Abstract

Chronic infections are frequently driven by biofilms, surface-associated bacterial communities encased in a protective matrix that limits antibiotic penetration and immune clearance. Their persistence is largely regulated by quorum sensing (QS), a cell–cell communication process that coordinates virulence and biofilm formation. The autoinducer-2 (AI-2) pathway, catalyzed by the metalloenzyme LuxS in both Gram-positive and Gram-negative bacteria, presents a promising antivirulence target. Inhibiting LuxS offers a strategy to disrupt QS and weaken biofilm defenses without applying the selective pressure associated with conventional antibiotics.

To support this approach, we developed a platform for LuxS expression, purification, and functional characterization. LuxS was heterologously expressed in Escherichia coli using cobalt-supplemented minimal media and purified via fast protein liquid chromatography (FPLC). Inductively coupled plasma mass spectrometry (ICP-MS) confirmed predominant cobalt incorporation, with minor zinc and iron present. Enzymatic activity was assessed using UV–Vis spectrophotometry and Ellman’s reagent to monitor homocysteine production.

Despite successful purification, LuxS exhibited lower activity than expected, indicating sensitivity to metal incorporation and protein stability. UV–Vis analysis suggested incomplete or improper cofactor coordination. Current work focuses on improving cofactor retention during purification and probing active-site function through site-directed mutagenesis (Cys→Ala). This optimized system is now being used to screen small-molecule inhibitors targeting LuxS, providing a foundation for evaluating quorum sensing disruption as an antivirulence strategy against biofilm-associated infections.

Keywords

quorum sensing, QS, biofilms, biofilm, biochemistry, metalloenzyme, LuxS, autoinducer-2, (AI-2), cobalt, SRH, S-ribosylhomocysteinase, S-ribosylhomocysteine

How to Cite