【作者】 蒋建军；

【导师】 剡根强； 方维焕；

【作者基本信息】 石河子大学 ， 动物遗传育种与繁殖， 2011， 博士

【摘要】 单核细胞增多症李斯特菌(Listeria monocytogenes, LM)是重要的人畜共患食源性病原菌,广泛存于自然环境和食品加工环境,耐酸、耐盐,低温下可繁殖,能引起人和动物的脑膜炎、败血症和胃肠炎,致死率达20-30%,给人及动物健康带来较大危害。不同来源的单核细胞增多症李斯特菌的毒力差异很大,自上世纪80年代以来李斯特菌的毒力因子一直是人们研究的热点。内化素C2(InlC2)是LM内化素家族中众多内化素的成员之一。它存在于主要的致病性李斯特菌菌株中,为LM所独有,常与iinlD等内化素基因形成基因簇,发挥其功能,近来研究表明,InlC2与InlA一起在李斯特菌病的体液免疫中发挥重要作用。为了解InlC2在单核细胞增多症李斯特菌感染宿主细胞过程中的作用以及与其他重要内化素的关系。我们从以下几个方面对其进行研究。研究结果如下：1、采用PCR的方法检测了134株来源不同的单核细胞增多症李斯特菌inlA、inlB、inlC、inlC2、inlD、inlE、inlG内化素的分布以及ascB-dapE内化素岛的内化素的构成情况,结果：除1/2b型的菌株S10不含inlA和inlB,其余都含有inlA和inlB。inlC存在于所有谱系Ⅰ、Ⅱ(121/121)和53.8%(7/13)谱系Ⅲ菌株。inlD存在于所有谱系Ⅰ100%(55/55)、谱系Ⅱ25.7%(17/66)和谱系Ⅲ69.2%(9/13)。inlE存在于所有谱系Ⅰ、Ⅱ(121/121)和谱系Ⅲ30.7%(4/13)。inlG在谱系Ⅰ中不存在(0/55),谱系Ⅱ74.2%(49/66)和谱系Ⅲ30.7%(4/13)。134株中有110株(82.1%)含有inlC2。所有谱系Ⅰ(血清型1/2b和4b)都含有inlC2基因,除一个在inlGC2DE基因群中以外,其余都在inlC2DE中,在谱系Ⅱ中49株血清型1/2a的其中47株菌含有inlC2,在inlC2DE(17/47)或inlGC2DE(30/47)基因群中,16株血清型1/2c中仅有一株含有inlC2基因,存在于inlGC2DE基因群中,谱系Ⅲ中的13株菌其中6株inlC2存在于inlGC2DE(4/6)基因群或别的结构中(2/6),此外,其余5个种无inlC2基因。结果表明：LM特有的内化素基因inlC2出现在所有谱系Ⅰ和大多数血清型1/2a的菌株中。谱系Ⅲ中存在相对较少。inlC2与其他内化素基因inlD、inlE,有时inlG一起在ascB-dapE内化素岛中组成不同的内化素基因簇。使ascB-dapE内化素岛具有较大的多态性,这可作为区分LM谱系/血清型的分子标志。2、PCR扩增inlC2基因两端的同源臂,融合后,使用同源重组的方法构建LM标准株681和L10的△inlC2缺失突变株。经氯霉素筛选突变株,旁侧引物扩增和测序方式验证获得△inlC2缺失突变株。3、通过LM681和L10与相应△inlC2突变株的生长培养、BALB/c小鼠增殖试验,ICR小鼠毒力试验,致死病理解剖和常规组织染色法,确定△inlC2突变株对LM毒力的影响。结果：亲本株与突变株生长培养结果之间差异不显著。在小鼠体内增殖试验中△inlC2突变株与亲本株之间差异明显,△inlC2突变株在脾脏中的存活数是亲本株的10倍(P<0.01),在肝脏中是亲本株的7倍。ICR小鼠毒力试验LD50差异不显著。组织染色结果显示△inlC2突变株引起的病理变化比亲本株要明显,特别是在脑、脾脏和肝脏。结果表明：inlC2与单核细胞增多症李斯特菌对小鼠的致病能力的大小有关。△inlC2突变株对小鼠的致病力增强了。4、通过LM681、Ll0及其△inlC2突变株对Hela细胞的粘附、侵袭和细胞内的增殖试验,确定inlC2基因的缺失是否会影响LM对细胞的感染作用。结果：△inlC2突变株比它们的亲本株的对HeLa细胞的粘附能力增强1.3倍(P<0.05),侵袭力约2.7倍(P<0.01),细胞内增殖是亲本株的1.7倍。这些数据结果表明：inlC2基因的缺失增强了LM对HeLa细胞的内化作用。5、分别通过qRT-PCR(?)Iwestern blot评估inlA和inlB的转录和转录后水平,以确定单核细胞增多症李斯特菌△inlC2突变株内化作用的增强是否与inlA和inlB有关。结果：inlA和inlB的转录水平并没有增加,但产量却有很大提高。结果表明：inlC2基因的缺失增加了InlA的产量但没有改变inlA的转录水平,InlB也同样如此。6、通过qRT-PCR检测对比人工胃液(pH 2.5)和BHI中单核细胞增多症李斯特菌的inlA, inlB、inlC2、inlD和inlE转录水平。确定inlC2是否与感染有关。转录结果：在人工胃液中inlC2、inlA和inlB转录水平相对于在BHI中(pH 7：P<0.01)显著增加。相反inlD和inlE转录水平没有变化。结果表明：在人工胃液中inlC2被诱导表达,表明它可能是另外一个在感染中起作用LPXTG内化素基因,再者说明它的表达是单顺反子,独立于inlD和inlE外的单独表达。所有以上结果表明inlC2基因缺失增强了LM的致病性,LM的内化作用是以不同的内化素之间相互作用的复杂的网络结构来调节。并且可能与其他感染相关因子(例如分选酶A)与LPTGX基序支撑蛋白的易位有关。研究结果为深入探索单核细胞增多症李斯特菌的功能基因及其调控机制、致病性和环境适应性的分子机制等方面具有重要的理论与实践意义。更多还原

【Abstract】 Listeria monocytogenes(L. monocytogenes, LM) can cause significant foodborne zoonosis. It exists generally in natural environment and food processing environment and resists the acid and salt, which can breed under low temperature. Because it can cause meningitis, septicaemia, gastroenteritis of humans and animals and its mortality approaching 30%, that brings major harm to humans and animals health. The virulence diversity of different source listeria monocytogenes is large, so the study of virulence factor of LM is always the hot spot since The 1980s. Internlin C2 (InlC2) is one of the L. monocytogenes internlin family and exists in the major pathogenic L. monocytogenes. The internlin gene inlC2 is L. monocytogenesspecific and forms an internalin cluster with inlD, inlE and, in some cases, inlG between ascB and dapE to play its function. The recent study shows that InlC2 plays important action in humoral immune together with InlA. To understand the affect of InlC2 in L. monocytogenes infection and the relation with other important internlin gene, the several aspects were studied. The results are as below:1、A PCR-based approach revealed the distribution of internlin gene inlA, inlB, inlC, inlC2, inlD, inlE, and inlG and the structure of the internalin cluster between ascB and dapE from 134 source different L. monocytogenes strains. The results showed that internlin gene inlA and inlB existed in all serovars L. monocytogenes but serovars 1/2b strain S10. Internlin gene inlC existed in all lineageⅠ,Ⅱ(121/121) strains and 53.8%(7/13) lineageⅢstrains. Internlin gene inlD existed in all lineageⅠ(55/55) strains,25.7% (17/66)lineageⅡstrains and 69.2%(9/13)lineageⅢstrains. Internlin gene inlE existed in all lineageⅠ,Ⅱ(121/121) strains and 30.7%(4/13) lineageⅢstrains.InlG did not exist in lineageⅠ(0/55) strains and 74.2%(49/66) lineageⅡstrains and 30.7%(4/13) lineageⅢstrains contained this gene.Among 134 strains L. monocytogenes there were 110 strains (82.1%) harbored inlC2. All lineageⅠ(serovars 1/2b and 4b) strains contained inlC2 which was embedded into the inlC2DE cluster except one serovar 1/2b strain bearing inlGC2DE.Among lineageⅡstrains,47 of the 49 serovar 1/2a strains carried inlC2 within inlC2DE (17/47) or inlGC2DE (30/47) clusters, while only 1 of the 16 serovar 1/2c strains harbored this gene within inlGC2DE. Of 13 lineageⅢstrains,6 contained inlC2 within inlGC2DE cluster (4/6) or other structures (2/6). In addition, other five Listeria species lacked this gene. Overall, the L. monocytogenes-specific internalin gene inlC2 presents in all lineageⅠand majority of serovar 1/2a strains, and forms internalin cluster with other internalin genes inlD, inlE and, in some cases, inlG. The polymorphism of ascB-dapE internalin cluster may be molecule marker for L. monocytogenes lineage/serovar distinguish.2、The homology arms of both ends of inlC2 gene were amplified and fused. InlC2 deletion mutant of L. monocytogenes parent strain LM681 and L10 were constructed by homologous recombination and verified by amphemycin screening, side primer amplification and sequencing.3、To examine the influence of inlC2 deletion mutant to L. monocytogenes virulence,we detected the growth cultivation of L. monocytogenes strain LM681 and L10 and their corresponding inlC2 deletion mutant, the proliferation in BALB/c mouse, ICR mouse virulence test, pathologic anatomy and routine tissue staining. The results revealed that the growth cultivation between parent strain and mutant had no obvious diversity,but the survival in mouse spleen of inlC2 mutant was ten fold than parent strain (P<0.01) and about seven fold increment in the liver than parent strain. The LD50 disparity was not significant in ICR mouse virulence test. The tissue stain results displayed that pathological change caused by inlC2 mutate was obvious than that of parent strain, especially in brain, spleen and liver. This indicated that inlC2 was related with L. monocytogenes pathogenecity to mouse and the loss of inlC2 appeared to enhance the pathogenecity.4.To investigate whether the putative infection-related internalin gene inlC2 contributed to internalization of L. monocytogenes by epithelial cells, we compared the adhesion and invasion abilities of LM681, L10 and LM681-ΔinlC2 and L10-ΔinlC2 in HeLa cells. Interestingly adherence ofΔinlC2 to HeLa cells was 1.3-fold higher than that of the parent strain (P<0.05), and invasion of DinlC2 into HeLa cells was around 2.7-fold higher than that of its parent strain (P<0.01;). A 1.7-fold increase in intracellular growth of inlC2 deletion mutant was also observed. These data indicated that the loss of inlC2 appeared to enhance the internalization of L. monocytogenes in HeLa cells.5. To determine whether the increased internalization of inlC2 mutate was related with the level of inlA and inlB, we evaluated expression of inlA at the transcriptional level by qRT-PCR and at posttranscriptional level by western blot.The results suggested that disruption of inlC2 increased the production of InlA and inlB without changing inlA and inlB transcript level.6. In order to clarify whether inlC2 was infection-related, we tested the transcriptional levels of inlA, inlB, inlC2, inlD and inlE when exposed to synthetic human gastric fluid (PH 2.5) and BHI. Transcriptional analysis showed that levels of inlC2, inlA and inlB transcripts in synthetic human gastric fluid significantly increased as compared to those in BHI (pH 7; P\0.01). In contrast, inlD and inlE transcripts remained unchanged These data support that (1) inlC2 is induced in human gastric fluid, representing another LPXTG internalin gene possibly invovled in the infection, and (2) its expression is monocistronic, independently of inlD and inlE expression.Overall, this study presents supportive evidence that deletion of inlC2 gene may strengthen the pathogenicity of L. monocytogenes and its internalization is a complex network through the interplay of distinct internalins and possibly other infection-related factors, e.g. sortase A (encoded by strA) that is implicated in translocation of proteins bearing LPTGX motif.These study have important significance in theory and practice for exploring deeply the molecule mechanism of functional gene, regulation mechanism, pathogenicity and environmental suitability of L. monocytogenes.更多还原