耐异烟肼和链霉素的结核分枝杆菌临床分离株与敏感株差异蛋白表达研究

Quantitative proteomic analyses of isoniazid-and streptomycin-resistant and sensitive clinical isolates and H37Rv of Mycobacterium tuberculosis

目的鉴定结核分枝杆菌对异烟肼和链霉素耐药相关的潜在蛋白。方法以药物敏感株(01105)和标准株H37Rv为对照,核素标记相对和绝对定量(isobaric tags for relative and absolute quantitation,iTRAQ)结合Nano液相色谱-串联质谱分析仪(LC-MS-MS)技术和生物信息学,鉴定并相对定量结核分枝杆菌对异烟肼与链霉素耐药的临床分离株02166菌体蛋白。结果 02166菌株分别与01105菌株和H37Rv菌株比较差异表达蛋白为153个和130个,02166菌株与01105菌株和H37Rv比较差异表达蛋白均为86个(共同差异表达蛋白)。差异表达蛋白理论相对分子质量和等电点分布广泛,相对分子质量从7.63～326.22,等电点从3.74～12.48,其主要参与中间代谢、呼吸作用和脂类代谢。共同差异表达蛋白:9个核糖体蛋白(Rv0056、Rv0651、Rv0652、Rv0701、Rv0719、Rv1630、Rv2785c、Rv2909c和Rv3458c)在02166菌株中表达下调;5个蛋白(Rv0234c、Rv2466c、Rv2626c、Rv2986c和Rv3118)在02166菌株中呈显著差异表达,其中琥珀酸半醛脱氢酶(Rv0234c)和假定未知蛋白(Rv2466c)在02166菌株中表达上调倍数>1.2,DNA结合蛋白HU同系物hupB(Rv2986c)、假定未知蛋白(Rv2626c和Rv3118)在02166菌株中表达下调倍数<0.5。结论 iTRAQ发现了耐异烟肼和链霉素的结核分枝杆菌临床分离株差异表达蛋白,为进一步探讨结核分枝杆菌异烟肼或链霉素耐药机制奠定了基础。

Objective To identify the proteins differentially expressed in isoniazid- and streptomycin-resistant Mycobacterium tuberculosis clinical isolate （INH/Sr isolate） compared with drug-sensitive clinical isolate （INH/S isolate） and H37Rv. Methods Whole cellular proteins were extracted from the INH/Sr isolate 02166, the INH/Sisolate 01105 and H37Rv of M. tubereulosis, respectively. The proteins were digested with trypsin. The peptides were labeled, separated and identified by isobaric tags for relative and absolute quantitation （iTRAQ） combined with Nano LC-MS-MS technology. The bioinformatics were used to identify and quantify the proteins. Results One hundred and fifty three and 130 proteins were found differential expression in 02166 strain compared with 01105 strain and H37Rv, respectively, including 86 proteins in 02166 strain compared with both 01105 strain and H37Rv. The theoretical molecular weight and isoelectric point of differentially expressed proteins ranged from 7.63 to 326.22 and from 3.74 to 12.48, respectively. Differentially expressed proteins were mainly associated with intermediary metabolism, respiration, and lipid metabolism. Nine ribosomal proteins （Rv0056, Rv0651, Rv0652, Rv0701, Rv0719, Rv1630, Rv2785c, Rv2909c and Rv3458c） were commonly down-regulated in 02166 strain compared with both 01106 and H37Rv. Succinate〉semialdehyde dehydrogenase （Rv0234c） and putative uneharacterized protein （Rv2466c） were common up-regulation （the ratios〉1. 2）, and probable DNA-binding protein HU homolog hupB （Rv2986c） and putative uncharacterized protein （Rv2626c and Rv3118） were down-regulation （the ratios 〈0.5） in 02166 strain compared with both 01105 strain and H37Rv. Conclusion Differentially expressed proteins were identified in INH/S isolate compared with INH/S isolate and H37Rv using iTRAQ. Further study will focus on the above proteins playing role in INH or S resistanee.