Development of a Microbial Biosensor for Detection of Inflammation-Associated Microenvironments

Clinical monitoring of inflammatory bowel disease (IBD) has advanced in recent years due to the validation of calprotectin, a neutrophil-source antimicrobial peptide involved in zinc sequestration, as a biomarker for IBD-associated inflammation. Fecal calprotectin assays, which have become an inexpensive diagnostic used in IBD maintenance, have marked sensitivity: <50 ug/mL calprotectin represents a negative test and ≥150 ug/mL is indicative of intestinal inflammation. 50-150 ug/mL represents a range of borderline, subclinical inflammatory levels that require re-testing. However, re-testing is problematic due to lack of compliance in collecting fecal material for the test, limiting the ability to actively predict and prevent symptomatic flares. Thus, we are developing a microbial biosensor for calprotectin that will involve sensing the presence of inflammatory levels of calprotectin via the identification and engineering of calprotectin-sensitive bacterial promoters. This non-invasive, sensitive diagnostic would allow patients to self-monitor for potential flares before they become problematic, and reduce the need for repeated clinical contact for re-tests on borderline results.

To build these sensors, we are utilizing Lactobacillus reuteri LJO1 and Escherichia coli Nissle 1917, probiotic microbes that have exhibited survivability within the human gastrointestinal tract. Using RNA-seq technology we identified calprotectin-sensitive promoters within L. reuteri and E. coli Nissle. In order to optimize the promoters’ response to human calprotectin, we cloned the promoters into plasmids wherein promoter activity was coupled with green fluorescent protein (GFP) expression as the primary output reading. Sensors were incubated in subclinical levels of human calprotectin in vitro and GFP output was measured via flow cytometry. Co-culture with human calprotectin yielded increases in GFP expression of 1.60- to 3.00-fold for E. coli Nissle sensors and 1.30- to 4.50-fold for L. reuteri sensors, indicating the sensor constructs were sensitive to calprotectin. To identify how the promoters functioned in the context of fecal communities, the sensors were co-cultured in fecal slurries obtained from either healthy subjects or IBD patients. E. coli Nissle sensors incubated with IBD fecal slurries containing between 110 and 2560 ug/mL calprotectin, a range representing subclinical to severe inflammation, have 3.00- to 5.00-fold increase in GFP expression compared to those co-cultured in fecal slurries with <110 ug/mL calprotectin. Likewise, L. reuteri sensors had an increase of 4.50- to 8.50-fold in GFP expression when co-cultured with an IBD sample compared to a healthy sample. Preliminary analysis suggests that the promoters are activated in response to metal starvation spurred by specific binding of zinc by calprotectin.

These results demonstrate that our prospective sensors are currently activated by subclinical levels of calprotectin in vitro, as well as IBD patient fecal samples containing an array of calprotectin concentrations ranging from intermediate up to inflammatory levels. The data provide proof of principle as we work towards fine-tuning promoter sensitivity. Successful completion of bacterial biosensors sensitive to inflammatory grades of calprotectin will provide patients a non-invasive method of at-home disease monitoring and would represent a practical and novel mechanism for detection of human disease.