A novel genetic toolkit may improve the study of how Clostridia bacteria in the gut contribute to disease and reveal new molecular mechanisms that may serve as effective therapeutic targets, as detailed in a recent study published in Nature Biotechnology.
Among the many microorganisms within the gut microbiome is Clostridiaceae, a family of the bacterial class Clostridia, which helps maintain gut microbiota homeostasis but are also associated with disease.
While Clostridia bacterial genes and metabolites associated with disease can be easily identified with current methods, evaluating their precise function has remained challenging.
“This is especially true for many gut Clostridia, which are important members of the microbiome but historically have been difficult to genetically manipulate,” said Yaping Liu, PhD, assistant professor of Biochemistry and Molecular Genetics and co-author of the study.
To address this challenge, Liu and investigators led by Chun-Jun Guo, PhD, associate professor in the Jill Roberts Institute (JRI) for Research in Inflammatory Bowel Disease at Weill Cornell Medicine, developed a new modular toolkit to better understand gut Clostridia’s role in disease development and outcomes.
The toolkit features strong constitutive promoters, or DNA sequences that drive continuous transcription of their associated genes, as well as an inducible gene-expression system and CRISPR-based gene deletion tools. Liu’s lab specifically contributed to the bioinformatics analysis used to prioritize candidate promoters by performing the analysis of human gut metatranscriptomic and metagenomic datasets.
“We normalized bacterial gene expression by the abundance of the corresponding bacterial strains and evaluated how strongly and consistently candidate genes were expressed across human stool samples. This helped prioritize regulatory sequences for experimental testing by the Guo laboratory,” said Liu, who is also a professor in the Ken and Ruth Davee Department of Neurology’s Division of Multiple Sclerosis and Neuroimmunology and a member of the Center for Human Immunobiology.
The candidate promoters were experimentally tested across multiple gut Clostridia and the strongest candidate promoters were incorporated into inducible and CRISPR-based systems. The toolkit was also used to manipulate biologically relevant microbial metabolites, including trimethylamine and deoxycholic acid, in a controlled and reversible manner in mice.
The new toolkit can help gut microbiome research move from correlation to identifying precise mechanisms that contribute to disease, Liu said.
The next steps for the research, according to Liu, include expanding the toolkit to more patient-derived gut bacteria and using it to systematically study microbial genes and pathways associated with human disease.
“Instead of simply observing that a bacterial gene or metabolite is associated with disease, researchers can directly increase, decrease, or eliminate that function and determine its effect on the host. That makes it possible to test whether specific microbial genes and metabolites actually contribute to inflammation, metabolism, cancer or other disease processes,” Liu said
Jiayan Ma, a student in the Driskill Graduate Program in Life Sciences (DGP), was a co-author of the study.
This work was supported by a pilot award from the American Gastroenterological Association; National Institutes of Health grants 1DP2HD101401-01, DK135816-01, CA299862, AI178683, AI172027 and DK132244; the W. M. Keck Foundation; the Kenneth Rainin Foundation and the Regional Agricultural Promotion Program funding from the Weill Cornell Medicine; and National Institutes of Health National Institute of General Medical Sciences grant R35GM147283.
