Formatted Title
Multiomics Insights into Fungal-Mediated PFAS Precursor Biotransformation
Background/Objectives
Per- and polyfluoroalkyl substances (PFAS) are extensively found in the environment due to their widespread use in industrial and consumer products. They are also categorized as emerging contaminants and potential endocrine-disruptors and carcinogens due to which a collective effort to identify PFAS degradation mechanisms is underway. At AFFF-impacted sites, fungi are more frequently found in the vadose zones of soil. Early work in the Mahendra Laboratory at UCLA has reported that these isolated fungi are able to biotransform PFAS, but the relevant enzymes and biochemical pathways involved in the biotransformation are yet to be fully elucidated. The objective of this work was to conduct a multiomics analysis on fungi exposed to PFAS, to identify differentially expressed enzymes and transformation intermediates that may help determine specific PFAS biotransformation pathways.
Approach/Activities
Whole genomic sequencing analyses were performed to predict and annotate protein coding genes for a well-characterized fungus, and for fungi isolated from AFFF-impacted regions. To study the differential expression of proteins, a well-characterized strain of a wood-decaying fungus was cultivated in different growth media under aerobic conditions. Disappearance of 10 mg/L of 6:2 FTS and PFOA was measured in triplicate experimental flasks over 30 days. Abiotic, killed and PFAS free controls were also monitored simultaneously. The sample’s pellet and supernatant were separated via centrifugation and the total intracellular and extracellular protein content in both conditions was quantified using the Lowry assay. The pellets were washed with deionized water prior to freezing. The proteomics analysis was then conducted by the UCLA Proteomics Center through Mass Spectroscopy, and data analysis was performed using MaxQuant, followed by functional enrichment analysis using the annotated whole genomes to characterize these proteins. Lastly, in 6:2 FTOH biotransformation and metabolomics experiments, fungi were incubated in modified Kirk medium in the presence and absence of sulfate for 60 days at 30C. Control groups include abiotic and killed controls. At day 0, 30, and 60, pellets were collected and stored in -80 C. The samples are analyzed by liquid chromatograph (LC) coupled to a high-resolution mass spectrometer (MS) via electrospray ionization.
Results/Lessons Learned
Forty-seven oxidoreductases and eight peroxidases were differentially expressed in fungal secretome of fungi cultivated in PFAS exposed conditions as opposed to those grown in conditions without PFASs. Approximately 20 putative P450 proteins appeared to be candidates of interest among the intracellular protein components. Comprehensive data reporting their induction mechanisms and specific roles in PFAS biotransformation are still being characterized via proteomics enrichment analysis and will be presented at the conference. Results from ongoing metabolomics experiments will identify intracellular and extracellular products of 6:2 FTS biotransformation and will be valuable in assembling the biodegradation pathways. These results will be valuable in deepening our understanding of fungal transformation of PFAS precursors that have been proposed to not result in the production of terminal perfluoroalkyl acids.