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

• 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
• 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

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，我们申请资金购买第二个灵生鼠架，以扩大我们现有的 试验性住房能力，以充分实现#年提出的目标的研究潜力和影响 R35GM142685。 简而言之，我们资助的R35 Mira计划侧重于增加我们对一种重要的细菌间 由人类肠道微生物区系中最突出的类群之一--类杆菌--编码的防御途径。 对细菌间相互作用如何影响细菌的组成和稳定性的机械理解 人体肠道微生物群目前缺乏，但对微生物区系靶向治疗的前景至关重要。 将在影响人类健康方面实现。我们和其他人之前已经证明，依赖于接触 拮抗途径在人类肠道细菌基因组中广泛存在并高度丰富。在……里面 特别是，革兰氏阴性拟杆菌编码VI型分泌系统的变体，更好地 在铜绿假单胞菌和霍乱弧菌等病原体中已知。这条途径调节了接触- 依赖于向目标细菌输送抑菌和杀菌蛋白毒素。直到最近，它一直是这样 在任何细菌中都是未知的，目标细菌是否或如何防御 挥舞着T6SS的竞争对手。我们发现，超过50%的人类衍生类杆菌基因组中含有 与编码酪氨酸重组酶的基因相邻的免疫因子的广泛排列--我们将这些命名为 重组酶相关的获得性细菌间防御(RAID)系统。建议的目标是在资助 R35试图确定生物发生、体内动力学和该途径对 微生物组组成，以及对其调节的洞察，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)在体外和硅胶中使用 人-粪便衍生的元基因组测序数据集，包括来自NIH支持的人类微生物组的数据 项目，我们最终要求动物模型在仔细控制的实验下询问假说 条件。对于微生物组研究，最有效的方法是使用诺生菌小鼠。我们 建议利用灵知生菌小鼠来探索突袭系统对 以具有或不具有T6SS的特定类杆菌菌群定植的小鼠。我们也 建议采用类似的方法研究空袭系统的规则，并研究空间结构 用荧光记者检测小鼠肠道中T6SS和RAID的活性。我们计划扩大我们原来的 通过对RAID酪氨酸重组酶功能的体外机械洞察来验证的范围 单克隆化灵知生菌小鼠实验。重要的是，由于共同预言而产生的笼子效应是众所周知的。 这对微生物组研究来说是有问题的。为了解决笼子效应，我们通常每个笼子里放两只老鼠， 用现实的N和多个小组进行实验，我们几乎超出了目前的能力。 在我到达之前，达特茅斯没有灵知生菌设施。我从达特茅斯谈妥了启动资金 大学，我用来购买所有现有的设备，雇用和培训员工，并排除运营故障。之后 三年来，我们已经成功地建立了一个可操作的灵知菌设施，并保持了 快一年了。该设施由两个专用的单独使用房间组成。这些房间中有一间是我们养的 菌落，在柔性塑料双层隔离器中。我们的第二个房间是我们的试验性手术，包括 一个单独的35笼Tecniplast IsoCage P机架和一个用于处理处于无菌状态下的动物的生物安全柜 条件。尽管到目前为止，这已经足够了，特别是在我们执行故障排除和优化时 按照标准操作程序，我们发现需要扩展实验机架容量以 更好地实现实验的并行化，避免长时间的延迟。此外，我们不能利用我们目前所有的35个笼子 机架同时由于我们需要有无菌保持架来更换保持架。而不是购买许多 额外的Tecniplast笼子，我们认为购买额外的机架要实际得多，因为它将 将我们的容量扩大两倍以上，并增加灵活性。 拟议中的新设备来自另一家制造商Allentown，而不是Tecniplast。我们 更换制造商有几个原因。首先，Allentown机架占用的空间要小得多， 因为鼓风机单元(通过高效空气过滤器吸入室内空气并进入每个笼子)位于顶部 而不是放在边上)。这一点很重要，因为我们的房间很小，有必要保存 在标准操作期间，有足够的空间让员工在不违反无菌条件的情况下自信地移动。第二， Allentown单元带有来自Lynn Hajjar博士的强烈个人推荐，该主任 克利夫兰诊所勒纳研究所的灵知生物设施，他为 我在2020年提交的R35。她之前在华盛顿大学，在那里我遇到了她， 在她的工厂接受培训，那里使用Tecniplast设备。在搬到勒纳后，她完全改变了 制造商，现在完全使用Allentown。用她的话说，产品有了实质性的增长 可靠和可靠是灵知生物的一切。 关于支付这台设备的任何经常性成本的计划，我们打算使用来自 R35，或用于支付日常维修和维护的可自由支配资金。将提供有关新单位的培训 作为购买的一部分，后续的培训将由我们的灵知生物设施进行 经理，达琳·罗伊斯。

项目成果

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
• 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。

• DOI: 10.1038/s41564-023-01569-7
• 发表时间: 2024
• 期刊: Nature microbiology
• 影响因子: 28.3
• 作者: Ross,BenjaminD