Salman, Mohammed (2025) The Role of LuxR/LuxI-Quorum Sensing based System in P. gessardii in the Regulation of Proteolytic Activity: Insights into QS Inhibition as a Strategy for Control of Spoilage Activity. [Tesi di dottorato]

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Abstract

Quorum sensing (QS) is a bacterial signaling mechanism regulating the expression of genes associated with spoilage and virulence. In dairy products, psychrotrophic Pseudomonas spp. can proliferate during cold storage and produce thermostable proteases that compromise milk quality and processing. This study aimed to characterize the acyl-homoserine lactone (AHL)-mediated QS system in Pseudomonas gessardii isolated from raw milk and to investigate the potential of bioactive polyphenols and postbiotics, to mitigate proteolytic spoilage activity by QS inhibition. Raw milk samples were screened for highly proteolytic bacteria, which were identified by MALDI-TOF mass spectrometry and 16S rRNA sequencing. The AHL profile of the isolates was characterized using biosensor strains, thin layer chromatography-overlay assay, and LC-MS analysis. Whole-genome sequencing provided insights into the presence of LuxRI-type QS systems. The AHL-degrading enzyme lactonase was heterologously expressed in the strains to investigate the contribution of AHL signals to spoilage phenotype and proteolytic activity in comparison with wild-type strains. Fifty polyphenols were virtually screened by molecular docking analysis for their ability to interact with the 3D model of LuxR protein, which was built from the amino acid sequences of P. gessardii obtained from whole genome sequencing. The compounds that scored higher the reference ligand of the LuxR (C4-HSL) were tested for their inhibitory effect on proteolytic activity at 4 °C and 25 °C in skim milk. Six highly proteolytic isolates were identified as P. gessardii. They were found to produce short chain AHL, mainly C4-HSL. Whole genomic sequencing revealed for the first time presence of diverse LuxI and LuxR solo genes in P. gessardii. Conjugated P. gessardii strains with the expression of the AHL-degrading enzyme lactonase confirmed the regulatory role of AHLs in proteolytic activity. In addition, construction of mutant in the AHL-synthase genes reported that rhlI-like gene is involved in the regulation of proteolytic activity. Cinnamaldehyde and salicylic acid were found to compete with C4-HSL to bind with the LuxR. These 2 compounds showed significant inhibition of proteolytic activity without antibacterial effects. Furthermore, postbiotic compounds from Lactobacillus acidophillus LA-5 were also shown to exert anti-proteolytic activity without antimicrobial effect. Docking analysis supporting their function as quorum sensing inhibitors by bind with the LuxR receptors. This work provides new insights into QS-regulated spoilage mechanisms in dairy-associated Pseudomonas and demonstrates the potential of polyphenols and postbiotics to stabilize raw milk by restricting proteolytic activity. These results highlight a sustainable strategy to preserve food quality and nutritional value.

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