ACACIAIN PEPTIDASE OF ACACIA CAVEN (MOL.) MOLINA, ANTIVIRULENCE CAPACITY AGAINST FOOD CONTAMINANTS AND HUMAN PATHOGENIC BACTERIA

QUIROGA, HG; BARBERIS, SE; RIVEROS,L; DANILOVICH, MARIANA; ARENA, MARIO; ALBERTO, MARIA ROSA

San Juan, Argentina

Congreso; XLI Reunión Científica Anual de la Sociedad de Biología de Cuyo; 2023

Sociedad de Biología de Cuyo

ACACIAIN PEPTIDASE OF ACACIA CAVEN (MOL.) MOLINA, ANTIVIRULENCE CAPACITYAGAINST FOOD CONTAMINANTS AND HUMAN PATHOGENIC BACTERIAQuiroga HG1, Barberis SE1,2, Riveros L1, Danilovich M3, Arena ME2,3, Alberto MR2,31Laboratorio de Control de Calidad y Desarrollo de Bromatología, Universidad Nacional de San Luis, San Luis, Argentina. 2ConsejoNacional de Investigaciones Científicas y Técnicas (CONICET). 3Instituto de Biotecnología Farmacéutica y Alimentaria, Universidad Nacionalde Tucumán, San Miguel de Tucumán, Argentina. E-mail: soniaebarberis@gmail.comAcacia caven (Mol.) Molina is an autochthonous and plentiful plant of South America. The aims of this work are to characterize the mainproteolytic enzyme of the pollen from Acacia caven (Mol.) Molina, and to study its influence on virulence factor production byStaphylococcus aureus ATCC 6538. A. caven pollen was obtained from natural crops of San Luis, Argentina. Pollen extract was prepared bystirring 6 g of pollen into 100 mL of 50 mM Tris-HCl buffer pH 7.4 overnight at room temperature, centrifuged at 12,000 rpm for 15 min at4ºC, the supernatant was filtered (f: 0.45 μm) and purified. Proteins content was measured according to Bradford method. The proteolyticactivity of acaciain peptidase (215 μg protein/mL) was performed using Boc-L-alanine-4-nitrophenyl ester (Boc-L-Ala-ONp) as substrate.The optimum pH of acaciain peptidase was measured at 30°C and at different pH values, during 30 min. The optimum temperature wasmeasured at pH 8 in the range of 15 - 60°C, during 30 min. The pH stability was determined after diluting the enzyme (1:2) in the appropriatedbuffer (pH 4-11) and incubating at 30°C for 0 - 12 h. For testing thermal stability, the enzyme was incubated at pH 8 and from 25 to 60°Cfor 0 – 12 h. This enzyme exhibited the highest specific proteolytic activity at 25°C and pH 8, but when the temperature was increased from25 to 60 °C only 35 % of that maximum activity was maintained. The enzyme was almost completely inactivated by heating at 40 - 65°Cduring 3 h. The effect of the enzyme on the production of virulence factors of S. aureus was also evaluated. At 20 and 40 μg/ml (MIC) ofacaciain peptidase did not significantly influence on the growth of the pathogen, but the bacterial biofilm formation index (DO biofilm/DOgrowth ratio) decreased until 32% and 67%, indicating an antipathogenic effect at those concentrations. Ciprofloxacin (control) reduced thatindex by 32%. When 5, 10, 20 and 40 μg/ml of the enzyme were added to the culture medium, a decrease in the hemolysin production rate(hemolysin OD/growth OD ratio) of 25%, 95%, 96% and 97%, respectively was observed. As a control, Triton X100 was used, whichinduced 100% hemolysis. Acaciain peptidase into the culture medium also resulted in a decrease in the production of coagulase, anothervirulence factor of S. aureus. This fact was evidenced by a delay in clot formation compared to the control without enzyme, with a delay of30 and 90 min in the presence of 5 and 10 μg/ml of enzyme. At 20 and 40 μg/ml, the clot did not form until 240 min, indicating completeinhibition of bacterial coagulase production. Acaciain peptidase inhibited the formation of virulence factors by S. aureus at subinhibitoryconcentrations, which could have significant implications in the development of new therapeutic strategies against to S. aureus