(M1030-01-07) E. coli Nissle 1917-Based Bacterial Cell Therapy Helps Restore Intestinal Barrier and Improve Post-Myocardial Infarction Outcomes

Purpose: Myocardial infarction events result in intestinal hyperpermeability which is associated with leakage of intestinal bacteria & bacterial components into the systemic circulation as well as translocation of certain intestinal bacteria to the infarcted cardiac tissue. This phenomenon is associated with significant systemic and cardiac inflammation which results in adverse cardiovascular outcomes. The purpose of the study is to develop a live biotherapeutic drug based on the probiotic bacterium – E. coli Nissle 1917 (EcN) engineered to express MAM – a potent, commensal bacterium F. prausnitzii-derived anti-inflammatory protein known to reinforce intestinal integrity.

Methods: Sequence for the MAM protein was inserted into the pBbA19a plasmid under the control of a strong J23105 promoter. Resultant plasmid was transformed into EcN and used for further experiments. Immunoblotting was performed to confirm the synthesis of MAM protein by our bacterium. EcN-MAM was administered to C57BL/6 mice followed by which levels of intestinal tight junction proteins were evaluated using immunoblotting. Serum lipopolysaccharide binding protein (LBP) levels were determined by ELISA as a measure of body’s response to systemic bacteria and bacterial components into the systemic circulation. Echocardiographic studies were performed to assess cardiac function.

Results: Immunoblotting revealed that the developed construct indeed produces the anti-inflammatory MAM protein. Past studies have demonstrated that systemic leakage of intestinal bacterial products peaks between 48-72h post-MI. We found that levels of intestinal tight junction proteins occludin and zona occludens-1, which maintain the integrity of the intestinal barriers, were significantly higher in large intestines of mice treated with EcN-MAM vs. mice treated with control EcN (EcN-Lux). This observation was concomitant with systemic LBP levels which confirmed that EcN-MAM-treated mice had significantly lower levels of serum LBP compared to mice treated with EcN-Lux. LBP is an acute-phase protein that enhances host’s immunological response to lipopolysaccharides (LPS). Echocardiographic studies demonstrated that EcN-MAM-treated mice had overall better cardiac function 4 weeks post-MI compared to control and sham mice. EcN-MAM treated mice performed significantly better than control mice in various cardiac function parameters such as ejection fraction and fractional shortening. Additionally, mice treated with the developed therapeutic had significantly lower left ventricular volumes in systole and diastole indicating better cardiac performance than control mice.

Conclusion: The developed live biotherapeutic EcN-MAM was able to ameliorate intestinal hyperpermeability and resulted in significantly improved cardiac function 4 weeks post-MI.

References: 1. T. Madaan et al., 2022. Circulation Research (Conference Abstract). DOI: 10.1161/res.131.suppl_1.P1043
2. X. Zhou et al., 2018. Microbiome. DOI: 10.1186/s40168-018-0441-4
3. E. Quévrain et al., 2016. Gut. DOI: 10.1136/gutjnl-2014-307649


Fig. 1 (A) Gene circuit diagram of the developed EcN-MAM construct, (B) Immunoblotting of EcN-MAM extract confirms synthesis of MAM protein, (C) Immunoblotting of total protein extracted from large intestine indicates increased presence of tight junction proteins in EcN-MAM treated samples vs. controls, (D) Mice treated with EcN-MAM were found to have lower serum LBP levels than controls at 48h, (E-F) EcN-MAM treated mice had significantly increased cardiac ejection fraction and fractional shortening vs. controls in echocardiographic studies, (G-H) EcN-MAM treated mice had significantly lower systolic and diastolic ventricular volumes vs. controls, (I) Echocardiography images of mice treated with EcN-MAM and EcN-Lux.

Acknowledgements: All animal studies were approved by the Institutional Animal Care and Use Committee at the University of Cincinnati. TM and NK have filed a provisional patent on this research serial no. 63/406, 004. Grant

Funding: R21GM137321.