Mechanisms of adaptation to interbacterial antagonism by the human gut microbiota

人类肠道微生物群适应细菌间拮抗作用的机制

基本信息

批准号：
10797050
负责人：
Benjamin Ross
金额：
$ 6.39万
依托单位：
DARTMOUTH COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-02 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10797050
关键词：
Address Air Animal Model Animals Bacteria Bacteriocides Biogenesis Breeding Clinic Communities Data Set Dedications Direct Costs Equipment Funding Genes Genome Gnotobiotic Health Housing Human Human Microbiome Immunity In Vitro Intestines Knowledge Letters Maintenance Manufacturer Mediating Mediation Mus Names Pathway interactions Positioning Attribute Procedures Proteins Pseudomonas aeruginosa Recommendation Recurrence Regulation Reporter Research Research Institute Side Sterility Structure System Time Toxin Training Tyrosine United States National Institutes of Health Universities Variant Vibrio cholerae Washington antagonist college cost experimental study flexibility gut bacteria gut microbiome gut microbiota high efficiency particulate air filter in silico in vivo insight metagenomic sequencing microbiome microbiome composition microbiome research microbiota operation parallelization pathogen preservation recombinase repaired targeted treatment

项目摘要

RESEARCH STRATEGY Here, we request funds to purchase a second gnotobiotic mouse rack for expansion of our existing experimental housing capabilities in order to fully realize the research potential and impact of aims proposed in R35GM142685. Briefly, our funded R35 MIRA proposal focuses on increasing our knowledge of an important interbacterial defense pathway encoded by one of the most prominent taxa in the human gut microbiota, the Bacteroidales. A mechanistic understanding of how interbacterial interactions impact the composition and stability of the human gut microbiome is currently lacking yet will be essential for the promise of microbiota-targeted therapies to be actualized in impacting human health. We and others have previously shown that contact-dependent antagonism pathways are widespread and highly abundant in the genomes of human gut bacteria. In particular, the Gram-negative Bacteroidales encode a variant version of the type VI secretion system, better known in pathogens like Pseudomonas aeruginosa and Vibrio cholerae. This pathway mediates the contact- dependent delivery of bacteriostatic and bacteriocidal protein toxins to targeted bacteria. It had until recently been largely unknown in any bacterium if or how targeted bacteria could defend themselves against attack by T6SS-wielding competitors. We discovered that over 50% of all human-derived Bacteroidales genomes harbor extensive arrays of immunity factors adjacent to a gene encoding a tyrosine recombinase – we named these recombinase-associated acquired interbacterial defense (rAID) systems. The proposed aims in the funded R35 sought to determine mechanistic details of the biogenesis, in vivo dynamics, and impact of the pathway on microbiome composition, as well as insight into its regulation including experiments exploring biogeography. While some of the proposed research can (and has, very successfully) been done in vitro and in silico using human-fecal derived metagenomic sequencing datasets including from the NIH-supported Human Microbiome Project, we ultimately require animal models to interrogate hypotheses under carefully controlled experimental conditions. For microbiome research, the most powerful approach to do so is to use gnotobiotic mice. We proposed to utilize gnotobiotic mice to explore the impact of the rAID system on microbiome compostion in mice colonized with defined communities of Bacteroidales strains that possess or lack the T6SS. We also proposed to study the regulation of the rAID system using a similar approach and to study the spatial structure of T6SS and rAID activity in the mouse intestine using fluorescent reporters. We plan to expand our original scope to validate in vitro mechanistic insight into the function of the rAID tyrosine recombinase through monocolonized gnotobiotic mouse experiments. Importantly, cage effects due to coprophagy are well-known and problematic for microbiome studies. To address cage effects, we typically house two mice per cage and perform an experiment with a realistic N and multiple groups we are nearly exceeding our current capacity. Dartmouth did not have a gnotobiotic facility prior to my arrival. I negotiated startup funds from Dartmouth College that I used to purchase all existing equipment, hire and train staff, and troubleshoot operations. After three years, we have successfully established an operational gnotobiotic facility, with sterility maintained for nearly 1 year. This facility consists of two dedicated sole-use rooms. One of these rooms houses our breeding colonies, in flexible plastic double-tier isolators. Our second room houses our experimental operation, including a single 35-cage Tecniplast IsoCage P rack and a biosafety cabinet used for handling animals under sterile conditions. Though this has been sufficient so far, particularly as we performed troubleshooting and optimized standard operating procedures, we have found that we need to expand our experimental rack capacity in order to parallelize experiments better and avoid lengthy delays. Further, we cannot utilize all 35 cages of our current rack at the same time due to our need to have sterile cages for cage changes. Instead of purchasing many additional Tecniplast cages, we believe it is far more practical to purchase an additional rack since it will expand our capacity by more than two-fold and increase flexibility. The proposed new piece of equipment is from a different manufacturer, Allentown instead of Tecniplast. We have several reasons for switching manufacturer. First, the Allentown rack utilizes a much smaller footprint, since the blower unit (which draws room air through HEPA filters and into each cage) is positioned on the top of the rack instead of on the side). This is important because our room is small and it is necessary to preserve enough space for staff to confidently move without breaching sterility during standard operations. Second, the Allentown unit comes with strong personal recommendation from Dr. Lynn Hajjar, the Director of the Gnotobiotic Facility at the Lerner Research Institute at the Cleveland Clinic, who provided a letter of support for my R35 submission in 2020. She previously had been at the University of Washington, where I met her and trained in her facility, which used Tecniplast equipment. Upon moving to Lerner, she completely switched manufacturer and now entirely uses Allentown. In her words, there is a substantial increase in product reliability and reliability is everything for gnotobiotics. Regarding plans to cover any recurring costs for this piece of equipment, we intend to use direct costs from the R35, or discretionary funds to cover routine repair and maintenance. Training on the new unit will be provided by Allentown as part of the purchase, and subsequent training will be performed by our gnotobiotic facility manager, Darlene Royce.

研究策略在这里，我们请求资金购买第二个无菌小鼠架，以扩大我们现有的实验住房能力，以充分实现研究潜力和所提出目标的影响 R35GM142685。简而言之，我们资助的 R35 MIRA 提案侧重于增加我们对重要的细菌间化合物的了解防御途径由人类肠道微生物群中最重要的类群之一——拟杆菌目编码。从机理上理解细菌间相互作用如何影响细菌的组成和稳定性人类肠道微生物群目前尚缺乏，但对于微生物群靶向治疗的前景至关重要我们和其他人之前已经证明，接触依赖会影响人类健康。拮抗途径在人类肠道细菌的基因组中广泛且丰富。特别是，革兰氏阴性拟杆菌编码 VI 型分泌系统的变体版本，更好已知在铜绿假单胞菌和霍乱弧菌等病原体中，该途径介导接触。直到最近，这种方法仍然依赖于向目标细菌传递抑菌和杀菌蛋白毒素。在任何细菌中，对于目标细菌是否或如何能够防御来自细菌的攻击，目前还知之甚少。我们发现超过 50% 的人类来源拟杆菌基因组都含有 T6SS 竞争者。与编码酪氨酸重组酶的基因相邻的大量免疫因子——我们将它们命名为重组酶相关获得性细菌间防御（rAID）系统的拟议目标。 R35 试图确定生物发生的机制细节、体内动力学以及该途径对微生物组的组成，以及对其调节的深入了解，包括探索生物地理学的实验。虽然一些拟议的研究可以（并且已经非常成功地）在体外和计算机中使用人类粪便衍生的宏基因组测序数据集，包括来自 NIH 支持的人类微生物组项目中，我们最终需要动物模型在严格控制的实验下质疑假设对于微生物组研究，最有效的方法是使用无菌小鼠。提议利用无菌小鼠探索 rAID 系统对微生物组组成的影响我们还用具有或缺乏 T6SS 的特定拟杆菌菌株群落定殖于小鼠。提议使用类似的方法研究 rAID 系统的调节并研究空间结构使用荧光发生器检测小鼠肠道中的 T6SS 和 rAID 活性我们计划扩展我们最初的研究。范围通过体外验证 rAID 酪氨酸重组酶功能的机制重要的是，食粪引起的笼效应是众所周知的。为了解决笼子效应，我们通常在每个笼子里饲养两只小鼠。用现实的 N 和多个组进行实验，我们几乎超出了我们当前的能力。在我到达之前，达特茅斯没有无菌设施，我从达特茅斯协商启动资金。我用大学来购买所有现有设备、雇用和培训员工以及解决运营问题。三年来，我们成功建立了一个可运行的无菌设施，并保持无菌状态该设施由两个专用的房间组成，其中一间用于我们的饲养。我们的第二个房间是我们的实验操作室，包括菌落。一个 35 笼 Tecniplast IsoCage P 架和一个生物安全柜，用于处理无菌动物尽管到目前为止这已经足够了，特别是当我们进行故障排除和优化时。标准操作程序，我们发现我们需要扩大我们的实验架容量，以便为了更好地并行实验并避免长时间的延迟，我们无法利用当前的所有 35 个笼子。同时放置架子，因为我们需要有无菌笼子来更换笼子，而不是购买许多笼子。额外的 Tecniplast 笼子，我们认为购买额外的架子更为实际，因为它可以将我们的产能扩大两倍以上并提高灵活性。拟议的新设备来自不同的制造商 Allentown，而不是 Tecniplast We。更换制造商有几个原因：首先，阿伦敦机架占用的空间要小得多，因为鼓风机装置（通过 HEPA 过滤器吸入室内空气并进入每个笼子）位于顶部放在架子上而不是放在侧面）这很重要，因为我们的房间很小，有必要保存。在标准操作过程中，有足够的空间让工作人员自信地移动，而不会破坏无菌性。阿伦敦单位得到了该中心主任 Lynn Hajjar 博士的强烈个人推荐。克利夫兰诊所勒纳研究所的知生设施，他们提供了一封支持信我在 2020 年提交的 R35。她之前曾在华盛顿大学，我在那里认识了她，在她使用 Tecniplast 设备的设施中接受培训后，她就完全转变了。制造商现在完全使用阿伦敦，用她的话说，产品大幅增加。对于gnotobiotics来说，可靠性和可靠性就是一切。关于支付该设备的任何经常性成本的计划，我们打算使用来自 R35，或用于新设备日常维修和维护培训的酌情资金。作为购买的一部分，由阿伦敦提供，后续培训将由我们的无菌设施进行经理达琳·罗伊斯。

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Persistent delay in maturation of the developing gut microbiota in infants with cystic fibrosis.

囊性纤维化婴儿正在发育的肠道微生物群的成熟持续延迟。

DOI：
10.1101/2023.05.02.539134
发表时间：
2023
期刊：
bioRxiv : the preprint server for biology
影响因子：
0
作者：
Salerno,Paige;Verster,Adrian;Valls,Rebecca;Barrack,Kaitlyn;Price,Courtney;Madan,Juliette;O'Toole,GeorgeA;Ross,BenjaminD
通讯作者：
Ross,BenjaminD

Bacteroides fragilis uses toxins for gut success.

脆弱拟杆菌利用毒素来维持肠道健康。