PrfA与SigB在单核细胞增生李斯特菌生物被膜形成中的作用
发布时间：2018-07-13 09:20
【摘要】：[目的]单核细胞增生李斯特菌(Listeria monocytogenes, LM)是重要的革兰氏阳性食源性致病菌,易在食品以及各种食品加工、运输和保藏设备的接触面形成生物被膜,从而具有更强的抗逆性而难以彻底清除,因此成为食品卫生安全的重要隐患。PrfA是LM在侵染宿主细胞时毒力基因转录表达的最重要的调控因子,而SigB是LM抵御外界不良环境时主要的压力应答因子,由于尚未有文献直接把PrfA和SigB--这个LM中调控绝大多数毒力基因在宿主细胞内转录表达的重要的蛋白因子和压力应答因子,与LM在宿主外的一种重要存在形式--生物被膜联系起来,所以我们的研究将为深入研究LM致病机理提供新的视点。 [方法]本文的实验方法可以主要分为三部分。一、对LM野生株(EGD)、基因缺失菌株(EGDeΔprfA, EGDeAsigB、EGDΔprfAΔsigB)和无害李斯特菌(LI)在BHI培养基比较了各菌株生物被膜形成能力的差别。二、对PrfA的研究本文主要通过两组对比实验即：1)在常用BHI培养基中,比较研究LM野生株(EGD和EGDe)、PrfA缺失株(EGDΔprfA和EGDeΔprfA)、无害李斯特菌(Listeria innocua, LI)、携带组成性表达PrfA蛋白的重组无害李斯特菌(LI-pERL3-prfA*)以及重组单核细胞增生李斯特菌(EGDeΔprfA-pERL3-prfA*),在生物被膜形成能力方面差异；2)比较研究LM野生株EGDe、PrfA缺失株EGDeΔprfA、以及携带组成性表达PrfA蛋白的重组单核细胞增生李斯特菌EGDeAprfA-pERL3-prfA*在不同碳源的培养基中,BHI(Brain Heart Infusion,复杂多糖作为碳源)和基础培养基(Minimal Essential Medium简称MM培养基,可添加PTS糖或非PTS糖作为碳源,本文采用的PTS糖为葡萄糖,非PTS糖为甘油)中生物被膜形成的差异,探讨LM重要的毒力调控蛋白PrfA在不同的糖成分作为碳源的培养基中对生物被膜形成的影响；三、比较了三种菌株LM野生株EGD、sigB缺失株EGDeAsigB和无害李斯特菌(Listeria innocua, LI)分别在BHI培养基和BHI培养基添加0.3%胆汁酸盐的培养基中,生物被膜形成能力的差异。 [结果]1)LM野生株具有较强的生物被膜形成能力,而LI形成生物被膜的能力最弱;PrfA和SigB的缺失均能降低LM生物被膜的形成能力;2)在营养丰富的常用BHI培养基中,组成性高量表达PrfA蛋白可以回复EGDeAprfA的生物被膜形成能力,但对LI没有增强作用。在不同糖类作为碳源的培养基中,各菌株在MM培养基中的被膜形成能力要显著性高于在BHI培养基中相应菌株的被膜形成能力；但无论在何种培养基中LM野生株(EGD或EGDe)均具有较强的生物被膜形成能力,PrfA的缺失均能降低LM生物被膜的形成能力；只是在BHI培养基中组成性高量表达PrfA蛋白可以回复EGDeAprfA的生物被膜形成能力；而在MM培养基中高量表达PrfA蛋白不能回复EGDeΔprfA的生物被膜形成能力,并且在以葡萄糖为唯一碳源的MM培养中,高表达PrfA菌株的生物被膜形成能力甚至比EGDeΔprfA还要低；3)EGD、EGDeAsigB和LI三种菌株无论在填不添加0.3%的胆汁酸盐的BHI培养基中均表现出：LI的被膜形成能力最差(几乎没有交联结构的出现)低于EGDeAsigB低于EGD的被膜形成能力,区别在于：在添加0.3%胆汁酸盐后,EGD的被膜形成能力较BHI中要提高一些,而EGDeAsigB却较BHI中被膜形成能力要降低一些,LI几乎不受影响。 [结论]1)LM在营养极端贫乏的MM培养基(无论添加PTS糖还是非PTS糖)中生物被膜的形成能力要远远高于在营养十分丰富的BHI培养基。但无论在哪种培养基中,PrfA均在LM生物被膜形成中具有重要的促进作用,缺失该基因均可降低LM的生物被膜的形成能力,但这种促进作用与培养基的成分(我们认为主要是糖成分不同)、PrfA数量与活性均有密切的关系。2)0.3%的胆汁酸盐可以在一定程度上促进LM的被膜形成,却不能促进EGDeAsigB的被膜形成,反而使其被膜形成的能力略有下降,表明：LM中对外界不良环境的抵抗能力一定程度上依赖于SigB因子,所以不利于细菌生存的外界条件可以在一定条件下通过调节SigB因子,而调控相关基因的表达,进而通过促进被膜的形成在一定程度上协助LM对抗不良环境。
[Abstract]:It is an important factor to control the expression of virulence genes in the host cells , and SigB is the most important factor for the expression of virulence genes in host cells .
In this paper , the differences of biofilm formation ability of different strains were compared between two groups : EGD , EGD , EGD and EGD .
2 ) The effect of LM on membrane formation in medium containing glucose and non - PTS sugar as carbon source was investigated .
Thirdly , the differences in the ability of biofilm formation were compared between EGD , sigB deletion strain EGD , sigB deletion strain EGDeAsigB and virulent innocua , LI in the medium supplemented with 0.3 % bile acid salt in BHI medium and BHI medium respectively .
( 1 ) LM wild strain has stronger ability of biofilm formation , but the ability of LI to form biofilm is the weakest , and the deletion of PrA and SigB can reduce the ability of the membrane formation of LM biofilm , but it has no enhancement effect on LI .
However , no matter what culture medium LM wild plant ( EGD or EGDe ) had stronger ability of biofilm formation , the deletion of PrA could reduce the ability of LM biofilm formation ;
Only in BHI medium the constitutive high expression Prin protein can respond to the biofilm formation ability of EGDeApra .
whereas in MM culture medium the high expression of Prin protein could not respond to the biofilm formation ability of EGDe . DELTA.prf , and in MM culture with glucose as the sole carbon source , the biofilm formation ability of the high - expression PrG strain was even lower than that of EGDe .
3 ) EGD , EGDeAsigB and LI showed that LI had the worst film formation ability ( almost no cross - linked structure ) than EGD in the BHI medium supplemented with 0.3 % bile acid salt . The difference was that after the addition of 0.3 % bile acid salt , the film formation ability of EGD was higher than that in BHI , while EGDeAsigB was less capable of being formed in BHI than in BHI , and LI was almost unaffected .
Conclusion : ( 1 ) The ability of LM to form the membrane is much higher than that in the nutrient - rich MM culture medium ( whether the PTS sugar is added to non - PTS sugar ) . However , the ability of the membrane to form the LM can be reduced to some extent .
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378
本文编号：2118893
[Abstract]:It is an important factor to control the expression of virulence genes in the host cells , and SigB is the most important factor for the expression of virulence genes in host cells .
In this paper , the differences of biofilm formation ability of different strains were compared between two groups : EGD , EGD , EGD and EGD .
2 ) The effect of LM on membrane formation in medium containing glucose and non - PTS sugar as carbon source was investigated .
Thirdly , the differences in the ability of biofilm formation were compared between EGD , sigB deletion strain EGD , sigB deletion strain EGDeAsigB and virulent innocua , LI in the medium supplemented with 0.3 % bile acid salt in BHI medium and BHI medium respectively .
( 1 ) LM wild strain has stronger ability of biofilm formation , but the ability of LI to form biofilm is the weakest , and the deletion of PrA and SigB can reduce the ability of the membrane formation of LM biofilm , but it has no enhancement effect on LI .
However , no matter what culture medium LM wild plant ( EGD or EGDe ) had stronger ability of biofilm formation , the deletion of PrA could reduce the ability of LM biofilm formation ;
Only in BHI medium the constitutive high expression Prin protein can respond to the biofilm formation ability of EGDeApra .
whereas in MM culture medium the high expression of Prin protein could not respond to the biofilm formation ability of EGDe . DELTA.prf , and in MM culture with glucose as the sole carbon source , the biofilm formation ability of the high - expression PrG strain was even lower than that of EGDe .
3 ) EGD , EGDeAsigB and LI showed that LI had the worst film formation ability ( almost no cross - linked structure ) than EGD in the BHI medium supplemented with 0.3 % bile acid salt . The difference was that after the addition of 0.3 % bile acid salt , the film formation ability of EGD was higher than that in BHI , while EGDeAsigB was less capable of being formed in BHI than in BHI , and LI was almost unaffected .
Conclusion : ( 1 ) The ability of LM to form the membrane is much higher than that in the nutrient - rich MM culture medium ( whether the PTS sugar is added to non - PTS sugar ) . However , the ability of the membrane to form the LM can be reduced to some extent .
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378
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