MULTI-OMICS ANALYSIS ON PROKARYOTIC MODEL TO EXPLORE THE TARGETS OF BIOACTIVE NATURAL MOLECULES

THE THREAT OF BACTERIAL RESISTANCE TO ANTIBIOTICS IS A MAJOR CONCERN, THUS MAKING THE DISCOVERY OF NEW MOLECULES WITH ANTIMICROBIAL ACTIVITY A CENTRAL GOAL OF BIOMEDICAL RESEARCH. THIS PHD PROJECT AIMS TO IDENTIFY A PLANT DERIVED MOLECULES WITH ANTIMICROBIAL ACTIVITY AND ELUCIDATE THEIR MECHANISMS OF ACTION. FIRSTLY, THE STUDY OF THE LABDANE DITERPENOID MANOOL, A SPECIALIZED METABOLITE OF SALVIA GENUS, LAMIACEAE, WAS CARRIED OUT. THE ANTIMICROBIAL ACTIVITY OF MANOOL WAS TESTED ON DIFFERENT BACTERIAL STRAINS, BOTH GRAM+ AND GRAM, AND THE MOST PROMISING ACTIVITY WAS OBSERVED TOWARDS STREPTOCOCCUS MUTANS. DRUG AFFINITY RESPONSIVE TARGET STABILITY (DARTS) ASSAY COUPLED WITH MASS SPECTROMETRY WAS USED AS A QUICK APPROACH TO IDENTIFY THE PUTATIVE MOLECULAR TARGET(S) OF MANOOL IN S. MUTANS. DARTS WAS PRELIMINARY PERFORMED ON PROTEIN EXTRACT (PE-DARTS) OF S. MUTANS IN PRESENCE OR NOT OF MANOOL, AND SUBSEQUENTLY IT WAS CARRIED OUT ON LIVE BACTERIA. HOWEVER, APPLYING DARTS TO BACTERIAL SYSTEMS PRESENTS INTRINSIC CHALLENGES DUE TO THE RAPID REPLICATION AND SHORT GENERATION TIMES OF PROKARYOTIC CELLS. THESE FAST DYNAMICS CAN QUICKLY TRIGGER REGULATORY RESPONSES, SUCH AS TRANSCRIPTIONAL AND TRANSLATIONAL CHANGES, WHICH CAN MAKE IT DIFFICULT TO INTERPRET DARTS RESULTS. SO, EXPERIMENTAL CONDITIONS WERE OPTIMIZED TO ENABLE THE EFFECTIVE APPLICATION OF DARTS IN BACTERIAL CELLS, TO PRESERVE THE INTEGRITY OF THE PROTEIN/LIGAND INTERACTION SNAPSHOT THAT DARTS AIMS TO OBTAIN. THESE APPROACHES ALLOWED THE IDENTIFICATION OF THE ATP-BINDING CASSETTE (ABC) SUPERFAMILY. IN DEEPER, ATP-BINDING CASSETTE PROTEINS FORM A LARGE FAMILY INVOLVED IN THE ACTIVE TRANSPORT OF SEVERAL MOLECULES, USING ATP TO MOVE SUBSTRATES ACROSS CELL MEMBRANES. SUBSEQUENTIALLY, THE PUTATIVE TARGETS OF MANOOL IN S. MUTANS WERE VALIDATED. TO CONFIRM ATP-BINDING CASSETTE (ABC) TRANSPORTERS AS POTENTIAL TARGETS OF MANOOL, AMINO ACID AND SUGAR UPTAKE ASSAYS WERE PERFORMED USING LC-MS AND NMR ANALYSES. SEVERAL STUDIES HAVE SHOWN THAT ABC TRANSPORTERS ARE INVOLVED IN THE TRAFFICKING OF VARIOUS SUBSTRATES ACROSS MEMBRANES, SUGGESTING THEY MAY PLAY A KEY ROLE IN BACTERIAL SUSCEPTIBILITY TO ANTIBIOTICS. THEREFORE, INHIBITION OF ABC TRANSPORTERS BY MANOOL COULD ENHANCE THE EFFICACY OF OTHER ANTIBACTERIAL DRUGS. TO TEST THIS HYPOTHESIS, LC-MS ANALYSIS OF THE SUPERNATANTS OF S. MUTANS INCUBATED WITH AND WITHOUT MANOOL IN COMBINATION WITH KANAMYCIN WAS PERFORMED, ALONG WITH EXPERIMENTS MEASURING THE FRACTIONAL INHIBITORY CONCENTRATION INDEX (FICI) FOR MANOOL AND KANAMYCIN. IN ORDER TO ELUCIDATE THE POSSIBLE STRESS RESPONSE AND THE PRINCIPAL PATHWAY INVOLVED IN MANOOL TREATMENT, S. MUTANS WAS SUBJECTED TO CHEMICAL PROTEOMICS STUDIES. THE RESULTS SHOWED REGULATION OF ABC TRANSPORTERS AND SEVERAL PROTEINS INVOLVED IN STRESS RESPONSES. BASED ON THESE RESULTS, THE DOWNSTREAM EFFECTS OF MANOOL IN S. MUTANS WERE INVESTIGATED. FIRSTLY, S. MUTANS WAS CULTURED IN CO-CULTURE WITH HUMAN GINGIVAL FIBROBLASTS (HGF-1). INCUBATING MANOOL AND S. MUTANS IN CO-CULTURE WITH EUKARYOTIC CELLS ENABLED THE ROLE OF THE DITERPENE IN THE ADHESION AND INVASION OF S. MUTANS IN HGF-1 TO BE STUDIED. SUBSEQUENTLY, AN EVOLUTION EXPERIMENT WAS CONDUCTED TO STUDY S. MUTANS' RESPONSE TO INCREASING CONCENTRATIONS OF MANOOL OVER LONG PERIODS. AFTER EIGHT WEEKS, PROTEOMICS AND GENOMICS ANALYSES WERE PERFORMED TO INVESTIGATE THE POSSIBLE MUTATIONS UNDER MANOOL TREATMENT. THE EXPERIMENTAL DESIGN USED FOR INVESTIGATION OF MANOOL'S TARGET ON S.MUTANS WAS APPLIED ON AMMORESINOL, A SESQUITERPENOID COUMARIN FROM FERULA AMMONIACUM (APIACEAE), THAT EXHIBITED AN INTERESTING ANTIMICROBIAL ACTIVITY. DARTS EXPERIMENTS ON PROTEIN EXTRACTS (PE-DARTS) AND BACTERIAL CELLS (BC-DARTS) IDENTIFIED PROTEIN TARGETS INVOLVED IN PEPTIDOGLYCAN SYNTHESIS AND BACTERIAL VIRULENCE. THE DARTS EXPERIMENTS IDENTIFIED PRFA AS A MAJOR TARGET. A TARGETED PROTEOMICS ASSAY WAS PERFORMED TO CONFIRM THE INTERACTION BETWEEN AMMORESINOL AND PRFA.