Characterization of the major autolysin in Staphylococcus aureus

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 Re e ences Ab aham, E. P., and E. 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S uc u al Basis o Cell Wall Clea age by a S aphylococcal Au olysin. PLoS Pa hogens, 6:e1. 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