线虫病原细菌与宿主肠道共生菌互作机制研究

Colonization in host intestines by pathogenic bacteria is an intense focus in global health and disease research area. The relationships between the pathogenic bacteria and the commensal microbes in intestines are key for successful colonization. In our previous study on the molecular mechanism on colonization of Bacillus nematocida B16, we found that commensal bacteria in intestines were important factors affecting colonization, and some commensal bacteria could inhibit B16 colonization by producing some antimicrobial factors, while others could help B16 remove this suppression. However, it is still not clear on the interactions between pathogen B. nematocida B16 and commensal bacteria in intestines of nematodes so far. In this project, we shall choose the pathogenic bacterium B. nematocida B16, a commensal bacterium Pediococcus sp. X7, which prevents B16 colonization and another commensal bacterium Pseudomonas sp. SCO6 antagonizing against X7 strain as our research system. We shall employ protein purification, circular dichroism spectra, immunofluorescence localization, 2D electrophoresis, construction and screening of mutant library, gene functional verification, real-time PCR, colonization test feeding restricted bacteria methods to systematically study the structure, function and targets of the protein capable of preventing B16 colonization in X7 strain; explore the mechanism on forming mixed biofilm in co-culture strains SCO6 and B16, and reveal the molecular mechanism on how the pathogen B16 successfully colonizes in combination with the commensal bacterium Pseudomonas sp. SCO6 and how they overcome the prevention of Pediococcus sp. X7. Our research has important scientific significance and will provide a molecular model on the interaction mechanism between pathogenic and commensal bacteria.

病原细菌在宿主肠道定殖是全球健康和疾病研究的热点, 病原细菌与宿主肠道共生菌互作是其能否定殖的关键。前期研究线虫病原细菌Bacillus nematocida B16定殖的分子机制发现：肠道共生菌是影响B16定殖的重要因素，一些共生菌能够产生抑菌因子抑制B16定殖，另一些则能够被B16利用来消除这种抑制，但具体的互作机制仍不清楚。本项目以病原细菌B16、抵抗B16定殖的共生菌Pediococcus sp. X7及帮助B16拮抗X7的共生菌Pseudomonas sp. SCO6为研究体系，采用蛋白纯化、圆二色谱、免疫荧光定位、2D电泳、突变文库构建与筛选、基因功能验证、定量PCR及限菌喂养试验等方法，研究X7抑菌蛋白的结构、功能及作用靶点，探索B16与SCO6混菌生物膜形成机理，揭示B16联合SCO6战胜X7的分子机制，为病原细菌与宿主共生菌的互作机制提供一个分子模型，具有重要的科学意义。

我们前期在筛选分离秀丽隐杆线虫（Caenorhabditis elegans）肠道伴生菌的过程中发现：自然界中的C. elegans肠道菌比实验室喂养的更具有多样性，因此我们将研究对象扩大至自然界中的线虫C. elegans，从而更好地揭示其生态学意义。（1）无论C. elegans来源于土壤、腐烂水果还是实验室，当杀线虫芽孢杆菌B16入侵线虫后，线虫肠道菌群的结构和数量均发生了巨大改变；（2）从腐烂的水果和蔬菜中分离到C. elegans, 再将分离获得的C. elegans饥饿两天后，表面消毒、研磨，得到约52株肠道细菌；（3）杀线虫芽孢杆菌B16进入肠道后，自身通过抗菌肽抑制线虫体内共生菌W5的生长，B16占据生态位；（4）线虫内生菌SCO41，BX15和BC7显示了强抑制杀线虫芽孢杆菌B16在线虫体内的定殖能力，可见，线虫并不是被动被B16杀死。系统的分类鉴定结果表明：三株菌SCO41，BX15和BC7是同一个菌种，均属于Phytobacter属。以Phytobacter sp. SCO41为代表研究其定殖抗性的分子机制：SCO41菌株鞭毛相关蛋白多、运动能力强、游离性高、黏附能力强、综合铁离子运输竞争等机制来抑制B16的定殖。此外，从肠道内生菌SCO41菌株中分别纯化出两个具有协同性抑制B16定殖的鞭毛蛋白P1和功能未知蛋白 P2，这两个蛋白协同抑制B16的定植；（5）当病原细菌B16和SCO6联合培养时，形成混菌生物膜，SCO41无法作用到B16的靶点，从而SCO41对B16的定殖抑制被解除。本课题的研究丰富了线虫肠道内细菌种类，报道了线虫肠道这一特殊生境下细菌特有的生命形态，揭示了线虫肠道内的微生物食物环，对今后深入探索病原细菌、肠道共生菌及其宿主三者之间的互作机制奠定了良好的基础。该研究内容已经正式发表SCI论文6篇，另有2篇SCI论文仍在投稿中，正式发表中文核心期刊2篇，同时授权国家发明专利4项，还有4项在实审阶段。培养硕士研究生15名。申请人获得河南省学术技术带头人，河南省优秀硕士学位论文指导教师，河南省优秀教师、优秀共产党员的称号，并被评为“南阳师范学院卧龙学者”，完成了既定目标。本课题的研究对今后深入探索病原菌、宿主内生菌及宿主三者的互作机制奠定了良好的基础。