Evaluation of bioactive molecules as inhibitors for atioxidant system of typical 2-Cys peroxiredoxins (AhpC) in baceria

• Grant number: 17/06263-4
• Support type: Scholarships in Brazil - Doctorate
• Effective date (Start): July 01, 2017
• Effective date (End): June 02, 2020
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Cooperation agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
• Principal researcher: Marcos Antonio de Oliveira
• Grantee: Melina Cardoso dos Santos
• Home Institution: Instituto de Biociências (IB-CLP). Universidade Estadual Paulista (UNESP). Campus Experimental do Litoral Paulista. São Vicente , SP, Brazil
• Associated research grant: 13/07937-8 - Redoxome - Redox Processes in Biomedicine, AP.CEPID

Abstract

The resistance to antibiotics by microorganisms has increased dramatically and experts have warned about the rapid inefficacy of drugs against super-resistant bacteria strains. Recent studies indicate that bacterial resistance to antibiotics may be directly related to oxidative stress, since distinct antibiotics have the convergent ability to produce reactive oxygen species (ROS), which cause damage to macromolecules, especially DNA, which may contribute to the appearance of super-resistant strains. ROS are also produced by the host immune system to combat pathogens and studies indicate that inhibition of the pathogen antioxidant enzymes expression is able to generate a significant ROS intracellular increase, which substantially interferes with the pathogens survival. It has recently been shown that a molecule is able to inhibit the peroxidatic activity of peroxidases called peroxiredoxins (Prx), resulting in tumor cell death but presenting low toxicity to normal cells. Prx are able to decompose hydroperoxides using a cysteine residue (CP) which, after peroxide decomposition, is generally reduced by the enzymes Trx and TrxR. It should be noted that the high CP reactivity is achieved by interactions with the residues of Thr / Ser and Arg, absolutely conserved among the Prx. Together, CP, Thr / Ser and Arg, are called catalytic triad (CT). Prx are also present in bacteria and are called AhpC, being considered one of the fundamental defense fronts for the pathogen resistance to the oxidative explosion process promoted by the host immune system. However, in these organisms, AhpC reduction is effected mainly by an enzyme called AhpF, although the bacterial Trx and TrxR can also perform this function. In a previous project, we evaluated the effects of CT amino acid substitutions on the structure and function of Prx. We show that the substitution of a threonine (Thr) residue, present in Prx of eukaryotes, by serine (Ser) leads to functional and structural alterations of these proteins. In my master's degree, we demonstrated that the inhibitor mentioned above has a higher affinity for enzymes containing Ser on CT, and that this substitution exists essentially in prokaryotes, especially in bacteria. Cytotoxicity assays indicated that this molecule has low toxicity over Escherichia coli, which possess a Thr on AhpC CT, whereas toxicity is high for those having Ser, as is the case of the pathogens Staphylococcus aureus and Staphylococcus epidermidis. However, although this molecule presents great potential for use as a bactericide, additional studies are necessary for a better understanding of its activity. In this project, we will initially evaluate the inhibition of this compound over the peroxidatic activity AhpC from bacteria that have Thr as part of the TC and that naturally have the substitution of Thr-Ser, as well as the effect of the toxicity of this inhibitor over the bacteria. Subsequently, we will investigate the action of this molecule using AhpC and AhpCT44S proteins from E. coli. We will evaluate in vitro its capacity of inhibition on these proteins using two reducing systems involved in the reduction of AhpC (Trx-TrxR and AhpF) in E. coli lines ”ahpc, ”trxa, ”txR and ”ahpf. To assess whether the inhibition is Ser- or Thr-dependent on CT, E. coli ”hpc lines will be transformed with plasmids carrying the gene encoding the wild-type E. coli AhpC enzyme (pPROEX-ahpc) or the AhpCT44S mutant enzyme (pPROEX - ahpct44s). Finally, it will be selected commercial and extracted from Brazil plants molecules that present structural and functional similarities with the compound in question and will be evaluated the inhibitory potential over AhpC enzymes containing Thr or Ser. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

DE OLIVEIRA, MARCOS ANTONIO; TAIRUM, CARLOS A.; SOARES NETTO, LUIS EDUARDO; PIRES DE OLIVEIRA, ANA LAURA; ALEIXO-SILVA, ROGERIO LUIS; MONTANHERO CABRERA, VITORIA ISABELA; BREYER, CARLOS A.; DOS SANTOS, MELINA CARDOSO. Relevance of peroxiredoxins in pathogenic microorganisms. Applied Microbiology and Biotechnology, v. 105, n. 14-15 JUL 2021. Web of Science Citations: 0.

TAIRUM, CARLOS A.; SANTOS, MELINA CARDOSO; BREYER, CARLOS ALEXANDRE; PIRES DE OLIVEIRA, ANA LAURA; MONTANHERO CABRERA, VITORIA ISABELA; TOLEDO-SILVA, GUILHERME; MORI, GUSTAVO MARUYAMA; TOYAMA, MARCOS HIKARI; SOARES NETTO, LUIS EDUARDO; DE OLIVEIRA, MARCOS ANTONIO. Effects of Serine or Threonine in the Active Site of Typical 2-Cys Prx on Hyperoxidation Susceptibility and on Chaperone Activity. ANTIOXIDANTS, v. 10, n. 7 JUL 2021. Web of Science Citations: 1.

DE PAULA, CARLA PERES; DOS SANTOS, MELINA CARDOSO; TAIRUM, CARLOS A.; BREYER, CARLOS ALEXANDRE; TOLEDO-SILVA, GUILHERME; TOYAMA, MARCOS HIKARI; MORI, GUSTAVO MARUYAMA; DE OLIVEIRA, MARCOS ANTONIO. Glutaredoxin-like protein (GLP)-a novel bacteria sulfurtransferase that protects cells against cyanide and oxidative stresses. Applied Microbiology and Biotechnology, v. 104, n. 12 APR 2020. Web of Science Citations: 0.