C. elegans model of B. anthracis immunity & pathogenesis

描述（由申请人提供）：炭疽芽孢杆菌是一种普遍存在的土壤细菌，也是引起炭疽的NIAID a类病原体。虽然研究已经揭示了对这种致命细菌的重要见解（例如，三方毒素的活性），但炭疽的发病机制尚未得到令人满意的解释，我们还有很多不了解的地方。对致病性起重要作用的染色体因素还没有得到充分的研究，我们对先天免疫系统如何防止炭疽感染知之甚少。我们已经证明了炭疽芽胞杆菌可以在土壤线虫和模式无脊椎动物秀丽隐杆线虫中产生致命感染。这一发现开启了在前所未有的规模上使用正向和反向遗传学研究炭疽感染的可能性。炭疽杆菌可以在线虫体内感染和增殖，最终由内而外吞噬线虫。然而，秀丽隐杆线虫具有强大的先天免疫系统，与哺乳动物有许多相同的途径和效应机制。在某些情况下，线虫似乎能够对炭疽杆菌产生有效的免疫反应。本提案将利用这一新颖的秀丽隐杆线虫- b。在前所未有的规模上鉴定(i)参与对炭疽杆菌感染的先天防御的宿主基因和（ii）参与在完整动物中建立感染和规避先天防御的炭疽杆菌基因。Affymetrix微阵列将用于鉴定对炭疽杆菌感染反应上调的潜在宿主免疫基因。这些基因将在秀丽隐杆线虫中使用RNAi系统地敲除。治疗后的动物将被筛选是否对炭疽杆菌感染过敏，以确定参与先天防御的新基因。对炭疽芽孢杆菌感染过敏的秀丽隐杆线虫突变体的大规模基因筛选也将进行。在病原体方面，将生成随机转座子和/或质粒整合的炭疽杆菌突变文库，并在高通量秀丽隐杆线虫感染试验中筛选毒力衰减。对小鼠巨噬细胞内生长的无毒突变体进行筛选，作为对哺乳动物致病性潜在降低的初步评估。这项R21研究的结果将产生新的试剂和假设，在未来的R-01或P-01提案中探索炭疽芽胞杆菌毒力机制和相关宿主先天防御，包括直接扩展到哺乳动物模型系统。

DESCRIPTION (provided by applicant): Bacillus anthracis is a ubiquitous soil bacterium and NIAID category A pathogen that causes anthrax. Although research has revealed important insights into this deadly bacterium (e.g., the activity of the tripartite toxins), the pathogenesis of anthrax has yet to be satisfactorily explained and there is much we do not understand. Chromosomal factors important for pathogenicity have been understudied, and we know very little about how the innate immune system acts to protect against anthrax infection. We have demonstrated that B. anthracis can produce a lethal infection in the soil nematode and model invertebrate C. elegans. This discovery opens up the possibility to study anthrax infection using forward and reverse genetics on an unprecedented scale. B. anthracis can infect and proliferate inside the nematode, eventually devouring it from the inside out. C. elegans, however, has a potent innate immune system that shares many pathways and effector mechanisms with that of mammals. The nematode appears capable in some cases of mounting an effective immune response against B. anthracis. This proposal will exploit this novel C. elegans-B. anthracis system to identify, on an unprecedented scale, (i) host genes involved in innate defense against B. anthracis infection and (ii) B. anthracis genes involved in establishing infection and circumventing innate defenses in an intact animal. Affymetrix microarrays will be used to identify potential host immunity genes up-regulated in response to B. anthracis infection. These genes will be systematically knocked down in C. elegans using RNAi. Treated animals will be screened for hypersensitivity to B. anthracis infection in order to identify novel genes involved in innate defense. A large-scale genetic screen for C. elegans mutants hypersensitive to B. anthracis infection will also be performed. On the pathogen side of the equation, random transposon and/or plasmid integrational mutant libraries of B. anthracis will be generated and screened for virulence attenuation in a high-throughput C. elegans infection assay. Avirulent mutants will be screened for growth within murine macrophages as a preliminary assessment of potential reduced pathogenicity in mammals. Results from this R21 study will generate novel reagents and hypotheses to be explored in a future R-01 or P-01 proposal on B. anthracis virulence mechanisms and relevant host innate defenses, including direct extension into mammalian model systems.