单核细胞增生性李斯特菌prfA缺失株及其亲本株胞外蛋白的比较蛋白质组学

Comparative proteomics analysis of extracellular proteins from Listeria monocytogenes and its isogenic prfA deletion mutant

【目的】PrfA蛋白对单核细胞增生性李斯特菌致病过程中毒力基因的表达起着重要调控作用,本文从蛋白质水平上初步探讨了PrfA的调控功能。【方法】对LM4及LM4ΔprfA的胞外蛋白采用双向凝胶电泳结合基质辅助激光解析电离飞行时间质谱鉴定技术,进行比较蛋白质组学研究。【结果】发现差异表达的有31个蛋白点,质谱鉴定成功19个点,对应12种蛋白,其中已知的毒力相关蛋白有:InlC、ActA、LLO,此外还发现丙氨酸丙氨酰羧肽酶、GW重复表面蛋白、假定转录调节因子、天冬氨酸半醛脱氢酶和一些假定蛋白。采用实时荧光定量PCR方法对蛋白质组学方法获得的结果进行了验证,结果显示hly、actA、inlC的基因表达量显著下降,丙氨酰氨羧肽酶、GW重复表面蛋白的mRNA转录水平降低。【结论】PrfA蛋白对毒力岛LIPI-I和毒力岛LIPI-II中毒力基因的表达具有重要的调控作用,新发现的转录调控因子和假定蛋白的功能有待于进一步深入研究。

[Objective] Positive regulatory factor A （PrfA） protein plays a key role in the pathogenicity of Listeria monocytogenes by regulating the expression of virulence genes.We studied the regulation functions of PrfA and its role in Listeria monocytogenes （Lm） virulence. [Method]Extracellular proteins were obtained from the supernatants of parental strain LM4 and mutant strain LM4ΔprfA cultured in minimal medium. We used two-dimensional gel electrophoresis and matrix associated laser dissociation/ionization time of flight mass spectrometry （MALDI- TOF-MS） to analyze the differences of secreted proteins between LM4 and LM4ΔprfA.[Results]The electrophoresis results show that 31 different spots,19 spots corresponding 12 proteins were identified by MALDI- TOF-MS. Some virulence related proteins were verified,such as InlC,ActA and LLO. Some new proteins that are regulated by PrfA include D-alanyl-D-alanine carboxypeptidase,dipeptide Glycine and Trytophan （GW） repeat-containing surface protein,transcriptional regulator and some hypothetical proteins with unknown functions. Real-time quantitative PCR was conducted to verify the proteomics results. The mRNA expression level of hly,actA and inlC gene was significantly reduced,and that of D-alanyl-D-alanine carboxypeptidase and GW repeat-containing surface protein＇s synthesis also had a reduction in LM4ΔprfA strain.[Conclusion]PrfA plays key roles on the regulation of genes in LIPI- I and LIPI- II.