【作者】 栗冬梅；

【导师】 张建中；

【作者基本信息】 中国疾病预防控制中心 ， 病原生物学， 2009， 博士

【摘要】 猫抓病（cat-scratch disease,CSD）主要是由汉赛巴尔通体引起的人类新发人兽共患病。猫是这种巴尔通体的自然宿主,通过抓或咬伤将巴尔通体传染给人,人感染这些细菌后在伤口处会出现红斑丘疹或化脓性丘疱疹,继而发生以局部淋巴结炎为典型症状的CSD,少数患者还可并发心内膜炎、杆菌性血管瘤、骨髓炎和脑膜炎等全身性疾病。人们对汉赛巴尔通体的致病机理和宿主的免疫保护机制还不十分清楚。本研究拟应用蛋白质组学和生物信息学的研究方法对汉赛巴尔通体外膜蛋白质组进行研究,筛选可能用于诊断和疫苗研制的候选蛋白,以及发现可能与细胞粘附、侵入及毒力相关的膜蛋白成分。实验根据外膜蛋白组分不能溶解在十二烷基肌氨酸钠（简称SLS）和碳酸钠（Na₂CO₃）溶液中的特性,用2种分离方法制备汉赛巴尔通体ATCC 49882 Houston-1株外膜蛋白样品。采用双向电泳（2-DE）,即进行pH 3-10非线性IPG干胶条进行第一向等电聚焦电泳、12.5%凝胶第二向SDS-PAGE电泳、考马斯亮蓝G-250染色、图像扫描及分析;比较上述2种方法制备的外膜蛋白样品2-DE图谱差异,并对不同裂解液溶解的蛋白样品结果进行比较。在获得SLS法和碳酸钠法在外膜蛋白样品制备中存在互补性的条件下,同时用两种方法制备了另外3种致病性巴尔通体（伊丽莎白巴尔通体、文森巴尔通体博格霍夫亚种和格拉汉姆巴尔通体）的外膜蛋白样品,进行2-DE实验分析。应用SLS制备的蛋白样品上样量为600μg,获得约368个蛋白质点,大量高丰度蛋白集中在pⅠ值4.3～6.5之间。等电点范围为4.0～10.0,分子量范围为15 kDa～150 kDa。其中大部分高丰度蛋白分布在pⅠ值5～6和8～9之间。应用碳酸钠的蛋白样品上样量为800μg,获得约471个蛋白质点,高丰度蛋白集中在pⅠ值5.0～6.3之间。等电点范围为4.8～10.0,分子量范围为10 kDa～180 KDa。在裂解液中增加ASB-14等两性离子去污剂使蛋白样品溶解性增强,检出蛋白点增多,碱性端检出蛋白点明显增加。重复性实验2种方法均获得稳定、一致的2-DE图谱。应用基质辅助激光解析-飞行时间质谱技术鉴定了上述2种方法检出的全部蛋白点,并对鉴定的蛋白进行信号肽、亚细胞定位和结构域搜索等生物信息学分析。SLS法制备的样品中鉴定出176点代表94种蛋白,外膜蛋白有6种;碳酸钠法制备的样品中鉴定出259点代表139种蛋白,外膜蛋白有9种;2种方法共鉴定出10种外膜蛋白,2种是本研究中首次证实存在的外膜蛋白,包括孔蛋白、血红素结合蛋白、表面抗原蛋白、药物排出相关蛋白和脂蛋白等。对汉赛巴尔通体基因组中预测出的外膜蛋白应用生物信息学方法初步预测结构和功能,结果发现43个外膜蛋白中与铁离子吸收相关毒力因子有5个、细胞粘附相关的蛋白5个、溶血素4个、血凝素6个、自转运蛋白7个、药物外排相关蛋白2个、运输相关蛋白2个、抵抗有机溶剂蛋白1个及一些与膜结构相关的蛋白;有2个细菌表面抗原蛋白,均已被分离鉴定出来,可能具有免疫保护性,需要进一步研究证实。本研究建立了巴尔通体外膜蛋白2-DE技术,并发现两种外膜蛋白样品制备方法有一定的互补性,SLS法表现出对外膜上高表达的孔蛋白的富集,Na₂CO₃法对低丰度、碱性蛋白表现出较好的分离效果,为今后巴尔通体外膜蛋白质组研究提供了参考。新发现的蛋白提示巴尔通体可能存在药物外排系统,对抗菌药物和耐药机制产生的影响有待进一步研究。外膜蛋白的质谱鉴定及生物信息学分析提示在汉赛巴尔通体外膜上存在大量与细胞粘附、侵袭等致病相关的蛋白,对汉赛巴尔通体外膜蛋白质组的全面评估不仅对今后开展诊断和疫苗技术研究有重要意义,而且为巴尔通体致病机制研究指出了新的方向。更多还原

【Abstract】 Cat scratch disease (CSD) is a bacterial disease caused by Bartonella henselae which is an emerging zoonotic pathogen causing a wide range of disease manifestations in humans. Most people with CSD have been bitten or scratched by a cat and developed a mild infection at the point of injury. Lymph nodes, especially those around the head, neck, and upper limbs, become swollen. Additionally, a person with CSD may experience fever, headache, fatigue, and a poor appetite. Rare complications of B. henselae infection are bacillary angiomatosis, endocarditis and Parinaud’s oculolandular syndrome.Despite there are significant advances in recent years, our understanding of Bartonella pathogenesis is still incomplete. Little is known about the disease pathogenesis and immunopathogenesis of Bartonella, diagnosis and immunoprophylaxis of human infection remains extremely challenging. To address this paucity of knowledge, we sought to identify potential membrane-associated virulence factors for adhesion, invasion and secretions of proteins, as well as protective and diagnostically relevant B. henselae antigens, by characterizing the outer membrane fraction and bioinformatic analysis of outer membrane proteome of B. henselae.We isolated the outer membrane proteins of B. henselae by using the ionic detergent lauryl sarcosine (SLS) and sodium carbonate, purification by two-dimensional electrophoresis with immobilized pH gradients (IPG Strips pH 3-10) in the first dimension, followed by protein identification using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Reference maps of OMPs were established and analyzed by software. It is possible to see that solubilization buffer with ASB-14 and ZWITTERGENT 3-10 can reveal significantly more spots especially in the base region of the 2-DE profiles, as shown by the increased number of spots with good resolution. Total 368 spots were calculated on the gel from the sarcosine-insoluble outer membrane fraction, and 94 distinct protein species from 176 spots were identified. In the outer membrane fraction by applying carbonate incubation, 471 spots were calculated on the gel and 259 were identified which represent 139 protein entries. There are 6 outer membrane proteins from the the sarcosine-insoluble outer membrane fraction comparing with 9 outer membrane proteins from samples of carbonate incubation. Among the total 10 outer membrane proteins variably expressed outer membrane protein porin, iron ion uptake associated proteins, surface antigens, putative drug efflux proteins, lipoprotein and peptidyl-prolyl cis-trans-isomerase. In an additional demonstration we show the value of bioinformatic analysis for 44 outer membrane proteins by prediction of domains and tertiary structures document the potential virulence factors comprising 5 iron ion adorption proteins, 5 adhesion proteins, 4 hemolysins, 6 aggruglobins, 7 autotransporter proteins, 2 drug efflux proteins, and 2 bacteria surface antigens.In summary, we established the 2-DE reference maps of the outer membrane subproteome of B. henselae by using the two different extraction methods which were complementary to each other in partly. Sodium carbonate extraction isolated low abundance and basic proteins better than the SLS extraction which preferred to enrich high abundance porins. Some efflux systems may present on B. henselae cells warranting further consideration as the potential ability to limit the access of antimicrobial agents to their targets. The identification and bioinformatic evaluation of these B. henselae outer membrane proteins should not only aid in the development of better diagnostic tests and better disease prevention but also provide insight into the pathogenesis of Bartonella.更多还原