以agrA為靶點(diǎn)的反義鎖核酸抗MRSA活性研究
發(fā)布時(shí)間：2018-07-28 18:21
【摘要】：目的 金黃色葡萄球菌是一種重要的人類致病菌，能夠引起皮膚和軟組織感染，膿毒血癥，中毒性休克和壞死性肺炎等一系列中度或重度感染性疾病。近些年來，由于耐藥菌株的出現(xiàn)，特別是耐甲氧西林金黃色葡萄球菌(methicillin-resistant S.aureus,MRSA)的快速播散，以及耐萬古霉素金黃色葡萄球菌(vancomycin-resistantMRSA strains，VRSA)，甚至是多重耐藥MRSA的不斷檢出，使臨床上可用于治療金黃色葡萄球菌感染的抗菌藥物屈指可數(shù)。同時(shí)MRSA有形成生物膜的能力，從而阻礙抗生素發(fā)揮抗菌作用，這又進(jìn)一步加劇了抗MRSA感染治療的難度。而MRSA感染的死亡率要遠(yuǎn)遠(yuǎn)高于甲氧西林敏感金黃色葡萄球菌(methicillin-susceptible S.aureus,MSSA)感染的死亡率,這些迫使我們必須探索新的抗MRSA策略。近年來，阻斷群體感受系統(tǒng)(Quorum sensing，QS)能夠有效降低細(xì)菌的致病力、減小生存壓力和不易誘發(fā)耐藥，因而成為倍受青睞的抗菌新策略之一。本研究的目的是利用反義技術(shù)抑制金黃色葡萄球菌agr群體感受系統(tǒng)中的關(guān)鍵分子agrA的表達(dá)，阻斷agr信號通路，從而降低金黃色葡萄球菌的致病力，為抗MRSA感染提供一個(gè)潛在的靶點(diǎn)和一種新思路。 方法 1.針對agrA mRNA反義寡核苷酸的設(shè)計(jì)合成：在NCBI的Nucleotide數(shù)據(jù)庫中檢索agrA mRNA的序列，，并利用BLAST工具比對agrA在不同葡萄球菌菌屬中的同源性。首先應(yīng)用Primer Premier5.0軟件，預(yù)測與agrA mRNA特異結(jié)合的反義寡核苷酸序列(18～24bp)；然后再用RNAstructure4.5軟件預(yù)測agrA的mRNA的二級結(jié)構(gòu)并計(jì)算反義寡核苷酸與agrA mRNA各靶點(diǎn)結(jié)合時(shí)的自由能；最后，綜合PrimerPremier5.0和RNA structure4.5分析結(jié)果選取參數(shù)較優(yōu)的反義寡核苷酸，進(jìn)行鎖核酸修飾，并與透膜肽(KFF)3K共價(jià)連接，制備成與透膜肽偶聯(lián)的鎖核酸(Cell penetratepeptide conjugated locked neucleic acid, PLNA)。 2.抗agrA反義鎖核酸PLNA34和PLNA522對MRSA生長的影響：以社區(qū)獲得性耐甲氧西林金黃色葡萄球菌(CA-MRSA)菌株LAC(USA300)作為實(shí)驗(yàn)菌株，通過觀察細(xì)菌的生長曲線對篩選的兩條PLNA(PLNA34和PLNA522)的抗菌活性進(jìn)行評價(jià)。實(shí)驗(yàn)中PLNA34和PLNA522的藥物終濃度分別為12.5μM，25μM和40μM，同時(shí)設(shè)PBS為空白對照組。 3.抗agrA反義鎖核酸PLNA34和PLNA522降低MRSA毒力基因表達(dá)的研究：將LAC菌液與PLNA34和PLNA522共孵育，5h后收集菌體和上清。利用熒光定量PCR檢測PLNA對agrA及受其調(diào)控的毒力因子psmα和psmβ表達(dá)的抑制作用，和agr的效應(yīng)分子RNAⅢ及其調(diào)控的毒力基因hla和pvl表達(dá)的抑制作用；利用westernblot檢測PLNA對hla編碼的α-溶血素的抑制效果；利用溶血試驗(yàn)檢測細(xì)菌培養(yǎng)上清中溶血素的分泌情況；通過人中性粒細(xì)胞裂解和趨化實(shí)驗(yàn)分析細(xì)菌培養(yǎng)上清中PSMs的分泌情況。實(shí)驗(yàn)中PLNA34和PLNA522的藥物終濃度分別為3.125μM，6.25μM和12.5μM，同時(shí)設(shè)PBS為空白對照組。 4.抗agrA反義鎖核酸PLNA34的體內(nèi)活性研究：構(gòu)建C57BL/6J小鼠皮下感染模型，將PLNA34與細(xì)菌菌液混合后立即注射到小鼠皮下，觀察PLN34對C57BL/6J小鼠膿腫創(chuàng)面大小的影響；通過測定C57BL/6J小鼠皮下膿腫中細(xì)菌CFU，觀察PLNA34對機(jī)體清除細(xì)菌的促進(jìn)作用；通過C57BL/6J小鼠皮下膿腫的HE染色，觀察PLNA34對小鼠皮膚組織的病理保護(hù)作用。PLNA34的藥物終濃度為40μM，PBS作為空白對照組。 結(jié)果 1.針對agrA mRNA反義寡核苷酸的設(shè)計(jì)合成：Nucleotide數(shù)據(jù)庫中提供的金黃色葡萄球菌agrA的序列就是其mRNA序列；BLAST結(jié)果顯示agrA在金黃色葡萄球菌中的同源性很高，甚至與某些表皮葡萄球菌的相似度也達(dá)到80%，表明agrA可作為特異性的反義靶點(diǎn)。結(jié)合Primer Premier5.0和RNAstructure4.5分析結(jié)果優(yōu)選取2條潛在的反義寡核苷酸序列，進(jìn)行鎖核酸修飾后與透膜肽(KFF)3K共價(jià)連接，分別為PLNA34和PLNA522。 2.抗agrA反義鎖核酸對MRSA生長的影響：PLNA34組，PLNA522組和PBS對照組在各個(gè)時(shí)間點(diǎn)的吸光值基本一致，繪制的生長曲線也幾乎完全重合，說明PLNA34和PLNA522不影響LAC的生長，體外無抗菌活性，這與設(shè)計(jì)理論相符。 3.抗agrA反義鎖核酸PLNA34和PLNA522降低MRSA毒力基因表達(dá)：熒光定量PCR結(jié)果顯示，與PBS空白對照組相比，在藥物濃度均為12.5μM時(shí)PLNA34和PLNA522都能夠顯著抑制靶基因agrA mRNA (P0.001)和agr效應(yīng)分子RNAⅢ(P0.001)的表達(dá)，同時(shí)能明顯抑制被RNAⅢ調(diào)控的毒力基因hla (P均0.001)和pvl (P均0.001)，也顯著下調(diào)受AgrA調(diào)控的重要毒力基因psmα(P均0.001)和psmβ的表達(dá)(PLNA34,P0.001；PLNA522，P0.01)。但在相同濃度(均為12.5μM)時(shí)， PLNA34比PLNA522能更有效地抑制上述6個(gè)基因的轉(zhuǎn)錄(P0.001)，且具有劑量依賴效應(yīng)。Western blot結(jié)果顯示，PLNA34組α-溶血素蛋白的表達(dá)受到了明顯的抑制且呈劑量依賴性，12.5μM PLNA34組細(xì)菌幾乎未表達(dá)α-溶血素，而PLNA522組α-溶血素蛋白的表達(dá)僅受到了輕度的抑制。溶血試驗(yàn)表明，PLNA522組與PBS空白對照組相比無顯著性差異，不同濃度的PLNA34均能顯著降低溶血率(P0.01)且具有劑量依賴性。相同濃度(均為12.5μM)作用下， PLNA34組較PLNA522組能有效降低溶血率(P0.01)。中性粒細(xì)胞裂解和趨化實(shí)驗(yàn)表明，PLNA522組與PBS空白對照組相比無顯著性差異，6.25μM和12.5μM PLNA34組顯著降低細(xì)胞的裂解程度(P0.05)，不同濃度PLNA34組顯著降低細(xì)胞的趨化程度(3.125μM組與6.25μM組，P0.01；12.5μM組，P0.001)。以上結(jié)果初步說明PLNA34的反義抑制效果優(yōu)于PLNA522。 4.抗agrA反義鎖核酸PLNA34的體內(nèi)活性研究：在C57BL/6J小鼠皮下感染模型中，PLNA34能夠顯著減小感染傷口的大小，抑制膿腫的形成；能夠明顯降低感染部位的細(xì)菌滴度(P0.001)；減輕LAC對皮膚組織的損傷。 結(jié)論 1．抗agrA mRNA的PLNA34和PLNA522能夠選擇性抑制LAC agrA的表達(dá)，但PLNA34的抑制效果更好，能有效地降低細(xì)菌毒力，促進(jìn)機(jī)體對體內(nèi)細(xì)菌的清除。 2．AgrA可作為抗MRSA感染的新靶點(diǎn)，通過抑制關(guān)鍵分子AgrA的表達(dá)或使其失活從而阻斷agr群體感受系統(tǒng)，為抗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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R515
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R515
【共引文獻(xiàn)】
相關(guān)期刊論文 前10條
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2 Debasmita Dubey;Shakti Rath;Mahesh C.Sahu;Subhrajita Rout;Nagen K.Debata;Rabindra N.Padhy;;A report on infection dynamics of inducible clindamycin resistance of Staphylococcus aureus isolated from a teaching hospital in India[J];Asian Pacific Journal of Tropical Biomedicine;2013年02期
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7 吳e
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