Castaldo, Margherita (2025) Identification and assessment of alternative pathways for bioremediation of polyhalogenated compounds polluted matrices. [Tesi di dottorato]

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Abstract

The aim of this PhD project was to identify new sources of enzymes for the bioremediation of matrices contaminated with Pentachlorophenol (PCP). The project was based on the use of a Drosophila melanogaster strain resistant to PCP (selected in the IPSP laboratory), with the objective to discover new degradation systems and identify active molecules involved in PCP breakdown. Insect resistance can also depend on microorganisms residing in the gut. Therefore, the role of the intestinal microbiota in the resistance of D.melanogaster was evaluated. Lines deprived of gut microbiota and lines with a known microbiome were tested in the presence of PCP. The effect of PCP exposure on the composition of the gut microbiome was studied using metagenomic and metatranscriptomic approaches. Furthermore, PCP degradation assays were conducted to confirm the degradative capacity of the resistant strain and to compare degradation levels between the resistant strain and the resistant axenic line. RNA-seq analysis was conducted to identify genes constitutively expressed in the resistant strain and those induced by PCP exposure. The most promising genes, known to be involved in detoxification pathways, were expressed in E. coli to correlate overexpression with PCP degradation. Degradation tests with the recombinant enzymes extracts and the whole bacterial cells expressing the enzymes were carried out to evaluate their ability to degrade PCP. Overall, the results demonstrated that Drosophila melanogaster resistance to pentachlorophenol (PCP) is not mediated by its gut microbiota but rather by intrinsic mechanisms of the host. Although the resistant strain exhibited a significant ability to degrade PCP, the microbiome composition and activity remained largely unaffected by exposure to the contaminant. Transcriptomic data indicated that PCP exposure strongly induced detoxification-related genes, particularly Cytochrome P450s and Glutathione S-transferases. Among these, CYP6W1, CYP6D5, and GSTD2 were expressed in E. coli. In vitro degradation assays showed no direct PCP degradation activity of the singular extracts of each enzyme. Preliminary evidence of cooperative interactions among these enzymes suggests a possible multi-enzyme detoxification mechanism in D. melanogaster. In conclusion, these findings enhance the understanding of insect responses to persistent pollutants and open new perspectives for the use of Drosophila as a model in environmental toxic-genomics and bioremediation research.

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