【摘要】 目的研究表皮葡萄球菌trap基因表达与生物膜形成的相关性及其对agr系统的活化作用。方法采用微量板半定量法检测表皮葡萄球菌生物膜表型,二维电泳和高灵敏度的基质辅助激光解吸附飞行时间质谱分析比较表皮葡萄球菌标准株蛋白质表达谱,实时定量逆转录PCR检测trap基因和RNAⅢ的转录水平,CLUSTAL X对表皮葡萄球菌trap基因与金黄色葡萄球菌序列进行分析,Mega软件构建进化树。结果比较分析形成生物膜的表皮葡萄球菌ATCC 35984株与不形成生物膜的ATCC 12228株生长中期的蛋白质组,发现ATCC 35984株的TRAP蛋白量明显高于ATCC 12228株。转录水平的检测显示ATCC 12228株和生物膜阳性临床SE1457、SE671株的trap基因均低转录,但RNAⅢ的转录水平未降低。SE1457株agr系统突变后,RNAⅢ的转录明显降低,trap基因的转录无变化。RNAⅢ的转录随细菌生长变化而增加,trap基因转录保持不变。trap序列分析显示其在表皮葡萄球菌与金黄色葡萄球菌中较为保守,但存在明显的进化距离。结论在本论文所研究的表皮葡萄球菌菌株中,trap基因的转录和翻译与细菌生物膜的形成不存在直接的调控相关性;并提示TRAP蛋白不是表皮葡萄球菌agr系统活化所必需的细菌产物;表皮葡萄球菌trap基因序列与金黄色葡萄球菌之间存在差异,可能与其功能差异相关。更多还原

【Abstract】 Purpose To investigate the relationship of trap expression and biofilm formation and examine the function of trap in activating agr system in Staphylococcus epidermidis. Methods S.epidermidis biofilm was measured by microtiter-plate test.Proteome of S.epidermidis standard strains was analyzed by 2-D and MALDI-TOF-MS.Transcription level of trap and RNAⅢ was detected by real time RT-PCR.The trap genes of S.epidermidis clinic strains were sequenced and compared with that of S.aureus by Align X.The polygenetic tree was constructed by Mega software. Results Proteomic comparison of S.epidermidis ATCC 35984(bifilm positive) and ATCC 12228(biofilm negative) at mid-exponential phase demonstrated that TRAP expression in ATCC 35984 was much higher than that in ATCC 12228.At transcription level,the trap of ATCC 12228 and other biofilm positive clinic strains SE1457 and SE671 were very low compared to ATCC 35984,but the RNAⅢ was relative excessive in all strains,except in SE1457 △agr.RNAⅢ increased with the bacteria growth,and reached the maximum at post-exponential phase,while the transcription level of trap did not change.Sequences and phylogenic tree analysis showed that there existed obvious molecular divergence between S.epidermidis and S.aureus. Conclusions There is no direct regulatory relationship in both transcriptional and translational levels of trap and biofilm formation for the S.epidermidis strains.It indicated that in S.epidermidis,TRAP probably is not an essential protein for activating agr system.The functional difference of TRAP between S.epidermidis and S.aureus may relate to the gene sequence difference.更多还原