粘附素及其他致病相关因子在中间普氏菌与人上皮细胞互作中的作用研究
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摘要
牙周病是常见的口腔疾病,在世界范围内有很高的发病率。中间普氏菌(Prevotella intermedia)是一种与牙周病相关的病原菌,在牙周病的发生和发展中起到重要的作用。中间普氏菌能粘附和侵入多种宿主细胞,在这一过程中细菌粘附素及其他致病相关因子起了很重要的作用。目前该过程的分子机制以及所参与蛋白的功能仍有待进一步的研究。本论文利用中间普氏菌17全基因组序列,应用生物信息学、基因组学和蛋白组学方法来研究中间普氏菌与人上皮细胞相互作用过程中相关基因和蛋白的功能及其机制。
生物信息学研究中间普氏菌全基因组序列发现其具有两个富含亮氨酸重复序列的潜在粘附素,AdpD和AdpE.分别把编码这两个蛋白的基因PI1564和PI1571克隆到pET30a(+)原核表达载体上,并将重组的表达载体转化进入大肠杆菌表达菌株BL21(DE3)中诱导AdpD和AdpE融合蛋白的表达。酶联免疫吸附(ELISA)分析表明AdpD特异结合纤维蛋白原,而AdpE特异结合胶原蛋白。Western印迹表明AdpD和AdpE分布在中间普氏菌细胞外膜上。额外加入的融合蛋白AdpD和AdpE能抑制中间普氏菌粘附到口腔上皮细胞HN4。这些结果表明,AdpD和AdpE可能在中间普氏菌粘附到口腔上皮细胞的过程中起到一定的作用。
运用实时荧光定量PCR (qRT-PCR)技术来检测中间普氏菌粘附素及其他致病相关因子在中间普氏菌与HeLa细胞相互作用后的基因表达情况。并成功运用自已设计的qRT-PCR阵列,采用相对定量的方法检测172个中间普氏菌基因在HeLa细胞环境中的表达情况。当中间普氏菌与HeLa细胞相互作用24 h后中间普氏菌基因表达水平发生明显变化,其中31个基因的表达变化在2倍以上。与此不同的是,与常规细胞培养基相比,中间普氏菌在HeLa细胞条件培养液培养24 h后的基因表达比较稳定,只有2个基因在HeLa细胞条件培养液中表达下降。在中间普氏菌与HeLa细胞相互作用后表达显著上升的基因中挑选基因PIO137做进一步的生物学功能分析。其原因是基因PIO137在这个过程中表达显著上升了3.1倍,而且生物信息学分析显示PIO137编码的蛋白可能是一个具有粘附作用的凝集素样蛋白前体。因此基因PIO137被分子克隆并在大肠杆菌中原核表达其编码的蛋白。ELISA结果表明PIO137编码的蛋白特异结合凝集素。这些研究表明qRT-PCR列阵是一种快速有效筛选互作基因的方法。
通过研究中间普氏菌生长在分泌有细胞因子的上皮细胞条件培养液中的蛋白和基因表达水平来分析中间普氏菌在宿主体内环境中的生长情况。准备了三种中间普氏菌生长的培养液分别为上皮细胞HN4和HeLa细胞培养了24 h后收集的细胞条件培养液,以及常规培养HN4和HeLa的细胞培养基,并应用二维凝胶电泳(2-DE)结合液相色谱-串联质谱(LC-MS/MS)技术分析中间普氏菌在这三种不同培养液中的蛋白表达水平。中间普氏菌全蛋白以及外膜蛋白在三种不同培养液下的2-DE图谱总体上比较相似,有一些蛋白在HN4和HeLa条件培养液中发生了显著的变化。用质谱分析了40个差异表达的蛋白点,从中鉴定了15个中间普氏菌外膜蛋白。其中PIO932编码的血红素结合蛋白、PI1599和PIO779编码的具有TPR结构域的蛋白的表达水平在具有HN4和HeLa细胞分泌因子的条件培养液中明显上升。这些结果表明中间普氏菌在适应宿主细胞内环境过程中其整体的蛋白表达水平发生了变化,那些受细胞分泌因子影响而表达明显发生变化的蛋白可能对中间普氏菌在宿主环境内生存起了重要作用。
Periodontal disease is an oral infectious disease with a high prevalence worldwide. The oral bacterium Prevotella intermedia mediates the development of periodontal disease and other oral infections. P. intermedia is able to adhere to and invade various host cells. Although adhesins and other virulence factors are critical for bacteria to successfully adhere, invade and colonize their host, the molecular mechanisms and relative protein roles in this process remain unclear. The availability of the genomic sequence of P. intermedia 17 allowed us to apply bioinformatics, genomics and proteomic approaches to identify genes and proteins that maybe mediating in the process of the interaction between bacteria and human epitherial cells.
Using bioinformatics analysis, we identified and characterized the adhesin candidates AdpD and AdpE, which are members of the leucine-rich family of proteins. Gene PI 1564 and PI 1571 were cloned into expression vector pET30a(+) and transformed into E. coli BL21(DE3) cells to generate recombinant proteins. Enzyme-linked immunosorbent assay analysis (ELISA) revealed that AdpD bound specifically to fibrinogen and AdpE bound specifically to collagen. Furthermore, Western blot showed that AdpD and AdpE localized in the outer membrane fraction of P. intermedia. Recombinant AdpD and AdpE inhibited adherence of P. intermedia to oral epithelial cells HN4. These results suggested that AdpD and AdpE play roles in P. intermedia adherence to oral epithelial cells.
We used real-time quantitative reverse transcription PCR (qRT-PCR) to examine the gene expression of P. intermedia adhesins and other virulence factors upon exposure to HeLa cells. We applied relative quantitation qRT-PCR in our homemade array to 172 P. intermedia genes. Out of the 172 bacterial genes tested,31 genes were regulated by greater than two fold after P. intermedia exposure to HeLa cells for 24 h. In contrast, the transcription patterns between bacteria grown in plain HeLa medium and in conditioned medium for 24 h were not markedly different (only two genes were down-regulated). After we identified the significant regulated genes through qRT-PCR, we examined the biological function of genes involved in the interaction between P. intermedia and HeLa cells. The lectin-like precursor gene PI0137, which was up-regulated by 3.1 fold, was cloned and induced to express recombinant protein in vitro. ELISA showed that PI0137- encoded protein specifically binds to lectin. Thus, this study showed that qRT-PCR array is a rapid and effective way to screen interesting gene candidates.
We prepared sets of experimental conditions designed to reflect important features of host cell environments. P. intermedia was grown in the conditioned medium of HN4 and HeLa and plain HN4 and HeLa cell medium. Two-dimensional gel electrophoresis (2-DE) with LC-MS/MS were used to analyze the proteome of P. intermedia grown in these three conditions. The 2-DE patterns of P. intermedia whole proteins and its outer membrane proteins were generally similar across three conditions. However, some proteins showed significant difference in expression in conditioned cell medium. We sampled 40 of the differentially-expressed protein spots to be analyzed by mass spectrum, and identified 15 P. intermedia outer membrane proteins from these 40 spots. Some proteins are up-regulated in the presence of HN4 and HeLa cell components, including a PI0932-encoded hemin receptor and two TPR-repeating-containing proteins, encoded by PI0779 and PI1559 respectively. These results suggest that adaptation to a host cell environment induces a shift in the expressed proteome of P. intermedia, and the regulated proteins may play important roles for the survival of P. intermedia in the host environment.
生物信息学研究中间普氏菌全基因组序列发现其具有两个富含亮氨酸重复序列的潜在粘附素,AdpD和AdpE.分别把编码这两个蛋白的基因PI1564和PI1571克隆到pET30a(+)原核表达载体上,并将重组的表达载体转化进入大肠杆菌表达菌株BL21(DE3)中诱导AdpD和AdpE融合蛋白的表达。酶联免疫吸附(ELISA)分析表明AdpD特异结合纤维蛋白原,而AdpE特异结合胶原蛋白。Western印迹表明AdpD和AdpE分布在中间普氏菌细胞外膜上。额外加入的融合蛋白AdpD和AdpE能抑制中间普氏菌粘附到口腔上皮细胞HN4。这些结果表明,AdpD和AdpE可能在中间普氏菌粘附到口腔上皮细胞的过程中起到一定的作用。
运用实时荧光定量PCR (qRT-PCR)技术来检测中间普氏菌粘附素及其他致病相关因子在中间普氏菌与HeLa细胞相互作用后的基因表达情况。并成功运用自已设计的qRT-PCR阵列,采用相对定量的方法检测172个中间普氏菌基因在HeLa细胞环境中的表达情况。当中间普氏菌与HeLa细胞相互作用24 h后中间普氏菌基因表达水平发生明显变化,其中31个基因的表达变化在2倍以上。与此不同的是,与常规细胞培养基相比,中间普氏菌在HeLa细胞条件培养液培养24 h后的基因表达比较稳定,只有2个基因在HeLa细胞条件培养液中表达下降。在中间普氏菌与HeLa细胞相互作用后表达显著上升的基因中挑选基因PIO137做进一步的生物学功能分析。其原因是基因PIO137在这个过程中表达显著上升了3.1倍,而且生物信息学分析显示PIO137编码的蛋白可能是一个具有粘附作用的凝集素样蛋白前体。因此基因PIO137被分子克隆并在大肠杆菌中原核表达其编码的蛋白。ELISA结果表明PIO137编码的蛋白特异结合凝集素。这些研究表明qRT-PCR列阵是一种快速有效筛选互作基因的方法。
通过研究中间普氏菌生长在分泌有细胞因子的上皮细胞条件培养液中的蛋白和基因表达水平来分析中间普氏菌在宿主体内环境中的生长情况。准备了三种中间普氏菌生长的培养液分别为上皮细胞HN4和HeLa细胞培养了24 h后收集的细胞条件培养液,以及常规培养HN4和HeLa的细胞培养基,并应用二维凝胶电泳(2-DE)结合液相色谱-串联质谱(LC-MS/MS)技术分析中间普氏菌在这三种不同培养液中的蛋白表达水平。中间普氏菌全蛋白以及外膜蛋白在三种不同培养液下的2-DE图谱总体上比较相似,有一些蛋白在HN4和HeLa条件培养液中发生了显著的变化。用质谱分析了40个差异表达的蛋白点,从中鉴定了15个中间普氏菌外膜蛋白。其中PIO932编码的血红素结合蛋白、PI1599和PIO779编码的具有TPR结构域的蛋白的表达水平在具有HN4和HeLa细胞分泌因子的条件培养液中明显上升。这些结果表明中间普氏菌在适应宿主细胞内环境过程中其整体的蛋白表达水平发生了变化,那些受细胞分泌因子影响而表达明显发生变化的蛋白可能对中间普氏菌在宿主环境内生存起了重要作用。
Periodontal disease is an oral infectious disease with a high prevalence worldwide. The oral bacterium Prevotella intermedia mediates the development of periodontal disease and other oral infections. P. intermedia is able to adhere to and invade various host cells. Although adhesins and other virulence factors are critical for bacteria to successfully adhere, invade and colonize their host, the molecular mechanisms and relative protein roles in this process remain unclear. The availability of the genomic sequence of P. intermedia 17 allowed us to apply bioinformatics, genomics and proteomic approaches to identify genes and proteins that maybe mediating in the process of the interaction between bacteria and human epitherial cells.
Using bioinformatics analysis, we identified and characterized the adhesin candidates AdpD and AdpE, which are members of the leucine-rich family of proteins. Gene PI 1564 and PI 1571 were cloned into expression vector pET30a(+) and transformed into E. coli BL21(DE3) cells to generate recombinant proteins. Enzyme-linked immunosorbent assay analysis (ELISA) revealed that AdpD bound specifically to fibrinogen and AdpE bound specifically to collagen. Furthermore, Western blot showed that AdpD and AdpE localized in the outer membrane fraction of P. intermedia. Recombinant AdpD and AdpE inhibited adherence of P. intermedia to oral epithelial cells HN4. These results suggested that AdpD and AdpE play roles in P. intermedia adherence to oral epithelial cells.
We used real-time quantitative reverse transcription PCR (qRT-PCR) to examine the gene expression of P. intermedia adhesins and other virulence factors upon exposure to HeLa cells. We applied relative quantitation qRT-PCR in our homemade array to 172 P. intermedia genes. Out of the 172 bacterial genes tested,31 genes were regulated by greater than two fold after P. intermedia exposure to HeLa cells for 24 h. In contrast, the transcription patterns between bacteria grown in plain HeLa medium and in conditioned medium for 24 h were not markedly different (only two genes were down-regulated). After we identified the significant regulated genes through qRT-PCR, we examined the biological function of genes involved in the interaction between P. intermedia and HeLa cells. The lectin-like precursor gene PI0137, which was up-regulated by 3.1 fold, was cloned and induced to express recombinant protein in vitro. ELISA showed that PI0137- encoded protein specifically binds to lectin. Thus, this study showed that qRT-PCR array is a rapid and effective way to screen interesting gene candidates.
We prepared sets of experimental conditions designed to reflect important features of host cell environments. P. intermedia was grown in the conditioned medium of HN4 and HeLa and plain HN4 and HeLa cell medium. Two-dimensional gel electrophoresis (2-DE) with LC-MS/MS were used to analyze the proteome of P. intermedia grown in these three conditions. The 2-DE patterns of P. intermedia whole proteins and its outer membrane proteins were generally similar across three conditions. However, some proteins showed significant difference in expression in conditioned cell medium. We sampled 40 of the differentially-expressed protein spots to be analyzed by mass spectrum, and identified 15 P. intermedia outer membrane proteins from these 40 spots. Some proteins are up-regulated in the presence of HN4 and HeLa cell components, including a PI0932-encoded hemin receptor and two TPR-repeating-containing proteins, encoded by PI0779 and PI1559 respectively. These results suggest that adaptation to a host cell environment induces a shift in the expressed proteome of P. intermedia, and the regulated proteins may play important roles for the survival of P. intermedia in the host environment.
引文
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