HOI N G H ! KHOA HOC C O N G N G H E SINH HOC T O A N Q U O C 2013
K H A O S A T sy HIEN DIEN C O A C A C GEN MA H 6 A PROTEIN DOC T 6 G A N TREN T H A N H T ^ B A O STREPTOCOCCUS SUIS P H A N LAP TLf BENH N H A N VIEM M A N G N A O M O d VIET NAM
Phan Nha Uyen, T r i n Thj Bich Chieu, Nguyen Van Vinh Chau, Ngo Thj Hoa*
Dcm V) Nghien cCni LSm siing Dai hoc Oxford T 6 M TAT
Sirepiococcus suis {S. suis) Ik vi khuan co hpi tren heo co kha nang gay benh nguy hiem tren heo va nguoi. Nhimg nguoi co tiep xuc vdi heo ho^c cdc san phim tu heo benh co nguy co bi nhiem S. suis cao. Do vay nhiem S. suis dugc xem la nguy co benh ngh^ nghiep cho nhihig Dgu£ri tiep xiic vdi heo, san pham heo va heo l)?nh Viem mang nao raii (VMNM), nhilm trung huylt la cac trieu chiing thudng gap tren ngucri b?nh do nhilm 5. suis. Nham phdt triin phuang phap tam soat huySt flianh hgc tr6n nh6m d6i tirgng nguy co chung toi tiin , hanh tim hilu chlic nang ctia cac nhom protein be mat te bao 5'. suisvakhao sat su hi^n dien ciia chiing trong 153 chung .S'. ^uuphan lap tit b§nh nhSn VMNM. Ket qud cho thay 8 protein gan m ^ g da dirge nhan dien nho vao cac trinh ti,r dSu hi?u d^c tnmg cua protein b€ mat ti bdo cua vi khuan gram duong (YSIRK va LPXTG). Kk qud phan tich trinh tu loan bg bg gene cvia cac chung S. suis cho thiy c6 sp da dgng trong kieu gen ctia cac protein nay tren cdc chiing phdn lap nguoi bfiih, heo benh va heo. Do vgy, khao sdt cdc gen nay se giiip hieu dugc vai tid ciia cdc chung tiong dgc l\rc ciia chiing vd khd ndng ihig dung trong cdm soat huyit thanh hgc.
Tir khda: dgc lyc, LPXTG, protem bi mat, protein gdn tfidnh. Streptococcus suis, viem mdng nao mii.
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Slrsptococcus suis (S. suis) Id vi khuin gram du-omg cd kha n5ng gSy b?nh nguy hiem tren ngu^i vk b^n heo. Ct heo, S. suis la vi khuin ca h$i gSy c5c b^nh nhir viem mang n§o mu (VMNM), viem khdp, viem mSng n$i t3m thit. nhilm trdng mau, '' viem phdi. s l y thai v.v. gay thi^t hgi kh6ng nho cho ngdnh chdn nu6i heo hen the gi6i. Nguy ca lay nhi§m cho ngu^i duwc
xdc dinh thong qua tilp xOc tn/c tilp viS-i heo v^ san phim cua heo hay nhilm S. suis chira dutyc nau chin (Nghia et al., a 2011). B$nh ph6 bien nhat tr§n ngu'6'i do S. su/sldVMNM, nhilm trung huylt vilm mang tim trong (Gottschalkefa/.. 2010).
Trong 35 kilu huyet thanh cOa S. suis, S. suis huylt thanh 2 (SS2) du^c phdn l|p nhilu nhit ti> benh nhan VMNM. Cho den
^, nay, gin 800 ca b^nh nhilm S. suis Ok Oieiyc bao cao trSn t h i gi6i, tuy hiu h i t cdc ca b$nh xdy ra rdi rac, tgi Trung Qudc, SS2 dd di^o^ bdo cdo Id tdc nhdn gay 2 Otyl djch b#nh tr§n ngireri vao ndm 1998 vd 2005. Dgi djch b§nh nam 2005 da Idm 215 ngu^i nhdp vi^n trong v6ng mgt thdng voi tl l§ 10 vong id 18% do s6c suy da co' quan. Tuy nhien, Tnii/c ddy, nguy cc Idy nhilm S. suis a ngi/o'i, nhit Id i/ nh6m nghe nghiep cd tilp xtic vdi heo chira du^c quan tdm tim hiiu diing mi,rc do chira c6 phirang phdp chin dodn hi^u qud. VI v$y, phu'ang phap chin doan nhanh va vaccine phong b^nh do S suis c i n du'oc phdt tnln de giup tam sodt vd ngdn ngi>a nhilm S. suis a nhdm nguy ca cao.
Cdc protein gan tren thanh t l bdo hi^n du'Q'c xem Id d l i tifong chinh de nghien ciru ve ddc tinh va ddp irng miln djch cua vi khuin len v§t chii do chung cd nhilu kha ndng tilp xijc ven te bdo vgt chu de xdm nhgp cung nhir kich thich cac phan irng miln djch (Gottschalk ef al., 2001). Do vay ddy se Id d l i tirg-ng dtrpc d i u tu' nghifin ci>u d l Ccng dyng vdo cdc ky thu^t t i m sodt vd chan dodn khdng huylt thanh va sdn xuat vaccine t h i h^ md-i. Vai si,r phdt triin cCia cdc ky thuat phan hr hign dgi nhu" giai ma trinh tir DNA b$ gene, ky thu§t proteomics, ky thugt gidi ma d u tnic tinh t h i protein.v.v., ngdy cdng cd nhieu protein ddc IM'C b l m$t te bdo diro'c phdt hi$n vd md ta chirc nang. Cdc protein b l mgt t l bdo ndy cd dac diem nhan dien chung la dogn trinh ty LPXTG d d i u carboxyl (C) trong trinh ty protein. Vj tri b l mgt ndy cung Id vj tri diroc enzyme sortase i!3(SrtA) nh^n dign vd gdn k i t vao thdnh t l bdo, hinh thanh Idp protein be mgt, thOc d i y qud trinh xdm nhdp va gdy benh cua Atvi khuin. Cdc nhdm nghien ciru d$c l|p tren the gi6i da cdng bo han 10 protein gan tren thanh t l bdo S. suis cd d$c lyc nha ipsurface protein 1 (SAO) (Li ef al., 2006), muramidase released protein (MRP) , opacity factor (OFS) (Baums ef al., 2006).
Wihdm protein chira lactor H-receptor cd khd nang ching Igi s y tan cdng cua dgi thyc bdo (Vaillancourt ef al., 2013) , pStreptococus suis Dnase (SsnA) (Fontaine ef al., 2004),v.v. Cac nghien ciru cOng cho thiy co sy da dgng ve kilu gene cua ir'cdc ylu t l ddc lyc trong nhiing chung 5. 5i;/s gay benh a heo, ngircn ho|c khdng gdy b^nh tCr dd du'a den gia thuylt ve vai isHrd cCia cdc y l u td ddc lyc b l m$t te bdo len dpc tinh ciia nhiing chung S. suis khdc nhau. Trong nghien ciru nay, chiing tdi ,fise khdo sdt sy hi$n di$n cua cdc y l u t l ddc lyc b l mat n§u trSn trong thu' vien chung S. suis chiing tdi da phan Igp tir benh
#ihan VMNM do S. suis a Vigt Nam, vd so sdnh vdi trinh ty cua cdc gen nay thudc cdc chiing dirge phdn l$p tt> heo b#nh.
ilib^nh nhdn d Trung Quoc vd Vi§t Nam (PI/7, SC84. BM467) da du-yc gidi ma trinh ty DNA bd gene vd liru trO" tren ngan aihdng gene t h i gidi (Genbank). K i t qua cOa nghien ciru nay se gdp phan danh gid m^l cdch hg thong khd nang ang dyng
cCia cdc y l u td ddc lyc b l mgt t l bao ndy trong chin dodn vd phdng ngira benh Idy nhilm do S. suis.
NGUVeN U£U VA PHU'O'NG P H A P
Phdn l$p DNA b$ gene cua cdc chung S. suis tir bgnh nhan VMNM
Trong tdng sd 153 chOng, 128 chung S. suis dayc phdn Igp tir miu mdu cua benh nhdn VMNM tai B#nh vien Bgnh Nhiet Dd'i TpHCM vd 25 chung dirp^ phdn l§p tir b#nh nhdn d- Hue tir ndm 1997 den 2010. S. suis trong mdu benh pham diroc ang smh trong mdi trwdyng Todd Hewitt Brcith (BD. #249240) va dircrc phdn lap tren mdi trud-ng thach mau BA (Oxoid,
¥CM0271). DNA by gene dgng mgch vdng cua S. suis daoc tdch chilt si> dyng kit va theo hadng din cua nha san xuat promega, #A1125). Trinh ty DNA bp gen dayc gidi ma va kit qua daoc gi>i ve tir vi^n Sanger, Anh Qudc.
HOI NGH! KHOA HQC C 6 N G N G H ^ SINH HQC TOA^^ CUOtfTamTf-
Phdn tfch k i t q u i g i i i trtnh t y DNA cOa 153 chOng S. suis
Thu$t todn tbiastn cua phan m i m Bioedit diryc sa dyng d l dich ma cho 8 protein b l mgt ^ong 153 t;;"^ t ^ ^ cSig cy interpro cua trang web EBI doge sir dyng d l phdn tich cdc doan tin h,0u va phdng dodn chirc ndng cua cdc p b l m ^ L
Dinh danh protein b l mgt t l bdo vi Ithuan S. suis
PI/7 8084 doyc sir dyng Idm trinh h^ DNA bp gen tidu bilu cho S suis tCr heo yd tCr ngirdi BM407 cOng diryc sir dtfngbS dg. d i j n ^ o Tsuis pKdn lap tirngirdri a Viet Nam do mang nhilu d#c diim khac bi&t trong c^" ^ ^ JDNA bO g e n ^ protein b l mat diryc phdn lap tO ca 3 bd gen tieu chuin dya hdn sy h'g"d,en cua dogn gan thdnh LPXTG. Cdc profen dayc so sanh bdng phirong phdp gidng cOt d l xdc djnh cdc protein b l mat tidu bilu cho cd 3 bp gen chuSn.
KfiT QUA VA T H A O LUAN
Protein gan thdnh t l bdo 6- Sbvptococcus suis: doc 1y« va i>ng dyng
hflnh 1. M6 ph6ng 4 birOc trong cc chl sin xuit protein gin thtoh te bdo cua vl khuin gram dirarng. (BirOc 1) protein tli m$t dirgt; vSn chuyin tCr n^ bdo ra ngodi qua con duimg v^n chuyin Soc, dogn tin hi$u Sig di/yc cit bd. (Birdc 2) Sortase nhjn di$n vd cit rdri protdn be mjt khfll mdng t l bdo tgi mofif LPXTG vd gidi phdng diu carboxyl ty do. {Bir6c 3) protein tmng gian Lipkl 11 se lien kit vin protein b l m$t tgi ddu caiboxy ty do cua protein ndy. {Birdc 4) Phtrc hyp ndy se tham gta vdo bd mdy ling hyp thArih t l bdo cua vi khudn d l gin ch$t pmtein b l m$t vdo thdnh t l bdo.
<(m mm^ M^mmimmm-. J l'T??gd
0 vi khuin, protein b l m$t Id cdng cy chfnh giiip t l bao vi khuin thich nghi vai cdc thay ddi cua mdi froimg vd mang Unli d§c thii cho mdi chiing bdi. Dya trdn cau biic trinh t y amino acW cua 8 protein mang motif gdn mdng dap^ phdn l$p Kf i * Chung S. suis tidu chuin phin ldp h> heo vd ngu-di (PI/7. SC84, BM407), cdc protein ndy duyc phdn thdnh 2 nhdm: i ^ chira cd hai dogn tin higu YSIRK va LPXTG vd nhdm chi chi>a mot dogn tin hiiu LPXTG (Bang 1). Mpt sd protein ffi CT d l c$p qua cdc nghien ciru tnrdrc ddy. Ddng chu y nhat Id MRP (m^t trong 3 protein ddc lyc dayc sa dyng phd biln d i J logi Chung S. suis), nhdm DNase vd nucleotidase (SsnA) vd nhdm peptidase (SspA). Nhung protein ndy cd d$c diim am Id Cd khd ndng sinh miln djch tdt vd cd d$c lyc (Zhang ef ai., 2008; Luo e( al., 2009; Bonifait ef al., 2010). Ngodi ra, c h O d da nhSn di^n dayc thdm 10 protein b l mgt mdi hong bO gen cOa chiing BM407 (chung tip Vigt Nam).
Bdng 1. Protein gin thdnh te bao cua S. suis Kfch thvd'c
(amino ackl) Chirc nSng dy dodn YSIRK
(xSxRKxxxGxxS) LPXTG (xPxTG) MRP
SspA SSUBM407 1843 SAO-L OFS SsnA SSUBM407_0574, SSUBM407_1432
1260/1314 1585 1692 670
^ S 1041 607 674
Muramidase Peptidase Peptidase Isopeptide linker von Willebrand factor Dnase Peptidase Nucleotidase
FSIRKYHFGAAS FSLRKYKIGTVS FSIKKLSVGVAS
LPNTG LPNTG LPQTG LPVTG LPSTG LPKTG LPSTG LPNTG
So sdnh trinh ty amino acid cua cdc protein ndy trong tha vidn diCing cua chung tdi (153 chung) cho thiy cdc P " ^ dayc phdn bd rgng rdi chimg td ddy Id nhirng protein doyc bdo tdn vd ddng vai trd quan trpng trong sy tdn tgi cda S S vi khuan gram doang ndi chung hay S. suis ndi rieng, protein b l mgt diryc gan vd'i thdnh t l bdo thdng qua lidn kit ( » tri giO'a gdc carboxyl cua protein b l m|t vd c i u peptidoglycan cua thdnh t l bdo. Qua trinh ndy diryc xiic tdc bdi wM (SrtA). D l cd t h i doyc sortase nhgn di|n. cdc protein b l m§t mang motif a§c tnrng Id LPXTG fr duoi carboxyl (C) ciili
HOI NGHI KHOA HQC C O N G N G H ^ SINH HQC T O A N Q U O C 2013
ty amino acid (Hlnh 1).
Hlnh 2. Mo phong d u tnic ciJa cdc protein gan thdnh t l bdo. cua S suis. (a) N h ^ protein mang cd hai doan gin thdnh (d vudng tim) vd dogn tin hi$u vdn chuyin xuydn mdng (d vudng vdng). (b) Nhdm pmtein chi mang doan gin thdnh (diu cartioxyl).
Cf diu N' Id cdc dogn tin hi#u hd try cho qua trinh v$n chuyin protein tir t l bdo chit ra ngodi t l bdo. Cdc kdnh v$n chuyin khdc nhau sd nh$n di|n cdc dogn tin hi$u khdc nhau. Vf dy nha kenh Sec se nh$n dign va v$n chuyin protein mang dogn tin higu YSIRK (Bae ef al., 2003). trong khi kdnh Tat se hd try v§n chuyin cho tin hi#u arginine kdp (RR). Cd 6 trong cdc protein b l m$t ciia S. suia mang cd hai dogn tin
Bdng 2. Sy hifn difn cda 8 protein gan thdnh ta^n 153 chiing S.
suis phdn l^p ti> ngwM bfnh
STT Protein
Tongc$ng(n=153) S6loyng
MRP OFS SAO-L SspA SsnA SSUBM407_0574 SSUBM407_1843 SSUBM407_1432 hi|u ndy trong d u tnic bdc 1 cOa protein (Hlnh 2).
Trang 8 protein b l m$l cfla 5. suis doyc tim thay, phin Id'n Id cdc enzyme peptidase (MRP, SspA, SSUBM407_1843, SSUBM407_0574). Trong nhilu logi khuin b|nh, peptidase, enzyme xiic ldc phdn irng OiGy phdn c i u peptide da dayc biit din Id cdc yeu td ddc lyc quan trgng. Cf S. suis, ddc lyc cua SspA da dayc chii-ng minh qua vai trd cua protein ndy trong vigc gidp S. suis thodt khdi qud itrlnh thyc bdo, mdt qud trinh trong hd thdng miln Idjch trdn chudt vd trgn ngad'i (Bonifait etal., 2010).
ID(lc lyc cOa SspA cdn doyc chirng minh qua khd mdng tdc dyng Idn qud trinh sdn sinh cytokine cua jdgi thyc bdo, gdy ra Id phdn i>ng vidm trdn nao vd din din gdy VMNM trdn ngud'i va heo. Nhirng thi nghidm taong t y trdn cdc ddi tayng peptidase cdn Igi se gidp hieu rd han vai trd ciia nhdm protein b l m|l ndy.
Mdt nhdm protein dang chii"} khdc cung hign dign tren be mgt t l bao S. suis Id cdc enzyme nuclease vd nucleotidase (SsnA, SSUBM407_1432). Cf cac khuin bdnh gram dirang nhir Streptococcus sp. va Staphylococcus sp., hogt dOng xuc tac phdn huy DNA ciia cdc Dnase ndy d l sir dung ngudn oligonucleotide ciia v$t chu lidn quan d i n ddc tinh cOa b^nh khuan. Cf S.
suis, SsnA Id mpt nuclease dayc tilt ra ngodi t l bdo vd cd hogt tinh thdy phdn DNA ngogi bdo vd cSn trd* sy phdn bdo cua cdc t l bdo Idn c^n (Fontaine ef ai. 2004). Cf myt nghiin cipu khdc, SsnA diryc chirng minh Id cd kha nang sinh m i ^ dich cao nhit hen chudt so vdi cdc protein b l m§t vd cac protein ngogi bdo khac, do dd SsnA cd tilm nang d l sir dyng ldm khdng nguydn trong phirong phdp t i m sodt huylt thanh hpc ma chiing tdi dang phat triin.
Nhijng nd lyc tRrdc ddy si> dyng nguyin bdo d l gdy ddp irng miln djch tren heo vd chupt khdng cho dp dgc hieu cao do anh hadng cOa phdn irng miln djch khdng d§c higu vdi cdc khdng nguyen khdc. Do dd gan day, vi^c SIP dyng protein tdng hyp Idm khdng nguyin dayc ddnh gid se khac phyc dtryc hgn che nay vd cho dd nhgy cung nha dd dgc hieu cao han trong phdp thir khdng huylt thanh cho S. suis.
Sy da dgng kieu gene ma hoa cho protein b l mgt t l bdo S. suis tir ngird'i bgnh VMNIVI do S. suik cr Viet Nam Cdc protein b l mgt d l cgp trong nghien ciru nay hign dien tren hau h i t (69% - 100%) d' tat ca cdc chung S. suis tir bdnh nhdn VMNM (Bang 2). SsnA, SSUBM407 0574 vd SSUBM407J432 la cac protein hign dign h^n 100% chOng cho thiy day
^'d cdc nhdm cdc protein dayc bdo tdn nhJt d' S. suis SS2 phdn l§p tir ngad'i cung nha SS9 phdn l§p tir heo (kit qua tu" mpt ''nghidn ciru khdc). Protein b l m§t SAO-L chl dayc tim thiy trong 69% ting so chung do ngodi SAO-L cdc chiing S. suis cdn
^;d t h i mang hai biln t h i khdc Id SAO-M vd SAO-S. Cdc protein SAO diu mang dogn be m|it LPVTG vd dogn amino acid '^irc ndng nhang chi khdc nhau a cdc dogn i$p Igi. Taong ty vdl protein SAO, MRP cung cd hon mdt phidn bdn nen ty lg brotein hi|n didn trin S. suis se cao han con sd 91%. Diem can lau •) Id t i t ca cdc chiing deu mang trinh t y DNA ma hda
HOI NGHI KHOA HOC C O N G N G H ^ SINH HQC TO«w r
cho 8 protein b l mgt tuy nhien cdc dpt biln xdy ra trong qua trinh xdm nhidm va nhdn ban b#nh khuin (ldi s ^ j t j chromosome, chen dogn do vims, v.v.) da gdy doan mgch do dd cac protein taong i>ng se khong dayc sdn xudt troif^^
sd chiing. ^ KfTLUAN
Ddy Id nghidn ciru diu tien tong hop vd md t l sy hidn dign ciia cac protein b l mgt t l bao S. suis phdn Up tir b§nh nti|
VMNM * Vidt Nam Kit qud cho thay ddy Id nhdm protein doyc phdn bd rpng rai va dac higu cho 3. suis. VI v$y cdc prot^
nay Id ddi tiryng phii hyp tdm protein chf thi d l t i m sodt vd chin dodn Ssuis. Ngodi ra, nghidn cii-u cung nh#n thlya nhirng sy da dgng kilu gen a cdc protein nay tr&n cdc chung phdn ldp ngad'i bdnh so vd'i cdc diOng tidu bilu tip ngirdliji heo. Do v#y, khdo sat cdc gen nay se giiip hiiu dayc vai trd cOa chiing trong khd ndng xdm nhilm va gdy bdnh cda chi^
va kha nang ung dyng bong t i m sodt huylt thanh hgc.
L M cdm o n : Chung tdiancamansifhStrpcHa Vien Sanger. Vaang Quoc Anh. gidi trinh t^ cac chung S. suis. Di tai dufK hd Irp idi chbik mQi phan tir tS chuc WeUcome Trust, Vuong QuSc Anh va cua Quy Phai Tnin Khoa hgc va Cong Nghf Quoc Gia cho TS Ngo Thi Hoa (N/
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Bonifait L, de la Cmz Dominguez-Punaro M, Vaillancourt K, Bart C. Slater J , Frenette M, Gottschalk M, Grenier D (2010) The cell envelcfc subtilisin-like pmleinase ts a virulence determinant for Streptococcus suis. BMC Microbiol 10:42
Fontaine MC, Perez-Casal J , WiHson PJ (2004) Investigation of a novel DNase of Sfnspfococcus sujs serotype 2. Infect tmmun 72 (2):774-ei Gottschalk M. Segura M, Chariand N, Latonde M, Lacouture S (2001) Interactions between Straptococcus suis serotype 2 and phagocjft;
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Gottschalk M. Xu J , Calzas C, Segura M (2010) Streptococcus suis: a new emerging or an old neglected zoonotic pathogen? FiM M ; C T O 6 / O / 5 ( 3 ) 3 7 1 - 9 1 .
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Zhang A. Xie C, Ctien H. J n M (2008) Identification of immunogenic cell wall-associated proteins of Streptococcus suis serotjpf
Proteomics 8 (17):3506-15 >
CHARACTERISATION OF CELL WALL-ANCHORED VIRULENCE FACTORS IN STREPTOCOCCUS SUIS ISOLATED FROM VIETNAMESE MENINGITIS PATIEh
Phan Nha Uyen, Tran Thi Bich Chieu, Nguyen Van Vinh Chau and Ngo Thi Hoa*
Oxidrd University Clinical Research Unit, 764 Vo Van Kiet St, Ward 1, District 5, Ho Chi Minh City, Viet Nam
SUMMARY
Streptococcus suis (S. suis) is an opportunistic pathogen of swine that can cause acute symtoms in pigs and humans. People aaso widi direct exposure to infected pigs or undercooked infected porcine products are more subjected to S. suis infection. Thus, S. m been considered an occupational threat for this high nsk group. Of the acute symtoms in S suis infected human, meningm septicemia arc most commonly seen. In order to develop a surveillance serological method for the high risk group, we have chj^"
celt wall-anchored proteins in 153 virulent strains isolated from meningitis patients in Viet Nam. As a result, we have identifi^ftn harbonng conserved transmembrane secretion and cell wall-anchored motife which are YSIRK. (in the N-termini) and L P X T M | tcmimi), respectively. Careful analyses ofthe whole genome sequences of these strains and those of avimlent shains f r o m l 3 have revealed diffenl variants in the genotypes of these cell wall-anchored proteins. Thus, fiirther characterization of these proieiBi provide more insights on their roles in the virulence ofthe pathogen and their potential applicahons in serological techniques.
Kenvords: bacterial meningitis, cell wall-anchored protein, LPXTG, Streptococcus suis, surface protein, virulence 'Corresponding author. Tel. +84 8 39241761; Email: [email protected]
