Khosrow Mohammadi, Per Erik Joakim Saris

Abstract: Internalin proteins localized at the surface of pathogenic Listeria monocytogenes interfere with E-cadherin to adhere and internalize into mammalian cells. E-cadherin has five extracellular, immunoglobulin-like domains (EC1 to EC5), of which the first domain is sufficient to mediate L. monocytogenes invasion. Immunomagnetic beads employ antibodies that react either with pathogenic or non-pathogenic Listeria. To obtain cost-effective and easy-to-produce beads for detecting pathogenic L. monocytogenes, we cloned and efficiently expressed human E-cadherin domains 1 and 2 (hEcad1/2) in the Bacillus subtilis spore coat. The cDNA sequence encoding hEC1/2 protein 23.7 kDa (MH511517.1) was inserted in pET22b and expressed and purified from Escherichia coli BL21. We used the CotY as a significant structural component of the B. subtilis spore coat to express hEcad1/2. We constructed a recombinant plasmid p1CSV-CotY-N-hEcad1/2 incorporating the cotY-hEcad1/2 gene under the control of the cotY promoter. The constructed plasmid was transformed into B. subtilis KO7 by double cross-over method and an amylase-inactivated mutant was generated. After spore induction, the developed sporobeads showed a high binding affinity for L. monocytogenes 4b. This result demonstrated the potential of human E-cadherin ectodomain 1 and 2 in a practical application involving the detection and capture of pathogenic L. monocytogenes.

Keywords: Listeria monocytogenes, detection, E-cadherin, expression, Bacillus subtilis, sporobead.

Date Published: October 16, 2023
DOI: https://jbeb.avestia.com/2023/006.html

View Article