Kinetics caracterization of Ohr from Acinetobacter baumannii, a protein involved in bacterial resistence to antibiotics

• Grant number: 24/03340-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: June 01, 2024
• End date: May 31, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Enzymology
• Principal Investigator: Luis Eduardo Soares Netto
• Grantee: Melissa Siolin Martins
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 13/07937-8 - Redoxome - Redox Processes in Biomedicine, AP.CEPID

Abstract

Ohrs (organic hydroperoxide resistance proteins) are peroxidases involved in the defense of bacteria against, mainly peroxynitrite and organic hydroperoxides, such as fatty acid peroxides, being absent in vertebrates and vascular plants, but present in a wide range of microorganisms (including some pathogens) with high structure conservation (Meireles et al., 2017). Therefore, an inhibitory molecule can specifically. Ohrs from pathogens, possibly not affecting hosts with interest in medicine and/or agriculture. This project focus on Ohr from Acinetobacter baumanii, a bacterium of high risk to global health that is on the World Health Organization (WHO) list of organisms super resistant to antibiotics. Therefore, the group led by Dr. Wei Huang (Shenzhen People's Hospital, China) identified a molecule (6-fluorobenzo[d]isothiazol-3(2H)-one = 6D1) that inhibited Acinetobacter baumannii growth. Notably, mutant strains that developed resistance to 6D1 presented a mutation in a catalytic residue (Arg15) in the plasmid-derived ohrB gene. Therefore, this project aims to obtain information about the enzymatic mechanisms underlying the involvement of OhrB in the inhibition of Acinetobacter baumannii by 6D1 compound. Therefore, wild-type and OhrBR15C were expressed in Escherichia coli and purified by Nickel affinity chromatography through their His tag. Lipoamide-Lipoamide desidrogenase coupled assays were undertaken and preliminary results indicated that the R15C mutations provoked a marked decrease in the OhrB activity. Then, we intend to carry out enzymatic assays to investigate a possible inhibitory effect of the 6D1 molecule on OhrB.