万古霉素诱导下粪肠球菌V583及临床分离株V309的比较蛋白质组学研究

发布时间：2018-07-07 20:11

本文选题：肠球菌 + 万古霉素耐药　；参考：《中国人民解放军军事医学科学院》2011年硕士论文

【摘要】：肠球菌通常为条件致病菌。当发生异位寄生时,肠球菌可引起多种感染,如泌尿系感染、心内膜炎、脑膜炎、败血症、伤口感染、呼吸道感染等,严重的可危及生命。与其他革兰氏阳性菌相比,肠球菌属具有更强的天然耐药性,且易被抗生素诱导产生新的耐药性。近年来,由于免疫抑制剂在临床治疗中广泛应用,以及广谱抗生素的过度使用及不合理应用等因素导致肠球菌属感染和耐药性日益增多,已成为院内感染的主要致病菌。随着耐万古霉素肠球菌(Vancomycin-Resistant Enterococci,VRE)的出现,临床治疗耐药菌感染面临着更大的困难。VRE的耐药基因可通过质粒转移给其他致病菌,随着近年来报道的由VRE导致的耐万古霉素金黄色葡萄球菌( Vancomycin-Resistant Staphylococcus aureus,VRSA)的出现,人们对VRE的耐药机制更加关注。本文对临床分离到的耐万古霉素粪肠球菌(Enterococcus Faecalis V309,EF V309)菌株进行了分型鉴定,特性研究及比较蛋白质组学研究,进一步阐明了粪肠球菌对万古霉素的耐药机制,并首次对万古霉素对耐药基因诱导的特异性和可逆性及万古霉素诱导下菌体蛋白磷酸化修饰作用进行了研究,为控制耐药基因的传播和指导临床用药奠定基础。首先,利用蛋白质双向电泳技术,对万古霉素耐药粪肠球菌标准菌株EFV583及临床分离耐万古霉素粪肠球菌菌株EFV309在加药诱导前后及加药后去除药物等条件下的菌体蛋白表达情况进行了对比分析。通过ImageMaster软件分析各组电泳图谱,找出差异蛋白点,对表达量差异3倍以上的蛋白质点进行质谱鉴定。利用多功能串联飞行时间质谱(MALDI-TOF/TOF-MS),共鉴定到57个有意义的蛋白,其中大多数蛋白质点的pI和分子量与理论值相符,但也有一些存在差异。本研究在V309中鉴定到20个上调蛋白、6个下调蛋白及4个发生位移的蛋白,在V583中鉴定到28个上调蛋白、8个下调蛋白及1个发生位移的蛋白。利用半定量RT-PCR在反转录水平上也验证了这些蛋白编码基因表达量的相应变化。其中包括部分已知耐药相关蛋白VanA、VanX(V309)及VanB、VanXB(V583),它们在万古霉素诱导下均有20倍以上的上调表达,且存在翻译后修饰。值得关注的是在V309中VanA、VanX在未经万古霉素诱导时也表达,这与V583不同,表明两者耐药的分子机制不尽相同。此外,还鉴定到一些耐药机制尚不十分明确的蛋白,包括毒力相关因子(EF2076、EF0577、EF3256)、应激蛋白(EF1308、EF1744、EF0080)、代谢相关蛋白、翻译相关蛋白、接合转移相关蛋白及一些假想蛋白。这些蛋白可能与细胞粘附机制、耐药性的转移与表达、耐药基因的获取与基因重组修复、菌体内酶的活化与抑制有密切关系。我们对这些蛋白在菌体中可能发挥的功能进行了分析。在对万古霉素对耐药基因诱导的特异性的研究中,将EFV583、EFV309菌株在万古霉素诱导培养8h、12h、16h后,随着药物诱导时间的延长,耐药相关蛋白VanA、VanB、VanX的表达量也随之明显升高,说明了药物对这些蛋白的诱导是特异性的。在对万古霉素对耐药基因诱导的可逆性研究中,将EFV583、EFV309菌株在不加和加万古霉素诱导培养6h后,分别去除和不去除万古霉素诱导,继续培养6h。结果显示当药物诱导因素去除后,在诱导培养时表达量升高的耐药相关蛋白VanA、VanB、VanX表达量又明显降低,说明了药物对这些蛋白的诱导是可逆性的。通过RT-PCR的检测,证实耐药相关蛋白编码基因在各个条件下表达量的变化与对应蛋白的变化一致。此外,有6个蛋白可能存在翻译后修饰(Gpm、Ldh、Gap2、RpsB、EF2076、EF3256)。菌体中存在的蛋白翻译后修饰,通常是由于氨基酸修饰或肽段断裂引起的蛋白电荷或分子量的改变,表现为蛋白点丰度或位置的迁移。本研究中发现V583及V309分别有1个及4个蛋白在万古霉素诱导培养后发生了位移,即Esa (EF2076)及Pgm1 (EF0195)、Ldh (EF0255)、Gap-2 (EF1964)、RpsB (EF2398),推测可能是磷酸化修饰或其他修饰引起分子量及等电点变化的结果。利用Pro-Q染色及Western Blots技术共鉴定到3个磷酸化蛋白Ldh、Gap-2、cAD1,通过PHOSIDA数据库进行同源性比对,确定了其可能的磷酸化位点。这也是首次在粪肠球菌中鉴定到磷酸化蛋白。其中性激素cAD1前体脂蛋白(EF3256)有5个同源异构体,通过Pro-Q染色发现它存在明显的磷酸化翻译后修饰,且在经万古霉素诱导培养后的菌体中其磷酸化程度均明显增强,Western Blot检测证实在EF3256中同时存在丝氨酸和苏氨酸的磷酸化。但性激素cAD1前体脂蛋白既没有已知的磷酸化功能域(Phosphorylation motif, PMF)也无法在相关磷酸化肽段数据库(PHOSIDA)中找到其同源性结构。已知许多耐药因子的水平传播归因于性激素依赖的质粒转移,但目前有关性激素cAD1前体脂蛋白的磷酸化及其在细胞信号调节中的作用都未有过报道,因此需要进一步对其功能进行研究。在粪肠球菌V583中,性激素cAD1前体脂蛋白由309个氨基酸组成,在信号序列中有一个22个氨基酸组成的带有半胱氨酸残基的脂质区,最后的8个氨基酸构成了cAD1,位于脂蛋白信号序列之后的Sec依赖的输出部位为假定的信号肽切割位点。由于另一种能够将性激素cCF10转移至胞内的性激素转运蛋白(OppA-like protien)在万古霉素诱导的V309菌株中明显上调,因此胞外的cAD1应是与OppA-like膜表面脂蛋白结合提高了受体菌的感受性,并经由ABC肽转运系统吸收。这暗示了万古霉素能增加质粒发生接合转移的可能性,并增强对外源细菌的感受性。我们推测V583和V309中磷酸化性激素cAD1前体脂蛋白可能是一种激活状态,在性激素在细胞信号传导中起重要作用。通过诱导毒力相关基因表达及质粒的接合转移,万古霉素使粪肠球菌更容易获得或传播质粒,尤其对于带有耐药基因的菌株。粪肠球菌的耐药性,尤其是对万古霉素的获得性耐药是与其基因组特征紧密相关的。目前,可能已有难以估量的毒力相关及耐药移动元件被粪肠球菌基因组整合。本文中对粪肠球菌V583和V309的比较蛋白质组学研究丰富了对其生理特性的研究,同时也支持由Paulsen等提出的移动DNA元件作为万古霉素耐药性的转移因子及糖肽类耐药分子机制的假设。更重要的是,通过该研究确定了部分万古霉素耐药相关蛋白,并且证实了一些在万古霉素调节作用下差异性表达的抗生素耐药蛋白激发了内源信号调整因子、粘附因子及代谢相关基因的表达。这一系列反应都使得粪肠球菌能够在万古霉素压力下继续生存和引起发病。本研究结果对揭示粪肠球菌耐药的分子机制,从而找到相应的控制策略具有重要的意义。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

【参考文献】