基于比较蛋白质组学技术的牙髓卟啉单胞菌毒力因子分析

Analysis of virulence factors of Porphyromonas endodontalis based on comparative proteomics technique

目的 分析牙髓卟啉单胞菌（Porphyromonas endodontalis,Pe）和不同毒力牙龈卟啉单胞菌（Porphyromonas gingivalis,Pg）表达量相近的蛋白,筛选Pe潜在的毒力因子.方法 将Pe ATCC35406分别与Pg高毒力株PgW83、低毒力株Pg ATCC33277进行全细胞蛋白质组学比较,采用相对和绝对定量同位素标记（isobaric tags for relative and absolute quantitation,iTRAQ）与纳升液相色谱串联质谱（nano liquid chromatography-tandem mass spectrometry,Nano-LC-MS/MS）相结合的比较蛋白质组学技术,通过同位素标记肽段、质谱检测和生物信息学分析等方法,分析Pe ATCC35406分别与PgW83、Pg ATCC33277之间表达相近的蛋白数及蛋白的生物学功能.结果 Pe ATCC35406和Pg W83比较时共鉴定到l 210个蛋白,其中表达量相近的蛋白130个（占蛋白总数的10.74％）,包括89个功能蛋白和41个未知功能蛋白;Pe ATCC35406和Pg ATCC33277比较时共鉴定到1 223个蛋白,其中表达量相近的蛋白110个（占蛋白总数的8.99％）,包括72个功能蛋白和38个未知功能蛋白.Pe ATCC35406与不同毒力的Pg表达相近的蛋白集中在外膜蛋白和蛋白酶,包括有致病作用的重组活化基因A（recombination activation geneA,RagA）、脂蛋白、伴侣蛋白Dnak、Clp家族蛋白（ClpC和ClpX）以及多种铁结合蛋白等,主要行使催化活性和结合功能,参与代谢和细胞进程.Pe ATCC35406与Pg W83表达相近的毒力蛋白种类和数目相对多于与低毒力Pg ATCC33277表达相近的蛋白.结论 脂蛋白、氧气耐受蛋白、铁结合蛋白等可能为Pe ATCC35406潜在的毒力因子,Pe的致病能力与高毒力Pg更接近.

Objective To analyze the protein expression profile and the potential virulence factors of Porphyromonas endodontalis （Pe） via comparison with that of two strains of Porphyromonas gingivalis （Pg）with high and low virulences,respectively.Methods Whole cell comparative proteomics of Pe ATCC35406 was examined and compared with that of high virulent strain Pg W83 andlow virulent strain Pg ATCC33277,respectively.Isobaric tags for relative and absolute quantitation （iTRAQ） combined with nano liquid chromatography-tandem mass spectrometry （Nano-LC-MS/MS） were adopted to identify and quantitate the proteins of Pe and two strains of Pg with various virulences by using the methods of isotopically labeled peptides,mass spectrometric detection and bioinformatics analysis.The biological functions of similar proteins expressed by Pe ATCC35406 and two strains of Pg were quantified and analyzed.Results Totally 1 210 proteins were identified while Pe compared with Pg W83.There were 130 proteins （10.74% of the total proteins） expressed similarly,including 89 known functional proteins and 41 proteins of unknown functions.Totally 1 223 proteins were identified when Pe compared with Pg ATCC33277.There were 110 proteins （8.99% of the total proteins） expressed similarly,including 72 known functional proteins and 38 proteins of unknown functions.The similarly expressed proteins in Pe and Pg strains with various virulences mainly focused on catalytic activity and binding function,including recombination activation gene （RagA）,lipoprotein,chaperonin Dnak,Clp family proteins （ClpC and ClpX） and various iron-binding proteins.They were involved in metabolism and cellular processes.In addition,the type and number of similar virulence proteins between Pe and high virulence Pg were higher than those between Pe and low virulence Pg.Conclusions Lipoprotein,oxygen resistance protein,iron binding protein were probably the potential virulence factors of Pe ATCC35406.It was speculated that pathogenicity of Pe was more similar to high virulence Pg than that to low virulence strain.