uisite atho*ogics eci%icity in "*istering on*y the su er%icia* e i!ermis. @e show that these to;ins act asserine roteases with e;treme*y %ocuse! mo*ecu*ar s eci%icity to c*ea e mouse an!human !esmog*ein 1 6+sg17 once a%ter g*utamic aci! resi!ue &81 "etween e;trace**u*ar!omains & an! . Mutation o% the re!icte! cata*ytica**y acti e serine to a*aninecom *ete*y inhi"its c*ea age. The mutate! =Ts "in! s eci%ica**y to +sg1 "yimmuno%*uorescence co*oca*ization an! "y coimmuno reci itation. Thus, =Ts, throughs eci%ic recognition an! roteo*ytic c*ea age o% one structura**y critica* e ti!e "on! inan a!hesion mo*ecu*e, cause its !ys%unction an! a**ow S. aureus to s rea! un!er thestratum corneum, the main "arrier o% the skin, e; *aining how, a*though they circu*ate

through the entire "o!y in SSSS, they cause atho*ogy on*y in the su er%icia* e i!ermis.

Introduction-n!erstan!ing the atho*ogy resu*ting %rom Staphylococcus aureus in%ection is o% greatinterest in me!icine "ecause o% the organismAs common an! increasing re a*ence inhumans, its growing "acteria* resistance, an! its a"i*ity to cause serious an! *i%e9


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threatening !isease 6 17. To;ins contri"ute in a ma$or way to the athogenicity o% S.aureus , as they !o with many other "acteria 6 27.

S. aureus %re?uent*y in%ects the skin. Bn %act, the most common "acteria* in%ection o%chi*!ren is im etigo, which accounts %or a ro;imate*y 15 o% a** skin ro"*ems in

chi*!ren 6&7. D% these im etigo atients, a"out &5 ha e "u**ous im etigo, which iscause! "y S. aureus strains that ro!uce e;%o*iati e to;ins 6=Ts7. Bn atients !ischarge!%rom the new"orn nursery, o er &5 may "e co*onize! with S. aureus , an! organismsthat ro!uce =Ts are the ma$or cause o% neonata* nursery out"reaks o% sta hy*ococca*!isease 6 1, 7. New"orns, young chi*!ren, an! a!u*ts with rena* %ai*ure an!Eor who areimmunocom romise! may !e e*o a genera*ize! %orm o% "u**ous im etigo ca**e!sta hy*ococca* sca*!e! skin syn!rome 6SSSS7. Bn this !isease, "u**ae an! erosions!e e*o o er a *arge area o% the skin sur%ace, resu*ting in the !eath o% ro"a"*y *ess than& o% a%%ecte! chi*!ren "ut u to F5 o% a%%ecte! a!u*ts 6 3, F7.

The now9c*assic e; eriments o% Me*ish et a*. in the ear*y 14


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su er%ami*y 614, 257 6T. Yamaguchi et a*., un u"*ishe! o"ser ations7. +sg1, "ut not thec*ose*y re*ate! +sg& or =9ca!herin, is c*ea e! "y =Ts. urthermore, +sg1 is a re*e antsu"strate "ecause anti"o!ies in the autoimmune !isease em higus %o*iaceus6/ 7 target+sg1 an! cause c*inica* an! histo*ogic "*isters in mouse an! man i!entica* to those seenin "u**ous im etigo an! SSSS. These !ata im *y that targeting o% +sg1 can resu*t in the

atho*ogy characteristic o% "u**ous im etigo an! SSSS.

Here we !emonstrate that =T', =T0, an! =T+ act as g*utamic aci! s eci%ic serine roteases with unusua**y %ocuse! s eci%icity that resu*ts in a sing*e c*ea age o% +sg1,*ea!ing to its %unctiona* inacti ation. The atho hysio*ogy o% "u**ous im etigo an! SSSScan thus "e attri"ute! to hy!ro*ysis o% a sing*e e ti!e "on!, resuma"*y in a %unctiona**ycritica* area in +sg1.

Methods

Recombinant ETs. The e; resse! roteins, ectors, an! their hosts are summarize! inTa"*e 1. Shutt*e ector C=15 , containing =T' an! =T' C mu, was %rom /atrickSch*ie ert 6+e artment o% Micro"io*ogy, -ni ersity o% Minnesota Me!ica* Schoo*,Minnea o*is, Minnesota, -S'7 6 1 7. =T' ' mu was constructe! %rom these ectors withthe KuikChange Site9+irecte! Mutagenesis #it 6Stratagene, La (o**a, Ca*i%ornia, -S'7.=T0 ' mu was c*one! "y /C) using C=15 9=T0 6 257 as a tem *ate. /C) was a*so use!to a!! nuc*eoti!es enco!ing e ti!e tags, J3His 6Bn itrogen Cor ., Car*s"a!, Ca*i%ornia,-S'7 or & L'G 6Sigma9'*!rich, St. Louis, Missouri, -S'7, to the & en! o% these =Tconstructs. =T'9J3His, =T' C mu J3His, =T' ' mu J3His, =T09J3His, =T0 &L'G, an! =T0 ' mu J3His were su"c*one! into the Hin !BBB an! Eco )B sites o% C=15 .+N' was trans%orme! into the S. aureus strain )N 225 6 ro i!e! "y /atrick Sch*ie ert7

ia e*ectro o*ation. The su ernatant %rom )N 225 trans%orme! with these *asmi!s was reci itate! with 83 ammonium su*%ate. /reci itate! =Ts were !ia*yze! against /0Swith 1 mM CaC* 2 6/0S9Ca7. His9tagge! =Ts were uri%ie! on Ni9NT' co*umns6KB'G=N Bnc., Ja*encia, Ca*i%ornia, -S'7 using the manu%acturerAs roce!ure an! then!ia*yze! against /0S9Ca. =T concentrations were estimate! with a rotein assay kit %rom0io9)a! La"oratories Bnc. 6Hercu*es, Ca*i%ornia, -S'7.

Ta"*e 1

Jectors an! hosts use! %or e; ression o% =Ts an! !esmog*eins

=T+ was c*one! "y /C) %rom a atient sam *e 6T. Yamaguchi et a*., un u"*ishe! !ata7,an! =T+ ' mu was c*one! "y /C) mutagenesis. The am *i%ie! +N' %ragments werec*one! into K=


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Coomassie "*ue staining o% S+S9/'G= ge*s in which the =Ts were the ma$or "an! an!accounte! %or o er 45 o% the tota* rotein.

igure 2

/oint mutation o% serine 143 6chymotry sin num"ering7, the resume! cata*ytica**yacti e serine, o% =Ts inhi"its c*ea age o% +sg1. 6 a7 'nti =9tag anti"o!y immuno"*ot o%S+S9/'G= o% h+sg1= incu"ate! with )N 225 sta hy*ococca* ector su ernatant 6)N7,wi*!9ty e 6@T7 =T', =T' C mu 6serine 143 mutate! to cysteine7, an! =T' ' mu 6serine143 mutate! to a*anine7 shows marke!*y !ecrease! c*ea age with =T' C mu com are!with wi*!9ty e =T'. =T' ' mu shows no cata*ytic acti ity. Horizonta* *ines in!icatemigration o% mo*ecu*ar weight markers o% 8& k+a 6to 7 an! &2 k+a. 6 b7 'nti L'G9tagimmuno"*ots o% anti L'G9tag immuno reci itates o% e;tracts o% m+sg19 L'Ga!eno irus trans!uce! ce**s that were incu"ate! with =T0 or =T0 ' mu. =T0 ' mu showsno c*ea age. Horizonta* *ines, %rom to , in!icate migration o% mo*ecu*ar weight markerso% 25& k+a, 113 k+a, an! 4& k+a. 6 c7 'nti =9tag anti"o!y immuno"*ot o% S+S9/'G= o%

h+sg1= incu"ate! with =T+ an! =T+ ' mu. =T+ ' mu !oes not c*ea e h+sg1. Horizonta**ines, %rom to , in!icate migration o% mo*ecu*ar weight markers o% 8& k+a an! & k+a.-nc*ea e! +sg1 6o en arrowhea!7 an! its car"o;y9termina* c*ea age ro!uct 6%i**e!arrowhea!7 are in!icate!.

Cell culture and transduction with adenovirus vectors. HaCaT ce** keratinocytescu*ture! in +M=M with 15 0S were trans!uce! with recom"inant a!eno irus ectors';9m+sg1 an! ';9m+sg& 6 257, enco!ing mouse +sg1 an! +sg&, res ecti e*y, withcar"o;y9termina* L'G tags 6Ta"*e 17. '%ter 2 hours, the ce**s were incu"ate! withrecom"inant =T', =T0, or =T+ in cu*ture me!ia %or 15 minutes. Then the ce**s werewashe! with /0S9Ca, e;tracte!, an! use! %or immuno reci itation or immuno"*otting.

In vitro digestion of recombinant Dsg1 with ETs. The entire e;trace**u*ar !omain o%human or mouse recom"inant +sg1 with an = tag 6h+sg1= an! m+sg1=7 on the car"o;y*terminus was ro!uce! as a secrete! rotein "y "acu*o irus, as re ious*y !escri"e! 6 217.6These constructs a*so containe! a His tag on the car"o;y terminus.7 ' ro;imate*y 1 go% mouse or human +sg1 uri%ie! with a T'LDN 60+ 0iosciences C*ontech, San (ose,Ca*i%ornia, -S'7 or =9tag co*umn 6'mersham 0iosciences Bnc., /iscataway, New (ersey,-S'7 was incu"ate! with recom"inant =T', =T0, or =T+ in Tris9"u%%ere! sa*ine with 1mM CaC* 6T0S9Ca7 %or 1 hour at &


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%or immuno"*otting an! immunohistochemistry. H)/9con$ugate! anti9BgG anti"o!ies o%the a ro riate s eci%icity 60io9)a! La"oratories Bnc.7 were use! %or immuno"*otting.'nti9human anti"o!y con$ugate! with '*e;a *uor 43 an! anti9mouse anti"o!ycon$ugate! with '*e;a *uor 345 6Mo*ecu*ar /ro"es Bnc., =ugene, Dregon, -S'7 wereuse! %or immunohistochemistry.

Ex vivo binding of ETA A mu to mouse epidermis and immunohistochemistr!. Mousetai* or aw skin was incu"ate! in +M=M at a ratio o% 1 1 with 15 0S an! =T' ' mu J3His 6 8& M7 in /0S9Ca at OC %or 12 hours an! then cryosectione!. '%ter "eing%i;e! with ara%orma*!ehy!e in /0S9Ca, s ecimens were "*ocke! with 1 0S' in/0S9Ca. 0*ocke! s ecimen was incu"ate! with anti9J3 anti"o!y %o**owe! "y anti9mouseanti"o!y con$ugate! with '*e;a *uor 345, an! then staine! with / serum %o**owe! "yanti9human anti"o!y con$ugate! with '*e;a *uor 43. Staine! sections were

hotogra he! using con%oca* microsco y 6M)C 152 : 0io9)a! La"oratories Bnc.7. orcontro*s, skin was %irst incu"ate! with wi*!9ty e =T' or an irre*e ant to;in6sta hy*ococca* enteroto;in 0 with a J3 tag7, then staine! with / serum or anti9J3

anti"o!y, res ecti e*y. These contro*s showe! no staining 6not shown7.

Immunoprecipitation. '%ter incu"ation with =Ts, a!eno irus9in%ecte! ce**s weree;tracte! with 1 Triton 9155 in T0S9Ca with rotease inhi"itors 62 gEm* a rotinin, 2gEm* *eu e tin, an! 1 mM /MS 7 %or immuno reci itation. Su ernatants o% High i einsect ce**s 6Bn itrogen Cor .7 trans!uce! with "acu*o irus enco!ing h+sg1= wereincu"ate! with =Ts, then use! !irect*y %or immuno reci itation. L'G9tagge! roteinswere reci itate! with anti9 L'G agarose 6Sigma9'*!rich7, an! =9tagge! roteins were

reci itate! with anti =9tag Se harose 6'mersham 0iosciences Bnc.7 at OC %or 1 hour.Bmmuno reci itates were washe! ten times with 1 Triton 9155 in T0S9Ca, then e*ute!with Laemm*i sam *e "u%%er at 155OC.

Immunoblotting. Ce** e;tracts or immuno reci itates in Laemm*i sam *e "u%%er werese arate! "y 25 or 15 Tris9g*ycine S+'9/'G= 60io9)a! La"oratories Bnc. orBn itrogen Cor .7, then trans%erre! to nitroce**u*ose sheets 6Trans90*ot: 0io9)a!La"oratories Bnc.7. The sheets were incu"ate! %or 1 hour in "*ocking "u%%er 63 %at9%reemi*k ow!er in /0S7. The %irst anti"o!y, !i*ute! in "*ocking "u%%er, was a *ie! %or 1hour at room tem erature. '%ter two washes with 5.1 Tween 25 in /0S, the sheets wereincu"ate! with H)/9con$ugate! secon!ary anti"o!y !i*ute! in "*ocking "u%%er. Bn somee; eriments, on*y one anti"o!y, H)/9con$ugate! anti9=T', was use!. '%ter the "*otswere washe!, the signa*s were !etecte! with chemi*uminescence 6=CL or =CL /*us,'mersham 0iosciences Bnc.7.

Results ETs cleave human and mouse Dsg1 at a uni"ue site. /re ious stu!ies o% roteo*ysis o%+sg1 "y =Ts i!enti%ie! on*y a sma** car"o;y9termina* %ragment, *ea ing the e;tent o%!egra!ation uncertain. To !etermine whether there is one s eci%ic c*ea age site aso ose! to more genera* roteo*ytic !egra!ation, the e;trace**u*ar !omain o% human


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+sg1, containing an = tag 6h+sg1=7 on the car"o;y terminus, was uri%ie! an! c*ea agewas characterize! "y S+S9/'G= an! amino9termina* se?uence ana*ysis. h+sg1= was

ro!uce! "y "acu*o irus in High i e insect ce**s 6 217. The recom"inant rotein wasiso*ate! %rom insect ce** su ernatant with anti =9tag Se harose an! e*ution with e;cess =

e ti!e. Coomassie "*ue staining o% the e ti!e9e*ute! materia* reso* e! "y S+S9/'G=

6 igure 1a, *e%t7 in!icates a c*ose*y s ace! !ou"*et at the re!icte! mo*ecu*ar weight o%8 k+a. The iso*ate! h+sg1= was incu"ate! %or 1 hour with =T' at &


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consistent with structura* mo!e*s that suggest that =T' an! =T0 might c*ea e a su"stratea%ter a g*utamic aci! resi!ue.

'mino termina* se?uence ana*ysis o% the s*ower migrating 359k+a "an! %orme! "yc*ea age o% h+sg1= 6 igure 1a, arrow marke! 17 re ea*e! the amino terminus o% h+sg1=

*acking the signa* e ti!e 6which was resuma"*y rocesse! "y the insect ce**s7 "ut sti**containing the ro e ti!e 6 igure 1!7. Se?uencing o% the "an! shown imme!iate*y "e*owthe 359k+a "an! 6 igure 1a, arrow marke! 27 in!icate! that it was the amino terminus o%the h+sg1= mature rotein 6 igure 1!7. These !ata show that the signa* e ti!e o%h+sg1= ro!uce! "y "acu*o irus in insect ce**s is ro er*y rocesse!, an! con%irm thatthe se?uence re ious*y re!icte! "y com uter mo!e*ing to "e the signa* e ti!e inh+sg1 6 igure 1!, shown in green7 is correct. The !ata a*so show that the ro rotein

ro!uce! "y "acu*o irus in insect ce**s is not e%%icient*y rocesse!. Howe er, "oth the recursor an! mature roteins are e%%icient*y c*ea e! "y =T' at the same site.

Taken together, the !ata in igure 1 !emonstrate that =T', =T0, an! =T+ c*ea e h+sg1

at the same site, an! that =T' c*ea es m+sg1 at the homo*ogous site. These =Ts areknown not to c*ea e the c*ose*y re*ate! +sg& 6 14, 257. Se?uence a*ignment o% human an!mouse +sg1 an! +sg& 6 igure 1e7 in!icate! marke! homo*ogy 61< o% 18 i!entica*resi!ues7 o% mouse an! human +sg1, "ut !i ergence o% +sg&, aroun! the c*ea age site.

#erine 1$% of ETs is critical for proteol!sis of Dsg1. The in%erence %rom structura*stu!ies o% =T' an! =T0 is that the serine resi!ue at osition 143 in =T' an! 18F in =T0corres on!s to the cata*ytica**y acti e serine 143 o% chymotry sin. urthermore, *ikechymotry sin, =T' an! =T0 ha e a %unctiona* cata*ytic tria! consisting o% serine143E18FE143, histi!ine


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on*y in the su er%icia* e i!ermis. @e ha e hy othesize! that this s eci%icity is !ue tos eci%ic recognition an! c*ea age o% a sing*e target, +sg1.

B% =T' "in!s to +sg1, an! that "in!ing !oes not re?uire the cata*ytic serine, then =T'' mu shou*! "e a com etiti e inhi"itor o% wi*!9ty e =T'. =sta"*ishing the a"i*ity o% =T'

' mu to rotect +sg1 %rom c*ea age "y =T' wou*! a*so ro i!e an estimate o% the!issociation constant 6 K ! 7 %or the reaction. To !etermine the K ! %or the interaction "etween =T' ' mu an! +sg1, +sg1 was incu"ate! with increasing concentrations o% =T'' mu, with =T' ' mu a*ways in *arge e;cess o% +sg1. ree +sg1 was e a*uate! "yre*ati e*y ra i! treatment with wi*!9ty e =T' an! "y using S+S9/'G= to estimate the%raction c*ea e! an! the %raction rotecte! %rom c*ea age 6 igure &7. 't concentrations o%1& M an! a"o e, =T' ' mu most*y inhi"ite! c*ea age, "ut at concentrations o% M an!

"e*ow, it !i! not. ' ro;imate*y ha*% o% the +sg1 was o"ser e! c*ea e! at an =T' ' mu concentration o% 8 M, a**owing K ! to "e rough*y estimate! as %o**ows

igure &

=T' ' mu inhi"its c*ea age o% +sg1 "y =T'. 'nti =9tag immuno"*ot o% h+sg1= 6o enarrowhea!7 an! its car"o;y9termina* c*ea age ro!uct 6%i**e! arrowhea!7. =T' ' mu at aconcentration o% 1& M incu"ate! with h+sg1= inhi"its c*ea age "y su"se?uent a!!itiono% wi*!9ty e =T', "ut at M !oes not. 't 8 M, a"out ha*% o% the +sg1 is %ree to "ec*ea e!. K ! can then "e rough*y estimate! to "e 8 M. =T' ' mu in the concentrationsshown 6a** in *arge e;cess o% that o% h+sg1=7 was incu"ate! with h+sg1 at 23OC %or F5minutes, then wi*!9ty e =T' was a!!e! %or a 259minute incu"ation "e%ore S+S9/'G=.

1

The magnitu!e o% this K ! is within the range o% a hy!ro*ytic reaction whose s eci%icity is!ue to re*ati e*y strong "in!ing in the cata*ytic site with a re*ati e*y *ow K m, "ut is ro"a"*y not consistent with strong "in!ing to an site outsi!e the su"strate "in!ing site.

@e %urther showe! that mutant =T' "in!s the e i!ermis where it causes a "*ister. Thiswas !emonstrate! "y incu"ating mouse skin with =T' ' mu with a J3 e ito e tag on thecar"o;y terminus at concentrations in e;cess o% K ! . Bmmuno%*uorescence with anti"o!iesagainst the J3 tag showe! "in!ing o% =T' ' mu to e i!ermis in a attern i!entica* to thato% +sg1 6 igure 7.

igure

Bmmuno%*uorescence co*oca*ization o% =T' ' mu an! +sg1 in e i!ermis. Mouse skin wasincu"ate! with =T' ' mu with a J3 tag. +ou"*e immuno%*uorescence staining withanti"o!ies against J3 tag 6re! %*uorescence7 an! +sg1 6green %*uorescence7 showe!co*oca*ization. Note that immuno%*uorescence *oca*ization o% =T' ' mu shows the ty ica*

attern o% +sg1 *oca*ization, with *ess intense staining in the "asa* *ayer o% the e i!ermis Q a attern o osite that o% +sg&, which is seen most intense*y in the "asa* *ayer 6 &F7.
http://www.jci.org/articles/view/15766#F3http://www.jci.org/articles/view/15766#F3http://www.jci.org/articles/view/15766/figure/3http://www.jci.org/articles/view/15766#F4http://www.jci.org/articles/view/15766/figure/4http://www.jci.org/articles/view/15766#B36http://www.jci.org/articles/view/15766#F3http://www.jci.org/articles/view/15766/figure/3http://www.jci.org/articles/view/15766#F4http://www.jci.org/articles/view/15766/figure/4http://www.jci.org/articles/view/15766#B36


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ina**y, we con%irme! "y coimmuno reci itation that =Ts "in! +sg1. Bncu"ation o%acti e =Ts with +sg1 showe! co reci itation that ?uick*y !ecrease! with time asc*ea age rogresse! 6 igure 3a7. The ra i! change in the a"i*ity to co reci itate =T'with +sg1 in!icates that =T' !issociates %rom +sg1 a%ter hy!ro*ysis o% the targete!

e ti!e "on!, consistent with enzyme recyc*ing. Su orting this conc*usion are stu!ies

with =T' C mu, which hy!ro*yzes +sg1 at a much s*ower rate. Consistent with the s*owerc*ea age rate, co reci itation o% =T' C mu with +sg1 was o"ser e! o er a *onger time, "ut sti** !ecrease! with increasing hy!ro*ysis 6 igure 3 "7. ina**y, =T' ' mu, which !oesnot c*ea e +sg1 at a**, showe! increase! "in!ing with time.

igure 3

Co reci itation o% =Ts with +sg1. 6 a7 Bmmuno reci itation %o**owe! "y immuno"*otting.Bncu"ation o% =T0 6marke! with a L'G e ito e tag7 an! =T+ with +sg1 showe!co reci itation at 1 minute that was marke!*y !iminishe! at F5 minutes, a%ter increase!c*ea age o% +sg1. Simi*ar resu*ts were %oun! %or =T' 6!ata not shown7. 6 b7 0in!ing o%

+sg1 with =T' C mu an! =T' ' mu. =T' C mu, which s*ow*y c*ea es +sg1, showe!!ecrease! "in!ing with time. 6 c7 =T' an! =T0 6marke! with His e ito e tags7 "in! to+sg1 "ut not +sg&. These =Ts !i! not c*ea e or "in! to +sg&, which is high*yhomo*ogous in amino aci! se?uence to +sg1. Simi*ar resu*ts were %oun! with =T+ 6!atanot shown7. B/, immuno reci itation: B0, immuno"*ot.

To show s eci%icity o% "in!ing in these stu!ies, we a*so incu"ate! +sg& with =Ts an!showe! that =Ts !i! not c*ea e or "in! to +sg&, which is high*y homo*ogous in aminoaci! se?uence to +sg1 6 igure 3c7.

Discussion ETs are serine proteases that specificall! bind to and cleave Dsg1 at a uni"ue site aftera glutamic acid residue. /re ious stu!ies ha e suggeste! arious ossi"*e sites an!mechanisms o% action o% =Ts, such as "in!ing to gang*iosi!es, causing re*ease o%

roteases "y keratinocytes: acting as su erantigens in stimu*ating the skinAs immunesystem: an! acting as *i ases 6re iewe! in re%. 17. Howe er, recent in%erences %romcrysta* structures o% =T' an! =T0 suggest that they might "e aty ica* serine roteases61 1


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interaction with a s eci%ic su"strate or rece tor that wou*! im art the re*ati e*y sma**energy necessary to %*i this "on!.

+es ite these re!ictions, on*y one rotein su"strate, me*anocyte9stimu*ating hormone,was re orte! to "e c*ea e! "y =Ts unti* recent*y, a*though an ester su"strate was re orte!

62 , 237. The "io*ogica* signi%icance o% this rotein su"strate, howe er, was not a arent.

)ecent*y we showe! that +sg1 is c*ea e! "y =T' an! =T0 6 14, 257. Here we !eterminethe mo*ecu*ar mechanism o% that c*ea age an! o% its e;?uisite s eci%icity. The =Ts 6=T',=T0, an! =T+7 s eci%ica**y "in!, an! consistent with the re!ictions %rom amino aci!se?uence an! crysta* structure, act as g*utamic aci! s eci%ic serine roteases to c*ea e+sg1 at a uni?ue site. These !ata are a*so consistent with re ious %in!ings o% !i%%erentia*accumu*ation o% =T' in new"orn mouse skin com are! with "*oo! an! other tissues62F7.

/roteo*ysis o% this one e ti!e "on! *ea!ing to !ys%unction o% +sg1 an! the !esmosome

wou*! e; *ain the atho hysio*ogy o% "*ister %ormation in "u**ous im etigo an! SSSS6see "e*ow7. Bt is there%ore *ike*y that this e ti!e "on! is critica**y im ortant to the ro er %unction o% +sg1. Bts site in re*ation to the !omain structure o% +sg1 is shown inigure F. Bnteresting*y, the =Ts c*ea e at the "or!er "etween e;trace**u*ar ca!herin!omains 6=Cs7 & an! , $ust towar! the car"o;y termina* %rom one o% the re!icte!ca*cium9"in!ing !omains in =C&. '*though three9!imensiona* structures o% !esmog*einsha e not "een so* e!, there is marke! homo*ogy in "oth ca*cium9"in!ing sites an! =Cs

"etween =9 an! N9ca!herins an! +sg1. Structura* stu!ies o% =9 an! N9ca!herin ha esuggeste! that the "or!er "etween =Cs, through ca*cium "in!ing, sta"i*izes the rigi!ity o%the mo*ecu*e an! %i;es its orientation, an! is critica* %or %unction 6 2< &17. The resu*ts we

resent here suggest that this "or!er "etween =Cs in +sg1 is a*so critica* %or their ro er

%unction, since the c*ea age o% one e ti!e "on! in this region causes !ramatic!ys%unction o% this !esmosoma* ca!herin.

igure F

Schematic !iagram o% !omains an! =T c*ea age site in +sg1. Jertica* *ines in!icate resum ti e ca*cium9"in!ing sites. 'rrowhea! shows =T c*ea age site. S, signa* e ti!e:/, ro e ti!e se?uence: TM, transmem"rane region.

'athoph!siolog! of blister locali(ation in ') bullous impetigo and ####* inactivationof Dsg1 with Dsg+ compensation. Bn "u**ous im etigo an! SSSS, "*isters occur in the

su er%icia* e i!ermis %rom *oss o% keratinocyte a!hesion in the granu*ar *ayer. Striking*y,an autoanti"o!y9me!iate! !isease, / , has ery simi*ar histo atho*ogy to that seen in "u**ous im etigo an! SSSS. / is cause! "y anti9+sg1 autoanti"o!ies 6 &2 & 7.

'*though these anti"o!ies are thought to inacti ate +sg1, which is %oun! throughout thee i!ermis an! in mucous mem"ranes, the "*ister occurs on*y in the su er%icia* e i!ermis.This "*ister *oca*ization has "een e; *aine! "y the !esmog*ein com ensationhy othesisI 6 &37. This hy othesis states that in areas o% e ithe*ium where "oth +sg& an!
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+sg1 are e; resse!, a s ontaneous "*ister wi** not occur when anti9+sg1 anti"o!iesinacti ate +sg1, "ecause +sg& can com ensate. Howe er, i% on*y +sg1 is resent, a

"*ister wi** occur. This hy othesis has "een a*i!ate! "oth "y c*inica* o"ser ation an!e; erimenta**y. Bn mucous mem"ranes, "oth +sg1 an! +sg& are %oun! throughout thee ithe*ia: there%ore, no "*isters are seen in / , e en though anti9+sg1 anti"o!ies "in! to

mucous mem"ranes. Bn e i!ermis, +sg1 is %oun! throughout, "ut +sg& is on*y in the !eee i!ermis. There%ore, / anti"o!ies cause on*y su er%icia* "*isters where +sg1 is notcom ensate! %or "y +sg&. Simi*ar*y, mothers with / assi e*y trans%er anti9+sg1anti"o!ies to their neonates: howe er, these neonates !o not !e e*o / "ecause neonata*skin, un*ike a!u*t skin, e; resses +sg& throughout a** *ayers 6 &F7.

=; erimenta* e i!ence %or the !esmog*ein com ensation hy othesis has "een o"taine! inthe neonata* mouse mo!e* o% / in which assi e*y trans%erre! anti"o!ies %rom /

atients cause c*inica**y an! histo*ogica**y ty ica* !isease 6 &37. Norma* neonata* micethat ha e +sg& in the !ee e i!ermis 6simi*ar to a!u*t human e i!ermis7 an! throughoutthe ora* mucous mem"ranes !e e*o on*y su er%icia* e i!erma* "*isters when in$ecte!

with / BgG, "ut Dsg3 knockout mice, which ha e no +sg& to com ensate, !e e*o "*isters in the !ee e i!ermis an! in ora* mucous mem"ranes when simi*ar*y in$ecte!.urthermore, mice that e; ress +sg& in the su er%icia* e i!ermis 6%rom a transgenecontaining +sg& c+N' !ri en o%% an in o*ucrin romoter7 are not susce ti"*e to

"*istering %rom in$ecte! / BgG 6 &F7. These !ata ro i!e an e; *anation %or anti"o!yinacti ation o% +sg1 resu*ting in "*isters on*y in the su er%icia* e i!ermis.

=Ts cause "*isters with atho*ogy i!entica* to those occurring in / . '*though =Tscircu*ate throughout the "o!y in SSSS, as !o anti9+sg1 anti"o!ies in / , "*isters occuron*y in the su er%icia* e i!ermis, not in the !ee e i!ermis or in mucous mem"ranes,i!entica* to the %in!ings in / . Such i!entica* atho*ogy can "e e; *aine! "y the *oss o%

%unction o% +sg1 cause! "y anti"o!ies in the case o% / , an! "y =T c*ea age in the caseo% SSSS or "u**ous im etigo. Loss o% %unction o% +sg1 "y c*ea age with =Ts is a*soconsistent with re ious immuno%*uorescence e; eriments in which =Ts cause ce**u*arinterna*ization an! *oss o% ce** sur%ace staining o% +sg1 6147.

ETs are s,in-smart. ' ma$or hysio*ogic %unction o% skin is to ro i!e a "arrier againstin%ection. Much o% that "arrier resi!es in the stratum corneum. S. aureus , through the useo% =Ts, has e o* e! an e%%icient an! e;treme*y %ocuse! mechanism o% ro*i%erating an!s rea!ing un!er that "arrier. Dnce intro!uce!, the "acteria can s rea! e%%icient*y "y using=T to ro!uce a c*ea age *ane right un!er the stratum corneum. To !o so, the "acteriumhas !e e*o e! a to;in that s eci%ica**y "in!s an! c*ea es a mo*ecu*e that is critica* toa!hesion in this area an! c*ea es it at a s eci%ic site that !estroys its %unction.
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Staphylococcal Exfoliative Toxin B Specifically CleavesDesmoglein 1

Masayuki 'magai, Takayuki Yamaguchi R, Yasushi Hanakawa , #o$i Nishi%u$i,Motoyuki Sugai R an! (ohn ) Stan*ey

1. +e artment o% +ermato*ogy, #eio -ni ersity Schoo* o% Me!icine, Tokyo, (a an:2. R+e artment o% 0acterio*ogy, Hiroshima -ni ersity Gra!uate Schoo* o%

0iome!ica* Sciences, Hiroshima, (a an:&. +e artment o% +ermato*ogy, -ni ersity o% /ennsy* ania Schoo* o% Me!icine,

/hi*a!e* hia, -.S.'.

Corres on!ence +r Masayuki 'magai, +e artment o% +ermato*ogy, #eio -ni ersitySchoo* o% Me!icine, &3 Shinanomachi, Shin$uku9ku, Tokyo 1F598382, (a an. =mai*amagai>sc.itc.keio.ac.$

)ecei e! 1& No em"er 2551: )e ise! 2< +ecem"er 2551: 'cce te! & (anuary 2552.

To o% age

Abstract

Sta hy*ococca* sca*!e! skin syn!rome an! its *oca*ize! %orm, "u**ous im etigo, showsu er%icia* e i!erma* "*ister %ormation cause! "y e;%o*iati e to;in ' or 0 ro!uce! "yStaphylococcus aureus . )ecent*y we ha e !emonstrate! that e;%o*iati e to;in 's eci%ica**y c*ea es !esmog*ein 1, a !esmosoma* a!hesion mo*ecu*e, that wheninacti ate! resu*ts in "*isters. Bn this stu!y we !etermine the target mo*ecu*e %or

e;%o*iati e to;in 0. =;%o*iati e to;in 0 in$ecte! in neonata* mice cause! su er%icia*e i!erma* "*isters, a"o*ishe! ce** sur%ace staining o% !esmog*ein 1, an! !egra!e!!esmog*ein 1 without a%%ecting !esmog*ein & or =9ca!herin. @hen a!eno irus9trans!uce!cu*ture! keratinocytes e; ressing e;ogenous mouse !esmog*ein 1 or !esmog*ein & wereincu"ate! with e;%o*iati e to;in 0, !esmog*ein 1, "ut not !esmog*ein &, was c*ea e!.urthermore, ce** sur%ace staining o% !esmog*ein 1, "ut not that o% !esmog*ein &, wasa"o*ishe! when cryosections o% norma* human skin were incu"ate! with e;%o*iati e to;in0, suggesting that *i ing ce**s were not necessary %or e;%o*iati e to;in 0 c*ea age o%!esmog*ein 1. ina**y, in vitro incu"ation o% the recom"inant e;trace**u*ar !omains o%!esmog*ein 1 an! !esmog*ein & with e;%o*iati e to;in 0 !emonstrate! that "oth mousean! human !esmog*ein 1, "ut not !esmog*ein &, were !irect*y c*ea e! "y e;%o*iati e

to;in 0 in a !ose9!e en!ent %ashion. These %in!ings !emonstrate that e;%o*iati e to;in 'an! e;%o*iati e to;in 0 cause "*ister %ormation in sta hy*ococca* sca*!e! skin syn!romean! "u**ous im etigo "y i!entica* mo*ecu*ar atho hysio*ogic mechanisms.

Keywords:

ca!herin, im etigo, em higus %o*iaceus, skin in%ection, SSSS
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Abbreviations:

+sg1, !esmog*ein 1: =T, e;%o*iati e to;in: =T', e;%o*iati e to;in ': =T0, e;%o*iati eto;in 0: SSSS, sta hy*ococca* sca*!e! skin syn!rome

Sta hy*ococca* sca*!e! skin syn!rome 6SSSS7 is a genera*ize! "*istering skin !iseasein!uce! "y the e;%o*iati e 6e i!ermo*ytic7 to;in 6=T7 o% Staphylococcus aureus 6Me*ish an!G*asgow, 14


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c*inica* an! histo*ogic "*isters simi*ar to those seen in "u**ous im etigo an! SSSS 6 Stan*ey,144&:'magai, 1444 :Mahoney et al , 1444 7. These o"ser ations *e! us to hy othesize an! ro e that =T's eci%ica**y c*ea es +sg1 6 'magai et al , 2555 7.

Bn this stu!y, we !etermine that the mo*ecu*ar atho hysio*ogy o% "*istering cause! "y

=T0 is i!entica* to that o% =T', name*y the !irect c*ea age o% +sg1.

To o% age

Materials and methods

Construction of recombinant !"

=T09 ro!ucing S. aureus TY chromosoma* +N' was re are! as !escri"e! re ious*y6Sugai et al , 1448 :Yamaguchi et al , 2551 7 an! use! as a o*ymerase chain reaction 6/C)7 tem *ate. '

rimer set, 3 9''GCTTCC'CCT''T'CCCT''T''TC9& an! 3 9

GG'TCC'C'G'GGTTC''CTC'TGGTT9& , was !esigne! to generate a 11 8 "+N' %ragment containing the entire etb gene 6M1


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co**ecte!. Those %ractions were co**ecte! an! %urther a *ie! to 0iosca*e CHT29Bhy!ro;ya atite H/LC co*umn 60io9)a!,


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!igeste! recom"inant +sg1, in!icating that )N 225 !i! not ro!uce any =T 6!ata notshown7.

To o% age

Results

%roduction of recombinant !"

c+N' %or =T0 was /C)9am *i%ie! %rom =T09 ro!ucing S. aureus TY , su"c*one! intoan E. coli-S. aureus shutt*e ector 6 TY1&&7, an! use! to trans%orm S. aureus )N 2256TY21& 7. TY21& ro!uce! =T0 in cu*ture su ernatant, which was recognize! as asing*e re!ominant "an! o% 2< k+a "y Coomassie 0*ue. N9termina* se?uencing o% the

rotein "an! i!enti%ie! that it was the correct*y rocesse! %orm o% recom"inant =T0 6!atanot shown7. ina**y &F5 g recom"inant =T0 was uri%ie! to homogeneity %rom 1 *iter o%cu*ture su ernatant as !escri"e! in Materials and Methods 6&igure $a 7.

)igure 1.

%roduction of recombinant !" with e'foliative activity( Coomassie 0*ue staine!S+S9/'G= shows recom"inant =T0 was ro!uce! as a 2< k+a e ti!e 6 A7. Neonata*mice in$ecte! with =T0 showe! e;tensi e "*isters 2 h a%ter in$ection 6 B7, whereas

neonata* mice in$ecte! with sa*ine a*one !i! not "*ister 6 7.

u** %igure an! *egen! 6F1#7

To con%irm the e;%o*iati e acti ity o% the recom"inant =T0, 25 g o% =T0 in 155 * o%/0S was su"cutaneous*y in$ecte! into neonata* mice. The mice starte! to show gross

"*isters as soon as 2 & h a%ter in$ection aroun! the in$ecte! site 6 &igure $b : com are tocontro* &igure $c 7.

Dsg$) but not Dsg* or +cadherin) was degraded in vivo in neonatalmouse skin in,ected with !"

To in estigate the mo*ecu*ar mechanism o% the "*ister %ormation "y =T0 an! to!etermine whether =T0 s eci%ica**y a%%ects +sg1 as !oes =T' 6 'magai et al , 2555 7, wee;amine! the skin %rom neonata* mice in$ecte! with =T0 "y immuno%*uorescence withanti"o!ies to +sg1, +sg&, an! =9ca!herin. @hen the skin was e;amine! 1 h a%ter =T0in$ection, the ce** sur%ace staining o% +sg1 was marke!*y !iminishe! 6 &igure -b :
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com are to contro* &igure -a 7. Bn the same area, the ce** sur%ace staining o% +sg& or =9ca!herin was una%%ecte! 6&igure -c.f 7.

)igure .

Immunofluorescence staining of cadherins in the epidermis of mice in,ected with!"( Neonata* mice were in$ecte! with /0S 6 A, , E 7 or =T0 6 B, D, ! 7 an! staine! %or+sg1 6 A, B7, +sg& 6 , D7, an! =9ca!herin 6 E , ! 7. +sg1 staining showe! that the ce**sur%ace staining o% +sg1 was remo e! "y in$ection o% =T0. The stainings o% +sg& an! =9ca!herin were not a%%ecte! "y in$ection o% =T0. Arro"heads in!icate the *ocation o% the

"asement mem"rane zone. Scale bar 35 m

u** %igure an! *egen! 6128#7

To !etermine whether this change in +sg1 staining was cause! "y !egra!ation o% +sg1 inthe neonata* mouse skin, e;tracts o% the skin %rom the mice in$ecte! with =T0 or /0Swere su"$ecte! to immuno"*ot ana*ysis %or +sg1, +sg&, an! =9ca!herin. ' 1F5 k+a "an!%or +sg1 was !egra!e! into a "an! o% a ro;imate*y 11& k+a in a** three mice in$ecte!with =T0, whereas +sg& an! =9ca!herin were not !egra!e! 6 &igure * 7.

)igure +.

Immunoblot analysis of skin e'tracts from neonatal mice in,ected with %"/ or !"(+sg1 was !egra!e! "y =T0 in$ection, whereas +sg& an! =9ca!herin were not a%%ecte!.=ach *ane re resents an e;tract %rom a !i%%erent mouse. Arro"heads an! arro"s in!icateintact +sg1 an! c*ea e! +sg1, res ecti e*y.

u** %igure an! *egen! 62


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'ogenous Dsg$) but not Dsg*) was degraded by addition of !" totransduced 0aCa! cells

To %urther !emonstrate s eci%ic c*ea age o% +sg1 "y =T0, we trans!uce! HaCaT ce**s, ahuman keratinocyte ce** *ine, with recom"inant a!eno irus containing c+N' enco!ing

mouse +sg1 or +sg& with a L'G e ito e tag on their C9termini. @hen 35 g er m* o%=T0 was a!!e! to the cu*ture me!ium o% these ce**s an! incu"ate! %or 1 h, +sg1 was!egra!e! whereas +sg& was not 6 &igure 1a 7. @hen these trans!uce! HaCaT ce**s wereincu"ate! with arious amounts o% =T0 65, 5.53, 5.13, an! 5.23 g er m*7 %or 15 min or1 h, +sg1 was c*ea e! in a !ose9 an! time9!e en!ent %ashion to a 11& k+a ro!uct6&igure 1b 7.

)igure /.

Immunoblot analysis of e'tracts from cultured keratinocytes incubated with !"(6 A7 HaCaT ce**s were trans!uce! with recom"inant a!eno irus with mouse +sg1 6+sg17or mouse +sg& 6+sg&7 or contro* a!eno irus without any insert 6C7, an! incu"ate! with35 g er m* =T0 %or 1 h. =;ogenous +sg1 an! +sg& was isua*ize! anti9 L'G taganti"o!y. +sg1, "ut not +sg&, was !egra!e! "y =T0. Arro"heads , asteris# , an! arro"s in!icate intact +sg1, intact +sg&, an! c*ea e! +gs1. 6 B7 HaCaT ce**s trans!uce! withrecom"inant a!eno irus with +sg1 was incu"ate! with arious amounts o% =T0 %or15 min or 1 h. +sg1 was !egra!e! "y =T0 in a !ose9 an! time9!e en!ent %ashion.


Dsg$ cleavage by !" is a direct effect

The in vivo %in!ings !escri"e! a"o e !o not ro e that =T0 !irect*y !egra!es +sg1, aso ose! to, %or e;am *e, !egra!ation through acti ation o% other roteases in *i ing ce**s.There%ore, to %irst !emonstrate that *i ing ce**s are not necessary %or this inacti ation o%+sg1, we incu"ate! cryosections o% norma* human skin with =T0, =T', or /0S, as acontro*, an! staine! them with anti"o!ies against arious !esmosoma* com onents6&igure 2 7. The ce** sur%ace staining o% +sg1 "y em higus %o*iaceus sera, which reactwith the e;trace**u*ar !omain o% +sg1 6 'magai et al , 1443 7, was remo e! "y =T0, whereas that

o% +sg& "y 3H15, which reacts with the amino termina* e;trace**u*ar !omain o% +sg&6/ro"y et al , 2555 7, was not a*tere! at a** 6&igure 2a) l) d) f 7. These e%%ects "y =T0 on the +sg1an! +sg& staining were i!entica* to those "y =T' 6 &igure 2b) e 7. =T0 treatment !i! nota%%ect the staining attern "y monoc*ona* anti"o!y +G&.15, which recognizes thecyto *asmic !omain o% +sg1 6 &igure 2g 7. =T0 treatment !i! not a*ter the staining o%!esmoco**in "y 329&+ nor that o% !esmo *akin "y 1193 6 &igure 2h) i 7. =T' treatment!i! not change the staining "y +G&.15, 329&+, or 1193 , either 6!ata not shown7. These
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%in!ings in!icate that =T0 as we** as =T' s eci%ica**y a%%ect the e;trace**u*ar !omain o%+sg1 in the a"sence o% *i ing ce**s, resuma"*y "y c*ea age.

)igure %.

In vitro !" treatment of cryosectioned normal human skin( Non9%i;e!cryosectione! norma* human skin was incu"ate! with =T' 6 B, E 7, E$B 6 , ! , %, H , & 7or/0S a*one 6 A, D7, an! staine! %or e;trace**u*ar !omain o% +sg1 6 A, B, 7, e;trace**u*ar!omain o% +sg& 6 D, E , ! 7, cyto *asmic !omain o% +sg1 6 %7, cyto *asmic !omain o%!esmoco**in 6+sc, H 7or !esmo *akin 6+/, & 7. The staining %or the e;trace**u*ar !omain o%+sg1 was remo e! "y =T0 6 7as seen with =T' 6 B7, whereas the staining %or +sg& wasnot a%%ecte!. The staining %or the cyto *asmic !omain o% +sg1, !esmoco**ins, or!esmo *akin was not a%%ecte!. Scale bar 35 m

u** %igure an! *egen! 6&14#7

To !emonstrate !irect roteo*ysis o% the e;trace**u*ar !omain o% +sg1 "y =T0, weincu"ate! a so*u"*e recom"inant %orm o% the e;trace**u*ar !omain o% +sg1 an! +sg& with=T0 in vitro 6&igure 3 7. =T0 c*ea e! the 81 k+a recom"inant mouse +sg1 !own to a &k+a e ti!e in a !ose9!e en!ent %ashion, whereas =T0 !i! not c*ea e mouse +sg& at a**.Bn the same way, =T0 c*ea e! the recom"inant human +sg1, "ut not human +sg&. These%in!ings in!icate that =T0 s eci%ica**y recognizes an! c*ea es the e;trace**u*ar !omain o%

"oth mouse an! human +sg1.

)igure 0.

In vitro !" treatment of recombinant e'tracellular domain of Dsg$ and Dsg*(

Mouse 6 A7 or human 6 B7 +sg1 an! +sg& e;trace**u*ar !omains ro!uce! in "acu*o iruswere incu"ate! with arious amounts o% =T0 6 A lane ' , 15 g er m*: lane ( , & g erm*: lane 3 , 1 g er m*: lane ) , 5.& g er m*: lane * , 5.1 g er m*: lane + , 5 g er m*:

B lane ' , 1 g er m*: lane ( , 5.& g er m*: lane 3 , 5.1 g er m*: lane ) , 5.5& g er m*:lane * , 5.51 g er m*: lane + , 5 g er m*7, an! su"$ecte! to immuno"*ots an! isua*ize!with anti9=9tag monoc*ona* anti"o!y. The e;trace**u*ar !omain o% +sg1, "ut not that o%+sg&, was c*ea e! "y =T0 in a !ose9!e en!ent %ashion. Arro"heads an! arro"s in!icate
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the intact recom"inant +sg1 an! c*ea e! ro!uct, res ecti e*y. Bars on the *e%t si!ein!icate mo*ecu*ar weight stan!ar!s 235, 155, 35, an! 23 k+a, %rom to to "ottom.


To o% ageDiscussion

The ma$or hysio*ogic %unction o% skin is to %orm a rotecti e "arrier that ham ers the enetration o% microorganisms an! inhi"its the *oss o% water. This "arrier has "een shownto resi!e in the stratum corneum. Bn "u**ous im etigo, S. aureus is %oun! in a "*isterca ity $ust "eneath the stratum corneum, thus circum enting the "arrier. This "*isterca ity is known to "e cause! "y =T, re*ease! "y the atho*ogic organisms. ' simi*ar

"*ister occurs in atients with the autoimmune !isease em higus %o*iaceus. Bn that!isease BgG autoanti"o!ies against +sg1 "*ock the ce** a!hesion %unction o% +sg1 with

resu*tant su er%icia* "*isters in the e i!ermis where +sg1 is e; resse! withoutcoe; resse! +sg& 6 'magai, 1444 :Mahoney et al , 1444 7. '*though +sg1 is a*so e; resse! in the !eee i!ermis an! mucous mem"ranes, "*isters !o not occur in these areas "ecause +sg& iscoe; resse! an! can com ensate %or the anti"o!y9in!uce! *oss o% %unction o% +sg1.0ecause em higus %o*iaceus shows i!entica* tissue s eci%icity an! histo*ogy to SSSSan! "u**ous im etigo, which are cause! "y =T, we re ious*y sus ecte! that the targetmo*ecu*e %or a ma$or ty e o% =T, =T', might "e +sg1 an! ro e! that =T' s eci%ica**yc*ea es +sg1 6 'magai et al , 2555 7.

Bn this stu!y, we hy othesize! that another ty e o% =T that causes SSSS an! "u**ousim etigo, =T0, a*so c*ea es +sg1. @e ha e shown that in$ection o% recom"inant =T0into neonata* mice cause! the s eci%ic remo a* o% ce** sur%ace staining o% +sg1 an! the!egra!ation o% +sg1 in vivo with resu*tant "*ister %ormation, whereas +sg& an! =9ca!herin were not a%%ecte!. Simi*ar*y, when =T0 was a!!e! to cu*ture! keratinocytese; ressing tagge! +sg1 or +sg&, =T0 !igeste! +sg1 without a%%ecting +sg&. S eci%ic*oss o% immuno%*uorescence o% +sg1, "ut not other ce** sur%ace mo*ecu*es, a%terincu"ation o% norma* human skin with =T0, suggeste! that inacti ation o% +sg1 "y =T0!i! not re?uire *i ing ce**s an! was ro"a"*y a !irect e%%ect. ina**y, we ha e!emonstrate! that =T0 s eci%ica**y c*ea e! the recom"inant e;trace**u*ar !omain o%+sg1, "ut not that o% +sg&, in vitro . These %in!ings in!icate that =T0 recognizes thee;trace**u*ar !omain o% +sg1 an! c*ea es it !irect*y.

Histo*ogic stu!ies suggeste! that =T "in!s to a rece tor in the granu*ar *ayer o% thee i!ermis, a*though to keratohya*in granu*es, not to the ce** sur%ace 6 Smith et al , 1484 7. Dtherstu!ies ha e suggeste! "in!ing to an e i!erma* GM 9*ike g*yco*i i! 6 Sakurai an! #on!o, 14


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Bt is hy othesize! that =T' an! =T0 are serine roteases. =T' an! =T0 are 23i!entica* to the sta hy*ococca* serine rotease J8. Structura* stu!ies show articu*ar*ystriking homo*ogy in the region o% the serine9as artic aci!9histi!ine cata*ytic tria! that%orms the acti e site o% try sin9*ike serine roteases 6 +ancer et al , 1445 7. Bn a!!ition, the 9raycrysta* structure o% =T' an! =T0 showe! signi%icant structure simi*arity with known

g*utamate9s eci%ic try sin9*ike serine roteases 6Jath et al , 144< :1444

7. Bt is interesting that =T'an! =T0 share i!entica* "in!ing s eci%icity an! c*ea age s eci%icity a*though o era**i!entity is on*y 5 . @e sus ect that amino aci! resi!ues that are res onsi"*e %or thiss eci%icity shou*! "e conser e!. The %ina* i!enti%ication o% =T' an! =T0 as g*utamate9s eci%ic serine roteases has to await the !etermination o% the c*ea age site o% +sg1 "y=T' an! =T0.

+sg1 is targete! "y =T' as we** as =T0 in SSSS an! "u**ous im etigo, resu*ting in a "*ister $ust "e*ow the stratum corneum, the ma$or "arrier in skin. These to;ins, then,a**ow the "acteria to circum ent this "arrier an! s rea! $ust "eneath it. These %in!ings

ro i!e an im ortant %ramework to un!erstan! the mo*ecu*ar mechanism %or "*ister

%ormation in these !iseases as we** as ce**9ce** a!hesion o% keratinocytes in the e i!ermis.

)ecom"inant Staphylococcus aureus =;%o*iati e To;ins 're Not 0acteria* Su erantigensLisa ). @. /*ano, 1 +e*ia M. Gutman, 2 Markus @oischnik, 2 an! Car*een M. Co**ins 2R

+e artments o% /e!iatrics 1 an! Micro"io*ogy an! Bmmuno*ogy, 2 -ni ersity o% MiamiSchoo* o% Me!icine, Miami, *ori!a &&151=!itor (. T. 0ar"ieriRCorres on!ing author. Mai*ing a!!ress +e artment o% Micro"io*ogy an! Bmmuno*ogy,/.D. 0o; 51F4F5 6)91&87, Miami, L &&151. /hone 6&537 2 &9F118. a; 6&537 2 &9F2&. =9mai* cco**ins>me!.miami.e!u .)ecei e! No em"er 2, 1444: )e isions re?ueste! +ecem"er 13, 1444: 'cce te!

e"ruary &, 2555.This artic*e has "een cite! "y other artic*es in /MC. DTH=) S=CTBDNSX

'0ST)'CTSta hy*ococca* sca*!e!9skin syn!rome is an e;%o*iati e !ermatitis characterize! "y these aration o% the e i!ermis at the stratum granu*osum. This !isru tion is me!iate! "y oneo% twoStaphylococcus aureus e;oto;ins, e;%o*iati e to;ins ' an! 0 6=T' an! =T07.0oth =T' an! =T0 ha e "een re orte! to "e "acteria* su erantigens. ' contro ersye;ists, howe er, as other !ata in!icate that these e;oto;ins are not su erantigens. Here we!emonstrate that recom"inant e;%o*iati e to;ins ro!uce! in Escherichia coli !o not actas T9ce** mitogens an! thus are not "acteria* su erantigens. These !ata %it the c*inica*

ro%i*e o% the !isease, which is not associate! with the c*assic sym toms o% asu erantigen9me!iate! syn!rome.

DTH=) S=CTBDNSX

Sta hy*ococca* sca*!e!9skin syn!rome 6SSSS7 is an e;%o*iati e !ermatitis o% in%ants an!chi*!ren that resu*ts %rom in%ection with e;%o*iati e9to;in9 ro!ucing Staphylococcus
http://www.nature.com/jid/journal/v118/n5/full/5601482a.html#bib8http://www.nature.com/jid/journal/v118/n5/full/5601482a.html#bib8http://www.nature.com/jid/journal/v118/n5/full/5601482a.html#bib8http://www.nature.com/jid/journal/v118/n5/full/5601482a.html#bib36http://www.nature.com/jid/journal/v118/n5/full/5601482a.html#bib36http://www.nature.com/jid/journal/v118/n5/full/5601482a.html#bib36http://www.nature.com/jid/journal/v118/n5/full/5601482a.html#bib37http://www.nature.com/jid/journal/v118/n5/full/5601482a.html#bib37mailto:[email protected]://www.ncbi.nlm.nih.gov/pmc/articles/PMC97528/citedby/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC97528/citedby/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC97528/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC97528/http://www.nature.com/jid/journal/v118/n5/full/5601482a.html#bib8http://www.nature.com/jid/journal/v118/n5/full/5601482a.html#bib36http://www.nature.com/jid/journal/v118/n5/full/5601482a.html#bib37mailto:[email protected]://www.ncbi.nlm.nih.gov/pmc/articles/PMC97528/citedby/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC97528/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC97528/


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aureus 61, 137. SSSS is characterize! "y the %ormation o% *arge "u**ae withoutin%*ammatory ce** in%i*trate an! se aration o% e;ten!e! areas o% the e i!ermis at thestratum granu*osum *ea ing the keratinocytes intact. Two "io*ogica**y an! sero*ogica**y!istinct S. aureus e;oto;ins are res onsi"*e %or the skin mani%estations o% SSSS inhumans, e;%o*iati e to;in ' 6=T'7 an! e;%o*iati e to;in 0 6=T07 6 237. =T' 62F.4 k+a7

is enco!e! on the "acteria* chromosome an! shares 5 amino aci! i!entity with the *asmi!9"orne =T0 62


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an =T'9 ro!ucing S. aureus strain 6!ata not shown7. Thus, r=T' an! r=T0, "ut notr=T'9S143', were a"*e to ro!uce the characteristic an! !iagnostic c*ea age o% SSSS inthis anima* mo!e*.

&I5( $Histo*ogica* e;amination o% neonata* mouse skin e; ose! to r=T' an!

/0S. Sections are %rom the "ases o% the tai*s o% 29!ay9o*! mice sacri%ice!at 2 h ostin$ection an! were staine! with hemato;y*in an! eosin.Magni%ication, [ 5. 6'7 Contro* mouse 6more ...7

+etermination o% su erantigen acti ity.r=T', r=T0, an! r=T'9S143' were assaye! %or mitogenic acti ity using human

eri hera* "*oo! mononuc*ear ce**s 6/0MCs7. Mitogenicity assays were er%orme! as!escri"e! re ious*y 6 127. 0rie%*y, he arinize! who*e "*oo! %rom human a!u*ts was%ractionate! on ico**9/a?ue 6/harmacia 0iotech, /iscataway, N.(.7 an! the /0MCs werehar este!. Ce**s 615 37 were a!!e! to 4F9we** -9"ottom *ates in )/MB 1F 5su *emente! with 15 %eta* ca*% serum an! were incu"ate! %or


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cata*ytic tria! o% this %ami*y, an! su"stitution o% an '*a resi!ue %or any one o% theseresi!ues in =T' resu*ts in to;in una"*e to cause e;%o*iation in the neonata* mouse mo!e*621, 22: L. ). @. /*ano an! C. M. Co**ins, un u"*ishe! !ata7. 0oth =T' an! =T0 ha eestero*ytic acti ity, which is associate! with serine roteases 6 &7. There%ore, it iscommon*y "e*ie e! that =T' an! =T0 are serine roteases. =ither they ha e a high*y

s eci%ic target 6*ike*y, gi en that the stratum granu*osum is the on*y site o% !amage seenin an into;icate! anima*7, or the con!itions re?uire! %or their cata*ysis ha e not "eeni!enti%ie!.Bt was suggeste! in the ear*y 1485s "y Mor*ock et a*. that the e;%o*iati e to;ins weremitogens 6 1


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im etigo, orang yang kontak !engan orang yang terkena im etigo segera mencuci tangan !engansa"un !an air menga*ir, mencuci akaian, han!uk !an s rei !ari en!erita im etigo ter isah !ariyang *ani**a 6cuci !engan air anas !an keringkan !i "awah sinar matahari atau engering yang

anas7, !an gunakan sarung tangan saat mengo*eskan anti"iotik to ika* !i tem at yang terin%eksi!an cuci tangan sete*ah itu.Tera i %armako*ogis yang !igunakan yaitu menggunakan anti"iotikto ika* atau anti"iotik er9ora*. /enggunaan anti"iotik er9ora* !i"erikan $ika asien sensiti%terha!a anti"iotik to ika* !an kon!isi enyakit atau *esi yang !itim"u*kan su!ah arah 6*esi *e"ih*uas7. 'nti"iotik to ika* yang !a at !igunakan yaitu mu irocin !an asam %usi!at. 'nti"iotik er9ora* yang !a at !igunakan yaitu eritromisin !an %*uk*oksasi*in.

%ilihan obat

Antibiotik topikal

Mu irocin

Nama Generik Mu irocin

Nama aten 0'CT)D0'N 6G*a;oSmith#*ine7

0ran! name 0acto!erm 6Bka harmin!o7

Bn!ikasi in%eksi ku*it rimer akut, misa*nya im etigo, %o*iku*itis, %urunku*osis

#ontrain!ikasi hi ersensiti% terha!a mu irocin

0entuk se!iaan sa*e !an krim

+osis sa*e `o*eskan &;Ehr se*ama 15 hari,

krim`o*eskan &;Ehr, $ika er*u !aerah yang !io"ati !itutu !engan kasa

*akukan e a*uasi $ika ti!ak a!a res on k*inis !a*am &93 hari

=%ek sam ing rasa ter"akar, gata*, rasa tersengat, kemerahan

/eringatan hin!ari kontak !engan mata. Hati9hati enggunaan a!a gangguan gin$a*se!ang sam ai "erat, hami*, *akatasi. Hentikan enggunaan $ika ter$a!i reaksi sensiti itasatau reaksi kimia. Ti!ak untuk !igunakan a!a ermukaan mukosa. /enggunaan $angka

an$ang menye"a"kan ertum"uhan "er*e"ihan !ari mikroorganisme yang ti!ak!iinginkan.

'sam usi!at

Nama Generik 'sam usi!at


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0ran! name '%uci! 6 erron7, usycom 6Com"i har7, u*a!ic 6Guar!ian7, uta!erm6Bnter"at7

Bn!ikasi Bm etigo kontagiosum, %o*iku*itis su er%is!ia*, %urunku*osis, sikosis "ar"ae, hi!ra!enitis akse*aris, a"ses, aronikia, eritrasma

#ontrain!ikasi hi ersensiti% terha!a asam %usi!at.

0entuk se!iaan sa*e 6Na %usi!at7 !an krim 6asam %usi!at7

+osis tan a em"a*utEkasa steri* gunakan &9 ;Ehari

!engan em"a*utEkasa steri* gunakan *e"ih sering *ama tera i kurang*e"ih < hari.

=%ek sam ing reaksi sensiti%itas misa*nya ruam ku*it, urtikaria, iritasi

/eringatan hin!ari enggunaan a!a "agian mata. /enggunaan $angka !a atmeningkatkan resiko sensitisasi ku*it !an resistensi "akteri. Hami* trimester ertama.0ayi "aru *ahir.

Antibiotik per+oral

=ritromisin

Nama Generik =ritromisin

Nama aten =)YTH)DCBN 6'""ott7

0ran! name Corsatrocin 6Corsa7.

Bn!ikasi in%eksi sa*uran na%as "agian atas !an "awah tonsi*itas, a"ses eritonsi*er,%aringitis, *aringitis, sinusitis, in%eksi sekun!er a!a !emam !an %*u, trakeitis, "ronkitis akut !ankronis, neunomia, "ronkiektaksis. Bn%eksi te*inga otitis me!ia !an eksterna*, mastoi!itis.Bn%eksi ora* gingi itis, angina incenti. Bn%eksi mata "*e%aritis. Bn%eksi ku*it !an $aringan *unak%urunke* !an kar"unke*, aronikia, a"ses, akne ustu*aris, im etigo, se*u*itis, erisi e*as.

#ontrain!ikasi hi ersensiti% terha!a eritromisin, enyakit hati.

0entuk se!iaan ta"*et atau ka su*

+osis !ewasa 192gEhr tia F, 8 atau 12 $am. Bn%eksi "erat gEhr !a*am !osis ter"agi.

'nak &5935 mgEkg00Ehr tia F, 8 atau 12 $am.

0ayi92tahun 123mg ;Ehr, 298tahun 235 mg ;Ehr atau 355 mg tia 12$am


30/30

Se"e*um atau a!a waktu makan.

=%ek sam ing $arang he atotoksik, ototoksik.

Gangguan GB mua*, muntah, nyeri erut,!iare.

-rtikaria, ruam !an reaksi a*ergi *ainya.

/eringatan gangguan gin$a*, gangguan %ungsi hati, or%iria, kehami*an 6ti!ak!iketahui e%ek "uruknya7 menyusui 6se$um*ah keci* masuk ke 'SB7

*uk*oksasi*in

Nama Generik %*uk*oksasi*in Na monohi!rat

0ran! name LD '/=N 6G*a;oSmith#*ine7

Bn!ikasi in%eksi "akteri gram6W7 termasuk yang resisten enisi*in.

Bn%eksi karena sta i*okokus terutama a!a ku*it 6im etigo, se*u*itis7

#ontrain!ikasi hi ersensiti% terha!a enisi*in, "ayi yang *ahir !ari i"u yanghi ersensiti% enisi*in.

0entuk se!iaan ka su* 6235 mg, 355mg7

+osis !ewasa 2359355 mg tia 8 $am 6&;Ehr7.

'nak U2tahun F2,3mg &;Ehr 6tia 8 $am7, 2915tahun 123 mg &;Ehr 6tia 8 $am7

=%ek sam ing mua*, muntah, nyeri erut, !iare.-rtikaria, ruam ku*it, ka!ang ter$a!ireaksi ana%i*aktik.

/eringatan hi ersensiti% enisi*in, gangguan gin$a*, *eukimia *im%atik.

molecular mechanisms of blister formation in bullous impetigo and staphylococcal scalded skin...

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