口腔中過氧化氫的來源及在微生態(tài)平衡中的作用

章可可 周學(xué)東 徐欣

口腔疾病研究國(guó)家重點(diǎn)實(shí)驗(yàn)室，國(guó)家口腔疾病臨床研究中心，四川大學(xué)華西口腔醫(yī)院，成都 610041

口腔中過氧化氫的來源及在微生態(tài)平衡中的作用

章可可 周學(xué)東 徐欣

口腔疾病研究國(guó)家重點(diǎn)實(shí)驗(yàn)室，國(guó)家口腔疾病臨床研究中心，四川大學(xué)華西口腔醫(yī)院，成都 610041

過氧化氫（H2O2）作為口腔中一種重要的抑菌物質(zhì)，在口腔微生態(tài)平衡中發(fā)揮著重要作用。在口腔齦上菌斑定植的先鋒菌中，約有80%細(xì)菌屬于口腔鏈球菌屬，這些口腔鏈球菌可以利用不同的氧化酶合成H2O2，同時(shí)H2O2可通過抑制對(duì)H2O2敏感的微生物參與菌間競(jìng)爭(zhēng)拮抗，使氧耐受菌得以存活，從而調(diào)節(jié)生物膜的發(fā)展過程；H2O2還可通過影響微生物細(xì)胞外DNA的釋放和感受態(tài)細(xì)胞的形成進(jìn)而影響基因的水平轉(zhuǎn)移，調(diào)節(jié)生物膜對(duì)口腔環(huán)境的適應(yīng)能力；同時(shí)口腔微生物也可通過一系列機(jī)制耐受H2O2。此外，H2O2在微生物與宿主的關(guān)系中也發(fā)揮著重要作用。本文就口腔中H2O2的來源及其在口腔微生態(tài)平衡的作用兩方面的研究進(jìn)展進(jìn)行綜述。

過氧化氫； 口腔微生物； 口腔微生態(tài)； 生物膜； 微生物與宿主

口腔是一個(gè)復(fù)雜的微生態(tài)系統(tǒng)，過氧化氫（H2O2）是這個(gè)復(fù)雜生態(tài)系中一種重要的非特異性抗菌物質(zhì)。H2O2可由乳桿菌和牙菌斑早期定植的鏈球菌產(chǎn)生，也可來源于宿主，在調(diào)控微生態(tài)平衡中發(fā)揮重要作用。H2O2可通過抑制對(duì)其敏感的微生物參與微生物間的競(jìng)爭(zhēng)拮抗，使得對(duì)H2O2較為耐受的細(xì)菌獲得競(jìng)爭(zhēng)優(yōu)勢(shì)，從而在生物膜形成過程中，特別是在生物膜形成初期發(fā)揮重要的菌群調(diào)節(jié)作用。H2O2可通過介導(dǎo)細(xì)胞外DNA（extracellular DNA，eDNA）的釋放，參與細(xì)菌間基因的水平轉(zhuǎn)移[1]，影響生物膜對(duì)口腔復(fù)雜環(huán)境的適應(yīng)能力；口腔內(nèi)微生物也可通過一系列耐受機(jī)制減少H2O2對(duì)自身的傷害[2-3]。此外，H2O2在微生物和宿主的交互關(guān)系中也發(fā)揮著重要作用[4-5]。本文就口腔中H2O2的來源及其在口腔微生態(tài)中的作用進(jìn)行綜述。

1 口腔中H2O2的來源

口腔中的H2O2主要來源于細(xì)菌和宿主[6]。乳桿菌和口腔鏈球菌很早就被發(fā)現(xiàn)可以產(chǎn)生H2O2[7-8]。定性和定量研究[9-12]發(fā)現(xiàn)，口腔鏈球菌、緩癥鏈球菌、表兄鏈球菌、血鏈球菌、戈登鏈球菌、寡發(fā)酵鏈球菌、副血鏈球菌等均能產(chǎn)生H2O2。這些鏈球菌均可以從人口腔牙菌斑中分離，且在牙菌斑中具有較高豐度[13]。體外實(shí)驗(yàn)[8-9,11]表明，細(xì)菌H2O2的產(chǎn)生與其培養(yǎng)環(huán)境條件密切相關(guān)?？谇患?xì)菌主要依賴丙酮酸氧化酶[14]、乳酸氧化酶[15]、煙酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide，NADH）氧化酶[16]和L-氨基酸氧化酶[17]等途徑來產(chǎn)H2O2。

1.1 丙酮酸氧化酶途徑

血鏈球菌和戈登鏈球菌依賴丙酮酸氧化酶產(chǎn)生H2O2，該酶由spxB基因編碼，可催化丙酮酸鹽、無機(jī)磷酸鹽和氧分子生成H2O2[14,18]。研究者通過構(gòu)建血鏈球菌和戈登鏈球菌的spxB基因的突變株和spxB基因的補(bǔ)償菌株，并比較了野生株、spxB基因突變株及spxB基因補(bǔ)償株與變異鏈球菌的競(jìng)爭(zhēng)關(guān)系，證實(shí)spxB基因編碼丙酮酸氧化酶合成H2O2是血鏈球菌和戈登鏈球菌與變異鏈球菌細(xì)菌間相互競(jìng)爭(zhēng)的重要分子機(jī)制[14]。

血鏈球菌和戈登鏈球菌H2O2的產(chǎn)生受細(xì)菌生長(zhǎng)環(huán)境影響和自身相關(guān)基因的調(diào)控。Barnard等[11]系統(tǒng)研究了外環(huán)境因素對(duì)戈登鏈球菌H2O2產(chǎn)生的影響，結(jié)果發(fā)現(xiàn)，培養(yǎng)基中葡萄糖和蔗糖、金屬離子、pH、細(xì)菌生長(zhǎng)期和氧分壓均會(huì)對(duì)戈登鏈球菌H2O2合成造成顯著影響。當(dāng)培養(yǎng)基中蔗糖、葡萄糖含量有限時(shí)（0.1 mmol·L-1左右），戈登鏈球菌產(chǎn)生H2O2和乳酸的能力相當(dāng)，而當(dāng)培養(yǎng)基中蔗糖和葡萄糖較為富足時(shí)，H2O2合成顯著減少。由于口腔中的各種碳源代謝底物有限，戈登鏈球菌為了獲取有限的生存空間和營(yíng)養(yǎng)，可能通過產(chǎn)生H2O2來抑制其他H2O2敏感菌的生長(zhǎng)，進(jìn)而在生物膜群落中獲得生長(zhǎng)優(yōu)勢(shì)。Zheng等[19]發(fā)現(xiàn)，戈登鏈球菌中的分解代謝物控制蛋白CcpA可結(jié)合到spxB的啟動(dòng)子，抑制spxB基因表達(dá)，從而調(diào)控戈登鏈球菌H2O2的合成。

血鏈球菌H2O2的合成也受到一系列基因的調(diào)控。Zheng等[20]研究發(fā)現(xiàn)，血鏈球菌分解代謝物控制蛋白CcpA同樣能調(diào)控spxB的表達(dá)，血鏈球菌ccpA突變株spxB表達(dá)量較野生株上調(diào)約6倍，提示血鏈球菌CcpA也可通過抑制spxB基因的表達(dá)調(diào)控H2O2的產(chǎn)生。與戈登鏈球菌不同的是，血鏈球菌spxB的表達(dá)受培養(yǎng)基中葡萄糖含量的影響并不顯著，推測(cè)血鏈球菌CcpA蛋白依賴抑制作用受到其他代謝物（例如輔脫氫酶Ⅱ）的控制。Chen等[21]通過突變血鏈球菌全局性調(diào)控因子編碼基因spxA1和spxA2，研究了這兩個(gè)基因?qū)ρ溓蚓鶫2O2產(chǎn)生的影響。研究發(fā)現(xiàn)，spxA1突變株H2O2產(chǎn)量顯著減少，spxA1補(bǔ)償株H2O2的產(chǎn)生約為野生株的80%，但spxA2突變株H2O2的產(chǎn)量與野生株相比無明顯差異，提示血鏈球菌的全局性調(diào)控因子SpxA1參與控制spxB的表達(dá)，而spxA2對(duì)該細(xì)菌H2O2的產(chǎn)生無明顯影響。Chen等[22]另一研究表明，血鏈球菌ackA、spxR和tpk三個(gè)基因的突變株與野生株相比，產(chǎn)H2O2明顯減少。ackA和spxR的突變株中spxB的表達(dá)量減少而tpk的突變株中spxB的表達(dá)量增多，推測(cè)ackA和spxR可能為spxB表達(dá)的正調(diào)節(jié)基因，而tpk為spxB表達(dá)的輔因子。Zhu等[23]設(shè)計(jì)spxB通用引物對(duì)口腔菌斑spxB的動(dòng)態(tài)表達(dá)進(jìn)行定量檢測(cè)，結(jié)果發(fā)現(xiàn)，個(gè)體菌斑中spxB的表達(dá)量隨時(shí)間的改變相對(duì)穩(wěn)定，提示spxB在口腔菌斑中具有功能性作用。

1.2 乳酸氧化酶途徑

寡發(fā)酵鏈球菌除可通過丙酮酸氧化酶合成H2O2外[15]，還可通過乳酸氧化酶參與H2O2的合成。Tong等[10]發(fā)現(xiàn)，寡發(fā)酵鏈球菌可通過乳酸氧化酶利用變異鏈球菌產(chǎn)生的乳酸合成H2O2。在細(xì)菌生長(zhǎng)的不同周期，主要負(fù)責(zé)合成H2O2的基因不同。丙酮酸氧化酶主要負(fù)責(zé)細(xì)菌生長(zhǎng)潛伏期和對(duì)數(shù)期H2O2的產(chǎn)生，而乳酸氧化酶主要負(fù)責(zé)細(xì)菌生長(zhǎng)平臺(tái)期H2O2的合成。

1.3 NADH氧化酶和L-氨基酸氧化酶途徑

Higuchi等[16]在變異鏈球菌耐氧菌株NCIB11723中分離純化獲得了可產(chǎn)生H2O2的NADH氧化酶Nox-1。該酶由4個(gè)相對(duì)分子質(zhì)量約為5.6×104的亞基組成，黃素腺嘌呤二核苷酸可促進(jìn)該酶H2O2合成活性。Tong等[17]發(fā)現(xiàn)，寡發(fā)酵鏈球菌乳酸氧化酶Lox突變株在蛋白胨富集時(shí)對(duì)變異鏈球菌仍具有顯著抑制作用。進(jìn)一步體外研究發(fā)現(xiàn)，氨基酸氧化酶LAAO可通過7種氨基酸產(chǎn)生H2O2，該蛋白編碼基因aaoSo突變株在上述氨基酸存在條件下也不產(chǎn)生H2O2，證實(shí)寡發(fā)酵鏈球菌還可通過氨基酸氧化酶LAAO合成H2O2。

1.4 宿主來源的H2O2

H2O2是活性氧（reactive oxygen species，ROS）的一種[24]。宿主來源的H2O2可來自眾多途徑。線粒體可通過呼吸作用產(chǎn)生ROS，包括H2O2[25-27]。細(xì)胞內(nèi)的清除系統(tǒng)有效地保護(hù)宿主免受ROS造成的傷害，其中產(chǎn)生的H2O2可能不會(huì)離開黏膜細(xì)胞，但這個(gè)途徑產(chǎn)生的H2O2的量大到足以在口腔微生態(tài)中發(fā)揮作用[5]。H2O2產(chǎn)生的另一個(gè)途徑來自巨噬細(xì)胞的氧化爆發(fā)。ROS的穩(wěn)定產(chǎn)生（包括H2O2）是巨噬細(xì)胞氧化爆發(fā)的一部分，巨噬細(xì)胞產(chǎn)生的ROS是針對(duì)巨噬細(xì)胞外對(duì)宿主有侵害的病原菌，因此可以自由擴(kuò)散到病原菌的附近[28]。但有研究[29-30]表明，個(gè)體巨噬細(xì)胞的含量處于一個(gè)逐日變化的過程，因此由巨噬細(xì)胞產(chǎn)生的H2O2量很難確定。Geiszt等[31]發(fā)現(xiàn)了另一種宿主產(chǎn)H2O2的途徑，他們發(fā)現(xiàn)表達(dá)雙氧化酶2的唾液腺細(xì)胞能持續(xù)地給唾液供應(yīng)H2O2，并且產(chǎn)生的H2O2能直接隨著唾液進(jìn)入到口腔中，這可能是唾液中H2O2的宿主來源的最主要途徑。

2 H2O2在口腔中的生物學(xué)作用

2.1 H2O2對(duì)生物膜的調(diào)節(jié)作用

H2O2可在牙菌斑生物膜形成初期影響微生物的早期定植。細(xì)菌的初始黏附在生物膜形成過程中具有重要作用。在初始黏附時(shí)，鏈球菌是主要的早期定植菌，通過特異性表面蛋白與唾液中蛋白結(jié)合，黏附定植于牙齒表面[32]。鏈球菌本身的表面蛋白也為其他口腔細(xì)菌提供了黏附和聚集的位點(diǎn)[33]。因此，初始黏附的細(xì)菌（主要是鏈球菌）為生物膜的成熟奠定了基礎(chǔ)。臨床研究發(fā)現(xiàn)，血鏈球菌與低齲風(fēng)險(xiǎn)相關(guān)[34-35]；寡發(fā)酵鏈球菌與齲病發(fā)生呈負(fù)相關(guān)[36]，提示在生物膜形成初期，早期黏附的鏈球菌可通過產(chǎn)生H2O2抑制齲病的機(jī)會(huì)致病菌（如變異鏈球菌）的生長(zhǎng)和定植，對(duì)早期生物膜健康發(fā)展具有重要意義。

H2O2可通過對(duì)微生物的殺傷作用在生物膜形成過程中調(diào)節(jié)生物膜的組成。在生物膜形成過程中，細(xì)菌間的距離是一個(gè)重要因素[37]。Liu等[38]通過測(cè)量距戈登鏈球菌生物膜不同距離H2O2的濃度發(fā)現(xiàn)，在距生物膜表面100 μm處H2O2濃度為1.4 mmol·L-1，而在距離生物膜表面200 μm處H2O2的濃度為0.4 mmol·L-1。1.4 mmol·L-1是一個(gè)可以抑制H2O2敏感細(xì)菌的濃度，提示細(xì)菌產(chǎn)生的H2O2主要作用于那些與其相距較近的細(xì)菌。生物膜形成過程中H2O2濃度梯度的存在提示H2O2可通過對(duì)不同H2O2敏感性的微生物進(jìn)行篩選，調(diào)節(jié)生物膜的組成。H2O2可穿過細(xì)菌細(xì)胞膜，對(duì)細(xì)菌的殺傷作用機(jī)制包括破壞細(xì)菌的酶活性或直接氧化細(xì)菌大分子（蛋白質(zhì)和DNA）[39]。H2O2的氧自由基可與Fe2+發(fā)生反應(yīng)，破壞對(duì)酶活性進(jìn)行調(diào)控的鐵硫簇[4Fe-4S]結(jié)構(gòu)[40]。此外，細(xì)菌細(xì)胞內(nèi)H2O2的積累會(huì)導(dǎo)致在精氨酸、賴氨酸、脯氨酸及蘇氨酸殘基的側(cè)鏈中引入羰基引起不可逆的蛋白質(zhì)羰基化，介導(dǎo)蛋白質(zhì)的靶向降解[41-43]。

口腔細(xì)菌可以通過產(chǎn)生H2O2影響細(xì)菌eDNA的釋放和細(xì)菌感受態(tài)的形成，從而影響基因的水平轉(zhuǎn)移，進(jìn)而調(diào)節(jié)生物膜對(duì)口腔多變環(huán)境的適應(yīng)能力。口腔細(xì)菌為適應(yīng)多變的口腔環(huán)境進(jìn)化出了一系列的應(yīng)對(duì)措施，包括基因的水平轉(zhuǎn)移[1,44-45]。接合、轉(zhuǎn)導(dǎo)和轉(zhuǎn)化是微生物基因水平轉(zhuǎn)移的3種形式[46]。轉(zhuǎn)化是處于感受態(tài)的細(xì)菌獲取eDNA進(jìn)行DNA同源交換的過程[47]。通過同源交換，細(xì)菌可以獲取新的基因適應(yīng)環(huán)境（如抗性基因、代謝相關(guān)基因等），同時(shí)也可修復(fù)突變的基因以維持其遺傳穩(wěn)定性[48]。細(xì)菌eDNA的釋放依賴胞外細(xì)菌素、胞壁酸水解酶、胞內(nèi)細(xì)菌素和H2O2等途徑[49]。Kreth等[14]發(fā)現(xiàn)，戈登鏈球菌和血鏈球菌染色體DNA的釋放與H2O2產(chǎn)生密切相關(guān)，SpxB缺陷株H2O2產(chǎn)生減少，染色體DNA的釋放也相應(yīng)減少。Itzek等[50]研究發(fā)現(xiàn)，戈登鏈球菌在厭氧環(huán)境下H2O2合成降低，其染色體DNA的釋放也減少；在厭氧條件下加入H2O2會(huì)導(dǎo)致DNA的釋放增加。盡管H2O2導(dǎo)致細(xì)菌DNA釋放的具體機(jī)制尚不清楚，但該研究指出H2O2并不是通過介導(dǎo)細(xì)菌裂解進(jìn)而促使DNA的釋放。細(xì)菌H2O2的產(chǎn)生也伴隨著細(xì)菌感受態(tài)的產(chǎn)生[50-51]，可以推測(cè)細(xì)菌在感知到H2O2所介導(dǎo)的DNA損傷后，可通過一些未知的分子機(jī)制釋放DNA并形成感受態(tài)細(xì)胞，通過轉(zhuǎn)化修復(fù)基因損傷與突變。

口腔微生物能通過一系列機(jī)制減少H2O2的損害。部分口腔細(xì)菌能利用過氧化氫酶減少H2O2對(duì)其的傷害?！案甑擎溓蚓蛢?nèi)氏放線菌”是口腔細(xì)菌相互作用的經(jīng)典研究模型，戈登鏈球菌可產(chǎn)生H2O2抑制內(nèi)氏放線菌[52-53]。值得注意的是，戈登鏈球菌不能產(chǎn)生過氧化氫酶緩解H2O2對(duì)自身的傷害。Jakubovics等[3,53]發(fā)現(xiàn)，內(nèi)氏放線菌可通過自身過氧化氫酶不斷消耗H2O2，從而減少H2O2對(duì)自身和戈登鏈球菌的傷害。另有研究表明，口腔細(xì)菌中的很多基因與耐受H2O2密切相關(guān)。“血鏈球菌和變異鏈球菌”以及“戈登鏈球菌和變異鏈球菌”是經(jīng)典的口腔相互競(jìng)爭(zhēng)研究模型，血鏈球菌和戈登鏈球菌主要產(chǎn)生H2O2抑制變異鏈球菌，而變異鏈球菌可產(chǎn)生變鏈素抑制血鏈球菌和戈登鏈球菌[14,54-55]。Xu等[2]分別觀察了血鏈球菌和戈登鏈球菌dps、trxB和sodA三個(gè)基因突變株對(duì)H2O2的耐受情況，發(fā)現(xiàn)血鏈球菌和戈登鏈球菌Dps突變株對(duì)H2O2極為敏感；血鏈球菌的TrxB的突變株和戈登鏈球菌SodA突變株對(duì)H2O2的敏感也顯著增加；而血鏈球菌SodA和戈登鏈球菌TrxB突變株對(duì)H2O2的耐受無明顯變化，提示戈登鏈球菌Dps和SodA及血鏈球菌Dsp和TrxB在相關(guān)細(xì)菌耐受H2O2過程中發(fā)揮了重要作用。另外，該研究還發(fā)現(xiàn)戈登鏈球菌比血鏈球菌更耐受H2O2，推測(cè)戈登鏈球菌在牙菌斑中的豐度較低，因此需要更強(qiáng)的耐受力進(jìn)而在牙菌斑中獲得生長(zhǎng)優(yōu)勢(shì)。Zheng等[56]發(fā)現(xiàn)，與野生株相比，變異鏈球菌谷胱甘肽合成酶基因缺失株對(duì)H2O2更敏感；進(jìn)一步通過血鏈球菌和變異鏈球菌雙菌種實(shí)驗(yàn)證明谷胱甘肽合成酶在變異鏈球菌耐受H2O2過程中有重要作用。此外，產(chǎn)H2O2細(xì)菌為了減少H2O2對(duì)自身的傷害，可通過Mn2+代替[4Fe-4S]結(jié)構(gòu)中的Fe2+，減少細(xì)胞內(nèi)依賴鐵硫簇調(diào)控活性的蛋白質(zhì)[57-58]。

2.2 H2O2在微生物與宿主交互關(guān)系中的作用

H2O2參與了微生物與宿主之間的交互作用。口腔微生物與宿主口腔細(xì)胞相互接近，微生物與宿主間存在頻繁的交互作用。微生物可產(chǎn)生對(duì)宿主有毒害作用的H2O2，對(duì)宿主造成損害。Ramsey等[59]發(fā)現(xiàn)，戈登鏈球菌產(chǎn)生的H2O2誘導(dǎo)伴放線菌嗜血菌表達(dá)基因katA和apiA，促使伴放線菌嗜血菌逃脫宿主的免疫系統(tǒng)。Okahashi等[60]發(fā)現(xiàn)，血鏈球菌可產(chǎn)生H2O2導(dǎo)致人類巨噬細(xì)胞死亡，抑制血鏈球菌ROS的產(chǎn)生可有效減少血鏈球菌對(duì)巨噬細(xì)胞的毒害作用。Okahashi等[61]進(jìn)一步研究了口腔鏈球菌SpxB對(duì)人類巨噬細(xì)胞的作用，闡明了口腔鏈球菌產(chǎn)生H2O2導(dǎo)致人類巨噬細(xì)胞死亡的機(jī)制。Okahashi等[4]還發(fā)現(xiàn)，口腔鏈球菌產(chǎn)生的H2O2可誘導(dǎo)宿主上皮細(xì)胞白細(xì)胞介素-6的表達(dá)，引起上皮細(xì)胞死亡。宿主細(xì)胞也能通過合成H2O2，起到殺菌或化學(xué)屏障的作用[28,62]。例如巨噬細(xì)胞針對(duì)病原菌的氧化爆發(fā)過程產(chǎn)生大量的ROS（包括H2O2），可殺滅病原菌或?qū)⒉≡帘卧谟蒆2O2組成的化學(xué)屏障外，進(jìn)而保護(hù)宿主。有意思的是，細(xì)菌還可以通過產(chǎn)生H2O2減少條件致病菌對(duì)宿主的侵害，乳桿菌可以通過產(chǎn)生H2O2抑制白假絲酵母菌對(duì)上皮細(xì)胞的黏附[63]。

在口腔中，生物膜中的微生物持續(xù)處于“戰(zhàn)爭(zhēng)與和平”的狀態(tài)，致病菌與宿主之間也時(shí)刻展開著“斗爭(zhēng)”。微生物可通過一系列途徑產(chǎn)生H2O2，限制有限生存資源內(nèi)的競(jìng)爭(zhēng)對(duì)手，并采取措施減少H2O2對(duì)自身的損害。H2O2是研究產(chǎn)H2O2菌株與對(duì)H2O2敏感菌間交互關(guān)系的良好切入口。H2O2在微生物與宿主間的關(guān)系中也發(fā)揮著重要的調(diào)節(jié)作用。系統(tǒng)研究H2O2在口腔微生態(tài)中的作用，揭示H2O2在維持口腔微生態(tài)平衡中的更多功能，有利于更好地理解口腔微生態(tài)系統(tǒng)。

[1] Roberts AP, Kreth J. The impact of horizontal gene transfer on the adaptive ability of the human oral microbiome[J]. Front Cell Infect Microbiol, 2014, 4:124.

[2] Xu Y, Itzek A, Kreth J. Comparison of genes required for H2O2resistance inStreptococcus gordoniiandStreptococcus sanguinis[J]. Microbiology, 2014, 160(Pt 12):2627-2638.

[3] Jakubovics NS, Gill SR, Vickerman MM, et al. Role of hydrogen peroxide in competition and cooperation betweenStreptococcus gordoniiandActinomyces naeslundii[J]. FEMS Microbiol Ecol, 2008, 66(3):637-644.

[4] Okahashi N, Sumitomo T, Nakata M, et al. Hydrogen peroxide contributes to the epithelial cell death induced by the oral mitis group of streptococci[J]. PLoS ONE, 2014, 9(1): e88136.

[5] Starkov AA. The role of mitochondria in reactive oxygen species metabolism and signaling[J]. Ann N Y Acad Sci, 2008, 1147:37-52.

[6] Halliwell B, Clement MV, Long LH. Hydrogen peroxide in the human body[J]. FEBS Lett, 2000, 486(1):10-13.

[7] Hillier SL, Krohn MA, Klebanoff SJ, et al. The relationship of hydrogen peroxide-producing lactobacilli to bacterial vaginosis and genital microflora in pregnant women[J]. Obstet Gynecol, 1992, 79(3):369-373.

[8] Ryan CS, Kleinberg I. Bacteria in human mouths involved in the production and utilization of hydrogen peroxide[J]. Arch Oral Biol, 1995, 40(8):753-763.

[9] García-Mendoza A, Liébana J, Castillo AM, et al. Evaluation of the capacity of oral streptococci to produce hydrogen peroxide[J]. J Med Microbiol, 1993, 39(6):434-439.

[10] Tong H, Chen W, Merritt J, et al.Streptococcus oligofermentansinhibitsStreptococcus mutansthrough conversion of lactic acid into inhibitory H2O2: a possible counteroffensive strategy for interspecies competition[J]. Mol Microbiol, 2007, 63(3):872-880.

[11] Barnard JP, Stinson MW. Influence of environmental conditions on hydrogen peroxide formation byStreptococcus gordonii[J]. Infect Immun, 1999, 67(12):6558-6564.

[12] Scoffield JA, Wu H. Oral streptococci and nitrite-mediated interference ofPseudomonas aeruginosa[J]. Infect Immun, 2015, 83(1):101-107.

[13] Lucas VS, Beighton D, Roberts GJ. Composition of the oral streptococcal flora in healthy children[J]. J Dent, 2000, 28 (1):45-50.

[14] Kreth J, Zhang Y, Herzberg MC.Streptococcal antagonismin oral biofilms:Streptococcus sanguinisandStreptococcus gordoniiinterference withStreptococcus mutans[J]. J Bacteriol, 2008, 190(13):4632-4640.

[15] Liu L, Tong H, Dong X. Function of the pyruvate oxidaselactate oxidase cascade in interspecies competition betweenStreptococcus oligofermentansandStreptococcus mutans[J]. Appl Environ Microbiol, 2012, 78(7):2120-2127.

[16] Higuchi M, Shimada M, Yamamoto Y, et al. Identification of two distinct NADH oxidases corresponding to H2O2-formingoxidase and H2O-forming oxidase induced inStreptococcus mutans[J]. J Gen Microbiol, 1993, 139(10):2343-2351.

[17] Tong H, Chen W, Shi W, et al. SO-LAAO, a novel L-amino acid oxidase that enablesStreptococcus oligofermentansto outcompeteStreptococcus mutansby generating H2O2from peptone[J]. J Bacteriol, 2008, 190(13):4716-4721.

[18] Ramos-Monta?ez S, Tsui HC, Wayne KJ, et al. Polymorphism and regulation of the spxB (pyruvate oxidase) virulence factor gene by a CBS-HotDog domain protein (SpxR) in serotype 2Streptococcus pneumoniae[J]. Mol Microbiol, 2008, 67 (4):729-746.

[19] Zheng L, Itzek A, Chen Z, et al. Environmental influences on competitive hydrogen peroxide production inStreptococcus gordonii[J]. Appl Environ Microbiol, 2011, 77(13):4318-4328.

[20] Zheng L, Chen Z, Itzek A, et al. Catabolite control protein A controls hydrogen peroxide production and cell death inStreptococcus sanguinis[J]. J Bacteriol, 2011, 193(2):516-526.

[21] Chen L, Ge X, Wang X, et al. SpxA1 involved in hydrogen peroxide production, stress tolerance and endocarditis virulence inStreptococcus sanguinis[J]. PLoS ONE, 2012, 7 (6):e40034.

[22] Chen L, Ge X, Dou Y, et al. Identification of hydrogen peroxide production-related genes inStreptococcus sanguinisand their functional relationship with pyruvate oxidase[J]. Microbiology, 2011, 157(Pt 1):13-20.

[23] Zhu L, Xu Y, Ferretti JJ, et al. Probing oral microbial functionality—expression of spxB in plaque samples[J]. PLoS ONE, 2014, 9(1):e86685.

[24] Kanta J. The role of hydrogen peroxide and other reactive oxygen species in wound healing[J]. Acta Medica, 2011, 54(3):97-101.

[25] Lambert AJ, Brand MD. Reactive oxygen species production by mitochondria[J]. Methods Mol Biol, 2009, 554:165-181.

[26] Murphy MP. How mitochondria produce reactive oxygen species[J]. Biochem J, 2009, 417(1):1-13.

[27] Turrens JF. Mitochondrial formation of reactive oxygen species[J]. J Physiol, 2003, 552(Pt 2):335-344.

[28] Leto TL, Geiszt M. Role of Nox family NADPH oxidases in host defense[J]. Antioxid Redox Signal, 2006, 8(9/10): 1549-1561.

[29] Ozmeric N. Advances in periodontal disease markers[J]. Clin Chim Acta, 2004, 343(1/2):1-16.

[30] Vidovi? A, Vidovi? Juras D, Vu?i?evi? Boras V, et al. Determination of leucocyte subsets in human saliva by flow cytometry[J]. Arch Oral Biol, 2012, 57(5):577-583.

[31] Geiszt M, Witta J, Baffi J, et al. Dual oxidases represent novel hydrogen peroxide sources supporting mucosal surface host defense[J]. FASEB J, 2003, 17(11):1502-1504.

[32] Kolenbrander PE, Palmer RJ Jr, Rickard AH, et al. Bacterial interactions and successions during plaque development[J]. Periodontol 2000, 2006, 42:47-79.

[33] Nobbs AH, Lamont RJ, Jenkinson HF.Streptococcus adherenceand colonization[J]. Microbiol Mol Biol Rev, 2009, 73(3):407-450.

[34] Nyvad B, Kilian M. Comparison of the initial streptococcal microflora on dental enamel in caries-active and in cariesinactive individuals[J]. Caries Res, 1990, 24(4):267-272.

[35] Becker MR, Paster BJ, Leys EJ, et al. Molecular analysis of bacterial species associated with childhood caries[J]. J Clin Microbiol, 2002, 40(3):1001-1009.

[36] Zhang J, Tong HC, Dong XZ, et al. A preliminary study of biological characteristics ofStreptococcus oligofermentansin oral microecology[J]. Caries Res, 2010, 44(4):345-348. [37] Kolenbrander PE, Palmer RJ Jr, Periasamy S, et al. Oral multispecies biofilm development and the key role of cellcell distance[J]. Nat Rev Microbiol, 2010, 8(7):471-480.

[38] Liu X, Ramsey MM, Chen X, et al. Real-time mapping of a hydrogen peroxide concentration profile across a polymicrobial bacterial biofilm using scanning electrochemical microscopy[J]. Proc Natl Acad Sci USA, 2011, 108(7):2668-2673.

[39] Imlay JA. Pathways of oxidative damage[J]. Annu Rev Microbiol, 2003, 57:395-418.

[40] Jang S, Imlay JA. Micromolar intracellular hydrogen peroxide disrupts metabolism by damaging iron-sulfur enzymes [J]. J Biol Chem, 2007, 282(2):929-937.

[41] Nystr?m T. Role of oxidative carbonylation in protein quality control and senescence[J]. EMBO J, 2005, 24(7):1311-1317.

[42] Stadtman ER, Levine RL. Free radical-mediated oxidation of free amino acids and amino acid residues in proteins[J]. Amino Acids, 2003, 25(3/4):207-218.

[43] Grune T, Merker K, Sandig G, et al. Selective degradation of oxidatively modified protein substrates by the proteasome [J]. Biochem Biophys Res Commun, 2003, 305(3):709-718.

[44] Fux CA, Costerton JW, Stewart PS, et al. Survival strategies of infectious biofilms[J]. Trends Microbiol, 2005, 13(1):34-40.

[45] Hannan S, Ready D, Jasni AS, et al. Transfer of antibiotic resistance by transformation with eDNA within oral biofilms[J]. FEMS Immunol Med Microbiol, 2010, 59(3):345-349.

[46] Gal-Mor O, Finlay BB. Pathogenicity islands: a molecular toolbox for bacterial virulence[J]. Cell Microbiol, 2006, 8 (11):1707-1719.

[47] Smillie CS, Smith MB, Friedman J, et al. Ecology drives a global network of gene exchange connecting the human microbiome[J]. Nature, 2011, 480(7376):241-244.

[48] Kn?ppel A, Lind PA, Lustig U, et al. Minor fitness costs in an experimental model of horizontal gene transfer in bacteria [J]. Mol Biol Evol, 2014, 31(5):1220-1227.

[49] Jakubovics NS, Burgess JG. Extracellular DNA in oral microbial biofilms[J]. Microbes Infect, 2015, 17(7):531-537.

[50] Itzek A, Zheng L, Chen Z, et al. Hydrogen peroxide-dependent DNA release and transfer of antibiotic resistance genes in Streptococcus gordonii[J]. J Bacteriol, 2011, 193(24):6912-6922.

[51] Prudhomme M, Attaiech L, Sanchez G, et al. Antibiotic stress induces genetic transformability in the human pathogenStreptococcus pneumoniae[J]. Science, 2006, 313(5783): 89-92.

[52] Stacy A, Everett J, Jorth P, et al. Bacterial fight-and-flight responses enhance virulence in a polymicrobial infection[J]. Proc Natl Acad Sci USA, 2014, 111(21):7819-7824.

[53] Jakubovics NS, Gill SR, Iobst SE, et al. Regulation of gene expression in a mixed-genus community: stabilized arginine biosynthesis inStreptococcus gordoniiby coaggregation withActinomyces naeslundii[J]. J Bacteriol, 2008, 190(10): 3646-3657.

[54] Kreth J, Merritt J, Shi W, et al. Competition and coexistence betweenStreptococcus mutansandStreptococcus sanguinisin the dental biofilm[J]. J Bacteriol, 2005, 187(21):7193-7203.

[55] Ge Y, Caufield PW, Fisch GS, et al.Streptococcus mutansandStreptococcus sanguiniscolonization correlated with caries experience in children[J]. Caries Res, 2008, 42(6): 444-448.

[56] Zheng X, Zhang K, Zhou X, et al. Involvement of gshAB in the interspecies competition within oral biofilm[J]. J Dent Res, 2013, 92(9):819-824.

[57] Jakubovics NS, Smith AW, Jenkinson HF. Oxidative stress tolerance is manganese Mn2+regulated inStreptococcus gordonii[J]. Microbiology, 2002, 148(Pt 10):3255-3263.

[58] Tseng HJ, McEwan AG, Paton JC, et al. Virulence ofStreptococcus pneumoniae: PsaA mutants are hypersensitive to oxidative stress[J]. Infect Immun, 2002, 70(3):1635-1639.

[59] Ramsey MM, Whiteley M. Polymicrobial interactions stimulate resistance to host innate immunity through metabolite perception[J]. Proc Natl Acad Sci USA, 2009, 106(5):1578-1583.

[60] Okahashi N, Okinaga T, Sakurai A, et al.Streptococcus sanguinisinduces foam cell formation and cell death of macrophages in association with production of reactive oxygen species[J]. FEMS Microbiol Lett, 2011, 323(2):164-170.

[61] Okahashi N, Nakata M, Sumitomo T, et al. Hydrogen peroxide produced by oral streptococci induces macrophage cell death[J]. PLoS ONE, 2013, 8(5):e62563.

[62] Allaoui A, Botteaux A, Dumont JE, et al. Dual oxidases and hydrogen peroxide in a complex dialogue between host mucosae and bacteria[J]. Trends Mol Med, 2009, 15(12):571-579.

[63] Morales DK, Hogan DA.Candida albicansinteractions with bacteria in the context of human health and disease[J]. PLoS Pathog, 2010, 6(4):e1000886.

（本文編輯 吳愛華）

The origin of hydrogen peroxide in oral cavity and its role in oral microecology balance

Zhang Keke, Zhou Xuedong, Xu Xin. (State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China)

Hydrogen peroxide, an important antimicrobial agent in oral cavity, plays a significant role in the balance of oral microecology. At the early stage of biofilm formation, about 80% of the detected initial colonizers belong to the genus Streptococcus. These oral streptococci use different oxidase to produce hydrogen peroxide. Recent studies showed that the produced hydrogen peroxide plays a critical role in modulating oral microecology. Hydrogen peroxide modulates biofilm development attributed to its growth inhibitory nature. Hydrogen peroxide production is closely associated with extracellular DNA(eDNA) release from microbe and the development of its competent cell which are critical for biofilm development and also serves as source for horizontal gene transfer. Microbe also can reduce the damage to themselves through several detoxification mechanisms. Moreover, hydrogen peroxide is also involved in the regulation of interactions between oral microorganisms and host. Taken together, hydrogen peroxide is an imperative ecological factor that contributes to the microbial equilibrium in the oral cavity. Here we will give a brief review of both the origin and the function in the oral microecology balance of hydrogen peroxide.

hydrogen peroxide; oral microbe; oral microecology; biofilm; microorganisms and host

R 780.2

A

10.7518/hxkq.2017.02.020

Supported by: The National Natural Science Foundation of China (81170959, 81200782, 81430011, 81371135). Correspondence: Xu Xin, E-mail: nixux1982@hotmail.com.

2016-10-15；

2017-01-12

國(guó)家自然科學(xué)基金（81170959，81200782，81430011，81371135）

章可可，博士，E-mail：593572773@qq.com

徐欣，副教授，博士，E-mail：nixux1982@hotmail.com