PrfA與SigB在單核細胞增生李斯特菌生物被膜形成中的作用
發(fā)布時間：2018-07-13 09:20
【摘要】：[目的]單核細胞增生李斯特菌(Listeria monocytogenes, LM)是重要的革蘭氏陽性食源性致病菌,易在食品以及各種食品加工、運輸和保藏設備的接觸面形成生物被膜,從而具有更強的抗逆性而難以徹底清除,因此成為食品衛(wèi)生安全的重要隱患。PrfA是LM在侵染宿主細胞時毒力基因轉(zhuǎn)錄表達的最重要的調(diào)控因子,而SigB是LM抵御外界不良環(huán)境時主要的壓力應答因子,由于尚未有文獻直接把PrfA和SigB--這個LM中調(diào)控絕大多數(shù)毒力基因在宿主細胞內(nèi)轉(zhuǎn)錄表達的重要的蛋白因子和壓力應答因子,與LM在宿主外的一種重要存在形式--生物被膜聯(lián)系起來,所以我們的研究將為深入研究LM致病機理提供新的視點。 [方法]本文的實驗方法可以主要分為三部分。一、對LM野生株(EGD)、基因缺失菌株(EGDeΔprfA, EGDeAsigB、EGDΔprfAΔsigB)和無害李斯特菌(LI)在BHI培養(yǎng)基比較了各菌株生物被膜形成能力的差別。二、對PrfA的研究本文主要通過兩組對比實驗即：1)在常用BHI培養(yǎng)基中,比較研究LM野生株(EGD和EGDe)、PrfA缺失株(EGDΔprfA和EGDeΔprfA)、無害李斯特菌(Listeria innocua, LI)、攜帶組成性表達PrfA蛋白的重組無害李斯特菌(LI-pERL3-prfA*)以及重組單核細胞增生李斯特菌(EGDeΔprfA-pERL3-prfA*),在生物被膜形成能力方面差異；2)比較研究LM野生株EGDe、PrfA缺失株EGDeΔprfA、以及攜帶組成性表達PrfA蛋白的重組單核細胞增生李斯特菌EGDeAprfA-pERL3-prfA*在不同碳源的培養(yǎng)基中,BHI(Brain Heart Infusion,復雜多糖作為碳源)和基礎培養(yǎng)基(Minimal Essential Medium簡稱MM培養(yǎng)基,可添加PTS糖或非PTS糖作為碳源,本文采用的PTS糖為葡萄糖,非PTS糖為甘油)中生物被膜形成的差異,探討LM重要的毒力調(diào)控蛋白PrfA在不同的糖成分作為碳源的培養(yǎng)基中對生物被膜形成的影響；三、比較了三種菌株LM野生株EGD、sigB缺失株EGDeAsigB和無害李斯特菌(Listeria innocua, LI)分別在BHI培養(yǎng)基和BHI培養(yǎng)基添加0.3%膽汁酸鹽的培養(yǎng)基中,生物被膜形成能力的差異。 [結(jié)果]1)LM野生株具有較強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)在營養(yǎng)豐富的常用BHI培養(yǎng)基中,組成性高量表達PrfA蛋白可以回復EGDeAprfA的生物被膜形成能力,但對LI沒有增強作用。在不同糖類作為碳源的培養(yǎng)基中,各菌株在MM培養(yǎng)基中的被膜形成能力要顯著性高于在BHI培養(yǎng)基中相應菌株的被膜形成能力；但無論在何種培養(yǎng)基中LM野生株(EGD或EGDe)均具有較強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培養(yǎng)基中組成性高量表達PrfA蛋白可以回復EGDeAprfA的生物被膜形成能力；而在MM培養(yǎng)基中高量表達PrfA蛋白不能回復EGDeΔprfA的生物被膜形成能力,并且在以葡萄糖為唯一碳源的MM培養(yǎng)中,高表達PrfA菌株的生物被膜形成能力甚至比EGDeΔprfA還要低；3)EGD、EGDeAsigB和LI三種菌株無論在填不添加0.3%的膽汁酸鹽的BHI培養(yǎng)基中均表現(xiàn)出：LI的被膜形成能力最差(幾乎沒有交聯(lián)結(jié)構(gòu)的出現(xiàn))低于EGDeAsigB低于EGD的被膜形成能力,區(qū)別在于：在添加0.3%膽汁酸鹽后,EGD的被膜形成能力較BHI中要提高一些,而EGDeAsigB卻較BHI中被膜形成能力要降低一些,LI幾乎不受影響。 [結(jié)論]1)LM在營養(yǎng)極端貧乏的MM培養(yǎng)基(無論添加PTS糖還是非PTS糖)中生物被膜的形成能力要遠遠高于在營養(yǎng)十分豐富的BHI培養(yǎng)基。但無論在哪種培養(yǎng)基中,PrfA均在LM生物被膜形成中具有重要的促進作用,缺失該基因均可降低LM的生物被膜的形成能力,但這種促進作用與培養(yǎng)基的成分(我們認為主要是糖成分不同)、PrfA數(shù)量與活性均有密切的關系。2)0.3%的膽汁酸鹽可以在一定程度上促進LM的被膜形成,卻不能促進EGDeAsigB的被膜形成,反而使其被膜形成的能力略有下降,表明：LM中對外界不良環(huán)境的抵抗能力一定程度上依賴于SigB因子,所以不利于細菌生存的外界條件可以在一定條件下通過調(diào)節(jié)SigB因子,而調(diào)控相關基因的表達,進而通過促進被膜的形成在一定程度上協(xié)助LM對抗不良環(huán)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 .
【學位授予單位】：華中師范大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378
本文編號：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 .
【學位授予單位】：華中師范大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378
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