Rice University researchers say they have engineered a bacterium with the necessary capabilities for diagnosing a human disease. The engineered strain of the gut bacteria E. coli senses pH and glows when it encounters acidosis, an acidic condition that often occurs during flareups of inflammatory bowel diseases like colitis, ileitis, and Crohn’s disease, according to the scientists.
Researchers at the University of Colorado (CU) School of Medicine used the Rice-created organism in a mouse model of Crohn’s disease to show acidosis activates a signature set of genes. The corresponding genetic signature in humans has previously been observed during active inflammation in Crohn’s disease patients. The study (“Mucosal acidosis elicits a unique molecular signature in epithelia and intestinal tissue mediated by GPR31-induced CREB phosphorylation”) is available online in the Proceedings of the National Academy of Sciences (PNAS).
“Metabolic changes associated with tissue inflammation result in significant extracellular acidosis (EA). Within mucosal tissues, intestinal epithelial cells (IEC) have evolved adaptive strategies to cope with EA through the up-regulation of SLC26A3 to promote pH homeostasis. We hypothesized that EA significantly alters IEC gene expression as an adaptive mechanism to counteract inflammation,” write the investigators.
“Using an unbiased RNA sequencing approach, we defined the impact of EA on IEC gene expression to define molecular mechanisms by which IEC respond to EA. This approach identified a unique gene signature enriched in cyclic AMP response element-binding protein (CREB)-regulated gene targets. Utilizing loss- and gain-of-function approaches in cultured epithelia and murine colonoids, we demonstrate that EA elicits prominent CREB phosphorylation through cyclic AMP-independent mechanisms that requires elements of the mitogen-activated protein kinase signaling pathway.
“Further analysis revealed that EA signals through the G protein-coupled receptor GPR31 to promote induction of FosB, NR4A1, and DUSP1. These studies were extended to an in vivo murine model in conjunction with colonization of a pH reporter Escherichia coli strain that demonstrated significant mucosal acidification in the TNFΔARE model of murine ileitis. Herein, we observed a strong correlation between the expression of acidosis-associated genes with bacterial reporter sfGFP intensity in the distal ileum.
“Finally, the expression of this unique EA-associated gene signature was increased during active inflammation in patients with Crohn’s disease but not in the patient control samples. These findings establish a mechanism for EA-induced signals during inflammation-associated acidosis in both murine and human ileitis.”
Study co-author Jeffrey Tabor, PhD, whose lab engineered the pH-sensing bacterium, said it could be reprogrammed to make colors that show up in the toilet instead of the fluorescent tags used in the CU School of Medicine experiments.
“We think it could be added to food and programmed to turn toilet water blue to warn patients when a flareup is just beginning,” explained Tabor, an associate professor of bioengineering in Rice’s Brown School of Engineering.
Over billions of year, bacteria have evolved specific and sensitive genetic circuits to sense their surroundings. Tabor and colleagues developed a biohacking toolkit that allows them to mix and match the inputs and outputs of these bacterial sensors. The pH-sensing circuit was discovered by Rice doctoral student Kathryn Brink in a 2019 demonstration of the plug-and-play toolkit.
PNAS study co-authors Sean Colgan, PhD, the director of the CU School of Medicine’s mucosal inflammation program, and Ian Cartwright, PhD, a postdoctoral fellow in Colgan’s lab, read about the pH sensor and contacted Tabor to see if it could be adapted for use in a mouse model of Crohn’s disease.
“It turns out that measuring pH within the intestine through noninvasive ways is quite difficult,” said Colgan, the Levine-Kern Professor of Medicine and Immunology in the CU School of Medicine.
Brink spent a few weeks splicing the necessary sensor circuits into an organism and sent it to Colgan’s lab.
“Normally, the pH in your intestines is around seven, which is neutral, but you get a lot of inflammation in Crohn’s disease, and pH goes to something like three, which is very acidic,” Tabor said.
Colgan and colleagues have studied the genes that are turned on and off under such conditions and “needed a tool to measure pH in the intestine to show that the things they were observing in in vitro experiments were also really happening in a live animal,” Tabor said.
“Colonizing this bacterial strain was the perfect biological tool to monitor acidosis during active inflammation,” added Colgan. “Correlating intestinal gene expression with the bacterial pH sensing bacteria proved to be a useful and valuable set of biomarkers for active inflammation in the intestine.”
Tabor believes the pH-sensing bacterium could potentially be advanced for human clinical trials in several years.