In:
Science, American Association for the Advancement of Science (AAAS), Vol. 376, No. 6594 ( 2022-05-13)
Abstract:
Bacteria within the gut continuously adapt their gene expression to environmental conditions that are associated with diet, health, and disease. Noninvasive measurements of bacterial gene expression patterns throughout the intestine are important to understand in vivo microbiota physiology and pathophysiology. Current methods do not offer sufficient information about transient or proximal events within the intestine without using indirect or invasive approaches that disturb normal physiology and are inapplicable to clinical practice. RATIONALE Transcriptional recording by CRISPR spacer acquisition from RNA (Record-seq) enables engineered bacteria to continuously record the history of gene expression in a population of bacteria. Over time, snippets of intracellular RNA are converted into DNA and integrated as a historical record of spacer sequences within CRISPR arrays through the action of an integration complex that contains a reverse transcriptase Cas1 fusion protein (RT-Cas1) and Cas2. Here, using a refined Record-seq methodology, we used transcriptional recording Escherichia coli sentinel cells to reveal intestinal and microbiota physiology under different dietary and disease contexts along the length of the unmanipulated mouse intestine. RESULTS We used transcriptional recording sentinel cell technology in the gastrointestinal tract of germ-free and gnotobiotic mice to assess how the DNA record of spacer sequences in fecal samples uncovered distinct transcriptional records during passage from the proximal to the distal intestine, which depended on diet, inflammation, and microbe-microbe interactions. Sentinel cells retrieved from feces of stably colonized germ-free mice accumulated new spacers over time and during intestinal transit. Fecal Record-seq profiles were distinct for mice on chow diet versus starch or fat diets. Transcriptional records of these diets were preserved in fecal spacer sequences even 2 weeks after a dietary switch, whereas diet-specific fecal bacterial RNA-sequencing (RNA-seq) profiles were rapidly lost. Direct measurements showed that Record-seq efficiently captured proximal transient transcriptional events. This included evidence of different carbon source preferences and bacterial hexuronate metabolism through the Entner-Doudoroff pathway under conditions of restricted carbon source availability, which was then verified through competitive colonization of wild-type (WT) E. coli and a ∆ idnK/ ∆ gntK mutant defective in hexuronate catabolism. In addition to information about carbon source preference and metabolism, the transcriptome-scale records also provided evidence of an acid-stress response that was associated with a lowered pH in the cecum of starch-fed mice. In a dextran sulfate sodium (DSS) colitis model, sentinel cells recorded transcriptional alterations consistent with reduced anaerobic metabolism, a stringent response, and increased oxidative and membrane stress. Cocolonization with Bacteroides thetaiotaomicron revealed likely cross-feeding of E. coli from records of uptake and metabolism of fiber-derived saccharides liberated by B. thetaiotaomicron glycoside hydrolases. Record-seq was also able to capture diet-specific signatures in mice colonized with a 12-organism model microbiota. Moreover, by using barcoded CRISPR arrays, we could show that Record-seq can be multiplexed in several strains of the same bacterial species that cocolonize the intestine, thus elucidating the compensatory response of a single-gene mutant to competition with the WT strain. CONCLUSION Transcriptional recording sentinel cells function in vivo in the mouse intestine and record transcriptome-scale information about diet, disease, and microbial interactions integrated along the length of the intestinal tract over time. Transcriptional recording enables noninvasive measurement of the intestinal tract with potential for biomedical research and future biomedical diagnostic applications. Transcriptional recording sentinel cells noninvasively report interactions with diet, host, other microbes, and pathological environments. Throughout intestinal transit, sentinel cells capture information about transient mRNA expression into plasmid-encoded CRISPR arrays through the action of a reverse transcriptase Cas1-Cas2 complex. This information is retrieved by means of fecal sampling and deep sequencing followed by computational analyses. Barcoded CRISPR arrays enable transcriptional profiling of isogenic bacteria coinhabiting the intestine.
Type of Medium:
Online Resource
ISSN:
0036-8075
,
1095-9203
DOI:
10.1126/science.abm6038
Language:
English
Publisher:
American Association for the Advancement of Science (AAAS)
Publication Date:
2022
detail.hit.zdb_id:
128410-1
detail.hit.zdb_id:
2066996-3
detail.hit.zdb_id:
2060783-0
SSG:
11
Bookmarklink
