CAREER: Experimental manipulation of host-microbe associations to reveal key features promoting symbiosis

许多动物依靠微生物共生体来帮助它们获得足够的营养，保护它们免受捕食者和病原体的侵害，或者让它们生活在其他不适宜生存的环境中。研究这些关系可能很困难，因为通常不可能将微生物与其宿主分开。因此，由于无法独立操纵宿主和共生体，对这些关系背后的机制的理解受到限制。接吻虫是一种以脊椎动物血液为食的昆虫，是共生细菌的宿主，帮助它们成功地发育和繁殖。这些细菌也可以从接吻虫中清除，并被新的细菌感染。共生细菌也可以在昆虫体外生长，并可以进行基因改造。因此，该系统提供了一个独特的机会来实验操纵宿主和共生体，以确定和表征支持共生的因素。研究重点包括参与寄主共生体定植的基因、参与寄主与共生体营养物质交换的基因、寄主免疫系统与共生体的相互作用等。鉴于无处不在的动物-微生物共生，这项研究将阐明在其他不太容易处理的系统中可能控制宿主-微生物相互作用的机制。除了提出的研究外，本项目还将为高中教师提供研究经验和培训。这一经验将与课程开发相结合，将宿主-微生物的相互作用纳入教师的课堂。宿主-微生物系统的实验操作由于无法分离高度整合的伙伴或难以在高度复杂的群落中解开个体成员的影响而受到限制。锥蝽吻虫的肠道中有必要的共生体，这些共生体每一代都是在环境中获得的，允许产生无细菌、无菌的昆虫，然后可以用细菌进行实验接种。这项工作利用了该系统的独特特征——能够产生无性和非生物昆虫、低复杂性微生物组和分子工具——来描绘共生的机制基础。转座子诱变将用于鉴定共生体罗得红球菌中共生所必需的基因，然后产生缺乏共生促进基因的罗得红球菌敲除菌株。敲除菌株将用于生物测定，以表征感兴趣基因在关系中的作用。宿主免疫系统在调节细菌丰度中的作用将被评估，检查对共生体的体液和细胞免疫反应。脂质代谢基因，包括脂肪酸合成酶和脂肪酸辅酶a还原酶，已被确定为差异表达在无菌和无菌昆虫。液相色谱和质谱将用于了解共生细菌如何塑造吻虫的脂质组，而特定脂质基因的沉默将揭示这些基因在脂质代谢和宿主适应性中的作用。提出的工作将为宿主-共生体相互作用的演变提供新的线索，并作为探索不太适合实验操作的系统的框架。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Many animals rely on microbial symbionts to help them gain sufficient nutrients, provide protection from predators and pathogens, or allow them to live in otherwise inhospitable environments. Studying these relationships can be difficult as it is often impossible to separate the microbes from their host. As a result, understanding of the mechanisms that underly these relationships has been limited by the inability to manipulate both host and symbiont independently. Kissing bugs are insects that feed exclusively on vertebrate blood and serve as host to symbiotic bacteria, that help them successfully develop and reproduce. These bacteria also can be cleared from the kissing bugs and infected with new bacteria. The symbiont bacteria can also be grown outside of the insect and can be genetically altered. This system, thus, provides a unique opportunity to experimentally manipulate the host and symbiont, to identify and characterize factors that support the symbiosis. The research will focus on genes involved in symbiont colonization of the host, host and symbiont genes involved in the exchange of nutrients, and interactions of the host immune system and the symbiont. Given the ubiquity of animal-microbe symbioses, this research will illuminate mechanisms that may govern host-microbe interactions in other less-tractable systems. Along with the proposed research, this project will also provide research experience and training to high school teachers. This experience will be paired with curriculum development to incorporate host-microbe interactions into the teachers’ classrooms. Experimental manipulation of host-microbe systems has been limited by inability to separate highly integrated partners or difficulties disentangling the effects of individual members in highly complex communities. Triatomine kissing bugs harbor essential symbionts in their gut which are environmentally acquired each generation, allowing for generation of bacteria-free, axenic insects, which can then be experimentally inoculated with bacteria. This work leverages the unique features of this system – ability to generate axenic and gnotobiotic insects, low-complexity microbiomes, and molecular tools – to delineate the mechanistic basis of symbiosis. Transposon mutagenesis will be used to identify genes in the symbiont Rhodococcus rhodnii that are essential for symbiosis followed by generation of knockout strains of R. rhodnii lacking symbiosis-promoting genes. Knockout strains will be used for bioassays to characterize the role of the gene-of-interest in the relationship. The role of the host immune system in regulating bacterial abundance will be assessed, examining both humoral and cellular immune responses to symbionts. Lipid metabolism genes including fatty acid synthases and fatty acid CoA reductases have been identified as differentially expressed in axenic and gnotobiotic insects. Liquid chromatography and mass spectroscopy will be applied to understand how symbiotic bacteria shape the lipidome of kissing bugs while silencing of specific lipid genes will reveal the role of these genes in lipid metabolism and host fitness. The proposed work will shed new light on the evolution of host-symbiont interactions and serve as a framework for exploring systems which are less amenable to experimental manipulation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.