本文選題：腸球菌 + 萬(wàn)古霉素耐藥��； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2011年碩士論文

【摘要】：腸球菌通常為條件致病菌。當(dāng)發(fā)生異位寄生時(shí),腸球菌可引起多種感染,如泌尿系感染、心內(nèi)膜炎、腦膜炎、敗血癥、傷口感染、呼吸道感染等,嚴(yán)重的可危及生命。與其他革蘭氏陽(yáng)性菌相比,腸球菌屬具有更強(qiáng)的天然耐藥性,且易被抗生素誘導(dǎo)產(chǎn)生新的耐藥性。近年來(lái),由于免疫抑制劑在臨床治療中廣泛應(yīng)用,以及廣譜抗生素的過(guò)度使用及不合理應(yīng)用等因素導(dǎo)致腸球菌屬感染和耐藥性日益增多,已成為院內(nèi)感染的主要致病菌。隨著耐萬(wàn)古霉素腸球菌(Vancomycin-Resistant Enterococci,VRE)的出現(xiàn),臨床治療耐藥菌感染面臨著更大的困難。VRE的耐藥基因可通過(guò)質(zhì)粒轉(zhuǎn)移給其他致病菌,隨著近年來(lái)報(bào)道的由VRE導(dǎo)致的耐萬(wàn)古霉素金黃色葡萄球菌( Vancomycin-Resistant Staphylococcus aureus,VRSA)的出現(xiàn),人們對(duì)VRE的耐藥機(jī)制更加關(guān)注。 本文對(duì)臨床分離到的耐萬(wàn)古霉素糞腸球菌(Enterococcus Faecalis V309,EF V309)菌株進(jìn)行了分型鑒定,特性研究及比較蛋白質(zhì)組學(xué)研究,進(jìn)一步闡明了糞腸球菌對(duì)萬(wàn)古霉素的耐藥機(jī)制,并首次對(duì)萬(wàn)古霉素對(duì)耐藥基因誘導(dǎo)的特異性和可逆性及萬(wàn)古霉素誘導(dǎo)下菌體蛋白磷酸化修飾作用進(jìn)行了研究,為控制耐藥基因的傳播和指導(dǎo)臨床用藥奠定基礎(chǔ)。 首先,利用蛋白質(zhì)雙向電泳技術(shù),對(duì)萬(wàn)古霉素耐藥糞腸球菌標(biāo)準(zhǔn)菌株EFV583及臨床分離耐萬(wàn)古霉素糞腸球菌菌株EFV309在加藥誘導(dǎo)前后及加藥后去除藥物等條件下的菌體蛋白表達(dá)情況進(jìn)行了對(duì)比分析。通過(guò)ImageMaster軟件分析各組電泳圖譜,找出差異蛋白點(diǎn),對(duì)表達(dá)量差異3倍以上的蛋白質(zhì)點(diǎn)進(jìn)行質(zhì)譜鑒定。利用多功能串聯(lián)飛行時(shí)間質(zhì)譜(MALDI-TOF/TOF-MS),共鑒定到57個(gè)有意義的蛋白,其中大多數(shù)蛋白質(zhì)點(diǎn)的pI和分子量與理論值相符,但也有一些存在差異。 本研究在V309中鑒定到20個(gè)上調(diào)蛋白、6個(gè)下調(diào)蛋白及4個(gè)發(fā)生位移的蛋白,在V583中鑒定到28個(gè)上調(diào)蛋白、8個(gè)下調(diào)蛋白及1個(gè)發(fā)生位移的蛋白。利用半定量RT-PCR在反轉(zhuǎn)錄水平上也驗(yàn)證了這些蛋白編碼基因表達(dá)量的相應(yīng)變化。其中包括部分已知耐藥相關(guān)蛋白VanA、VanX(V309)及VanB、VanXB(V583),它們?cè)谌f(wàn)古霉素誘導(dǎo)下均有20倍以上的上調(diào)表達(dá),且存在翻譯后修飾。值得關(guān)注的是在V309中VanA、VanX在未經(jīng)萬(wàn)古霉素誘導(dǎo)時(shí)也表達(dá),這與V583不同,表明兩者耐藥的分子機(jī)制不盡相同。此外,還鑒定到一些耐藥機(jī)制尚不十分明確的蛋白,包括毒力相關(guān)因子(EF2076、EF0577、EF3256)、應(yīng)激蛋白(EF1308、EF1744、EF0080)、代謝相關(guān)蛋白、翻譯相關(guān)蛋白、接合轉(zhuǎn)移相關(guān)蛋白及一些假想蛋白。這些蛋白可能與細(xì)胞粘附機(jī)制、耐藥性的轉(zhuǎn)移與表達(dá)、耐藥基因的獲取與基因重組修復(fù)、菌體內(nèi)酶的活化與抑制有密切關(guān)系。我們對(duì)這些蛋白在菌體中可能發(fā)揮的功能進(jìn)行了分析。 在對(duì)萬(wàn)古霉素對(duì)耐藥基因誘導(dǎo)的特異性的研究中,將EFV583、EFV309菌株在萬(wàn)古霉素誘導(dǎo)培養(yǎng)8h、12h、16h后,隨著藥物誘導(dǎo)時(shí)間的延長(zhǎng),耐藥相關(guān)蛋白VanA、VanB、VanX的表達(dá)量也隨之明顯升高,說(shuō)明了藥物對(duì)這些蛋白的誘導(dǎo)是特異性的。在對(duì)萬(wàn)古霉素對(duì)耐藥基因誘導(dǎo)的可逆性研究中,將EFV583、EFV309菌株在不加和加萬(wàn)古霉素誘導(dǎo)培養(yǎng)6h后,分別去除和不去除萬(wàn)古霉素誘導(dǎo),繼續(xù)培養(yǎng)6h。結(jié)果顯示當(dāng)藥物誘導(dǎo)因素去除后,在誘導(dǎo)培養(yǎng)時(shí)表達(dá)量升高的耐藥相關(guān)蛋白VanA、VanB、VanX表達(dá)量又明顯降低,說(shuō)明了藥物對(duì)這些蛋白的誘導(dǎo)是可逆性的。通過(guò)RT-PCR的檢測(cè),證實(shí)耐藥相關(guān)蛋白編碼基因在各個(gè)條件下表達(dá)量的變化與對(duì)應(yīng)蛋白的變化一致。 此外,有6個(gè)蛋白可能存在翻譯后修飾(Gpm、Ldh、Gap2、RpsB、EF2076、EF3256)。菌體中存在的蛋白翻譯后修飾,通常是由于氨基酸修飾或肽段斷裂引起的蛋白電荷或分子量的改變,表現(xiàn)為蛋白點(diǎn)豐度或位置的遷移。本研究中發(fā)現(xiàn)V583及V309分別有1個(gè)及4個(gè)蛋白在萬(wàn)古霉素誘導(dǎo)培養(yǎng)后發(fā)生了位移,即Esa (EF2076)及Pgm1 (EF0195)、Ldh (EF0255)、Gap-2 (EF1964)、RpsB (EF2398),推測(cè)可能是磷酸化修飾或其他修飾引起分子量及等電點(diǎn)變化的結(jié)果。利用Pro-Q染色及Western Blots技術(shù)共鑒定到3個(gè)磷酸化蛋白Ldh、Gap-2、cAD1,通過(guò)PHOSIDA數(shù)據(jù)庫(kù)進(jìn)行同源性比對(duì),確定了其可能的磷酸化位點(diǎn)。這也是首次在糞腸球菌中鑒定到磷酸化蛋白。 其中性激素cAD1前體脂蛋白(EF3256)有5個(gè)同源異構(gòu)體,通過(guò)Pro-Q染色發(fā)現(xiàn)它存在明顯的磷酸化翻譯后修飾,且在經(jīng)萬(wàn)古霉素誘導(dǎo)培養(yǎng)后的菌體中其磷酸化程度均明顯增強(qiáng),Western Blot檢測(cè)證實(shí)在EF3256中同時(shí)存在絲氨酸和蘇氨酸的磷酸化。但性激素cAD1前體脂蛋白既沒(méi)有已知的磷酸化功能域(Phosphorylation motif, PMF)也無(wú)法在相關(guān)磷酸化肽段數(shù)據(jù)庫(kù)(PHOSIDA)中找到其同源性結(jié)構(gòu)。已知許多耐藥因子的水平傳播歸因于性激素依賴的質(zhì)粒轉(zhuǎn)移,但目前有關(guān)性激素cAD1前體脂蛋白的磷酸化及其在細(xì)胞信號(hào)調(diào)節(jié)中的作用都未有過(guò)報(bào)道,因此需要進(jìn)一步對(duì)其功能進(jìn)行研究。 在糞腸球菌V583中,性激素cAD1前體脂蛋白由309個(gè)氨基酸組成,在信號(hào)序列中有一個(gè)22個(gè)氨基酸組成的帶有半胱氨酸殘基的脂質(zhì)區(qū),最后的8個(gè)氨基酸構(gòu)成了cAD1,位于脂蛋白信號(hào)序列之后的Sec依賴的輸出部位為假定的信號(hào)肽切割位點(diǎn)。由于另一種能夠?qū)⑿约に豤CF10轉(zhuǎn)移至胞內(nèi)的性激素轉(zhuǎn)運(yùn)蛋白(OppA-like protien)在萬(wàn)古霉素誘導(dǎo)的V309菌株中明顯上調(diào),因此胞外的cAD1應(yīng)是與OppA-like膜表面脂蛋白結(jié)合提高了受體菌的感受性,并經(jīng)由ABC肽轉(zhuǎn)運(yùn)系統(tǒng)吸收。這暗示了萬(wàn)古霉素能增加質(zhì)粒發(fā)生接合轉(zhuǎn)移的可能性,并增強(qiáng)對(duì)外源細(xì)菌的感受性。我們推測(cè)V583和V309中磷酸化性激素cAD1前體脂蛋白可能是一種激活狀態(tài),在性激素在細(xì)胞信號(hào)傳導(dǎo)中起重要作用。通過(guò)誘導(dǎo)毒力相關(guān)基因表達(dá)及質(zhì)粒的接合轉(zhuǎn)移,萬(wàn)古霉素使糞腸球菌更容易獲得或傳播質(zhì)粒,尤其對(duì)于帶有耐藥基因的菌株。 糞腸球菌的耐藥性,尤其是對(duì)萬(wàn)古霉素的獲得性耐藥是與其基因組特征緊密相關(guān)的。目前,可能已有難以估量的毒力相關(guān)及耐藥移動(dòng)元件被糞腸球菌基因組整合。本文中對(duì)糞腸球菌V583和V309的比較蛋白質(zhì)組學(xué)研究豐富了對(duì)其生理特性的研究,同時(shí)也支持由Paulsen等提出的移動(dòng)DNA元件作為萬(wàn)古霉素耐藥性的轉(zhuǎn)移因子及糖肽類耐藥分子機(jī)制的假設(shè)。更重要的是,通過(guò)該研究確定了部分萬(wàn)古霉素耐藥相關(guān)蛋白,并且證實(shí)了一些在萬(wàn)古霉素調(diào)節(jié)作用下差異性表達(dá)的抗生素耐藥蛋白激發(fā)了內(nèi)源信號(hào)調(diào)整因子、粘附因子及代謝相關(guān)基因的表達(dá)。這一系列反應(yīng)都使得糞腸球菌能夠在萬(wàn)古霉素壓力下繼續(xù)生存和引起發(fā)病。本研究結(jié)果對(duì)揭示糞腸球菌耐藥的分子機(jī)制,從而找到相應(yīng)的控制策略具有重要的意義。
[Abstract]:Enterococci are usually conditional pathogens. Enterococcus can cause a variety of infections, such as urinary tract infection, endocarditis, meningitis, septicemia, wound infection, respiratory infection, and serious danger and life. Compared with other Gram-positive bacteria, Enterococcus has stronger natural resistance and is easily induced by antibiotics. In recent years, the widespread use of immunosuppressive agents in clinical treatment, as well as the excessive use of broad-spectrum antibiotics and irrational use of antibiotics have led to the increasing infection and resistance of Enterococcus, which has become the main pathogen of nosocomial infection. With vancomycin resistant Enterococcus (Vancomycin-Resistant Enteroco) The emergence of CCI, VRE), the clinical treatment of drug-resistant bacteria is facing greater difficulties..VRE resistance genes can be transferred to other pathogenic bacteria by plasmid. With the emergence of VRE Staphylococcus aureus (Vancomycin-Resistant Staphylococcus aureus, VRSA), which has been reported in recent years, the drug resistance mechanism of VRE is more closely related to the emergence of VRE. Note.
In this paper, the clinical isolates of vancomycin resistant Enterococcus faecalis (Enterococcus Faecalis V309, EF V309) were identified, characterized and compared with proteomics, and the resistance mechanism of Enterococcus faecalis to vancomycin was further clarified, and the specificity and reversibility of vancomycin on resistance genes were first introduced. The effect of fosamycin on the phosphorylation of protein was studied in order to lay a foundation for controlling the spread of drug-resistant genes and guiding clinical medication.
First, the protein expression of the standard strain EFV583 of vancomycin resistant Enterococcus faecalis and the clinical isolation of vancomycin resistant Enterococcus EFV309 were compared and analyzed by the ImageMaster software, and the electrophoresis of each group was analyzed by ImageMaster software. To identify the difference protein points and identify the protein points with more than 3 times the difference in the expression, 57 meaningful proteins were identified by the multifunction tandem time of flight mass spectrometry (MALDI-TOF/TOF-MS). The pI and molecular weight of most of the protein points were in accordance with the theoretical values, but there were some differences.
In this study, 20 up - regulated proteins, 6 down-regulated proteins and 4 displaced proteins were identified in the study. 28 up - regulated proteins, 8 down regulated proteins and 1 displaced proteins were identified in V583. The corresponding changes in the expression of these proteins were also verified by semi quantitative RT-PCR at the reverse transcriptional level, including some of them. VanA, VanX (V309) and VanB, VanXB (V583) are known to be up to 20 times up - regulated by vancomycin, and there are post-translational modifications. It is worth paying attention to the expression of VanA in V309 without vancomycin induction, which is different from V583, indicating that the molecular mechanisms of resistance are not the same. Some proteins, including EF2076 (EF0577, EF3256), stress protein (EF1308, EF1744, EF0080), metabolic related proteins, translation related proteins, conjugative transfer related proteins and some hypothetical proteins, are determined. These egg white may be associated with cell adhesion mechanism, transfer and expression of drug resistance, resistance genes It is closely related to the activation and inhibition of enzymes in the bacteria. We analyzed the possible functions of these proteins in the bacteria.
In the study of the specificity of vancomycin on the induction of resistance genes, EFV583, EFV309 strain was induced by vancomycin in the induction of 8h, 12h, and 16h, with the prolongation of drug induction time, the expression of drug resistance related protein VanA, VanB, VanX also increased obviously, indicating that the induction of these proteins was specific. In the reversible study of antibiotic resistance gene induction, EFV583, EFV309 strain was removed and no vancomycin induction after induction of 6h without adding vancomycin, and no vancomycin induction was removed. The results of continuous culture of 6h. showed that when the drug induction factors were removed, the expression of drug resistance related protein VanA, VanB, and VanX expression increased in the induction culture. The expression of VanX was clear. It was shown that the induction of these proteins was reversible. Through the detection of RT-PCR, it was proved that the changes in the expression of the resistant protein coding gene in each condition were in accordance with the changes of the corresponding protein.
In addition, 6 proteins may have post-translational modifications (Gpm, Ldh, Gap2, RpsB, EF2076, EF3256). The post-translational modification of protein in the mycelium, usually due to changes in the charge or molecular weight caused by amino acid modification or peptide fragment, shows the migration of protein abundance or location. In this study, there were 1 and 4 V309, respectively, of V583 and V309, respectively. After the induction of vancomycin, Esa (EF2076) and Pgm1 (EF0195), Ldh (EF0255), Gap-2 (EF1964), RpsB (EF2398), may be the result of the changes of molecular weight and isoelectric point caused by phosphorylation or other modification. 3 phosphorylated proteins were identified by Pro-Q dyeing and Western. D1, homology comparisons were made through PHOSIDA database, and the possible phosphorylation sites were identified. This is the first time that phosphorylated proteins have been identified in Enterococcus faecalis.
The sex hormone cAD1 precursor lipoprotein (EF3256) has 5 homologous isomers. Through Pro-Q staining, it is found that it has obvious phosphorylation posttranslational modification, and its phosphorylation degree is obviously enhanced in the mycelium induced by vancomycin, and Western Blot tests confirmed the phosphorylation of serine and threonine in EF3256. Sex hormone cAD1 precursor lipoproteins have neither the known phosphorylation domain (Phosphorylation motif, PMF) nor the homologous structure in the related phosphorylated peptide database (PHOSIDA). The horizontal transmission of a number of drug resistant factors is known to the plasmid transfer of sex hormone dependent plasmids, but at present, the phosphorus of the sex hormone cAD1 precursor lipoprotein is present. Acidification and its role in cellular signal regulation have not been reported, so we need to further study its function.
In Enterococcus faecalis V583, the sex hormone cAD1 precursor lipoprotein is composed of 309 amino acids. In the signal sequence, there is a lipid region with a cysteine residue of 22 amino acids. The final 8 amino acids constitute cAD1. The output site of the Sec dependence after the lipoprotein signal sequence is the presumed signal peptide cutting site. Another type of sex hormone transporter (OppA-like protien), which can transfer sex hormone cCF10 to the intracellular, is obviously up-regulated in the V309 strain induced by vancomycin. Therefore, the extracellular cAD1 should be combined with the OppA-like membrane surface lipoprotein to enhance receptor sensitivity and be absorbed through the ABC peptide transport system. This suggests that vancomycin can be increased. We speculate that the phosphorylated sex hormone cAD1 precursor lipoprotein in V583 and V309 may be an active state and plays an important role in the signal transduction of the sex hormone in the cell signal transduction. Cocci are more likely to acquire or transmit plasmids, especially for strains with resistance genes.
The drug resistance of Enterococcus faecalis, especially the acquired resistance to vancomycin, is closely related to its genomic characteristics. At present, there may be inestimable virulence related and drug resistant mobile elements being integrated with the genome of Enterococcus faecalis. In this paper, the comparative proteomic study of Enterococcus faecalis V583 and V309 enriches its physiological characteristics The study also supports the hypothesis that mobile DNA components, such as Paulsen, as the transfer factor of vancomycin resistance and the molecular mechanism of glycopeptide resistance. More importantly, some vancomycin resistance related proteins have been identified, and some of the differentially expressed antibiotics under the regulation of vancomycin are confirmed. The resistance protein stimulates the expression of endogenous signal regulating factors, adhesion factors and metabolic related genes. This series of reactions make Enterococcus faecalis survive and cause disease under the pressure of vancomycin. The results of this study are of great significance to reveal the molecular mechanism of resistance to Enterococcus faecalis and to find a corresponding control strategy.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R378

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本文編號(hào)：2106131