【作者】 朱晶；

【导师】 赵立平； Joshua Gong； Parviz Sabour；

【作者基本信息】 上海交通大学 ， 微生物学， 2011， 博士

【摘要】 产肠毒素大肠杆菌(enterotoxigenic Escherichia coli, ETEC)是引起动物和人体发生腹泻的重要病原菌之一。这种病原菌分泌在表面的黏附素可以介导细菌向肠道黏膜附着,随后细菌产生肠毒素而引起宿主腹泻。乳酸菌作为一类益生菌能够帮助宿主改善肠道菌群微生态平衡,维护宿主的肠道健康。已有的体外研究报道ETEC可以引起某些肠上皮细胞的死亡,并且乳酸菌能够通过和ETEC的相互作用而保护宿主细胞。因此,研究ETEC对肠上皮细胞致病的分子机制,以及乳酸菌保护宿主细胞的机理,对于益生菌在防治产肠毒素大肠杆菌引起的腹泻疾病方面有着重要的作用。本研究将一株猪小肠上皮细胞系IPEC-J2细胞和两种浓度的产肠毒素大肠杆菌ETEC K88菌株JG280共培养后,发现108 CFU/ml的ETEC对IPEC-J2细胞的细胞毒性显著性高于109 CFU/ml,这一结果提示ETEC对IPEC-J2的细胞毒性根据细菌浓度的不同而产生差别,并且细菌的群体感应(quorum sensing)可能在ETEC的致病机理中发挥重要的作用。研究发现在108 CFU/ml的ETEC和IPEC-J2细胞共培养过程中,ETEC产生的AI-2(autoinducer-2,自体诱导物)活性和IPEC-J2的细胞死亡呈正相关,而与ETEC的毒力基因estA(编码大肠杆菌耐热肠毒素a)和estB(编码大肠杆菌耐热肠毒素b)表达呈负相关。为了进一步地研究ETEC群体感应的机理,我们将ETEC K88菌株JG280的luxS基因(该基因的产物催化AI-2的生物合成)克隆,并在大肠杆菌E. coli DH5α中过量表达。将载有过量表达的luxS基因的E. coli DH5α的无菌培养上清液(内含高活性的AI-2)和IPEC-J2细胞及108 CFU/ml的ETEC共培养后发现,AI-2可以显著性地降低ETEC对IPEC-J2的细胞毒性并抑制estA基因的表达。以上结果共同提示,由AI-2介导的群体感应在ETEC的致病机理中起重要作用,并且AI-2可能是通过对大肠杆菌耐热肠毒素a的负向调控来实现的。我们还研究了13株从猪体内分离到的乳酸菌是否能够保护IPEC-J2细胞免受ETEC K88菌株JG280的侵染,及其作用的分子机制。本研究首先发现一株非产肠毒素大肠杆菌E. coli K88 JFF4在浓度为108和109 CFU/ml时,均不会对IPEC-J2产生细胞毒性,这一结果提示肠毒素对于引起肠上皮细胞死亡(或损伤)的重要作用。研究还发现,这13株乳酸菌中有5株能够显著地降低ETEC对IPEC-J2的细胞毒性,进而保护IPEC-J2细胞。通过对6株乳酸菌CL9、CL11、CL12、K67、S33和S64的进一步研究表明,这6株乳酸菌均能够降低ETEC诱导IPEC-J2分泌促炎因子白细胞介素-8 (interleukin-8, IL-8),并且除了K67之外的5株乳酸菌均能够促进IPEC-J2分泌抗炎因子白细胞介素-10 (interleukin-10, IL-10)。用实时定量PCR的方法研究发现,一株对IPEC-J2有保护作用的乳酸菌CL9能够抑制ETEC的estA和estB基因表达。乳酸菌S8能够显著性地促进ETEC分泌的AI-2活性,同时它还可以降低ETEC对IPEC-J2的细胞毒性。以上结果共同提示,某些乳酸菌可能通过抑制ETEC毒力基因的表达,另一些乳酸菌可能通过分泌某些物质影响ETEC群体感应信号分子的作用,进而降低ETEC肠毒素的产生,从而保护宿主细胞。同时,IPEC-J2细胞分泌的细胞因子IL-8和IL-10在乳酸菌的作用机制中起重要作用。为了进一步研究乳酸菌的作用机理,我们还需更加深入的研究。更多还原

【Abstract】 Enterotoxigenic Escherichia coli (ETEC) are often associated with the outbreaks of diarrhea in animals and human beings worldwide. The adhesins of ETEC mediate bacterial adherence to the intestine, after binding of the fimbriae to enterocytes, ETEC proliferate rapidly to attain massive numbers and produce one or more types of enterotoxin, which stimulate fluid and electrolyte secretion by intestinal cells, thus leading to diarrhea. Some lactic acid bacteria were identified as probiotics, which can help the host with the improvement of gut microbial ecology and confer health benefits to animals and humans. Previous report showed that ETEC induced cell death of several intestinal epithelial cells in vitro, and lactic acid bacteria may protect the intestinal epithelial cells through the interactions with ETEC. However, the mechanisms of the actions are largely undefined. Therefore, understanding the ETEC pathogenesis of infecting host cells, and the potential protecting mechanisms of lactic acid bacteria would provide new insights into the study of how lactic acid bacteria protect the host.In this study, we incubated pig intestinal IPEC-J2 cells with two concentrations of ETEC K88 strain JG280. We observed that 108 CFU/ml of ETEC K88 strain JG280 caused more death of IPEC-J2 cells than did 109 CFU/ml, suggesting that ETEC-induced cell death was cell density-dependent and that quorum sensing (QS) may play a role in pathogenesis. Subsequent investigations demonstrated a positive correlation between autoinducer 2 (AI-2) activity of ETEC K88 strain JG280 and death of IPEC-J2 cells during the infection up to 3 hours. However, there was a negative correlation between AI-2 activity and expression of the ETEC K88 strain JG280 enterotoxin genes estA and estB when IPEC-J2 cells were exposed to the pathogen at 108 CFU/ml. To further understand the QS mechanisms of ETEC, we therefore cloned the luxS gene (responsible for AI-2 production) from ETEC K88 strain JG280, and overexpressed it in E. coli DH5α. Addition of culture fluid from E. coli DH5αwith the overexpressed luxS reduced cell death of IPEC-J2 by 108 CFU/ml JG280. The addition also reduced the estA expression of ETEC K88 strain JG280. These results suggest the involvement of AI-2-mediated quorum sensing in K88+ ETEC pathogenesis, possibly through a negative regulation of STa production.In this study, we have also investigated possible protection of thirteen previously selected lactic acid bacteria (LAB) strains from swine, against intestinal cell damage by ETEC K88 strain JG280 infection and the underlying mechanisms. In previous study, we observed that the non-pathogenic K88+ E. coli strain JFF4 (toxins-negative) caused no cytotoxicity to IPEC-J2 cells at both concentrations of 108 CFU/ml and 109 CFU/ml, implying the effect of enterotoxins on cell damage/death. Five out of thirteen LAB strains were able to prevent the ETEC-induced cytotoxicity to IPEC-J2 cells. Further investigations on six LAB strains CL9, CL11, CL12, K67, S33 and S64 showed that IL-8 (interleukin-8, pro-inflammatory cytokine) production was decreased by these six strains, and IL-10 (interleukin-10, anti-inflammatory cytokine) production was increased by five strains, except K67. Quantitative PCR analysis showed that expression of two enterotoxin genes estA and estB of ETEC were reduced by CL9 (an effective LAB strain) during incubation. Subsequent investigations showed that LAB strain S8 significantly increased the AI-2 activity produced by ETEC, and meanwhile inhibited the ETEC cytotoxicity to IPEC-J2 cells. Together, our results indicate that some LAB strains inhibit the intestinal cell damage caused by ETEC through inhibiting the enterotoxin gene expression of ETEC, and some LAB might secrete a molecule(s) that interfere with ETEC quorum sensing signaling. Meanwhile inflammatory cytokines IL-8 and IL-10 may involve in the lactic acid bacteria-reduced inflammation of IPEC-J2. To determine the molecular mechanism of the protection offered by the LAB strains, further studies are required.更多还原