Weill Cornell Medicine investigators have developed a set of molecular tools to regulate the activity of specific genes in one of the most common classes of intestinal bacteria. The ability to genetically manipulate these microbes could lead to a better understanding of the role they play in human physiology and diseases.

In healthy individuals, species of Clostridia comprise half of the bacteria found in the gut. Many produce metabolites that are likely beneficial for human health and immune regulation, but some are associated with disease – from botulism and inflammatory intestinal conditions to metabolic disorders such as diabetes or even cancer.

Exactly how Clostridia contribute to disease onset or progression is not well understood. For example, do the bacteria produce a specific toxin or disease-affecting metabolite? Or could changes in their abundance alter other gut bacteria and influence health or disease?

“There is no way to answer this question without having a genetic toolset that will allow us to study the Clostridia obtained from clinical strains,” said Chun-Jun “C.J.” Guo, associate professor of immunology in medicine in the Division of Gastroenterology and Hepatology and a scientist at the Jill Roberts Institute for Research in Inflammatory Bowel Disease at Weill Cornell Medicine.

The toolsets that Guo and his team developed are described in a study published Aug. 25 in Nature Biotechnology. Ting-Ting Li and Xu Chen, both postdoctoral associates in the Guo lab at the time of the study, were the lead authors.

Taking control of Clostridial genes

To develop a tool for precisely controlling specific Clostridial genes, Guo and his team turned to the DNA sequences, also known as promoters, that normally regulate gene expression activity in these microbes. They tested 67 different regulatory sequences isolated from one strain of Clostridia and identified the one that directed the highest level of gene activity. They then took this strong gene “promoter” and attached to it a “switch” that would allow them to turn the promoter on or off using a chemical inducer.

The researchers – including collaborator Matthew Sorbara from the University of Guelph in Ontario – then inserted this controllable promoter into Clostridia and found that they could use their chemical inducer to regulate the levels of selected bacterial metabolic genes. This manipulation worked both in culture and when they introduced the promoter-bearing Clostridia into mice.

As a test case, the researchers tweaked production of trimethylamine, a compound that Guo said acts as a “double-edged sword.” Although some studies report that high levels of the trimethylamine N-oxide, the downstream product of trimethylamine, track with an increased risk of blood clots, Guo said that others indicate that the increased level of this metabolite can increase the efficacy of immunotherapy.

The ability to precisely control how much trimethylamine resident Clostridia produce could help minimize its level in healthy individuals while selectively boosting it in people undergoing immunotherapy. The researchers were similarly able to manipulate the levels of the metabolite deoxycholic acid, which can suppress the body’s natural anti-tumor immune reaction in colorectal cancer, but can enhance some forms of immunotherapy. 

Engineering biological therapeutics

The team also developed a CRISPR system – another powerful tool for controlling or eliminating gene activity – that they could deploy in Clostridia isolated from clinical samples. The team could use this tool to systematically knock out numerous metabolic genes to determine which, if any, could play a role in either enhancing intestinal immunity or precipitating disease.

“We still don’t really understand what role microbiota play in complex diseases like IBD or cancer,” Guo said. “If we find that any of these metabolites are beneficial, we could engineer living therapeutics that could deliver these metabolites in a well-controlled way to a patient to achieve therapeutic effects.”

This work was supported by grants from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, the National Institute of Diabetes and Digestive and Kidney Diseases, the National Cancer Institute, the National Institute on Aging, and the National Institute of General Medical Sciences, all part of the National Institutes of Health. Additional support was provided by a pilot award from the American Gastroenterological Association; the W.M. Keck Foundation; the Kenneth Rainin Foundation; and the RAPP funding from the Weill Cornell Medicine.

Karen Hopkin is a freelance writer for Weill Cornell Medicine.