Molecular mechanisms of blister

formation in bullous impetigo and

staphylococcal scalded skin syndrome
Yasushi 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 to1 (ohn ). Stan*ey, +e,artment o% +ermato*ogy, -ni.ersity o%
/ennsy*.ania, Schoo* o% Me!icine, 211 C*inica* )esearch 0ui*!ing, 213 Curie 0ou*e.ar!,
/hi*a!e*,hia, /ennsy*.ania 14152, -S'. /hone1 62137 8489&225: a;1 62137 3<&925&&:
=9mai*1 $rstan>mai*.me!.u,enn.e!u.
/u"*ishe! (u*y 1, 2552
)ecei.e! %or ,u"*ication ',ri* 22, 2552, an! acce,te! in re.ise! %orm May 24, 2552.
0u**ous im,etigo !ue to Staphylococcus aureus is one o% the most common "acteria*
in%ections o% man, an! its genera*ize! %orm, sta,hy*ococca* sca*!e! skin syn!rome
6SSSS7, is a %re?uent mani%estation o% sta,hy*ococca* e,i!emics in neonata* nurseries.
0oth !iseases are me!iate! "y e;%o*iati.e to;ins 6=Ts7, which show e;?uisite ,atho*ogic
s,eci%icity in "*istering on*y the su,er%icia* e,i!ermis. @e show that these to;ins act as
serine ,roteases with e;treme*y %ocuse! mo*ecu*ar s,eci%icity to c*ea.e mouse an!
human !esmog*ein 1 6+sg17 once a%ter g*utamic aci! resi!ue &81 "etween e;trace**u*ar
!omains & an! 2. Mutation o% the ,re!icte! cata*ytica**y acti.e serine to a*anine
com,*ete*y inhi"its c*ea.age. The mutate! =Ts "in! s,eci%ica**y to +sg1 "y
immuno%*uorescence co*oca*ization an! "y coimmuno,reci,itation. Thus, =Ts, through
s,eci%ic recognition an! ,roteo*ytic c*ea.age o% one structura**y critica* ,e,ti!e "on! in
an a!hesion mo*ecu*e, cause its !ys%unction an! a**ow S. aureus to s,rea! un!er the
stratum corneum, the main "arrier o% the skin, e;,*aining how, a*though they circu*ate
through the entire "o!y in SSSS, they cause ,atho*ogy on*y in the su,er%icia* e,i!ermis.
Introduction
-n!erstan!ing the ,atho*ogy resu*ting %rom Staphylococcus aureus in%ection is o% great
interest in me!icine "ecause o% the organismAs common an! increasing ,re.a*ence in
humans, its growing "acteria* resistance, an! its a"i*ity to cause serious an! *i%e9
threatening !isease 617. To;ins contri"ute in a ma$or way to the ,athogenicity o% S.
aureus, as they !o with many other "acteria 627.
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 15C o% a** skin ,ro"*ems in
chi*!ren 6&7. D% these im,etigo ,atients, a"out &5C ha.e "u**ous im,etigo, which is
cause! "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 &5C may "e co*onize! with S. aureus, an! organisms
that ,ro!uce =Ts are the ma$or cause o% neonata* nursery out"reaks o% sta,hy*ococca*
!isease 61, 27. New"orns, young chi*!ren, an! a!u*ts with rena* %ai*ure an!Eor who are
immunocom,romise! may !e.e*o, a genera*ize! %orm o% "u**ous im,etigo ca**e!
sta,hy*ococca* sca*!e! skin syn!rome 6SSSS7. Bn this !isease, "u**ae an! erosions
!e.e*o, o.er a *arge area o% the skin sur%ace, resu*ting in the !eath o% ,ro"a"*y *ess than
&C o% a%%ecte! chi*!ren "ut u, to F5C o% a%%ecte! a!u*ts 63, F7.
The now9c*assic e;,eriments o% Me*ish et a*. in the ear*y 14<5As esta"*ishe! that =T
,ro!uce! "y S. aureus causes the "*istering in "u**ous im,etigo an! SSSS 6<G47. /assi.e
trans%er o% the to;in to neonata* mice cause! the same skin *esions seen in humans,
name*y a "*ister in the su,er%icia* *i.ing e,i!ermis cause! "y se,aration o% the
keratinocytes in the granu*ar ce** *ayer !ue to s,*itting o% the !esmosomes as either a
,rimary or secon!ary e.ent 615, 117. /resuma"*y, this ty,e o% "*ister a**ows the "acteria
to s,rea! un!er an! circum.ent the stratum corneum, a ma$or "arrier that ,re.ents
in%ection o% the skin. Two ma$or =Ts, =T' an! =T0, which share 25C i!entity in amino
aci! se?uence, ha.e "een i!enti%ie! an! c*one! 612, 1&7. )ecent*y, a thir! =T 6terme!
=T+7 that causes i!entica* e,i!erma* "*isters has "een i!enti%ie! 6T. Yamaguchi et a*.,
un,u"*ishe! o"ser.ations7. Bn "u**ous im,etigo, "*isters occur at the site o% in%ection with
S. aureus, an! are cause! "y *oca* ,ro!uction o% =T, whereas in SSSS the to;in is
,ro!uce! at the site o% in%ection 6not necessari*y in the skin7 an! causes genera*ize!
"*istering !ue to its systemic circu*ation. The *atter !isease un!erscores the e;?uisite
s,eci%icity o% =Ts that, e.en when they circu*ate through the entire "o!y, cause ,atho*ogy
on*y in the su,er%icia* e,i!ermis.
The ,atho,hysio*ogic "asis o% this s,eci%icity has "een a mystery %or o.er &5 years, e.en
though recent crysta* structure an! amino aci! se?uences o% =T' an! =T0 ha.e
suggeste! that they are Haty,ica*I g*utamate9s,eci%ic serine ,roteases. The term
Haty,ica*I was use! "ecause some crysta* structures o% =T' an! =T0 ha.e shown that
the ,resume! cata*ytic site is not con%igure! ,ro,er*y to "e acti.e, ,erha,s re?uiring
acti.ation "y "in!ing to a s,eci%ic su"strate or rece,tor 612G1<7. Dn the other han!, it has
"een argue! that the to;icity o% =Ts may not "e as serine ,roteases at a** 6e.g., !ata has
suggeste! that these =Ts are ,oor*y inhi"ite! "y serine ,rotease inhi"itors7, "ut as
su,erantigens or through *ess9!irect mechanisms such as re*ease o% ,roteases "y the
e,i!ermis 6re.iewe! in re%s. 1, 187. urthermore, unti* .ery recent*y, ,roteo*ysis o% a
re*e.ant "io*ogica* target ha! not "een !emonstrate!.
Jery recent*y, we i!enti%ie! a re*e.ant su"strate o% =T', =T0, an! =T+ to "e
!esmog*ein 1 6+sg17, a transmem"rane g*yco,rotein o% !esmosomes in the ca!herin gene
su,er%ami*y 614, 257 6T. Yamaguchi et a*., un,u"*ishe! o"ser.ations7. +sg1, "ut not the
c*ose*y re*ate! +sg& or =9ca!herin, is c*ea.e! "y =Ts. urthermore, +sg1 is a re*e.ant
su"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 seen
in "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!Gs,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! SSSS
can thus "e attri"ute! to hy!ro*ysis o% a sing*e ,e,ti!e "on!, ,resuma"*y in a %unctiona**y
critica* area in +sg1.
Methods
Recombinant ETs. The e;,resse! ,roteins, .ectors, an! their hosts are summarize! in
Ta"*e 1. Shutt*e .ector ,C=152, containing =T' an! =T' C
mu
, was %rom /atrick
Sch*ie.ert 6+e,artment o% Micro"io*ogy, -ni.ersity o% Minnesota Me!ica* Schoo*,
Minnea,o*is, Minnesota, -S'7 6127. =T' '
mu
was constructe! %rom these .ectors with
the KuikChange Site9+irecte! Mutagenesis #it 6Stratagene, La (o**a, Ca*i%ornia, -S'7.
=T0 '
mu
was c*one! "y /C) using ,C=1529=T0 6257 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 L'G 6Sigma9'*!rich, St. Louis, Missouri, -S'7, to the &M en! o% these =T
constructs. =T'9J3His, =T' C
mu
GJ3His, =T' '
mu
GJ3His, =T09J3His, =T0G&L
L'G, an! =T0 '
mu
GJ3His were su"c*one! into the Hin!BBB an! Eco)B sites o% ,C=152.
+N' was trans%orme! into the S. aureus strain )N2225 6,ro.i!e! "y /atrick Sch*ie.ert7
.ia e*ectro,o*ation. The su,ernatant %rom )N2225 trans%orme! with these ,*asmi!s was
,reci,itate! with 83C ammonium su*%ate. /reci,itate! =Ts were !ia*yze! against /0S
with 1 mM CaC*
2
6/0S9Ca7. His9tagge! =Ts were ,uri%ie! on Ni9NT' co*umns
6KB'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 %rom
0io9)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 were
c*one! into ,K=<5 in or!er to e;,ress the %usion ,rotein with a F;His tag se?uence on the
car"o;y terminus in E. coli. The recom"inant ,roteins were ,uri%ie! using Ni9NT' resin
accor!ing to the manu%acturerAs ,rotoco* 6KB'G=N Bnc.7.
or com,arison o% mutant to wi*!9ty,e =Ts 6see igure 27, the su,ernatants %rom
trans%orme! )N2225 sta,hy*ococci were use!, with the amount o% =Ts norma*ize! "y
Coomassie "*ue staining o% S+S9/'G= ge*s in which the =Ts were the ma$or "an! an!
accounte! %or o.er 45C o% the tota* ,rotein.
igure 2
/oint mutation o% serine 143 6chymotry,sin num"ering7, the ,resume! cata*ytica**y
acti.e serine, o% =Ts inhi"its c*ea.age o% +sg1. 6a7 'ntiG=9tag anti"o!y immuno"*ot o%
S+S9/'G= o% h+sg1= incu"ate! with )N2225 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
6serine
143 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!icate
migration o% mo*ecu*ar weight markers o% 8& k+a 6to,7 an! &2 k+a. 6b7 'ntiGL'G9tag
immuno"*ots o% antiGL'G9tag immuno,reci,itates o% e;tracts o% m+sg19L'G
a!eno.irusGtrans!uce! ce**s that were incu"ate! with =T0 or =T0 '
mu
. =T0 '
mu
shows
no c*ea.age. Horizonta* *ines, %rom to,, in!icate migration o% mo*ecu*ar weight markers
o% 25& k+a, 113 k+a, an! 4& k+a. 6c7 'ntiG=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! &2 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** keratinocytes
cu*ture! in +M=M with 15C 0S were trans!uce! with recom"inant a!eno.irus .ectors
';9m+sg1 an! ';9m+sg& 6257, enco!ing mouse +sg1 an! +sg&, res,ecti.e*y, with
car"o;y9termina* L'G tags 6Ta"*e 17. '%ter 22 hours, the ce**s were incu"ate! with
recom"inant =T', =T0, or =T+ in cu*ture me!ia %or 15 minutes. Then the ce**s were
washe! 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! 6217.
6These constructs a*so containe! a His tag on the car"o;y terminus.7 ',,ro;imate*y 1 Ng
o% 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 1
mM CaC* 6T0S9Ca7 %or 1 hour at &<OC. The !igeste! sam,*es were su"$ecte! to S+S9
/'G=, trans%erre! to /J+ mem"ranes, an! staine! using Coomassie "ri**iant "*ue.
Staine! "an!s were cut out an! se?uence! "y =!man !egra!ation at the /rotein
Microchemistry aci*ity o% The @istar Bnstitute 6/hi*a!e*,hia, /ennsy*.ania, -S'7.
Antibodies. 'ntiG=9tag mouse monoc*ona* anti"o!y con$ugate! with horsera!ish
,ero;i!ase 6H)/: 'mersham 0iosciences Bnc.7, antiGJ39tag mouse monoc*ona* anti"o!y
6Bn.itrogen Cor,.7, antiGHis9tag mouse monoc*ona* anti"o!y 6KB'G=N Bnc.7, antiG
L'G9tag mouse monoc*ona* anti"o!y 6M2: Sigma9'*!rich7, anti9=T' shee, ,o*yc*ona*
anti"o!y 6To;in Techno*ogy Bnc., Sarasota, *ori!a, -S'7, anti9=T+ ra""it ,o*yc*ona*
anti"o!y 6T. Yamaguchi, un,u"*ishe! o"ser.ations7, an! / serum 6anti9+sg17 were use!
%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 243 an! anti9mouse anti"o!y
con$ugate! with '*e;a *uor 345 6Mo*ecu*ar /ro"es Bnc., =ugene, Dregon, -S'7 were
use! %or immunohistochemistry.
Ex vivo binding of ETA A
mu
to mouse epidermis and immunohistochemistr!. Mouse
tai* or ,aw skin was incu"ate! in +M=M at a ratio o% 111 with 15C 0S an! =T' '
mu
G
J3His 6 8& NM7 in /0S9Ca at 2OC %or 12 hours an! then cryosectione!. '%ter "eing
%i;e! with 2C ,ara%orma*!ehy!e in /0S9Ca, s,ecimens were "*ocke! with 1C 0S' in
/0S9Ca. 0*ocke! s,ecimen was incu"ate! with anti9J3 anti"o!y %o**owe! "y anti9mouse
anti"o!y con$ugate! with '*e;a *uor 345, an! then staine! with / serum %o**owe! "y
anti9human anti"o!y con$ugate! with '*e;a *uor 243. Staine! sections were
,hotogra,he! using con%oca* microsco,y 6M)C 1522: 0io9)a! La"oratories Bnc.7. or
contro*s, skin was %irst incu"ate! with wi*!9ty,e =T' or an irre*e.ant to;in
6sta,hy*ococca* enteroto;in 0 with a J3 tag7, then staine! with / serum or anti9J3
anti"o!y, res,ecti.e*y. These contro*s showe! no staining 6not shown7.
Immunoprecipitation. '%ter incu"ation with =Ts, a!eno.irus9in%ecte! ce**s were
e;tracte! with 1C Triton L9155 in T0S9Ca with ,rotease inhi"itors 62 NgEm* a,rotinin, 2
NgEm* *eu,e,tin, an! 1 mM /MS7 %or immuno,reci,itation. Su,ernatants o% High i.e
insect ce**s 6Bn.itrogen Cor,.7 trans!uce! with "acu*o.irus enco!ing h+sg1= were
incu"ate! with =Ts, then use! !irect*y %or immuno,reci,itation. L'G9tagge! ,roteins
were ,reci,itate! with anti9L'G agarose 6Sigma9'*!rich7, an! =9tagge! ,roteins were
,reci,itate! with antiG=9tag Se,harose 6'mersham 0iosciences Bnc.7 at 2OC %or 1 hour.
Bmmuno,reci,itates were washe! ten times with 1C Triton L9155 in T0S9Ca, then e*ute!
with Laemm*i sam,*e "u%%er at 155OC.
Immunoblotting. Ce** e;tracts or immuno,reci,itates in Laemm*i sam,*e "u%%er were
se,arate! "y 2G25C or 15C Tris9g*ycine S+'9/'G= 60io9)a! La"oratories Bnc. or
Bn.itrogen Cor,.7, then trans%erre! to nitroce**u*ose sheets 6Trans90*ot: 0io9)a!
La"oratories Bnc.7. The sheets were incu"ate! %or 1 hour in "*ocking "u%%er 63C %at9%ree
mi*k ,ow!er in /0S7. The %irst anti"o!y, !i*ute! in "*ocking "u%%er, was a,,*ie! %or 1
hour at room tem,erature. '%ter two washes with 5.1C Tween 25 in /0S, the sheets were
incu"ate! with H)/9con$ugate! secon!ary anti"o!y !i*ute! in "*ocking "u%%er. Bn some
e;,eriments, on*y one anti"o!y, H)/9con$ugate! anti9=T', was use!. '%ter the "*ots
were washe!, the signa*s were !etecte! with chemi*uminescence 6=CL or =CL /*us,
'mersham 0iosciences Bnc.7.
Results
ETs cleave human and mouse Dsg1 at a uni"ue site. /re.ious stu!ies o% ,roteo*ysis o%
+sg1 "y =Ts i!enti%ie! on*y a sma** car"o;y9termina* %ragment, *ea.ing the e;tent o%
!egra!ation uncertain. To !etermine whether there is one s,eci%ic c*ea.age site as
o,,ose! to more genera* ,roteo*ytic !egra!ation, the e;trace**u*ar !omain o% human
+sg1, containing an = tag 6h+sg1=7 on the car"o;y terminus, was ,uri%ie! an! c*ea.age
was characterize! "y S+S9/'G= an! amino9termina* se?uence ana*ysis. h+sg1= was
,ro!uce! "y "acu*o.irus in High i.e insect ce**s 6217. The recom"inant ,rotein was
iso*ate! %rom insect ce** su,ernatant with antiG=9tag Se,harose an! e*ution with e;cess =
,e,ti!e. Coomassie "*ue staining o% the ,e,ti!e9e*ute! materia* reso*.e! "y S+S9/'G=
6igure 1a, *e%t7 in!icates a c*ose*y s,ace! !ou"*et at the ,re!icte! mo*ecu*ar weight o%
82 k+a. The iso*ate! h+sg1= was incu"ate! %or 1 hour with =T' at &<OC, an! the
,ro!ucts were se,arate! "y S+S9/'G= 6igure 1a, right7. Two ma$or "an!s o%
a,,ro;imate*y 35 k+a 6arrow marke! 17 an! &2 k+a 6%i**e! arrowhea!7 were !etecte! "y
Coomassie "*ue staining, suggesting that h+sg1= was c*ea.e! at a sing*e site.
Bmmuno"*otting with antiG=9tag anti"o!ies o% h+sg1= !igests i!enti%ie! the &29k+a "an!
as the car"o;y9termina* %ragment 6igure 1"7. 'mino termina* ,e,ti!e se?uencing o% this
%ragment shown in igure 1a 6right si!e, arrowhea!7 was then use! to !etermine the e;act
c*ea.age site, which was a%ter g*utamic aci! resi!ue &81 6as counte! %rom the initiating
methionine o% h+sg17 6igure 1e7. ' simi*ar stu!y showe! that =T' c*ea.e! mouse +sg1
at the same site 6igure 1e7.
igure 1
C*ea.age site o% human an! mouse +sg1 "y =Ts. 6a7 Coomassie "ri**iant "*ue staining o%
S+S9/'G= o% ,uri%ie! h+sg1= 6*e%t ,ane*7 an! its =T' c*ea.age ,ro!ucts 6right ,ane*7.
D,en arrowhea! shows a c*ose*y s,ace! !ou"*et at the ,re!icte! mo*ecu*ar weight o% 82
k+a, re,resenting the ,ro,rotein an! mature ,rotein. C*ea.age resu*ts in ,ro!ucts with
a,,ro;imate mo*ecu*ar weights o% 35 k+a 6arrow 17, 23 k+a 6arrow 27, an! &2 k+a
6%i**e! arrowhea!7. 6b7 Bmmuno"*otting with antiG=9tag anti"o!ies o% h+sg1= c*ea.e! "y
=T' an! =T0 i!enti%ies the &29k+a "an! 6%i**e! arrowhea!7 as the car"o;y9termina*
%ragment. D,en arrowhea! in!icates unc*ea.e! h+sg1=. 6c7 Coomassie "*ue staining o%
S+S9/'G= o% the car"o;y9c*ea.age %ragments o% h+sg1= 6%i**e! arrowhea!s7 c*ea.e! "y
=T0 or =T+ an! ,uri%ie! on an antiG=9tag Se,harose co*umn. 6d7 /e,ti!e se?uencing
6resi!ues %o**owing P1P in *ight gray7 o% the 359k+a c*ea.age ,ro!uct 6arrow 1 in a7
showe! the amino aci! se?uence o% the amino terminus o% h+sg1= *acking the signa*
,e,ti!e 6resi!ues in green7 "ut sti** containing the ,ro,e,ti!e 6resi!ues in "*ue7. /e,ti!e
se?uencing 6resi!ues %o**owing P2P in *ight gray7 o% the 239k+a ,e,ti!e 6arrow 2 in a7
i!enti%ies the amino terminus o% mature h+sg1 6resi!ues in "*ack7. 6e7 C*ea.age site in
+sg1. 'mino9termina* ,e,ti!e se?uencing 6resi!ues in gray7 o% the &29k+a car"o;y
terminus o% h+sg1= 6%i**e! arrowhea!s in a an! c7 an! m+sg1= 6S+S9/'G= not shown7
c*ea.e! "y =Ts. /e,ti!e se?uence in "*ack in!icates mouse an! human +sg1 an! +sg&
aroun! the c*ea.age site. Sha!ing shows ,e,ti!e i!entity. '', amino aci!.
To !etermine the site o% c*ea.age "y =T0 an! =T+ in h+sg1, we incu"ate! un,uri%ie!
h+sg1= 6o"taine! !irect*y %rom "acu*o.irus su,ernatant7 with these =Ts, then ,uri%ie!
the car"o;y terminus with antiG=9tag Se,harose. Coomassie "*ue staining o% the resu*ting
,uri%ie! car"o;y9c*ea.age %ragments se,arate! on S+S9/'G= is shown in igure 1c.
'mino9termina* ,e,ti!e se?uencing o% these %ragments in!icate! that the =T0 an! =T+
c*ea.age site in +sg1 was i!entica* to that ,ro!uce! "y =T' 6igure 1e7. These !ata are
consistent with structura* mo!e*s that suggest that =T' an! =T0 might c*ea.e a su"strate
a%ter a g*utamic aci! resi!ue.
'mino termina* se?uence ana*ysis o% the s*ower migrating 359k+a "an! %orme! "y
c*ea.age o% h+sg1= 6igure 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 6igure 1!7. Se?uencing o% the "an! shown imme!iate*y "e*ow
the 359k+a "an! 6igure 1a, arrow marke! 27 in!icate! that it was the amino terminus o%
the h+sg1= mature ,rotein 6igure 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 that
the se?uence ,re.ious*y ,re!icte! "y com,uter mo!e*ing to "e the signa* ,e,ti!e in
h+sg1 6igure 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 are
known not to c*ea.e the c*ose*y re*ate! +sg& 614, 257. Se?uence a*ignment o% human an!
mouse +sg1 an! +sg& 6igure 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 =T0
corres,on!s to the cata*ytica**y acti.e serine 143 o% chymotry,sin. urthermore, *ike
chymotry,sin, =T' an! =T0 ha.e a %unctiona* cata*ytic tria! consisting o% serine
143E18FE143, histi!ine <2EF3E3<, an! as,artic aci! 125E112E152 6=T'E=T0Echymotry,sin
num"ering: su"se?uent num"ering wi** "e %or chymotry,sin on*y7. To !emonstrate that
this serine is critica* in the c*ea.age o% h+sg1, it was mutate! to a cysteine 6resu*tant
mutant =T' C
mu
7. Bncu"ation o% =T' C
mu
with +sg1 at &<OC %or 1 hour !emonstrate! a
marke!*y !ecrease! rate o% c*ea.age com,are! with wi*!9ty,e =T' 6igure 27. 'ssuming
this acti.ity was *ike*y me!iate! "y the cysteine su*%hy!ry* grou,, simi*ar to what has
"een %oun! in a simi*ar mutant o% try,sin 6227, we mutate! serine 143 to a*anine 6=T'
'
mu
7. The =T' '
mu
!i! not c*ea.e +sg1 6igure 27. @e simi*ar*y showe! that mutation o%
serine 143 to a*anine in =T0 an! =T+ inhi"ite! c*ea.age o% +sg1 6igure 27.
The o"ser.ations that su"stitution o% serine 143 with "oth cysteine an! a*anine inhi"it
c*ea.age, an! that the mutant =Ts sti** s,eci%ica**y "in! +sg1 6see "e*ow7, !emonstrate
that serine 143 is necessary %or e%%icient cata*ytic c*ea.age o% +sg1.
&inding of ETs to Dsg1. Crysta* structura* stu!ies o% =T' ha.e suggeste! that it may "e
an inacti.e enzyme !ue to an ina,,ro,riate a*ignment o% certain resi!ues %orming the
acti.e site. Bt has "een ,ostu*ate! that as a conse?uence, =T' may ha.e to "in! to its
s,eci%ic su"strate or to a rece,tor in or!er to "ecome cata*ytic 612G1F, 2&7. 6Howe.er, %or
an o,,osing .iew, see re%. 1<.7 Bn SSSS an! in mouse mo!e*s o% SSSS, =Ts !i%%use
through the entire "o!y, yet ha.e e;?uisite s,eci%icity in causing ,atho*ogy 6e.g., "*isters7
on*y in the su,er%icia* e,i!ermis. @e ha.e hy,othesize! that this s,eci%icity is !ue to
s,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 6K
!
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! "y
re*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 6igure &7. 't concentrations o%
1& NM an! a"o.e, =T' '
mu
most*y inhi"ite! c*ea.age, "ut at concentrations o% 2 NM 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 NM, a**owing K
!
to "e rough*y estimate! as %o**ows1
igure &
=T' '
mu
inhi"its c*ea.age o% +sg1 "y =T'. 'ntiG=9tag immuno"*ot o% h+sg1= 6o,en
arrowhea!7 an! its car"o;y9termina* c*ea.age ,ro!uct 6%i**e! arrowhea!7. =T' '
mu
at a
concentration o% 1& NM incu"ate! with h+sg1= inhi"its c*ea.age "y su"se?uent a!!ition
o% wi*!9ty,e =T', "ut at 2 NM !oes not. 't 8 NM, a"out ha*% o% the +sg1 is %ree to "e
c*ea.e!. K
!
can then "e rough*y estimate! to "e 8 NM. =T' '
mu
in the concentrations
shown 6a** in *arge e;cess o% that o% h+sg1=7 was incu"ate! with h+sg1 at 23OC %or F5
minutes, 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. This
was !emonstrate! "y incu"ating mouse skin with =T' '
mu
with a J3 e,ito,e tag on the
car"o;y terminus at concentrations in e;cess o% K
!
. Bmmuno%*uorescence with anti"o!ies
against the J3 tag showe! "in!ing o% =T' '
mu
to e,i!ermis in a ,attern i!entica* to that
o% +sg1 6igure 27.
igure 2
Bmmuno%*uorescence co*oca*ization o% =T' '
mu
an! +sg1 in e,i!ermis. Mouse skin was
incu"ate! with =T' '
mu
with a J3 tag. +ou"*e immuno%*uorescence staining with
anti"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.
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 as
c*ea.age ,rogresse! 6igure 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*ower
c*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 6igure 3"7. ina**y, =T' '
mu
, which !oes
not c*ea.e +sg1 at a**, showe! increase! "in!ing with time.
igure 3
Co,reci,itation o% =Ts with +sg1. 6a7 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. 6b7 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. 6c7 =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*y
homo*ogous in amino aci! se?uence to +sg1. Simi*ar resu*ts were %oun! with =T+ 6!ata
not 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 amino
aci! se?uence to +sg1 6igure 3c7.
Discussion
ETs are serine proteases that specificall! bind to and cleave Dsg1 at a uni"ue site after
a 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 immune
system: an! acting as *i,ases 6re.iewe! in re%. 17. Howe.er, recent in%erences %rom
crysta* structures o% =T' an! =T0 suggest that they might "e aty,ica* serine ,roteases
612G1<7. These =Ts ha.e a structura* homo*ogy to the chymotry,sin %ami*y o% serine
,roteases. The ,resum,ti.e acti.e site contains the c*assic cata*ytic tria! o% serine 143,
histi!ine 3<, an! as,artic aci! 152. Bn a!!ition, homo*ogy to the Streptomyces griseus
,rotease G*u9SG/ suggests that =T' an! =T0 c*ea.e a%ter a g*utamic aci! 6or ,ossi"*y
as,artic aci!7 resi!ue that wou*! "e sta"i*ize! in the acti.e site "y histi!ine 21&,
threonine 145, an! *ysine 21F. Howe.er, the =Ts are aty,ica* "ecause in =T', an!
,ossi"*y =T0, the o;yanion ho*e, which he*,s to sta"i*ize the transitiona*9state com,*e;
o% cata*ysis, is not ,ro,er*y %orme!. This is "ecause, as in!icate! "y crysta* structures, the
,e,ti!e "on! "etween ,ro*ine 142 an! g*ycine 14& is %*i,,e! 185O re*ati.e to ty,ica*
serine ,roteases. This o"ser.ation has *e! to s,ecu*ation ,ro,osing the re?uirement %or
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!
622, 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 614, 257. Here we !etermine
the 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!Gs,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 tissues
62F7.
/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! SSSS
6see "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 in
igure F. Bnteresting*y, the =Ts c*ea.e at the "or!er "etween e;trace**u*ar ca!herin
!omains 6=Cs7 & an! 2, $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*eins
ha.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.e
suggeste! 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 62<G&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 ####* inactivation
of 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&2G&27.
'*though these anti"o!ies are thought to inacti.ate +sg1, which is %oun! throughout the
e,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 H!esmog*ein com,ensation
hy,othesisI 6&37. This hy,othesis states that in areas o% e,ithe*ium where "oth +sg& an!
+sg1 are e;,resse!, a s,ontaneous "*ister wi** not occur when anti9+sg1 anti"o!ies
inacti.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 the
e,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 !ee,
e,i!ermis. There%ore, / anti"o!ies cause on*y su,er%icia* "*isters where +sg1 is not
com,ensate! %or "y +sg&. Simi*ar*y, mothers with / ,assi.e*y trans%er anti9+sg1
anti"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! in
the 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* mice
that ha.e +sg& in the !ee, e,i!ermis 6simi*ar to a!u*t human e,i!ermis7 an! throughout
the 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$ecte!.
urthermore, mice that e;,ress +sg& in the su,er%icia* e,i!ermis 6%rom a transgene
containing +sg& c+N' !ri.en o%% an in.o*ucrin ,romoter7 are not susce,ti"*e to
"*istering %rom in$ecte! / BgG 6&F7. These !ata ,ro.i!e an e;,*anation %or anti"o!y
inacti.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 =Ts
circu*ate throughout the "o!y in SSSS, as !o anti9+sg1 anti"o!ies in /, "*isters occur
on*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 case
o% SSSS or "u**ous im,etigo. Loss o% %unction o% +sg1 "y c*ea.age with =Ts is a*so
consistent with ,re.ious immuno%*uorescence e;,eriments in which =Ts cause ce**u*ar
interna*ization an! *oss o% ce** sur%ace staining o% +sg1 6147.
ETs are s,in-smart. ' ma$or ,hysio*ogic %unction o% skin is to ,ro.i!e a "arrier against
in%ection. Much o% that "arrier resi!es in the stratum corneum. S. aureus, through the use
o% =Ts, has e.o*.e! an e%%icient an! e;treme*y %ocuse! mechanism o% ,ro*i%erating an!
s,rea!ing un!er that "arrier. Dnce intro!uce!, the "acteria can s,rea! e%%icient*y "y using
=T to ,ro!uce a c*ea.age ,*ane right un!er the stratum corneum. To !o so, the "acterium
has !e.e*o,e! a to;in that s,eci%ica**y "in!s an! c*ea.es a mo*ecu*e that is critica* to
a!hesion in this area an! c*ea.es it at a s,eci%ic site that !estroys its %unction.
Staphylococcal Exfoliative Toxin B Specifically Cleaves
Desmoglein 1
Masayuki 'magai, Takayuki Yamaguchi
R
, Yasushi Hanakawa
S
, #o$i Nishi%u$i,
Motoyuki Sugai
R
an! (ohn ) Stan*ey
S
1. +e,artment o% +ermato*ogy, #eio -ni.ersity Schoo* o% Me!icine, Tokyo, (a,an:
2.
R
+e,artment o% 0acterio*ogy, Hiroshima -ni.ersity Gra!uate Schoo* o%
0iome!ica* Sciences, Hiroshima, (a,an:
&.
S
+e,artment o% +ermato*ogy, -ni.ersity o% /ennsy*.ania Schoo* o% Me!icine,
/hi*a!e*,hia, -.S.'.
Corres,on!ence1 +r Masayuki 'magai, +e,artment o% +ermato*ogy, #eio -ni.ersity
Schoo* o% Me!icine, &3 Shinanomachi, Shin$uku9ku, Tokyo 1F598382, (a,an. =mai*1
amagai>sc.itc.keio.ac.$,
)ecei.e! 1& No.em"er 2551: )e.ise! 2< +ecem"er 2551: 'cce,te! & (anuary 2552.
To, o% ,age
Abstract
Sta,hy*ococca* sca*!e! skin syn!rome an! its *oca*ize! %orm, "u**ous im,etigo, show
su,er%icia* e,i!erma* "*ister %ormation cause! "y e;%o*iati.e to;in ' or 0 ,ro!uce! "y
Staphylococcus aureus. )ecent*y we ha.e !emonstrate! that e;%o*iati.e to;in '
s,eci%ica**y c*ea.es !esmog*ein 1, a !esmosoma* a!hesion mo*ecu*e, that when
inacti.ate! resu*ts in "*isters. Bn this stu!y we !etermine the target mo*ecu*e %or
e;%o*iati.e to;in 0. =;%o*iati.e to;in 0 in$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 & were
incu"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 &, was
a"o*ishe! when cryosections o% norma* human skin were incu"ate! with e;%o*iati.e to;in
0, 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 mouse
an! 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!rome
an! "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
Abbreviations:
+sg1, !esmog*ein 1: =T, e;%o*iati.e to;in: =T', e;%o*iati.e to;in ': =T0, e;%o*iati.e
to;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 !isease
in!uce! "y the e;%o*iati.e 6e,i!ermo*ytic7 to;in 6=T7 o% Staphylococcus aureus 6
Me*ish an!
G*asgow, 14<5:Me*ish et al, 14<2
7. Bt was a*so known in the ,ast as )itterTs !isease, !ermatitis
e;%o*iati.a neonatorum, or ,em,higus neonatorum. Bt is a !isease ,rimari*y a%%ecting
in%ants an! young chi*!ren, "ut a!u*ts can a*so "e a%%ecte!. Bts c*inica* mani%estations
"egin a"ru,t*y with %e.er, skin ten!erness, an! erythema, %o**owe! "y *arge sheets o%
e,i!erma* se,aration in.o*.ing the entire skin sur%ace within hours to !ays. The morta*ity
is sti** &C in chi*!ren 6
Geme**, 1443
7, an! o.er 35C in a!u*ts with un!er*ying !iseases !es,ite
anti"iotic treatment 6
Cri"ier et al, 1482
7. SSSS cause! "y anti"iotic9resistant strains o% S. aureus
has recent*y emerge! as an e.en more serious ,ro"*em 6
Yokota et al, 144F:'c*an! et al, 1448
7. The
,athogenic ro*e o% =T in SSSS is we** esta"*ishe!. or e;am,*e, =T in$ecte! into neonata*
mice causes e;tensi.e "*isters simi*ar to the !isease mani%estations in human neonates
6
Me*ish an! G*asgow, 14<5
7. Bn SSSS, S. aureus is ,resent at !istant %oci such as the ,haryn;, nose,
ear, or con$uncti.a, an! =T ,ro!uce! "y S. aureus gets into circu*ation an! causes
e;%o*iation at remote sites, whereas in "u**ous im,etigo, a *oca*ize! %orm o% SSSS, S.
aureus is ,resent in the *esions.
=T has two ma$or seroty,es ' an! 0 6=T' an! =T07. Bn the -.S.'. an! =uro,e more
than 85C o% to;in9,ro!ucing S. aureus ,ro!uce =T' 6
Cri"ier et al, 1482
7, whereas in (a,an there
is a ,re!ominance o% =T09,ro!ucing strains 6
Murono et al, 1488
7. The gene enco!ing =T' is
*ocate! on the chromosome whereas the gene enco!ing =T0 is %oun! on a *arge ,*asmi!.
The genes %or =T' an! =T0 ha.e "een c*one! an! their amino aci! se?uences ha.e "een
!e!uce! 6
DTToo*e an! oster, 148F:Lee et al, 148<
7. The mature ,roteins o% =T' an! =T0 are 222 an!
22F resi!ues, res,ecti.e*y, a%ter their signa* se?uences are c*ea.e!. The =T' an! =T0
amino aci! se?uences are a"out 25C i!entica* to each other.
'*though ,re.ious u*trastructura* stu!ies ha.e shown interce**u*ar c*ea.age with s,*it
!esmosomes a%ter in$ection o% =T into neonata* mice, it was not c*ear whether !isru,tion
o% !esmosomes is an initia* e.ent or a secon!ary e.ent that occurs on*y a%ter e!ema
cause! "y other interce**u*ar ,atho*ogy 6
Li**i"ri!ge et al, 14<2:Me*ish et al, 14<2:=*ias et al, 14<3:+imon! et al, 14<<
7.
=Ts ha.e a*so "een re,orte! to "e su,erantigens 6
Choi et al, 1484:#a,,*er et al, 1484
7, which "in! to
ma$or histocom,ati"i*ity com,*e; c*ass BB mo*ecu*es on antigen9,resenting ce**s an! to
the .aria"*e ,arts o% the T ce** rece,tor, resu*ting in ,o*yc*ona* T ce** acti.ation. Dther
in.estigators ha.e argue!, howe.er, that the ,re.ious !emonstration o% su,erantigenic
acti.ity with =Ts was ,ro"a"*y !ue to contamination with other sta,hy*ococca*
enteroto;ins 6
*eischer an! 0ai*ey, 1442:*eischer et al, 1443
7. There%ore, unti* recent*y, the mo*ecu*ar
mechanism o% the e,i!erma* se,aration "y =T ha! "een unc*ear, e.en &5 y a%ter the
,athogenic ro*e o% =T was !emonstrate! using neonata* mice in 14<5 6
La!hani et al, 1444
7. The
c*ue to how =T' might cause the "*ister was ,ro.i!e! "y stu!ies o% the ,atho,hysio*ogy
o% ,em,higus %o*iaceus, an autoimmune "*istering !isease, in which inacti.ation o% the
!esmosoma* ca!herin !esmog*ein 1 6+sg17 "y autoanti"o!ies was shown to cause
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 TY2 chromosoma* +N' was ,re,are! as !escri"e! ,re.ious*y
6
Sugai et al, 1448:Yamaguchi et al, 2551
7 an! use! as a ,o*ymerase chain reaction 6/C)7 tem,*ate. '
,rimer set, 3T9''GCTTCC'CCT''T'CCCT''T''TC9&T an! 3T9
GG'TCC'C'G'GGTTC''CTC'TGGTT9&T, was !esigne! to generate a 1128 ",
+N' %ragment containing the entire etb gene 6M1<&287 6
(ackson an! Ban!o*o, 148F
7 with termina*
Hin!BBB an! BamHB sites. The /C) ,ro!uct was c*one! into ,G=M9T =asy .ector to
generate ,TY2&1. The ,TY2&1 was !igeste! with Hin!BBB an! BamHB, an! the insert was
c*one! into E. coli-S. aureus shutt*e .ector ,CL8 to generate ,TY1&&. S. aureus )N2225
6NCTC8&2392r97, which is a stan!ar! strain with no ,ro!uction o% =T' or =T0, was then
trans%orme! with ,TY1&&, an! one o% the trans%ormants was !esignate! TY21&2. The
,hysica* ma, an! genetic !eterminants o% NCTC8&23 in!icate that this strain !oes not
,ossess eta or etb 6
Ban!o*o, 2555
7. The nuc*eoti!e se?uence o% the etb gene was con%irme! "y
+N' se?uencing.
Staphylococcus aureus TY21&2 e;,onentia**y growing in Try,ticase soy "roth 60ecton
+ickinson Micro"io*ogy Systems, Cockeys.i**e, M+7 was inocu*ate! in 1 *iter o% the
same %resh me!ium an! incu"ate! with continuous agitation "y a rotary shaker %or 22 h at
&<OC unti* the ce**s reache! the stationary ,hase. The cu*ture was centri%uge! at 15,555
g %or 25 min at 2OC. Concentrate! cu*ture %i*trate was ,re,are! "y 85C saturate!
ammonium su*%ate ,reci,itation o% the cu*ture su,ernatant. ' so!ium !o!ecy* su*%ate
,o*yacry*ami!e ge* e*ectro,horesis 6S+S9/'G=7 an! su"se?uent Coomassie 0ri**iant
0*ue staining o% concentrate! cu*ture %i*trate re.ea*e! a sing*e ma$or ,rotein "an! o%
2< k+a, which was not ,resent in the concentrate! cu*ture %i*trate o% )N2225 ,CL8. @e
there%ore use! Coomassie 0ri**iant 0*ue staining o% S+S9/'G= ge* %or !etection o% the
=T09,ositi.e %raction with the 2< k+a "an! as the marker. Concentrate! cu*ture %i*trate
!ia*yze! against 15 mM ,hos,hate "u%%er 6,H F.87 6"u%%er 17 was a,,*ie! to a
hy!ro;ya,atite co*umn 6@ako, 13 mm 45 mm7, which was e?ui*i"rate! with "u%%er 1.
The co*umn was washe! with "u%%er 1 unti* most o% the un"oun! ,roteins ,asse! through.
0oun! ,roteins were e*ute! "y ste,wise e*ution with 155 mM, 235 mM, an! 355 mM
,hos,hate "u%%er 6,H F.87, res,ecti.e*y. =*uate with 155 mM ,hos,hate "u%%er 6,H F.87
was !ia*yze! against 15 mM ,hos,hate "u%%er 6,H F.87 6"u%%er 27 an! concentrate! to
355 *. The sam,*e was *oa!e! onto TS#ge* S@&555
LL
6Tosoh, <.3 mm &55 mm7 an!
e*ute! with "u%%er 2 at a %*ow rate o% 5.3 m* ,er min, an! the =T09,ositi.e %ractions were
co**ecte!. Those %ractions were co**ecte! an! %urther a,,*ie! to 0iosca*e CHT29B
hy!ro;ya,atite H/LC co*umn 60io9)a!, <.3 mm 32 mm7, an! e*ute! with a *inear
gra!ient %rom 15 mM to 355 mM ,hos,hate "u%%er 6,H F.87 at a %*ow rate o% 1 m* ,er
min. The =T09,ositi.e %ractions were co**ecte! an! e;tensi.e*y !ia*yze! against
,hos,hate9"u%%ere! sa*ine 6/0S7. This ,uri%ie! =T0 was use! %or incu"ation with
cryosections o% norma* human skin 6see "e*ow7 an! recom"inant +sg1 an! +sg& 6see
"e*ow7.
@e a*so c*one! the etb gene "y a simi*ar a,,roach using an =T09,ro!ucing S. aureus
iso*ate! %rom a ,atient as a /C) tem,*ate. ' ,rimer set, 3T9
'CCCT''T''TCC'''''C'G9&T an! 3T9C'C'G'GGTTC''CTC'TGGT9&T, was
use! to am,*i%y the etb gene an! ,romoter se?uence, an! the /C) ,ro!ucts were *igate!
into the ,C)BB .ector an! then su"c*one! into the ,C=152 .ector 6a kin! gi%t %rom +r.
/atrick Sch*ie.ert7 6
Jath et al, 144<
7. The su,ernatant %rom S. aureus )N2225 trans%orme! with
this ,*asmi! containe! =T0 as the ma$or ,rotein, which was greater than 45C ,ure as
!etermine! "y S+S9/'G=. This ,artia**y ,uri%ie! =T0 was use! %or incu"ation with
trans!uce! HaCaT ce**s 6see "e*ow7 an! in$ecte! into neonata* mice ,rior to
immuno%*uorescence an! immuno"*otting o% e,i!ermis 6see "e*ow7.
#eonatal mouse study with !"
To e.a*uate the e;%o*iati.e acti.ity o% recom"inant =T0, neonata* BC) or 0'L0EC mice
6U 22 h o% age7 were in$ecte! su"cutaneous*y with a !esignate! amount o% =T0 in 25G
35 * o% /0S, an! the skin was ana*yze! gross*y an! microsco,ica**y 1G22 h a%ter
in$ection.
Mouse "ack skin was homogenize! on !ry ice, an! then e;tracte! with Laemm*i sam,*e
"u%%er. Sam,*es with e?ua* amounts o% ,rotein 6,rotein assay kit: 0io9)a! La"oratories,
Hercu*es, C'7 were se,arate! "y FC Tris9g*ycine /'G= 6No.e; ge*s: Bn.itrogen,
Car*s"a!, C'7, an! then trans%erre! to nitroce**u*ose mem"ranes 6Trans90*ot: 0io9)a!
La"oratories7. The mem"ranes were incu"ate! with ra""it antiserum against mouse +sg1
an! +sg&, an! =CC+92 rat monoc*ona* anti"o!y against mouse =9ca!herin 6a kin! gi%t
%rom M. Takeichi7 6
Shirayoshi et al, 148F
7.
Cell culture study with !"
To trans!uce ce**s with constructs enco!ing mouse +sg1 an! mouse +sg& with L'G
tag, recom"inant a!eno.irus was constructe!. The cosmi! cassette ,';C'w, contro* '!
';1w, an! the ,arent .irus '!39!LL were a** kin! gi%ts %rom +r. Bzumi Saito 6Tokyo
-ni.ersity, (a,an7 6
Miyake et al, 144F
7. c+N' enco!ing mouse +sg19L'G an! mouse +sg&9
L'G 6
'magai et al, 2555
7 were su"c*one! into the '! cosmi! cassette ,';C'w. '!eno.irus
containing C' ,romoter an! c+N' enco!ing mouse +sg19L'G an! mouse +sg&9
L'G 6';m+sg1 an! ';m+sg&7 were generate! "y the CDS9T/C metho! 6
Miyake et al,
144F
7 as %o**ows. The cosmi! +N' was mi;e! with the EcoT22B9!igeste! +N'9termina*
,rotein com,*e; o% '!39!LL an! use! to cotrans%ect to 24& ce**s in which recom"inant
.iruses were generate! through homo*ogous recom"ination. Jirus stocks were ,re,are!
using stan!ar! ,roce!ures 6
Miyake et al, 144F
7, an! were concentrate! "y the CsC* gra!ient
metho!. The .irus titer was checke! with a ,*a?ue %ormation assay.
HaCaT ce**s were cu*ture! in a 129we** ,*ate an! trans!uce! with ';m+sg1 an!
';m+sg&. '%ter 22 h the ce**s were incu"ate! with 5, 5.53, 5.13, an! 5.23 g ,er m* o%
recom"inant =T0 in cu*ture me!ia an! incu"ate! %or 15 min or 1 h. Then the ce**s were
washe! with /0S, an! e;tracte! with 255 * 2 S+S Laemm*i sam,*e "u%%er 60io9)a!
La"oratories7. The trans!uce! +sg1 an! +sg& were .isua*ize! "y immuno"*otting with
anti9L'G tag ra""it anti"o!y 6Vyme!, San rancisco, C'7.
Immunofluorescence
orma*in9%i;e!, ,ara%%in9em"e!!e! skin %rom neonata* mice in$ecte! with =T0 or sa*ine
was use! %or in!irect immuno%*uorescence to *oca*ize +sg1, +sg&, an! =9ca!herin, as
,re.ious*y !escri"e! 6
'magai et al, 2555
7. ' ra""it antiserum against e;trace**u*ar !omain 3 o%
mouse +sg& was raise! an! a%%inity ,uri%ie! on the antigenic ,e,ti!e. ' ra""it antiserum,
raise! simi*ar*y against e;trace**u*ar !omain 3 o% mouse +sg1, an! =CC+92 were a*so
use!. Staine! sections were ,hotogra,he! using con%oca* microsco,y 6Leica TCS 2+,
@etz*ar, Germany7.
Cryosections o% non%i;e! norma* human skin were incu"ate! with 5.1 g ,er m* o% =T'
6To;in Techno*ogy, Sarasota, L7 in /0S with 1 mM CaC*
2
6/0S9Ca7, 5.1 g ,er m* o%
recom"inant =T0 in /0S9Ca, or /0S9Ca a*one %or 1 h at room tem,erature. The sections
were then staine! with anti9+sg1 sera o"taine! %rom ,atients with ,em,higus %o*iaceus,
anti9+sg& mouse monoc*ona* anti"o!y, 3H15, which reacts with the e;trace**u*ar !omain
6
/ro"y et al, 2555
7, anti9+sg1 W 2 mouse monoc*ona* anti"o!y, +G&.15, which reacts with the
cyto,*asmic !omain 6
#och et al, 1445
7, anti!esmoco**in mouse monoc*ona* anti"o!y, 329&+,
which reacts with the cyto,*asmic !omain o% +sc1G& 6a kin! gi%t %rom +r. +. ). Garro!7
6
Co**ins et al, 1441
7, an! anti!esmo,*akin mouse monoc*ona* anti"o!y, 1193 6a kin! gi%t %rom
+r. +. ). Garro!7. Staining with +G&.15 in this stu!y re,resents the e;,ression o% +sg1
"ecause there is no !etecta"*e e;,ression o% +sg2 in norma* human skin.
In vitro digestion of recombinant Dsg$ with !"
The entire e;trace**u*ar !omain o% mouse an! human recom"inant +sg1 an! +sg&, with
an =9tag on the car"o;y* terminus, were ,ro!uce! as a secrete! ,rotein "y "acu*o.irus
e;,ression as ,re.ious*y !escri"e! 6
Bshii et al, 144<:'magai et al, 2555
7. These recom"inant human
+sg1 an! +sg& ,roteins ha.e "een shown to retain their nati.e con%ormations enough to
a!sor" out a** immunoreacti.ity o% ,em,higus %o*iaceus an! .u*garis sera, res,ecti.e*y
6
Bshii et al, 144<
7. High i.e ce**s 6Bn.itrogen, San +iego, C'7 cu*ture! in serum9%ree =L Ce**
253 me!ium 6()H 0ioscience, Lene;a, #S7 were in%ecte! with the recom"inant .iruses
an! incu"ate! %or & !. Cu*ture su,ernatant containing each recom"inant +sg was
incu"ate! with the in!icate! amount o% =T0 %or 1 h at &<OC, an! su"se?uent*y su"$ecte!
to immuno"*ot ana*ysis with anti9=9tag mouse monoc*ona* anti"o!y 6/harmacia 0iotech,
-,,sa*a, Swe!en7 %or !etection o% the intact as we** as !igeste! recom"inant ,roteins.
Cu*ture su,ernatant o% )N2225 trans%ecte! with etb, "ut not that o% )N2225 itse*%,
!igeste! recom"inant +sg1, in!icating that )N2225 !i! not ,ro!uce any =T 6!ata not
shown7.
To, o% ,age
Results
%roduction of recombinant !"
c+N' %or =T0 was /C)9am,*i%ie! %rom =T09,ro!ucing S. aureus TY2, su"c*one! into
an E. coli-S. aureus shutt*e .ector 6,TY1&&7, an! use! to trans%orm S. aureus )N2225
6TY21&27. TY21&2 ,ro!uce! =T0 in cu*ture su,ernatant, which was recognize! as a
sing*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!ata
not 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 $a7.
)igure 1.
%roduction of recombinant !" with e'foliative activity( Coomassie 0*ue staine!
S+S9/'G= shows recom"inant =T0 was ,ro!uce! as a 2< k+a ,e,ti!e 6A7. Neonata*
mice in$ecte! with =T0 showe! e;tensi.e "*isters 2 h a%ter in$ection 6B7, whereas
neonata* mice in$ecte! with sa*ine a*one !i! not "*ister 67.
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$ecte! into neonata* mice. The mice starte! to show gross
"*isters as soon as 2G& h a%ter in$ection aroun! the in$ecte! site 6&igure $b: com,are to
contro* &igure $c7.
Dsg$) but not Dsg* or +cadherin) was degraded in vivo in neonatal
mouse 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, we
e;amine! the skin %rom neonata* mice in$ecte! with =T0 "y immuno%*uorescence with
anti"o!ies to +sg1, +sg&, an! =9ca!herin. @hen the skin was e;amine! 1 h a%ter =T0
in$ection, the ce** sur%ace staining o% +sg1 was marke!*y !iminishe! 6&igure -b:
com,are to contro* &igure -a7. Bn the same area, the ce** sur%ace staining o% +sg& or =9
ca!herin was una%%ecte! 6&igure -c.f7.
)igure ..
Immunofluorescence staining of cadherins in the epidermis of mice in,ected with
!"( Neonata* mice were in$ecte! with /0S 6A, , E7 or =T0 6B, D, !7 an! staine! %or
+sg1 6A, B7, +sg& 6, D7, an! =9ca!herin 6E, !7. +sg1 staining showe! that the ce**
sur%ace staining o% +sg1 was remo.e! "y in$ection o% =T0. The stainings o% +sg& an! =9
ca!herin were not a%%ecte! "y in$ection o% =T0. Arro"heads in!icate the *ocation o% the
"asement mem"rane zone. Scale bar1 35 m
u** %igure an! *egen! 6128#7
To !etermine whether this change in +sg1 staining was cause! "y !egra!ation o% +sg1 in
the neonata* mouse skin, e;tracts o% the skin %rom the mice in$ecte! with =T0 or /0S
were 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$ection, whereas +sg& an! =9ca!herin were not a%%ecte!.
=ach *ane re,resents an e;tract %rom a !i%%erent mouse. Arro"heads an! arro"s in!icate
intact +sg1 an! c*ea.e! +sg1, res,ecti.e*y.
u** %igure an! *egen! 62<#7
These resu*ts in!icate that +sg1, "ut not +sg& or =9ca!herin, was c*ea.e! in vivo in
neonata* mouse skin a%ter in$ection with =T0.
'ogenous Dsg$) but not Dsg*) was degraded by addition of !" to
transduced 0aCa! cells
To %urther !emonstrate s,eci%ic c*ea.age o% +sg1 "y =T0, we trans!uce! HaCaT ce**s, a
human keratinocyte ce** *ine, with recom"inant a!eno.irus containing c+N' enco!ing
mouse +sg1 or +sg& with a L'G e,ito,e tag on their C9termini. @hen 35 g ,er m* o%
=T0 was a!!e! to the cu*ture me!ium o% these ce**s an! incu"ate! %or 1 h, +sg1 was
!egra!e! whereas +sg& was not 6&igure 1a7. @hen these trans!uce! HaCaT ce**s were
incu"ate! with .arious amounts o% =T0 65, 5.53, 5.13, an! 5.23 g ,er m*7 %or 15 min or
1 h, +sg1 was c*ea.e! in a !ose9 an! time9!e,en!ent %ashion to a 11& k+a ,ro!uct
6&igure 1b7.
)igure /.
Immunoblot analysis of e'tracts from cultured keratinocytes incubated with !"(
6A7 HaCaT ce**s were trans!uce! with recom"inant a!eno.irus with mouse +sg1 6+sg17
or mouse +sg& 6+sg&7 or contro* a!eno.irus without any insert 6C7, an! incu"ate! with
35 g ,er m* =T0 %or 1 h. =;ogenous +sg1 an! +sg& was .isua*ize! anti9L'G tag
anti"o!y. +sg1, "ut not +sg&, was !egra!e! "y =T0. Arro"heads, asteris#, an! arro"s
in!icate intact +sg1, intact +sg&, an! c*ea.e! +gs1. 6B7 HaCaT ce**s trans!uce! with
recom"inant a!eno.irus with +sg1 was incu"ate! with .arious amounts o% =T0 %or
15 min or 1 h. +sg1 was !egra!e! "y =T0 in a !ose9 an! time9!e,en!ent %ashion.
u** %igure an! *egen! 625#7
Dsg$ 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, as
o,,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 a
contro*, an! staine! them with anti"o!ies against .arious !esmosoma* com,onents
6&igure 27. The ce** sur%ace staining o% +sg1 "y ,em,higus %o*iaceus sera, which react
with 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) f7. These e%%ects "y =T0 on the +sg1
an! +sg& staining were i!entica* to those "y =T' 6&igure 2b) e7. =T0 treatment !i! not
a%%ect the staining ,attern "y monoc*ona* anti"o!y +G&.15, which recognizes the
cyto,*asmic !omain o% +sg1 6&igure 2g7. =T0 treatment !i! not a*ter the staining o%
!esmoco**in "y 329&+ nor that o% !esmo,*akin "y 1193 6&igure 2h) i7. =T' treatment
!i! not change the staining "y +G&.15, 329&+, or 1193, either 6!ata not shown7. These
%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' 6B, E7, E$B 6, !, %, H, &7or
/0S a*one 6A, D7, an! staine! %or e;trace**u*ar !omain o% +sg1 6A, B, 7, e;trace**u*ar
!omain o% +sg& 6D, E, !7, cyto,*asmic !omain o% +sg1 6%7, cyto,*asmic !omain o%
!esmoco**in 6+sc, H7or !esmo,*akin 6+/, &7. The staining %or the e;trace**u*ar !omain o%
+sg1 was remo.e! "y =T0 67as seen with =T' 6B7, whereas the staining %or +sg& was
not a%%ecte!. The staining %or the cyto,*asmic !omain o% +sg1, !esmoco**ins, or
!esmo,*akin was not a%%ecte!. Scale bar1 35 m
u** %igure an! *egen! 6&14#7
To !emonstrate !irect ,roteo*ysis o% the e;trace**u*ar !omain o% +sg1 "y =T0, we
incu"ate! a so*u"*e recom"inant %orm o% the e;trace**u*ar !omain o% +sg1 an! +sg& with
=T0 in vitro 6&igure 37. =T0 c*ea.e! the 81 k+a recom"inant mouse +sg1 !own to a &2
k+a ,e,ti!e in a !ose9!e,en!ent %ashion, whereas =T0 !i! not c*ea.e mouse +sg& at a**.
Bn the same way, =T0 c*ea.e! the recom"inant human +sg1, "ut not human +sg&. These
%in!ings in!icate that =T0 s,eci%ica**y recognizes an! c*ea.es the e;trace**u*ar !omain o%
"oth mouse an! human +sg1.
)igure 0.
In vitro !" treatment of recombinant e'tracellular domain of Dsg$ and Dsg*(
Mouse 6A7 or human 6B7 +sg1 an! +sg& e;trace**u*ar !omains ,ro!uce! in "acu*o.irus
were incu"ate! with .arious amounts o% =T0 6A1 lane ', 15 g ,er m*: lane (, & g ,er
m*: lane 3, 1 g ,er m*: lane ), 5.& g ,er m*: lane *, 5.1 g ,er m*: lane +, 5 g ,er m*:
B1 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"$ecte! to immuno"*ots an! .isua*ize!
with anti9=9tag monoc*ona* anti"o!y. The e;trace**u*ar !omain o% +sg1, "ut not that o%
+sg&, was c*ea.e! "y =T0 in a !ose9!e,en!ent %ashion. Arro"heads an! arro"s in!icate
the intact recom"inant +sg1 an! c*ea.e! ,ro!uct, res,ecti.e*y. Bars on the *e%t si!e
in!icate mo*ecu*ar weight stan!ar!s 235, 155, 35, an! 23 k+a, %rom to, to "ottom.
u** %igure an! *egen! 6122#7
To, o% ,age
Discussion
The ma$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 shown
to resi!e in the stratum corneum. Bn "u**ous im,etigo, S. aureus is %oun! in a "*ister
ca.ity $ust "eneath the stratum corneum, thus circum.enting the "arrier. This "*ister
ca.ity is known to "e cause! "y =T, re*ease! "y the ,atho*ogic organisms. ' simi*ar
"*ister occurs in ,atients with the autoimmune !isease ,em,higus %o*iaceus. Bn that
!isease BgG autoanti"o!ies against +sg1 "*ock the ce** a!hesion %unction o% +sg1 with
resu*tant su,er%icia* "*isters in the e,i!ermis where +sg1 is e;,resse! without
coe;,resse! +sg& 6
'magai, 1444:Mahoney et al, 1444
7. '*though +sg1 is a*so e;,resse! in the !ee,
e,i!ermis an! mucous mem"ranes, "*isters !o not occur in these areas "ecause +sg& is
coe;,resse! an! can com,ensate %or the anti"o!y9in!uce! *oss o% %unction o% +sg1.
0ecause ,em,higus %o*iaceus shows i!entica* tissue s,eci%icity an! histo*ogy to SSSS
an! "u**ous im,etigo, which are cause! "y =T, we ,re.ious*y sus,ecte! that the target
mo*ecu*e %or a ma$or ty,e o% =T, =T', might "e +sg1 an! ,ro.e! that =T' s,eci%ica**y
c*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**ous
im,etigo, =T0, a*so c*ea.es +sg1. @e ha.e shown that in$ection o% recom"inant =T0
into 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! =9
ca!herin were not a%%ecte!. Simi*ar*y, when =T0 was a!!e! to cu*ture! keratinocytes
e;,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%ter
incu"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 the
e;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% the
e,i!ermis, a*though to keratohya*in granu*es, not to the ce** sur%ace 6
Smith et al, 1484
7. Dther
stu!ies ha.e suggeste! "in!ing to an e,i!erma* GM29*ike g*yco*i,i! 6
Sakurai an! #on!o, 14<4:Tana"e
et al, 1443
7. Dur stu!ies !emonstrate !irect c*ea.age o% recom"inant +sg1 in so*ution,
howe.er, suggesting that any e,i!erma* rece,tor other than +sg1 is unnecessary %or
acti.ation o% =TTs ,roteo*ytic acti.ity.
Bt is hy,othesize! that =T' an! =T0 are serine ,roteases. =T' an! =T0 are 23C
i!entica* to the sta,hy*ococca* serine ,rotease J8. Structura* stu!ies show ,articu*ar*y
striking 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 L9ray
crysta* structure o% =T' an! =T0 showe! signi%icant structure simi*arity with known
g*utamate9s,eci%ic try,sin9*ike serine ,roteases 6
Jath 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 25C. @e sus,ect that amino aci! resi!ues that are res,onsi"*e %or this
s,eci%icity shou*! "e conser.e!. The %ina* i!enti%ication o% =T' an! =T0 as g*utamate9
s,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,erantigens
Lisa ). @. /*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% Miami
Schoo* o% Me!icine, Miami, *ori!a &&151
=!itor1 (. T. 0ar"ieri
R
Corres,on!ing author. Mai*ing a!!ress1 +e,artment o% Micro"io*ogy an! Bmmuno*ogy,
/.D. 0o; 51F4F5 6)91&87, Miami, L &&151. /hone1 6&537 22&9F118. a;1 6&537 22&9
2F2&. =9mai*1 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)'CT
Sta,hy*ococca* sca*!e!9skin syn!rome is an e;%o*iati.e !ermatitis characterize! "y the
se,aration o% the e,i!ermis at the stratum granu*osum. This !isru,tion is me!iate! "y one
o% two Staphylococcus 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.ersy
e;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 act
as 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% a
su,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
aureus 61, 137. SSSS is characterize! "y the %ormation o% *arge "u**ae without
in%*ammatory ce** in%i*trate an! se,aration o% e;ten!e! areas o% the e,i!ermis at the
stratum 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 in
humans, e;%o*iati.e to;in ' 6=T'7 an! e;%o*iati.e to;in 0 6=T07 6237. =T' 62F.4 k+a7
is enco!e! on the "acteria* chromosome an! shares 25C amino aci! i!entity with the
,*asmi!9"orne =T0 62<.& k+a7 62, 1&, 147. +es,ite e;tensi.e stu!ies, the e;act
mechanism res,onsi"*e %or the skin !isru,tion is not known.
L9ray crysta**ogra,hic structures o% =T' an! =T0 63, 25, 2&, 227 suggest that the to;ins
are mem"ers o% the try,sin9*ike serine ,rotease %ami*y. /rotease acti.ity has not "een
!emonstrate! %or either to;in in .itro, "ut "oth =T' an! =T0 ha.e intrinsic esterase
acti.ity, which is associate! with serine ,roteases 6&7. Thus, it is *ike*y that "oth to;ins
are ,roteases. Bn a!!ition to ha.ing ,ossi"*e ,rotease acti.ity, "oth =T' an! =T0 are
re,orte! to "e "acteria* su,erantigens 612, 1F, 1<, 227. 0acteria* su,erantigens are a
%ami*y o% ,roteins a"*e to "in! simu*taneous*y to the ma$or histocom,ati"i*ity com,*e;
an! to the T9ce** rece,tor 6TC)7, resu*ting in stimu*ation o% a *arge num"er o% T ce**s
e;,ressing s,eci%ic JY su"sets o% the TC) re,ertoire 6127.
/re.ious*y, *eischer an! 0ai*ey re,orte! that recom"inant =T' e;,resse! in a
su,erantigen9%ree S. aureus "ackgroun! !oes not ha.e mitogenic acti.ity 6<7. They
conc*u!e that the acti.ity seen "y others is !ue to contamination o% the to;in ,re,arations
with other su,erantigens. Howe.er, since this re,ort, a!!itiona* *iterature has a!!resse!
the su,erantigenic acti.ity o% =T' an! =T0. Here we !emonstrate that recom"inant
e;%o*iati.e to;ins ,ro!uce! in an Escherichia coli "ackgroun! !o not act as T9ce**
mitogens an! thus are not "acteria* su,erantigens.
Bso*ation, e;,ression, an! ,uri%ication o% recom"inant e;%o*iati.e to;ins.
+N' %ragments enco!ing =T' an! =T0 were o"taine! "y uti*izing /C) an! +N' %rom
e;%o*iati.e9to;in9,ro!ucing "acteria* strains. To;ins were e;,resse! an! ,uri%ie! using
the No.agen 6Ma!ison, @is.7 ,=T e;,ression system. Bn "rie%, a <859", +N' %ragment
enco!ing mature =T' was o"taine! using the o*igonuc*eoti!e ,rimers =T'9& 63M9
GCGCCTCG'GGTTTC'GC'G''G'''T''''9&M7 an! =T'92 63M 9 GCGCC T
CG'G T 'T''''C'TCC'CGG'T T T T 9 &M7 an! chromosoma* +N' o% an =T'9
,ro!ucing S. aureus strain. ' <<39", +N' %ragment enco!ing mature =T0 was
am,*i%ie! %rom ,*asmi! ,B(552 6generous*y ,ro.i!e! "y /eter McNamara7 using
o*igonuc*eoti!e ,rimers =T09& 63M9GCGCC'T'TG'''G''T'C'GCGC'9&M7 an!
=T092 63M9CGCGGG'TCC'T'TTG'''T'TT''9&M7. The am,*i%ications were
,er%orme! using $a, +N' ,o*ymerase 60ethes!a )esearch La"oratories, Gaithers"urg,
M!.7 or -.u Tur"o +N' ,o*ymerase 6Stratagene, La (o**a, Ca*i%.7. /rimers were !esigne!
to am,*i%y the co!ing se?uences %or the mature ,roteins without the amino9termina*
signa* se?uences. They were a*so !esigne! to contain restriction en!onuc*ease sites that
%aci*itate! insertion o% the %ragments into the E. coli e;,ression .ector ,=T913"
6No.agen7. The +N' se?uences o% "oth the =T'9 an! =T09enco!ing %ragments were
!etermine! an! shown to "e i!entica* to the Gen0ank !ata"ase se?uences 6eta, accession
num"ers L23&<2 an! M25&<1: etb, accession num"ers M1<&28 an! M1&<<37. Bn or!er to
generate an a,,ro,riate negati.e contro*, the =T'9enco!ing %ragment was mutate! to
co!e %or mature =T' with an a*anine resi!ue re,*acing the ,utati.e acti.e9site serine
resi!ue at ,osition 143 621, 227, using o.er*a, e;tension mutagenesis 6157. The com,*ete
nuc*eoti!e se?uence o% "oth stran!s o% the mutate! %ragment was !etermine! to con%irm
the ,resence o% the !esire! mutation.
=;%o*iati.e9to;in9enco!ing +N' %ragments were inserte! into the e;,ression .ector ,=T9
13", ,re.ious*y mo!i%ie! to co!e %or kanamycin resistance. The recom"inant to;ins were
e;,resse! in E. coli as !escri"e! ,re.ious*y 6,=T system manua*, 2th e!., No.agen7.
/uri%ie! to;ins were !igeste! with throm"in to remo.e the amino9termina* histi!ine9rich
*ea!er ,e,ti!e, an! the histi!ine9rich *ea!er ,e,ti!e was se,arate! %rom the to;in "y
!ia*ysis against ,hos,hate9"u%%ere! sa*ine 6/0S7. Throm"in was remo.e! %rom the to;in
,re,arations "y chromatogra,hy o.er Z9amino"enzami!ine9agarose 6Sigma Chemica*s,
St. Louis, Mo.7. The resu*tant recom"inant =T' 6r=T'7 an! recom"inant =T'9S143'
6r=T'9S143'7 consist o% the mature %orms o% nati.e =T' an! =T'9S143' with %i.e
a!!itiona* N9termina* amino aci!s 6GSHML7. The resu*tant recom"inant =T0 6r=T07
consists o% the mature %orm o% nati.e =T0 with %our a!!itiona* N9termina* amino aci!s
6GSHM7. The recom"inant to;ins ran as sing*e "an!s o% a,,ro;imate*y 2< k+a on staine!
so!ium !o!ecy* su*%ate9,o*yacry*ami!e ge* e*ectro,horesis ge*s. r=T' a,,eare! as a
sing*e "an! "y @estern "*ot ana*ysis. /rotein concentrations were !etermine! using a
"icinchonic aci! ,rotein assay kit 6/ierce, )ock%or!, B**.7.
)ecom"inant e;%o*iati.e to;ins ,ro!uce the sym,toms o% SSSS in the neonata* mouse
assay.
The recom"inant e;%o*iati.e to;ins were teste! %or acti.ity in neonata* mice 6137. Dne9
!ay9o*! 0'L0Ec mice were in$ecte! su"cutaneous*y at the na,e o% the neck with
increasing concentrations o% r=T', r=T0, r=T'9S143', /0S 6negati.e contro*7, or =T'9
,ro!ucing S. aureus 6,ositi.e contro*7 an! o"ser.e! at .arious times ,ostin$ection %or
sym,toms o% SSSS. '** mice were returne! to *actating mothers an! were o"ser.e! at
hour*y inter.a*s %or gross a,,earance. They were gra!e! on a,,earance an! tacti*e
e;amination 6Ta"*e 6Ta"*e17. 1 7. Mice !ie! !uring the course o% the e;,eriment or were
sacri%ice! at the en! o% the 229h o"ser.ation ,erio!. '%ter on*y 2 h, the mice in$ecte! with
the highest !ose o% r=T' 623 NgEg o% "o!y weight7 showe! o".ious signs o% e;%o*iation
6Ta"*e 6Ta"*e17. 1 7. r=T0 an! r=T' e;hi"ite! simi*ar acti.ities, whi*e r=T'9S143' !i!
not cause any .isi"*e signs o% e;%o*iation e.en at 15 times the !osage that e*icite! a
,ositi.e res,onse with either r=T' or r=T0.
!A"4 $
'cti.ities o% r=T', r=T0, an! r=T'9S143' in the
neonata* mouse
To con%irm that the recom"inant to;ins were causing the characteristic c*ea.age at the
stratum granu*osum, skin sam,*es %rom re,resentati.e anima*s were ,re,are! %or
histo*ogica* e;amination. 't the time o% !eath, anima*s were ,*ace! in 15C %orma*in %or
%i;ation an! em"e!!e! in ,ara%%in wa; %or ,re,aration o% skin sections. Sections were
staine! with hemato;y*in an! eosin an! e;amine! .ia *ight microsco,y %or se,aration at
the stratum granu*osum. Bn$ection o% "oth r=T' 6ig. 6ig.17 1 7 an! r=T0 6!ata not
shown7 resu*te! in the !iagnostic skin c*ea.age, whi*e r=T'9S143' 6!ata not shown7 !i!
not. The c*ea.age was i!entica* to that o"ser.e! in mice in$ecte! in a simi*ar manner with
an =T'9,ro!ucing S. aureus strain 6!ata not shown7. Thus, r=T' an! r=T0, "ut not
r=T'9S143', were a"*e to ,ro!uce the characteristic an! !iagnostic c*ea.age o% SSSS in
this 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 22 h ,ostin$ection an! were staine! with hemato;y*in an! eosin.
Magni%ication, [25. 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 6127. 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 were
har.este!. Ce**s 615
3
7 were a!!e! to 4F9we** -9"ottom ,*ates in )/MB 1F25
su,,*emente! with 15C %eta* ca*% serum an! were incu"ate! %or <2 h with .arious
concentrations o% one o% the %o**owing1 r=T', r=T0, r=T'9S143', rS,e'1 6,uri%ie! in
this *a"oratory as !escri"e! ,re.ious*y \12]7, sta,hy*ococca* J8 ,rotease 6/romega,
Ma!ison, @is.7, an! /0S. Sta,hy*ococca* J8 ,rotease is a serine ,rotease structura**y
simi*ar to the e;%o*iati.e to;ins an! was use! as a negati.e contro*: the stre,tococca*
su,erantigen rS,e'1 was use! as a ,ositi.e contro*. Dne microcurie o% \
&
H]thymi!ine
6BCN 0iochemica*s, Costa Mesa, Ca*i%.7 was a!!e! to each we**, an! ce**s were incu"ate!
%or an a!!itiona* 22 h an! har.este!: the \
&
H]thymi!ine u,take was then ?uanti%ie!. or
each to;in or contro*, three to %i.e !istinct !onors were use!. 'ssays were ,er%orme! a
minimum o% three times ,er recom"inant to;in with !i%%erent !onor ce**s each time. The
human /0MCs res,on!e! as e;,ecte! to "oth rS,e'1 6ig. 6ig.27 2 7 an! the anti9C+&
monoc*ona* anti"o!y D#T&, which ser.e! as a nons,eci%ic T9ce** mitogen 6!ata not
shown7. Howe.er, there was no !etecta"*e mitogenic acti.ity when /0S, r=T', r=T0,
r=T'9S143', or J8 ,rotease was a!!e! to the ce**s. To !etermine i% the *ack o% acti.ity
was !ue to the age o% the !onor, %resh*y iso*ate! human um"i*ica* cor! "*oo! was
,re,are! as !escri"e! a"o.e, as a source o% neonata* mononuc*ear ce**s. Neonata*
mononuc*ear ce**s ha! the same mitogenic res,onse to r=T', rS,e'1, D#T&, an! /0S as
a!u*t /0MCs 6!ata not shown7.
&I5( -
Mitogenic acti.ities o% r=T', r=T0, r=T'9S143', sta,hy*ococca* J8
,rotease 6J87, an! rS,e'1. Shown are the resu*ts o"taine! %rom one
re,resentati.e e;,eriment. '** e;,eriments ha! simi*ar resu*ts.
't this time a contro.ersy e;ists o.er whether these to;in are su,erantigens. The L9ray
crysta* structures o% "oth to;ins 63, 25, 2&, 227 in!icate that =T' an! =T0 are mem"ers
o% the try,sin9*ike serine ,rotease %ami*y. 0oth to;ins contain the signature His9Ser9's,
cata*ytic tria! o% this %ami*y, an! su"stitution o% an '*a resi!ue %or any one o% these
resi!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.e
estero*ytic acti.ity, which is associate! with serine ,roteases 6&7. There%ore, it is
common*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 seen
in an into;icate! anima*7, or the con!itions re?uire! %or their cata*ysis ha.e not "een
i!enti%ie!.
Bt was suggeste! in the ear*y 1485s "y Mor*ock et a*. that the e;%o*iati.e to;ins were
mitogens 61<7. Bn 1484 #a,,*er et a*. re,orte! stimu*ation o% s,eci%ic human TC) JY9
chain9e;,ressing T9ce** ,o,u*ations in res,onse to e;%o*iati.e to;in 6117. Bn the %o**owing
years e;%o*iati.e to;in was re,orte! to stimu*ate a!!itiona* human JY9 an! mouse JY9
e;,ressing T9ce** ,o,u*ations 6F, 87 an! to in!uce cutaneous *ym,hocyte9associate!
antigen e;,ression in ,eri,hera* T *ym,hocytes 62F7. Howe.er, !uring these ear*y stu!ies
in.estigators were una"*e to !emonstrate "in!ing o% =T' to ma$or histocom,ati"i*ity
com,*e; c*ass BB rece,tors, a re?uirement %or su,erantigen acti.ity 647.
Bn more recent work, Jath an! coworkers re,ort that wi*!9ty,e =T' an! an =T' acti.e9
site mutant are mitogens 6227. Bn a %o**ow9u, stu!y Mon!ay et a*. re,ort e;,ansion o%
s,eci%ic JY9e;,ressing human T9ce** ,o,u*ations not ,re.ious*y cite! %or =T' or =T0
"ut %ai* to show e;,ansion o% the ,o,u*ations which were origina**y !escri"e! %or the
e;%o*iati.e to;ins 61F7. They conc*u!e that the e;%o*iati.e to;ins are *ess ,otent in
in!ucing T9ce** ,ro*i%eration an! *ess to;ic in a ra""it mo!e* than the con.entiona*
sta,hy*ococca* su,erantigens.
Bn contrast to the resu*ts %rom the a"o.e9mentione! stu!ies, others ha.e not seen
su,erantigenic acti.ity with these to;ins. Bn their 1442 re,ort *eischer an! 0ai*ey cou*!
not !emonstrate a mitogenic res,onse %rom r=T' e;,resse! in S. aureus 6<7. They
conc*u!e that the acti.ity seen "y the other grou,s was cause! "y contamination o% the
commercia* ,re,arations use!. Ca.are**i et a*. re,orte! that the ,uri%ie! to;in they use! to
generate an L9ray structure was not a"*e to stimu*ate cutaneous *ym,hocyte9associate!
antigen e;,ression in T ce**s 637.
Here we !emonstrate that r=T' an! r=T0 e;,resse! an! ,uri%ie! %rom E. coli !o not act
as su,erantigens. These recom"inant to;ins are a"*e to ,ro!uce a** the characteristic signs
o% SSSS in the neonata* mouse mo!e*, an! the histo,atho*ogy o% skin sam,*es %rom mice
in$ecte! su"cutaneous*y with either ,uri%ie! r=T' or r=T0 is i!entica* to that o% mice
in$ecte! with =T'9,ro!ucing S. aureus. There%ore, whi*e these to;ins ha.e s*ight
mo!i%ication at the amino9termina* en! com,are! to wi*!9ty,e =T' an! =T0, they are
%u**y acti.e as e;%o*iatins. Howe.er, in our stan!ar! T9ce** ,ro*i%eration assay neither
recom"inant to;in was mitogenic. No acti.ity was o"ser.e! a"o.e the "ackgroun! *e.e*
generate! "y "u%%er a*one or "y the contro* ,rotein sta,hy*ococca* J8 ,rotease. The
rS,e'1 ,rotein use! as a ,ositi.e contro* was e;,resse! in the same E. coli e;,ression
system as use! %or the r=Ts an! a*so has a s*ight*y mo!i%ie! N9termina* se?uence.
The L9ray structures o% the e;%o*iati.e to;ins !o not resem"*e the structures o% the other
su,erantigens %rom gram9,ositi.e "acteria 63, 257: there%ore, the e;%o*iati.e to;ins !o not
"e*ong to that %ami*y. rom this %in!ing, an! the *ack o% mitogenic acti.ity seen on our
assays, we conc*u!e that =T' an! =T0 are not "acteria* su,erantigens. '!mitte!*y, it
can "e argue! that the %act that our recom"inant to;ins are mo!i%ie! at the amino
terminus e;,*ains why they are not mitogens. '*so, regar!ing the structura* !ata, there
are su,erantigens with a secon! acti.ity 62, 187, an! ,ossi"*y the e;%o*iati.e to;ins are
,roteases with a secon! acti.ity. Howe.er, our main reason %or arguing that =T' an!
=T0 are not su,erantigens is that SSSS !oes not resem"*e a su,erantigen9me!iate!
!isease. C*assic su,erantigen9me!iate! !iseases, such as the to;ic shock syn!romes, are
associate! with erythematous rash, hy,otension, mu*tiorgan %ai*ure, an! high morta*ity
rates. Bn contrast, among young SSSS ,atients, morta*ity is *ow with a,,ro,riate
anti"iotic thera,y, an! systemic mani%estations o% the in%ection genera**y are not ,resent,
with the e;ce,tion o% the e;%o*iati.e rash. This rash is !ue to the !irect e%%ect o% the to;in
on the skin an! !oes not resem"*e the erythematous rash o% the to;ic shock syn!romes.
Hy,otension an! ,ossi"*e organ %ai*ure can "e %oun! in SSSS on*y in se.ere cases where
there are e;tensi.e areas o% !enu!e! skin with signi%icant %*ui! *oss or with the onset o%
se,sis with either S. aureus or a secon!ary in%ecting organism.
Morta*ity is associate! with SSSS in the a!u*t e.en with a,,ro,riate anti"iotic thera,y,
"ut it is *ike*y secon!ary to "acteria* se,sis associate! with the un!er*ying c*inica* state o%
these ,atients, who are o%ten immunocom,romise!, age!, or a%%*icte! with other c*inica*
,ro"*ems, such as rena* com,romise.
Jiewing the !ata as a who*e, we conc*u!e that SSSS is not a su,erantigen9me!iate!
!isease an! that the e;%o*iati.e to;ins are not su,erantigens. The !ata an! the c*inica*
,icture o% SSSS su,,ort our thesis that these to;ins are most *ike*y uni?ue serine
,roteases which act on an unknown target in the u,,er e,i!ermis.
%enatalaksanaan !erapi
%enyakit Impetigo
/oste! on +ecem"er 28, 255< ^ Lea.e a comment
o*eh 1 Jincensius 'n$ar Tri*aksono, S.arm 5<81135&F
Bm,etigo meru,akan suatu in%eksi ku*it su,er%isia* 6ku*it "agian atas7 yang !ise"a"kan o*eh
"akteri stre,tokokus atau "akteri sta%i*okokus. /enyakit im,etigo !itan!ai !engan a!anya "u*a
yaitu "en$o*an ,a!a ku*it !engan !iameter _5,3 cm !an "erisi cairan yang meru,akan pustula
6,enum,ukkan nanah !a*am ku*it7. Gam"aran k*inis !ari ,enyakit ini yaitu "u*a yang "er!in!ing
ti,is sehingga mu!ah ,ecah akan menim"u*kan krusta 6koreng7 ,a!a ku*it.
/engo"atan in%eksi ini !a,at !igunakan anti"iotik secara to,ika* !an ora*. Tu$uan tera,inya yaitu
mengo"ati in%eksi, mencegah ,enu*aran, menghi*angkan rasa ti!ak nyaman, !an mencegah
ter$a!inya kekam"uhan. Sasaran tera,inya yaitu in%eksi "akteri stre,tokokus atau sta%i*okokus.
Tera,i non %armako*ogis untuk ,engo"atan im,etigo yaitu menghi*angkan krusta !engan cara
man!i se*ama 259&5 menit !isertai menge*u,askan krusta !engan han!uk "asah !an "i*a ,er*u
o*esi !engan zat anti"akteri, mencegah menggaruk !aerah *ecet atau !a,at !i*akukan !engan
menutu, !aerah yang *ecet !engan ,er"an tahan air !an memotong kuku, *an$utkan ,engo"atan
sam,ai semua *uka *ecet sem"uh. Tera,i non %armako*ogis untuk ,encegahan ,enyakit im,etigo
yaitu man!i teratur !engan sa"un !an air 6sa"un antise,tik !a,at !igunakan, namun !a,at
mengiritasi ,a!a se"agian ku*it orang yang ku*it sensiti%7, men$aga ke"ersihan yang "aik 6cuci
tangan teratur, men$aga kuku $ari teta, ,en!ek !an "ersih7, $auhkan !iri !ari orang !engan
im,etigo, orang yang kontak !engan orang yang terkena im,etigo segera mencuci tangan !engan
sa"un !an air menga*ir, mencuci ,akaian, han!uk !an s,rei !ari ,en!erita im,etigo ter,isah !ari
yang *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"iotik
to,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 ,er9
ora* yang !a,at !igunakan yaitu eritromisin !an %*uk*oksasi*in.
%ilihan obat
Antibiotik topikal
Mu,irocin
Nama Generik 1 Mu,irocin
Nama ,aten 1 0'CT)D0'N 6G*a;oSmith#*ine7
0ran! name 1 0acto!erm 6Bka,harmin!o7
Bn!ikasi 1 in%eksi ku*it ,rimer akut, misa*nya im,etigo, %o*iku*itis, %urunku*osis
#ontrain!ikasi 1 hi,ersensiti% terha!a, mu,irocin
0entuk se!iaan 1 sa*e, !an krim
+osis 1 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 $ika ti!ak a!a res,on k*inis !a*am &93 hari
=%ek sam,ing 1 rasa ter"akar, gata*, rasa tersengat, kemerahan
/eringatan 1 hin!ari kontak !engan mata. Hati9hati ,enggunaan ,a!a gangguan gin$a*
se!ang sam,ai "erat, hami*, *akatasi. Hentikan ,enggunaan $ika ter$a!i reaksi sensiti.itas
atau 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 1 'sam usi!at
0ran! name 1 '%uci! 6erron7, usycom 6Com"i,har7, u*a!ic 6Guar!ian7, uta!erm
6Bnter"at7
Bn!ikasi 1 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 1 hi,ersensiti% terha!a, asam %usi!at.
0entuk se!iaan 1 sa*e,6Na %usi!at7 !an krim 6asam %usi!at7
+osis 1 tan,a ,em"a*utEkasa steri* 1 gunakan &92;Ehari
!engan ,em"a*utEkasa steri* 1 gunakan *e"ih sering *ama tera,i kurang
*e"ih < hari.
=%ek sam,ing 1 reaksi sensiti%itas misa*nya ruam ku*it, urtikaria, iritasi
/eringatan 1 hin!ari ,enggunaan ,a!a "agian mata. /enggunaan $angka !a,at
meningkatkan resiko sensitisasi ku*it !an resistensi "akteri. Hami* trimester ,ertama.
0ayi "aru *ahir.
Antibiotik per+oral
=ritromisin
Nama Generik 1 =ritromisin
Nama ,aten 1 =)YTH)DCBN 6'""ott7
0ran! name 1 Corsatrocin 6Corsa7.
Bn!ikasi 1 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 !an
kronis, ,neunomia, "ronkiektaksis. Bn%eksi te*inga1 otitis me!ia !an eksterna*, mastoi!itis.
Bn%eksi ora* 1 gingi.itis, angina .incenti. Bn%eksi mata1 "*e%aritis. Bn%eksi ku*it !an $aringan *unak1
%urunke* !an kar"unke*, ,aronikia, a"ses, akne ,ustu*aris, im,etigo, se*u*itis, erisi,e*as.
#ontrain!ikasi 1 hi,ersensiti% terha!a, eritromisin, ,enyakit hati.
0entuk se!iaan 1 ta"*et atau ka,su*
+osis 1 !ewasa 192gEhr tia, F, 8 atau 12 $am. Bn%eksi "erat 2gEhr !a*am !osis ter"agi.
'nak &5935 mgEkg00Ehr tia, F, 8 atau 12 $am.
0ayi92tahun 123mg 2;Ehr, 298tahun 235 mg 2;Ehr atau 355 mg tia,12$am
Se"e*um atau ,a!a waktu makan.
=%ek sam,ing 1 $arang1 he,atotoksik, ototoksik.
Gangguan GB 1 mua*, muntah, nyeri ,erut,!iare.
-rtikaria, ruam !an reaksi a*ergi *ainya.
/eringatan 1 gangguan gin$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 1 %*uk*oksasi*in Na monohi!rat
0ran! name 1 LDL'/=N 6G*a;oSmith#*ine7
Bn!ikasi 1 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 1 hi,ersensiti% terha!a, ,enisi*in, "ayi yang *ahir !ari i"u yang
hi,ersensiti% ,enisi*in.
0entuk se!iaan 1 ka,su* 6235 mg, 355mg7
+osis 1 !ewasa 2359355 mg tia, 8 $am 6&;Ehr7.
'nak U2tahun F2,3mg &;Ehr 6tia, 8 $am7, 2915tahun 123 mg &;Ehr 6tia, 8
$am7
=%ek sam,ing 1 mua*, muntah, nyeri ,erut, !iare.-rtikaria, ruam ku*it, ka!ang ter$a!i
reaksi ana%i*aktik.
/eringatan 1 hi,ersensiti% ,enisi*in, gangguan gin$a*, *eukimia *im%atik.