CHARACTERIZATION OF THE MAJOR AUTOLYSIN IN
STAPHYLOCOCCUS AUREUS
JOANA FILIPA GOMES DA SILVA
DISSERTATION PRESENTED TO OBTAIN A MASTER DEGREE IN
MEDICAL MICROBIOLOGY
OCTOBER 2015
CHARACTERIZATION OF THE MAJOR AUTOLYSIN IN
STAPHYLOCOCCUS AUREUS
JOANA FILIPA GOMES DA SILVA
DISSERTATION PRESENTED TO OBTAIN A MASTER DEGREE IN
MEDICAL MICROBIOLOGY
Supe iso : D . Ri a Sob al, UCIBIO-FCT/UNL
Co-supe iso : P o . Ana Madalena Ludo ice, DCV-FCT/UNL;
ITQB/UNL
OCTOBER 2015
ii
iii
Bibliog aphic elemen s esul ing om his disse a ion
Sil a, J., I. G ilo, A. M. Ludo ice, H. de Lencas e, R. G. Sob al. Cha ac e iza ion o
he majo pep idoglycan hyd olase o S aphylococcus au eus. Mic oBio ec’15 –
Po uguese Cong ess o Mic obiology and Bio echnology 2015. P230, pg. 354.
Decembe 10-12, 2015.

iii
i
Ag adecimen os
À Dou o a Ri a Sob al, exp esso o meu p o undo ag adecimen o pela o ien ação, pela
disponibilidade pa a o deba e de ideias e apoio que mui o ele a am os meus
conhecimen os cien í icos e, sem dú ida, mui o es imula am o meu desejo de que e
sabe mais e a on ade cons an e de que e aze melho .
Ag adeço, ambém, à P o esso a Ana Madalena Ludo ice, pela co-o ien ação nes e
p oje o. Mui o ob igada pelo p o issionalismo e pela o al disponibilidade que semp e
e elou pa a comigo.
Aos meus colegas de labo a ó io, Bá ba a, Raquel, Rica do e Vanessa, um mui o
ob igado pela ossa amizade, companhei ismo e ajuda, a o es mui o impo an es na
ealização des a ese e que me pe mi i am que cada dia osse enca ado com um so iso e
pa icula mo i ação.
À Inês G ilo, com quem i e o p i ilégio de colabo a , ag adeço oda a disponibilidade
e paciência que e e pa a comigo nas semanas que es i e no ITQB/UNL.
Aos colegas do UCIBIO, que o ag adece a disponibilidade pa a pa ilha de ideias, pelo
auxílio e pelos momen os que elemb o com um so iso nos lábios, em pa icula à
Cyn hia Ba oco, ao João Caço, ao João B i o, ao Tiago Dias e à Nicole.
Ao José Dias, um ag adecimen o especial pelo apoio e ca inho diá ios, pelas pala as
doces e pela ansmissão de con iança e de o ça, em odos os momen os.
Ag adeço, ambém aos meus amigos que semp e me apoia am.
À minha amília, em especial aos meus pais, um eno me ob igada po ac edi a em
semp e em mim, pelo es o ço e dedicação que semp e i e am pa a que eu pudesse
e mina es a e apa e po odos os ensinamen os de ida. À minha ia Dulce e às minhas
p imas-manas Inês e Viole a pelo apoio e pela o ma como me acolhe am e in eg a am
em Lisboa.
Po im, mas não po úl imo, que ia ag adece a Deus pela opo unidade e pela manei a
como guiou es a ese, que ac edi o e sido uma bênção.
i
Abs ac
Fo bac e ial cells o enla ge and di ide, pep idoglycan mus be clea ed by speci ic
hyd olases so ha new subuni s can be inco po a ed in o he ma u e cell wall. The mos
impo an mu ein hyd olase o he oppo unis ic human pa hogen S aphylococcus
au eus is ATL, a 137.5 kDa bi unc ional p o ein wi h wo domains: AM and GL ha
a e ex acellula ly p ocessed and bind o he s aphylococcal su ace a p ecise loca ions
o he equa o ial su ace ings. Based on obse a ions ha showed ha a l mu an s,
cons uc ed in di e en S. au eus gene ic backg ounds, a e no all impai ed in bio ilm
o ma ion, and ha he GL-DNA in e ac ion impac s bio ilm o ma ion in a s ain
speci ic way, we hypo hesized ha he physiological oles o ATL may be s ain-
speci ic. Di e en app oaches we e used o cha ac e ize ATL in di e en S. au eus
s ains: (i) he a l gene was sequenced o iden i y amino acid di e ences in he p o ein
ha could change i s ac i i y o unde go di e en p o eoly ic clea age; (ii) he size o
he di e en ATL p ocessed o ms and he cell compa men whe e hey accumula e
was assessed; (iii) he exp ession o ATL was analyzed o e ime by wes e n blo ing;
(i ) he impac o DNA on GL ly ic ac i i y was de e mined o hea -inac i a ed cells,
cell wall and pep idoglycan, h ough ly ic assays wi h he GL pu i ied p o ein.
The esul s ob ained allowed o iden i y dis inc pa e ns o ATL p o ein exp ession and
o p o eoly ic clea age ha may be he basis o he p ima y pheno ypic di e ences.
Keywo ds: Au olysin; S aphylococcus au eus; ly ic ac i i y; ATL; Glucosaminidase;
p o ein exp ession.
xiii
Ni-NTA
Nickel Ni ile-T iace ic Acid
Nm
Nanome e
NMR
Nuclea Magne ic Resonance
OD
Op ical Densi y
O/N
O e nigh
PBP
Penicillin-Binding-P o ein
PBS
Phospha e Bu e ed Saline
PCR
Polyme ase Chain Reac ion
pI
Isoelec ic Poin
PP
P opep ide
K
Kel in
KH2PO4
Monopo assium Phospha e
kDa
Kilodal on
Rpm
Re olu ions pe minu e
RT
Room Tempe a u e
SCCmec
S aphylococcal casse e ch omosome mec
SDS
Sodium Dodecyl Sul a e
SDS-PAGE
SDS-Polyac ilamide Gel Elec opho esis
SNP
Single-nucleo ide Polymo phism
TA
Teichoic Acids
TCA
T ichlo oace ic Acid
TSA
T yp ic Soy B o h Aga
TSB
T yp ic Soy B o h
UV
Ul a iole
V
Vol
WTA
Wall Teichoic Acids

1
Chap e I - In oduc ion
1. S aphylococcus au eus
S aphylococcus au eus a e G am-posi i e cocci wi h low DNA G+C con en ha
usually occu as g ape-like clus e s, and o m a ai ly la ge yellow colony on ich
medium and a e o en hemoly ic on blood aga . S aphylococci a e acul a i e anae obes
ha g ow by ae obic espi a ion o by e men a ion ha yields p incipally lac ic acid.
These bac e ia a e ca alase-posi i e (con e s hyd ogen pe oxide o wa e ) and oxidase-
nega i e, and can g ow a a empe a u e ange o 15 o 45 deg ees and a NaCl
concen a ions as high as 15 pe cen . They a e coagulase posi i e, a ma ke ha allows
he dis inc ion be ween S. au eus and o he S aphylococcus (Kloos, 1997).
1.1. Pa hogenici y
S. au eus a e equen ly ound as a commensal in he espi a o y ac o humans
(Kluy mans e al, 1997) bu can also ac as an oppo unis ic human pa hogen. They a e
he majo cause o nosocomial in ec ions wo ldwide (P alle e al, 1988).
In coloniza ion, he ela ionship wi h he hos is benign and asymp oma ic, bu b eak
o he cu aneous ba ie allows he bac e ia o in e nalize and cause diseases such as
skin and so issue in ec ions (nonin asi e in ec ions). As a pa hogen, i is conside ed
e sa ile bac e ia, as i can cause a wide spec um o in ec ions om impe igo and
olliculi is o li e- h ea ening in asi e in ec ions, such as bac e emia, pneumonia o
endoca di is. In addi ion, in ake o oxins om ood p oduc s colonized by S. au eus,
can cause acu e gas oen e i is (Bouche e al, 2010; Lee, 2003; P ojan & No ick,
1997).
1.2. Vi ulence
S. au eus exp esses many po en ial i ulence ac o s including oxins, immune-
modula o y ac o s, and exoenzymes (Wa kins e al, 2012).
Mos o hese i ulence ac o s a e cell-su ace-associa ed, helping o a oid
phagocy osis and consequen ly allowing he e asion o hos de enses. They usually a e
in ol ed in one o he ollowing p ocesses: i) adhe ence o S. au eus o su aces, such
as adhesins, coagulase, ib inogen-binding p o eins, clumping ac o , and bio ilm
2
polysaccha ides; ii) escaping o he hos immune sys em, such as en e o oxins, p o ein
A and leukocidins; and iii) damage o he hos including hemolysins, phospholipase C
and α- oxin. Toxins, p o eases and supe an igens, p e en he de elopmen o a s ong
an ibody esponse by p omo ing he bac e ial a ack o he hos , p e en ing he
de elopmen o an an ibody esponse, comp omising he immune memo y (Fos e ,
2005; P ojan e al, 1997).
S. au eus is also insensi i e o lysozyme (pep idoglycan hyd olase), a bac e icidal
p o ein p oduced by he inna e immune sys em ha is p esen in mos human body
luids such as sali a, swea and ea s, and which p oduc ion is inc eased du ing
in ec ion (Be a e al, 2005; Le y , 2000; Schindle e al, 1997).
The ac ha S. au eus has he abili y o easily acqui e gene ic in o ma ion also
con ibu es o i ulence, mainly h ough he acquisi ion o mobile gene ic islands ha
ca y i ulence de e minan s.
1.2.1. Mul i-d ug esis ance
S. au eus, he pa adigm among he bac e ia o his na u al phenomenon, has always
been a challenge o an i-mic obial chemo he apy (Chambe s, 2001).
Ini ially, s aphylococci in ec ions we e ea ed wi h penicillin, a β-lac am an ibio ic
disco e ed in 1928 imp o ing he p ognosis o pa ien s wi h s aphylococcal in ec ions,
dec easing he ex emely high mo ali y a e. β-lac ams inhibi he las s ep o he cell
wall pep idoglycan biosyn hesis, inac i a ing he penicillin binding p o eins (PBPs) due
o he simila i y o hei subs a e – pep idoglycan e minal D-ala-D-ala. The acquisi ion
o a plasmid encoding o a β-lac amase p o ein (penicillinase) led o penicillin
esis ance (Ab aham and Chain, 1940); his β-lac amase hyd olyzes he β-lac am ing
and consequen ly inac i a es he an ibio ic (Ghuysen, 1991). In o de o o e come he
acqui ed esis ance o his na u al an ibio ic, semi-syn he ic compounds (de i a i es o
penicillin), modi ied o esis o β-lac amase ac ion, we e de eloped o comba S. au eus
penicillin esis an in ec ions: me hicillin and i s de i a i es oxacillin, na cillin, among
o he s (Pla a e al, 2009; Moelle ing, 2012).
Me hicillin was in oduced clinically in 1959, howe e only wo yea s la e , he i s
s ains o me hicillin esis an S. au eus (MRSA) we e iden i ied, ca ying he
3
exogeneous mecA gene (Je ons, 1961). The MRSA pheno ype is a mul i ac o ial
p ocess ha occu s by he acquisi ion o a s aphylococcal ch omosome casse e
(SCCmec) and he exp ession o se e al housekeeping auxilia y genes. Besides β-
lac ams an ibio ics (penicillins, cephalospo in and ca bapenems), MRSA s ains also
de eloped esis ance o i ually all o he classes o an ibio ics ha we e in oduced in o
clinical p ac ice, such as mac olides, chlo amphenicol and e acycline, ha a ge
p o ein syn hesis o luo oquinolones and i ampicin, ha a ge nucleic acid syn hesis
(Bambeke e al, 2003).
The majo esis ance elemen o SCCmec is he mecA gene. This gene codes o an
ex a PBP, PBP2a (Reynolds & B own, 1985). PBP2a has a e y low a ini y o β-
lac ams, allowing cell wall biosyn hesis o p oceed in he p esence o he an ibio ic
(Ha man and Tomasz, 1984). The mecA gene is no na i e o S. au eus bu was
acqui ed om ano he species, mos p obably S. sciu i (Cou o e al 1996; Rolo e al
2013), by an unknown mechanism. (Beck e al, 1986) Al hough mecA is he main
gene ic de e minan o me hicillin esis ance, ecen ly, a highly di e gen mecA gene,
mecC, was iden i ied wi h ela i ely low p e alence a es (Sho e e al, 2011).
The me hicillin- esis an pheno ype also depends on he exp ession o mo e han 32
housekeeping genes - auxilia y ac o s, equen ly associa ed wi h he cell wall
pep idoglycan biosyn hesis and deg ada ion (Roeme e al, 2013; Be ge -Bächi e al,
1992; de Lencas e e al, 1999).
1.2.2. MRSA epidemiology
The e a e se e al p edominan clonal lineages o S. au eus; each clonal lineage is
de ined as a esul o i s speci ic gene ic backg ound and o he geog aphic si e o i s
i s iden i ica ion (Johnson e al, 2005). S udies based on he gene ic analysis o MRSA
isola es om di e en coun ies e ealed ha mos cases o Hospi al-acqui ed MRSA
(HA-MRSA) in ec ions a e caused by a small g oup o epidemic MRSA (EMRSA)
clones, which a e highly dissemina ed wo ldwide (Tomasz & de Lencas e, 1997;
Oli ei a e al, 2002). Each MRSA clonal lineage ecei ed a designa ion and some o he
mos success ul and well dissemina ed a e he Ibe ian, B azilian, Huga ian, New
Yo k/Japan, Pedia ic and epidemic clones: EMRSA-16, EMRSA-15 and Be lin.
4
In con as o MRSA in ec ions a hospi al se ings, in which in ec ed pa ien s ha e
p edisposing isk ac o s such as an immunocomp omised immune sys em, speci ic
MRSA clones eme ged in he communi y, in he mid and la e 1990’s, in heal hy
indi iduals wi hou hospi aliza ion his o y – he communi y acqui ed MRSA (CA-
MRSA) (DeLeo e al, 2010; Da id & Daum, 2010). CA-MRSA s ains a e usually mo e
i ulen and mo e ansmissible, howe e less esis an o an ibio ics in compa ison wi h
HA-MRSA. Mo eo e , CA-MRSA s ains ca y he smalle SCCmec elemen s o ypes
IV and V which we e associa ed wi h lowe i ness cos s, hus p omo ing inc eased
oxin p oduc ion and he eby inc eased i ulence (Chambe s & DeLeo 2009; DeLeo e
al. 2010; O o 2012).
Se e al CA-MRSA backg ounds eme ged and sp ead di e en ly in sepa a e
geog aphical a eas, howe e , nowadays CA-MRSA a e no es ic ed o a speci ic
geog aphic egion. USA400 clone is p esen in Asia, Eu ope and he USA, USA300 in
he USA and Eu ope, he Sou hwes Paci ic clone in Aus alia, Eu ope and Sou h
Ame ica, he ST59-V clone in Asia and he USA, he Eu opean clone in Eu ope, Asia
and he Middle Eas , and ST398 clone, i s associa ed wi h coloniza ion in pigs in
F ance, is cu en ly dissemina ed wo ldwide, no only in animals bu also in humans
(Media illa e al, 2012; Monecke e al, 2011; Uhlemann e al, 2012).
The expanding communi y ese oi o CA-MRSA has led o he ine i able
in il a ion o CA-MRSA in o hospi als. This phenomenon has become a majo public
heal h h ea and i is pos ula ed ha CA-MRSA will become he dominan MRSA
s ain in hospi als, wi h compe i i e exclusion o he adi ional HA-MRSA s ain
(Seybold e al, 2006).
2. Cell Wall
One o he c ucial bac e ial s uc u es is he cell en elope and i s in eg i y has o be
gua an eed.
The G am-posi i e cell en elope consis s o wo unc ional laye s: a cy oplasmic
memb ane, su ounded by a hick cell wall. The cell wall is a complex and a highly
o ganized s uc u e ha allows bac e ia o in e ac wi h he en i onmen bu also
p o ec s hem agains hos ile insul s. The G am-posi i e cell wall is composed by
5
di e se s uc u es, being he pep idoglycan hei majo componen (up o 50%) and he
eichoic acids (TAs) he key mul i- unc ional componen s o he cell wall. Many G am-
posi i e bac e ia, such as S. au eus, con ain wo ypes o TAs; wall eichoic acids
(WTA), which a e co alen ly linked o he pep idoglycan laye and lipo eichoic acids
(LTA), which a e embedded in he memb ane ia a lipid ancho (Reichmann &
G undling, 2011; Xia e al 2010). Besides pep idoglycan and eichoic acids, he cell
wall ha bo s a a ie y o di e en polysaccha ides, polyme s and p o eins, like he
PBPs.
2.1. Pep idoglycan Biosyn hesis
Pep idoglycan, also called mu ein, is a polyme ha consis s o long glycan chains
o al e na ed disaccha ide uni s (N-ace yl-glucosamine and N-ace yl-mu amic acid) ha
a e c oss-linked ia lexible pep ide b idges o o m a s ong bu elas ic s uc u e ha
p o ec s om lysis due o he high in e nal osmo ic p essu e (Ehle & Hol je, 1996;
Hol je, 1998; Nanninga, 1998; Schlei e & Kandle , 1972) (Figu e 1).
Figu e 1 - Chemical s uc u e o S. au eus pep idoglycan: disaccha ide uni s c oss-linked h ough
an in e -pep ide b idge consis ing o i e glycines o connec he ε-amino g oup o L-Lys in he hi d
posi ion o one s em (b idge-link, highligh ed) o he D-Ala in he ou h posi ion o he connec ed s em
(c oss-link) wi h he concomi an clea age o he e minal D-Ala. (Zhou & Cegelski, 2012)
Pep idoglycan and i s biosyn he ic pa hway is he a ge o se e al an ibio ic classes
including β-lac ams and glycopep ides (Pla a e al. 2009).

6
In S. au eus, pep idoglycan syn hesis begins in he cy oplasm whe e he p ecu so
UDP-Mu NAc-pen apep ide is assembled. I consis s o a uni o N-ace yl-mu amic acid
(Mu NAc) ha is a ached o a pen ape ide chain L-alanine-D-glu ama e-L-lysine-D-
alanyl-D-alanine (Vollme e al, 2008). Then, UDP-Mu NAc-pen apep ide is ans e ed
o a memb ane-bound lipid ca ie by he ac ion o M aY, o ming lipid I. UDP-GlcNAc
is added o lipid I by he ac ion o Mu G, leading o he o ma ion o lipid II ( an
Heijenoo , 2007), ha is anspo ed h ough he memb ane by he ac ion o F sW
lippase and is polyme ized h ough ansglycosyla ion ( o ex end he glycan chains)
and anspep ida ion (c osslinking be ween s em pep ides o di e en glycan s ands). In
S. au eus mos o he pen apep ide chains o adjacen mac omolecules a e linked by
pen aglycine in e b idges be ween he penul ima e D-alanine o one pep ide chain and
he ee amino g oup o he lysine o he o he chain. This is achie ed h ough he
anspep ida ion ac i i y o he so-called penicillin-binding p o eins (PBP’s), which also
ca alyze he ansglycosyla ion eac ion. (Go in e al, 1998) The pep idyl ans e ases
(FemX, FemA and FemB) a e esponsible o he addi ion o he i e glycine esidues o
he b idge, in a eac ion ca alyzed a he memb ane le el. (Figu e 2)
Addi ional modi ica ions o he pep idoglycan s uc u e occu in many bac e ial
species including modi ica ions o he glycan chains, modi ica ions o he s em pep ide,
such as he amida ion o he glu ama e esidue by he p o eins Mu T and Ga D, and
inco po a ion o cell wall polyme s (Figuei edo e al, 2012).
The hickness o he mu ein laye , in S. au eus, a ies be ween 20 o 400 nm
(50% o he cell wall). I is a dynamic mac o-molecule ha su e s pe manen
biosyn hesis, ma u a ion, ecycling, assembly and disassembly in o de o main ain he
cell shape, and allow o cellula g ow h and di ision. (Figu e 2)
7
Figu e 2 – Schema ic ep esen a ion o coo dina ed cell wall biosyn hesis and cell di ision in S.
au eus (adap ed om Roeme e al, 2013).
2.2. Hyd olases
Syn hesized pep idoglycan uni s a e inco po a ed in o he in ac cell wall laye a e
clea age by pep idoglycan hyd olases. A p ope balance be ween pep idoglycan
syn hesis and deg ada ion du ing bac e ial g ow h is essen ial. In gene al, pep idoglycan
hyd olases a e hough o play an impo an ole in cell wall u no e , cell di ision, and
cell sepa a ion, and in he lysis o bac e ia induced by he β-lac am an ibio ics (Biswas
e al, 2006).
S. au eus p oduces se e al pep idoglycan hyd olases, such as N-
ace ylglucosaminidases, N-ace ylmu amidases, N-ace ylmu amyl-L-alanine amidases,
ly ic ansglycosylases and endopep idases. Only he genes a l, sleI and ly M and hei
p oduc s ha e been cha ac e ized (Figu e 3).
8
Figu e 3 – Mu ein hyd olases a ge s wi hin S. au eus pep idoglycan. The a ows indica e he
clea age si es (adap ed om Szweda e al, 2012).
SleI is a 32kDa p o ein wi h N-ace ylmu amyl-L-alanine ac i i y and is in ol ed in
cell sepa a ion a e di ision in S. au eus (Heilmann e al, 2005). Ly M is a 32 kDa
p o ein wi h glycylglycine endopep idase ac i i y, being able o hyd olyse he glycyl-
glycine bonds o S. au eus c oss b idges. Ramadu ai (1997 and 1999) epo ed ha
Ly M plays a ole in cell g ow h as i is dis ibu ed on he cell su ace uni o mly. O he
pep idoglycan hyd olases wi h N-ace ylmu amyl-L-alanine amidases ac i i y we e
desc ibed in S. au eus, including Ly A (23 kDa), Ly H (33kDa) and Ly N (46kDa).
2.3. ATL – The majo au olysin o S aphylococcus au eus
The mos p ominen mu ein hyd olase o S. au eus is ATL, a 137.5 kDa
bi unc ional p o ein (Oshida, 1995). ATL p o ein consis s o a signal pep ide, a p o-
pep ide, a ca aly ic domain wi h N-ace ylmu amyl-L-alanine amidase ac i i y (AM),
h ee epea s (R1-R3), and a C- e minal ca aly ic domain wi h N-ace ylglucosaminidase
ac i i y (GL). A e sec e ion, he p ecu so p o ein is p ocessed ex acellula ly o yield
he ma u e amidase (AM) and glucosaminidase (GL) p o eins. (Figu e 4)
9
Figu e 4 – Domain a angemen o he bi unc ional ATL p ecu so p o ein. A ows (ligh ning)
indica e he pos - ansla ional clea age si es. SP- signalpep ide; PP- p opep ide; ca - ca aly ic domains;
R1 R2 R3- epea domains.
The amidase (63.3 kDa) clea es he amide bond be ween he N-ace yl mu amic
acid in he glycan backbone and L-alanine in he s em pep ide, con ains an enzyma ic
domain and wo epea domains in ol ed in localiza ion and subs a e ecogni ion (R1
and R2, ha can each be u he di ided in o an a- ype and a b- ype subuni ) (Biswas,
2006; Ma ino e al, 2002). The amidase epea s R1R2 a e esponsible o a aching he
enzyme o he cell wall and do no con ibu e o ly ic ac i i y (Oshida, 1995; Biswas,
2006). The s uc u e o AM om S aphylococcus epide midis is al eady de e mined
(Figu e 5). This domain, wi hou epea s, adop s a globula , mixed α/β old, wi h six
s anded, cen al β-shee su ounded by se en α-helices (Zoll e al, 2010). In he cen e
o he ecessed a ea is a zinc ion. R2ab esembles a hal -open β-ba el o med by a
semi-ci cula , ou s anded β-shee , and he wo subuni s a e a anged in a simila
o ien a ion.
Figu e 5 – (A) S uc u e o he ca aly ic domain o AmiE amidase (wi hou epea s R1,2) o S.
epide midis A lE. Helices and s ands a e shown in g een and pink espec i ely. (B) S uc u e o he A l
epea s R2ab. R2a and R2b ha e a simila β-s uc u e ha is connec ed wi h a lexible linke wi h R1a.
(Gö z e al, 2013)
B
16
2. DNA Me hods
Ch omosomal DNA om s ains was ex ac ed using Wiza d Genomic DNA
Pu i ica ion ki (P omega, USA) as sugges ed by he manu ac u e , wi h some
modi ica ions, namely, an ini ial cell lysis s ep, pe o med in T is pH8 supplemen ed
wi h 10 mg/ml o lysos aphin (AMBI PRODUCTS LLC, USA) and 30 µg/ml o RNase
(SIGMA, USA).
2.1. PCR and sequencing
Rou ine PCR (polyme ase chain eac ion) amplifica ion was pe o med wi h
NZYTaq DNA polyme ase (Nzy ech, Po ugal). The p ime s used a e lis ed in Table 2.
PCR p oduc s we e pu ified wi h DNA Clean & Concen a o TM-5 (Zymo Resea ch,
USA).
Table 2 – P ime s used o PCR ampli ica ion.
P ime
Sequence
n
PAM w BamHI
CCAGGATCCGCTTCAGCACAACCAAGATCAG
31
pGL XhoI
CCACTCGAGTTTATATTGTGGGATGTCG
28
P eAM w
ATGAATGCCCAATGTCATGC
20
AM
AGTAGTTACTTTAGGTGTCGC
21
PcompATL w SalI
CGAGTCGACGATTTGTCACGTCACC
25
PAMR2 SalI
CCAGTCGACTTAGGTAGTTGTAGATTGCG
29
PR1R2R3GL w NcoI
GCTCCATGGCTCCTACTACACCATCAAAACC
31
PAM SalI
CCAGTCGACTTATTTTACAGCTGTTTTTGG
30
PR2R3GL w NcoI
GCACCATGGCTCCTACACCAACACCTAAGCC
31
pGLSH3 SalI
CCTGTCGACTTAATGCTTAACATCATTAAAGTTAG
C
36
PGL w BamHI
CGTGGATCCGCTTATACTGTTACTAAACC
29
PGL SalI
CCAGTCGACTTATTTATATTGTGGGATGTCG
31
PGLSH3 w
GATGTTAAGCATGCAATGGATACG
24
PosGL
ACGTTGCGAATTGATTGAAGC
21
PCR p oduc s we e sequenced a STAB VIDA (Po ugal), and he sequence
aces we e analyzed using he so wa e DNAs a Lase gene SeqMan P o (Ve sion:
7.1.0).

17
3. F ac iona ion o cul u e con en s
Cul u e samples we e aken o e ime co esponding o he ollowing OD620nm:
0.1, 0.2, 0.4, 0.6, 1, 2, 3, 4, 6 and a la e s a iona y phase (≈24h). Cells we e ha es ed
by cen i uga ion (10 000g, o 10 minu es a 4ºC) and he supe na an and he pelle
we e sepa a ely s o ed a -20ºC and la e p ocessed as ollows.
Supe na an
The supe na an p o ein p ecipi a ion was pe o med using 1/10 olume o
TCA, du ing 17h, a -20ºC. The p o ein ac ion was collec ed by cen i uga ion a
13 000 pm 4ºC, 10 minu es (SIGMA 3-16K, 12155 o o , Sa o ius, Ge many). The
p o ein pelle was insed wi h ice-cold ace one and cen i uged a 13 000 pm 4ºC o
10 minu es. The supe na an was disca ded and he d ied pelle essuspended in
500µl PBS 1x.
Pelle
In o de o ob ain he same cell numbe , di e en olumes o cul u e we e
used, as showed in Table 3. The pelle was washed in 1ml o ice-cold 50mM T is-
HCl (pH7.5)-150mM NaCl and cen i uged a 10 000 pm, 4ºC o 10 minu es. Then,
he memb ane-associa ed p o eins we e ex ac ed by essupending he pelle in 100µl
o 4% SDS and incuba ing a oom empe a u e (RT) o 30 minu es wi h s i ing.
The SDS suspensions we e cen i uged a 13 000 pm (Bio uge Pico He aeus), o 15
minu es a RT. The supe na an s we e s o ed in aliquo s a -20ºC.
Table 3 – Cul u e olumes aken a di e en OD’s620nm.
OD620nm
Volume (mL)
0.1
50
0.2
25
0.4
12.5
0.6
8.3
1
5
2
2.5
3
1.7
4
1.25
6
0.83
18
3.1. P o ein analysis by SDS-PAGE and Wes e n Blo
To e i y he in eg i y o he p o ein ex ac s, p o ein samples we e analyzed
unde dena u ing condi ions using SDS-polyac ylamide gel elec opho esis (PAGE),
wi h he mini-PROTEAN sys em (BIO-RAD, USA). Be o e elec opho esis, he
samples we e mixed in a a io o 1:2 wi h 19:1 (Laemmli: β-me cap oe hanol) solu ion,
incuba ed a 95ºC o 5 min and hen 10 min on ice. Elec opho esis was pe o med in
unning bu e (24 mM T is-base, 191 mM Glycine, 3.46 mM SDS) a 30 mA o 1-1.5
h. Samples we e analyzed in 10% SDS-PAGE along wi h molecula weigh ma ke
(Colo Bu s Elec opho esis Ma ke , Sigma o P ecision Plus P o einTM All Blue
S anda ds, BIO-RAD).
P o eins we e ans e ed o a ni ocellulose memb ane (Ame sham Hybond
ECL Ni ocellulose, 0.45 µm om GE Heal hca e, UK) using he Mini T ans-blo
elec opho e ic ans e cell (BIO-RAD) and ans e solu ion (25 mM T is-base, 192
mM Glycine, 10% E hanol). Blo ing was pe o med a 4ºC o 90 min a 100 V wi h
agi a ion. A e blo ing, he memb ane was incuba ed o e nigh in blocking solu ion
(PBS-Tween and 5% w/ low- a milk), washed wi h PBS-Tween and p obed wi h he
p ima y an ibody an i-GL o an i-AM (G ilo e al, unpublished), in a a io o 1:1500 o
2 h and 1:1000 o 5h, espec i ely. The memb ane was hen washed, imme sed in esh
blocking bu e and incuba ed wi h seconda y an ibody (an i- abbi IgG) (Pe kin Elme ,
USA) in a a io o 1:20000 o 30min. A e a inal washing s ep, he memb ane was
incuba ed wi h chemiluminescence de ec ion solu ion o 1 min (Wes e n Ligh ning
Plus-ECL, Pe kinElme , USA) and exposed o au o adiog aphic ilm (Ame sham
Hype ilmTM ECL GE Heal hca e) o app op ia e pe iods o ime. The ilm was
p ocessed manually by imme sion in de eloping and ixing eagen s.
4. GL-media ed Lysis Assays
Th ee di e en subs a es we e used o analyze he ly ic ac i i y o GL: (i) hea -
inac i a ed cells, (ii) cell wall, and (iii) pep idoglycan. Samples we e p epa ed as
desc ibed in he nex sec ions (4.1 – 4.3). Hea -inac i a ed cells we e dilu ed o an
OD600≈ 0.4 in T is pH7.5. Cell wall and pu i ied pep idoglycan we e p epa ed T is
pH7.5 o an ini ial concen a ion o 4mg/mL. Low-molecula weigh salmon spe m
DNA (Sigma) (0.5 and 0.05 mg/mL) and pu i ied p o ein ATL-C (GL domain wi hou
19
he epea egion, G ilo e al, 2014) (5ng/µl) we e added o he wells, as needed.
Mu anolysin (5µg/mL) and lysos aphin (5µg/mL) we e used as posi i e con ols o
ly ic ac i i y.
Lysis assays we e pe o med in s e ile non ea ed 96-well mic opla es
(B andpla es®, B and, Po ugal) a 37ºC wi h shaking o 10h, aking eadings (600nm)
wi h 10 minu es in e al in a mic opla e eade Sp ec a Max190 (Molecula de ices,
USA).
4.1. Pu i ica ion o hea -inac i a ed cells
S. au eus s ains we e g own a 37°C wi h s i ing o an OD o ≈0,3 and cells we e
ha es by cen i uga ion a 10 000 pms a 4ºC o 10 minu es (So all RC-5C 19 Plus,
SLA-150 o o , Kend o Labo a o y P oduc s New own, USA). The pelle s we e
essuspended in cold wa e and cells we e boiled in SDS ( o a inal 4% SDS
concen a ion) o 30 minu es. The cul u es we e kep a RT O/N. The SDS was
emo ed by washing he cells wi h ho wa e un il no SDS is de ec ed in he
essuspended pelle h ough he Hayashi me hod. The pelle cells we e kep in H2O wi h
0,05% NaN3.
4.2. Cell wall ex ac ion
To ex ac he cell walls, he p ocedu e o sec ion 4.1. was pe o med and
subsequen ly he cells we e b oken by glass beads (Glass beads, acid-washed, 425-600
μm, Sigma) using he Fas p ep appa a us (Fas p ep FP120, Bio 101 Sa an , F ance), 3
imes, 40 seconds a speed 6. The samples we e cooled on ice be ween uns. Glass
beads we e emo ed by il a ion using a accum il e (po osi y 3). The il a e was
cen i uge in co ex ubes o 5 minu es a 2 000 pm, RT, o emo e unb oken cells and
la ge cellula deb is. The supe na an was cen i uged o 15minu es a 15 000 pm, RT.
Pelle s we e essuspended in 100mM T is (pH 7.5).
To pu i y he cell walls, he samples we e incuba ed wi h MgSO4 (20 mM),
DNAse and RNAse (10 and 50 µg/ml, espec i ely) a 37ºC o 2h. A e wa ds, CaCl2
(10 mM) and ypsin (100µg/ml) we e added and he samples we e incuba ion
p oceeded O/N wi h agi a ion. To inac i a e he enzymes, SDS was added o a inal
concen a ion o 1% and he samples boiled o 15minu es.
20
SDS was emo ed by 2 washes wi h H2O, cen i uging a 15 000 pm o 15
minu es. The pelle was incuba ed in 8M LiCl2, o 30 minu es a 37ºC and cen i uged
o 15 minu es a 15 000 pm a RT. The pelle was incuba ed in 0.1M EDTA (pH 7.0),
o 30 minu es a 37ºC and again cen i uged. A e 4 washings wi h H2O he pelle was
lyophilized O/N in Speed ac (Sa an TM Speed acTM Concen a o , USA).
4.3. Pu i ica ion o Pep idoglycan
Pep idoglycan pu i ica ion was pe o med ollowing he p ocedu es o sec ions
4.1. and 4.2.. The lyophilized pelle (sec ion 4.2) was ea ed wi h 48% hyd o luo ic
acid, o 48h a 4ºC wi h agi a ion. A e incuba ion, H2O was added and a pelle was
ob ained by cen i uga ion (45min a 20 000 pm, 4ºC). This s ep was epea ed. The
pelle was essuspended in 10mM T is (pH 7.0), ollowed by cen i uga ion. Th ee H2O
washing s eps we e pe o med. The pelle was lyophilized O/N in Speed ac.
5. P o ein exp ession and pu i ica ion
ATL-C p o ein (GL domain wi hou he epea egion) (G ilo e al, 2014) was
exp essed using wo di e en p ocedu es, acco ding o he inal objec i e: i) p o ein o
he lysis assays, and ii) p o ein o he NMR assays.
5.1. Exp ession o ATL-C p o ein in complex medium o diges ion
assays
ATL-C p o ein was exp essed in E. coli BL21(DE3)+PET28a-ATL-C. Cells
ans o med wi h he app op ia e ecombinan plasmid we e g own in LA medium
supplemen ed wi h Kanamycin (30 μg/ml o Km) a 37ºC.
A colony was inocula ed in 500mL o au o-induc ion medium (LB medium
supplemen ed wi h 2mM MgSO4, 10mL 50x5052 (0.5% glyce ol, 0.05% glucose and
0.2% lac ose), 25mL 20xNPS (see annex 3) and Kanamycin 30µg/ml), g own a
37ºC o 17h and cells we e ha es ed (12000 pm o 10 min) (So all RC-5C 19
Plus) and essuspended in 20mL lysis bu e (50mM Na2HPO4; 300mM NaCl;
10mM Imidazole; pH8) and 10 UmL-1 benzonase (No agen, Ge many) (4µL).
A e cell dis up ion pe o med wi h a F ench P ess (FA-032 (40k) s anda d
cell a 12000 psi om The mo Elec on Co po a ion), and emo al o cellula deb is
21
and memb anes by cen i uga ion (12000 pm o 1 h), he lysa e was subsequen ly
pu i ied as desc ibed in he nex sec ion.
5.1.1. Manual p o ein pu i ica ion using Ni-NTA ma ix
The column was cha ged wi h Ni-NTA aga ose (Qiagen, USA) and equilib a ed
wi h 20mL o wa e and 20mL o lysis bu e . The lysa e was loaded on o he column
and an aliquo was collec ed ( low- h ough). 20mL o wash bu e (50mM Na2HPO4;
300mM NaCl; 30mM Imidazole; pH8) was used o he washing s eps and 2mL o
Elu ion Bu e (50mM Na2HPO4; 300mM NaCl; 250mM Imidazole; pH8) o collec ing
5 elu ions. Finally, he column was washed wi h wa e and 30% e hanol and s o ed a
4ºC. Aliquo s we e collec ed a each s ep.
To e i y he p o ein pu i y le el, he p o ein samples we e analyzed unde
dena u ing condi ions using SDS-polyac ylamide gel elec opho esis (PAGE), as
desc ibed be o e, in sec ion 3.1.
5.2. Exp ession o ATL-C p o ein in Minimal Medium o NMR analysis
ATL-C p o ein was exp essed in E. coli BL21(DE3)+PET28a-ATL-C. To es
he exp ession condi ions, he assays we e pe o med in small-scale, using 200 ml o
bac e ial cul u e, while, o ob ain high quan i ies o p o ein, exp ession was pe o med
in la ge-scale, using 500mL o 1L o bac e ial cul u e. The me hod o cell dis up ion
adop ed was mechanical dis up ion wi h F ench P ess as be o e.
Small-scale:
A colony was inocula ed in 10mL o non-induc ion minimal medium (50mM
Na2HPO4, 50mM KH2PO4, 5mM Na2SO4, 50mM NH4Cl, 2mM MgSO4, 0.2x ace
me als (see annex 3), 0.5% glucose) and g own a 37ºC o 7h. Subsequen ly, 2% o he
olume was inocula ed in 25 mL o esh non-induc ion minimal medium and he
cul u e was g own a 37ºC o 17h. Then, 2% o he cul u e was ans e ed o 200mL o
induc ion minimal medium (50mM Na2HPO4, 50mM KH2PO4, 5mM NaSO4, 50mM
NH4Cl, 2mM MgSO4, 0.2x ace me als, 200µL 50x5052). A e 24 h o incuba ion a
37ºC, cells we e ha es ed (12000 pm o 10 min) (So all RC-5C 19 Plus) and
essuspended in 20mL lysis bu e and 10 UmL-1 benzonase (adap ed om S udie ,
2005).

22
A e cell dis up ion pe o med using a F ench P ess and emo al o cellula
deb is and memb anes by cen i uga ion, he lysa e was subsequen ly pu i ied as
desc ibed in 5.1.1 sec ion and analyzed by SDS-PAGE as desc ibed in sec ion 5.1.2..
La ge-scale:
The p ocedu e was he same as o small-scale, wi h he co esponding olumes
scalled-up o 1L o bac e ial cul u e.
5.2.1. Exp ession o ATL-C p o ein in Minimal Medium o 15N NMR
analysis
The p o ein exp ession p ocedu e was pe o med as op imized o minimal medium
(sec ion 5.2), wi h he excep ion ha ins ead o 50mM NH4Cl, labeled 15NH4Cl (Sigma)
was added o he same concen a ion, wi h he co esponding olumes scalled-up o
500mL o bac e ial cul u e.
5.3. Desal ing and p o ein concen a ion
Desal ing and bu e exchange we e pe o med using PD-10 desal ing columns (GE
Heal hca e) o 100mM T is (pH7.5). PD-10 Desal ing Columns con ain Sephadex G-25
Medium, which allows sepa a ion o high molecula weigh subs ances om low
molecula weigh subs ances; small molecules like sal and o he impu i ies a e
e icien ly sepa a ed om he high molecula weigh subs ances o in e es .
The samples we e concen a ed using Amicon® Ul a-4 (10 k) Cen i ugal Fil e
Uni s (Me ck Millipo e, USA). This de ice p o ides e icien concen a ion and
desal ing o mac omolecules by ul a il a ion using Millipo e’s Ul acel® YM
egene a ed cellulose aniso opic memb anes. Cen i ugal o ce d i es sol en s and low
molecula weigh solu es h ough he memb ane while he mac omolecules emain
inside he sample ese oi .
5.4. P o ein quan i ica ion
The o al amoun o p o ein p esen in each ac ion collec ed was es ima ed by
UV abso p ion a 280 nm (NanoD op ND-1000, Fishe Scien i ic, Spain), using
ex inc ion coe icien and p o ein molecula weigh calcula ed wi h he online ools
23
P o Pa am and Compu e pI/MW (ExPASy, Bioin o ma ics Resou ce Po al) speci ic o
he a ge p o ein (ATL-C: ε= 64860 cm-1/M; MW = 39639.6641 Da).
P o ein concen a ion om he ex ac s (sec ion 3) was measu ed wi h BCA assay
(Pie ce) in a mic opla e eade .
6. NMR analysis
NMR expe imen s we e pe o med a 298 K in a A ance II+ 600-MHz spec ome e
(B uke , Ge many) equipped wi h 5-mm TCI c yop obe. P o on chemical shi s we e
e e enced agains ex e nal DSS while ni ogen chemical shi s we e e e enced
indi ec ly o DSS using he absolu e equency a io. Da a was p ocessed using he
Topspin 3.1 package (B uke ).
A 1 mM solu ion o 15N-labeled ATL-C in 25mM T is-DCl bu e (10% 2H2O,
pH=7.5) 75mM NaCl, was i a ed.
24
25
Chap e III – Resul s
P elimina y esul s (see annex1) sugges ha he physiological oles o ATL
au olysin and in pa icula , i s associa ion wi h DNA (G ilo e al, 2014) is dependen on
he gene ic backg ound o S. au eus. Thus, wi h he pu pose o cha ac e ize he S.
au eus ATL p o ein, ega ding s ain speci ici y, di e en app oaches we e designed
and di e en gene ic backg ounds we e used, namely s ains COL (HA-MRSA, a chaic
clone), WIS (CA-MRSA, Taiwan clone), HDE288 (HA-MRSA, pedia ic clone),
UAMS-1 (MSSA, , JE2 (CA-MRSA, USA300), NCTC8325 (MSSA, labo a o y s ain)
and MW2 (CA-MRSA, USA400).
1. De e mina ion o a l gene SNPs
In o de o compa e he nucleo ide sequence o a l gene o he di e en s ains, he
a l gene was ully sequenced o s ains o which he genome sequence was s ill
unde e mined, namely WIS, HDE288 and JE2.
The sequence leng h o a l gene, including he p omo e egion, is app oxima ely
4000 bps; ampli ica ion o his egion was pe o med in 6 sepa a e DNA agmen s
( agmen s A o F), as shown on Table 4 and Figu e 7.
Table 4 – a l gene agmen s ampli ied and p ime s used o PCR ampli ica ion.
DNA agmen
P ime s
Leng h (bps)
A
PcompATL wSalI + PAMR2 SalI
1431
B
PR1R2R3GL wNcoI + PAM SalI
1053
C
PR2R3GL wNcoI + pGLSH3 SalI
1392
D
PGL wBamHI + PGL SalI
1446
E
P eAM w + AM
941
F
PGLSH3 w + PosGL
915
32
4221
AG
778
TA
4416
GA
1022
TI
4424
GA
1025
AT
1051
GD
2659
ACCATCAAC
-
255
KPS
WIS
1109
AG
-
-
-
-
-
1197
GA
-
-
-
-
-
HDE288
1802
AC
-
-
-
152
NH
1868
AG
174
TA
-
-
1868-1992
176-184
-
1946
GA
199
TA
2942
CT
-
-
-
531
PS
3042
AT
-
-
-
563
QL
3147
CT
-
-
-
599
TI
3802
AG
-
-
-
874
KR
4133
GT
-
-
-
928
AS
4416
CT
-
-
-
1022
TI
4424
GA
-
-
-
1025
AT
4503
GA
-
-
-
1051
GD
1071
G
-
-
-
JE2
2619
CG
-
-
-
252
TS
2686
TA
-
-
-
256
KT
2743
AT
-
-
-
597
TA
2942
CT
-
-
-
615
SA
2998
TC
-
-
-
742
FY
3042
AT
-
-
-
752
VI
3147
CT
-
-
-
773
AV
4156
TC
-
-
-
776
AD
4424
GA
-
-
-
1025
AT
4503
GA
-
-
-
1051
GD

33
3. ATL p o ein exp ession along g ow h
In o de o analyze he ATL p o ein exp ession o e ime and o de e mine i he
exp ession pa e n and he p o eoly ic p o ile a ies om s ain o s ain, he ela i e
amoun o AM and GL was assessed by Wes e n blo ing, o s ains COL, NCTC 8328,
WIS, HDE288, JE2, UAMS-1 and MW2. Wes e n blo ing (o p o ein immunoblo ) is
an analy ical echnique ha can be used o de ec speci ic p o eins in a complex ex ac ,
using speci ic an ibodies. We used he p e iously a ailable an i-GL and an i-AM aised
an ibodies aised agains p o eins GL-C (GL wi hou epea s domain) and ATL-H (AM
wi hou epea s domain) espec i ely (G ilo e al, unpublished). The amoun o ATL
p o ein was assessed in he cell supe na an ac ion (spen medium) (Figu e 11, panel
A) and also in he cell wall ac ion (Figu e 11, panel B).
Cul u e samples we e aken a disc e e ime poin s along he g ow h cu e (Figu e
10).
Figu e 10 – G ow h cu e o s ains COL, WIS, HDE288, UAMS-1, JE2, NCTC8325 and MW2 in
complex medium a 37ºC.
0
1
2
3
4
5
6
7
0 2 4 6 8 10
OD620nm
Time (h)
JE2
UAMs-1
WIS
HDE 288
COL
NCTC 8325
MW2
34
3.1.Exp ession analysis using an i-GL an ibody
The p o ein ex ac s we e sepa a ed by SDS-PAGE and ans e ed o a ni ocellulose
memb ane ha was hen hyb idized wi h he an i-GL an ibody. By analyzing he
appa en molecula weigh o he bands ob ained, he an i-GL an ibody was obse ed o
hyb idize wi h he ull ATL p o ein (137.5 kDa), he ATL wi hou he PP (121 kDa) and
also wi h mo e han one p ocessed o m o he GL domain, he ull o m R3GL (53.6
kDa), and GL (40.5 kDa).
35
Figu e 11 – GL exp ession along ime. Wes e n blo ing was pe o med o p o ein ex ac s om s ains
WIS, HDE288, JE2, UAMS-1, COL, NCTC8325 and MW2, using an i-GL speci ic an ibody. (A) Cell
supe na an ; (B) Cell wall ac ion. A- OD 0.2, B- OD 0.4, C- OD 0.6, D- OD 1, E- OD 2, F- OD 3, G-
OD 4, H- OD 6, I- OD 11.
Di e ences no only in he amoun o ATL p o ein p oduced along ime, bu also in
he numbe and molecula weigh o he p o ein bands ob ained, we e obse ed be ween
he 7 s ains analyzed.
O e all, o mos s ains, ATL p o ein ( he unp ocessed o m o he AM-R1R2R3-
GL o m) seemed o accumula e i s ly in he supe na an (a OD~0.2-0.4) and only la e
was he p ocessed GL domain a ge ed o he cell wall (s a ing a OD~0.4).
WIS, HDE 288, JE2, UAMS-1, NCTC 8325 and MW2 showed a simila ATL
exp ession pa e n in he supe na an , wi h a g adual inc ease along ime, while COL
showed a cons an ATL exp ession pa e n. The ATL unp ocessed o m and he AM-
36
R1R2R3-GL o m we e no obse ed in he cell wall ac ion o he pe iod o g ow h
analyzed.
Rega ding GL domain, se e al p ocessed o ms accumula ed oge he wi h he ATL
ull p o ein in he supe na an o mos s ains excep COL. Fo s ains HDE288 and JE2,
GL domain began o accumula e in he supe na an du ing exponen ial phase (OD-0.1)
and la e (OD-4) o s ains WIS, UAMS-1 and NCTC8325. Fo s ain MW2, GL
domain was only de ec ed du ing la e s a iona y phase (OD-11). A he cell wall le el,
GL domain was p esen in one single o m (co esponding o one band) o s ains
HDE288 and MW2 o as se e al p ocessed bands (co esponding o mul iple bands) o
s ains WIS, JE2, UAMS-1, COL and NCTC8325. These dis inc GL bands may
co espond o di e en clea age e en s o GL p o ein.
Rega ding he amoun o GL domain p esen in he cell wall ac ion, i was
in e es ing o obse e h ee di e en pa e ns: o s ains WIS, HDE288, JE2 and
UAMS-1, GL domain showed an accumula ion peak a exponen ial phase (OD-1)
ollowed by a apid dec ease a OD-2 and inally a s eady inc ease; o s ains COL and
NCTC8325, GL domain only s a ed o accumula e a la e exponen ial phase (OD~3-4),
while o s ain MW2, i cons an ly accumula ed along ime.
3.2. Exp ession analysis using an i-AM an ibody
The same p o ein ex ac s we e sepa a ed in SDS-PAGE and ans e ed o a new
ni ocellulose memb ane ha was hyb idized wi h an i-AM an ibody. By analyzing he
appa en molecula weigh o he bands ob ained, we obse ed ha he an i-AM
an ibody was no able o ecognize he unp ocessed o m o ATL (137.5 KDa), only he
AM domain (63.3 kDa). The Wes e n blo ing esul s a e shown in Figu e 12, panel A
(supe na an ac ion) and panel B (cell wall ac ion).
37

38
Figu e 12 – AM exp ession along ime. Wes e n blo ing was pe o med o p o ein ex ac s om
s ains WIS, HDE288, JE2, UAMS-1, COL, NCTC8325 and MW2, using an i-AM speci ic an ibody. (A)
Cell supe na an ; (B) Cell wall ac ion. A- OD 0.2, B- OD 0.4, C- OD 0.6, D- OD 1, E- OD 2, F- OD 3,
G- OD 4, H- OD 6, I- OD 11.
Di e ences in he exp ession pa e n o AM domain along ime and amongs s ains
was e en mo e s iking han o GL domain.
While WIS, COL and UAMS-1 showed low and poo ly consis en amoun s o AM in
he supe na an , NCTC8325 and MW2 only showed AM p esence a la e s a iona y
phase (OD 6 o 11). HDE288 did no p esen AM in he supe na an un il la e
exponen ial phase (OD 1), and JE2 did no p esen AM in he supe na an in he
s a iona y phase.
Rega ding he amoun o AM domain p esen in he cell wall ac ion, i was
in e es ing o obse e, as o GL domain, h ee di e en pa e ns: o s ains WIS,
HDE288 and UAMS-1, AM domain showed an accumula ion peak a exponen ial phase
(OD-1) ollowed by a apid dec ease a OD-2 and inally a s eady inc ease; o s ains
COL, NCTC8325 and MW2, AM domain did no accumula e a he cell wall ac ion;
o s ain JE2, i cons an ly accumula ed along ime.
4. The associa ion be ween DNA and GL ly ic ac i i y
The in e ac ion be ween DNA and GL seems o ha e an impo an ole in he bio ilm
o ma ion. In o de o unde s and i he GL-DNA associa ion also has impo ance in
ly ic ac i i y, ly ic assays we e pe o med using hea -inac i a ed cells, cell wall ac ion
and pu i ied pep idoglycan as subs a e o GL in he p esence o added DNA. To
de e mine i he cell wall o he pep idoglycan composi ion o he s ain would in luence
GL ac i i y, hese we e es ed o all he s ains unde s udy.
39
ATL-C ecombinan His- agged p o ein (GL domain wi hou he epea egion) was
exp essed o he diges ion assays, and hen pu i ied using a Ni-NTA column. The Ni-
NTA (nickel ni ile- iace ic acid) column co-pu i ica ion is based in he high a ini y
be ween polyhis idine agged p o eins and he nickel ions p esen in he ma ix o he
Ni-NTA columns. P o eins bound o he esin a e elu ed by compe i ion wi h imidazole.
The p o ein pu i y le el was e i ied by SDS-PAGE as shown a igu e 13.
Figu e 13 - Pu i ica ion o ATL-C ecombinan His- agged p o ein. Lane 1: o al ex ac ion lysa e;
Lane 2: low- h ough; Lane 3 and 4: washes; Lane 5-9: elu ions 1-5; Lane 10: p o ein ma ke (Low
Molecula Weigh P o ein Ma ke ). G een a ows: ATL-C (GL wi hou R3).
In o de o de ine he bes ly ic ac i i y condi ions, di e en bu e s we e es ed
(Sodium Phospha e Bu e pH 7.4; Sodium Phospha e Bu e pH 5.5; T is-HCl pH 8;
T is-HCl pH 7.4; T is-HCl pH 5.5) and di e en GL p o ein concen a ions (5ng/µl;
10ng/µl; 20ng/µl). The eac ion condi ions ha e ie ed highe GL pep idoglycan ly ic
ac i i y le els we e bu e T is pH 7.5 and a p o ein concen a ion o 5ng/µl. The
sample was incuba ed a 37ºC wi h agi a ion and he OD600 was moni o ized o 2h wi h
5 minu e in e als eads.
As posi i e con ols, mu anolysin (a mu aly ic enzyme ha clea es he N-
ace ylmu amyl-β-N-ace ylglucosamine linkage o he bac e ial cell wall polyme
pep idoglycan-polysaccha ide) and lysos aphin (enzyme ha clea es he c osslinking
pen aglycin b idges o S. au eus pep idoglycan) we e used; bo h enzymes we e able o
40
deg ade he hea -inac i a ed cells, he cell wall ac ion and he pu i ied pep idoglycan
samples. To de e mine i he salmon spe m DNA added o he samples would in luence
he measu emen s, con ols we e pe o med only wi h subs a e and added DNA; no
signi ican di e ences we e obse ed when compa ed o he samples wi hou DNA.
Di e ences we e obse ed in he ly ic ac i i y o GL when using subs a es om
di e en s ains: pu i ied pep idoglycan, cell wall and hea -inac i a ed cells (Figu e 14).
The ollowing g aphics a e p esen ed wi h ep esen a i e alues o ime, he g aphics
wi h all ime poin s a e p esen ed in annex 2.
The ly ic ac i i y o GL was highe o he pep idoglycan o s ains WIS, HDE288,
UAMS-1 and MW2. In WIS and MW2 GL-DNA wi h 0.05 and 0.5 mg/mL,
espec i ely, has a highe diges ion o pep idoglycan.
Rega ding he cell wall, i is obse ed mo e ly ic ac i i y o GL in he s ains
HDE288 and MW2. The e seems o be no signi ican di e ence in GL ac i i y when
associa ed wi h DNA.
In he inac i a ed cells he e is a g adual dec ease in OD600 when GL is added,
howe e no di e ences a e obse ed in he p esence o DNA.
GL showed no ly ic ac i i y o he hea -inac i a ed cells o all se en s ains.
Fu he mo e, GL also did no show ly ic ac i i y o he cell ac ion o he pu i ied
pep idoglycan o s ain COL. The ly ic ac i i y o GL was highe o he cell wall
ac ion o s ains MW2 and HDE288; acco dingly, o hei pep idoglycan ac ion as
well. Fo all o he s ains (WIS, UAMS-1, JE2 and NCTC8325), GL only showed ly ic
ac i i y in he pep idoglycan ac ion.
Rega ding he e ec o DNA addi ion o he lysis eac ion, con a y e ec s we e
obse ed o he ly ic ac i i y o GL agains WIS and MW2 pep idoglycan: he 0.05
mg/ml concen a ion was ound o ac i a e GL ly ic ac i i y on WIS pep idoglycan and
inhibi he same ac i i y on MW2 pep idoglycan. In con as , he highe DNA
concen a ion, 0.5 mg/mL, was esponsible o inhibi ing GL ac i i y on WIS
pep idoglycan and ac i a es i on MW2 pep idoglycan.
41
0
0,2
0,4
0,6
0,8
1
1,2
1,4
OD (600 nm)
(A) COL
5 min. 25 min. 50 min. 75 min. 100 min. 120 min.
0
0,2
0,4
0,6
0,8
1
1,2
1,4
OD (600 nm)
(B) WIS
48

49
Chap e IV – Discussion and Conclusions
The main objec i e o his p ojec was o cha ac e ize he ATL p o ein ega ding
s ain speci ici y. Fo his pu pose, ou asks we e p oposed: (i) s udy he di e en
p ocessed o ms and he cell compa men whe e he clea age occu s, in di e en S.
au eus backg ounds; (ii) analyze he exp ession o ATL along ime in he di e en
s ains; (iii) analyze he impac o DNA on GL ly ic ac i i y on he pep idoglycan o
se e al S. au eus lineages; (i ) cha ac e ize GL-DNA in e ac ion by NMR.
S aphylococcus au eus is a e y clonal mic oo ganism ha is able o dissemina e
wo ldwide. Among he se e al clonal lineages o S. au eus, in his s udy we included
s ains ep esen ing MSSA, CA-MRSA and HA-MRSA clonal lineages ha showed
di e en ATL-dependen bio ilm o ma ion pa e ns.
COL is a hospi al acqui ed MRSA wi h a sccmec ype I, membe o he
“a chaic” clone o MRSA and pe haps he mos s udied MRSA s ain. COL was
isola ed om a pa ien in Colindale, Uni ed Kingdom in 1960 (Je ons, 1961). This
s ain is a membe o he mos success ul o all MRSA lineages, which nowadays no
only includes hospi al bu also communi y-associa ed s ains. NCTC 8325 and UAMS-1
a e bo h MSSA; NCTC 8325 was isola ed om a co neal ulce , and he o iginal genome
map o S. au eus was based on his s ain (No ick, 1991), UAMS-1 was isola ed om
an os eomyeli is pa ien . MW2, a communi y acqui ed MRSA wi h sccmec ype IV, was
isola ed in 1998 in he USA and caused a al sep icemia and sep ic a h i is (Baba,
2002). I belongs o he USA400 clone, one o he mos common lineages in he Uni ed
S a es. WIS is a CA-MRSA wi h sccmec ype V, isola ed in Taiwan. HDE288 is a HA-
MRSA wi h sccmec ype VI, a pedia ic clone isola ed in Nica agua. JE2 is a CA-
MRSA ha belongs o USA300 clone, isola ed om skin and so issue in ec ions in
USA.
1. a l gene SNPs a ec he p o ein sequence
Single nucleo ide polymo phisms, equen ly called SNPs, a e he mos common
ype o gene ic a ia ion. SNPs wi hin a coding sequence do no necessa ily change he
amino acid sequence o he p o ein.
50
The SNPs obse ed in he a l egion o he s ains in his s udy p o ided in e es ing
in o ma ion. No al e a ions we e obse ed in he p omo o egion, indica ing ha any
exp ession di e ences be ween he s ains do no esul om mu a ions in his egion.
Some SNPs esul ed in amino acid esidue subs i u ions and only one was loca ed in he
icini y o he ac i e si e o GL domain, E1128 (V1126I) in MW2 s ain. The sequence
o ATL p o ein o UAMS-1 showed mo e di e ences, mainly in he p opep ide egion,
when compa ed wi h he o he s ains, sugges ing an al e ed p o eoly ic clea age
pa e n. The ATL sequence o HDE288 and JE2 also showed se e al amino acid
subs i u ions, mo e dispe sed along he ATL p o ein domains (PP, AM, R epea s and
GL). The only ATL sequence ha was iden ical o COL was he one o WIS s ain.
2. Di e ences in he exp ession o ATL p o ein along g ow h
ATL is 137.5 kDa mu ein hyd olase, wi h wo ca aly ic domains: AM (63.3 kDa),
ha con ains an enzyma ic domain and wo epea domains (R1 and R2), and GL (53.6
kDa), con aining an enzyma ic domain and a single epea domain (R3).
To analyze he ATL p o ein exp ession o e ime and de e mine i he exp ession
pa e n a ies om s ain o s ain, he ela i e amoun o ATL was assessed by Wes e n
blo ing, using speci ic an ibodies agains AM and GL domains. The an i-GL an ibody
was ound o ecognize he ATL comple e p o ein while also ecognizing GL domain.
A he beginning o exponen ial phase, ATL p o ein was p esen only in he supe na an ,
sugges ing ha he p o ein accumula ed in he cell ex e io and only a e a p o ein
eshold limi i was hen a ge ed o he cell wall, al eady in a clea ed o m. The
comple e o m o ATL (137 kDa) was no p esen in he cell wall ac ion.
The p esence o GL was mo e e iden in he pelle , sugges ing ha a e clea age,
GL domain is a ge ed o he cell wall. These esul s suppo he ecen ly desc ibed
unc ion o his hyd olase in bio ilm de elopmen : GL may p o ide an a achmen
poin be ween he cell su ace and he bio ilm ma ix.
Rega ding AM domain, COL and NCTC8325 s ains seem no o a ge AM domain o
he cell wall. Howe e , p esence o AM a he cell wall was obse ed in he o he
s ains, in acco dance wi h he li e a u e desc ip ion o R1R2 epea s being esponsible
51
o a ge ing AM domain o he pelle (Biswas, 2006; Ma ino e al, 2002). These
obse a ions demons a e how di e en s ains can ha e di e en le els o AM
associa ed o he cell wall and aise wo hypo heses: AM is no loca ed a he cell wall in
some s ains, o AM is only a ge ed o he cell wall a a la e g ow h phase. Two
possible mechanisms a e desc ibed o he speci ic localiza ion o ATL a he sep al
si es o he cell su ace. One is ha ATL p o ein is syn hesized, ansloca ed a he cell
di ision si e, and localized wi h an ancho ing componen . The o he is ha ATL is
sec e ed in o he cul u e medium and eabso bed o an ancho ing componen ia ligand-
ecep o in e ac ion (Schlag e al, 2010; Yamada e al, 1995). Di e ences in he
s uc u e o he epea domains o AM and GL migh e lec he di e ences o he
ecogni ion si es on s aphylococcal cell walls. Ou esul s con i m ha a ge ing o AM
and GL occu s h ough independen mechanisms: GL was p esen in he cell wall o all
s ains, in con as o AM. Fu he mo e, he ac ha no AM was obse ed in he cell
wall o some s ains s eng hens he second hypo hesis ha he ATL domains a e
sec e ed and subsequen ly eabso bed.
Di e en ATL, GL and AM exp ession pa e ns along he g ow h cu e we e
obse ed o he di e en s ains and also dis inc p o eoly ic pa e ns, illus a ed by he
occu ence o bands o di e en sizes. Howe e , no di ec ela ion wi h he amino acid
subs i u ions iden i ied p e iously was possible o es ablish. Fo example, he ATL
sequence o WIS and COL s ains a e iden ical, howe e , hei ATL, GL and AM
exp ession p o iles we e comple ely dis inc .
3. The impac o DNA on GL ly ic ac i i y
ATL is he mos p edominan pep idoglycan hyd olase in s aphylococci. Al hough
se e al s udies ha e ocused on he unc ion o ATL, he indi idual con ibu ions o he
AM and GL domains a e no known. Recen s udies e eal he impo ance o he
in e ac ion be ween DNA and GL in bio ilm o ma ion. This ac aises he ques ion i
he in e ac ion be ween DNA and GL may also be impo an o he majo unc ion o
ATL, he pep idoglycan ly ic ac i i y.
52
The esul s ob ained showed no signi ican di e ences in he ly ic ac i i y o GL
when DNA is added, excep o s ains WIS and MW2. The e ec o bo h DNA
concen a ions on he ly ic ac i i y o GL o he pep idoglycan o hese wo s ains, was
con adic o y: i had apposi e inhibi o y and ac i a ing e ec s. Di e ences in he
pep idoglycan composi ion o hese s ains may explain he di e en ly ic ac i i y o GL
p o ein and he pep idoglycan o hese s ains should be analyzed in u u e expe imen s.
Fu he mo e, all he assays we e pe o med wi h GL p o ein om COL s ain. I is
in e es ing o obse e no ly ic e ec agains COL inac i a ed cells, cell wall o e en
pep idoglycan, sugges ing ha GL may ac as a weapon agains o he S. au eus s ains,
al hough no ha ing ly ic ac i i y agains i s own su ace polyme s.
Rega ding he hea -inac i a ed cells, no e ec o GL ly ic ac i i y was obse ed;
as GL is expec ed o lyse he pep idoglycan mesh a disc e e loca ions, his beha io
was expec ed. Also, o he cell su ace associa ed ac o s/enzymes may also bind o
DNA, in his way compe ing wi h GL. Conce ning he esul s o he cell wall ac ion,
he p esence o WTAs, which p e en s S. au eus om au olysis (Schlag e al, 2010),
can be, also, he explana ion o he non-signi ican ly ic ac i i y. I is belie ed ha
a ge ing o amidase R1R2 epea domains is a he based on an a oidance s a egy by
WTA, which p e en s binding o ATL. As WTAs a e abundan in he old cell wall bu
no a he c oss-wall egion, ATL can bind o he sep al egion.
I would be in e es ing o analyze he ly ic ac i i y o AM-R1R2 in hese s ains, in
he p esence o added DNA, since he DNA-binding ac i i y o he AM domain appea s
o be es ic ed o he epea s (G ilo e al, 2014).
4. NMR
Nuclea magne ic esonance (NMR) spec oscopy is a me hodology ha e eals he
a omic s uc u e o mac omolecules in solu ion in highly concen a ed samples (app ox.
1 mM) (Bha i and Roy, 2012). The echnique is based on he ac ha ce ain a omic
nuclei a e in insically magne ic. Only a limi ed numbe o iso opes display his
p ope y, called spin (½), such as 1H, 15N, 13C. A spinning p o ein in α s a e can be
aised o an exci ed s a e (β s a e) applying a pulse elec omagne ic adia ion (a adio-
53
equency). The spin will change om α o β and esonance will be ob ained. A
esonance spec um o a molecule can be ob ained a ying he magne ic ield a a
cons an equency o elec omagne ic adia ion o keeping he magne ic ield cons an
a ying elec omagne ic adia ion. Wi h his echnique, one, wo and ee-dimension
spec a (1H-spec um, 1H-15N-spec um and 1H15N-13C-spec um) can be ob ained in
o de o de e mine he p o ein s uc u e (Mon elione e al., 2000). To bea he low
na u al abundance o 15N and 13C o ob aining wo and ee-dimension spec a,
inco po a ion o such iso opes mus be o ced by exp essing he p o ein in minimal
media supplemen ed wi h he iso opes.
In his s udy, ATL-C, GL domain wi hou epea s, was exp essed in 15N labeled
minimal medium and hen analyzed by NMR. The 15N-HSQC spec um ob ained
e ealed ha he p o ein is olded since a high signal dispe sion is obse ed. These
esul s p o ided impo an con ibu ions o he cha ac e iza ion o GL by NMR. The
s uc u e o GL domain has no been elucida ed ye , in con as wi h AM domain. I is
necessa y in he u u e o op imize he condi ions o he exp ession o GL o ob ain a
13C spec um essen ial o he s udy o he in e ac ion be ween GL and DNA.
5. Conclusion
This s udy allowed o iden i y dis inc pa e ns o ATL p o ein exp ession and
p o eoly ic clea age ha may be he basis o he p ima y pheno ypic di e ences
associa ed o ATL p o ein. GL and AM exp ession a ies om s ain o s ain, and GL
and AM we e shown o be he a ge ed o he cell wall h ough di e en mechanisms.
Ou esul s suppo he hypo hesis o ATL being sec e ed in o he cul u e medium and
hen e a ge ed o he cell wall. The e ec o DNA-GL binding in ly ic ac i i y o GL
was ound o be, as al eady obse ed o bio ilm o ma ion, s ain dependen . The
s uc u al cha ac e iza ion o GL-DNA associa ion may cla i y he mechanisms behind
hese obse a ions.
The hyd olysis o pep idoglycan by hyd olases esul s in a s ong bac e icidal e ec
which makes his g oup o enzymes an al e na i e an ibac e ial weapon, o example a

54
possible accina ion scheme wi h ATL-AM has been s udied and his accine was
shown o induce Th1 and Th2 immune esponse (Nai e al, 2015).
Due o he impo ance al eady demons a ed o he ATL in S aphylococcus au eus
and he possibili y o being a po en ial weapon agains his mic oo ganism, i is
impo an o s udy and cha ac e ize his p o ein.
55
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65
Annex
1. Impac o he GL-DNA associa ion in bio ilm o ma ion. (G ilo e al, 2014)
I has been shown ha a l mu an s do no o m bio ilm p esumably due o he ole o
he au olysin in genomic DNA elease and also in assis ing he a achmen o cells o he
su ace du ing he i s phases o bio ilm o ma ion.
1.1. In ol emen o ATL in bio ilm o ma ion o s ain MW2
These p elimina y s udies es ed he independen and combined oles o AM and GL
domains in bio ilm o ma ion, using a S. au eus s ain, MW2, ha o m bio ilm.
In o de o unde s and bio ilm o ming capaci y o he a l mu an , an assay wi h
di e en ecombinan ATL p o eins was pe o med. (Figu e 18)
Figu e 18 – Bio ilm o ma ion o s ain MW2, i s isogenic a l mu an MW2L9, and MW2L9 wi h
added ecombinan p o eins. A l e e s o he AMR1-3GL ecombinan p o ein, N-His-GL e e s o
R3GL, and AM e e s o AMR1-2. Th ee expe imen s we e pe o med, a leas in duplica es.
P o ein AMR1-2 was able o complemen he bio ilm o ming capaci y o he
mu an , owing o he inc ease o ex acellula DNA p esen in he media due o he ly ic
ac i i y o he added ecombinan p o ein. When AMR1-2 and R3GL p o eins we e added
oge he , as well he comple e p o ein AMR1-3GL, he bio ilm o ming capaci y was
also complemen ed, al hough only o highe p o ein amoun s. Howe e , when R3GL
66
was added alone no complemen a ion o bio ilm o ma ion occu ed. In some cases, he
bio ilm o ming capaci y o he mu an wi h R3GL was lowe han he mu an alone, and
no concen a ion dependence was seen.
1.2. In ol emen o ATL in bio ilm o ma ion in di e en s ains.
To analyze he impo ance o ATL in bio ilm o ma ion s ains we e chosen due o
hei high bio ilm o ming capaci y and a l mu an s we e ob ain (by ansduc ion o he
RUSAL9 ansposi ion mu an wi h phage 80α). The esul ing isogenic a l mu an s we e
es ed by Wes e n Blo . Di e en pa e ns and amoun s o GL p o ein we e ound
be ween he di e en s ains as shown a Figu e 19.
Figu e 19 – De ec ion o GL p o ein pe o med by Wes e n Blo using an i-GL aised an ibody
agains cellula ex ac s o di e en s ains and hei espec i e isogenic a l mu an s.
When s a ic bio ilm assays we e pe o med on hese s ains and hei a l mu an s,
only MW2L9 and WISL9 showed a dec ease in bio ilm o ma ion when compa ed o
he espec i e pa en al s ains (Figu e 20).
67
Figu e 20 – Bio ilm o ma ion by s ains MW2, HDE 288, WIS and NCTC 8325 and hei
espec i e isogenic a l mu an s. Values shown a e a e age alues e e ing o he pe cen age o bio ilm
when compa ed o MW2 s ain. Two expe imen s we e pe o med, a leas in iplica es.
These esul s we e unexpec ed; he lack o a l is expec ed o esul in a dec ease in
bio ilm o ma ion in S. au eus. Howe e , his suppo s he impo ance o he gene ic
backg ound on bio ilm o ma ion, and he di e en pa e ns ound in he GL Wes e n
Blo could also jus i y he di e en impac ha he lack o ATL has in bio ilm
o ma ion, which is s ain-dependen .
Also, he s ains wi hou a l has a diminished bio ilm (MW2 and WIS) and simila
Wes e n pa e ns, wi h many smalle bands pe haps co esponding o ex ensi e
p ocessing o GL p o ein.
1.3. In ol emen o GL in bio ilm o ma ion in s ain WIS
To s udy he bio ilm o ming capaci y o s ain WIS and i s a l mu an , di e en ATL
ecombinan p o eins in di e en concen a ions we e added. The bio ilm o ming
capaci y o s ain WIS was p og essi ely es o ed, achie ing ull complemen a ion a a
p o ein concen a ion o 10 µg. The addi ion o AMR1-2 and R3GL ecombinan
p o eins oge he showed a highe bio ilm o ming capaci y when compa ed o he
addi ion o only one o hese p o eins, sugges ing ha he wo p o eins may wo k
syne gis ically in p omo ing bio ilm o ma ion. (Figu e 21)
O he a ia ions we e obse ed be ween WIS and MW2, showing ha he di e en
gene ic backg ound may be in ol ed.
68
Figu e 21 - Bio ilm o ma ion o S. au eus s ain WIS and i s isogenic a l mu an WISL9 wi h
added ecombinan p o eins. Bio ilms we e g own wi h di e en amoun s o ex acellula ly added
ecombinan GL domain (R3GL), AM domain (AMR1-2), and en i e A l ecombinan p o ein lacking he
SP and PP sequences (AMR1-3GL).
1.3.1. ATL-DNA associa ion
The addi ion o ex acellula DNA was able o comple e he pa ial complemen a ion
achie ed when added a less p o ein concen a ion (5 µg), howe e , ull es o a ion was
no achie ed un il AMR1-2 was added. This is consis en wi h a c i ical ole o
ex acellula DNA in he GL-dependen bio ilm o ma ion p ocess. (Figu e 22)
Figu e 22 - Bio ilm o ma ion o S. au eus s ain WIS and i s isogenic a l mu an WISL9 wi h
added ecombinan p o eins. Bio ilms we e g own wi h 1 µg o he di e en ecombinan p o eins and
complemen ed wi h 100 µg o low molecula weigh salmon spe m DNA o 50 µg/ml o DNase I.
Addi ion o complemen ing amoun s o any o he ecombinan p o eins oge he
wi h DNase I o he mu an esul ed in an almos comple e block o bio ilm o ma ion
69
(Figu e 23). This e ec was less dis inc when only AMR1-2 was added, suppo ing he
sugges ion ha he GL domain o ATL plays a mo e impo an ole han he AM
domain o his p o ein in he DNA-dependen o ma ion o bio ilm.
Figu e 23 - Bio ilm o ma ion o S. au eus s ain WIS and i s isogenic a l mu an WISL9 wi h
added ecombinan p o eins. Bio ilms we e g own wi h 10 µg o he di e en ecombinan p o eins,
and addi ion o DNase I a 50 µg/ml dis up ed bio ilm o ma ion. The amoun o bio ilm p oduced was
calcula ed as a pe cen age o he bio ilm p oduced by he pa en al WIS s ain.

70
2. Lysis assay
The esul s ob ained in he lysis assay o pep idoglycan a e shown in he nex
g aphics (Figu e 24), he e a e no ele an di e ences in he ly ic ac i i y o GL when
DNA is added.
0
0,5
1
1,5
2
050 100 150
OD600nm
Time (minu es)
COL GL(5ng/µl)
GL + DNA (0,05mg/mL) GL + DNA (0,5mg/mL)
A
0
1
2
050 100 150
OD600nm
Time (minu es)
WIS GL(5ng/µl)
GL + DNA (0,05mg/mL) GL+ DNA (0,5mg/mL)
B
0
0,5
1
1,5
2
050 100 150
OD600nm
Time (minu es)
HDE 288 GL(5ng/µl)
GL + DNA (0,5mg/mL) GL + DNA (0,05mg/mL)
C
0
0,5
1
1,5
2
050 100 150
OD600nm
Time (minu es)
UAMS-1 GL(5ng/µl)
GL + DNA (0,05mg/mL) GL + DNA (0,5mg/mL)
D
0
0,5
1
1,5
2
050 100 150
OD600nm
Time (minu es)
JE2 GL(5ng/µl)
GL + DNA (0,05mg/mL) GL + DNA (0,5mg/mL)
E
71
0
0,5
1
1,5
2
050 100 150
OD600nm
Time (minu es)
MW2 GL(5ng/µl)
GL + DNA (0,05mg/mL) GL + DNA (0,5mg/mL)
Figu e 24 – Ly ic ac i i y o GL p o ein in pep idoglycan and DNA-GL associa ion in di e en
s ains. (A) COL; (B) WIS; (C) HDE288; (D) UAMS-1; (E) JE2 (F) NCTC8325; (G) MW2. Pu ple:
pep idoglycan wi hou GL; Blue: pep idoglycan wi h GL 5ng/µl; Red: pep idoglycan wi h GL 5ng/µl and
DNA(0,05mg/mL); G een: pep idoglycan wi h GL 5ng/µl and DNA(0,5mg/mL).
The esul s ob ained in he lysis assay o cell wall a e shown in he nex g aphics
(Figu e 25), he e a e no ele an di e ences in he ly ic ac i i y o GL when DNA is
added.
A
0
1
2
050 100 150
OD600nm
Time (minu es)
GL(5ng/µl) GL + DNA (0,05mg/mL)
GL + DNA (0,5mg/mL) COL
B
0
1
2
050 100 150
OD600nm
Time (minu es)
WIS GL(5ng/µl)
GL + DNA (0,05mg/mL) GL + DNA (0,5mg/mL)
0
0,5
1
1,5
2
050 100 150
OD600nm
Time (minu es)
NCTC 8325 GL(5ng/µl)
GL + DNA (0,05mg/mL) GL + DNA (0,5mg/mL)
F
G
72
Figu e 25- Ly ic ac i i y o GL p o ein in cell wall and DNA-GL associa ion in di e en s ains. (A)
COL; (B) WIS; (C) HDE288; (D) UAMS-1; (E) JE2 (F) NCTC8325; (G) MW2. Pu ple: pep idoglycan
wi hou GL; Blue: pep idoglycan wi h GL 5ng/µl; Red: pep idoglycan wi h GL 5ng/µl and
DNA(0,05mg/mL); G een: pep idoglycan wi h GL 5ng/µl and DNA(0,5mg/mL).
C
0
1
2
050 100 150
OD600nm
Time (minu es)
HDE 288 GL(5ng/µl)
GL + DNA (0,05mg/mL) GL + DNA (0,5mg/mL)
D
0
1
2
050 100 150
OD600nm
Time (minu es)
GL(5ng/µl) GL + DNA (0,05mg/mL)
GL + DNA (0,5mg/mL) UAMS-1
E
0
0,5
1
1,5
2
050 100 150
OD600nm
Time(minu es)
JE2 GL(5ng/µl)
GL + DNA (0,05mg/mL) GL + DNA (0,5mg/mL)
F
0
0,5
1
1,5
2
050 100 150
OD600nm
Time (minu es)
NCTC 8325 GL(5ng/µl)
GL + DNA (0,05mg/mL) GL + DNA (0,5mg/mL)
G
0
1
2
050 100 150
OD600nm
Time (minu es)
mw2 GL(5ng/µl)
GL + DNA(0,05mg/mL) GL + DNA (0,5mg/mL)
73
The esul s ob ained in he hea inac i a ed cells ly ic assay a e shown in he nex
g aphics (Figu e 26).
0
0,5
1
1,5
2
050 100 150
OD600nm
Time (minu es)
GL(5ng/µ)l GL + DNA (0,05mg/mL)
GL + DNA (0,5mg/mL) COL
A
0
0,5
1
1,5
2
050 100 150
OD600nm
Time (minu es)
GL(5ng/µl) GL + DNA (0,05mg/mL)
GL + DNA (0,5mg/mL) WIS
B
0
0,5
1
1,5
2
050 100 150
OD600nm
Time (minu es)
GL(5ng/µl) GL+DNA (0,05mg/mL)
GL + DNA (0,5mg/mL) HDE 288
C
0
0,5
1
1,5
050 100 150
OD600nm
Time (minu es)
GL(5ng/µl) GL+ DNA (0,05mg/mL)
GL + DNA (0,5mg/mL) UAMS-1
D
0
0,5
1
1,5
2
050 100 150
OD600nm
Time (minu es)
GL(5ng/µl) GL+ DNA(0,05mg/mL)
GL + DNA (0,5mg/mL) JE2
E
0
0,5
1
1,5
2
050 100 150
OD600nm
Time (minu es)
GL(5ng/µl) GL + DNA (0,05mg/mL)
GL + DNA (0,5 mg/mL) NCTC 8325
F