implementing multiple resistant mechanisms. Among processes that bacteria implement to contribute to the evolution of antibiotic resistance, the SOS pathway plays a central role. The SOS response is orchestrated mainly by two enzymes: the first, RecA, is the stress sensor, which promote self-cleavage of the second protein, LexA, involved in the system as transcriptional repressor and SOS regulator: LexA cleavage results in induction of the SOS effectors. The activation of the SOS response is directly related to bacteria ability to evolve into resistant bacteria by development of intrinsic resistance and/or acquisition of antimicrobial resistance genes. RecA is a highly conserved protein that plays a critical role in homologous recombination and in LexA activation. Structurally, RecA consists of three domains with a central core surrounded by smaller regulatory domains. RecA monomers can form large nucleoprotein filaments on ssDNA. RecA binds ATP, which is required for filament formation, at the monomer-monomer interface. The nucleofilament is the prerequisite for LexA activation via allosteric modulation. Thus ATP or ssDNA binding sites are potential targets for inhibition. At this purposes several secondary metabolites form lichens belonging to depside, depsidone and acyl-itaconic acid derivatives where investigated for their ability to inhibit RecA. All compounds where characterized to be pure at least at 99% as demonstrated by TLC and H1 NMR. recA gene, previously His-tagged at N-terminus, has been cloned in an overexpression vector (pET 28b) and the protein purified by Hi-Trap. ATPase activity has been monitored by following the production of free phosphate by malachite green. The percentage of inhibition was calculated at 100 µM of each compound dissolved in 0.01% of Triton-X. From 30 tested compounds only 9 showed a percentage of inhibition above 50%. The IC50 calculated for each of them was ranging from 14 µM for protolichesterinic acid to 42 µM for sphaerophorin. We further investigated protolichesterinic acid in order to assess the mechanism of inhibition and the potential site of inhibition. First of all the ATPase activity of RecA was monitored at several concentration of ATP (from 30 to 600 µM) and fix concentration of ssDNA (polydT 36 mer). The plot of IC50 as function of [ATP]/KMATP showed a non-competitive mechanism of inhibition. However, when the same experiment has been performed at different concentration of polydT (from 9.8×10-4 to 2 µM) and 600 µM of ATP, the plot of IC50 as function of [polydT]/KMpolydT showed a possible competitive mechanism of inhibition. Since RecA is able to hydrolyse ATP only when is bound to ssDNA, its reduced ATPase activity in presence of protolichesterinic acid can be ascribed to its ability to bind the ssDNA binding site. Further investigations on the mechanism of action of protolichesterinic acid and the other most active compounds are actually carried on. Keywords: SOS response; RecA protein, lichen compounds
SOS response in bacteria: inhibitory activity of lichen secondary metabolites against RecA protein / Di Pietro, Letizia; Bellio, Pierangelo; Mancini, Alessia; Tondi, Donatella; Spyrakis, Francesca; Cendron, Laura; Nicoletti, Mario; Piovano, Mario; Perilli, Mariagrazia; Celenza, Giuseppe. - (2016). ((Intervento presentato al convegno IV International Conference on Antimicrobial Research – ICAR2016 tenutosi a Torremolinos-Malaga (Spain) nel 29 June – 1 July 2016.