2型豬鏈球菌中國(guó)強(qiáng)毒株及其covR基因突變株的蛋白質(zhì)組學(xué)研究

倪　華，張　劍，鄭　峰，胡　丹，李先富，王長(zhǎng)軍，潘秀珍

?

2型豬鏈球菌中國(guó)強(qiáng)毒株及其covR基因突變株的蛋白質(zhì)組學(xué)研究

倪華1,2，張劍1,2，鄭峰2，胡丹2，李先富2，王長(zhǎng)軍2，潘秀珍1,2

1.南京師范大學(xué)生命科學(xué)學(xué)院，南京210023；2.南京軍區(qū)軍事醫(yī)學(xué)研究所，南京210002

摘要：目的通過比較2型豬鏈球菌中國(guó)強(qiáng)毒株05ZYh13及其covR基因突變株△covR蛋白表達(dá)譜差異，質(zhì)譜鑒定差異表達(dá)蛋白，分析CovR在蛋白表達(dá)調(diào)控中的作用。方法首先將05ZYh13和△covR在THB培養(yǎng)基中培養(yǎng)至對(duì)數(shù)期，然后裂解細(xì)菌制備蛋白樣品。第一向等電聚焦電泳在ph1～10的IPG膠條上完成后進(jìn)行SDS-PAGE電泳，電泳膠經(jīng)掃描分析后選取蛋白點(diǎn)進(jìn)行質(zhì)譜鑒定。結(jié)果05ZYh13和△covR全菌蛋白裂解液經(jīng)二維電泳分別得到559和491個(gè)蛋白點(diǎn)，經(jīng)比對(duì)發(fā)現(xiàn)兩菌株蛋白表達(dá)量差異3倍以上蛋白點(diǎn)40個(gè)，經(jīng)質(zhì)譜鑒定出15個(gè)蛋白，主要涉及細(xì)胞代謝的酶類如谷氨酸脫氫酶、腺苷酸激酶、PTS系統(tǒng)成分等，以及分子伴侶蛋白如GroEL和Dnak等；電泳分離得到△covR特異蛋白點(diǎn)124個(gè)，質(zhì)譜鑒定出15個(gè)，主要為參與細(xì)胞糖代謝過程中的酶，如磷酸甘油酸激酶、甘油醛-3-磷酸脫氫酶、丙酮酸激酶等；質(zhì)譜鑒定05ZYh13特異表達(dá)蛋白5個(gè)。結(jié)論鑒定05ZYh13和△covR差異表達(dá)蛋白35個(gè)，部分蛋白涉及細(xì)菌毒力、宿主細(xì)胞粘附、細(xì)胞分裂等生命過程，同時(shí)蛋白分子伴侶在△covR中的表達(dá)變化說(shuō)明CovR的調(diào)控可能發(fā)生在蛋白修飾水平，為研究CovR在調(diào)控細(xì)菌毒力中的作用和分子機(jī)制奠定基礎(chǔ)。

摘要：目的通過比較2型豬鏈球菌中國(guó)強(qiáng)毒株05ZYh13及其covR基因突變株△covR蛋白表達(dá)譜差異，質(zhì)譜鑒定差異表達(dá)蛋白，分析CovR在蛋白表達(dá)調(diào)控中的作用。方法首先將05ZYh13和△covR在THB培養(yǎng)基中培養(yǎng)至對(duì)數(shù)期，然后裂解細(xì)菌制備蛋白樣品。第一向等電聚焦電泳在ph1～10的IPG膠條上完成后進(jìn)行SDS-PAGE電泳，電泳膠經(jīng)掃描分析后選取蛋白點(diǎn)進(jìn)行質(zhì)譜鑒定。結(jié)果05ZYh13和△covR全菌蛋白裂解液經(jīng)二維電泳分別得到559和491個(gè)蛋白點(diǎn)，經(jīng)比對(duì)發(fā)現(xiàn)兩菌株蛋白表達(dá)量差異3倍以上蛋白點(diǎn)40個(gè)，經(jīng)質(zhì)譜鑒定出15個(gè)蛋白，主要涉及細(xì)胞代謝的酶類如谷氨酸脫氫酶、腺苷酸激酶、PTS系統(tǒng)成分等，以及分子伴侶蛋白如GroEL和Dnak等；電泳分離得到△covR特異蛋白點(diǎn)124個(gè)，質(zhì)譜鑒定出15個(gè)，主要為參與細(xì)胞糖代謝過程中的酶，如磷酸甘油酸激酶、甘油醛-3-磷酸脫氫酶、丙酮酸激酶等；質(zhì)譜鑒定05ZYh13特異表達(dá)蛋白5個(gè)。結(jié)論鑒定05ZYh13和△covR差異表達(dá)蛋白35個(gè)，部分蛋白涉及細(xì)菌毒力、宿主細(xì)胞粘附、細(xì)胞分裂等生命過程，同時(shí)蛋白分子伴侶在△covR中的表達(dá)變化說(shuō)明CovR的調(diào)控可能發(fā)生在蛋白修飾水平，為研究CovR在調(diào)控細(xì)菌毒力中的作用和分子機(jī)制奠定基礎(chǔ)。

關(guān)鍵詞：2型豬鏈球菌；毒力調(diào)控因子CovR；二維電泳；蛋白質(zhì)組

2型豬鏈球菌(Streptococcussuisserotype 2，S.suis2)是一種世界性廣泛分布的人獸共患病病原菌[1-3]。

細(xì)菌致病過程是與宿主細(xì)胞相互作用的復(fù)雜過程，依靠復(fù)雜而精密的信號(hào)系統(tǒng)感受、傳導(dǎo)和響應(yīng)外界環(huán)境變化，進(jìn)而調(diào)節(jié)相應(yīng)的基因表達(dá)以作出適應(yīng)性反應(yīng)[4]。二元信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)(two-component signal transduction system，TCS)，是細(xì)菌中普遍存在的一種跨膜信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)[5-6]。

1材料與方法

1.1材料

1.1.1菌株與培養(yǎng)條件05ZYh13本實(shí)驗(yàn)室保存[12]；△covR本實(shí)驗(yàn)室構(gòu)建[11]。將05ZYh13和△covR菌株凍存菌分別接種于5%羊血哥倫比亞血平板，挑單菌落于THB(Todd-Hewitt Broth)液體培養(yǎng)基在37 ℃搖床震蕩培養(yǎng)至平臺(tái)期，1%比例轉(zhuǎn)接至THB后培養(yǎng)至對(duì)數(shù)晚期；△covR菌株THB培養(yǎng)基中加入100 μg/mL鹽酸壯觀霉素。

1.1.2儀器和試劑水化緩沖液：8 mol/L脲，2% CHAPS，0.2% Bio-Lyte 4/7，50 mMDTT，0.01%溴酚藍(lán)；平衡緩沖液：6 mol/L脲，2% SDS，20%甘油，375 mM Tris-HCl pH 8.8，含2% DTT或者2.5%碘乙酸銨；IPG干膠條(Ready Strip TMIPG Strip, ph1～10,17 cm)、蛋白定量試劑盒、CHAPS(Bio-Rad)；等電聚焦儀、垂直板電泳儀、蛋白轉(zhuǎn)印系統(tǒng)(Bio-Rad)；掃描儀Powerlook 2100XL；質(zhì)譜儀4700型MALDI-TOF/MS。

1.2實(shí)驗(yàn)方法

1.2.1全菌蛋白的制備05ZYh13和△covR培養(yǎng)至穩(wěn)定生長(zhǎng)期，6 000 g離心15 min收集菌體。菌體在液氮中研磨后加入蛋白裂解液1 mL/100 mg，蛋白樣品制成勻漿液后超聲破碎5 min，10 ℃下40 000g離心30 min，取上清，分裝后于-80 ℃凍存。改良Bradford 法測(cè)定蛋白含量。

1.2.2二維電泳及凝膠染色將05ZYh13和△covR蛋白樣品各取500 μg溶解于400 μL水化緩沖液中，蛋白樣載入17 cm IPG膠條后進(jìn)行等電聚焦電泳，50 V，20 ℃主動(dòng)水化12 h，S1 250 V 30 min，S2 1 000 V 1 h，S3 10 000 V 5 h，S4 10 000 V 60 000伏時(shí)聚焦，S5 500 V 任意時(shí)間。聚焦完成后的膠條分別用DTT和碘乙酸銨平衡緩沖液平衡15 min，10%的聚丙烯酰胺凝膠分離蛋白。每個(gè)蛋白樣品均平行進(jìn)行3次。

1.2.3差異點(diǎn)分析及質(zhì)譜鑒定TYPHOON SCANNE掃膠后用Image Master4.01進(jìn)行圖像分析，獲取蛋白質(zhì)點(diǎn)位置坐標(biāo)和表達(dá)量等信息。以05ZYh13作為對(duì)照，分析△covR蛋白表達(dá)譜，統(tǒng)計(jì)有或無(wú)差異點(diǎn)以及蛋白表達(dá)Ratio≥3 的蛋白點(diǎn)，SPSS12.0 進(jìn)行統(tǒng)計(jì)學(xué)分析。MALDI-TOF/MS獲得候選蛋白的肽質(zhì)量指紋圖譜(Peptide Mass Fingerprint, PMF)，Mascot軟件將PMF數(shù)據(jù)在NCBI非冗余蛋白數(shù)據(jù)庫(kù)進(jìn)行比對(duì)搜索，搜索參數(shù)(搜索類型：PMF；消化酶：胰蛋白酶；固定化修飾：Carbamidomethyl；可變修飾：Oxidation；質(zhì)量值：?jiǎn)我煌凰?；肽質(zhì)量偏差：±100 ppm；Max missed cleavages: 1)。Mowse分值的概率P評(píng)價(jià)搜索結(jié)果的質(zhì)量，分值大于51表明差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。

2結(jié)果

2.1二維電泳結(jié)果05ZYh13和△covR菌株全菌蛋白經(jīng)二維電泳分離后蛋白點(diǎn)多集中于pp～7，分子量多集中于30～70 kD，等電點(diǎn)(pI)多在3～6 之間，結(jié)果見圖1。

圖1　05ZYh13與突變株△covR全菌蛋白二維電泳圖譜Fig.1　Two-dimensional gel map of proteins from 05ZYh13 and △covR

2.2凝膠圖像分析結(jié)果對(duì)二維凝膠電泳結(jié)果進(jìn)行掃膠比較分析，結(jié)果發(fā)現(xiàn)05ZYh13分離得到559個(gè)蛋白點(diǎn)，△covR菌株分離得到491個(gè)蛋白點(diǎn)，其共有的匹配蛋白點(diǎn)數(shù)367個(gè)。對(duì)367個(gè)匹配蛋白點(diǎn)表達(dá)量變化分析發(fā)現(xiàn)，3倍以上差異蛋白點(diǎn)共40個(gè)(圖2)，其中15個(gè)蛋白點(diǎn)表達(dá)下調(diào)，25個(gè)蛋白點(diǎn)表達(dá)上調(diào)。對(duì)未匹配蛋白點(diǎn)分析發(fā)現(xiàn)，△covR特異表達(dá)蛋白點(diǎn)124個(gè)(圖3)，05ZYh13特異蛋白點(diǎn)192個(gè)(圖4)。

注：綠色圈表示ΔcovR較05ZYh13菌株3倍以上差異蛋白點(diǎn)，紅色箭頭表示質(zhì)譜鑒定蛋白點(diǎn)

Note: The green circles represent more than 3 ratios changed proteins of 05ZYh13 and △covRstrains. The red arrow represent identified proteins by mass spectrometry

圖205ZYh13與△covR菌株差異表達(dá)蛋白點(diǎn)分析圖

Fig.2Differentially expressed proteins analysis of 05ZYh13 and △covR

注：綠色圈表示△covR特異蛋白點(diǎn)，藍(lán)色箭頭表示質(zhì)譜鑒定蛋白點(diǎn)

Note: The green circles represent specific proteins of △covRstrain. The blue arrow represent identified proteins by mass spectrometry

圖3△covR菌株特異蛋白點(diǎn)分析圖

Fig.3Specific expressed proteins of △covRmutant strain

注：綠色圈表示05ZYh13菌株特異蛋白點(diǎn)，粉色箭頭指示質(zhì)譜鑒定蛋白點(diǎn)Note: The green circles represent specific proteins of 05ZYh13 strain. The pink arrow represent identified proteins by mass spectrometry 圖4　05ZYh13菌株特異蛋白點(diǎn)分析圖Fig.4　Specific expressed proteins of 05ZYh13 strain

2.3質(zhì)譜鑒定結(jié)果

05ZYh13和△covR差異表達(dá)蛋白MALDI-TOF/MS質(zhì)譜鑒定35個(gè)蛋白質(zhì)，其中3倍以上差異表達(dá)蛋白15個(gè)，△covR菌株7個(gè)蛋白下調(diào)表達(dá)，8個(gè)蛋白上調(diào)表達(dá)。其中NADPH依賴的谷氨酸脫氫酶上調(diào)表達(dá)9.29倍，蛋白點(diǎn)Spot43是一未知功能蛋白，上調(diào)9倍；另外分子伴侶GroEL和DnaK分別上調(diào)表達(dá)5.59倍和6.78倍，在核苷酸代謝過程中具有重要作用的腺苷酸激酶上調(diào)表達(dá)3.13倍。在下調(diào)表達(dá)蛋白中50S核糖體蛋白L10下調(diào)7.79倍，下調(diào)變化最大；糖代謝相關(guān)酶6-磷酸果糖激酶和乳酸脫氫酶分別下調(diào)4.17、4.65倍；細(xì)胞壁合成密切相關(guān)的N-乙酰葡萄糖胺磷酸轉(zhuǎn)移酶下調(diào)表達(dá)3.36倍。

質(zhì)譜鑒定△covR菌株特異蛋白15個(gè)，主要是涉及代謝過程中的酶和物質(zhì)轉(zhuǎn)運(yùn)過程。如在糖代謝過程中的甘油醛-3-磷酸脫氫酶、磷酸甘油酸激酶、磷酸甘油酸變位酶、丙酮酸激酶都是糖酵解途徑中重要的催化酶；Spot99蛋白點(diǎn)是3-酮乙酰-ACP還原酶，該酶是脂肪酸合酶的重要成分之一，催化脂肪酸合成；Spot5蛋白點(diǎn)是尿嘧啶DNA糖基化酶催化含尿嘧啶的單鏈和雙鏈DNA釋放游離尿嘧啶，Spot36蛋白點(diǎn)是脫氧核糖醛縮酶，能夠催化5-磷酸-2-脫氧-D-核糖分解成3-磷酸-D-甘油醛和乙醛，并可催化其逆反應(yīng)，這兩個(gè)酶都是參與核苷酸代謝過程的重要酶類；Spot36蛋白點(diǎn)是二氫吡啶二羧酸還原酶，Spot312蛋白點(diǎn)是二氫碟酸合酶，二者都與氨基酸代謝過程緊密聯(lián)系；另外支鏈氨基酸轉(zhuǎn)運(yùn)ATP酶和ABC型精氨酸轉(zhuǎn)運(yùn)子都是物質(zhì)轉(zhuǎn)運(yùn)相關(guān)蛋白；鑒定的蛋白中還發(fā)現(xiàn)了一些結(jié)構(gòu)蛋白如30S核糖體蛋白，膜錨定脂蛋白等。對(duì)05ZYh13特異蛋白點(diǎn)進(jìn)行質(zhì)譜鑒定共得到5個(gè)對(duì)應(yīng)蛋白，分別是跨膜蛋白、核糖體蛋白、翻譯延伸因子、葡萄糖-1-磷酸胸腺嘧啶轉(zhuǎn)移酶以及丙酮酸脫氫酶。結(jié)果見表1。

表105ZYh13與△covR菌株差異蛋白點(diǎn)質(zhì)譜鑒定結(jié)果

Tab.1Mass spectrum identification results of differentiated proteins from 05ZYh13 and △covR strains

Spotno.Gino.No.of05ZYH33geneProteinfunctionannotationTheoreticalMr/pIExperimentalMr/pIMascotscoreFoldchangesCOGΔcovR較05ZYH33表達(dá)差異3倍以上蛋白Morethan3ratiosdifferentialproteinsbetween05ZYH33andΔcovR15Gi:81177339SSU05_0252NAD(P)H-dependentglu-tamatedehydrogenase48780/5.3949119/5.77789.29Metabolism21Gi:500942565SSU05_18033-ketoacyl-ACPreductase25588/5.4821063/5.11984.35Metabolism40Gi:500275857SSU05_098450SRibosomalproteinL1018580/6.6725074/4.87118-7.79Informationstor-ageandprocessing43Gi:500941448SSU05_1092Hypotheticalprotein7400/6.298363/6.05589.00uncharacterized115Gi:145690806SSU05_0148ChaperoninGroEL23200/4.7435658/4.60665.59Cellularproces-sesandsignaling125Gi:223897437SSU05_0506PeptidaseU3246953/5.0436419/4.79533.36Cellularproces-sesandsignaling161Gi:500274274SSU05_0300ChaperoneproteindnaK64787/4.6238564/4.80576.78Cellularproces-sesandsignaling180Gi:489026222SSU05_0091Adenylatekinase23763/4.8839981/4.59683.13Metabolism317Gi:637064337SSU05_1076Lactatedehydrogenase35400/5.0553084/4.88161-4.65Metabolism334Gi:500942541SSU05_1778PTSmannosetransportersubunitIIAB35310/4.9556013/4.671313.94Metabolism344Gi:500274979SSU05_0543ATP-dependent6-phospho-fructokinase35526/5.5155816/5.40240-4.17Metabolism365Gi:145691966SSU05_1298dTDP-glucose4,6-de-hydratase44617/5.9058844/5.28156-6.62Metabolism476Gi:500941609SSU05_1172F0F1ATPsynthasesub-unitbeta51976/4.9670296/4.59256-3.03Metabolism478Gi:656224244SSU05_0852Serinehydroxymethyl-transferas45590/5.6570358/5.61200-3.56Metabolism495Gi:500942369SSU05_1854N-acetylglucosamine-1-phosphateuridyltransferase50075/5.8473783/5.5558-3.36MetabolismΔcovR菌株特異蛋白Specificexpressedproteinsby△covRmutantstrain5Gi:489025217SSU05_1460Uracil-DNAglycosylase22039/9.317203/4.2652Informationstor-ageandprocessing24Gi:500942332SSU05_1543ABC-typebranched-chainaminoacidtransportsys-tems,ATPasecomponent25533/5.6121742/5.4457Metabolism26Gi:500943265SSU05_2067ArginineABCtransporterATP-bindingprotein27119/5.6421934/4.97117Metabolism36Gi:489024864SSU05_1085Deoxyribose-phosphateal-dolase23188/4.5924100/6.11105Metabolism44Gi:146318482SSU05_0828Dihydrodipicolinatereduc-tase27349/5.0025185/5.9795Metabolism

注：Score分?jǐn)?shù)大于51具有統(tǒng)計(jì)學(xué)意義，P<0.05

Note: More than 51 scores showed the data has significant differences.P<0.05

3討論

病原菌在致病過程由多種致病因子共同參與，如莢膜多糖、溶血素、胞外因子、溶菌酶釋放蛋白等，也包括一些生長(zhǎng)及代謝相關(guān)的相互作用因子[4]。本文對(duì)CovR基因缺失株與中國(guó)強(qiáng)毒株05ZYh13蛋白質(zhì)譜的比較分析后，用MADLI-TOF-MS方法鑒定出35個(gè)差異表達(dá)蛋白。為進(jìn)一步研究毒力負(fù)調(diào)控因子CovR在調(diào)控蛋白表達(dá)的分子機(jī)制奠定基礎(chǔ)。

糖酵解是生物代謝的重要部分，能夠?yàn)榧?xì)胞提供能量，參與該過程的很多酶類定位于鏈球菌或其它細(xì)菌表面[13-14]。其中甘油醛-3-磷酸脫氫酶(GAPDH)、磷酸甘油酸激酶、磷酸甘油醛變位酶、丙糖磷酸異構(gòu)酶和烯醇化酶(Enolase)除參與糖代謝過程外，還具有纖溶酶/纖溶酶原蛋白結(jié)合能力，能夠增加細(xì)菌對(duì)組織的侵入能力[15]。Brassard等[16]克隆并純化獲得S.suis2的GAPDH重組蛋白，并證明其與宿主細(xì)胞的粘附相關(guān)。Quessy等[17]構(gòu)建S.suis2的gapdh基因缺陷株，發(fā)現(xiàn)GAPDH表達(dá)缺失株對(duì)宿主氣管表皮細(xì)胞的粘附能力降低。另外Tsugaw等[18]對(duì)類志賀氏菌的研究發(fā)現(xiàn)GroEL能增強(qiáng)細(xì)菌對(duì)宿主細(xì)胞的粘附。Singh等[19]對(duì)金黃色葡萄球菌的研究發(fā)現(xiàn)DnaK表達(dá)缺陷導(dǎo)致，細(xì)菌合成生物被膜減少，對(duì)宿主細(xì)胞的粘附減弱。本研究發(fā)現(xiàn)GAPDH(Spot63)、磷酸甘油酸激酶(Spot57)、磷酸甘油醛變位酶(Spot208)、GroEL(Spot115)和DnaK(Spot161)在△covR中表達(dá)均明顯增加，此類與粘附作用相關(guān)蛋白表達(dá)的增加可能與△covR菌株對(duì)宿主細(xì)胞的粘附增強(qiáng)有關(guān)。

丙酮酸激酶(Spot59)，其同源蛋白在肺炎鏈球菌中與毒力密切相關(guān)[20]，Burall等[21]在綿羊病原菌Chlamydiaabortus的研究中發(fā)現(xiàn)，當(dāng)丙酮酸激酶表達(dá)缺陷時(shí)該菌毒力減弱。Wang等[22]在S.suis2二元信號(hào)系統(tǒng)VirR/VirS的蛋白質(zhì)組研究發(fā)現(xiàn)，△virRS菌株丙酮酸激酶表達(dá)下調(diào)1.7倍。蛋白點(diǎn)Spot15是SSU05_0252基因編碼的NADPH依賴的谷氨酸脫氫酶(NAD(P)H-dependent glutamate dehydrogenase, GDH)，該蛋白位于S.suis2的細(xì)胞表面，能夠刺激機(jī)體產(chǎn)生高滴度的抗體，作為一個(gè)毒力蛋白已經(jīng)引起廣泛關(guān)注[23]。GDH在△covR菌株中上調(diào)9.29倍，丙酮酸激酶為特異性表達(dá)蛋白，提示CovR可能通過調(diào)節(jié)GDH和丙酮酸激酶的表達(dá)而影響細(xì)菌毒力，然而CovR與GDH和丙酮酸激酶的調(diào)控關(guān)系仍需要進(jìn)一步研究證實(shí)。

PTS是一個(gè)多蛋白系統(tǒng)，在許多細(xì)菌的糖代謝過程中起關(guān)鍵作用，參與細(xì)菌代謝和翻譯過程的調(diào)節(jié)。在S.mutans[24]和L.monocytogenes[25]病原菌中PTS甘露糖轉(zhuǎn)運(yùn)子亞單位IIAB在碳代謝抑制和毒力基因下調(diào)中承擔(dān)重要作用。另外腺苷酸激酶(Adk)可能也參與細(xì)菌致病過程，在肺炎鏈球菌中SpAdk為肺炎鏈球菌生長(zhǎng)必須基因，其細(xì)胞內(nèi)ATP水平隨SpAdk增加成比例增加，SpAdk影響細(xì)胞內(nèi)能荷穩(wěn)態(tài)，其突變體導(dǎo)致細(xì)胞分裂發(fā)生缺陷[26]。在鼠疫耶爾森氏菌的研究中，AKyp的突變導(dǎo)致細(xì)菌毒力顯著下降[27]。在本研究中PTS甘露糖轉(zhuǎn)運(yùn)子亞單位IIAB(Spot334)和Adk(Spot180)在△covR菌株中的表達(dá)分別上調(diào)表達(dá)3.94和3.13倍，但CovR是否對(duì)這兩個(gè)蛋白具有調(diào)控作用仍需進(jìn)一步研究。

本文對(duì)S.suis2中國(guó)強(qiáng)毒株05ZYh13與△covR菌株蛋白質(zhì)譜比較鑒定了35個(gè)差異表達(dá)蛋白，結(jié)果表明CovR可能通過對(duì)細(xì)胞代謝過程中關(guān)鍵酶如GAPDH、磷酸甘油酸激酶、磷酸甘油醛變位酶、GDH、丙酮酸激酶以及分子伴侶蛋白GroEL和DnaK等多方面的表達(dá)調(diào)控，從而影響細(xì)菌的毒力及致病性。但是由于本研究采用全菌蛋白裂解法制備樣品，所制樣品中多為胞漿蛋白，而胞膜蛋白含量低、缺乏分泌蛋白。因此未鑒定出莢膜多糖、溶血素(Sylisin)、溶菌酶釋放蛋白(MRP)、胞外因子(EF)以及血清渾濁因子(OFS)等分泌蛋白或胞膜蛋白類的S.suis2毒力因子；另外由于質(zhì)譜鑒定蛋白點(diǎn)偏少，也可能造成一些差異表達(dá)的毒力因子未被發(fā)現(xiàn)。后續(xù)課題組將分別制備細(xì)菌分泌蛋白、胞膜蛋白和胞漿蛋白樣品，從而獲得△covR菌株更全面的蛋白表達(dá)信息，為進(jìn)一步研究CovR調(diào)控S.suis2毒力的分子機(jī)制奠定基礎(chǔ)。

參考文獻(xiàn)：

[1] Feng Y, Zhang H, Wu Z, et al.StreptococcussuisInfection: an emerging/reemerging challenge of bacterial infectious diseases?[J]. Virulence, 2014, 5(4): 477-497.DOI：10.4161/yiru.28595.

[2] Pan Z, Ma J, Dong W, et al. Novel variant serotype ofStreptococcussuisisolated from piglets with meningitis[J]. Appl Environm Microbiol, 2014, 81(3): 976-985. DOI: 10.1128/AEM.02962-14.

[3] Gottschalk M, Xu J, Calzas C, et al.Streptococcussuis: a new emerging or an old neglected zoonotic pathogen?[J]. Future Microbiol, 2010, 5(3): 371-391. DOI: 10.2217/fmb.10.2.

[4] Calva E, Oropeza R. Two-component signal transduction systems, environmental signals, and virulence[J]. Microbial Ecol, 2006, 51(2): 166-176.

[5] Han H, Liu C, Wang Q, et al. The two-component system Ihk/irr contributes to the virulence ofStreptococcussuisserotype 2 strain 05ZYh13 through alteration of the bacterial cell metabolism[J]. Microbiology, 2012, 158(7): 1852-1866. DOI: 10.1099/mic.0.057448-0.

[6] Li J, Chen T, Yang Z, et al. The two-component regulatory system ciarh contributes to the virulence ofStreptococcussuis2[J]. Vet Microbiol, 2011, 148(1): 99-104. DOI: 10.1016/j.vetmic.2010.08.005.

[7] Mitrophanov AY, Churchward G, Borodovsky M. Control ofStreptococcuspyogenesvirulence: modeling of the CovR/S signal transduction system[J]. J Theoret Biol, 2007, 246(1): 113-128.

[8] Jiang SM, Cieslewicz MJ, Kasper DL, et al. Regulation of virulence by a two-component system in Group BStreptococcus[J]. J Bacteriol, 2005, 187(3): 13-1105.

[9] Dmitriev A, Mohapatra SS, Chong P, et al. CovR-controlled global regulation of gene expression inStreptococcusmutans[J]. PLoS One, 2011, 6(5): e20127. DOI: 10.1371/journal.pone.0020127.

[10] Trihn M, Ge X, Dobson A, et al. Two-component system response regulators involved in virulence ofStreptococcuspneumoniaeTIGR4 in infective endocarditis[J]. PLoS One, 2013, 8(1): e54320. DOI: 10.1371/journal.pone.0054320.

[11] Pan XZ, Ge JC, Li M, et al. The orphan response regulator CovR: a globally negative modulator of virulence inStreptococcussuisserotype2[J]. J Bacteriol, 2009, 191(8): 2601-2612. DOI: 10.1128/JB.01309-08.

[12] Chen C, Tang J, Dong W, et al. A glimpse of Streptococcal toxic shock syndrome from comparative genomics ofS.suis2 Chinese isolates[J]. PLoS One 2007，2(3): e315.

[13] Jones MN, Holt RG. Cloning and characterization of an α-enolase of the oral pathogen streptococcus mutans that binds human plasminogen[J]. Biochem Biophysic Res Communicat, 2007, 364(4): 924-929.

[14] Hughes MJ, Moore JC, Lane JD, et al. Identification of major outer surface proteins ofStreptococcusagalactiae[J]. Infect Immun, 2002, 70(3): 1254-1259.

[15] Kinnby B, Booth NA, Svensater G. Plasminogen binding by oral streptococci from dental plaque and inflammatory lesions[J]. Microbiology, 2008, 154(Pt 3): 924-931. DOI: 10.1099/mic.0.2007/013235-0.

[16] Brassard J, Gottschalk M, QuessyS.Cloningand purification of theStreptococcussuisserotype 2 glyceraldehyde-3-phosphate dehydrogenase and its involvement as an adhesin[J]. Vet Microbiol, 2004, 102(1/2): 87-94.

[17] Brassard J, Gottschalk M, QuessyS.Decreaseof the adhesion ofStreptococcussuisserotype 2 mutants to embryonic bovine tracheal cells and porcine tracheal rings[J]. Canad J Vet Research, 2001, 65(3): 156-160.

[18] Tsugawa H, Ito H, Ohshima M, et al. Cell adherence-promoted activity of Plesiomonas shigelloides groEL[J]. J Med Microbiol, 2007, 56(1): 23-29.

[19] Singh VK, Syring M, Singh A, et al. An insight into the significance of the Dnak heat shock system inStaphylococcusaureus[J]. Int J Med Microbiol, 2012, 302(6): 242-252. DOI: 10.1016/j.ijmm.2012.05.001.

[20] Yesilkaya H, Spissu F, Carvalho SM, et al. Pyruvate formate lyase is required for pneumococcal fermentative metabolism and virulence[J]. Infect Immun, 2009, 77(12): 5418-5427. DOI: 10.1128/IAI.00178-09.

[21] Burall LS, Rodolakis A, Rekiki A, et al. Genomic analysis of an attenuatedChlamydiaabortuslive vaccine strain reveals defects in central metabolism and surface proteins[J]. Infect Immun, 2009, 77(9): 4161-4167. DOI: 10.1128/IAI.00189-09.

[22] Wang H, Shen X, Zhao Y, et al. Identification and proteome analysis of the two-component VirR/virS system in epidemicStreptococcussuisserotype 2[J]. FEMS Microbiol Lett, 2012, 333(2): 160-168. DOI: 10.1111/j.1574-6968.2012.02611.x.

[23] Silva LM, Baums CG, Rehm T, et al. Virulence-associated gene profiling ofStreptococcussuisisolates by PCR[J]. Vet Microbiol, 2006, 115(1/3): 117-127.

[24] Abranches J, Candella MM, Wen ZT, et al. Different roles of EIIABMan and EIIGlc in regulation of energy metabolism, biofilm development, and competence inStreptococcusmutans[J]. J Bacteriol, 2006, 188(11): 3748-3756.

[25] Vu-Khac H, Miller KW. Regulation of mannose phosphotransferase system permease and virulence gene expression inListeriamonocytogenesby the EII(t)Man transporter[J]. Appl Environm Microbiol, 2009, 75(21): 6671-6678. DOI: 10.1128/AEM.01104-09.

[26] Thach TT, Luong TT, Lee S, et al. Adenylate kinase fromStreptococcuspneumoniaeis essential for growth through its catalytic activity[J]. FEBS Open Bio, 2014, 4(1): 672-682. DOI: 10.1016/j.fob.2014.07.002.

[27] Munier-Lehmann H, Chenal-Francisque V, Ionescu M, et al. Relationship between bacterial virulence and nucleotide metabolism: a mutation in the adenylate kinase gene rendersYersiniapestisavirulent[J]. Biochem J, 2003, 373(2): 515-522.

DOI:10.3969/j.issn.1002-2694.2016.05.001

通訊作者：潘秀珍，Email:panxiuzhen_2004@163.com

中圖分類號(hào)：R378

文獻(xiàn)標(biāo)識(shí)碼：A

文章編號(hào)：1002-2694(2016)05-417-07

Corresponding author:Pan xiu-zhen, Email: panxiuzhen_2004@163.com

收稿日期：2015-12-16修回日期：2016-03-18

Comparative proteomic research between theStreptococcussuisserotype 2 Chinese highly virulent strain and thecovRisogenic mutant

NI Hua1,2，ZHANG Jian1,2, ZHENG Feng2, HU Dan2,LI Xian-fu2, WANG Chang-jun2, PAN Xiu-zhen1,2

(1.CollegeofLifeSciences,NanjingNormalUniversity,Nanjing210023,China;2.InstituteofMilitaryMedicalSciences,NanjingCommand,Nanjing210002,China)

Abstract:In order to search for the virulence factor regulated by CovR, the proteomics of the whole-cell protein were compared between Streptococcus suis serotype 2 wild strain 05ZYh13 and an isogenic mutant strain △covR by two-dimensional gel electrophoresis. The 05ZYh13 and △covR were cultured in Todd-Hewitt Broth medium then the whole-cell proteins sample were extracted. The 2-DE gel was conducted using the ph1-10 IPG strip for the first dimension IEF and followed by SDS-PAGE. After electrophoresis, the gels were stained and analyzed. Results showed that the 05ZYh13 and △covR had 559 and 491 protein spots respectively. Compared with the 05ZYh13, the mutant strain had 40 proteins more than 3 folds changed which identified 15 proteins by mass spectrum. Those proteins major involved in cell metabolic enzymes, such as adenylate kinase, glutamate dehydrogenase and PTS system components, etc., as well as molecular chaperone proteins GroEL and Dnak. The △covR had about 124 specific protein spots in which 15 proteins were identified by mass spectrum. Those proteins were participate in the cell process of sugar metabolism, such as phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase, etc. Beside of those 5 specific proteins of 05ZYh13 was identified by mass spectrum. Appraisement of 35 differentially expressed proteins involved in bacterial virulence, host cell adhesion and cell division, etc., and the molecular chaperone up-regulated expression showed that the regulation may be occurred in the profile of protein modification. These results provided better understanding on pathogenic mechanisms of Streptococcus suis type 2 at the level of protein expression.

Key words：Streptococcus suis serotype 2; CovR; two-dimensional gel electrophoresis; proteome Funded by the National Natural Science Foundation of China (Nos. 81571965 and 81471920), the Natural Science Foundation of Jiangsu Province (No. BK20151091) and the 333 Engineering Science Foundation of Jiangsu Province (No. BRA2014363)

國(guó)家自然科學(xué)基金(Nos. 81571965, 81471920)、江蘇省自然科學(xué)基金(No. BK20151091)和江蘇省333工程科研資助項(xiàng)目(BRA2014363)聯(lián)合資助