以agrA为靶点的反义锁核酸抗MRSA活性研究

发布时间：2018-07-28 18:21

【摘要】：目的金黄色葡萄球菌是一种重要的人类致病菌，能够引起皮肤和软组织感染，脓毒血症，中毒性休克和坏死性肺炎等一系列中度或重度感染性疾病。近些年来，由于耐药菌株的出现，特别是耐甲氧西林金黄色葡萄球菌(methicillin-resistant S.aureus,MRSA)的快速播散，以及耐万古霉素金黄色葡萄球菌(vancomycin-resistantMRSA strains，VRSA)，甚至是多重耐药MRSA的不断检出，使临床上可用于治疗金黄色葡萄球菌感染的抗菌药物屈指可数。同时MRSA有形成生物膜的能力，从而阻碍抗生素发挥抗菌作用，这又进一步加剧了抗MRSA感染治疗的难度。而MRSA感染的死亡率要远远高于甲氧西林敏感金黄色葡萄球菌(methicillin-susceptible S.aureus,MSSA)感染的死亡率,这些迫使我们必须探索新的抗MRSA策略。近年来，阻断群体感受系统(Quorum sensing，QS)能够有效降低细菌的致病力、减小生存压力和不易诱发耐药，因而成为倍受青睐的抗菌新策略之一。本研究的目的是利用反义技术抑制金黄色葡萄球菌agr群体感受系统中的关键分子agrA的表达，阻断agr信号通路，从而降低金黄色葡萄球菌的致病力，为抗MRSA感染提供一个潜在的靶点和一种新思路。方法 1.针对agrA mRNA反义寡核苷酸的设计合成：在NCBI的Nucleotide数据库中检索agrA mRNA的序列，，并利用BLAST工具比对agrA在不同葡萄球菌菌属中的同源性。首先应用Primer Premier5.0软件，预测与agrA mRNA特异结合的反义寡核苷酸序列(18～24bp)；然后再用RNAstructure4.5软件预测agrA的mRNA的二级结构并计算反义寡核苷酸与agrA mRNA各靶点结合时的自由能；最后，综合PrimerPremier5.0和RNA structure4.5分析结果选取参数较优的反义寡核苷酸，进行锁核酸修饰，并与透膜肽(KFF)3K共价连接，制备成与透膜肽偶联的锁核酸(Cell penetratepeptide conjugated locked neucleic acid, PLNA)。 2.抗agrA反义锁核酸PLNA34和PLNA522对MRSA生长的影响：以社区获得性耐甲氧西林金黄色葡萄球菌(CA-MRSA)菌株LAC(USA300)作为实验菌株，通过观察细菌的生长曲线对筛选的两条PLNA(PLNA34和PLNA522)的抗菌活性进行评价。实验中PLNA34和PLNA522的药物终浓度分别为12.5μM，25μM和40μM，同时设PBS为空白对照组。 3.抗agrA反义锁核酸PLNA34和PLNA522降低MRSA毒力基因表达的研究：将LAC菌液与PLNA34和PLNA522共孵育，5h后收集菌体和上清。利用荧光定量PCR检测PLNA对agrA及受其调控的毒力因子psmα和psmβ表达的抑制作用，和agr的效应分子RNAⅢ及其调控的毒力基因hla和pvl表达的抑制作用；利用westernblot检测PLNA对hla编码的α-溶血素的抑制效果；利用溶血试验检测细菌培养上清中溶血素的分泌情况；通过人中性粒细胞裂解和趋化实验分析细菌培养上清中PSMs的分泌情况。实验中PLNA34和PLNA522的药物终浓度分别为3.125μM，6.25μM和12.5μM，同时设PBS为空白对照组。 4.抗agrA反义锁核酸PLNA34的体内活性研究：构建C57BL/6J小鼠皮下感染模型，将PLNA34与细菌菌液混合后立即注射到小鼠皮下，观察PLN34对C57BL/6J小鼠脓肿创面大小的影响；通过测定C57BL/6J小鼠皮下脓肿中细菌CFU，观察PLNA34对机体清除细菌的促进作用；通过C57BL/6J小鼠皮下脓肿的HE染色，观察PLNA34对小鼠皮肤组织的病理保护作用。PLNA34的药物终浓度为40μM，PBS作为空白对照组。结果 1.针对agrA mRNA反义寡核苷酸的设计合成：Nucleotide数据库中提供的金黄色葡萄球菌agrA的序列就是其mRNA序列；BLAST结果显示agrA在金黄色葡萄球菌中的同源性很高，甚至与某些表皮葡萄球菌的相似度也达到80%，表明agrA可作为特异性的反义靶点。结合Primer Premier5.0和RNAstructure4.5分析结果优选取2条潜在的反义寡核苷酸序列，进行锁核酸修饰后与透膜肽(KFF)3K共价连接，分别为PLNA34和PLNA522。 2.抗agrA反义锁核酸对MRSA生长的影响：PLNA34组，PLNA522组和PBS对照组在各个时间点的吸光值基本一致，绘制的生长曲线也几乎完全重合，说明PLNA34和PLNA522不影响LAC的生长，体外无抗菌活性，这与设计理论相符。 3.抗agrA反义锁核酸PLNA34和PLNA522降低MRSA毒力基因表达：荧光定量PCR结果显示，与PBS空白对照组相比，在药物浓度均为12.5μM时PLNA34和PLNA522都能够显著抑制靶基因agrA mRNA (P0.001)和agr效应分子RNAⅢ(P0.001)的表达，同时能明显抑制被RNAⅢ调控的毒力基因hla (P均0.001)和pvl (P均0.001)，也显著下调受AgrA调控的重要毒力基因psmα(P均0.001)和psmβ的表达(PLNA34,P0.001；PLNA522，P0.01)。但在相同浓度(均为12.5μM)时， PLNA34比PLNA522能更有效地抑制上述6个基因的转录(P0.001)，且具有剂量依赖效应。Western blot结果显示，PLNA34组α-溶血素蛋白的表达受到了明显的抑制且呈剂量依赖性，12.5μM PLNA34组细菌几乎未表达α-溶血素，而PLNA522组α-溶血素蛋白的表达仅受到了轻度的抑制。溶血试验表明，PLNA522组与PBS空白对照组相比无显著性差异，不同浓度的PLNA34均能显著降低溶血率(P0.01)且具有剂量依赖性。相同浓度(均为12.5μM)作用下， PLNA34组较PLNA522组能有效降低溶血率(P0.01)。中性粒细胞裂解和趋化实验表明，PLNA522组与PBS空白对照组相比无显著性差异，6.25μM和12.5μM PLNA34组显著降低细胞的裂解程度(P0.05)，不同浓度PLNA34组显著降低细胞的趋化程度(3.125μM组与6.25μM组，P0.01；12.5μM组，P0.001)。以上结果初步说明PLNA34的反义抑制效果优于PLNA522。 4.抗agrA反义锁核酸PLNA34的体内活性研究：在C57BL/6J小鼠皮下感染模型中，PLNA34能够显著减小感染伤口的大小，抑制脓肿的形成；能够明显降低感染部位的细菌滴度(P0.001)；减轻LAC对皮肤组织的损伤。结论 1．抗agrA mRNA的PLNA34和PLNA522能够选择性抑制LAC agrA的表达，但PLNA34的抑制效果更好，能有效地降低细菌毒力，促进机体对体内细菌的清除。 2．AgrA可作为抗MRSA感染的新靶点，通过抑制关键分子AgrA的表达或使其失活从而阻断agr群体感受系统，为抗MRSA提供新策略。
[Abstract]:objective
Staphylococcus aureus is an important human pathogen, which can cause a series of moderate or severe infectious diseases, such as skin and soft tissue infection, sepsis, toxic shock and necrotic pneumonia. In recent years, the emergence of drug resistant strains, especially methicillin resistant Staphylococcus aureus (methicillin-resistant S.aureus,) The rapid spread of MRSA, as well as the continuous detection of vancomycin-resistantMRSA strains (VRSA), and even multidrug-resistant MRSA, makes the clinically useful antibiotics for the treatment of Staphylococcus aureus infection, and MRSA has the ability to form a biofilm to prevent antibiotics from being antibiosis. This further exacerbates the difficulty of anti MRSA infection, and the mortality rate of MRSA infection is far higher than that of methicillin sensitive Staphylococcus aureus (methicillin-susceptible S.aureus, MSSA) infection, which compels us to explore new anti MRSA strategies. In recent years, the group receptor system (Quorum sensing, QS) has been blocked. The aim of this study is to inhibit the expression of the key molecule agrA in the agr colony sensing system of Staphylococcus aureus, block the agr signaling pathway and reduce the golden yellow globule. The pathogenicity of bacteria provides a potential target and a new idea for anti MRSA infection.
Method
1. design and synthesis of agrA mRNA antisense oligonucleotides: retrieving the sequence of agrA mRNA in the Nucleotide database of NCBI, and using BLAST tools to compare the homology of agrA in different Staphylococcus genera. First, Primer Premier5.0 software is used to predict the sequence of antisense oligonucleotides (18 ~) which are specifically combined with agrA mRNA. The RNAstructure4.5 software was used to predict the two stage structure of the agrA mRNA and to calculate the free energy of the combination of antisense oligonucleotides and agrA mRNA targets. Finally, comprehensive PrimerPremier5.0 and RNA structure4.5 analysis results were used to select the better antisense oligodeoxynucleotides, which were covalently linked with the transmembrane peptide (KFF) 3K, and were prepared and prepared. Cell penetratepeptide conjugated locked neucleic acid (PLNA) coupled with membrane permeation peptide.
2. effect of anti agrA antisense lock nucleic acid PLNA34 and PLNA522 on the growth of MRSA: using community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) strain LAC (USA300) as an experimental strain, the antimicrobial activity of selected two PLNA (PLNA34 and PLNA522) was evaluated by observing the growth curve of bacteria. The final concentrations were 12.5 M, 25 M and 40 M respectively, while PBS was used as blank control group.
3. anti sense agrA antisense lock nucleic acid PLNA34 and PLNA522 to reduce MRSA virulence gene expression: reincubating LAC bacteria with PLNA34 and PLNA522, collecting bacteria and supernatant after 5h. The inhibitory effect of PLNA on agrA and its regulated toxicity factor PSM alpha and beta expression by fluorescence quantitative PCR, and the virulence of the effector molecule III and its regulation The inhibitory effect of gene HLA and PVL expression; the inhibitory effect of PLNA on the alpha hemolysin encoded by HLA; the determination of the secretion of hemolysin in the bacterial culture supernatant by the hemolysis test, and the analysis of the secretion of PSMs in the bacterial culture supernatant by human neutrophilic lysis and chemotactic experiments. PLNA34 and PLNA52 in the experiment. The final concentration of 2 was 3.125 M, 6.25 M and 12.5 M respectively, while PBS was used as blank control group.
4. in vivo activity of anti sense agrA antisense PLNA34: construct C57BL/6J mouse subcutaneous infection model, injecting PLNA34 and bacterial liquid into mice immediately and observing the effect of PLN34 on the size of C57BL/6J mice abscess wound. By measuring the bacterial CFU in the subcutaneous abscess of C57BL/6J mice, the bacteria were scavenged by PLNA34 to the organism. To promote the effect, through the HE staining of subcutaneous abscess in C57BL/6J mice, the pathological protective effect of PLNA34 on the skin tissue of mice was observed. The final concentration of.PLNA34 was 40 u M, and PBS was used as a blank control group.
Result
1. design and synthesis of agrA mRNA antisense oligonucleotides: the sequence of Staphylococcus aureus agrA provided in the Nucleotide database is its mRNA sequence; BLAST results show that the homology of agrA in Staphylococcus aureus is very high, even with some Staphylococcus epidermidis similar to 80%, indicating that agrA can be used as a specific inverse. 2 potential antisense oligonucleotide sequences were selected from the results of Primer Premier5.0 and RNAstructure4.5 analysis, and were covalently linked with the transmembrane peptide (KFF) 3K after modified nucleic acid, PLNA34 and PLNA522., respectively.
The effect of 2. anti agrA antisense lock nucleic acid on the growth of MRSA: PLNA34 group, PLNA522 group and PBS control group were almost identical at all time points, and the growth curves were almost completely coincided. It shows that PLNA34 and PLNA522 do not affect the growth of LAC and have no antibacterial activity in vitro, which is in accordance with the design theory.
3. anti agrA antisense lock nucleic acid PLNA34 and PLNA522 reduced MRSA virulence gene expression: the fluorescence quantitative PCR results showed that, compared with PBS blank control group, both PLNA34 and PLNA522 could significantly inhibit the expression of agrA mRNA (P0.001) and inhibitory effect molecule III of target gene when the drug concentration was 12.5 mu M. The controlled virulence gene HLA (P 0.001) and PVL (P 0.001) also significantly lowered the expression of the important virulence gene PSM alpha (P all 0.001) and PSM beta regulated by AgrA (PLNA34, P0.001; PLNA522, P0.01). But when the same concentration (all 12.5 mu), the 6 genes were inhibited more effectively and had a dose dependent effect. The results of.Western blot showed that the expression of alpha hemolysin protein in group PLNA34 was significantly inhibited and dose-dependent. The 12.5 micron M PLNA34 group had almost no expression of alpha hemolysin, and the expression of alpha hemolysin protein in group PLNA522 was only slightly inhibited. The hemolysis test showed that there was no significant difference between the PLNA522 group and the PBS blank control group. Different concentrations of PLNA34 can significantly reduce the hemolysis rate (P0.01) and have a dose-dependent manner. Under the same concentration (all 12.5 u M), the PLNA34 group can effectively reduce the hemolysis rate (P0.01). Neutrophil lysis and chemotactic experiments show that there is no significant difference between the PLNA522 group and the PBS blank control group, 6.25 M and 12.5 mu M PLN. A34 group significantly reduced the degree of cell lysis (P0.05), and different concentrations of PLNA34 groups significantly reduced the chemotactic degree of cells (3.125 mu M group and 6.25 M group, P0.01; 12.5 mu M group, P0.001). The above results showed that the antisense inhibition effect of PLNA34 was better than PLNA522..
In vivo activity of 4. anti agrA antisense lock nucleic acid PLNA34: in C57BL/6J mice model of subcutaneous infection, PLNA34 can significantly reduce the size of infected wounds and inhibit the formation of abscess; it can obviously reduce the bacterial titer of the infected site (P0.001), and reduce the damage of LAC to skin tissue.
conclusion
1. the PLNA34 and PLNA522 of anti agrA mRNA can selectively inhibit the expression of LAC agrA, but the inhibition effect of PLNA34 is better, it can effectively reduce the bacterial virulence and promote the organism to remove the bacteria in the body.
2.AgrA can be used as a new target for anti MRSA infection. By inhibiting the expression of key molecule AgrA or inactivating it, it can block the sensory system of agr group, and provide a new strategy for anti MRSA.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R515

【共引文献】