Mechanisms of adaptation to interbacterial antagonism by the human gut microbiota

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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.