Data_Sheet_1_Bartonella taylorii: A Model Organism for Studying Bartonella Infection in vitro and in vivo.pdf

Bartonella spp. are Gram-negative facultative intracellular pathogens that infect diverse mammals and cause a long-lasting intra-erythrocytic bacteremia in their natural host. These bacteria translocate Bartonella effector proteins (Beps) into host cells via their VirB/VirD4 type 4 secretion system (T4SS) in order to subvert host cellular functions, thereby leading to the downregulation of innate immune responses. Most studies on the functional analysis of the VirB/VirD4 T4SS and the Beps were performed with the major zoonotic pathogen Bartonella henselae for which efficient in vitro infection protocols have been established. However, its natural host, the cat, is unsuitable as an experimental infection model. In vivo studies were mostly confined to rodent models using rodent-specific Bartonella species, while the in vitro infection protocols devised for B. henselae are not transferable for those pathogens. The disparities of in vitro and in vivo studies in different species have hampered progress in our understanding of Bartonella pathogenesis. Here we describe the murine-specific strain Bartonella taylorii IBS296 as a new model organism facilitating the study of bacterial pathogenesis both in vitro in cell cultures and in vivo in laboratory mice. We implemented the split NanoLuc luciferase-based translocation assay to study BepD translocation through the VirB/VirD4 T4SS. We found increased effector-translocation into host cells if the bacteria were grown on tryptic soy agar (TSA) plates and experienced a temperature shift immediately before infection. The improved infectivity in vitro was correlating to an upregulation of the VirB/VirD4 T4SS. Using our adapted infection protocols, we showed BepD-dependent immunomodulatory phenotypes in vitro. In mice, the implemented growth conditions enabled infection by a massively reduced inoculum without having an impact on the course of the intra-erythrocytic bacteremia. The established model opens new avenues to study the role of the VirB/VirD4 T4SS and the translocated Bep effectors in vitro and in vivo.

巴尔通体属（Bartonella spp.）为革兰氏阴性兼性胞内致病菌，可侵染多种哺乳动物，并在其自然宿主体内引发持久的红细胞内菌血症。此类细菌可通过VirB/VirD4 IV型分泌系统（T4SS）将巴尔通体效应蛋白（Bartonella effector proteins, Beps）转运至宿主细胞内，以颠覆宿主细胞的正常功能，进而导致先天免疫应答下调。目前针对VirB/VirD4 T4SS及Beps的功能研究，大多以主要人畜共患病原菌汉氏巴尔通体（Bartonella henselae）为对象，其体外感染方案已较为成熟。但其自然宿主猫并不适合作为实验感染模型。现有体内研究多局限于使用啮齿动物特异性巴尔通体菌种的啮齿动物模型，而针对汉氏巴尔通体建立的体外感染方案并不适用于此类病原菌。不同物种间体外与体内研究的差异，掣肘了学界对巴尔通体致病机制的深入理解。本研究介绍了小鼠特异性菌株泰勒氏巴尔通体IBS296（Bartonella taylorii IBS296）作为新型模式生物，可同时支撑细胞培养层面的体外研究与实验小鼠体内的病原致病机制探究。我们采用基于拆分式纳米荧光素酶（NanoLuc）的易位检测实验，探究BepD通过VirB/VirD4 T4SS的易位情况。研究发现，若细菌在胰蛋白胨大豆琼脂（TSA）平板上培养，并在感染前进行温度骤变处理，可显著提升效应蛋白向宿主细胞内的转运效率。体外感染活性的提升与VirB/VirD4 T4SS的上调表达呈正相关。通过优化后的感染方案，我们在体外实验中证实了BepD依赖的免疫调节表型。在小鼠体内，经优化的培养条件可使感染所需的接种量大幅降低，且不会影响红细胞内菌血症的病程进展。本研究建立的模型为体外及体内研究VirB/VirD4 T4SS及其转运的Beps效应蛋白的功能提供了全新的研究途径。