An N, S-acetylated L-cysteine-cysteamine conjugate hinders pyocyanin redox cycling to weaken Pseudomonas aeruginosa biofilm and dampens LPS-driven acute pulmonary inflammation

Abstract

The persistence of P. aeruginosa infections is largely driven by the secretion of several factors during invasion, including the redox-active phenazine pyocyanin (PYO), which promotes biofilm formation and oxidative stress. Biofilms contribute to chronic infections and antibiotic resistance, limiting the efficacy of conventional therapies. We found that a synthetic compound, I-152, a conjugate of N-acetyl-L-cysteine (NAC) and S-acetylcysteamine (also known as S-acetyl-{beta}-mercaptoethylamine; SMEA), effectively restored colistin susceptibility against P. aeruginosa by altering biofilm nanomechanical properties. These perturbations in matrix integrity were associated with I-152s ability to hinder the phenazine redox cycle, shifting PYO to a reduced state as well as enabling S-conjugate formation. The compound decreased PYO accumulation in bacterial cultures and PYO-generated reactive oxygen species (ROS) in macrophage cells. Together with PYO, LPS is another driver of ROS-dependent inflammatory signaling in the host, which leads to an uncontrolled cytokine response and organ damage, especially in patients with cystic fibrosis. I-152 treatment downregulated the expression of LPS-induced inflammatory cytokines, i.e., IL-6 and TNF-, in bone marrow-derived macrophages (BMDM) isolated from transgenic CFTR-/- and CFTR+/+ mice. Consistently, I-152 partially counteracted the inflammatory response in the P. aeruginosa LPS-induced acute lung injury murine model. Taken together, these results support I-152 as an adjunctive treatment for P. aeruginosa respiratory infections through a dual mechanism: combating antimicrobial resistance in biofilms and dampening host inflammation in the respiratory system. HighlightsO_LII-152 potentiates colistin activity against P. aeruginosa by compromising the biofilm surface C_LIO_LII-152 rewires the pyocyanin (PYO) redox state and forms covalent adducts with it C_LIO_LIPYO accumulation and PYO-induced ROS generation in macrophages is impaired by I-152 C_LIO_LIEx vivo, I-152 dampens excessive pro-inflammatory response to P. aeruginosa LPS in CFTR-/- and CFTR+/+ BM-derived macrophages C_LIO_LII-152 (140 mg/Kg) attenuates LPS-driven inflammation and lung damage in CFTR+/+ mice C_LI