The quorum sensing (QS) circuitry of Pseudomonas aeruginosa represents an attractive target to attenuate bacterial virulence and antibiotic resistance. In this context, phytochemicals harboring anti-virulent properties have emerged as an alternative medicine to combat pseudomonal infections. Hence, this study was undertaken to investigate the synergistic effects and quorum quenching (QQ) potential of cinnamaldehyde (CiNN) in combination with gentamicin (GeN) against P. aeruginosa. The QQ activity of this novel combination was evaluated using a QS reporter strain and synergism was studied using chequerboard assays. Further, the genotypic and phenotypic expression of pseudomonal virulence factors was examined alongside biofilm formation. The combination of CiNN and GeN exhibited synergy and promising anti-QS activity. This drug combination was shown to suppress AHL production and downregulate the expression of critical QS genes in P. aeruginosa PAO1. Molecular docking revealed strong interactions between the QS receptors and CiNN, asserting its QQ potential. Bacterial motility was compromised along with a significant reduction in pyocyanin (72.3%), alginate (58.7%), rhamnolipid (33.6%), hemolysin (82.6%), protease (70.9%), and elastase (63.9%) production. The drug combination successfully eradicated preformed biofilms and inhibited biofilm formation by abrogating EPS production. Our findings suggest that although GeN alone could not attenuate QS, but was able to augment the anti-QS potential of CiNN. To validate our results using an infection model, we quantified the survival rates of Caenorhabditis elegans following PAO1 challenge. The combination significantly rescued C. elegans from PAO1 infection and improved its survival rate by 54% at 96 h. In summary, this study is the first to elucidate the mechanism behind the QQ prospects of CiNN (augmented in presence of GeN) by abrogating AHL production and increasing the survival rate of C. elegans, thereby highlighting its anti-virulent properties.
SummaryThiol antioxidants disrupt the oxidative protein folding environment in the endoplasmic reticulum (ER), resulting in protein misfolding and ER stress. We recently showed that thiol antioxidants modulate the methionine-homocysteine cycle by upregulating an S-adenosylmethionine-dependent methyltransferase,rips-1, inCaenorhabditis elegans. However, the changes in cellular physiology induced by thiol-mediated reductive stress that modulate the methionine-homocysteine cycle remain uncharacterized. Here, using forward genetic screens inC. elegans, we discover that thiol reductive stress enhancesrips-1expression via the hypoxia response pathway. We demonstrate that thiol reductive stress activates the hypoxia response pathway and protects against hypoxic stress. Conversely, the hypoxia response pathway protects against thiol reductive stress. The activation of the hypoxia response pathway by thiol reductive stress is conserved in human cells. Finally, we show that enhanced cellular oxygen consumption by thiol reductive stress creates hypoxia. These studies reveal an intriguing interaction between thiol-mediated reductive stress and the hypoxia response pathway.
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