Biological detoxification of p-cresol by intestinal Enterococcus faecalis UTD-3 Vijayasarathy Muthu; Brindha Priyadarisini Venkatesan, Department of Microbial biotechnology, Bharathiar University - 641046 Background: Retention of dietary carcinogen such as p-cresol in the intestine of hemodialysis patients causes serious health concern related with chronic kidney disease(CKD), cardiovascular diseases (CVD) and Uremia. Several strategies are under progression for controlling pcresol toxicity, however, bacterial detoxification becomes the promising approach (Spanogiannopoulos et al. 2016). Rationale: Over the past decades, fecal microbiota transplantation(FMT) becomes the standard regimen in controlling various metabolic disorder arised due to retention of toxic metabolites (Kelly 2013). With this hypothesis, the rationale of present study was to explore the metabolic capability of intestinal bacteria for reducing p-cresol toxicity. Strategy: p-Cresol metabolizing bacteria was isolated from the human feces and screened based on p-cresol tolerance growth profile. The isolated potential strains evaluated for metabolic ability from its extracellular (Ext) and intracellular(Int) protein extract. Then the higher efficiency of p-cresol metabolism were noticed by optimizing the parameters such as pH, temp and incubation time in relevant to intestinal conditions. Further the molecular evidence of p-cresol metabolism was investigated through Sequencing analysis. Results: Based on the tolerance assay, the potential bacterial strains was screened and identified as Enterococcus faecalis and these results are published in our recent article (Vijayasarathy et al. 2020). Further the optimization studies shows that Ext extract possess higher efficiency of p-cresol reduction compare to the bacterial cultures. Moreover the next generation sequencing analysis evidenced with the regulation of gene expression pattern during p-cresol exposure. Graphical representation of p-cresol Toxicity (Chang et al. 2014) Pro-inflammatory effect Tissue fibrosis
Impaired angiogenesis
P-Cresol
Atherosclerosis
Vascular damage Leukocyte dysfunction
Uremia Endothelial dysfunction
Downregulated genes
Thrombosis
Workflow
1
• Screening of p-cresol tolerance
2
• Evaluation of p-cresol metabolizing property
3
• Optimization of p-cresol metabolism in Medium
4
• Optimization of p-cresol metabolism in Extract
5
• Gene expression Studies (NGS)
Results
Optimisation studies of p-cresol metabolism
ABC transporters - Glutamine ABC superfamily ATP binding cassette transporter, (membrane protein) - Amino acid ABC superfamily ATP binding cassette transporter, Upregulated genes (membrane protein) Phosphotransferase system - PTS family lactose/cellobiose - Glutamine ABC superfamily ATP binding cassette transporter, porter component IIC (binding protein) - PTS family porter, enzyme I -PTS family fructose/mannitol Purine metabolism - arcC2; carbamate kinase (fru) porter component IIA - agxt; aminotransferase Starch & sucrose metabolism - allB; allantoinase - bglA2; 6-phospho- β β-Lactam resistance glucosidase - oligopeptide ABC superfamily - celA4; PTS family porter, - ATP binding cassette transporter, enzyme I (binding protein) - treB; PTS family trehalose - oppA; oligopeptide ABC porter, IIBC component superfamily ATP binding cassette - yvdM; beta transporter,(binding protein) hosphoglucomutase Conclusion E.faecalis(UTD-3) capable of metabolizing p-cresol was isolated from human feces. Moreover, it is evident that compare to bacterial cultures metabolizing efficiency was higher with extracellular extract with active proteins or enzymes. Also these active agents with stability in varied pH and thermostability can foresee the tendency of p-cresol metabolism through gastro-intestinal tract. NGS results revealed that the complex gene expression pattern, involving induction of PTS system accompanied with inhibition in transporters were regulated for p-cresol metabolism and tolerance by E. faecalis. From this study, it is evidenced that the p-cresol metabolizing E. faecalis becomes a promising candidate in mitigating p-cresol toxicity in the hemodialysis pateints. References 1. Vijayasarathy,M., Kiran,G.K., Balaji, S et al.(2020).In vitro detoxification studies of pCresol by intestinal bacteria isolated from human feces.Curr microbial.77(10):30003012. 2. Chang, M. C., Chang, H. H., Chan, C. P., et al. (2014). p-Cresol affects reactive oxygen species generation, cell cycle arrest, cytotoxicity and inflammation/atherosclerosisrelated modulators production in endothelial cells and mononuclear cells. PloS one, 9(12):e114446. 3. Kelly CP. (2013) Fecal microbiota transplantation--an old therapy comes of age. N Engl J Med. 368(5):474-475. 4. Spanogiannopoulos, P., Bess, E. N., Carmody, R. N., et al. (2016). The microbial pharmacists within us: a metagenomic view of xenobiotic metabolism. Nat rev. Microbiol, 14(5), 273–287. www.postersession.com
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